Photocontrollable peroxynitrite generator based on N-methyl-N- nitrosoaminophenol for cellular application

Article abstract of DOI:10.1021/ja206744z

We designed and synthesized a photocontrollable peroxynitrite (ONOO ^-) generator, P-NAP, which has N-methyl-N-nitrosoaminophenol structure with four methyl groups introduced onto the benzene ring to block reaction of the photodecomposition prod

Full text of DOI:10.1021/ja206744z

Article
pubs.acs.org/JACS
Photocontrollable Peroxynitrite Generator Based on N-Methyl-N-
nitrosoaminophenol for Cellular Application
Naoya Ieda,† Hidehiko Nakagawa,*,†,§ Tao Peng,‡ Dan Yang,‡ Takayoshi Suzuki,† and Naoki Miyata*,†
†Graduate School of Pharmaceutical Science, Nagoya City University, 3-1, Tanabe-dori, Mizuho-ku, Nagoya, Aichi 467-8603, Japan
‡Morningside Laboratory for Chemical Biology and Department Chemistry, The University of Hong Kong, Pokfulam Road, Hong
Kong, People’s Republic of China
§PRESTO, Japan Science and Technology Agency, 4-1-8 Honcho Kawaguchi, Saitama 332-0012, Japan
S
* Supporting Information
ABSTRACT: We designed and synthesized a photocontrollable
peroxynitrite (ONOO−) generator, P-NAP, which has N-methyl-
N-nitrosoaminophenol structure with four methyl groups
introduced onto the benzene ring to block reaction of the
photodecomposition product with ONOO− and to lower the
semiquinoneimine’s redox potential. The semiquinoneimine
intermediate generated by photoinduced release of nitric oxide
(NO) reduces dissolved molecular oxygen to generate superoxide
radical anion (O2•−), which reacts with NO to afford ONOO−
under diffusion control (k = 6.7 × 109 M−1 s−1). NO release from
P-NAP under UV-A (330−380 nm) irradiation was confirmed by ESR spin trapping. Tyrosine nitration, characteristic of
ONOO−, was demonstrated by HPLC analysis of a photoirradiated aqueous solution of P-NAP and N-acetyl-L-tyrosine ethyl
ester. ONOO− formation was confirmed with a ONOO−-specific fluorogenic probe, HKGreen-3, and compared with that from 3-
(4-morpholinyl)sydnonimine hydrochloride (SIN-1), which is the most widely used ONOO− generator at present. The
photoreaction of P-NAP was influenced by superoxide dismutase, indicating that generation of O2•− occurs before ONOO−
formation. The quantum yield for formation of duroquinone, the main P-NAP photodecomposition product, was measured as
0.86 0.07 at 334 nm with a potassium ferrioxalate actinometer. Generation of ONOO− from P-NAP in HCT-116 cells upon
photoirradiation was successfully imaged with HKGreen-3A. This is the first example of a photocontrollable ONOO− donor
applicable to cultured cells.
We have previously reported DiP-DNB (1),7 having a 2,6-
1. INTRODUCTION
Peroxynitrite (ONOO−) is one member of a family of reactive
dimethylnitrobenzene structure, as a photocontrollable
ONOO− donor that generates NO by isomerization reaction
nitrogen species (RNS). It is formed in vivo by reaction
of the nitro group and subsequent homolysis of nitrite upon
between nitric oxide (NO) and superoxide (O2•−) under
photoirradiation.8 After release of NO from this compound, the
diffusion control (k = 6.7 × 109 M−1 s−1).1 Due to its potent
resulting semiquinone intermediate reduces O2 to generate an
oxidizing power and nitrating activity for biological molecules,
equivalent amount of O2•−, and reaction of the two generates
ONOO− is cytotoxic and is associated with the pathogenesis of
ONOO− (Chart 1a), as confirmed with the ONOO−-specific
various diseases.2 On the other hand, nitrated biomolecules,
probe HKGreen-3.9
such as nitrotyrosine- and nitrotryptophan-containing proteins,
However, DiP-DNB showed low water solubility and did not
induce tyrosine nitration, a characteristic reaction of ONOO−.1
8-nitroguanosine, and nitro fatty acids, are associated with
essential cell signaling pathways,3 so ONOO− may be involved
Further, HPLC analysis suggested that the stilbene quinone 2
in regulation of these pathways.4 Consequently, ONOO−
generators are expected to be useful tools for biological
research and also candidate anticancer drugs, due to the ability
of ONOO− to activate dendritic cells.5 The ONOO− generator
most widely used at present is 3-(4-morpholinyl)sydnonimine
hydrochloride (SIN-1), which generates equal amounts of O2•−
and NO spontaneously under physiological conditions, leading
to ONOO− formation.6 However, to study in detail the
physiological and pathophysiological roles of ONOO−, it would
be preferable to release ONOO− in a temporally and spatially
controlled manner.
generated from DiP-DNB could react with ONOO− to form
benzaldehyde derivatives (presumably this reaction would be
initiated by nucleophilic attack of ONOO− on the quinone
methide moiety of 2).
To overcome these weaknesses, we focused on the N-methyl-
N-nitrosoaniline structure, which shows high NO-releasing
efficiency in response to photoirradiation.10 By applying our
original concept for DiP-DNB to N-methyl-N-nitrosoaniline,
Received: July 19, 2011
Published: January 5, 2012
© 2012 American Chemical Society
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Chart 1. Photocontrollable ONOO− Generator and
Proposed Mechanisms of ONOO− Release from DiP-DNB
(a) and P-NAP (b)
traps NO to yield NO-Fe-MGD complex, which exhibits a
broad three-line signal around 330 mT in 1 GHz ESR
spectroscopy. An aqueous solution of P-NAP was irradiated
with UV-A light (without attenuation) under an argon
atmosphere to avoid any effect of O2•− in the presence of Fe-
MGD complex. The ESR spectrum of the irradiated solution
showed typical triplet signals assigned to the NO-Fe-MGD
complex (Figure 1a), suggesting that P-NAP released NO upon
we designed P-NAP (3), which would not generate stilbene
quinone but would instead be converted to a highly substituted
quinoneimine, 4 (Chart 1b). The four methyl groups were
introduced at the benzene ring not only to block reaction with
ONOO− due to steric hindrance but also to lower the redox
potential of the semiquinoneimine in order to increase the
efficiency of O2•− generation.
Figure 1. ESR spectra of solutions containing P-NAP (100 μM),
FeSO4 (1.5 mM), and N-methylglucamine dithiocarbamate (6 mM)
(in PBS, pH 7.0, containing 1% DMF) after photoirradiation with UV-
A (without attenuation) for 15 min. (a) A solution photoirradiated
under an argon atmosphere. (b) A solution photoirradiated under
aerobic conditions. (c) A solution containing SOD (1 kunit/mL)
photoirradiated under aerobic conditions.
2. RESULTS AND DISCUSSION
P-NAP (3) was synthesized as shown in Scheme 1. Nitration of
bromobenzene derivative 5 afforded the nitrobenzene deriva-
a
irradiation. When the solution was irradiated under aerobic
conditions, the triplet signals were not observed (Figure 1b).
Moreover, the irradiated solution of P-NAP containing
superoxide dismutase (SOD), which catalyzes the dismutation
of O2•− into O2 and H2O2 under diffusion control (k = 2.0 ×
109 M−1 s−1),13 showed the triplet signals even under aerobic
conditions (Figure 1c). These results are consistent with the
proposed mechanism of ONOO− generation. That is, NO
appeared to be consumed by reaction with O2•− under diffusion
control, under aerobic conditions.
Scheme 1. Synthesis of P-NAP (3)
Tyrosine nitration is a characteristic reaction of ONOO−.1
Therefore, an aqueous solution of P-NAP and N-acetyl-L-
tyrosine ethyl ester (Ac-Tyr-OEt) was irradiated with UV-A
(without attenuation) and analyzed by HPLC. As shown in
Figure 2b, a peak of N-acetyl-L-3-nitrotyrosine ethyl ester (Ac-
nitroTyr-OEt) was detected in the irradiated solution. This
peak was not detected in the solution irradiated under an argon
atmosphere (Figure 2c). Furthermore, neither the solution
irradiated without P-NAP nor that incubated in the dark with
P-NAP showed a peak of Ac-nitroTyr-OEt (Figure S1).
Interestingly, when the solution was irradiated in the presence
of SOD, the peak of Ac-nitroTyr-OEt was larger than that in
the absence of SOD (Figure 2d). This result is consistent with
the report by Beckman et al. that SOD promotes nitration of
tyrosine by ONOO−.14 On the other hand, SOD might
suppress ONOO− generation by our photocontrollable
ONOO− donor, which generates NO and O2•−, by eliminating
O2•−. We considered that our results could be explained as
follows. To obtain the results in Figure 2, we treated a high
concentration of P-NAP (1 mM) with Ac-Tyr-OEt (1 mM) in
order to get a clear peak of Ac-nitroTyr-OEt. Excess O2•− might
escape from SOD and combine with NO to form ONOO−.
SOD might then facilitate tyrosine nitration by the ONOO−
a
Reagents and conditions: (a) NO2BF4, MeCN; (b) KOH, Pd2dba3, 2-
di-tert-butylphosphino-2′,4′,6′-triisopropylbiphenyl, 1,4-dioxane, H2O;
(c) Raney Ni, MeOH, H2; (d) HCOOH, Ac2O, CH2Cl2; (e) LiAlH4,
THF; (f) NaNO2, AcOH, H2O. dba = dibenzylideneacetone.
tive 6, which was subjected to Pd-catalyzed hydroxylation11 to
afford the 4-nitrophenol derivative 7. This product was reduced
with Raney Ni to the 4-aminophenol 8, which in turn was
converted to the formamide 9 with Ac2O and HCOOH.
Formamide 9 was reduced with LiAlH4 to give the N-methyl-4-
aminophenol derivative 10, which was nitrosated with NaNO2
to afford the desired final product, P-NAP (3). Its structure and
purity were confirmed by 1H NMR, 13C NMR, mass
spectrometry, and elemental analysis. P-NAP (1 mM) was
readily soluble in water in the presence of 1% DMF, whereas
over 10% DMF was required to achieve the same concentration
of DiP-DNB in water. Therefore, P-NAP should be superior to
DiP-DNB in biological applications.
To detect NO release, the first step of ONOO− generation
upon photoirradiation of P-NAP, we used an ESR spin-trapping
method with iron N-methylglucamine dithiocarbamate complex
(Fe-MGD) under an argon atmosphere.12 Fe-MGD efficiently
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observed (data not shown). This result implies the importance
of the four methyl groups as blocking substituents to prevent
the undesired reaction of 11 with ONOO−. Further, although
1,4-benzoquinone derivatives are known to show cytotoxicity
due to ROS generation, duroquinone is one of the least
cytotoxic 1,4-benzoquinone derivatives, because its four methyl
groups prevent repeated redox-driven ROS generation and
nucleophilic attack by thiols.16
Despite the weak absorbance of P-NAP around 330−380 nm
(Figure S3), P-NAP is decomposed by irradiation as effectively
as DiP-DNB, which has strong absorbance around the UV-A
region. The quantum yield of duroquinone (11) formation,
Φ
DQ, was determined with a potassium ferrioxalate actino-
meter.17 The value of observed ΦDQ at 334 nm was 0.86 0.07
for P-NAP. This high value of ΦDQ is considered to explain the
efficient decomposition of P-NAP by UV-A light.
For further confirmation of ONOO− generation, we
employed HKGreen-3, a ONOO−-specific fluorogenic probe
which fluoresces following nucleophilic attack of ONOO− on
−
the trifluoromethyl ketone moiety, resulting in NO2
elimination. Slight decomposition of HKGreen-3 was observed
in control solutions (Figure 3a, i and iii), but the fluorescence
was strongly increased upon photoirradiation in the presence of
P-NAP (Figure 3a, iii and iv). Furthermore, the fluorescence
increase in the presence of P-NAP was suppressed by
irradiation under an argon atmosphere or by irradiation
under aerobic conditions in the presence of SOD, presumably
because O2•− generation was suppressed (Figure 3a, v and vi).
A fluorescence increase was also observed after 2 h incubation
in the presence of SIN-1 (Figure 3a, vii and viii). The
concentration of ONOO− generated by P-NAP (10 μM) after
irradiation for 15 min was determined to be 4.6 μM, while that
generated by SIN-1 (10 μM) after incubation for 2 h, when the
fluorescence increase seemed to be saturated (Figure S4), was
determined to be 5.1 μM from a standard curve. We also
measured the concentration of NO3− as the final product of
ONOO− by means of the 2,3-diaminonaphthalene fluorescence
method.18 The amount of NO3− in the irradiated solution of P-
NAP (10 μM) was determined to be 6.0 μM. When a solution
of P-NAP was irradiated for various time periods and at various
light intensities, the fluorescence increase was clearly dependent
on both irradiation time and light intensity (Figure 3b).
Although the concentration of ONOO− generated from P-NAP
was a little less than that from SIN-1, P-NAP allows the amount
released to be controlled, while this is not the case with SIN-1.
We also synthesized other N-methyl-N-nitrosoaminophenol
derivatives having reduced numbers of methyl groups at the
benzene moiety (Figure S5, compounds S1−S3) and examined
their efficiency of ONOO− generation upon photoirradiation by
using HKGreen-3 (Figure S6). S1−S3 showed lower levels of
fluorescence increase than P-NAP. Moreover, these three
compounds did not induce nitration of Ac-Tyr-OEt. We
considered that the methyl groups may serve not only as
blocking groups to prevent undesired reaction of the
decomposition product with the produced ONOO−, but also
as electron-donating groups to lower the redox potential of the
semiquinoneimine intermediate, thereby enhancing generation
of O2•−. It was not possible to determine the reduction
potentials of the N-methylquinoneimine derivatives, due to
their instability. However, the reported reduction potentials of
the corresponding 1,4-benzoquinone derivatives are as follows:
unsubstituted 1,4-benzoquinone, 92 mV; 2,5-dimethyl-1,4-
benzoquinone, −76 mV; and duroquinone, −244 mV, while
Figure 2. Reversed-phase HPLC chromatograms for detection of Ac-
nitroTyr-OEt (arrows). The detector was set at 358 nm. (a) A solution
of Ac-nitroTyr-OEt (100 μM). (b) A solution of P-NAP (1 mM) and
Ac-Tyr-OEt (1 mM) in PBS (pH 7.0, 2% DMF) after irradiation with
UV-A (without attenuation) for 15 min. (c) The same solution as (b),
with irradiation under an argon atmosphere. (d) The same solution as
(b), but with SOD (1 kunit/mL) after irradiation with UV-A (without
attenuation) for 15 min.
formed. When smaller amounts of P-NAP and Ac-Tyr-OEt
were used, Ac-nitroTyr-OEt formation was decreased by
addition of SOD (Figure S2). Presumably, O2•− was scavenged
by SOD, and it became difficult to generate ONOO−. Beckman
et al. also suggested that SOD can catalyze nitration efficiently
only with ONOO−, but not with NO, NO2−, or NO2. These
results strongly suggest that ONOO− generated from P-NAP in
response to irradiation caused nitration of Ac-Tyr-OEt.
The peak of the main photodecomposition product of P-
NAP around 15 min in Figure 2b−d was identified as 2,3,5,6-
tetramethyl-1,4-benzoquinone, also called duroquinone (11),
suggesting that quinoneimine 4, the expected photodecompo-
sition product of P-NAP, is hydrolyzed to 11 (Chart 2). In our
Chart 2. Expected Formation Pathway of Duroquinone, the
Main Photodecomposition Product of P-NAP
study on DiP-DNB, we have discovered that stilbene quinone
2, a main photodecomposition of DiP-DNB, reacts with
ONOO− to yield 4-hydroxybenzaldehyde derivatives. Further, it
was reported that unsubstituted 1,4-naphthoquinone reacts
with ONOO− to yield epoxide.15 These reactions are
undesirable for our purpose, because the generated ONOO−
would be consumed. However, when 11 was treated with an
equivalent amount of ONOO−, no decomposition was
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Figure 4. UV translumination photograph of DNA strand-breaking
assay after horizontal gel electrophoresis. Samples containing P-NAP
and pBR322 plasmid DNA (0.04 mg/mL) in PBS (pH 7.0, containing
0.3% DMF) were photoirradiated with UV-A light (without
attenuation) or incubated at room temperature in the dark for 15
min. (a) The solution after incubation in the dark without P-NAP. (b)
The solution after irradiation without P-NAP. (c) The solution after
irradiation with P-NAP (30 μM). (d) The solution after irradiation
with P-NAP (100 μM). (e) The solution after irradiation with P-NAP
(300 μM).
3A), which can permeate through the cell membrane. HCT-116
cells were treated with HKGreen-3A and P-NAP and then
irradiated with UV-A light (attenuated to 25%) for 15 min. A
clear fluorescence increase in the cells was observed by confocal
microscopy. In the absence of photoirradiation or P-NAP, little
fluorescence was observed in the cells. Further, we conducted
MTT assay after incubation in the presence of P-NAP or after
photoirradiation of HCT-116 cells. P-NAP showed no
cytotoxicity at concentrations less than 100 μM (IC50 = 462
μM). Irradiation for 15 min with UV-A attenuated to 25% did
show cytotoxicity. In order to obtain clear fluorescence images
in Figure 5, we nevertheless conducted irradiation at this level.
However, as shown in Figure 3b, ONOO− could be generated
from P-NAP by using light attenuated to the 1.5% level, which
was not cytotoxic.
Figure 3. Fluorescence measurement for detection of ONOO−
generation using HKGreen-3. Samples contained P-NAP (10 μM)
and HKGreen-3 (10 μM) in PBS, pH 7.4, with 0.2% DMF. The
fluorescence intensity of sample solutions was determined at 535 nm
with excitation at 520 nm. (a) The fluorescence intensity after
irradiation of P-NAP with UV-A light (without attenuation) or
incubation for 15 min: (i) solutions without P-NAP incubated in the
dark; (ii) solutions including P-NAP incubated in the dark; (iii)
solutions without P-NAP after irradiation; (iv) solutions including P-
NAP after irradiation; (v) solutions including P-NAP after irradiation
under an argon atmosphere; (vi) solutions including P-NAP and SOD
(1 kunit/mL) after irradiation; (vii) solutions without SIN-1 incubated
at 37 °C for 2 h; and (viii) solutions including SIN-1 (10 μM)
incubated at 37 °C for 2 h. p < 0.01 for (iv) versus (i)-(iii), (v), and
(vi) by multiple t test (n = 3). (b) Fluorescence intensity after
irradiation of P-NAP for various time periods with various light
P-NAP forms very stable white crystals, and the solution
(100 mM) in DMF is stable for at least 3 months when stored
at −30 °C in the dark, as determined with a UV−visible
photospectrometer. This stability of the compound is favorable
for biological applications of P-NAP as a ONOO− generator.
3. CONCLUSION
We have developed a novel controllable ONOO− photo-
generator, P-NAP, which does not suffer from the disadvan-
tages associated with DiP-DNB (poor water solubility and
undesired reaction of the decomposition product with
ONOO−). NO generation from P-NAP was confirmed by
ESR, and ONOO− generation was confirmed by the use of a
fluorogenic probe, HKGreen-3. Further, P-NAP-mediated
tyrosine nitration was detected by means of HPLC analysis.
Duroquinone (11), which is formed after photoirradiation of P-
NAP, is one of the least toxic quinone derivatives due to its four
methyl groups. The results of analysis under anaerobic
conditions and in the presence of SOD indicated that not
only NO but also O2•− is generated from P-NAP upon
irradiation. The results of DNA strand-breaking assay and
fluorescence imaging with HKGreen-3A in HCT-116 cells
indicated not only that P-NAP could release ONOO− in
cultured cells but also that the generated ONOO− could act on
biomolecules such as DNA. On the basis of these results, we
believe that P-NAP will be a useful tool for controllable
ONOO− generation in biological research.
●
intensities. Light intensity was not attenuated ( ) or attenuated to
■
□
○
25% ( ), 6% ( ), or 1.5% ( ). Error bars were calculated from three
independent measurements.
that of molecular dioxygen is −180 mV (vs standard hydrogen
electrode).19 The reduction potentials of the N-methylquino-
neimine derivatives are expected to be more negative than
those of the corresponding 1,4-benzoquinones due to lower
electronegativity of the nitrogen atom than that of the oxygen
atom, so it seems reasonable that the tetramethyl intermediate
can reduce molecular oxygen to produce O2•−
.
Next, to examine the DNA cleavage reactivity of P-NAP, an
aqueous solution of pBR322 plasmid DNA was photoirradiated
with UV-A light (without attenuation) in the presence of P-
NAP. The irradiated solution was subjected to 1% agarose gel
electrophoresis. After horizontal gel electrophoresis, the gel was
stained with ethidium bromide, with UV translumination to
visualize DNA. As shown in Figure 4, DNA strand breakage was
concentration-dependent and was not observed in the solution
irradiated without P-NAP.
4. EXPERIMENTAL SECTION
Finally, photogeneration of ONOO− from P-NAP in HCT-
116 cells was examined with acetylated HKGreen-3 (HKGreen-
General Methods. Melting point was determined with a Buchi
̈
545 melting point apparatus. Proton nuclear magnetic resonance
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Figure 5. Fluorescence images of ONOO− generation from P-NAP in HCT-116 cells in the presence of HKGreen-3A. Cultured HCT-116 cells were
treated with HKGreen-3A (10 μM) and P-NAP (10 μM). The dishes were then photoirradiated with UV-A light (attenuated to 25%) or incubated
in the dark for 15 min. The dishes were observed with a confocal microscope (a) after photoirradiation with P-NAP, (b) after incubation with P-
NAP in the dark, and (c) after photoirradiation without P-NAP.
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 Yanaco CHN CORDER NT-5 analyzer, and all
values were within 0.4% of the calculated values. The MS spectra
(EI-GC) were recorded on a JEOL JMS-SX102A mass spectrometer.
Ultraviolet−visible light absorbance spectra were recorded on an
Agilent 8453 spectrometer. All other reagents and solvents were
purchased from Aldrich, Tokyo Kasei Kogyo, Wako Pure Chemical
Industries, Nacalai Tesque, Kanto Kagaku, Kishida Kagaku, Junsei
Kagaku, and Dojindo and used without purification. Flash column
chromatography was performed using silica gel 60 (particle size
0.046−0.063 mm) supplied by Merck.
stirred for 20 min and then filtered through Celite. The filtrate was
evaporated in vacuo. Purification of the residue by silica gel flash
chromatography (n-hexane:AcOEt = 4:1) gave 215 mg (30%) of 10 as
a pale orange solid: H NMR (CDCl3, 500 MHz; δ, ppm) 4.40 (1H,
br), 2.63 (3H, s), 2.24 (6H, s), 2.18 (6H, s), 1.60 (1H, br).
1
Synthesis of P-NAP (3). A solution of 10 (195 mg, 1.09 mmol) in
AcOH (5 mL) was added to a solution of NaNO2 (75 mg, 1.09 mmol,
1.0 equiv) in water (6 mL) on an ice−water bath. The reaction
mixture was stirred on the ice−water bath for 10 min and then
quenched with saturated NaHCO3 and extracted with CHCl3. The
organic layer was washed with brine and dried over Na2SO4. Filtration,
evaporation of the filtrate in vacuo, and purification of the residue by
silica gel flash chromatography (n-hexane:AcOEt = 4:1) gave 147 mg
(65%) of 3 as a white solid. This product was recrystallized from
AcOEt/MeOH to afford 116 mg of 3 as white needles: mp 162.4−
Synthesis of 6. To a solution of 5 (1.81 g, 8.50 mmol) in MeCN
(30 mL) was added NO2BF4 (1.49 g, 11.2 mmol, 1.3 equiv). The
mixture was stirred at room temperature for 10 min. The reaction
mixture was quenched with water and extracted with CHCl3. The
organic layer was washed with brine and dried over Na2SO4. Filtration
and evaporation of the filtrate in vacuo gave 2.28 g (quant.) of 6 as a
1
163.9 °C; H NMR (CDCl3, 500 MHz; δ, ppm; 3:2 mixture of two
rotamers) (major) 4.77 (1H, s), 3.96 (3H, s), 2.16 (6H, s), 1.90 (6H,
s), (minor) 4.84 (1H, s), 3.31 (3H, s), 2.22 (6H, s), 2.01 (6H, s); 13C
NMR (CDCl3, 150 MHz; δ, ppm) 152.5, 152.5, 133.9, 132.4, 130.8,
130.7, 120.5, 120.3, 77.5, 40.4, 35.5, 14.9, 14.7, 12.3; MS (FAB) m/z
209 [(M+1)+]. Anal. Calcd for C11H16N2O2: C, 63.44; H, 7.74; N,
13.45. Found: C, 63.18; H, 7.58; N, 13.29.
1
white solid: H NMR (CDCl3, 500 MHz; δ, ppm) 2.44 (6H, s), 2.21
(6H, s).
Synthesis of 7. To a solution of 6 (2.28 g, 8.85 mmol) in 1,4-
dioxane (20 mL) were added Pd2dba3 (84 mg, 0.0917 mmol, 0.01
equiv) and 2-di-tert-butylphosphino-2′,4′,6′-triisopropylbiphenyl (75
mg, 0.177 mmol, 0.02 equiv), followed by 1 N KOH (20 mL). The
reaction mixture was stirred at 90 °C under a N2 atmosphere for 24 h.
After cooling, the reaction mixture was acidified with 2 N HCl and
filtered. The filtrate was extracted with AcOEt. The organic layer was
washed with brine and dried over Na2SO4. Filtration, evaporation of
the filtrate in vacuo, and purification of the residue by silica gel flash
chromatography (n-hexane:AcOEt = 9:1) gave 1.01 g (59%) of 7 as a
yellow solid: 1H NMR (CDCl3, 500 MHz; δ, ppm) 4.95 (1H, s), 2.18
(6H, s), 2.15 (6H, s).
Synthesis of 8. To a solution of 7 (1.01 g, 5.19 mmol) in MeOH
(15 mL) was added a catalytic amount of Raney Ni. The reaction
mixture was stirred at room temperature under a H2 atmosphere for 14
h and then filtered through Celite. The filtrate was evaporated in vacuo
to afford 650 mg (76%) of 8 as a brown solid: 1H NMR (CDCl3, 500
MHz; δ, ppm) 4.15 (1H, s), 3.35 (2H, s), 2.20 (6H, s), 2.17 (6H, s).
Synthesis of 9. A mixture of Ac2O (446 μL, 4.72 mmol, 1.2 equiv)
and HCOOH (594 μL, 15.7 mmol, 1.2 equiv) was stirred at room
temperature for 1 h. The mixture was added to a slurry of 8 (650 mg,
3.93 mmol) in CH2Cl2 (40 mL). The reaction mixture was stirred at
room temperature for 10 min, MeOH was added, and the mixture was
evaporated in vacuo to afford 770 mg (quant.) of 9 as a white solid: 1H
NMR (CDCl3, 500 MHz; δ, ppm) 9.21 (1H, s), 8.20 (1H, d, J = 1.2
Hz), 7.97 (1H, s), 2.08 (6H, s), 1.99 (6H, s).
Irradiation of Sample Solutions. Photoirradiation in the
ultraviolet-A range was performed by using the light source (100 W
Hg lamp) of a fluorescence microscope (Olympus BX60/BX-FLA)
with a WU filter (330−380 nm band-pass filter) at room temperature.
When required, the light intensity was attenuated to 25%, 6%, or 1.5%
by inserting attenuation filters.
ESR Analysis by Using Iron Dithiocarbamate Complex. A
solution (total volume 200 μL) of FeSO4·6H2O (1.5 mM), N-
methylglucamine dithiocarbamate (6 mM), and P-NAP (100 μM) in
PBS (pH 7.0, DMF 1%) was photoirradiated (without attenuation) at
room temperature for 15 min as a sample for ESR studies. Next, the
appropriate SOD (1 kunit/mL) was added or the solution was
irradiated under an argon atmosphere. ESR spectra were taken on a
JES-RE2X spectrometer (JEOL Co. Ltd., Tokyo, Japan). The
measurement conditions were 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.
HPLC Analysis for Detection of Ac-NitroTyr-OEt and
Duroquinone. Sample solutions (total volume 200 μL) of P-NAP
(1 mM) and N-acetyl-L-tyrosine ethyl ester (1 mM) in PBS (pH 7.0,
containing 2% DMF) were irradiated. An aliquot of each solution (20
μL) was loaded onto a Shenshu Pak C18 column (5 mm; 150 × 4.6
mm) fitted on a Shimadzu HPLC system, and the eluates were
monitored with a photodiode array detector. Milli-Q water containing
0.1% TFA (A) and MeCN containing 0.1% TFA (B) were used as
developing solvents. Gradient conditions were as follows: 0 min, A
70% and B 30% → 20 min, A 20% and B 80% → 30 min, A 20% and B
80% → 35 min, A 70% and B 30% → 40 min, A 70% and B 30%.
Fluorescence Measurement of HKGreen-3 in Vitro. Sample
solutions (total volume 1 mL) containing P-NAP (10 μM) or SIN-1
(10 μM) and HKGreen-3 (10 μM) in PBS (pH 7.4, containing 0.2%
Synthesis of 10. To a slurry of LiAlH4 (940 mg, 25.0 mmol, 6.3
equiv) in dry THF (20 mL) was added a slurry of 9 (770 mg, 3.99
mmol) on an ice−water bath. The reaction mixture was stirred at
reflux for 1 h and then allowed to cool, and water (1 mL), 2 N NaOH
(2 mL), and water (3 mL) were added successively. The mixture was
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Journal of the American Chemical Society
Article
DMF) were irradiated or incubated in the dark. The fluorescence
intensity was determined with an RF5300-PC (Shimadzu) at 535 nm,
with excitation at 520 nm. The slit width was set to 3 nm for both
excitation and emission.
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Assay for DNA Strand Breaks. Sample solutions (total volume
20 μL) of PBS (pH 7.0) containing 0.04 mg/mL of pBR 322 DNA,
0.3% DMF, and 0, 30, 100, or 300 μM P-NAP were photoirradiated
with UV-A (without attenuation) or incubated at room temperature in
the dark for 15 min. The reaction mixtures were treated with 5 μL of
loading buffer (100 mM TBE buffer, pH 8.3, containing 30% glycerol,
0.1% bromophenol blue). Horizontal gel electrophoresis was carried
out in 89 mM TBE buffer (pH 8.3) for 60 min at 100 V. The gel was
stained with ethidium bromide (1 mg/mL) for 30 min, destained in
TBE buffer for 30 min, and photographed with UV transillumination.
Detection of ONOO− in HCT-116 Cells. HCT-116 human colon
cancer cells were treated in the same manner as in the confocal
microscopy experiments. Briefly, the cells were plated on 3 cm glass-
bottomed dishes at 1.0 × 105 cells/dish with 2 mL of McCoy’s 5A
culture medium and then incubated at 37 °C in a humidified 5% (v/v)
CO2 incubator for 2 days. The cells were treated with 10 μM
HKGreen-3A and incubated at 37 °C in a humidified 5% (v/v) CO2
incubator for a further 2 h. The cells were treated with 10 μM P-NAP
for 15 min and subjected to photoirradiation for 15 min with the light
intensity attenuated to 25%. The dishes were subsequently washed
with DMEM three times, and the culture medium was replaced with 2
mL of DMEM. After washing, the cells were subjected to confocal
fluorescence microscopy (LSM 510, Carl Zeiss Japan Co. Ltd., Tokyo,
Japan).
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ASSOCIATED CONTENT
* Supporting Information
■
(12) Yoshimura, T.; Kotake, Y. Antioxidants Redox Signaling 2004, 6,
639−647.
S
Supporting figures, syntheses of S1−S3, 1H NMR spectra for
S1−S3 and P-NAP, and MTT assay. This material is available
free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
deco@phar.nagoya-cu.ac.jp; miyata-n@phar.nagoya-cu.ac.jp
■
(15) Nonoyama, N.; Oshima, H.; Shoda, C.; Suzuki, H. Bull. Chem.
Soc. Jpn. 2001, 74, 2385−2395.
ACKNOWLEDGMENTS
■
(16) Siraki, A. G.; Chan, T. S.; O’Brien, P. J. Toxicol. Sci. 2004, 81,
148−159.
This work was supported by the JST PRESTO program (H.N.)
from Japan Science and Technology Agency, as well as by a
Grant-in-Aid for Scientific Research on Innovative Areas
(Research in Proposed Research Area) (No. 21117514 to
H.N.) and a Grant-in-Aid for Scientific Research (No.
22590103 to H.N.) from the Ministry of Education, Culture,
Sports Science and Technology Japan, and a Sasagawa Scientific
Research Grant from the Japan Scientific Society (No. 23-322,
N.I.).
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New York, 1973; pp 119−123.
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(19) Song, Y.; Buettner, G. R. Free Radic. Biol. Med. 2010, 49, 919−
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