Nitric Oxide Turn-on Fluorescent Probe Based on Deamination of Aromatic Primary Monoamines

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}

*

ABSTRACT:

The stable, water-soluble, and non

fluorescent

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 speci

ficity 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 di

ffusion/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.

1. INTRODUCTION

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

diverse physiological and pathological pathways.

Although NO

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

techniques such as chemiluminescence,

colorimetry,

electron

paramagnetic resonance,

electrochemistry,

and

fluorimetry

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

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

responsive organic molecule based sensors. The

NO-reactive moiety acts as a modulator in the photoinduced

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

used NO-reactive moiety in organic

fluorescent probes.

The

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.

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 e

ffects of NO. The

NO-induced deamination of cytosine, adenine, guanine, and

5-methylcytosine indeed represents an important NO-induced

genotoxic mechanism.

In addition, previous research reported

that some aromatic amines react with NO or N

O

to give the

Received: February 20, 2012

Published: April 9, 2012

corresponding deaminated aromatics.

In addition, it was

reported that

fluorescence of a fluorescent molecule could be

adjusted by changing the electronic in

fluence of the nitrogen

electron lone pair.

Therefore, we have investigated the ability

of NO to deaminate the aromatic primary amino group,

thereby altering the electronic in

fluence 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 identi

fied,

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-FA-OMe were studied and compared. In order to

con

firm 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.

2. EXPERIMENTAL SECTION

2.1. Materials and Instruments. All chemicals were obtained from commercial suppliers and used as received without further purification. Hydrogen peroxide (H2O2), dehydroascorbic acid (DHA), ascorbic acid (AA), ethylenediaminetetraacetic acid (EDTA), lipopolysaccharide (LPS), and fluoresceinamine were purchased from Sigma-Aldrich. Sodium nitrite (NaNO2) and sodium nitrate (NaNO3) were purchased from Showa. Angeli’s salt (Na2N2O3) and sodium peroxynitrite (ONOONa, as a solution in 0.3 M NaOH) were purchased from Cayman Chemical Co. 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES, free acid, Ultrapure Bioreagent) was purchased from MP Biomedicals. Ferrous ammonium sulfate hexahydrate (Fe(NH4)2(SO4)2·6H2O) was pur-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% NO/90% N2) was purchased from SanFu. It was passed through an Ascarite II column to remove higher nitrogen oxides before use.14 Deionized water was produced by a Milli-Q reagent water purification system. Methanol was distilled over Mg/I2 and subsequently dried over 0.3 nm molecular sieves. Preparative thin-layer chromatographic (TLC) plates were prepared from silica gel (Macherey-Nagel).1_{H and} 13_{C NMR spectra were obtained on a Varian VXR-300 spectrometer at} 300 and 75 MHz and were referenced to the internal 1_{H and}13_C solvent peaks. ESI-LRMS spectra were recorded on a Micromass

Q-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 IX71fluorescence microscope equipped with a 100 W mercury lamp, B-2Afilters, and a color CCD camera system.

2.2. Preparation of 5-Amino-2-(6-hydroxy-3-oxo-3 H-xanth-en-9-yl)benzoic Acid Methyl Ester (FA-OMe). A modification of the synthetic procedure of FA-OMe was employed.15 A mixture of fluoresceinamine (200 mg, 0.576 mmol), MeOH (10 mL), and concentrated H2SO4 solution (0.2 mL) was heated for 48 h under reflux. The solution was poured on ice (300 g), and then a saturated NaHCO3solution was slowly added to adjust the pH to 6.5. An orange precipitate was separated by centrifugation and then subjected to TLC, with MeOH/CH2Cl2as eluant (1/12, v/v). A yellow band was isolated 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: m/z 362.1 ([M + H]+_{), M}+_{calculated 361.1.}1_{H NMR (DMSO-d} 6):δ 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, 2H), 3.49 (s, 3H).13_{C NMR (DMSO-d} 6):δ 178.4, 166.2, 157.3, 152.9, 149.7, 131.5, 130.8, 129.8, 122.8, 120.7, 117.0, 114.7, 111.1, 103.1, 51.9.

2.3. Reaction of FA-OMe with NO/O2. In 100 mL of an FA-OMe (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/ CH2Cl2as eluant (1/10, v/v). Thefirst bright yellow band was isolated 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}+ calculated 346.0841.1_{H NMR (DMSO-d} 6):δ 8.12 (d, J = 7.8 Hz, 1H), 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).13_{C NMR (DMSO-d} 6): δ178.7, 165.5, 157.2, 151.3, 134.8, 132.7, 130.7, 130.3, 129.8, 129.5, 129.3, 123.2, 110.3, 103.4, 52.2.

2.4. Reaction of Fluoresceinamine with NO/O2. In 45 mL of a 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/CH2Cl2 as eluant (1/10, v/v). The first bright yellow 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 methods.16 NO could be generated by slowly dropping 2.0 M H2SO4(aq) into a glass flask containing a saturated NaNO2 water solution. Since O2will rapidly oxidize NO to form NO2, all apparatus were carefully degassed with argon for 30 min to remove O2. The forming gas was passed twice through a 30% NaOH solution and once through water to trap NO2generated from the reaction of NO with traces of O2. To produce a saturated NO solution (1.8 mM, at 20°C) 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.3a Stock solutions were freshly prepared for each experiment and stored in a glassflask with a rubber septum.

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.

2.6.1. Fluorescence Quantum Yield Determination. Fluorescence quantum yields of FA-OMe and dA-FA-OMe were measured in 0.1 M NaOH(aq) by usingfluorescein (Φfl= 0.95 in 0.1 M NaOH(aq)) as a standard.12bFluorescence quantum yields were calculated by use of the following equation:Φunk=Φstd(Iunk/Aunk)(Astd/Istd)(nunk/nstd)2, where Figure 1.An NO turn-on probe through NO-induced deamination of

Φunk is the fluorescence quantum yield of the sample, Φstd is the fluorescence quantum yield of the standard, Iunk and Istd are the integrated emission intensities of the sample and the standard, respectively, Aunkand Astdare the absorbances of the sample and the standard at the excitation wavelength, respectively, and nunkand nstdare the refractive indexes of the corresponding solutions, respectively.

2.6.2. Stability Studies of FA-OMe, dA-FA-OMe, and Fluorescein-amine. Solutions of 20μM 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 20μM 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 (λex460 nm,λem524 nm).

2.6.3. Selectivity Studies. All thefluorescence 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 (λex 460 nm, λem 524 nm). The concentration of the ONOO−stock solution was calculated by using its molar extinction coefficient of 1670 M−1 cm−1 at 302 nm.17 The degassed aqueous solutions of Angeli’s salt were freshly prepared under anaerobic conditions and used as a nitroxyl (HNO) source.18Hydroxyl radical (•OH) was produced by reacting ferrous ammonium sulfate hexahydrate (Fe(NH4)2(SO4)2·6H2O) with H2O2 under anaerobic conditions.19Hydrogen peroxide (H2O2) was diluted promptly from the 30% H2O2solution and quantified by measuring its absorbance at 240 nm with a molar absorption coefficient of 43.6 M−1_cm−1_.20_The

aqueous solutions of NaNO2and NaNO3were freshly prepared and used as nitrite (NO2−) and nitrate (NO3−) sources, respectively. The

nitrogen-purged aqueous solutions of AA and DHA in 100 mM HEPES buffer at pH 7.4 containing 3 mM EDTA were freshly prepared before use.21

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 Gaussian 09 program.22

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% CO2. For endogenously generated nitric oxide detection studies, iNOS (inducible nitric oxide synthase) was induced in Raw 264.7 murine macrophages with 0.5μg mL−1of LPS for 4 h, and the cells were then coincubated with 10μM 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. Thefluorescence alterations were monitored by the fluorescence microscope. For FA-OMe diffusion/localization studie-s,13aRaw 264.7 murine macrophages were prestimulated with 1.25μg mL−1of LPS for 4 h, and then the cells were coincubated with 10μM 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 mediaflow, the PBS was removed, and Figure 2.1_{H NMR spectra of FA-OMe (A) and dA-FA-OMe (B) in d}

the cells were washed three times with 2 mL of fresh PBS and then bathed in 2 mL of fresh PBS while on thefluorescence microscope. This process was repeated every 10 min during imaging.

2.9. Cell Viability Studies. The Raw 264.7 murine macrophages were grown in 96-well plates at an initial density of 105_{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 mL−1) 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

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 puri

fied, 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.

The

H NMR spectra of FA-OMe and dA-FA-OMe are

shown in Figure 2. FA-OMe and dA-FA-OMe exhibit the

expected idealized C

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

ppm shows that the hydroxyls of FA-OMe and dA-FA-OMe

may be deprotonated in the solution. Thus, eight types of

protons in di

fferent 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 e

ffect, 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

C NMR spectra of FA-OMe and dA-FA-OMe

display 15 resonance signals, respectively, which indeed

correspond with the C

symmetry (Figure S1 in the

Supporting Information).

The reaction mechanism for deamination of FA-OMe is

proposed as shown in Scheme 1. First, the primary amino

group of FA-OMe is nitrosylated by N

O

, which forms by the

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

O

to form the N-nitroso

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

the corresponding radicals, nitrogen gas, and nitrate radical.

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

Scheme 1. Proposed Mechanism of FA-OMe with NO under Aerobic Conditions To Form dA-FA-OMe

(Figure S2 in the Supporting Information), the peaks

corresponding to the proposed intermediates 1, 2, and 5 (or

3

and 4) were certainly observed. These results supported the

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 speci

fic 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

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

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.

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 pro

files of FA-OMe and dA-FA-OMe are not

signi

ficantly 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.

3.4. Stability of FA-OMe and dA-FA-OMe. Since the

stability of probes under various pH conditions concerns the

biological applicability, the e

ffects of pH on the fluorescence

intensities of FA-OMe and dA-FA-OMe were investigated at

di

fferent pHs ranging from 6.5 to 10, as shown in Figure 5. A

signi

ficant 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 e

fficient fluorescent probe for the detection of

NO under physiological conditions.

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 bu

ffer at pH 7.4. To 50 μM FA-OMe solutions

were added di

fferent 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 di

fferent 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 in

fluence proportionally the turn-on fluorescent intensity

of FA-OMe. In addition, judging from Figure 6, the

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.

Table 1. Fluorescence Quantum Yields (

Φ

) and Absorption

and Emission Maxima (λ

,

λ

) of FA-OMe and

dA-FA-OMe in 0.1 M NaOH(aq) Determined by Comparison with

a Fluorescein Standard Solution (Φ

= 0.95 in 0.1 M

NaOH(aq))

compd λ_abs(nm) λ_em(nm) Φfl(%)

FA-OMe 488 518 1.5 dA-FA-OMe 490 514 88.8

Figure 4.Absorption spectra of FA-OMe (1μM, blue line) and dA-FA-OMe (1μM, red line) in 0.1 M NaOH(aq).

Figure 5.pH dependence of thefluorescence intensities of 20 μM FA-OMe (◆) and dA-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.

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

reported that the commercially available NO probe DAF-2

reacted with DHA or AA and turned on the probe under

physiological conditions.

Additionally, in the presence of

H

O

/peroxidase, OONO

, or

OH, the o-diaminophenyl

group is oxidized into an unstable arylaminyl radical, which can

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

Supporting Information). In addition, dA-FA-OMe formation

requires two N

O

molecules, as shown in Scheme 1, but the

formation of the triazole derivative DAF-2T only needs one

N

O

molecule.

This assertion may also explain why the

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

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.

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,

which may react with NO/O

to form dA-FA-OMe-carboxylate.

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

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

Figure 6.Fluorescence emission spectra of FA-OMe (50μM) upon reaction with various amounts of NO(aq) in 100 mM HEPES buffer of pH 7.4 at 25.0± 0.1 °C (λex460 nm,λem524 nm).

Figure 7. Time dependence of thefluorescence intensity of 50 μM FA-OMe in pH 7.4 100 mM HEPES buffer with different amounts of the NO stock solution. Thefluorescence intensities were detected at 524 nm with excitation at 460 nm at 25.0± 0.1 °C.

Figure 8.Selectivity of FA-OMe (20μM) for NO over other reactive oxygen and nitrogen species: normalizedfluorescence 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 (λex460 nm,λem524 nm).

of strongly

fluorescent fluorescein, which would transform into

dA-FA-OMe-carboxylate in aqueous solutions above pH 4.0, in

the reaction of

fluoresceinamine with NO/O

under neutral pH

conditions, FA-OMe-carboxylate can react with NO/O

to

generate dA-OMe-carboxylate in living cells. Therefore,

FA-OMe can be used as a

fluorescent probe for the detection of

NO formed in living cells.

4. SUMMARY

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-dA-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 signi

ficant

fluorescence enhancement. In addition, the use of a less

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 (10μM): (top) DIC images; (bottom) fluorescence. FA-OMe incubation with cells for (A) 8 h without LPS prestimulation, (B) 4 h with LPS (0.5μg mL−1) prestimulation for 4 h, and (C) 8 h with LPS (0.5μg mL−1) prestimulation for 4 h. The scale bar represents 50μm.

Figure 11.FA-OMe diffusion/localization in Raw 264.7 murine macrophages: (top) DIC images; (bottom) fluorescence. After each image, the cells were washed three times with 2 mL of PBS. [FA-OMe] = 10μM, [LPS] = 1.25 μg mL−1, scale bars 25μm.

electron rich NO-reactive moiety renders FA-OMe high

speci

ficity 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.

■

ASSOCIATED CONTENT

*

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.

■

AUTHOR INFORMATION

Corresponding Author

*Tel: 886-3-5712121 ext 56972. Fax: 886-3-5729288. E-mail:

[email protected].

Notes

The authors declare no competing

financial interest.

■

ACKNOWLEDGMENTS

Funding from the National Science Council of Taiwan (NSC

100-2627-M-009-002) and from National Chiao Tung

University and the Ministry of Education of Taiwan (Aim for

the Top University Plan) are gratefully acknowledged.

■

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