Nitric oxide turn-on fluorescent probe based on deamination of aromatic primary monoamines

Article abstract of DOI:10.1021/ic300379u

The stable, water-soluble, and nonfluorescent FA-OMe can sense nitric oxide (NO) and form the intensely fluorescent product dA-FA-OMe via reductive deamination of the aromatic primary amine. The reaction is accompanied by a notable increase of the fluorescent quantum yield from 1.5 to 88.8%. The deamination mechanism of FA-OMe with NO was proposed in this study. The turn-on fluorescence signals were performed by suppression of photoinduced electron transfer (PeT), which was demonstrated by density functional theory (DFT) calculations of the components forming FA-OMe and dA-FA-OMe. Furthermore, FA-OMe showed water solubility and good stability at physiological pHs. Moreover, the selectivity study indicated that FA-OMe had high specificity for NO over other reactive oxygen/nitrogen species. In an endogenously generated NO detection study, increasing the incubation time of FA-OMe with lipopolysaccharide (LPS) pretreated Raw 264.7 murine macrophages could cause an enhanced fluorescence intensity image. In addition, a diffusion/localization cell imaging study showed that FA-OMe could be trapped in Raw 264.7 cells. These cell imaging results demonstrated that FA-OMe could be used as a turn-on fluorescent sensor for the detection of endogenously generated NO.

Full text of DOI:10.1021/ic300379u

Article
pubs.acs.org/IC
Nitric Oxide Turn-on Fluorescent Probe Based on Deamination of
Aromatic Primary Monoamines
†
†
‡
†
‡
§
Tsun-Wei Shiue, Yen-Hao Chen, Chi-Ming Wu, Gyan Singh, Hsing-Yin Chen, Chen-Hsiung Hung,
∥
,†
Wen-Feng Liaw, and Yun-Ming Wang*
†Department of Biological Science and Technology, Institute of Molecular Medicine and Bioengineering, National Chiao Tung
University, 75 Bo-Ai Street, Hsinchu 300, Taiwan
‡
Department of Medicinal and Applied Chemistry, Kaohsiung Medical University, 100 Shih-Chuan first Road, Kaohsiung 807, Taiwan
Institute of Chemistry, Academia Sinica, 128 Academia Road Sec. 2, Nankang, Taipei 115, Taiwan
§
∥
Department of Chemistry, National Tsing Hua University, 101 Kuang-Fu Road Sec. 2, Hsinchu 300, Taiwan
*
S Supporting Information
ABSTRACT: The stable, water-soluble, and nonfluorescent FA-
OMe can sense nitric oxide (NO) and form the intensely fluorescent
product dA-FA-OMe via reductive deamination of the aromatic
primary amine. The reaction is accompanied by a notable increase of
the fluorescent quantum yield from 1.5 to 88.8%. The deamination
mechanism of FA-OMe with NO was proposed in this study. The
turn-on fluorescence signals were performed by suppression of
photoinduced electron transfer (PeT), which was demonstrated by
density functional theory (DFT) calculations of the components
forming FA-OMe and dA-FA-OMe. Furthermore, FA-OMe showed
water solubility and good stability at physiological pHs. Moreover,
the selectivity study indicated that FA-OMe had high specificity for
NO over other reactive oxygen/nitrogen species. In an endogenously
generated NO detection study, increasing the incubation time of FA-
OMe with lipopolysaccharide (LPS) pretreated Raw 264.7 murine macrophages could cause an enhanced fluorescence intensity
image. In addition, a diffusion/localization cell imaging study showed that FA-OMe could be trapped in Raw 264.7 cells. These
cell imaging results demonstrated that FA-OMe could be used as a turn-on fluorescent sensor for the detection of endogenously
generated NO.
8
1
. INTRODUCTION
electron transfer (PeT) mechanism. After reaction with NO or
the NO oxidized products, the PeT fluorescence quenching
property is suppressed and the fluorescence of the probe is
restored. The o-diaminophenyl group is the most commonly
Nitric oxide (NO), produced by nitric oxide synthases in the
biological systems, has been known as a ubiquitous signaling
molecule. Despite being a simple molecule, NO is involved in
9
1
used NO-reactive moiety in organic fluorescent probes. The
diverse physiological and pathological pathways. Although NO
electron-rich o-diaminophenyl group can react with NO under
aerobic conditions to produce fluorescent triazole derivatives.
However, there are some undesired handicaps, such as
complicated and low-yield synthetic procedures and blank
fluorescence, existing in these kinds of NO probes.
is a key vertebrate biological messenger, the mechanisms by
which it performs its diverse biological roles remain elusive. In
order to comprehend the diverse biological roles of NO, several
2
3
techniques such as chemiluminescence, colorimetry, electron
4
5
6
paramagnetic resonance, electrochemistry, and fluorimetry
In this study, the possibility of using an aromatic primary
monoamine moiety as an NO-reactive site for the modulation
of PeT was explored. The basic concept is outlined in Figure 1.
The study was inspired by the genotoxic effects of NO. The
NO-induced deamination of cytosine, adenine, guanine, and 5-
methylcytosine indeed represents an important NO-induced
have been developed. In view of its sensitivity, selectivity,
spatiotemporal resolution, and experimental feasibility, fluorim-
etry, which exploits fluorescent probes to monitor analytes of
interest under a fluorescence microscope, has been regarded as
a most promising method to detect endogenous NO. There
have been a number of small-molecule organic or inorganic
10
7
genotoxic mechanism. In addition, previous research reported
that some aromatic amines react with NO or N O to give the
fluorescent NO sensors reported to date. A two-component
system comprised of an NO-reactive moiety coupling to an
organic fluorophore has been widely investigated to develop
NO-responsive organic molecule based sensors. The NO-
reactive moiety acts as a modulator in the photoinduced
2
3
Received: February 20, 2012
Published: April 9, 2012
©
2012 American Chemical Society
5400
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Article
Tof mass spectrometer. EI-HRMS spectra were recorded on a
Finnigan/Thermo Quest MAT mass spectrometer. UV/vis absorption
spectra were recorded on a Hitachi U-3000 spectrophotometer.
Bright-field and fluorescence images were recorded on an Olympus
IX71 fluorescence microscope equipped with a 100 W mercury lamp,
B-2A filters, and a color CCD camera system.
2
.2. Preparation of 5-Amino-2-(6-hydroxy-3-oxo-3H-xanth-
en-9-yl)benzoic Acid Methyl Ester (FA-OMe). A modification of
Figure 1. An NO turn-on probe through NO-induced deamination of
15
the synthetic procedure of FA-OMe was employed. A mixture of
an aromatic primary amino group.
fluoresceinamine (200 mg, 0.576 mmol), MeOH (10 mL), and
concentrated H SO solution (0.2 mL) was heated for 48 h under
2
4
reflux. The solution was poured on ice (300 g), and then a saturated
1
1
corresponding deaminated aromatics. In addition, it was
reported that fluorescence of a fluorescent molecule could be
adjusted by changing the electronic influence of the nitrogen
NaHCO solution was slowly added to adjust the pH to 6.5. An orange
3
precipitate was separated by centrifugation and then subjected to TLC,
with MeOH/CH Cl as eluant (1/12, v/v). A yellow band was isolated
2
2
12
electron lone pair. Therefore, we have investigated the ability
of NO to deaminate the aromatic primary amino group,
thereby altering the electronic influence of the aromatic moiety
on the fluorophore. To the best of our knowledge, a NO turn-
on fluorescent probe through reductive deamination of an
aromatic primary monoamine has never been reported.
Among the various aromatic amines, we prefer aromatic
amino esters to be the NO-reactive moieties. The ester
functionality can enhance the cellular uptake of a probe and
ensure its trappability within the plasma membrane. In the
current study, 5-amino-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)-
benzoic acid methyl ester (FA-OMe) was synthesized as an NO
turn-on fluorescent probe. The reaction product (dA-FA-OMe)
of FA-OMe with NO under aerobic conditions was identified,
and a possible reaction mechanism was proposed. The DFT
calculations of dA-FA-OMe relative to FA-OMe were under-
taken to interpret the PeT phenomenon. For realizing the level
of fluorescence enhancement, the fluorescent properties of FA-
OMe and dA-FA-OMe were studied and compared. In order to
confirm their stability, the fluorescence intensities of FA-OMe
and dA-FA-OMe under various pH conditions were measured.
To comprehend the NO-detecting ability, time- and concen-
tration-dependent studies were also investigated. In addition,
the selectivity of FA-OMe for NO against other reactive oxygen
and nitrogen species was investigated. Furthermore, in vitro
fluorescence images were obtained.
and identified as 5-amino-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)-
benzoic acid methyl ester (FA-OMe; 184 mg, 88.5%). ESI-LRMS:
+
+
1
m/z 362.1 ([M + H] ), M calculated 361.1. H NMR (DMSO-d ): δ
6
7
.30 (s, 1H), 7.00 (d, J = 9.0 Hz, 1H), 6.90 (d, J = 9.0 Hz, 1H), 6.77
(
d, J = 9.0 Hz, 2H), 6.30 (d, J = 9.0 Hz, 2H), 6.23 (s, 2H), 5.81 (br s,
2
1
5
H), 3.49 (s, 3H). 13C NMR (DMSO-d ): δ 178.4, 166.2, 157.3, 152.9,
6
49.7, 131.5, 130.8, 129.8, 122.8, 120.7, 117.0, 114.7, 111.1, 103.1,
1.9.
2
.3. Reaction of FA-OMe with NO/O . In 100 mL of an FA-OMe
2
(30 mg, 0.083 mmol) solution in 100 mM HEPES buffer at pH 7.4,
10% NO gas was bubbled under aerobic conditions at room
temperature. The reaction was terminated using TLC to ensure the
consumption of all of FA-OMe. Then the solvent was removed on a
rotary evaporator. The residue was subjected to TLC, with MeOH/
CH Cl as eluant (1/10, v/v). The first bright yellow band was isolated
13
2
2
and identified as 2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzoic acid
methyl ester (dA-FA-OMe; 22.4 mg, 78.0%). ESI-LRMS: m/z 347.1
+
+
+
+
(
[M + H] ), M calculated 346.1. EI-HRMS: m/z 346.0836 (M ), M
1
calculated 346.0841. H NMR (DMSO-d ): δ 8.12 (d, J = 7.8 Hz, 1H),
6
7.80 (dd, J = 7.5 and 7.8 Hz, 1H), 7.71 (dd, J = 7.5 and 7.8 Hz, 1H),
7.40 (d, J = 7.5 Hz, 1H), 6.59 (d, J = 9.0 Hz, 2H), 6.30 (m, 4H), 3.54
(s, 3H). 13C NMR (DMSO-d
1
2
): δ178.7, 165.5, 157.2, 151.3, 134.8,
32.7, 130.7, 130.3, 129.8, 129.5, 129.3, 123.2, 110.3, 103.4, 52.2.
.4. Reaction of Fluoresceinamine with NO/O . In 45 mL of a
6
2
fluoresceinamine (10 mg, 0.029 mmol) solution in 100 mM HEPES
buffer at pH 7.4, 10% NO gas was bubbled under aerobic conditions at
room temperature. The reaction was terminated using TLC to ensure
the consumption of all of fluoresceinamine. Then the solvent was
removed on a rotary evaporator. The residue was subjected to TLC,
with MeOH/CH Cl as eluant (1/10, v/v). The first bright yellow
2
2
2. EXPERIMENTAL SECTION
band was isolated and identified as fluorescein (7.54 mg, 78.3%).
2.5. Preparation of NO Stock Solutions. The preparation of NO
and its stock solutions were carried out according to the reported
2
.1. Materials and Instruments. All chemicals were obtained
from commercial suppliers and used as received without further
1
6
purification. Hydrogen peroxide (H O ), dehydroascorbic acid
methods. NO could be generated by slowly dropping 2.0 M
2
2
(
(
DHA), ascorbic acid (AA), ethylenediaminetetraacetic acid
EDTA), lipopolysaccharide (LPS), and fluoresceinamine were
H SO (aq) into a glass flask containing a saturated NaNO water
2
4
2
solution. Since O will rapidly oxidize NO to form NO , all apparatus
2
2
purchased from Sigma-Aldrich. Sodium nitrite (NaNO ) and sodium
were carefully degassed with argon for 30 min to remove O . The
2
2
nitrate (NaNO ) were purchased from Showa. Angeli’s salt
forming gas was passed twice through a 30% NaOH solution and once
3
(
0
Na N O ) and sodium peroxynitrite (ONOONa, as a solution in
.3 M NaOH) were purchased from Cayman Chemical Co. 4-(2-
through water to trap NO generated from the reaction of NO with
2
2
3
2
traces of O . To produce a saturated NO solution (1.8 mM, at 20 °C)
2
Hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES, free acid,
Ultrapure Bioreagent) was purchased from MP Biomedicals. Ferrous
ammonium sulfate hexahydrate (Fe(NH ) (SO ) ·6H O) was pur-
as a stock solution, 10 mL of deoxygenated deionized water was
bubbled with NO for 30 min and kept under an NO atmosphere. The
saturation concentration was ascertained by using the Griess method.
Stock solutions were freshly prepared for each experiment and stored
in a glass flask with a rubber septum.
3a
4
2
4
2
2
chased from J. T. Baker. Dulbecco’s Modified Eagle Medium
DMEM) and sodium pyruvate were purchased from Cellgro. Fetal
(
bovine serum (FBS, 10%) was purchased from HyClone. NO(g) (10%
2.6. Fluorometric Analysis. Fluorescence spectra were recorded
by using a Hitachi F-7000 spectrophotometer. The slit widths were 5.0
and 2.5 nm for excitation and emission, respectively. The photon
multiplier voltage was 500 V. Spectra were routinely acquired at 25.0 ±
0.1 °C in quartz cuvettes with a volume of 3.5 mL.
NO/90% N ) was purchased from SanFu. It was passed through an
2
1
4
Ascarite II column to remove higher nitrogen oxides before use.
Deionized water was produced by a Milli-Q reagent water purification
system. Methanol was distilled over Mg/I and subsequently dried
2
over 0.3 nm molecular sieves. Preparative thin-layer chromatographic
2.6.1. Fluorescence Quantum Yield Determination. Fluorescence
quantum yields of FA-OMe and dA-FA-OMe were measured in 0.1 M
1
(
TLC) plates were prepared from silica gel (Macherey-Nagel). H and
1
3
C NMR spectra were obtained on a Varian VXR-300 spectrometer at
00 and 75 MHz and were referenced to the internal H and
NaOH(aq) by using fluorescein (Φ = 0.95 in 0.1 M NaOH(aq)) as a
fl
1
13
12b
3
C
standard. Fluorescence quantum yields were calculated by use of the
following equation: Φunk = Φ (Iunk/Aunk)(A /I )(n /n ) , where
2
solvent peaks. ESI-LRMS spectra were recorded on a Micromass Q-
std
std std
unk std
5
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1
Figure 2. H NMR spectra of FA-OMe (A) and dA-FA-OMe (B) in d -DMSO at 25.0 ± 0.1 °C.
6
Φ
is the fluorescence quantum yield of the sample, Φ is the
nitrogen-purged aqueous solutions of AA and DHA in 100 mM
unk
std
fluorescence quantum yield of the standard, I and I are the
HEPES buffer at pH 7.4 containing 3 mM EDTA were freshly
unk
std
2
1
integrated emission intensities of the sample and the standard,
respectively, Aunk and Astd are the absorbances of the sample and the
standard at the excitation wavelength, respectively, and nunk and nstd are
the refractive indexes of the corresponding solutions, respectively.
prepared before use.
2.7. Computational Methods. Density functional theory (DFT)
calculations at the B3LYP/6-31++G** level were used to obtain
optimized geometries and energy levels of frontier molecular orbitals
for methyl 3-aminobenzoate, methyl benzoate, and xanthene, the
components forming FA-OMe and dA-FA-OMe. The hydration effect
on energy levels was considered by performing single-point energy
calculations with the polarizable continuum model on gas-phase
optimized structures. All calculations were carried out with the
2
.6.2. Stability Studies of FA-OMe, dA-FA-OMe, and Fluorescein-
amine. Solutions of 20 μM FA-OMe in 100 mM HEPES in the pH
range 6−10 were prepared by mixing 0.2 mL of 500 μM solutions of
FA-OMe in deionized water with 4.8 mL of 100 mM HEPES whose
pH values were adjusted by hydrogen chloride and sodium hydroxide.
Solutions of 20 μM dA-FA-OMe and 20 μM fluoresceinamine in 100
mM HEPES in the pH range 6−10 were prepared as described above.
The fluorescence intensities of FA-OMe, dA-FA-OMe, and fluo-
resceinamine were measured after 2 h of mixing (λex 460 nm, λem 524
nm).
2
2
Gaussian 09 program.
2.8. Cell Culture and In Vitro Cell Imaging. Raw 264.7 murine
macrophage is organized from tumor ascites induced by intra-
peritoneal injection of Abselon Leukemia Virus (A-MuLV) in male
mice. Raw 264.7 murine macrophages were obtained from the
American Type Culture Collection (Manassas, VA). The cells were
cultured in DMEM and supplemented with 10% FBS, 1% sodium
pyruvate, and 1% MEM nonessential amino acids at 37 °C under a
humidified 5% CO2 atmosphere. For imaging studies, Raw 264.7
murine macrophages were passed and plated into poly-D-lysine coated
plates containing 2 mL of DMEM and incubated at 37 °C with 5%
2
.6.3. Selectivity Studies. All the fluorescence tests were performed
in 100 mM HEPES buffer at pH 7.4 and 25.0 ± 0.1 °C. The
fluorescence intensities were monitored after reacting 3 mL of 20 μM
FA-OMe (in 100 mM HEPES buffer at pH 7.4) with 100 equiv of
reactive oxygen species (ROS), reactive nitrogen species (RNS),
ascorbic acid (AA), and dehydroascorbic acid (DHA) for 2 h,
respectively (λ 460 nm, λem 524 nm). The concentration of the
ONOO stock solution was calculated by using its molar extinction
coefficient of 1670 M cm at 302 nm. The degassed aqueous
solutions of Angeli’s salt were freshly prepared under anaerobic
ex
CO . For endogenously generated nitric oxide detection studies, iNOS
2
−
(inducible nitric oxide synthase) was induced in Raw 264.7 murine
−1
−1
17
−1
macrophages with 0.5 μg mL of LPS for 4 h, and the cells were then
coincubated with 10 μM of FA-OMe for 4 and 8 h. Prior to imaging,
the cells were washed three times with 1 mL of PBS and then bathed
in 2 mL of PBS. The fluorescence alterations were monitored by the
fluorescence microscope. For FA-OMe diffusion/localization studie-
18
conditions and used as a nitroxyl (HNO) source. Hydroxyl radical
•
(
OH) was produced by reacting ferrous ammonium sulfate
hexahydrate (Fe(NH ) (SO ) ·6H O) with H O under anaerobic
conditions. Hydrogen peroxide (H O ) was diluted promptly from
the 30% H O solution and quantified by measuring its absorbance at
4
2
4
2
2
2
2
19
13a
s, Raw 264.7 murine macrophages were prestimulated with 1.25 μg
2
2
−1
mL of LPS for 4 h, and then the cells were coincubated with 10 μM
of FA-OMe for 8 h. The cells were washed three times with 1 mL of
PBS prior to imaging and then bathed in 2 mL of PBS during the
imaging procedure. To mimic media flow, the PBS was removed, and
2
2
−1
−1 20
240 nm with a molar absorption coefficient of 43.6 M cm . The
aqueous solutions of NaNO and NaNO were freshly prepared and
used as nitrite (NO ) and nitrate (NO ) sources, respectively. The
2
3
−
−
2
3
5
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Scheme 1. Proposed Mechanism of FA-OMe with NO under Aerobic Conditions To Form dA-FA-OMe
the cells were washed three times with 2 mL of fresh PBS and then
bathed in 2 mL of fresh PBS while on the fluorescence microscope.
This process was repeated every 10 min during imaging.
ppm shows that the hydroxyls of FA-OMe and dA-FA-OMe
may be deprotonated in the solution. Thus, eight types of
protons in different chemical environments for FA-OMe (a−h)
and dA-FA-OMe (a′−h′) are observed, respectively. As shown
in Figure 2B, b′ and c′ signals of dA-FA-OMe present the
splitting pattern of a doublet of doublets (dd, J = 7.5 and 7.8
Hz) at 7.80 and 7.71 ppm, respectively. In addition, the f′ signal
merging with the g′ signal is a doublet signal, like the e signal of
FA-OMe, as shown in Figure 2A. The primary amino group of
FA-OMe, because of its electron-donating effect, increases the
valence electron density around the protons of the phenyl ring.
Therefore, the protons (a−c) are shielded higher than those of
dA-FA-OMe (a′−d′) without an amino group. This result
makes the a−c signals shift upfield in comparison to a′−d′
signals. The 13C NMR spectra of FA-OMe and dA-FA-OMe
display 15 resonance signals, respectively, which indeed
correspond with the C2v symmetry (Figure S1 in the
Supporting Information).
2
.9. Cell Viability Studies. The Raw 264.7 murine macrophages
5
were grown in 96-well plates at an initial density of 10 cells per well
for 24 h. Subsequently, different concentrations of FA-OMe were
incubated with Raw 264.7 murine macrophages at 37 °C for 24 h.
After incubation, the supernatant was removed and the cells were
washed three times with PBS buffer solution. Cell viability was
evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo-
lium bromide (MTT) reduction assay. Briefly, MTT (20 μL, 5 mg
−1
mL ) was added to each well. After 4 h of incubation, each well was
treated with dimethyl sulfoxide (100 μL) with pipetting. The
absorbance at 570 nm was measured on a plate reader. Each treatment
was done in six wells, and the experiments were repeated three times.
Cell viability was calculated relative to the absorbance of the control
for each treatment. The viability of untreated cells was assumed to be
100%.
3. RESULTS AND DISCUSSION
The reaction mechanism for deamination of FA-OMe is
proposed as shown in Scheme 1. First, the primary amino
3
.1. Synthesis, Characterization, and Proposed Mech-
anism. FA-OMe was easily prepared by refluxing the MeOH
solution of fluoresceinamine with acid catalyst. To grossly
observe the optical behavior of FA-OMe, NO gas was bubbled
into an aqueous solution of FA-OMe. The solution
immediately changed from yellow to light green. This indeed
qualitatively establishes that FA-OMe is reactive toward NO.
The reaction product dA-FA-OMe was purified, and the
structure was elucidated using NMR spectroscopy and mass
spectrometry. Thus, FA-OMe can detect NO and produce
strongly fluorescent dA-FA-OMe through reductive deamina-
tion of FA-OMe.
group of FA-OMe is nitrosylated by N O , which forms by the
2
3
2
3
autoxidation of NO, to produce the N-nitrosoamine
intermediate 1. Subsequently, nitrite ion reacts with 1 to
produce the nitramine intermediate. Afterward, the secondary
amine of 2 is nitrosylated by N to form the N-nitroso
9g
2
4
O
3
2
nitramine intermediate 3, and then the N-nitroso nitramine
moiety of 3 rearranges into the diazo nitrate moiety to form the
diazo nitrate intermediate 5. It was reported that some diazo
nitrates could decompose by homolytic bond rupture to form
25
the corresponding radicals, nitrogen gas, and nitrate radical.
1
The H NMR spectra of FA-OMe and dA-FA-OMe are
Thus, the diazo nitrate intermediate 5 homolytically decom-
poses to give the fluoresceinyl radical 6, which readily abstracts
hydrogen from the solvent to give the deaminated product dA-
FA-OMe. According to the ESI-mass spectrum of FA-OMe
reacting with NO(aq) for 1.0 min under aerobic conditions
shown in Figure 2. FA-OMe and dA-FA-OMe exhibit the
expected idealized C2v symmetry in solution due to the
exchange process of the carbonyl and hydroxyl of the xanthene
moiety. The lack of an apparent broad signal at around 11.0
5
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Inorganic Chemistry
Figure S2 in the Supporting Information), the peaks
Article
(
corresponding to the proposed intermediates 1, 2, and 5 (or
and 4) were certainly observed. These results supported the
3
proposed mechanism. However, further experimental studies
are necessary to completely demonstrate our proposed
mechanism. In other words, the proposed intermediates or
their derivatives formed by a specific reagent (trap) should be
isolated and fully characterized. The related experimental
studies are currently under investigation.
3.2. Computations. A computational study was carried out
using the Gaussian 09 program to understand the theoretical
aspects of the change in fluorescence intensity. Density
functional theory (DFT) calculations at the B3LYP/6-31+
+
G** level were applied to obtain the optimized geometries
Figure 4. Absorption spectra of FA-OMe (1 μM, blue line) and dA-
FA-OMe (1 μM, red line) in 0.1 M NaOH(aq).
and energy levels of frontier molecular orbitals of the
components forming FA-OMe and dA-FA-OMe (methyl 3-
aminobenzoate, methyl benzoate, and xanthene). As shown in
Figure 3, the highest occupied molecular orbital (HOMO) of
3
.4. Stability of FA-OMe and dA-FA-OMe. Since the
stability of probes under various pH conditions concerns the
biological applicability, the effects of pH on the fluorescence
intensities of FA-OMe and dA-FA-OMe were investigated at
different pHs ranging from 6.5 to 10, as shown in Figure 5. A
Figure 3. Energy levels of the methyl 3-aminobenzoate, methyl
benzoate, and xanthene moieties. The values in parentheses were
derived from the calculations considering the aqueous environment.
the methyl 3-aminobenzoate moiety (electron donor) matches
that of the xanthene moiety (electron acceptor); therefore,
when the latter is photoexcited, the intramolecular electron
transfer from the methyl 3-aminobenzoate moiety to the
xanthene moiety is energetically favorable. In contrast, the
HOMO of the methyl benzoate moiety is significantly lower
than that of the xanthene moiety; consequently, the intra-
molecular electron transfer from the former to the latter is
energetically unfavorable. The DFT calculations indicate that
the behavior of the NO turn-on fluorescence of FA-OMe is
associated with the PeT phenomenon.
Figure 5. pH dependence of the fluorescence intensities of 20 μM FA-
OMe (◆) and dA-FA-OMe (●) in 100 mM HEPES buffer at 25.0 ±
.1 °C. The fluorescence intensities were detected at 524 nm with
0
excitation at 460 nm.
significant change in the fluorescence intensity of FA-OMe was
not observed with an increase in pH from 6.6 to 10. In contrast,
the gradually increasing fluorescence intensity of dA-FA-OMe
reached a maximum value at pH 7.4. However, the fluorescence
intensity ratio of dA-FA-OMe to FA-OMe shows high values
between pH 6.5 and 10. This result indicates that FA-OMe can
be used as an efficient fluorescent probe for the detection of
NO under physiological conditions.
3.3. Optical Properties of FA-OMe and dA-FA-OMe.
The fluorescence properties of FA-OMe and dA-FA-OMe are
summarized in Table 1. The ultraviolet−visible (UV/vis)
spectrum profiles of FA-OMe and dA-FA-OMe are not
significantly different, as shown in Figure 4. However, the
quantum yield of dA-FA-OMe is approximately 59-fold higher
than that of FA-OMe. Consequently, even a small amount of
dA-FA-OMe formed in the reaction of FA-OMe with NO can
produce remarkable fluorescence enhancement.
3
.5. Concentration and Time Dependence of FA-OMe
Reacting with NO. The concentration- and time-dependent
reactions between FA-OMe and NO were carried out in 100
mM HEPES buffer at pH 7.4. To 50 μM FA-OMe solutions
were added different amounts of the NO stock solution (1.8
mM). The fluorescence intensity at 524 nm is enhanced upon
increasing the amount of NO(aq), as shown in Figure 6.
Furthermore, the time-dependent fluorescence turn-on of FA-
OMe shows a highly uniform pattern with different amounts of
NO, as shown in Figure 7. The fluorescence intensities at 524
nm increase with time and reach a plateau within about 20 min.
These results indicate that the NO concentration and reaction
time influence proportionally the turn-on fluorescent intensity
of FA-OMe. In addition, judging from Figure 6, the
Table 1. Fluorescence Quantum Yields (Φ ) and Absorption
fl
and Emission Maxima (λ , λ ) of FA-OMe and dA-FA-
abs
em
OMe in 0.1 M NaOH(aq) Determined by Comparison with
a Fluorescein Standard Solution (Φ = 0.95 in 0.1 M
fl
NaOH(aq))
compd
λabs (nm)
λem (nm)
Φ (%)
fl
FA-OMe
488
490
518
514
1.5
dA-FA-OMe
88.8
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Figure 6. Fluorescence emission spectra of FA-OMe (50 μM) upon
reaction with various amounts of NO(aq) in 100 mM HEPES buffer of
pH 7.4 at 25.0 ± 0.1 °C (λex 460 nm, λem 524 nm).
Figure 8. Selectivity of FA-OMe (20 μM) for NO over other reactive
oxygen and nitrogen species: normalized fluorescence response after 2
h relative to the emission of the probe in 100 mM HEPES buffer at pH
7
.4 and 25.0 ± 0.1 °C (λex 460 nm, λem 524 nm).
Supporting Information). In addition, dA-FA-OMe formation
requires two N O molecules, as shown in Scheme 1, but the
2
3
formation of the triazole derivative DAF-2T only needs one
9g
N O molecule. This assertion may also explain why the
2
3
reactivity and fluorescent intensity of DAF-2 for NO are higher
than those of FA-OMe.
3
.7. Fluorescence Imaging of NO in Living Cells. The
cytotoxicity of FA-OMe was investigated by incubating various
concentrations of FA-OMe with Raw 264.7 murine macro-
phages. After 24 h, cell viability was examined by the 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
assay and the results demonstrated that FA-OMe displayed low
cytotoxicity to Raw 264.7 murine macrophages, as shown in
Figure 9. To evaluate the utility of FA-OMe for the detection of
endogenously generated NO, fluorescence microscopic imaging
of biologically produced NO in Raw 264.7 murine macrophages
was performed. The Raw 264.7 murine macrophages were
Figure 7. Time dependence of the fluorescence intensity of 50 μM
FA-OMe in pH 7.4 100 mM HEPES buffer with different amounts of
the NO stock solution. The fluorescence intensities were detected at
−1
524 nm with excitation at 460 nm at 25.0 ± 0.1 °C.
stimulated by lipopolysaccharide (LPS; 0.5 μg mL ) for 4 h
and then incubated with 10 μM FA-OMe for an additional 4
and 8 h. In a control experiment, cells were incubated for 8 h
with 10 μM FA-OMe in the absence of LPS. As shown in
Figure 10B,C, a visible increase in fluorescence was observed
over 4−8 h following FA-OMe incubation (8−12 h after
induction of iNOS), which was not observed in the control cells
(Figure 10A). These results indicate that FA-OMe can detect
endogenously produced NO. To test the cell trappability of FA-
OMe, Raw 264.7 murine macrophages prestimulated with 1.25
μg mL− LPS were incubated with 10 μM FA-OMe for 8 h and
then washed and imaged by the fluorescence microscope four
times over the course of 30 min, where a minimal change in
fluorescence intensity was observed, as shown in Figure 11.
enhancement in fluorescence intensity presents a linear
correlation with concentration. The detection limit for NO,
calculated as the concentration of NO at which the fluorescence
signal is equal to the blank value plus a 3-fold standard
deviation, was estimated as ∼44 nM. Therefore, FA-OMe could
be used for detecting endogenous NO in living cells.
3.6. Selectivity. The high specificity of the NO probe is
imperative for its application in understanding the diverse
biological roles played by NO. Therefore, the fluorescence
intensities of FA-OMe in the presence of a series of possible
competitive reactive oxygen species (ROS), reactive nitrogen
species (RNS), AA, and DHA at up to 100-fold excess were
examined under the same conditions. As shown in Figure 8, no
detectable responses for these agents were observed. It was
1
13a
Therefore, FA-OMe has cell-trappable characteristics. Since
the ester groups can be cleaved by intracellular esterases to give
carboxylates under physiological pH conditions to restrict
recrossing of the cell membrane, the cell trappability may be
caused by hydrolyzing FA-OMe into FA-OMe-carboxylate,
9b
reported that the commercially available NO probe DAF-2
reacted with DHA or AA and turned on the probe under
21
physiological conditions. Additionally, in the presence of
−
•
H O /peroxidase, OONO , or OH, the o-diaminophenyl
which may react with NO/O to form dA-FA-OMe-carboxylate.
2
2
2
group is oxidized into an unstable arylaminyl radical, which can
To prove that the turn-on fluorescence would be not achieved
by FA-OMe-carboxylate in living cells, pH-dependent studies of
FA-OMe and fluoresceinamine, which would transform into
26
directly combine with NO to form triazole. The better
selectivity of FA-OMe for NO, in contrast to that of DAF-2,
may be attributed to its less electron rich NO-reactive moiety.
However, because of its highly electron rich o-diaminophenyl
group, DAF-2 has a comparatively high reaction rate for NO
according to the time-dependent experiments of DAF-2 and
FA-OMe with 10 equiv of NO(aq) (Figure S3 in the
12a
FA-OMe-carboxylate in aqueous solutions above pH 4.0,
were undertaken in the pH range of 6.5−10, as shown in Figure
12. The fluorescence intensities are not apparently different at
this pH range. These results show that FA-OMe-carboxylate
cannot turn on the fluorescence. On the basis of the formation
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Figure 9. MTT assay on Raw 264.7 murine macrophages treated with different concentrations of FA-OMe for 24 h.
Figure 10. Endogenous NO detection in Raw 264.7 murine macrophages by FA-OMe (10 μM): (top) DIC images; (bottom) fluorescence. FA-
−1
OMe incubation with cells for (A) 8 h without LPS prestimulation, (B) 4 h with LPS (0.5 μg mL ) prestimulation for 4 h, and (C) 8 h with LPS
−1
(
0.5 μg mL ) prestimulation for 4 h. The scale bar represents 50 μm.
Figure 11. FA-OMe diffusion/localization in Raw 264.7 murine macrophages: (top) DIC images; (bottom) fluorescence. After each image, the cells
−1
were washed three times with 2 mL of PBS. [FA-OMe] = 10 μM, [LPS] = 1.25 μg mL , scale bars 25 μm.
of strongly fluorescent fluorescein, which would transform into
4. SUMMARY
dA-FA-OMe-carboxylate in aqueous solutions above pH 4.0, in
In this study, we have demonstrated the first example of an NO
turn-on fluorescent probe based on reductive deamination of an
aromatic primary monoamine. The DFT calculations of dA-FA-
OMe relative to FA-OMe suggest that the fluorescence
enhancement is associated with the PeT phenomenon. The
high quantum yield of deaminated product shows that even a
small deamination induced by NO can result in significant
fluorescence enhancement. In addition, the use of a less
the reaction of fluoresceinamine with NO/O under neutral pH
2
conditions, FA-OMe-carboxylate can react with NO/O to
2
generate dA-FA-OMe-carboxylate in living cells. Therefore, FA-
OMe can be used as a fluorescent probe for the detection of
NO formed in living cells.
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Figure 12. pH dependence of the fluorescence intensities of 20 μM fluoresceinamine (▲) and FA-OMe (◆) in 100 mM HEPES buffer at 25.0 ± 0.1
C. The fluorescence intensities were detected at 524 nm with excitation at 460 nm. Inset: comparison to that of 20 μM dA-FA-OMe (●) in 100
°
mM HEPES buffer at 25.0 ± 0.1 °C.
electron rich NO-reactive moiety renders FA-OMe high
specificity for NO over other reactive oxygen/nitrogen species
and substances that hinder the existing probes from sensing
NO. Furthermore, the aqueous solubility and high fluorescence
stability of FA-OMe and dA-FA-OMe over a wide range of pH
ensure its potential application for NO bioimaging, which was
successfully performed in Raw 264.7 murine macrophages. The
results obtained in the present study demonstrate the viability
of developing a series of NO probes based on reductive
deamination of an aromatic primary monoamine.
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■
*
S
Supporting Information
13
Figures giving C NMR spectra for FA-OMe and dA-FA-OMe,
ESI-mass spectrum of FA-OMe reacting with NO(aq), and
time-dependent studies of DAF-2 and FA-OMe. This material
is available free of charge via the Internet at http://pubs.acs.org.
2
(
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AUTHOR INFORMATION
Corresponding Author
Tel: 886-3-5712121 ext 56972. Fax: 886-3-5729288. E-mail:
ymwang@mail.nctu.edu.tw.
■
*
(
7) (a) Yang, Y.; Seidlits, S. K.; Adams, M. M.; Lynch, V. M.;
Schmidt, C. E.; Anslyn, E. V.; Shear, J. B. J. Am. Chem. Soc. 2010, 132,
3114. (b) Kim, J. H.; Heller, D. A.; Barone, P. W.; Song, C.; Zhang,
Notes
1
The authors declare no competing financial interest.
J.; Trudel, L. J.; Wogan, G. N.; Tannenbaum, S. R.; Strano, M. S. Nat.
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ACKNOWLEDGMENTS
Funding from the National Science Council of Taiwan (NSC
00-2627-M-009-002) and from National Chiao Tung
■
1
(f) Heiduschka, P.; Thanos, S. Neuroreport 1998, 9, 4051. (g) Gabe,
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