Synthesis, evaluation, and biological applications of visible-light-controllable nitric oxide releasers

Article abstract of DOI:10.1016/bs.mie.2020.04.002

Nitric oxide (NO) is biologically synthesized in human body and mediates various signal pathway. Because NO is too unstable to handle for biological assay, NO releasers had been developed for NO research. Among them, light-controllable NO releasers are quite useful tool because their NO release can be spatiotemporally controlled by light irradiation. This article shows how to synthesize visible-light controllable NO releasers based on N-nitrosoaminophenol structure, evaluate NO releasing efficiency in various methods, and apply them for biological experiments.

Full text of DOI:10.1016/bs.mie.2020.04.002

CHAPTER TWO
Synthesis, evaluation, and
biological applications of
visible-light-controllable nitric
oxide releasers
Naoya Ieda, Hidehiko Nakagawa∗
Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya, Aichi, Japan
∗Corresponding author: e-mail address: deco@phar.nagoya-cu.ac.jp
Contents
1. Introduction
2. Protocols: Synthesis
38
39
39
40
44
46
49
51
53
56
58
60
60
60
2.1 Materials, equipment, and reagents
2.2 Synthesis of NOBL-1
2.3 Synthesis of NO-Rosa5
2.4 Synthesis of NO-Rosa-Gal
3. Protocols: ESR spin trapping method with iron-dithiocarbamate complex
4. Protocols: NO electrode
5. Protocols: NO detection by fluorescence probes
6. Protocols: Determination of quantum yield
7. Protocols: Photocontrol of vasodilation using rat aorta strip.
8. Summary
Acknowledgment
References
Abstract
Nitric oxide (NO) is biologically synthesized in human body and mediates various signal
pathway. Because NO is too unstable to handle for biological assay, NO releasers had
been developed for NO research. Among them, light-controllable NO releasers are quite
useful tool because their NO release can be spatiotemporally controlled by light
irradiation. This article shows how to synthesize visible-light controllable NO releasers
based on N-nitrosoaminophenol structure, evaluate NO releasing efficiency in various
methods, and apply them for biological experiments.
#
Methods in Enzymology, Volume 640
ISSN 0076-6879
https://doi.org/10.1016/bs.mie.2020.04.002
2020 Elsevier Inc.
All rights reserved.
37

38
Naoya Ieda and Hidehiko Nakagawa
1. Introduction
Nitric oxide (NO) is biosynthesized in human body and plays important
roles to induce various biological events, such as vasodilation, neurotransmis-
sion, and immune response (Bodgan, 2001; Ignarro, Buga, Wood, & Byrns,
1987; Kyriakatos et al., 2006; Moncada, Palmer, & Higgs, 1991). However, it
is very difficult to handle NO because it is odorless, colorless, and short-life gas
under ambient condition. In order to control NO’s bioactivity, NO releasing
small compounds (NO releasers) have been developed so far (Wang et al.,
2002). Among them, light-controllable NO releasers allow to analyze the
spatiotemporally specific bioactivity of NO, and are also candidate chemother-
apeutic agents (Eroy-Reveles, Leung, Beavers, Olmstead, & Mascharak, 2008;
Hishikawa et al., 2009; Karaki, Kabasawa, Yanagimoto, & Umeda, 2012;
Makings & Tsien, 1994; Namiki, Arai, & Fujimori, 1997; Sortino, Petralia,
Compagnini, Conoci, & Condorelli, 2002).
We designed and synthesized blue- or green-light-controllable NO
releasers, which bear N-nitrosoaminophenol moieties for NO release tethered
to visible-light absorbing antenna moieties, BODIPY or rosamine (Hotta
et al., 2016; Ieda, Hotta, Kawaguchi, Kimura, & Nakagawa, 2019; Ieda,
Hotta, Miyata, Kimura, & Nakagawa, 2014; Ieda et al., 2019; Okuno
et al., 2017). Photoinduced electron transfer from N-nitrosoaminophenol
to the antenna moiety upon irradiation with relatively noncytotoxic visible
light should result in NO release with formation of a stable quinone
(Fig. 1). Based on this mechanism, we synthesized a blue-light controllable
NO releaser, NOBL-1 (around 500nm light), yellowish-green-light-
controllable one, NO-Rosa5 (around 560nm light), and NO-Rosa-Gal
which can work as a double-lock-type NO releaser in response to enzymatic
reaction of β-galactosidase and photochemical reaction of yellowish-green-
light (Fig. 2). NO release from these compounds can be confirmed by
Fig. 1 Plausible mechanism of photoinduced electron transfer induced nitric oxide
releaser.

Visible-light-controllable nitric oxide releasers
39
Fig. 2 Chemical structures of a blue-light-controllable NO releaser NOBL-1, a yellowish-
green-light controllable one NO-Rosa5, and a double-lock type NO releaser NO-Rosa-Gal.
ESR spin trapping, NO electrode, and fluorescence detection. Spatially
controlled NO release in cells can be observed with fluorogenic NO probes
and NO-Rosa-Gal can release NO in cells highly expressing β-glucosidase
specifically. Moreover, combination of these NO releasers and visible light
source can realize photo-vasodilation of rat aorta ex vivo condition. This article
shows how to synthesis, handle, and apply these compounds for biological
experiments.
2. Protocols: Synthesis
NOBL-1, NO-Rosa5, and NO-Rosa-Gal were synthesized accord-
ing to Scheme1–3. Uniformly, aniline derivatives were connected to
antenna moieties having formyl group by reductive amination followed
by N-nitrosylation with NaNO2. Generally, we did not take measures to
protect these compounds from room or nature light, for example covering
reaction flasks with tinfoil. Without any measures, remarkable degradation
was not observed during synthesizing these compounds. Just to be sure
quality of the final products, these compounds should be stored in dark
at ꢀ20 °C.
2.1 Materials, equipment, and reagents
Proton nuclear magnetic resonance spectra (1H NMR) and carbon nuclear
magnetic resonance spectra (13C NMR) were recorded on a JEOL JNM-
LA500, JEOL JNM-A500, Varian VNMRS 500, or Bruker AVANCE600
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 Yanaco CHN CORDER NT-5 analyzer, and
all values were within ꢁ0.4% of the calculated values. All other reagents
and solvents were purchased from Aldrich, Tokyo Kasei Kogyo,

40
Naoya Ieda and Hidehiko Nakagawa
FUJIFILM Wako Pure Chemical Corp., Nacalai Tesque, Kanto Kagaku,
Kishida Kagaku, Junsei Kagaku or Dojindo, and used without purification.
Flash column chromatography was performed using silica gel 60 supplied
by Taiko Shoji. MPLC purification was performed using YFLC-
Wprep2XY-S (Yamazen).
2.2 Synthesis of NOBL-1 (Scheme 1)
1. Preparation of 5: To a solution of 2-hydroxy-5-nitrobenzaldehyde (4)
(1.00g, 5.99mmol) and K2CO3 (997mg, 7.21mmol, 1.2 equiv.) in
DMF (30mL) was added benzyl bromide (860μL, 7.19mmol, 1.2
equiv.). The mixture was stirred for 2h at room temperature, and further
BnBr (215μL, 1.80mmol, 0.3 equiv.) was added to it. Stirring was
continued at room temperature for a further 1.5h, and the mixture
was diluted with AcOEt and washed with water. The organic layer was
washed with brine and dried over Na2SO4. Filtration, evaporation, and
purification of the residue by silica gel flash chromatography (n-hexane:
AcOEt¼3:1!2:1) gave 1.39g (90%) of 5 as a white solid: 1H NMR
(CDCl3, 500MHz, δ; ppm) 10.52 (1H, s), 8.73 (1H, d, J ¼2.8Hz), 8.41
(1H, dd, J ¼2.8 HZ, 9.2Hz), 7.45–7.38 (5H, m), 7.18 (1H, d,
J ¼9.3Hz), 5.33 (2H, s).
2. Preparation of 6: To a slurry of 5 (1.39g, 5.39mmol) and tert-butyl
bromoacetate (1.42mL, 9.74mmol, 1.8 equiv.) in sat. NaHCO3
(30mL) and THF (10mL) was added PPh3 (2.13g, 8.13mmol, 1.5
equiv.). The mixture was stirred at room temperature for 1.5h, then
diluted with water and extracted with CHCl3. The organic layer was
washed with brine and dried over Na2SO4. Filtration, evaporation in
vacuo, and purification of the residue by silica gel flash chromatography
(n-hexane:AcOEt¼8:1) gave 2.17g (q. y.) of 6 as a clear oil: 1H NMR
(CDCl3, 500MHz, δ; ppm, 10:7 mixture of two isomers) (trans) 8.44
(1H, d, J ¼2.8Hz), 8.18 (1H, dd, J ¼2.8Hz, 9.1Hz), 7.94 (1H, d,
J ¼16.2Hz), 7.42–7.37 (5H, m), 7.02 (1H, d, J ¼9.1Hz), 6.55 (1H, d,
J ¼16.1Hz), 5.27 (2H, s), 1.53 (9H, s), (cis) 8.39 (1H, d, J ¼2.6Hz),
8.18 (1H, dd, J ¼2.8Hz, 9.1Hz), 7.42–7.37 (5H, m), 7.03 (1H, d,
J ¼12.4Hz), 6.99 (1H, d, J ¼9.2Hz), 6.03 (1H, d, J ¼12.4Hz), 5.21
(2H, s), 1.40 (9H, s).
3. Preparation of 7: A slurry of 6 (164mg, 0.462mmol) and 5% Pd-C
(97mg) in MeOH (5mL) was stirred at room temperature under H2

Scheme 1 Synthetic scheme of NOBL-1. Reprinted with permission from Ieda, N., Hotta, Y., Miyata, N., Kimura, K., Nakagawa, H. (2014). Journal of
the American Chemical Society. 136, 7085–7091. Copyright 2014 American Chemical Society.

42
Naoya Ieda and Hidehiko Nakagawa
for 18h. The reaction mixture was filtered on Celite and the filtrate was
evaporated in vacuo. The residue was purified by silica gel flash chromatog-
raphy (n-hexane:AcOEt¼1:1) to obtain 86mg (79%) of 7 as a pale yellow
1
solid: H NMR (CDCl3, 500MHz, δ; ppm) 6.95 (1H, s), 6.72 (1H, d,
J ¼8.4Hz), 6.49 (1H, dd, J ¼2.8Hz, 8.4Hz), 6.45(1H, d, J ¼2.8Hz),
3.36 (2H, s), 2.77 (2H, t, J ¼6.3Hz), 2.60 (2H, t, J ¼6.3Hz), 1.41 (9H, s).
4. Preparation of 9: To a slurry of 8 (5.00g, 33.3mmol) in CH2Cl2
(200mL) was added N-methylmorpholine (3.66mL, 33.3mmol, 1.0
equiv.), followed by EDCꢂHCl (7.68g, 40.1mmol, 1.2 equiv.) and
NHMeOMeꢂHCl (3.60g, 36.9mmol, 1.1 equiv.). The mixture was
stirred at room temperature for 4.5h, and then evaporated in vacuo.
The residue was taken up in Et2O and the mixture was washed with
water and brine. The organic layer was dried over Na2SO4. Filtration
1
and evaporation in vacuo gave 4.31g (67%) of 9 as a yellow oil: H
NMR (CDCl3, 500MHz, δ; ppm) 10.07 (1H, s), 7.93 (2H, d,
J ¼8.4Hz), 7.81 (2H, d, J ¼8.2Hz), 3.54 (3H, s), 3.39 (3H, s).
5. Preparation of 10: An intermediate, 2,4-dimethyl-3-cyanopyrrole was
synthesized according to Urano’s paper (Ueno, Urano, Kojima, &
Nagano, 2006). To a solution of 9 (1.26g, 6.51mmol) and 2,4-
dimethyl-3-cyanopyrrole (1.55g, 12.9mmol, 2.0 equiv.) in CH2Cl2
(50mL) was added TFA (0.2mL). The mixture was stirred at room tem-
perature for 19h, and then DDQ (2.21g, 9.71mmol, 1.5 equiv.) was
added to it. Stirring was continued for a further 20min, then
BF3ꢂOEt2 (8.5mL, 32.4mmol, 5.0 equiv.) was added, followed by
diisopropylethylamine (6.0mL, 34.4mmol, 5.3 equiv.) on an ice-water
bath. Stirring was continued for a further 10min on the ice-water bath,
then the reaction mixture was quenched with water and filtered. The
organic layer was separated, and the water layer was extracted with
CHCl3. The combined organic layer was washed with brine and dried
over Na2SO4. Filtration, evaporation in vacuo, and purification of the res-
idue by silica gel flash chromatography (n-hexane:AcOEt¼1:1!1:2)
1
gave 1.92g (64%) of 10 as a dark red solid: H NMR (CDCl3,
500MHz, δ; ppm) 7.88 (2H, d, J ¼8.2Hz), 7.35 (2H, d, J ¼8.3Hz),
4.28 (4H, q, J ¼7.1Hz), 3.51 (3H, s), 3.42 (3H, s), 2.84 (6H, s), 1.68
(6H, s), 1.33 (6H, t, J ¼7.1Hz).
6. Preparation of 11: To a solution of 10 (206mg, 0.447mmol) in dry THF
(10mL) was added Cp2ZrHCl (142mg, 0.551mmol, 1.2 equiv.). The
mixture was stirred for 5min, and then quenched with water. The mix-
ture was extracted with CHCl3. The organic layer was washed with

Visible-light-controllable nitric oxide releasers
43
brine and dried over Na2SO4. Filtration, evaporation in vacuo, and puri-
fication of the residue by silica gel flash chromatography (n-hexane:
1
AcOEt¼2:1) gave 126mg (70%) of 11 as a red solid: H NMR
(CDCl3, 500MHz, δ; ppm) 10.37 (1H, s), 8.31 (2H, d, J ¼8.3Hz),
7.74 (2H, d, J ¼8.1Hz), 4.51 (4H, q, J ¼7.1Hz), 3.07 (6H, s), 1.87
(6H, s), 1.55 (6H, t, J ¼7.1Hz).
7. Preparation of 12: A solution of 7 (130mg, 0.549mmol, 1.0 equiv.), 11
(221mg, 0.550mmol), and AcOH (0.5mL) in CH2Cl2 (5mL) was
stirred at room temperature for 2h, then NaBH(OAc)3 (353mg,
1.67mmol, 3.0 equiv.) was added to it and stirring was continued for
10min. The reaction mixture was quenched with water and extracted
with CHCl3. The organic layer was washed with brine and dried over
Na2SO4. Filtration, evaporation and purification of the residue by silica
gel flash chromatography (n-hexane:AcOEt ¼2:1!3:2) gave 283mg
1
(83%) of 12 as a dark green solid: H NMR (CDCl3, 500MHz, δ;
ppm) 7.61 (2H, d, J ¼8.1Hz), 7.22 (2H, d, J ¼8.1Hz), 6.96
(1H, brs), 6.75 (1H, d, J ¼8.6Hz), 6.43 (1H, dd, J ¼2.9Hz, 8.5Hz),
6.38 (1H, d, J ¼2.8Hz), 4.43 (2H, s), 2.76 (2H, t, J ¼6.2Hz), 2.72
(6H, s), 2.58 (2H, t, J ¼6.2Hz), 1.59 (6H, s), 1.42 (9H, s).
8. Preparation of NOBL-1 (1): A solution of 12 (273mg, 0.438mmol) in
4N HCl AcOEt (7mL) and AcOEt (3mL) was stirred at room temper-
ature for 19.5h. The reaction mixture was neutralized with 2N NaOH
and sat. NaHCO3, and extracted with AcOEt. The organic layer was
washed with brine and dried over Na2SO4. Filtration, evaporation
and purification of the residue by silica gel flash chromatography
(CHCl3:MeOH¼100:1!30:1!10:1) gave 247mg (crude) of 13 as
a dark green solid. A solution of 13 (crude 247mg) in AcOH (6mL)
was added to a solution of NaNO2 (34mg, 0.493mmol) in water (6mL)
on an ice-water bath. Stirring was continued for 10min, then the mixture
was diluted with AcOEt and washed with 10% citric acid. The organic layer
was evaporated in vacuo. The residue was taken up in sat. NaHCO3 and
extracted with CHCl3. The organic layer was washed with brine and dried
over Na2SO4. Filtration, evaporation in vacuo and purification of the residue
by silica gel flash chromatography (n-hexane:AcOEt¼2:1!AcOEt only
! AcOEt:MeOH¼100:1!AcOEt:MeOH¼20:1) gave 113mg (44%)
of NOBL-1 as a red solid. This was recrystallized from n-hexane
and CHCl3 to obtain 75mg (66%) of green crystals: mp 195.3–198.3°C;
1H NMR (DMSO-d6, 500MHz, δ; ppm) 12.08 (1H, brs), 9.82 (1H, s),
7.36 (2H, d, J ¼8.2Hz), 7.32 (2H, d, J ¼8.2Hz), 7.29 (1H, d,

44
Naoya Ieda and Hidehiko Nakagawa
J ¼2.4Hz), 7.15 (1H, dd, J ¼2.7Hz, 8.7Hz), 6.82 (8.5Hz), 5.38 (2H, s),
2.75 (2H, t, J ¼7.8Hz), 2.63 (6H, s) 2.48 (2H, t, J ¼7.8Hz), 1.39 (6H, s);
13C NMR (DMSO-d6, 125MHz, δ; ppm) 173.7, 158.8, 154.9, 149.1,
147.1, 137.1, 132.0, 130.7, 130.7, 129.2, 127.7, 127.5, 123.3, 120.9,
114.9, 113.3, 105.4, 46.8, 33.1, 25.2, 13.5, 13.3; Anal.Calcd for
C31H27BF2N6O4ꢂ4/3H2O; C:60.01, H:4.82, N:13.55. Found; C:60.29,
H:4.79, N:13.28; MS (FAB): m/z 597 [(M+1)+].
2.3 Synthesis of NO-Rosa5 (Scheme 2)
1. Preparation of 15: An intermediate 14 was synthesized according to
Burgess’s paper (Wu & Burgess, 2008). A solution of 2-bromophenyl-
1,3-dioxolane (494μL, 3.30mmol, 3.3 equiv.) in dry THF (12mL) was
stirred at ꢀ78°C under an argon balloon for an hour, then 1.01M
sec-BuLi in n-hexane/cyclohexane (3.0mL, 3.03mmol, 3.0 equiv.) was
added dropwise. Stirring was continued at ꢀ78°C under an argon balloon
for an hour, and then a solution of 14 (366mg, 0.999mmol) in dry THF
(8mLꢃ2) was added. The reaction mixture was removed from the
low-temperature bath and stirred at room temperature for 30min. The
reaction was confirmed to be complete by ESI-MS, then the mixture
was quenched with AcOH (1mL), and evaporated in vacuo. The residue
was dissolved in MeCN (10mL) and 2N HCl (10mL). This solution was
stirred for 40min, then diluted with water (50mL), and extracted with
CH2Cl2 three times. The organic layer was dried over Na2SO4.
Filtration, evaporation in vacuo, and purification of the residue by silica
gel flash chromatography (CH2Cl2/MeOH¼16/1!10/1!5/1) gave
a mixture of aldehyde and methyl hemiacetal. To hydrolyze the hemi-
acetal, the mixture was dissolved in MeCN (10mL) and 2N HCl
(10mL), stirred for a few minutes, diluted with water (50mL), and
extracted with CH2Cl2 three times. The organic layer was dried over
Na2SO4. Filtration, and evaporation of the residue in vacuo gave 429mg
(87%) of 15 as a purple solid: 1H NMR (DMSO-d6, 500MHz, δ; ppm)
9.86 (1H, s), 8.26 (1H, dd, J ¼1.3Hz, 7.3Hz), 7.99–7.92 (2H, m), 7.56
(1H, dd, J ¼1.3Hz, 7.3Hz), 7.25 (2H, dd, J ¼2.4Hz, 9.6Hz), 7.18
(2H, d, J ¼2.4Hz), 6.97 (2H, d, J ¼9.6Hz), 3.76 (8H, t, J ¼5.2Hz),
1.73–1.64 (12H, m).
2. Preparation of 17: To a solution of 16 (362mg, 0.737mmol) in CH2Cl2
(15mL) was added 15 (190μL, 0.811mmol, 1.1 equiv.) and AcOH

Scheme 2 Synthetic scheme of NO-Rosa5.

46
Naoya Ieda and Hidehiko Nakagawa
(1.5mL). The mixture was stirred for an hour, and NaBH(OAc)3
(462mg, 2.18mmol, 3.0 equiv.) was added. Stirring was continued
for 15min, then the mixture was quenched with 0.1N HCl (50mL)
and extracted with CH2Cl2 three times. The organic layer was dried
over Na2SO4, filtered, and concentrated in vacuo. The residue was
dissolved into AcOH (8mL). To this solution was added a solution of
NaNO2 (56mg, 0.812mmol, 1.1 equiv.) in water (8mL) on an ice-
water bath. The mixture was stirred on an ice-water bath for 15min,
then quenched with 0.1N HCl (80mL) and extracted with CH2Cl2
three times. The organic layer was dried over Na2SO4. Filtration,
evaporation in vacuo, and purification of the residue by silica gel flash
chromatography (CH2Cl2/MeOH¼15/1!10/1!4/1) gave 354mg
1
(66%) of a mixture of 17 as a purple solid: H NMR (DMSO-d6,
500MHz, δ; ppm) 7.62–7.54 (2H, m), 7.36–7.32 (4H, m), 7.21
(2H, d, J ¼2.6Hz), 7.15 (2H, d, J ¼9.1Hz), 7.03 (2H, d, J ¼9.3Hz),
6.82 (2H, d, J ¼9.1Hz), 4.96 (2H, s), 3.79 (16H, brs), 0.95 (9H, s),
0.20 (6H, s).
3. Preparation of NO-Rosa5 (2): To a solution of 17 (354mg, 0.487mmol)
in MeCN (7mL) was added 0.1M NaF/HF buffer (7mL). The mixture
was stirred at room temperature for 2.5h, then water (35mL) and
brine (35mL) were added, and the whole was extracted with CH2Cl2
three times. The organic layer was dried over Na2SO4. Filtration, evap-
oration in vacuo, and purification of the residue by silica gel flash
chromatography (CH2Cl2/MeOH¼15/1!9/1!4/1) gave 216mg
(72%) of NO-Rosa5 as a purple solid: 1H NMR (DMSO-d6,
500MHz, δ; ppm) 9.85 (1H, s), 7.62–7.53 (2H, m), 7.21 (2H, d,
J ¼2.3Hz), 7.02 (2H, d, J ¼8.8Hz), 6.99 (2H, d, J ¼9.5Hz), 6.70
(2H, d, J ¼9.0Hz), 4.95 (2H, s), 3.79 (16H, s); 13C NMR (DMSO-
d6, 150MHz, δ; ppm) 157.58, 157.07, 156.82, 155.12, 133.05, 131.90,
130.90, 130.55, 130.27, 129.44, 127.88, 122.00, 115.54, 115.12,
113.95, 97.01, 65.72, 54.81, 47.00, 44.88; MS (ESI) m/z: 577 (M+),
547 ([M–NO]+); Anal. Calcd. for C34H33ClN4O5ꢂ5/2H2O: C, 62.05;
H, 5.82; N, 8.51. Found: C, 62.26; H, 5.73; N, 8.52.
2.4 Synthesis of NO-Rosa-Gal (Scheme 3)
1. Preparation of 19: Intermediate 18 was synthesized according to Wang’s
paper (Zheng, Qiao, Wan, Gao, & Wang, 2008). To a solution of 16

Scheme 3 Synthetic scheme of NO-Rosa-Gal.

48
Naoya Ieda and Hidehiko Nakagawa
(70.5mg, 0.144mmol) and 18 (76.5mg, 0.174mmol, 1.2 equiv.) in
CH2Cl2 (6mL) was added AcOH (0.5mL). The mixture was stirred
for an hour at room temperature, then NaBH(OAc)3 (93.5mg,
0.441mmol, 3.1 equiv.) was added. Stirring was continued for 22.5h
at room temperature. The reaction was confirmed to be complete
by ESI-MS, then the mixture was quenched with 2N HCl and
brine, and extracted with CH2Cl2 three times. The organic layer was
dried over Na2SO4. Filtration, evaporation in vacuo, and purification
of the residue on a Yamazen MPLC system (CH2Cl2/MeOH¼
94/6!90/10!80/20) gave 129mg (98%) of 19 as a purple solid:
1H NMR (DMSO-d6, 500MHz, δ; ppm) 7.48 (1H, d, J ¼9.0Hz),
7.25 (1H, t, J ¼7.5Hz), 7.13 (1H, t, J ¼7.8Hz), 6.69–6.65 (7H, m),
6.55–6.48 (2H, m), 6.43 (2H, d, J ¼10Hz), 5.25 (1H, d, J ¼3.5Hz),
5.13–5.05 (5H, m), 4.25 (1H, t, J ¼6.8Hz), 4.10–4.06 (1H, m),
4.01–3.97 (1H, m), 3.69 (8H, s), 3.09 (8H, s), 2.11 (3H, s), 2.00
(3H, s), 1.91 (3H, s), 1.86 (3H, s).
2. Preparation of 20: A solution of 19 (103mg, 0.113mmol) and NaOMe
(3.2mg, 0.0592mmol, 0.52 equiv.) in dry MeOH (10mL) was stirred at
room temperature under a N2 balloon. The mixture was stirred for 24h,
then 20% NaOEt in EtOH (10μL, 0.0232mmol, 0.21 equiv.) was
added. Stirring was continued for 3h. The reaction was confirmed
to be complete by ESI-MS, then the mixture was quenched with
brine, and extracted with CH2Cl2 three times. The organic layer
was dried over Na2SO4. Filtration, evaporation in vacuo and purification
of the residue on a Yamazen MPLC system (CH2Cl2/MeOH¼
94/6!90/10!80/20!70/30!0/100) gave 73mg (87%) of 20 as
1
a pale purple solid: H NMR (CDCl3, 500MHz, δ; ppm) 7.40 (1H,
d, J ¼7.1Hz), 7.21 (1H, t, J ¼7.8Hz), 7.12 (1H, t, J ¼8.0Hz), 6.80
(1H, d, J ¼7.7Hz), 6.77–6.72 (4H, m), 6.66 (2H, s), 6.49 (2H, d,
J ¼9.0Hz), 6.46–6.43 (2H, m), 5.05 (2H, s), 4.64 (1H, d, J ¼7.9Hz),
4.02 (1H, br), 3.98–3.94 (1H, m), 3.87–3.82 (10H, m), 3.64 (1H, s),
3.57 (1H, s), 3.14 (8H, s).
3. Preparation of NO-Rosa-Gal: 20 (73.3mg, 0.0982mmol) was dissolved
in AcOH (5mL), and a solution of NaNO2 (9.8mg, 0.142mmol, 1.4
equiv.) in water (5mL) was added. The mixture was stirred for an hour.
The reaction was confirmed to be complete by ESI-MS, then the
mixture was evaporated in vacuo. The crude product was purified
by reverse-phase HPLC (A:B¼80:20 to 20:80 (25min), A: 0.1%
TFA in MilliQ, B; 0.1% TFA in MeCN) to afford 38mg (49%) of

Visible-light-controllable nitric oxide releasers
49
1
NO-Rosa-Gal as a purple solid: H NMR (DMSO-d6, 500MHz, δ;
ppm) 7.61 (1H, dd, J ¼6.7Hz, 6.7Hz), 7.55 (1H, dd, J ¼7.4Hz,
7.4Hz), 7.42 (1H, d, J ¼7.8Hz), 7.32–7.26 (4H, m), 7.18 (2H, s),
7.09–6.99 (3H, m), 6.93 (2H, d, J ¼9.0Hz), 6.87 (1H, d, J ¼9.6Hz),
5.08 (1H, d, J ¼16Hz), 4.90 (1H, d, J ¼16Hz), 4.85 (1H, d,
J ¼7.7Hz), 3.79–3.71 (23H, m), 3.61–3.56 (3H, m); 13C NMR
(DMSO-d6, 150MHz, δ; ppm) 157.87, 157.61, 157.40, 157.13,
156.67, 156.35, 154.89, 134.01, 132.90, 130.78, 130.63, 130.42,
130.22, 129.36, 129.45, 127.85, 121.41, 116.36, 114.98, 113.76,
100.50, 96.96, 96.83, 75.39, 73.07, 69.98, 67.92, 65.65, 60.26, 46.90,
44.64; Anal. Calcd. for C42H43F3N4O12ꢂ2H2O: C, 52.70; H, 4.82;
N, 5.59. Found: C, 52.75; H, 4.65; N, 5.38.
3. Protocols: ESR spin trapping method with
iron-dithiocarbamate complex
ESR spin trapping method with iron-dithiocarbamate complex
(Fe-DTC) is one of conventional methods to detect NO (Yoshimura &
Kotake, 2004). Fe-DTC can trap NO rapidly to show three signals in
1GHz ESR measurements. The following method is for detection of
NO release from NOBL-1 in response to blue light.
Equipment
1. Visible light source, MAX-302 with a 470–500nm band pass filter
(Asahi Spectra)
2. Quartz cell (12.5ꢃ12.5ꢃ45mm)
3. JES-RE2X spectrometer (JEOL Co. Ltd., Tokyo, Japan)
4. ESR flat cell (#LLC-04B, LABOTEC)
Chemicals
1. N-Methylglucamine dithiocarbamate (MGD)
2. Iron (II) sulfate heptahydrate (FeSO4ꢂ7H2O)
3. Stock solution of NOBL-1 in DMF or DMSO (10mM)
4. Sodium phosphate buffer solution (200mM, pH7.0)
5. MilliQ water
Procedure
1. MGD was dissolved into MilliQ to prepare solution A (10mg
MGD/287 μL MilliQ water).
2. FeSO4ꢂ7H2O was dissolved into solution A to prepare solution B
(1.0mg FeSO4ꢂ7H2O /120 μL solution A).

50
Naoya Ieda and Hidehiko Nakagawa
Tips: This solution was vortexed until FeSO4ꢂ7H2O was dissolved
into the solution and the color turned into deep black to make
Fe-DTC firmly. The final concentration of solution B was 30mM
FeSO4, and 120mM MGD.
3. A sample solution was prepared by mixing 10mM solution of NOBL-1
in DMF (2 μL), 200mM phosphate buffer solution (pH7.0, 100 μL),
solution B (10 μL), and MilliQ (88 μL).
Tips: DMSO can be utilized to dissolve NOBL-1 instead of DMF.
4. The sample solution (total volume 200μL) of FeSO4ꢂ7H2O (1.5mM),
MGD (6mM) and NOBL-1 (100μM) in sodium phosphate buffer
(pH7.0, DMF 1%) was put in a quartz cell and the cell was photo-
irradiated (470–500nm, 200mW/cm2) at room temperature for
15min (Fig. 3A).
Tips: The light intensity was measured by using a 3664 optical power
meter (HIOKI, Nagano).
5. The irradiated solution was transferred to an ESR flat cell for ESR mea-
surement. ESR spectra were taken on a JES-RE2X spectrometer (JEOL
Co. Ltd., Tokyo, Japan). The measurement conditions were follows:
Fig. 3 (A) Actual photoirradiation setting for ESR study; (B) an ESR spectrum of a solution
containing NOBL-1 and Fe-MGD after blue light irradiation. Reprinted with permission
from Ieda, N., Hotta, Y., Miyata, N., Kimura, K., Nakagawa, H. (2014). Journal of the
American Chemical Society. 136, 7085–7091. Copyright 2014 American Chemical Society.

Visible-light-controllable nitric oxide releasers
51
microwave power, 10mW; frequency, 9.4200GHz; field, 330 mT;
sweep width, 7.5 mT; sweep time, 4min; modulation width, 0.125 mT;
time constant; 0.10s (Fig. 3B).
4. Protocols: NO electrode
Whereas NO electrode is a powerful tool for real-time determination
of NO concentration, there are some technical points to be concerned
(Fig. 4) (Ichimori, Ishida, Fukahori, Nakazawa, & Murakami, 1994).
Fig. 4 (A) Actual photoirradiation setting for NO electrode study; (B) pattern diagram of
the experimental setting.

52
Naoya Ieda and Hidehiko Nakagawa
Solution temperature should be paid attention because the electrode is very
thermosensitive. If the temperature of sample solution would be increased,
signal current is increased even in the absence of NO. The irradiating light
can affect accuracy of this NO electrode, too. Under our experimental con-
dition, blue-light irradiation (around 500nm light) induced signal current
increase of the electrode even in the absence of NO releasers whereas
>530nm light did not, so that we did not apply the NO electrode for
NO detection from a blue-light-controllable NO releaser, NOBL-1. In
spite of these concerns, NO electrode is very useful for monitoring accurate
change of NO concentration in the case of NO-Rosa5 (Fig. 5).
Equipment
1. Visible light source, MAX-303 with a 530–590nm band pass filter
(Asahi Spectra)
2. NO electrode, ISO-NOP and TBR1025 (World Precision Instruments)
3. Current recorder, PowerLab and LabChart7 (ADInstruments)
4. Incubator and magnetic stirrer (#5301B, YSI)
5. Stirring bar
6. Dark curtain
Fig. 5 Temporal control of NO release from NO-Rosa5. A solution of NO-Rosa5 (10 μM) in
HEPES buffer (100mM, pH7.3, DMSO 0.1%) was irradiated with a MAX-303 (Asahi Spectra)
fitted with a 530–590nm band-pass filter (light intensity: 70mW/cm2). © Springer Nature,
Ieda, N., Oka, Y., Yoshihara, T., Tobita, S., Sasamori, T., Kawaguchi, M., et al. (2019). Scientific
Reports. 9, 1430.

Visible-light-controllable nitric oxide releasers
53
Chemicals
1. A stock solution of NO-Rosa5 in DMSO (10mM)
2. A stock solution of NO-Rosa-Gal in MilliQ water (10mM)
3. 1M HEPES buffer (#345–06681, Dojindo)
4. Potassium nitrite and sulfuric acid for calibration of NO electrode
5. MilliQ water
Procedure
1. An NO electrode, ISO-NOP was calibrated with potassium nitrite in
acidic solution according to instructions of ISO-NOP.
2. A reaction vial was set inside of a dark curtain as shown in Fig. 4B.
3. The electrode was dipped into a stirring 100mM HEPES buffer
(1M HEPES solution 1mL+MilliQ 9mL) on a 37 °C circulating water
bath for 5–10min to stabilize the signal current.
4. A 10 μL solution of NO-Rosa5 in DMSO (10mM) was added to the
stirring 100mM HEPES buffer and keep stirring for further 1–2min
(The final concentration of NO-Rosa5 was 10 μM).
5. Light irradiation was conducted with a MAX-303 (Asahi Spectra)
equipped with 530–590nm band-pass filter from the side of water bath
(light intensity: 120mW/cm2).
6. For NO-Rosa-Gal, after stabilization of NO electrode in stirring Hank’s
buffer saline solution at 37 °C, a stock solution of NO-Rosa-Gal and
β-galactosidase (final 10units/mL) was added to the stirring solution.
After stirring for further 1h, the reaction was irradiated (light intensity:
120mW/cm2) (Ieda et al., 2019).
5. Protocols: NO detection by fluorescence probes
Fluorescence probes are useful to detect target molecules spatiotem-
porally. Various conjugates of o-phenylenediamine and fluorophores have
been developed as effective fluorescence NO probes (Kojima et al., 1999,
2001). An important point to choose fluorescence probes for light-
controllable NO releasers is their excitation wavelength. The wavelength
to excite a fluorescence NO probe should not be overlapped with that to
excite an NO releaser. Namely, a red fluorescence NO probe, DAR-4M
is suitable for NOBL-1, and a green fluorescence one, DAF-FM is for
NO-Rosa derivatives. For cellular experiments, acetoxymethyl esters of
these probes, DAF-FM AM and DAR-4MAM are suitable because they
can be easily uptaken into cells and hydrolyzed to form DAF-FM and
DAR-4M. By using DAR-4MAM, spatiotemporal NO release with

54
Naoya Ieda and Hidehiko Nakagawa
Fig. 6 Fluorescence imaging of NO release from NOBL-1 in HEK293 cells in the presence
of DAR-4MAM. Cultured HEK293 cells were treated with DAR-4MAM (5μM) and NOBL-1
(10μM). The dish was then photoirradiated inside the indicated circle using a confocal
microscope equipped with an argon laser. The dish was observed with the confocal
microscope.
NOBL-1 can be visualized (Fig. 6). NO-Rosa-Gal enabled cell-specific NO
release control in β-galactosidase-overexpressed HEK293T cells (Ieda et al.,
2019). The typical protocol to visualize spatially specific NO release control
using NOBL-1 by DAR-4MAM in HEK293 cells is as follows;
Equipment
1. Clean bench to treat cultured cells
2. CO2 incubator for cell culture
3. Confocal fluorescence microscope (LSM 510, Carl Zeiss Japan Co. Ltd.)
Chemicals
1. HEK293 cells
2. Glass bottom dish (#D11130H, Matsunami)
3. Dulbecco modified eagle medium (DMEM, #D5796, Sigma-Aldrich)
which was added 25mL of fetal bovine serum (#S1820–500,

Visible-light-controllable nitric oxide releasers
55
BioWest) and 5mL of Penicillin-Streptomycin, Liquid (#15070063,
Thermo Fisher Scientific)
4. Dulbecco’s Phosphate Buffered Saline (D-PBS, #8662, Sigma-Aldrich)
5. A stock solution of NOBL-1 in DMSO (10mM)
6. A stock solution of DAR-4MAM in DMSO (#SK1006–01, Goryo
Chemical)
Procedure
1. HEK293 cells were plated on 3.5cm glass dishes at 2.0ꢃ105 cells/dish
with 2mL of DMEM.
2. The cells were incubated at 37°C in a humidified atmosphere of 5%
(v/v) CO2 in air for 2days.
3. The medium was replaced with 2mL of fresh DMEM.
4. The cells were treated with 2μL of a stock solution of NOBL-1 in
DMSO or DMSO and incubated at 37°C in a humidified atmosphere
of 5% (v/v) CO2 in air for an hour.
5. The medium was replaced with 2mL of fresh DMEM, and the cells were
washed with 2mL of DMEM twice and treated with 2μL of a stock
solution of DAR-4MAM and incubated at 37°C in a humidified
atmosphere of 5% (v/v) CO2 in air for 30min.
6. The medium was replaced with 2mL of D-PBS. The cells were
washed with 2mL of D-PBS, and subjected to confocal fluorescence
microscopy.
7. A region of interest of a dish was selected and bleached with an argon
laser (488nm, output 70%, number of scans 1000, light intensity
attenuated to 30%).
These fluorescence probes can be also utilized to quantify NO in vitro. In
order to make a standard curve of NO and these probes, NOC-7 is suitable
as an NO resource which releases two equivalent NO efficiently and quickly
and is commercially available (#N377, Dojindo). The following procedure
shows how to make standard curve with DAR-4M and NOC-7.
Equipment
1. Fluorescence spectrometer (RF-5300PC, Shimadzu)
Chemicals
1. NOC-7 (#N377, Dojindo)
2. Stock solution of DAR-4M (5mM, #SK1005-01, Goryo Chemical)
3. Sodium phosphate buffer solution (100mM, pH7.4)
Procedure
1. NOC-7 (#N377, Dojindo) was dissolved into 0.1M NaOH to make a
10mM stock solution (1mg / 617 μL 0.1M NaOH).

56
Naoya Ieda and Hidehiko Nakagawa
Fig. 7 A standard curve made by reaction of NOC-7 and DAR-4M.
2. Sample solutions (1mL) were made by mixing 100μL of sodium phos-
phate buffer (100mM, pH7.4), 2μL of stock solution of DAR-4M, a
stock solution of NOC-7 (0.5, 1, 2.5, or 5μL) and diluting with
MilliQ water to 1mL.
3. Sample solutions were incubated in dark at room temperature for 2h.
4. The fluorescence intensity was determined with RF-5300PC
(Shimadzu) at 575nm, with excitation at 560nm. The slit width was
set to 1.5nm for both excitation and emission, and the sensitivity
was high
5. A standard curve was made from the fluorescence values (Fig. 7).
6. Protocols: Determination of quantum yield
Quantum yield shows how efficient is a photochemical reaction.
It can be calculated by division the amount of output (e.g., fluorescence,
NO, and decomposition) by the number of excited molecules. Whereas
the amount of NO can be determined by an NO electrode, the number
of irradiated photon number is necessary to calculate number of excited
molecules. In order to count the number of irradiated photon, we utilized
potassium ferrioxalate as a chemical actinometer because it is broadly appli-
cable to UV–visible light range (Hatchard, 1956). As a monochrome light
source, Xe lamp of a fluorescence spectrometer (RF-5300, Shimadzu)
equipped with a monochrometer was applicable instead of visible light lasers
(Fig. 8). After monitoring amount of NO by an NO electrode, the irradiated
photon number was calculated by using potassium ferrioxalate actinometer.

Visible-light-controllable nitric oxide releasers
57
Fig. 8 (A) Actual NO electrode setting of determination of NO concentration in order to
determine NO release quantum yield; (B) pattern diagram of this experimental setting.
Equipment
1. NO electrode, ISO-NOP and TBR1025 (World Precision Instruments)
2. Current recorder, PowerLab and LabChart7 (ADInstruments)
3. Quartz cell (12.5ꢃ12.5ꢃ45mm)
4. Fluorescence spectrometer (RF-5300PC, Shimadzu)
5. Absorption spectrometer (Agilent 8453)
Chemicals
1. Stock solution of NO-Rosa5 in DMSO (10mM)
2. 1M HEPES buffer (#345-06681, Dojindo)
3. Potassium ferrioxalate

58
Naoya Ieda and Hidehiko Nakagawa
How to prepare: 1.5M potassium oxalate (8.29g in 30mL MilliQ) and
1.0M iron (III) chloride (2.70g in 10mL MilliQ) were mixed and a little
bit water was evaporated, and then, a green precipitate was precipitated
soon. The precipitate was filtrated and dried under vacuum.
4. Sulfuric acid
5. Phenanthroline
Procedure: Measurement of the amount of released NO from
NOBL-1 in response to blue light
1. A solution of NO-Rosa5 (10μM) in 100mM HEPES buffer (pH7.3,
total volume: 3mL) containing 0.1% DMSO was placed in a quartz cell.
2. The cell was placed in a cell holder of a fluorescence spectrometer
RF-5300PC (Shimadzu).
3. The cell was irradiated at 510nm (band width was 10nm) for 1min with
the Xe lamp.
4. The amount of NO released was measured with an ISO-NOP (World
Precision Instruments) and recorded on LabChart7 (ADInstruments).
Procedure: Measurement of the number of irradiated photons
1. Potassium ferrioxalate (200mg) was dissolved in 5mL of 0.05M
sulfuric acid.
2. The solution (3mL) was irradiated under the same conditions as
mentioned above.
3. An aliquot (500μL) was mixed with 500μL of a buffer solution con-
taining phenanthroline (2.25g of sodium acetate, and 10mg of
phenanthroline were dissolved in 10mL of 0.5M sulfuric acid).
4. The absorption spectrum of the mixture was recorded on an Agilent
8453 spectrometer.
5. The absorption spectrum of the non-irradiated mixture was also recorded.
The photon number was calculated from the absorption difference.
7. Protocols: Photocontrol of vasodilation using rat
aorta strip.
NO can induce vasodilation in human body at 10ꢀ9 M range, which is
one of most important roles of NO. We demonstrated that light-
controllable NO releasers could manipulate vasodilation of rat aorta strip
by light irradiation to show utility of these compounds for control of
NO’s physiological events. The actual experimental set was described in
Fig. 9, and experimental procedure of NOBL-1 for photo-vasodilation
control is as follows.

Visible-light-controllable nitric oxide releasers
59
Fig. 9 (A) This experiment was performed in a dark room; (B) rat aorta strip in light-
irradiated organ bath. (C) pattern diagram of evaluation system. Light was irradiated
from outside the organ bath. ©The Korean Urological Association, Hotta, Y., Ieda, N.,
Fukumoto, A., Kataoka, K., Kawade, Y., Maeda, Y., et al. (2016). Investigative and
Clinical Urology. 57, 215–220.
Equipment
1. Visible light source, MAX-302 with a 470–500nm band pass filter
(Asahi Spectra)
2. Force transducer (ADInstruments, Bella Vista, Australia)
3. Bridge amp and PowerLab 4/26 (ADInstruments)
4. LabChart7 (ADInstruments)
5. Magnus tube
6. Dark curtain
Chemicals
1. Stock solution of NOBL-1 in DMSO (10mM)
2. Stock solution of L-NAME in MilliQ water (10mM)

60
Naoya Ieda and Hidehiko Nakagawa
3. Stock solution of ODQ in DMSO (10mM) for inhibition of vasodila-
tion signaling
4. Stock solution of noradrenaline hydrochloride in MilliQ water (10mM)
5. Krebs buffer (composed of 119mM sodium chloride, 4.6mM potassium
chloride, 1.5mM calcium chloride, 1.2mM magnesium chloride,
15mM sodium bicarbonate, 1.2mM sodium dihydrogen phosphate,
and 11mM glucose)
Procedure
1. A rat aortic strip was placed in a Magnus tube filled with Krebs buffer
(10mL) at 37 °C.
2. The tension was recorded on a LabChart7 (ADInstruments).
3. The strip was treated with L-NAME (10μM) or ODQ (10μM) before
addition of noradrenaline (10μM).
4. After equilibration, NOBL-1 (1or 10μM) or DMSO (10μL) was added
to the buffer.
5. The tube was irradiated with MAX-302 (14.5mW/cm2).
Tips: This system was very light-sensitive, even room light, so that
the Magnus tube must be inside of dark curtain after addition of
NOBL-1.
8. Summary
This article has described how to synthesize visible-light controllable
NO releasers and apply these compounds for biological applications. It is
expected that this method could be give some helps to develop further
functionalized or utilized not only light-controllable NO releasers but also
other photo-responsive compounds.
Acknowledgment
This work was supported by funds from JSPS KAKENHI Grant Number 18K14873 (N.I.),
and 19H03354 (H.N.), and The Tokyo Biochemical Research Foundation, and Takeda
Science Foundation, and CASIO Science Promotion Foundation.
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