A reductant-resistant and metal-free fluorescent probe for nitroxyl applicable to living cells

Article abstract of DOI:10.1021/ja404757s

Nitroxyl (HNO) is a one-electron reduced and protonated derivative of nitric oxide (NO) and has characteristic biological and pharmacological effects distinct from those of NO. However, studies of its biosynthesis and activities are restricted by the lack of versatile HNO detection methods applicable to living cells. Here, we report the first metal-free and reductant-resistant HNO imaging probe available for use in living cells, P-Rhod. It consists of a rhodol derivative moiety as the fluorophore, linked via an ester moiety to a diphenylphosphinobenzoyl group, which forms an aza-ylide upon reaction with HNO. Intramolecular attack of the aza-ylide on the ester carbonyl group releases a fluorescent rhodol derivative. P-Rhod showed high selectivity for HNO in the presence of various biologically relevant reductants, such as glutathione and ascorbate, in comparison with previous HNO probes. We show that P-Rhod can detect not only HNO enzymatically generated in the horseradish peroxidase-hydroxylamine system in vitro but also intracellular HNO release from Angeli's salt in living cells. These results suggest that P-Rhod is suitable for detection of HNO in living cells.

Full text of DOI:10.1021/ja404757s

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Article
A Reductant-Resistant and Metal-Free Fluorescent
Probe for Nitroxyl Applicable to Living Cells
Kodai Kawai, Naoya Ieda, Kazuyuki Aizawa, Takayoshi Suzuki, Naoki Miyata, and Hidehiko Nakagawa
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/ja404757s • Publication Date (Web): 18 Jul 2013
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A Reductant-Resistant and Metal-Free Fluorescent Probe for
Nitroxyl Applicable to Living Cells
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Kodai Kawai†, Naoya Ieda†, Kazuyuki Aizawa†, Takayoshi Suzuki†,¶, Naoki Miyata†, and Hidehi-
ko Nakagawa†,§,*
†Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya, Aichi
467-8603, Japan
§Japan Science and Technology Agency, PRESTO, 4-1-8 Honcho Kawaguchi, Saitama 332-0012, Japan.
KEYWORDS fluorescence, chemical probe, imaging, HNO, nitric oxide
ABSTRACT: Nitroxyl (HNO) is a one-electron-reduced and protonated derivative of nitric oxide (NO), and has character-
istic biological and pharmacological effects distinct from those of NO. However, studies of its biosynthesis and activities
are restricted by the lack of versatile HNO detection methods applicable to living cells. Here, we report the first metal-
free and reductant-resistant HNO imaging probe available for use in living cells, P-Rhod. It consists of a rhodol derivative
moiety as the fluorophore, linked via an ester moiety to a diphenylphosphinobenzoyl group, which forms an aza-ylide
upon reaction with HNO. Intramolecular attack of the aza-ylide on the ester carbonyl group releases a fluorescent rhodol
derivative. P-Rhod showed high selectivity for HNO in the presence of various biologically relevant reductants, such as
glutathione and ascorbate, in comparison with previous HNO probes. We showed that P-Rhod can detect not only HNO
enzymatically generated in the horseradish peroxidase-hydroxylamine system in vitro, but also intracellular HNO release
from Angeli’s salt in living cells. These results suggest that P-Rhod is suitable for detection of HNO in living cells.
Introduction
Fluorescence detection is recognized to have both high
sensitivity and high spatiotemporal resolution for visual-
izing biomolecules in vitro and in vivo. Several groups
have developed fluorescent probes for HNO, but they are
mostly based on the reduction of Cu (II) to Cu (I),10, 11 or
nitroxide to hydroxylamine, by HNO. 12 Therefore, those
probes are vulnerable to biological reductants, such as
glutathione (GSH) and ascorbate, which are abundant in
living cells.
Since the biosynthesis of nitric oxide (NO) in mamma-
lian systems was discovered in the late 1980’s, there has
been increasing interest in the biological roles of reactive
nitrogen species (RNS).1 NO mediates both physiological
and pathological processes, including cardiovascular sig-
naling in the circulatory system, neurotransmission in the
central nervous system, and rejection of foreign substanc-
es by the immune system.2 As for other RNS, peroxynitrite
(ONOO-) is thought to be involved in various diseases
and regulation of nitration of biomolecules,3 and dinitro-
To overcome the issue of poor selectivity, we focused
on the reaction between HNO and phosphine. It is known
that HNO reacts with triarylphosphines to give the corre-
sponding phosphine oxide and aza-ylide (Scheme 1, eq. 1).
In the presence of a properly situated electrophilic ester,
intramolecular nucleophilic attack of the aza-ylide on the
carbonyl carbon of the ester is expected to occur, leading
to the formation of alcohol and amide.13, 14 Phosphines are
abiotic and essentially unreactive toward biomolecules
inside or on the surface of cells, so that aza-ylide for-
mation by HNO is a bioorthogonal reaction.15 Moreover,
the reaction rate constant between HNO and phosphine
derivative is sufficiently high (estimated as 9×105 M-1s-1)
for the present purpose14, so we adopted tri-
phenylphosphine as the HNO detecting moiety.
-
gen trioxide (N2O3), nitrite (NO2 ), and nitrogen dioxide
(NO2) are involved in alkylation of DNA.4 Also, there is
increasing evidence that nitroxyl (HNO), the one-
electron-reduced form of NO, has biological effects dis-
tinct from those of NO.5 For example, HNO reacts direct-
ly with protein thiols to cause inhibition of aldehyde de-
hydrogenase,6 and it induces vasorelaxation through up-
regulation of calcitonin gene-related peptide.7
Several biochemical studies suggest that HNO can be
formed by two-electron oxidation of hydroxylamine cata-
lyzed by a heme enzyme.8 Further, hydrogen sulfide (H2S)
may be involved in its biosynthesis.9 However, studies of
the biological importance of HNO remain hampered by
the lack of efficient HNO detection methods available in
living cells.
Rhodol, 2-(3-amino-6-oxoxanthen-9-yl)benzoic acid,
has excellent photophysical properties, good solubility in
various solvents, high fluorescence quantum yield, and
good membrane permeability.16, 17 Further, it can adopt
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either of two tautomeric forms, the fluorescent xanthene
In the light of these facts, we designed a novel HNO
fluorescent probe, named P-Rhod (1), consisting of rhodol
acylated at its amino moiety, coupled to tri-
phenylphosphine moiety via an ester linker. Reaction be-
tween HNO and the phosphine moiety was expected to
form an aza-ylide, and then intramolecular nucleophilic
attack of the aza-ylide on carbonyl carbon would lead to
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4
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form (open form) and the non-fluorescent lactone form
(closed form). The closed form can be imposed on rhodol
by acylation of both the amino group and phenolic hy-
droxyl group at the xanthene moiety, while the open form
is in the preferred form of monoacylated rhodol (i.e., the
amide form of rhodol) in aqueous solution.18
a
7
release
of
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Scheme 1. Proposed mechanisms of the reaction of phosphine with HNO (eq. 1) and that of P-Rhod with HNO
(eq. 2)
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rhodol 2 in the fluorescent open form, together with (di-
phenylphosphonyl)benzamide 4 (Scheme 1, eq. 2). There-
fore, P-Rhod should exhibit turn-on type fluorogenic be-
havior in response to HNO, and we anticipated that it be
able to detect HNO with high sensitivity and selectivity in
living cells.
Synthesis of 6. To a suspension of fluorescein (50.0 g,
0.150 mol) and K2CO3 (62.4 g, 0.451 mol) in 210 mL of an-
hydrous DMF was added chloromethyl methyl ether (48.5
g, 0.602 mol) at 0 ºC under a nitrogen atmosphere. The
reaction mixture was stirred at room temperature for 24
h, diluted with CHCl3, washed with 0.5 N HCl solution,
water and brine, and then dried over Na2SO4. Filtration,
concentration in vacuo, and purification by silica gel flash
column chromatography (EtOAc:hexane = 1:1), followed
by purification by silica gel flash column chromatography
(CHCl3: MeOH = 20:1) gave 6.94 g (12%) of 6 as yellow
Experimental Section
General Methods. Melting points were determined us-
ing a Yanaco micro melting point apparatus or a Büchi B-
545 melting point apparatus and were left uncorrected.
Proton nuclear magnetic resonance (1H NMR) spectra and
carbon nuclear magnetic resonance (13C NMR) spectra
were recorded on a JEOL JNM-LA500 or JEOL JNM-A500
spectrometer in the indicated solvent. Chemical shifts ( )
are reported in parts per million (ppm) relative to the
internal standard, tetramethylsilane (TMS). Elemental
analysis was performed with a Yanaco CHN CORDER NT-
5 analyzer, and all values were within ±0.4% of the calcu-
lated values. Ultraviolet-visible light absorbance spectra
were measured on an Agilent 8453 spectrometer. GC-MS
analyses were performed on a Shimadzu GCMS-QP2010.
FAB-MS were recorded on a JEOL JMS-SX102A mass spec-
trometer. Angeli’s salt was obtained from Cayman Chemi-
cal and ONOO– in NaOH solution, NOC 7, NOR 1, SNAP,
and GSNO were obtained from Dojindo. All other rea-
gents and solvents were purchased from Aldrich, Tokyo
Kasei Kogyo, Wako Pure Chemical Industries, Nacalai
Tesque, Kishida Kagaku, Junsei Kagaku, and Kanto
Kagaku and used without further purification. Flash col-
umn chromatography was performed using silica gel 60
(particle size 0.046–0.063 mm) supplied by Taiko Shoji.
1
crystals: H NMR (CDCl3, 500 MHz, δ; ppm) 8.03 (1H, d, J
= 7.6 Hz), 7.65 (2H, m), 7.17 (1H, d, J = 7.5 Hz) , 6.97 (1H,
d, J = 2.0 Hz),6.71 (4H, m), 6.54 (1H, dd, J = 8.6 Hz, J = 2.5
Hz), 5.21 (2H, s), 3.49 (3H, s); MS(EI) m/z: 376 (M+).
Synthesis of 7. To a solution of 6 (6.00 g, 15.9 mmol)
and pyridine (6.31 g, 79.7 mmol) in CH2Cl2 was added
dropwise trifluoromethanesulfonic anhydride (13.5 g, 47.8
mmol) at 0 ºC under N2 gas. The resulting solution was
stirred at RT for 4 h, and then the reaction was quenched
with water. CH2Cl2 was added to the mixture and the or-
ganic layer was separated and washed with 1 N HCl, fol-
lowed by water and brine. The organic layer was dried
over Na2SO4 and concentrated. The residue was purified
by silica gel column chromatography (EtOAc:hexane = 1:1)
1
to gave 5.48 g (69%) of 7 as white crystals: H NMR
(CDCl3, 500 MHz, δ; ppm) 8.05 (1H, d, J = 7.9 Hz), 7.67
(2H, m), 7.23 (1H, s) , 7.18 (1H, d, J = 7.0 Hz), 6.95 (3H, m),
6.75 (2H, m), 5.21 (2H, s), 3.49 (3H, s); MS(EI) m/z: 508
(M+).
Synthesis of 8. An oven-dried flask was charged with
Pd(OAc)2 (0.484 g, 2.15 mmol), Xantphos (1.87 g, 3.23
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4
mmol) and Cs2CO3 (9.83 g, 30.2 mmol). A solution of 7
168.47, 165.92, 164.28 (d, JP-C = 1.8 Hz), 152.35, 151.51,
2
1
2
3
4
5
6
7
8
(5.48 g, 10.8 mmol) and benzamide (2.35 g, 19.3 mmol) in
dioxane was added, and the resulting mixture was stirred
under N2 gas at RT for 30 min and then at 100 ºC for 23 h.
At that time the reaction mixture was allowed to cool to
RT, diluted with CH2Cl2 and filtered through a pad of
Celite. The filter cake was washed with CH2Cl2. The fil-
trate was concentrated and the residue was purified by
silica gel column chromatography (MeOH:CHCl3 = 1:20)
150.93, 150.35, 141.45, 140.23 (d, JP-C = 27.8 Hz), 136.94 (d,
1JP-C = 11.0 Hz), 135.83, 134,37, 133.71, 133.44 (d, JP-C = 20.8
2
2
Hz), 133.03, 132.53 (d, JP-C = 19.2 Hz), 131.82, 131.15, 130.33,
129.03, 128.95, 128.84, 128.70 (d, JP-C = 7.2 Hz), 128.57,
3
128.36, 128.22, 127.67, 125.38, 124.84, 123.96, 118.02, 116.55
(d, JP-C = 5.3 Hz), 113.13, 110.36, 107.14, 81.242; 31P NMR
3
(DMSO, 243 MHz, δ; ppm) -5.041; MS (FAB) m/z: 724
(M+1)+; Anal. Calcd. for C46H30NO6 P･3/5 H2O : C, 75.22;
H, 4.28; N, 1.91. Found: C, 74.83; H, 4.38; N, 2.00.
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to gave 3.96 g (77%) of 8 as pale yellow crystals: H NMR
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(CDCl3, 500 MHz, δ; ppm) 8.44 (1H, s), 7.97 (1H, d, J = 7.6
Hz), 7.86 (3H, m) , 7.63 (2H, m), 7.50 (1H, dd, J = 7.3 Hz, J
= 7.3 Hz), 7.42 (2H, dd, J = 7.9 Hz, J = 7.9 Hz), 7.14 (2H,
m), 6.93 (1H, d, J = 2.0 Hz), 6.67 (3H, m), 5.18 (2H, s), 3.47
(3H, s); MS(FAB) m/z: 480 (M+1)+.
HPLC Analysis. HPLC analysis was performed on an
Inertsil ODS-3 (150 mm × Φ4.6 mm) column (Shenshu
Pak C18) using a Shimadzu HPLC system. Detection
wavelength was 254 nm. Injection volume was 20 µL. Flow
rate was 1.0 mL/min
Synthesis of Rhodol 2. To a solution of 8 (190 mg,
0.397 mmol) in CH2Cl2 (6 mL) was added dropwise tri-
fluoroacetic acid (6 mL) at 0 ºC under N2 gas. The result-
ing solution was stirred at RT for 16 h, then concentrated
in vacuo and azeotroped three times with toluene. Purifi-
cation by silica gel flash column chromatography (CHCl3:
MeOH = 20:1) gave 108 mg (63%) of crude product, which
was recrystallized from CH3Cl/hexane to afford 57 mg
Preparative HPLC. Preparative HPLC were performed
on an Inertsil ODS-3 (250 mm × Φ20 mm) column
(Shenshu Pak C18) using an HPLC system composed of a
pump and a detector (Shimadzu). Injection volume was
100-500 µL.
HPLC Analysis for Detection of Rhodol 2. Sample
solutions (total volume 1.0 mL) of 100 μM P-Rhod in 100
mM potassium phosphate buffer containing 1% DMF were
incubated for 30 min at 37ºC after addition of 100 or 2000
μM AS. An aliquot of each solution (20 μL) was loaded
onto a Shenshu Pak C18 column fitted on a Shimadzu
HPLC system, and the eluates were monitored with a
photodiode array detector and fluorescence detector. Mil-
li-Q water containing 0.1% TFA (A) and MeCN containing
0.1% TFA (B) were used as developing solvents. Gradient
conditions were as follows: A 15% and B 85% for 20 min,
then to A 60% and B 40% in 33 min and continued for 3
min.
1
(33%) of 2 as an orange solid: mp 180.4–182.7°C; H NMR
(CD3OD, 500 MHz, δ; ppm) 8.03 (1H, d, J = 7.7 Hz), 7.94
(3H, m), 7.79 (1H, dd, J = 7.4 Hz, J = 7.4 Hz), 7.72 (1H, dd, J
= 7.5 Hz, J = 7.5 Hz), 7.59 (1H, dd, J = 7.4 Hz, J = 7.4 Hz),
7.52 (2H, dd, J = 7.8 Hz, J = 7.8 Hz), 7.35 (1H, dd, J = 8.6
Hz, J = 2.1 Hz), 7.24 (1H, d, J = 7.6 Hz), 6.74 (2H, m), 6.61
(1H, d, J = 8.7 Hz), 6.58 (1H, dd, J = 8.7 Hz, J = 2.4 Hz); 13C
NMR (CD3OD, 150 MHz, δ; ppm) 171.51, 169.07, 161.28,
154.53, 153.99, 153.14, 142.38, 136.71, 136.08, 133.16, 131.25,
130.19, 129.72, 129.55, 129.36, 128.74, 128.02, 125.90, 125.31,
117.50, 115.99, 113.75, 111.11, 109.53, 103.66; HRMS (FAB)
Calcd. For C27H18NO5 436.11849, Found 436.11956; HPLC tR
= 8.10 min (purity 98.1 %); Anal. Calcd. for C27H17NO5･3/5
H2O : C, 72.67; H, 4.11; N, 3.14. Found: C, 72.60; H, 4.54; N,
3.26.
Fluorescence Measurement of P-Rhod in Vitro.
Fluorescence spectra were measured with a Shimadzu
RF5300-PC. The slit width was set to 5.0 nm for excitation
and emission. P-Rhod was freshly dissolved in DMF to
obtain 10 mM solution, and the solutions were diluted to
10 μM with 100 mM PBS buffer (pH 7.4). An aliquot of a
freshly prepared stock solution of reagents were added to
a buffered solution of the test compounds to give final
concentrations of 10 μM test compound and the indicated
concentrations of RNS, ROS, or biomolecules. The reac-
tion mixture (total volume 1 mL) was incubated for 30
min at 37°C, then the fluorescence spectrum (490-650
nm) was measured with excitation at 491 nm.
Synthesis of P-Rhod (1). 2 (1.11 g, 2.55 mmol), 2-
(diphenylphosphino)benzoic acid (782 mg, 2.55 mmol), 4-
(dimethylamino)pyridine (312 mg, 2.55 mmol), and 1-ethyl
3-(3-dimethylaminopropyl)carbodiimide
hydrochloride
(1.47 g, 7.66 mmol) were dissolved in THF. The reaction
mixture was stirred for 22 h at RT under N2 gas, and then
poured into 1 N HCl and extracted with CHCl3. The organ-
ic layer was separated, washed with sodium bicarbonate,
water, and brine, and dried over Na2SO4. Filtration, con-
centration in vacuo, and purification by silica gel flash
column chromatography (CHCl3: MeOH = 98:2 to CHCl3:
MeOH = 95:5) gave 1.51 g (82%) of crude product, which
was recrystallized from CH3Cl/hexane to afford 817 mg
(44%) of 6 as a pale yellow solid. The product was purified
by preparative HPLC: mp 154.5–156.4 °C; 1H NMR (DMSO,
500 MHz, δ; ppm) 10.55 (1H, s), 8.26 (1H, m), 8.06 (2H, m),
7.97 (2H, m), 7.82 (1H, dd, J = 7.4 Hz, J = 7.4 Hz), 7.76 (1H,
dd, J = 7.4 Hz, J = 7.4 Hz), 7.62 (3H, m), 7.55 (2H, dd, J =
7.7 Hz, J = 7.7 Hz), 7.49 (1H, dd, J = 8.6 Hz, , J = 2.0 Hz),
7.39 (7H, m), 7.23 (4H, m), 7.14 (1H, d, J = 2.3 Hz), 6.92
(1H, m), 6.83 (3H, m); 13C NMR (DMSO, 150 MHz, δ; ppm)
Detection of N2O by Gas Chromatography. A solu-
tion of hydroxylamine hydrochloride with or without
HRP in Tris buffer (500 mM, 0.2 mM EDTA, pH 7.4, 0.5
mL containing 0.1% DMSO as a cosolvent). These solu-
tions were placed in a 5-mL vial sealed with a rubber sep-
tum and flushed with nitrogen gas. To initiate the reac-
tion, hydrogen peroxide was added to the solution, and
the vials were then incubated for 2 h at 37°C. An aliquot
of the reaction headspace gas (50 μL) was injected into a
Shimadzu GC-2010 gas chromatograph equipped with a
mass spectrometer (QP2010) and a Rt-QPLOT column
(0.32 mm × 15 m) expanded with a 15-m inactivated fused
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silica tube (total 30 m). The GC injector was operated
with a split ratio of 0.1 at 200°C. The carrier gas (He) was
set at a flow rate of 2.2 mL/min. The GC oven was held at
35°C. The MS interface was set to 280°C.
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Detection of enzymatic HNO generation via HRP-
Mediated oxidation of hydroxylamine. A deoxygenat-
ed solution of P-Rhod (0.5 μL, 10 μM), hydroxylamine
hydrochloride (10 μL, 10 mM), and horseradish peroxidase
type I (10 μL, 20 μM) in Tris buffer (1.0 M, 0.4 mM EDTA,
pH 7.4, 0.25 mL) was prepared at ambient temperature in
a septum-sealed vial. Hydrogen peroxide (10 μL, 10 mM)
was added to initiate the reaction. After 2 h at 37°C, the
resulting solution (0.5 mL) was examined with a fluoro-
photometer to evaluate fluorescence enhancement. Con-
trol experiments involving exclusion of any one of the
reaction components did not exhibit fluorescence en-
hancement.
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Cell culture. A549 cells were purchased from the
RIKEN Cell Bank, and cultured in DMEM containing 10%
FBS and penicillin/streptomycin (100 U/ml) in a humidi-
fied incubator at 37°C. When the cells had reached con-
fluence, they were detached in trypsin solution, washed in
DMEM, centrifuged at 125 g for 2 min, and then resus-
pended and subcultured according to standard protocols.
αReagents and conditions: (a) CH3OCH2Cl, K2CO3, DMF;
(b) (CF3SO2)2O, pyridine, CH2Cl2; (c) Pd(OAc)2, 4,5’-
bis(diphenylphosphino)-9,9’-dimethylxanthene, Cs2CO3,
benzamide, dioxane; (d) CF3COOH, CH2Cl2; (e) 2-
(diphenylphosphino)benzoic acid, EDCI, DMAP, THF. DMF
=
N,N-dimethylformamide,
EDCI
=
1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide hydrochloride, DMAP =
N,N-dimethyl-4-aminopyridine, THF = tetrahydrofuran.
Detection of HNO in A549 Cells. A549 cells were
plated onto 3.5 cm glass-bottomed culture dishes at 1.0 x
105 cells/dish with 2.0 mL of DMEM containing penicillin
and streptomycin, supplemented with fetal bovine serum,
according to ATCC’s instructions, and incubated at 37 °C
in a humidified 5% (v/v) CO2 incubator for 1 day. The cul-
ture medium was then removed, and the cells were
washed once with 1 mL of Dulbecco’s PBS (D-PBS). They
were placed in 1 mL of D-PBS, and treated with 1 L of P-
Rhod (1 M) for 3 h, then washed twice with 1 mL of D-
PBS and placed in 1 mL of D-PBS. The cells were then
treated with 2 L of AS in 10 mM NaOH solution (200
M), 2 L of 10 mM NaOH solution, 1 L of NOC 7 in 10
mM NaOH solution (100 M), 10 L of GSNO in water
(100 M), or 1 L of NaClO in water (1 M), and subjected
to confocal microscopy. Imaging was performed with a
63x oil-immersion objective lens on a Zeiss LSM 510 laser
scanning microscope.
First, the photophysical properties of rhodol 2 were as-
sessed in a buffer solution (100 mM potassium phosphate
buffer, pH 7.4, 0.1% DMF). The compound was found to
have typical optical properties of a rhodol chromophore,
with an absorption band in the blue range centered at 491
nm (ε = 2.95 × 104 M-1 cm-1). The emission profile was also
typical of rhodol, showing green fluorescence with a max-
imum at 526 nm. The fluorescence quantum yield of 2 has
been reported.18 When rhodol 2 was esterified to afford P-
Rhod, the fluorescence intensity decreased by ca. 774-fold
(Figure 1). This remarkable change of the fluorescence
intensity can be attributed to the open-closed form con-
version of rhodol, due to acylation of the phenolic hy-
droxyl group of 2.
P-Rhod (10 μM) was readily soluble in water in the
presence of 0.1% DMF, suggesting that it might be suita-
ble for use in living cells. The absorption band of P-Rhod
at 491 nm was small (ε = 9.58 M-1 cm-1), and the fluores-
cence was too weak to be detected. Thus, P-Rhod was
essentially non-fluorescent, due to its unconjugated and
non-fluorescent closed form resulting from acylation of
the amino and hydroxyl groups at the xanthene moiety. It
is considered that an ester group is generally labile under
acidic or basic conditions. However, the fluorescence in-
tensity of P-Rhod, despite its ester group, was independ-
ent of pH over the range of pH 5.0-10.0 (see supporting
information, Figure S1). This would be highly advanta-
geous for biological applications, considering the different
pH conditions in various cellular compartments.
Results and Discussion
P-Rhod was synthesized as shown in Scheme 2, accord-
ing to Dan’s synthetic strategy.18 Briefly, the synthesis of
P-Rhod was started from commercially available fluores-
cein 5, which was first mono-protected with a methox-
ymethyl (MOM) group. The protected fluorescein 6 was
triflated to provide doubly protected fluorescein 7, which
was coupled with benzamide by using Pd catalyst to ob-
tain amide derivative 8. The MOM group of 8 was easily
removed under acidic conditions to afford a rhodol fluor-
ophore 2, which was subjected to esterification to yield P-
Rhod (1). The structure and purity of P-Rhod were con-
1
13
31
firmed by H NMR, C NMR, and P NMR spectroscopy,
mass spectrometry, and elemental analysis.
Scheme 2. Synthesis of P-Rhodα
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ing 10 μM P-Rhod was incubated at 37 °C for 30 min in the
500
400
300
200
100
0
Ex = 491 nm
1
2
3
4
5
6
7
8
presence of 200 μM Angeli’s salt (AS), which spontane-
-
Rhodol(2)
P-Rhod (1)
ously generates an equimolar ratio of HNO and NO2 un-
der physiological conditions19. After incubation, the fluo-
rescence intensity at 526 nm was significantly increased
(Figure 2), and reached maximum in ca. 20 min (Figure
S2). We assessed the selectivity of P-Rhod for HNO over
other reactive species present in the biological milieu.
RNS donors, reactive oxygen species (ROS) donors, and
-
oxidants (ONOO-, NO2 , NO3-, H2O2, ClO-, and FeCl3) did
9
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not induce significant fluorescence enhancement of P-
Rhod (Figure 3). It is particularly noteworthy that the
fluorescence intensity of P-Rhod was hardly affected by
biological reductants, including GSH, selenomethionine
(Sem), ascorbic acid (AA), and hydroxylamine (HA), as
well as sodium sulfide (Na2S), which works as a source of
H2S under physiological conditions. In this regard, P-
Rhod is greatly superior to the previously reported CuII–
based probes, which show substantial fluorescence in the
presence of large amounts of various biological reduct-
ants10a. When 10 μM P-Rhod in a buffered aqueous solu-
tion was treated with various amounts of AS, the fluores-
cence intensity increased concentration-dependently
(Figure 2), and 100 nM AS induced a 1.49-fold increase in
fluorescence intensity compared to the blank. Additional-
ly, even when a solution of AS and HNO was incubated in
the presence of various ROS or RNS, we successfully de-
tected sufficient fluorescence. (Figure S3). Such high sen-
sitivity for HNO is advantageous for the detection in
physiological condition.
500
520
540
560
580
600
Wavelength (nm)
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Figure 1. Fluorescence spectra of 10 μM rhodol 2 (top line)
and P-Rhod (bottom line).
180
Ex/Em = 491/526 nm
150
120
90
60
30
0
0.01eq 0.1eq
1eq
5eq
20eq
50eq 100eq
AS equiv.
Figure 2. Fluorescence responses of 10 μM P-Rhod to 0.1, 1, 10,
50, 200, 500 or 1000 μM AS in 100 mM potassium phosphate
buffer (pH 7.4) containing 1% DMF after 30 min incubation
at 37 ºC. Means ±SD.
We next examined whether P-Rhod could detect HNO
under physiological conditions. A buffer solution contain-
100
Ex/Em = 491/526 nm
20eq
80
60
40
20
20eq
20eq
20eq
20eq
1000eq 1000eq
300eq
1000eq
100eq
0
Figure 3. Fluorescence responses of 10 μM P-Rhod in 100 mM potassium phosphate buffer (pH 7.4) containing 0.1% DMF at 37 ºC
-
-
for 30 min after addition of the following substances: AS, NOC 7, NOR 1, SNAP, GSNO, ONOO-, NO2 , NO3 , H2O2, NaClO, FeCl3,
GSH, selenomethionine (Sem), ascorbic acid (AA), hydroxylamine (HA) and sodium sulfide (Na2S). Four hundred equivalents of
each analyte were added unless otherwise indicated in the figure. Bars represent the ratio of the observed intensity (FI) to the
control (FI0) emission at 526 nm.
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Figure 4. Reversed-phase HPLC chromatograms with fluorescence (left) and absorption (right) detection. The arrow indicates
rhodol 2. Excitation: 491 nm, emission: 526 nm. (a) An authentic solution of 1000 μM P-Rhod. (b) An authentic solution of 100
μM P-Rhod oxide 3. (c) An authentic solution of 100 μM rhodol 2. (d) A sample solution of 100 μM P-Rhod with 2000 μM AS in
100 mM potassium phosphate buffer (pH 7.4, 0.1% DMF) after incubation for 30 min at 37 ºC.
While P-Rhod did not react with various RNS and ROS,
as shown in Figure 3, SNAP and GSNO were found to in-
crease the fluorescence slightly (Figure S4). TXPTS,
(tris(4,6-dimethyl-3-sulfonatophenyl)phosphine trisodi-
um salt), has been reported to react with RSNO, generat-
ing phosphine oxide, aza-ylide, and S-phosphino adduct.20
Thus, there might be a direct reaction between P-Rhod
and these NO donors or nitroxyl anion (NO-) generated
by one-electron reduction of NO radical by phosphine.
However, the fluorescence increase of P-Rhod with SNAP
and GSNO was small, suggesting that the practical selec-
tivity of P-Rhod for HNO is sufficient. We speculate that
steric hindrance of the xanthene moiety of P-Rhod re-
duced the reactivity with RSNO groups. Also, RSNO
groups require three equivalents of phosphine to yield
aza-ylide, the key intermediate to form rhodol 2, while
HNO requires only two equivalents.
reaction mixture of AS and GSH. These results strongly
suggest that the turn-on response induced by AS is due to
HNO production, and not due to a by-product.
Scheme 3. Mechanism of HRP-mediated HNO for-
mation from NH2OH.
When the reaction of P-Rhod and AS was performed in
the presence of GSH, a scavenger of HNO,21 the fluores-
cence intensity decreased with increasing GSH concentra-
tion (Figure S5). Further, 1000 μM NaNO2, formed after
HNO release from AS, did not affect the fluorescence of 10
μM P-Rhod. Moreover, the fluorescent intensity of Rhodol
2 was not decreased after incubation with GSH or the
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(Figure 5). No marked fluorescence increase was observed
HRP
Compound I
2 H2O
NH2OH
1
2
3
in the absence of NH2OH, H2O2, or HRP in the incubation
mixture, indicating that the fluorescence increase de-
pended on the HRP system. These results demonstrate
that P-Rhod can efficiently detect enzymatically generat-
ed HNO.
H2O2
NH2O
4
5
6
7
8
HRP
Compound II
HRP (FeIII)
70
Ex/Em = 491/526 nm
60
**
9
50
40
30
20
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HNO
NH2O
2 P-Rhod (1)
P-Rhod oxide (3)
Aza-ylide
0
1
+
+
-
2
+
-
+
-
3
+
-
-
+
4a
-
5
+
-
+
+
6
+
+
-
7
+
+
+
-
8
+
+
+
+
Entry
P-Rhod
NH2OH・HCl
H2O2
-
-
+
Phosphonyl-
benzamide (4)
HRP
-
+
Figure 5. Fluorescence enhancement of P-Rhod (10 μM) in
the presence or absence of hydroxylamine hydrochloride (10
mM), horseradish peroxidase type I (20 μM), or hydrogen
peroxide (10 mM) in Tris buffer (500 mM, 0.2 mM EDTA, pH
Rhodol (2)
Next, to confirm that P-Rhod is converted to the ex-
pected product, rhodol 2, after incubation (30 min at 37
ºC) of a solution containing AS and P-Rhod, the sample
solution was analyzed by HPLC (Figure 4). The proposed
mechanism of the reaction of P-Rhod with HNO is shown
in eq. 2 of Scheme 1. P-Rhod reacts with HNO to give P-
Rhod oxide 3, phosphonylbenzamide 4, and rhodol 2. As
shown in Figure 4d, rhodol 2 and P-Rhod oxide 3 were
detected concomitantly with the decrease of P-Rhod in
the incubated solution, although phosphonylbenzamide 4
could not be detected, probably due to its extremely small
extinction coefficient. Although P-Rhod was slightly hy-
drolyzed in the solution without AS, as shown by weak
fluorescence of rhodol 2 in the control solution (Figure
4a), the fluorescence intensity was negligible as compared
with that of a solution of P-Rhod with AS. These results
support the proposed mechanism of reaction of P-Rhod
with HNO.
a
7.4) for 2 h at 37 °C. 0.1% DMSO was added instead of P-
Rhod in the solution of Entry 4. Means ±SD. **p<0.01
compared with entries 5, 6, and 7 by ANOVA and
Bonferroni-type multiple t-test (parametric, n=3).
Next, we applied P-Rhod for the detection of HNO in
living cells. Human alveolar basal epithelial A549 adeno-
carcinoma cells were incubated with 1 μM P-Rhod for 3 h
at 37 ºC. Under this condition, the cells showed little in-
tracellular fluorescence (Figure 6A), indicating that P-
Rhod was not susceptible to intracellular hydrolysis. Ad-
dition of 200 μM AS to the cells increased the intracellular
fluorescence 11-fold during a further 20 min incubation
(Figure 6b-d), as quantified by using Image J software.
The fluorescence intensity in the cells appeared to be
predominantly localized in cytoplasm. Although there are
plenty of GSH, a scavenger of HNO, in a living cell, we
successfully detected HNO from AS with P-Rhod. It might
be because the excellent sensitivity of P-Rhod enabled to
visualize HNO while the reaction of HNO with P-Rhod
could be minor compared to that with GSH. In the con-
trol experiment (vehicle treatment), no change in fluores-
cence intensity was seen (Figure S8). This result suggest-
ed that the ester moiety of P-Rhod was not so sensitive for
intracellular esterase, probably due to the bulkiness
around ester bond in this molecule. Treatment of P-Rhod-
preloaded A549 cells with NOC 7 (100 μM) or GSNO (100
μM) did not affect the observed fluorescence (Figure S9
and S10). Moreover, no fluorescence increase was induced
by addition of equimolar exogenous NaClO, an oxidant,
We next investigated whether P-Rhod could detect
HNO generated from an enzymatic system. A heme pro-
tein, horseradish peroxidase (HRP), was employed for the
detection of HNO generated enzymatically in vitro. One
of proposed biosynthetic pathways of HNO is two-
electron oxidation of NH2OH catalyzed by HRP (Scheme
3).8 Anaerobic incubation of NH2OH (10 mM) and H2O2
(10 mM) in the presence of HRP (20 μM) for 2 h at 37 ºC
afforded N2O, a dimerization product of HNO, as deter-
mined by GC-MS using N2O derived from AS as a positive
control. No N2O was produced in the incubation solution
in the absence of HRP (Figures S6, S7).14 P-Rhod was add-
ed to this enzymatic system before H2O2 addition. After
the addition of H2O2, the fluorescence intensity increased
to
the
cells
containing
P-Rhod
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Figure 6. HNO-induced fluorescence images of A549 cells stained with 1 μM P-Rhod before (a) and at 10 min (b), 15 min (c) and
20 min (d) after treatment with 2 μL of AS in 10 mM NaOH solution (200 μM): (top) FITC channels and (bottom) DIC images.
Scale bars represent 20 μm.
(Figure S11). Taken together, these results confirm that P-
AUTHOR INFORMATION
Rhod is available for detection of HNO in living cells.
Corresponding Author
*deco@phar.nagoya-cu.ac.jp
Conclusion
We designed a fluorogenic HNO probe, P-Rhod, by uti-
lizing the combination of aza-ylide formation via reaction
of triarylphosphine with HNO and the open-closed tau-
tomerism of rhodol fluorophore. The fluorescence in-
crease was AS concentration-dependent and the probe
was highly sensitive and selective for HNO over other
biologically relevant RNS and ROS. A key advantage of P-
Rhod is that it is unaffected by various biological reduct-
ants, in contrast to previously reported probes. Formation
of the expected decomposition product of P-Rhod with
AS was confirmed by HPLC analysis. P-Rhod could also
detect HNO generated in the NH2OH-HRP enzymatic
system, which is believed to operate for HNO generation
in vivo. P-Rhod successfully imaged HNO intracellularly
released from AS, with excellent selectivity over other
ROS and RNS. Thus, P-Rhod is the first metal-free fluo-
rescent molecular probe available for detection of HNO in
living cells, and it is expected to be a useful chemical tool
for studies of the biological roles of HNO.
Present Addresses
¶Graduate School of Medical Sciences, Kyoto Prefectural
University of Medicine, 13 Taishogun Nishitakatsukasa-cho,
Kita-ku, Kyoto 403-8334, Japan
ACKNOWLEDGMENT
This work was supported by JST PRESTO program (H.N.),
Grants-in-Aid for Scientific Research on Innovative Areas
(Research in a Proposed Research Area) (No. 21117514 to
H.N.), and Grants-in-Aid for Scientific Research (No.
22590103 to H.N.) from Ministry of Education, Culture,
Sports Science and Technology Japan.
REFERENCES
1.
Fukuto, J. M.; Bartberger, M. D.; Dutton, A. S.; Paolocci, N.;
Wink, D. A.; Houk, K. N. Chem. Res. Toxicol. 2005, 18, 790-
801.
2.
3.
4.
5.
Moncada, S.; Palmer, R. M. J.; Higgs, E. A. Pharmacol. Rev.
1991, 43, 109–142.
Ieda, N.; Nakagawa, H.; Peng, T.; Yang, D.; Suzuki, T.;
Miyata, N. J. Am. Chem. Soc. 2012, 134, 2563-2568.
Dedon, P. C.; Tannenbaum, S. R. Arch. Biochem. Biophys.
2004, 423, 12-22.
(a) Fukuto, J. M.; Cisneros, C. J.; Kinkade, R. L.; J. Inorg.
Biochem. 2013, 118, 201-208. (b) Paolocci, N.; Wink, D. A.
Am. J. Physiol. Heart Circ. Physiol. 2009, 296, H1217 -
H1220. (c) Miranda, K. M.; Paolocci, N.; Katori, T.; Thomas,
D. D.; Ford, E.; Bartberger, M. D.; Espey, M. G.; Kass, D. A.;
ASSOCIATED CONTENT
Supporting Information. Supporting figures, syntheses of 3
1
and 4, H NMR spectra for P-Rhod, 3 and 4. This material is
available free of charge via the Internet at
http://pubs.acs.org.
ACS Paragon Plus Environment

Page 9 of 10
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Feelisch, M.; Fukuto, J. M.; Wink, D. A. Proc. Natl. Acad.
1
2
3
4
5
6
7
8
Sci. U.S.A. 2003, 100, 9196–9201.
DeMaster, E. G.; Redfern, B.; Nagasawa, H. T. Biochem.
Pharmacol. 1998, 55, 2007–2015.
(a) Miranda, K. M.; Katori, T.; Torres de Holding, C. L.;
Thomas, L.; Rindnour, L. A.; McLendon, W. J.; Cologna, S.
M.; Dutton, A. S.; Champion, H. C. Mancardi, D.; Tocchet-
ti, C. G.; Saavedra, J. E.; Keefer, L. K.; Houk, K. N.; Fukuto,
J. M.; Kass, D. A.; Paolocci, N.; Wink, D. A. J. Med. Chem.
2005, 48, 8220-8228. (b) Paolocci, N.; Saavedra
6.
7.
9
8.
9.
W. F.; Miranda, K. M.; Martignani, C.; Isoda, T.; Hare, J. M.;
Espey, M. G.; Fukuto, J. M.; Feelisch, M.; Wink, D. A.; Kass,
D. A. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 10463-10468.
Donzelli, S.; Espey, M. G.; Flores-Santana, W.; Switzer, C.
H.; Yeh, G. C.; Huang, J.; Stuehr, D. J.; King, S. B.; Miranda,
K. M.; Wink, D. A. Free Radic Biol. Med. 2008, 45, 578-584.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
10. (a) Rosenthal, J.; Lippard, S. J. J. Am. Chem. Soc. 2010, 132,
5536-5537. (b) Royzen, M.; Wilson, J. J.; Lippard, S. J. J. In-
org. Biochem. 2013, 118, 162-170. (c) Zhou, Y.; Liu, K.; Li, J.
Y.; Fang, Y.; Zhao, T. C.; Yao, C. Org. Lett. 2011, 13, 1290-
1293.
11.
Kitamura, N.; Hiraoka, T.; Tanaka, K.; Chujo, Y. Bioorg.
Med. Chem. 2012, 20, 4668-4674.
12. M. R. Cline; J. P. Toscano J. Phys. Org. Chem. 2011, 24, 993–
998.
13. Reisz, J. A.; Klorig, E. B.; Wright, M. W.; King, S. B. Org.
Lett. 2009, 11, 2719–2721.
14. Reisz, J. A.; Zink, C. N.; King, S. B. J. Am. Chem. Soc. 2011,
133, 11675-11685.
15. Saxon, E.; Bertozzi, C. R. Science 2000, 287, 2007-2010.
16. Whitaker, J. E.; Haugland, R. P.; Ryan, D.; Hewitt, P. C.;
Haugland, R. P.; Prendergast, F. G. Anal. Biochem. 1992,
207, 267-279.
17. (a) Peng, T.; Yang, D. Org. Lett. 2010, 12, 4932-4935. (b)
Kamiya, M.; Asanuma, D.; Kuranaga, E.; Takeishi, A.; Sa-
kabe, M.; Miura, M.; Nagano, T.; Urano, Y. J. Am. Chem.
Soc. 2011, 133, 12960-12963.
18. Peng, T.; Yang, D. Org. Lett. 2010, 12, 496-499.
19. Liochev, S. I.; Fridovich, I. Free Radical Biol. Med. 2003, 34,
1399–1404.
20. (a) Bechtold, E.; Reisz, J. A.; Klomsiri, C.; Tsang, A. W.;
Wright, M. W.; Poole, L. B.; Furdui, C. M.;King, S. B. ACS
Chem. Biol. 2010, 5, 405-414. (b) Zhang, J.; Wang, H.; Xian,
M. J. Am. Chem. Soc. 2009, 131, 3854–3855. (c) Zhang, J. M.;
Wang, H.; Xian, M. Org. Lett. 2009, 11, 477–480.
21. Irvine, J. C.; Ritchie, R. H.; Favaloro, J. L.; Andrews, K. L.;
Widdop, R. E.; Kemp-Harper, B. K. Trends Pharmacol. Sci.
2008, 29, 601–608.
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