Photoinduced upregulation of calcitonin gene-related peptide in A549 cells through HNO release from a hydrophilic photocontrollable HNO donor

Article abstract of DOI:10.1248/cpb.c12-00348

Nitroxyl (HNO), a one-electron-reduced form of nitric oxide, has various biological activities, including a cardioprotective effect. Here, we first synthesized another, more hydrophilic photocontrollable HNO donor (3), which can release HNO in a spatially and temporally controlled manner, and then examined the properties of our series of compounds as practical HNO donors in a cellular system under photocontrol. We selected compound 2 as the preferred donor, and used it to show that calcitonin gene-related peptide (CGRP) can be upregulated in A549 cells via photocontrolled HNO release. This result demonstrates the suitability of this photocontrollable HNO donor for biological investigations.

Full text of DOI:10.1248/cpb.c12-00348

August 2012
Regular Article
Chem. Pharm. Bull. 60(8) 1055–1062 (2012)
1055
Photoinduced Upregulation of Calcitonin Gene-Related Peptide in A549
Cells through HNO Release from a Hydrophilic Photocontrollable HNO
Donor
Kazuya Matsuo, Hidehiko Nakagawa,* Yusuke Adachi, Eri Kameda, Kazuyuki Aizawa,
Hiroki Tsumoto,† Takayoshi Suzuki,‡ and Naoki Miyata*
Department of Organic and Medicinal Chemistry, Graduate School of Pharmaceutical Sciences, Nagoya City
University; 3–1 Tanabe-dori, Mizuho-ku, Nagoya, Aichi 467–8603, Japan.
Received April 16, 2012; accepted May 12, 2012
Nitroxyl (HNO), a one-electron-reduced form of nitric oxide, has various biological activities, includ-
ing a cardioprotective effect. Here, we first synthesized another, more hydrophilic photocontrollable HNO
donor (3), which can release HNO in a spatially and temporally controlled manner, and then examined the
properties of our series of compounds as practical HNO donors in a cellular system under photocontrol. We
selected compound 2 as the preferred donor, and used it to show that calcitonin gene-related peptide (CGRP)
can be upregulated in A549 cells via photocontrolled HNO release. This result demonstrates the suitability of
this photocontrollable HNO donor for biological investigations.
Key words nitroxyl; photo-uncaging; nitric oxide; retro hetero Diels–Alder reaction; chemical biology
Since nitric oxide (NO) was found to be biologically syn- previously reported HNO donors (Chart 1). Although Angeli’s
thesized in mammalian systems,1,2) many researchers have salt24,25) (Na2N2O3) is a widely used HNO donor and releases
studied its signaling pathways and bioactivities. The term ‘ni- HNO, together with a little NO, at physiological pH, its release
tric monoxide’ actually covers three different entities, which of HNO is spontaneous and not controllable, except by tem-
are nitrosyl cation (NO+), nitric oxide, and nitroxyl (anion).3–7) perature. Furthermore, Angeli’s salt releases NO, an oxidized
Nitroxyl (HNO), which is a one-electron-reduced form of NO, form of HNO, under acidic conditions, so that pH control is
has been less well studied than the former two species, but important. Piloty’s acid26) (PhSO2NHOH) and cyanamide27)
has attracted more interest in recent years. It can activate sar- (NH2CN) also offer only uncontrolled HNO release. King
coplasmic reticulum Ca2+-ATPase (SARCA)8) and ryanodine and colleagues reported a useful type of HNO donor, acyloxy
receptor (RyR),9,10) and induce positive inotropy.11) Its mecha- nitroso compounds,28) which have the property of slow release
nism of action is thought to be different from those of com- of HNO; they have been useful to elucidate HNO actions,29)
mon cardiovascular drugs. Furthermore, HNO was reported to but the HNO release is still uncontrollable.
have cardioprotective activity owing to a preconditioning-like
Photocontrol of HNO release permits precise temporal and
effect.12) Therefore, HNO is expected to be a candidate for spatial control of HNO treatment, and therefore photocontrol-
treatment of various heart disorders.13) Vasodilating action of lable HNO donors are expected to be superior tools to exam-
HNO has been reported; the mechanism is not clear, but there ine the biological effects of HNO, e.g., in cell cultures. Here,
are many possibilities, including soluble guanylate cyclase we developed another hydrophilic photocontrollable HNO
(sGC) upregulation,14,15) and K+ channel activation,16,17) which donor, compound 3, and examined the suitability of our series
are different from those in the case of NO. It has also been of compounds (Fig. 1) for use as practical HNO donors in cel-
suggested18,19) that the mechanism of vasodilation by HNO lular systems under photocontrol.
partly involves a pathway mediated by calcitonin gene-related
peptide (CGRP). CGRP is a neuropeptide consisting of 37
amino acids, produced by alternative mRNA splicing of the
Experimental
Synthesis of Compound 3. General Methods Melting
calcitonin gene, and shows vasodilatory action and ionotropic points were determined using a Yanaco micro melting point
and chronotropic activities. CGRP signaling is transduced apparatus or a Büchi B-545 melting point apparatus and
through G-protein-coupled receptor (GPCR), leading to pro- were left uncorrected. Proton nuclear magnetic resonance
duction of cAMP.11)
(1H-NMR) spectra and carbon nuclear magnetic resonance
Inspired by the cycloadduct formation between 9,10-di- (13C-NMR) spectra were recorded on a JEOL JNM-LA500
methylanthracene and acyl nitroso compounds reported by or JEOL JNM-A500 spectrometer in the indicated solvent.
King and colleagues,20,21) we have developed photocontrol- Chemical shifts (δ) are reported in parts per million (ppm)
lable HNO donors, compounds 1 and 222,23) (Fig. 1). These relative to the internal standard tetramethylsilane (TMS). Ele-
donors release HNO in response to photoirradiation, unlike mental analysis was performed with a Yanaco CHN CORDER
NT-5 analyzer, and all values were within 0.4% of the calcu-
lated values. UV/vis spectra were measured using an Agilent
The authors declare no conflict of interest.
8453 spectrometer. IR spectra were recorded on an Avatar360.
GC-MS analyses were performed on a Shimadzu GCMS-
QP2010. FAB-MS were recorded on a JEOL JMS-SX102A
mass spectrometer. Reagents and solvents were purchased
from Aldrich, Tokyo Kasei Kogyo, Wako Pure Chemical
† Present address: Kyoto University Graduate School of Pharmaceutical
Sciences; 46–29 Yoshida-Shimo-Adachi-cho, Sakyo-ku, Kyoto 606–8501,
Japan.
‡ Present address: Kyoto Prefectural University of Medicine; 13 Taisho-
gun Nishitakatsukasa-cho, Kita-ku, Kyoto 403–8334, Japan.
*To whom correspondence should be addressed. e-mail: deco@phar.nagoya-cu.ac.jp;
miyata-n@phar.nagoya-cu.ac.jp
© 2012 The Pharmaceutical Society of Japan

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Vol. 60, No. 8
mixture was stirred at room temperature for 1h. The reaction
mixture was concentrated by evaporation in vacuo and the
residue was suspended in n-hexane. Filtration and recrystal-
lization from tetrahydrofuran (THF)–n-hexane gave 591mg
1
(76%) of 7 as a yellow solid: H-NMR (DMSO, 500MHz, δ;
ppm) 12.33 (2H, br), 8.36 (4H, dd J=3.0, 7.0Hz), 7.59 (4H, dd,
J=3.2, 6.9Hz), 3.86 (4H, t, J=8.2Hz), 2.62 (4H, t, J=8.1Hz).
(S,S)-Di-tert-butyl
2-(3-{10-[2-(1-tert-Butoxycarbonyl-
methyl-2-isopropoxy-ethylcarbamoyl)ethyl]anthracen-9-yl}-
propionylamino)butanedioate (8) A solution of 7 (200mg,
0.621mmol), H-Asp(OtBu)–OtBu·HCl (350mg, 1.24mmol),
EDCI (357.1mg, 1.86mmol), HOBt·H2O (285.3mg, 1.86mmol),
and Et3N (0.17mL) in 35mL of DMF was stirred at room tem-
perature for 3h. The reaction mixture was poured into water
and extracted with AcOEt. The organic layer was separated,
washed with brine, and dried over Na2SO4. Filtration, evapo-
ration of the solvent in vacuo, and purification of the residue
by silica gel flash column chromatography (AcOEt–n-hexane=
1
0:1 to 1:1) gave 281.6mg (58%) of 8 as a yellow solid: H-
NMR (DMSO, 500MHz, δ; ppm) 8.42 (2H, d J=7.9Hz), 8.37
(4H, dd, J=3.3, 5.3Hz), 7.59 (4H, dd, J=3.0, 5.4Hz), 4.55 (2H,
q, J=7.3Hz), 3.79 (4H, t J=8.2Hz), 2.65 (2H, m), 2.55 (4H,
overlap), 1.42 (36H, s).
(S,S)-2-(3-{10-[2-(1-tert-Butoxycarbonylmethyl-2-iso-
propoxy-ethylcarbamoyl)ethyl]anthracen-9-yl}propionyl-
amino)butanedioic Acid (9) Two milliliters of TFA was
added to a solution of 8 (265.2mg, 0.34mmol) in 15mL of
CH2Cl2. The reaction mixture was stirred at room temperature
for 24h. The mixture was concentrated by evaporation in vac-
uo and the residue was dissolved in TFA (15mL). The solution
Fig. 1. Photocontrollable HNO Donors in This Study
Industries, and Kanto Kagaku and used without further purifi- was stirred at room temperature for 12h, then evaporated
cation. Flash chromatography was performed using Silica Gel in vacuo and the residue was recrystallized from THF–n-
1
60 (particle size 0.046–0.063mm) supplied by Merck.
hexane to give 151.9mg (74%) of 9 as a yellow solid: H-NMR
Di-tert-butyl 3,3′-(9,10-Anthracenediyl)bis(2-propenoate) (CD3OD, 500MHz, δ; ppm) 8.40 (4H, dd, J=3.3, 5.1Hz), 7.54
(5) A solution of 4 (3360mg, 10mmol), t-butyl acrylate (4H, dd, J=3.0, 5.0Hz), 4.75 (2H, t, J=6.9Hz), 3.95 (4H, t,
(29mL, 200mmol), Et3N (28mL, 200mmol), Pd(OAc)2 J=7.9Hz), 2.71 (4H, m), 2.70 (4H, m).
(246.96mg, 1.10mmol), and tri-o-tolylphosphine (669.6mg,
N-(Tetrahydropyran-2-yl)oxy-N′-(4-nitrophenyl)urea
2.20mmol) in N,N-dimethylformamide (DMF) (100mL) was (11) To a solution of NH2OTHP (936mg, 8mmol) in dehy-
heated at 120°C for 4h. After cooling, water and AcOEt were drated THF (16mL) was added dropwise 10 (1313mg, 8mmol)
added. The organic solution was evaporated under reduced at 0°C. The reaction mixture was stirred for 40min at 0°C.
pressure to give a residue, which was purified by silica gel The solvent was removed, and purification of the residue by
flash chromatography (AcOEt–n-hexane=1:10) to give 5 silica gel flash chromatography (AcOEt–n-hexane=2:3) gave
1
(4163mg, 97%) as a yellow solid: H-NMR (CDCl3, 500MHz, 2256mg (100%) of 11 as a yellow solid: 1H-NMR (CDCl3,
δ; ppm) 8.51 (2H, d, J=16.1Hz), 8.26 (4H, dd, J=3.2, 6.8Hz), 500MHz, δ; ppm) 8.63 (1H, s), 8.21 (2H, d, J=9.0Hz), 7.63
7.53 (4H, dd, J=3.2, 6.8Hz), 6.32 (2H, d, J=16.1Hz), 1.63 (2H, d, J=9.0Hz), 7.33 (1H, s), 4.86 (1H, m), 4.09 (1H, m),
(18H, s).
Di-tert-butyl 9,10-Anthracenedipropanoate (6) Pd(OAc)2
3.66 (1H, m), 1.90 (2H, m), 1.64 (4H, m).
N-Hydroxy-N′-(4-nitrophenyl)urea (12) To a solution
(89.8mg, 0.40mmol) was added to a stirred solution of 5 of 11 (1950mg, 6.9mmol) in MeOH (150mL) was added
(1722.2mg, 4.0mmol) in DMF (300mL) at 80°C, and HCOOK TsOH·H2O (119mg, 0.69mmol). The reaction mixture was
(1776.6mg, 21.1mmol) was added. The reaction mixture was stirred for 23h at room temperature. The solution was concen-
stirred for 29h. Then, AcOEt and water were added to the trated under reduced pressure, and purification of the residue
cooled mixture. The AcOEt layer was separated, washed with by silica gel flash chromatography (AcOEt–n-hexane=3:1)
1
brine, and dried over Na2SO4. The solvent was evaporated and gave 1068mg (79%) of 12 as a yellow solid: H-NMR (DM-
the residue was purified by silica gel flash column chroma- SO-d6, 500MHz, δ; ppm) 9.51 (1H, s), 9.29 (1H, s), 9.16 (1H,
tography (toluene–n-hexane=1:4) to give 1046.4mg (60%) of s), 8.16 (2H, d, J=9.5Hz), 7.92 (2H, d, J=9.5Hz).
1
6 as a yellow solid: H-NMR (CDCl3, 500MHz, δ; ppm) 8.33
(S, S)-9,10-[Dihydro-N-4-nitrophenyl-9,10-(epoxy-
(4H, dd J= 3.2, 6.8Hz), 7.54 (4H, dd, J=3.2, 6.8Hz), 3.92 (4H, imino)-11-carbamylanthracene-9,10-(propionylamino)-
t, J=8.51Hz), 2.69 (4H, t, J=8.51Hz), 1.49 (18H, s). butanedioic Acid (3) To suspension of (70mg,
a
9
9,10-Anthracenedipropanoic Acid (7) Eleven millili- 0.127mmol) and NaIO4 (54.2mg, 0.253mmol) in 10mL of
ters of trifluoroacetic acid (TFA) was added to a solution of acetone was added a solution of 12 (49.9mg, 0.253mmol) in
6 (1046mg, 2.41mmol) in 20mL of CH2Cl2. The reaction 7mL of acetone and 10mL of H2O over a period of 20min

August 2012
1057
Chart 1. Hetero Diels–Alder Reaction and Photo-Induced Retro Diels–Alder Reaction
at 0°C. The reaction mixture was stirred for 5h at 0°C, then for the designated time at room temperature. The cuvette
poured into water and extracted with AcOEt. The organic was then shaken and placed in an ice bath for 30min for
layer was washed with 4N HCl and brine, and dried over equilibration of N2O in the headspace of the cuvette without
Na2SO4. Filtration, concentration in vacuo, and precipitation further decomposition of the donor compound. An aliquot of
with n-hexane gave 48.5mg (51%) of 3 as a light yellow solid: the reaction headspace gas (50µL) was injected onto a Shi-
decomp. point 100.8–102.1°C; UV/Vis (DMSO–Milli Q=1:9): madzu GC-2010 gas chromatograph equipped with a mass
ε323nm=1.22×104 M−1 cm−1; 1H-NMR (DMSO-d6, 500MHz, spectrometer (QP2010, Shimadzu Corp., Kyoto, Japan) and an
δ; ppm) 9.39 (1H, br), 8.55 (1H, d, J=7.6Hz), 8.38 (1H, d, Rt-QPLOT column (0.32mm × 15m) (Restek, Bellefonte, PA,
J=8.2Hz), 8.09 (2H, d, J=9.1Hz), 7.76 (2H, d, J=9.1Hz), 7.55 U.S.A.) attached with a 15-m inactivated fused silica capillary
(4H, m), 7.30 (4H, m), 4.64 (2H, m), 2.50 (2H, overlap), 3.16 (total 30m). The GC injector was operated with a split ratio of
(2H, m), 2.55–2.78 (8H, m); 13C-NMR (DMSO-d6, 125MHz, 0.1 at 200°C. The carrier gas (He) flow rate was set at 2.2mL/
δ; ppm); 173.15, 172.58, 172.28, 171.90, 171.73, 171.55, 158.88, min. The GC oven was held at 35°C. The MS interface was
145.02, 141.89, 128.85, 127.28, 126.98, 126.39, 125.53, 124.66, set to 280°C. N2O formation was calculated based on the de-
118.54, 112.36, 81.13, 66.07, 48.96, 48.79, 36.11, 35.91, 30.99, composition of Angeli’s salt. On the chromatogram in GC-MS
28.96, 24.90, 22.22; MS (FAB) m/z: 748 ([M+H]+); Anal. analysis, the peak of N2O was partly overlapped with the large
Calcd for C35H33N5O14·3H2O: C, 52.43; H, 4.90; N, 8.74. CO2 peak in many cases. To obtain the peak area of N2O, the
Found: C, 52.46; H, 4.83; N, 8.63.
area of N2O on the tail of the CO2 peak was determined by
Photoirradiation Photoirradiation with UV A (UVA) was calculation.
performed by using the light source (100W mercury lamp) of
Reaction between HNO and 2-(4-Carboxyphenyl)-
a fluorescence microscope (Olympus BX60/BX-FLA) with a 4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide,
Sodium
WU filter (330–380nm band-pass filter). Photoirradiation in Salt (Carboxy-PTIO). N2O Detection in Fig. 3 A solution
vitro was performed under Ar- or He-purged anaerobic condi- of Angeli’s salt (1.5µmol) or compound 2 (1.5µmol) with car-
tions, and that in cellular experiments was done under aerobic boxy-PTIO (1.5µmol) in a mixed solution of DMSO (300µL)
conditions. The light intensity was attenuated to 1.5% with a and 50mM Tris buffer pH 7.5 (2700µL) was placed in a 4-mL
combination of a 6% ND filter and a 25% ND filter.
cuvette sealed with a rubber septum. The sample solution was
Detection of N2O by Gas Chromatography A solution of incubated for 30min at 37°C. The cuvette was then shaken
compound 1 (1.5µmol) in a mixed solution of dimethyl sulfox- and placed in an ice bath for 30min for equilibration of N2O
ide (DMSO) (2700µL) and 50mM Tris buffer pH 7.5 (300µL), in the headspace of the cuvette without further decomposition
a solution of compound 2 (1.5µmol) in DMSO (300µL) and of the donor compound. An aliquot of the reaction headspace
50mM Tris buffer pH 7.5 (2700µL), or a solution of compound gas was analyzed by the same method as described above.
3 (1.5µmol) in 50mM Tris buffer pH 7.5 (3000µL) was placed
Reaction of 1-Hydroxy-2-oxo-3-(N-methyl-3-amino-
in a 4-mL cuvette sealed with a rubber septum. For HNO propyl)-3-methyl-1-triazene (NOC7), Angeli’s Salt, or
scavenging experiments, 2-mercaptoethanol (1.05µL) was Compound 2 with Carboxy-PTIO in Vitro A solution of
added to the solution. The sample solution was photoirradi- NOC7 (1.5µmol), Angeli’s salt (1.5µmol), or compound 2
ated with UVA, or allowed to stand without photoirradiation (1.5µmol) with carboxy-PTIO (1.5µmol) in a mixed solution

1058
Vol. 60, No. 8
of DMSO (300µL) and 50mM Tris buffer pH 7.5 (2700µL)
was placed in a 5mL vial. The sample solutions were incu-
bated without photoirradiation for 30min at 37°C, and photo-
graphed.
Cell Culture A549 cells were obtained from RIKEN
Cell Bank and grown in Dulbecco’s modified Eagle’s medium
(DMEM) containing penicillin and streptomycin (100U/mL)
supplemented with 10% fetal bovine serum (FBS) at 37°C in
a humidified CO2 incubator. When confluent, cells were de-
tached in trypsin solution, washed with DMEM, centrifuged
at 125×g for 5min, and then resuspended and subcultured
according to the standard protocol. For the experiments, the
cells were washed with DMEM twice and maintained in
DMEM without FBS at 37°C for 4h, before treatment with
chemicals.
Detection of CGRP Release in A549 Cells in Response
to HNO Released from the Donors A549 cells were plated
at 2×105 per dish and cultured in 6-cm tissue culture dishes.
After reaching confluence, cells were treated with chemicals
(final DMSO concentration=0.09% in the culture medium)
and/or exposed to photoirradiation (UVA) for 10min, then
washed with DMEM without FBS, and incubated in the cul-
ture media in a humidified CO2 incubator at 37°C for 24h.
The treated cells were washed with D-PBS, homogenized in
2N acetic acid, heated at 90°C for 10min, and centrifuged.
The supernatant was dried in vacuo, and then dissolved in the
assay buffer. CGRP concentrations were determined using a
CGRP (human) EIA Kit from Cayman Chemical (Ann Arbor,
MI, U.S.A.).
Results
First, we modified our previous hydrophilic HNO donor
(compound 2),23) which has two carboxylate groups, to ex-
amine whether a compound bearing four carboxylate groups
(compound 3) would be superior in a cellular environment
(Fig. 1). Briefly, the hydrophilic anthracene (7) was synthe-
sized by the reported method,30) and conjugated with two
molecules of protected L-aspartic acid by condensation reac-
tion to obtain the highly hydrophilic anthracene (9) (Chart 2).
The N-hydroxyurea derivative with a p-nitrophenyl group was
converted into the corresponding acyl nitroso compound in
situ by oxidation, and then trapped with the synthesized an-
Fig. 2. Amounts of N2O Released from Compounds 1 (A), 2 (B), and 3
(C) Measured by GC-MS
Solid lines indicates the results obtained under photoirradiation, and broken lines
indicate those obtained in the dark.
thracene derivative via hetero Diels–Alder reaction to obtain extent of acceleration was less for compound 3 in comparison
compound 3 (Chart 3). Compounds 1–3 have a p-nitrophenyl with compounds 1 and 2 (Fig. 2). The conversion rates in the
group as the photo-absorbing moiety, and their absorp- dark and under photoirradiation were also calculated from the
tion spectra indicated that they would be activated by UVA absorption change at 400nm, which is characteristic for an-
(330–380nm) irradiation (see Supplemental information).
thracene derivatives, and we confirmed that photoirradiation-
We confirmed photoinduced release of HNO from com- induced conversion of compound 3 was less efficient, although
pounds 1–3 and compared their ability to release HNO in the conversion was accelerated by photoirradiation (see Fig.
vitro. The most distinctive feature of HNO chemistry is di- S1 in the Supplemental information). Considering these results
merization reaction to form N2O (nitrous oxide).31) N2O can and the solubility of the compounds in aqueous solution, we
be easily measured by gas chromatography-mass spectrometry selected compound 2 for further studies on the cellular effect
(GC-MS), and most studies on HNO donors have used N2O of photoinduced HNO release. Compound 2 was also found to
measurements to evaluate HNO production. So, we measured be the most stable among the synthesized donors in the dark.
N2O to compare HNO production from the compounds, as
To evaluate compound 2, as a controllable HNO donor in
previously reported.22) Compound 1 was evaluated in a so- biological systems, we focused on intracellular induction of
lution of DMSO–Milli Q water (=9:1), compound 2 in a CGRP. CGRP is produced in many primary-cultured cells, as
solution of DMSO–Milli Q water (=1:9), and compound 3 well as some cultured cell lines, including A549 cells.32) So,
in 50mM Tris buffer (pH 7.5), due to their different solubil- we treated A549 cells with compound 2 and evaluated the
ity characteristics. The formation of N2O from compounds intracellular level of CGRP in the cells. To measure CGRP
1–3 was found to be accelerated by photoirradiation, but the concentration, we employed a CGRP measurement kit from

August 2012
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(a) t-Butyl acrylate, o-tolylphosphine, Pd(OAc)2, Et3N, DMF, 120°C, 97% yield; (b) HCOOK, Pd(OAc)2, DMF, 60°C, 60% yield; (c) TFA, CH2Cl2, 76% yield; (d) H-
Asp(OtBu)-OtBu·HCl, HOBt·H2O, EDCI, DMF, 58% yield; (e) TFA, 74% yield. DMF=N,N-dimethylformamide, TFA=trifluoroacetic acid, HOBt=1-hydroxybenzotri-
azole, EDCI=1-ethyl-3-(3-dimethiaminopropyl)carbodiimide hydrochloride.
Chart 2
(a) NH2OTHP, dry THF, 100% yield; (b) TsOH·H2O, MeOH, 79% yield; (c) 9, NaIO4, acetone/H2O, 50% yield. THP=2-tetrahydropyranyl, TsOH=p-toluenesulfonic
acid.
Chart 3
SPI Bio (Cayman Chemical, Ann Arbor, MI, U.S.A.). Angeli’s compound 2 was less affected by carboxy-PTIO. Simultane-
salt was used as a positive control, and NOC7,33) a widely used ously, the experimental solution of Angeli’s salt with carboxy-
NO donor, was used to confirm the effect of NO on CGRP PTIO, which is originally blue, was bleached, probably due to
production. N-Acetyl-L-cysteine (NAC) was used as an HNO conversion of carboxy-PTIO to carboxy-PTI by one-electron
trap.34)
reduction, whereas the solution of compound 2 with carboxy-
We initially considered the use of carboxy-PTIO35) as a PTIO remained blue (Fig. 4). This result means that carboxy-
scavenger for NO to discriminate the effect of NO from that PTIO does not work as a pure NO scavenger, but is also a
of HNO itself. However, previous reports15,36) have suggested scavenger for HNO. So, we did not use it in our CGRP assay
that HNO from Angeli’s salt might react with carboxy-PTIO experiments.
to form NO. Therefore, we investigated whether carboxy-
The results of CGRP assay in A549 cells are shown in Figs.
PTIO is a suitable reagent for selectively trapping NO, before 5–8. First, we confirmed that HNO treatment of A549 cells
conducting cellular experiments. If HNO does not react with upregulates cellular CGRP. Angeli’s salt, a typical spontane-
carboxy-PTIO, this agent would be a powerful tool for selec- ous HNO donor, dramatically increased the level of CGRP
tively studying the effects of HNO. Angeli’s salt (0.50mM) (Fig. 5) at 50µM, but NOC7 was ineffective (see Supplemen-
or compound 2 (0.50mM) was incubated with or without car- tary information). Moreover, the increase of CGRP by An-
boxy-PTIO (0.50mM) for 30min at 37°C in the dark, and N2O geli’s salt was not observed in the presence of NAC (2mM), a
formation was determined by means of the GC-MS method scavenging reagent for HNO (Fig. 6). These results indicate
(Fig. 3). The amount of N2O derived from Angeli’s salt was that HNO induces CGRP production in A549 cells, but NO
found to be significantly reduced, but N2O derived from does not.

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Vol. 60, No. 8
Fig. 3. Amounts of N2O Release from Angeli’s Salt and Compound 2
in the Absence or Presence of Carboxy-PTIO Measured by GC-MS, as
a Percentage of the Amounts Corresponding to Complete Decomposition
Fig. 6. Change of CGRP Level in A549 Cells in the Presence of An-
geli’s Salt+NAC (2mM) (n=3)
Fig. 4. Photograph of Reaction Mixtures Containing Carboxy-PTIO
only (Far Left), Carboxy-PTIO+NOC7 (Second Left), Carboxy-
PTIO+Angeli’s Salt (Second Right), and Carboxy-PTIO+Compound 2
(Far Right)
Fig. 7. Change of CGRP Level in A549 Cells in the Presence of Com-
pound 2 (A) and Compound 2+NAC (B) in the Dark (n=3)
Discussion
Fig. 5. Effect of HNO on CGRP Level in A549 Cells Using Angeli’s
Salt as a HNO Generator
We synthesized a novel, highly hydrophilic photocontrolla-
ble HNO donor compound 3, based on our previously reported
compounds 1 and 2. Although the highly hydrophilic nature
of 3 was expected to be advantageous for biological applica-
tions, compound 3 was found to be unstable under ambient
**p<0.01 when compared with 0µM by ANOVA and Bonferroni-type multiple
t-test (parametric, n=3–6).
Next, our photocontrollable HNO donor, compound 2, was conditions even in the dark. This might be partly due to the
examined. In the dark, the CGRP level was not significantly linker length of the carboxyl groups. In the previous report,
changed (Fig. 7), but upon photoirradiation, it was increased we suggested that intramolecular interaction of the carboxylic
(Fig. 8A). These results indicate that compound 2 photoirradi- acid group with the heterocyclic part of the molecule would
ation-dependently induced CGRP upregulation. Furthermore, contribute to the unexpected stability of compound 2.23) But,
NAC abolished the effect of compound 2 (Fig. 8B).
in compound 3, the carboxylic acid might be located too far
Overall, these results indicate that HNO from compound 2, from the core part of the molecule to permit significant intra-
as well as Angeli’s salt, accelerated CGRP production in A549 molecular interaction. Therefore, we chose compound 2 for
cells. They also confirm that compound 2 can function as pho- further evaluation because of its stability in aqueous solution.
tocontrollable HNO donor under biological conditions.
HNO formation from the donor compounds in response to

August 2012
1061
an effective approach for in vitro mechanistic studies of the
biological effects of HNO and CGRP.
In our hands, carboxy-PTIO was unsuitable for HNO scav-
enging experiments. It has been suggested that the rate of
HNO release might influence the reactivity between carboxy-
PTIO and HNO, and a high HNO release rate might result
in a complex reaction.37) Angeli’s salt is a spontaneous HNO
releaser and its release rate of HNO is considered to be con-
centration-dependent, being high in the early stage and then
exponentially decreasing. The combination of Angeli’s salt
and carboxy-PTIO was often used in previous studies, but this
reactivity had not been taken into consideration, so at least
some of the reported findings might need to be reassessed.
Furthermore, Angeli’s salt might not work as an efficient HNO
donor in the presence of carboxy-PTIO. On the other hand,
unirradiated compound 2 did not show a significant reaction
with carboxy-PTIO, as indicated by the lack of color change.
Considering that compound 2 slowly releases HNO even in
the dark, as shown in Fig. 2, this might mean that controlled
HNO release from HNO donors such as compound 2 is com-
patible with carboxy-PTIO.
In conclusion, we confirmed that hydrophilic photocontrol-
lable HNO donors release HNO upon photoirradiation in an
aqueous environment. Further, we found that photoinduced
HNO release from compound 2 induces an increase of CGRP
production in cultured A549 cells. Therefore, compound 2 is
expected to be a useful chemical tool for in vitro studies of
the biological functions of HNO and CGRP.
Fig. 8. Change of CGRP Level in A549 Cells in the Presence of Com-
pound 2 as a HNO Generator (A) and in the Presence of Compound
2+NAC (B), under Photoirradiation
Acknowledgments This work was supported in part by
Grants-in-Aid for Scientific Research on Innovative Areas
(Research in a Proposed Research Area) (No. 21117514 to
*p<0.05 when compared with 0µM by ANOVA and Bonferroni-type multiple
t-test (parametric, n=3–6).
photoirradiation is believed to occur via retro Diels–Alder H.N.) and Grants-in-Aid for Scientific Research (No. 22590103
type reaction, followed by hydrolysis of the resulting acyl- to H.N.) from the Ministry of Education, Culture, Sports, Sci-
nitroso intermediate (Chart 1). Although retro Diels–Alder ence and Technology of Japan.
reaction as a photochemical process is forbidden according
to the Woodward–Hoffmann rule, we considered that the ab-
sorbed energy from photoirradiation could be partly converted
into thermal (internal) energy of the molecule, for example,
through a non-radiative transition-like process. We previously
showed that the conjugated system is important for this type
of retro Diels–Alder reaction.22) This seems to be a unique ex-
ample of a photo-triggered retro Diels–Alder reaction.
To assess the effect of HNO released from compound 2
on cellular CGRP regulation, we chose A549 human alveolar
basal epithelial carcinoma cells, which have been reported
to produce CGRP.32) Considering that compound 2 exhibited
HNO release upon photoirradiation in vitro (Fig. 2B) and An-
geli’s salt induced CGRP upregulation in A549 cells (Figs. 5,
6), the increase of CGRP in the cells treated with compound
2 in combination with photoirradiation was considered to have
been induced via HNO formation. Suppression of this effect
by NAC, an HNO trap, was considered to support this conclu-
sion although NAC may have several effects on the cells. The
results also imply that HNO might act as an important regula-
tory factor of pathways in which CGRP is involved.
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