Detection of Nitric Oxide and Nitroxyl with Benzoresorufin-Based Fluorescent Sensors

Article (PDF Available)inInorganic Chemistry 52(6) · March 2013with90 Reads
DOI: 10.1021/ic302793w · Source: PubMed
Abstract
A new family of benzoresorufin-based copper complexes for fluorescence detection of NO and HNO is reported. The copper complexes, CuBRNO1-3, elicit 1.5-4.8-fold emission enhancement in response to NO and HNO. The three sensors differ in the nature of the metal-binding site. The photophysical properties of these sensors are investigated with assistance from density functional theory calculations. The fluorescence turn-on observed upon reaction with HNO is an unexpected result that is discussed in detail. The utility of the new sensors for detecting HNO and NO in HeLa cells and RAW 264.7 macrophages is demonstrated.
Detection of Nitric Oxide and Nitroxyl with Benzoresorun-Based
Fluorescent Sensors
Ulf-Peter Apfel,
Daniela Buccella,
,
Justin J. Wilson,
and Stephen J. Lippard*
,
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States
Department of Chemistry, New York University, New York, New York 10003, United States
*
S
Supporting Information
ABSTRACT: A new family of benzoresorun-based copper
complexes for uorescence detection of NO and HNO is
reported. The copper complexes, CuBRNO13, elicit 1.54.8-
fold emission enhancement in response to NO and HNO. The
three sensors dier in the nature of the metal-binding site. The
photophysical properties of these sensors are investigated with
assistance from density functional theory calculations. The
uorescence turn-on observed upon reaction with HNO is an
unexpected result that is discussed in detail. The utility of the
new sensors for detecting HNO and NO in HeLa cells and RAW 264.7 macrophages is demonstrated.
INTRODUCTION
Reactive nitrogen (RNS) and oxygen species (ROS) have
numerous biological consequences.
1
Among these species,
nitric oxide (NO), initially identied as an endothelial-derived
relaxation factor,
2
has a broad variety of biological regulatory
and signaling functions.
38
Biological NO is generated by
oxidation of
L-arginine to L-citruline by a class of enzymes
known as nitric oxide synthases (NOS).
9
Among its many
functions, NO plays important roles in the control of smooth
muscle relaxation and vasodilation,
10
platelet aggregation in
vascular endothelial cells,
11
neurotransmission,
12
and regulation
of the immune response by macrophages.
13
Recently, it was
suggested that nitroxyl (HNO), the one-electron-reduced
congener of NO, is formed by NOS via oxidative degradation
of
L-arginine.
14,15
Some recent in vitro studies using HNO-
releasing molecules demonstrated that HNO increases the
contractility of heart cells,
16
leads to vasorelaxation in muscle
cells,
17
and decreases platelet aggregation.
18
Taken together,
these ndings suggest that HNO also plays a pivotal role in
biology.
In order to advance our insight into the physiological and
pathological roles of HNO and NO, our group has focused on
designing uorescent sensors that selectively respond to these
small molecules and aord both sp atial and tem poral
information regarding the natural occurrence of these species
at the cellular level. Various NO sensors, including o-
diaminouore scein, o-diaminonaphthalene, o-diam inocya-
nine,
1921
luminescent lanthanide complexes,
22
and 5-amino-
1-naphthonitrile (NO550),
23
detect NO in the presence of
oxygen. Sensors for HNO include metalloporphyrins,
24
thiols,
25
and phosphines.
26
In contrast to these reagents, copper-based
uorescent probes like CuFL1,
27,28
CuFL2E,
29
and CuSNFL
30
developed by our group (Scheme 1) and others
31,32
provide
direct, selective, and fast detection of NO both in vitro and in
vivo. We have also recently described a selective HNO sensor,
CuBOT1.
33,34
The emission intensity of these copper-based
sensors is modulated by the oxidation state of the copper ion,
which, in turn, can be modied by the RNS of interest. The
CuFL and CuSNFL sensors react with NO to form the
emissive nitrosamines FL-NO and SNFL-NO, with concom-
itant formation of Cu
I
ions that dissociate from the
complex.
30,35
The copper complex CuBOT1, on the other
hand, reacts selectively with HNO, leading to reduction of the
paramagnetic Cu
II
ion and uorescence enhancement.
33
The
dierent reactivity of CuBOT1 versus CuFL and CuSNFL
presumably arises from the lack of a secondary amine in the
Received: December 19, 2012
Published: March 5, 2013
Scheme 1. Small-Molecule Metal-Containing Sensors for NO
(Cu[FL1]) and HNO (Cu[BOT1]) as Well as Their
Reactions with Analytes
30,33
Article
pubs.acs.org/IC
© 2013 American Chemical Society 3285 dx.doi.org/10.1021/ic302793w | Inorg. Chem. 2013, 52, 32853294
former, which is the preferred site of attack on CuFL by NO.
35
A limitation of both uorescein- and BODIPY-based sensors is
their high-energy absorption and emission as well as their
moderate Stokes shifts of about 30 nm. These features can
cause problems associated with light scattering, which may lead
to a low signal-to-noise ratio. Additionally, for biological
imaging purposes, it is desirable to have sensors that emit far
into the red, because low-energy radiation penetrates tissue
more eectively.
In our continuing eorts to design improved sensors for NO
and HNO, we have functionalized a benzoresorun uorophore
with a Cu-binding site that contains a secondary amine, and we
investigated its ability to detect ROS and RNS. Resorun dyes
emit at wavelengths >600 nm with Stokes shifts up to 60 nm.
36
Accordingly, sensors based on resorun dyes should exhibit less
background absorption and emission from biological samples
and provide an advantage over previously reported green
emitters in terms of tissue penetration depth. The synthesis and
characterization of three benzoresorun-based sensors are
described herein. As with our previous systems, the Cu
II
/Cu
I
redox couple is used to modulate the emission response of the
sensors to NO and HNO. Despite the availability of a
secondary amine, a potential site of attack of NO, we found
that the benzoresorun-based probes show a better turn-on
response for HNO than for NO.
MATERIALS AND METHODS
Synthetic Materials and Methods. All reactions were carried out
under an N
2
atmosphere using standard Schlenk techniques.
1
H and
13
C{
1
H} NMR spectra were recorded with a Varian Mercury 300
NMR or a Varian Inova 500 NMR spectrometer at room temperature.
Peaks were referenced to residual
1
H signals from the deuterated
solvent and are reported in parts per million (ppm). 2-Methyl-4-
nitrosoresorcinol was synthesized by a literature procedure.
37
All other
compounds were obtained from commercial vendors and used without
further purication. Mass spectra were obtained with either an Agilent
5973 Network mass-selective detector connected to an Agilent 689N
Network GC system or an Agilent 1100 Series LC/MSA trap. High-
resolution mass spectral analyses were carried out at the Massachusetts
Institute of Technology (MIT) Department of Chemistry Instrumen-
tation Facility (DCIF). UVvis spectra were recorded with a Varian
Cary 1E spectrometer at 25 °C. Fluorescence spectra were obtained on
a Quanta Master 4 L-format scanning spectrouorimeter (Photon
Technology International) at 25 or 37 °C. X-band electron
paramagnetic resonance (EPR) spectra were collected with a Bruker
EMX spectrometer equipped with an ER4199HS cavity and a Gunn
diode microwave source. All solvents were dried prior to use according
to standard methods. Silica gel 60 321 (0.0150.040 mm) was used
for column chromatography. Thin-layer chromatography was
performed using Merck TLC aluminum sheets, silica gel 60 F254.
9-Hydroxy-8-methyl-5-benzo[a]phenoxazone (1). 2-Methyl-
4-nitrosoresorcinol (1.31 g, 7.68 mmol) and 1,3-dihydroxynaphthalene
(1.23 g, 7.68 mmol) were dissolved in n-butanol (20 mL) and heated
to 50 °C. To this solution was added concentrated sulfuric acid (2.6
mL), and the mixture was stirred for 15 min at 50 ° C. The mixture was
allowed to cool to room temperature, and after 12 h, a dark precipitate
formed. The precipitate was collected by ltration, washed with a
mixture of ethanol and n-butanol (1:1, 10 mL) and then water/ethanol
(1:1, 50 mL), and dried under vacuum. This material (2.11 g, 96%
yield) was used without further purication. ESI-MS. Calcd for
[C
17
H
11
NO
3
]
: 277.1. Found: 277.0.
8-Methyl-5-oxo-benzo[a]phenoxazin-9-yl acetate (2). Com-
pound 1 (1.41 g, 5.09 mmol) was dissolved in acetic anhydride (20
mL) and pyridine (2 mL). The mixture was heated for 3 h at 100 °C
and then left to stand at room temperature for 24 h. The resulting
dark-red crystalline solid was collected by ltration and washed with a
small amount of acetic anhydride and then a large volume of water.
The solid was dried under vacuum, yielding 1.06 g (65%) of an orange
crystalline material.
1
H NMR (CDCl
3
, 300 MHz, ppm): δ 8.718.67
(1H, m), 8.308.27 (1H, m), 7.787.73 (2H, m), 7.68 (1H, d,
3
J =9
Hz), 7.07 (1H, d,
3
J = 9 Hz), 6.46 (1H, s), 2.39 (3H, s), 2.27 (3H, s).
13
C NMR (CDCl
3
, 75 MHz, ppm): δ 184.1, 169.0, 151.2, 149.8, 132.4,
132.3, 132.0, 131.4, 130.9, 130.2, 127.8, 126.1, 124.9, 119.2, 118.8,
117.1, 107.7, 21.0, 9.1. ESI-MS. Calcd for [C
19
H
13
NO
4
+H
+
]
+
320.1.
Found: 320.2. Mp: 234235 °C.
6-Bromo-8-bromomethyl-5-oxo-benzo[a]phenoxazin-9-yl
acetate (3). Compound 2 (2.62 g, 8.2 mmol), 1,3-dibromo-5,5 -
dimethylhydantoin (5.16 g, 0.018 mol), and VAZO88 (0.72 g, 3.0
mmol) were dissolved in dry chlorobenzene (300 mL) under an N
2
atmosphere. Acetic acid (700 μL) was added, and the solution was
heated at 60 °C until
1
H NMR spectroscopy revealed full conversion
to the nal molecule (610 days). The hot solution was then washed
with hot water (60 °C, 2 × 50 mL) and brine (100 mL) and dried with
sodium sulfate. Evaporation of the organic fractions to dryness
aorded an orange solid, which was washed with diethyl ether/pentane
(1:1). The solid was collected by ltration and dried under vacuum to
aord 3.6 g (92%) of an orange solid.
1
H NMR (CDCl
3
, 300 MHz,
ppm): δ 8.718.69 (1H, m), 8.388.35 (1H, m), 7.867.76 (3H, m),
7.267.23 (1H, m), 4.73 (2H, s), 2.46 (3H, s). ESI-MS. Calcd for
[C
19
H
11
Br
2
NO
4
+Na
+
]
+
: 497.9. Found: 497.8. Mp: 221 °C.
6-Bromo-8-formyl-9-hydroxy-5-benzo[a]phenoxazone (4).
Compound 3 (1.5 g, 3.16 mmol) and sodium bicarbonate (1.8 g, 21
mmol) were dissolved in dimethyl sulfoxide (DMSO; 50 mL) and
heated at 150 °C for 2 h. After cooling to room temperature, the blue
solution was poured into 300 mL of HCl (4 M), and the resulting
mixture was stirred for 1 h. The obtained brown precipitate was
collected by ltration, washed with water, and dried under vacuum.
The crude material was puried by silica gel column chromatography
(dichloromethane to dichloromethane/methanol 100:2). The ob-
tained residue was washed with 20 mL of diethyl ether/pentane (1:1)
to aord 382 mg (29%) of a dark-orange solid.
1
H NMR (CDCl
3
, 300
MHz, ppm): δ 12.10 (1H, s), 10.71 (1H, s), 8.708.67 (1H, m),
8.408.37 (1H, m), 8.01 (1H, d,
3
J = 9 Hz), 7.857.74 (2H, m), 7.02
(1H, d,
3
J = 9 Hz). ESI-MS. Calcd for [C
17
H
8
BrNO
4
+H
+
]
+
: 370.0.
Found: 369.9. Mp: 254 °C.
6-Bromo-9-hydroxy-8-[[(2-methylquinolin-8-yl)amino]-
methyl]-5-benzo[a]phenoxazone (BRNO1). Compound 4 (25 mg,
0.068 mmol) and 8-amino-2-methyl-quinoline (11 mg, 0.07 mmol)
were dissolved in dry methanol (10 mL) and stirred at room
temperature for 1 h. The solution was cooled to 0 °C, and sodium
cyanoborohydride (102 mg, 1.62 mmol) was added in one portion.
The mixture was stirred for 36 h at room temperature and then poured
into 10 mL of aqueous 5 M HCl. The resulting precipitate was ltered
o and washed with copious amounts of water and then dichloro-
methane/hexane (10 mL, 1:1). Subsequently, the solid was dried
under vacuum to aord 14.2 mg (41%) of a dark-red compound.
1
H
NMR (CD
3
OD, 500 MHz, ppm): δ 12.33 (1H, s), 9.41 (1H, d,
3
J =6
Hz), 9.03 (1H, d,
3
J = 6 Hz), 8.958.91 (1H,m), 8.718.68 (1H, m),
8.648.61 (1H, m), 8.55 (1H, d,
3
J = 6 Hz), 8.20 (1H, d,
3
J = 6 Hz),
8.148.11 (1H, m), 7.95 (1H, d,
3
J = 6 Hz), 7.90 (1H, d,
3
J = 6 Hz),
7.87 (1H, d,
3
J = 6 Hz), 5.59 (2H, s), 3.46 (3H, s). ESI-HRMS. Calcd
for [C
27
H
18
N
3
BrO
3
H
+
]
: 510.0453. Found: 510.0439. Mp: 184 °C
(dec).
6-Bromo-9-hydroxy-8-[(quinolin-8-ylamin o)methyl]-5-
benzo[a]phenoxazone (BRNO2). Compound 4 (20 mg, 0.054
mmol) and 8-aminoquinoline (9.4 mg, 0.065 mmol) were dissolved in
dry methanol (10 mL) and stirred at room temperature for 2 h. The
solution was then cooled to 0 °C, and sodium cyanoborohydride (50
mg, 0.79 mmol) was added in one portion. The mixture was stirred for
72 h at room temperature and poured into a 20 mL solution of a
saturated aqueous sodium bicarbonate solution. The resulting
precipitate was ltered o and washed with water. Drying under
vacuum aorded 10.6 mg (39%) of a dark-purple solid.
1
H NMR
(DMSO-d
6
, 500 MHz, ppm): δ 8.70 (1H, d,
3
J = 6 Hz), 8.57 (1H, d,
3
J
= 6 Hz), 8.22 (1H, d,
3
J = 6 Hz), 8.18 (1H, d,
3
J = 6 Hz), 7.837.80
(1H, m), 7.727.69 (1H, m), 7.61 (1H, d,
3
J = 6 Hz), 7.487.44 (1H,
m), 7.367.33 (1H, m), 7.17 (1H, d,
3
J = 6 Hz), 7.05 (1H, d,
3
J =6
Inorganic Chemistry Article
dx.doi.org/10.1021/ic302793w | Inorg. Chem. 2013, 52, 328532943286
Hz), 6.83 (1H, d,
3
J = 6 Hz), 4.73 (2H, s), 2.62 (3H, s). ESI-HRMS.
Calcd for [C
26
H
16
N
3
BrO
3
H
+
]
: 496.0297. Found: 496.0293. Mp:
256 °C (dec).
6-Bromo-9-hydroxy-8-[[(pyridin-2-ylmethyl]amino]methyl)-
5-benzo[a]phenoxazone (BRNO3). Compound 4 (20 mg, 0.054
mmol) and 2-(aminomethyl)pyridine (11 μL, 0.11 mmol) were
dissolved in dry methanol (5 mL), and the resulting solution was
stirred at room temperature for 1 h. The solution was then cooled to 0
°C, and sodium cyanoborohydride (66 mg, 0.10 mmol) was added in
one portion. The mixture was stirred for 48 h at room temperature,
and a saturated aqueous solution of sodium bicarbonate (20 mL) was
added. The resulting precipitate was ltered o, washed with water (10
mL), and dried under vacuum to aord 21.4 mg (43%) of a purple
solid.
1
H NMR (CD
3
OD, 500 MHz, ppm): δ 8.72 (1H, d,
3
J = 6 Hz),
8.41 (1H, d,
3
J = 6 Hz), 8.34 (1H, d,
3
J = 6 Hz), 7.787.73 (2H, m),
7.677.63 (1H, m), 7.60 (1H, d,
3
J = 6 Hz), 7.51 (1H, d,
3
J = 6 Hz),
7.257.24 (1H, m), 6.79 (1H, d,
3
J = 6 Hz), 4.23 (2H, s), 4.03 (2H,
s). ESI-HRMS. Calcd for [C
23
H
16
N
3
BrO
3
H
+
]
: 460.0297. Found:
460.0293. Mp: 256 °C (dec).
Headspace EI-MS Studies. BRNO1 (0.95 mg, 1.86 μmol) was
dissolved in acetonitrile (1 mL), and 0.8 equiv of CuCl
2
·2H
2
O (0.2
mg, 1.18 μmol, in 1 mL acetonitrile) was added to this solution in a
custom-made, gastight cell in an inert-atmosphere glovebox. The
mixture was maintained at room temperature for 1 h, prior to addition
of 10 mg of Angelis salt. The cell was connected to a He gas-ow inlet
tube and to the mass spectrometer. The connecting copper tubing was
purged thoroughly with He prior to analysis of the reaction headspace.
Headspace analysis was performed with the mass spectrometer
operating in selective ion mode.
Cyclic Voltammetry. Cyclic voltammograms were measured in a
three-electrode cell with a 2.0-mm-diameter glassy carbon working
electrode, a platinum auxiliary electrode, and a Ag/AgNO
3
reference
electrode in acetonitrile. The solvent contained n-Bu
4
N(PF
6
) (0.05 M)
as the supporting electrolyte. The measurements were performed at
room temperature with a VersaSTAT3 (AMETE K) galvanostat.
Deoxygenation of the samples was accomplished by passing a stream
of N
2
through the solutions for 5 min prior to the measurements, and
the solutions were kept under N
2
for the duration of the study. All data
were referenced to the Fc/Fc
+
couple as an internal standard (E
1/2
=
+405 mV vs Ag/AgNO
3
reference electrode).
X-ray Data Collection and Structure Solution Renement.
Crystals of 2 and 3 were grown by slow evaporation of solutions of the
compounds dissolved in chloroform. Single crystals suitable for X-ray
analysis were coated with Paratone-N oil, mounted on a ber loop, and
placed in a cold, gaseous N
2
stream on a Bruker APEX CCD X-ray
diractometer performing φ and ω scans at 100(2) K. Diraction
intensities were measured using graphite-monochromated Mo Kα
radiation (λ = 0.71073 Å). Data collection, indexing, initial cell
renements, frame integration, and nal cell r enements were
accomplished with the program APEX2.
38
Absorption corrections
were applied using the program SADABS.
39
The structure was solved
by direct methods using SHELXS
40
and rened against F
2
on all data
by full-matrix least squares with SHELXL-97
41
following established
renement strategies. Crystallographic data collection and renement
parameters are presented in Tables S1 and S2 in the Supporting
Information (SI).
Density Functional Theory (DFT) Calculations. All calculations
were performed with the Gaussian 03 program package
42
using the
B3LYP functional.
43,44
Geometry optimizations were carried out in the
gas phase using the 6-31g(d,p) basis set.
45
Frequency calculations were
carried out at the same level of theory to ensure that geometries
converged to true local minima on the potential energy surface. The
30 lowest-energy singlet excited states were computed with time-
dependent DFT (TDDFT) calculations. For these calculations, the
larger 6-311++g(d,p) basis set was utilized. Solvent eects were
modeled with the conductor-like polarizable continuum model for
water.
46
Electron-density dierence maps (EDDMs) and calculated
UVvis absorbance spectra were generated with the program
GaussSum2.2.
47
Tables S3S12 in the SI contain the coordinates of
optimized structures and a summary of the lowest-energy singlet
excited states.
Spectroscopic Materials and Methods. Piperazine-N,N-bis(2-
ethanesulfonic acid) (PIPES; Calbiochem) and potassium chloride
(99.999%, Aldrich) were used to prepare buered solutions (50 mM
PIPES, 100 mM KCl, pH 7.0) in deionized water with resistivity 18
MΩ cm
1
, obtained using a Milli-Q water purication system. Nitric
oxide (NO) was purchased from Airgas and puried as previously
described.
48
S-Nitroso-N-acetylpenicillamine (SNAP), S-nitroso-L-
glutathione (GSNO), sodium peroxynitrite, and Angelis salt were
purchased from Cayman Chemical and stored at 80 °C when not in
use. NO and the other RNS were injected into buered solutions via a
gastight syringe. CuCl
2
·2H
2
O (99+%, Alfa Aesar) was used to prepare
7 mM CuCl
2
stock solutions in DMSO. Dye stock solutions were
prepared in DMSO (5 mmol/L) and stored at 80 °C when not in
use. Measurements were performed under inert-atmosphere con-
ditions. Quantum yields of BRNO13 were determined in 50 mM
PIPES buer (100 mM KCl, pH 7) using resorun(λ
em
= 585 nm, λ
ex
= 572 nm, and Φ = 0.74
49
) as the reference in water (pH 9.5).
Cell Culture. HeLa cells and Raw 264.7 murine macrophages were
cultured in Dulbeccos modied Eagle medium (DMEM; Cellgro,
MediaTek, Inc.), supplemented with 10% fetal bovine serum (FBS;
HyClone), 1% penicillinstreptomycin, 1% sodium pyruvate (Cellgro,
MediaTek, Inc.), 1% MEM nonessential amino acids (Sigma), and 1%
L-glutamine. For imaging studies, cells were grown to conuence,
passaged, and plated onto poly-
D-lysine-coated plates. The plates,
containing 2 mL of DMEM, were incubated at 37 °C with 5% CO
2
for
at least 12 h. The media were removed, the cells were washed with 5
mL of PBS buer, and solutions of the uorescent probes in 2 mL of
fresh DMEM were added. For all cell studies, the Cu
II
complexes were
generated in situ by combining a stock solution of the uorescent
sensor and CuCl
2
in a 1:1 molar ratio 1 h prior to addition to the cells.
Plates were prepared with identical volumes from the same cell stock
solution to provide an equal number of cells in each plate. For NO
detection studies, NO production by iNOS was induced in Raw 264.7
murine macrophages with 1.6 μg/mL lipopolysaccharide (LPS; Sigma)
and 4954950 U/mL of recombinant mouse interferon-γ (IFN-γ;BD
Biosciences). Cells were then incubated with 2.5 μM CuBRNO1 or 2.5
μM CuBRNO3 for 30 min. Then 5 μM HOECHST 33258 (Sigma)
was added, and the cells were incubated for a further 30 min. Prior to
imaging, cells were washed with 2 mL of PBS and then bathed with 1.5
mL of dye-free DMEM (Sigma). NO detection studies were
performed by addition of 1.25 mM GSNO. For HNO imaging
studies, HeLa and Raw 264.7 cells were treated as described above
before addition of 1.25 mM Angelis salt. Localization studies were
performed in both HeLa and Raw 264.7 cells in the presence of 2.5
μM CuBRNO1 or 2.5 μM CuBRNO3 and 1.25 mM Angelis salt.
Prior to uorescence imaging, the cells were incubated with either 3
μ M ER-Tracker Blue Blue-White DPX (Invitrogen), 13 μM
MitoTracker Green FM (Invitrogen), or CellLight Reagents BacMam
2.0 (Invitrogen) (10 parts per cell/16 h incubation) for 30 min.
Fluorescence Imaging. Fluorescence images were acquired on a
Zeiss Axiovert 200 M inverted epiuorescence microscope equipped
with an EM-CCD camera (Hamamatsu) and an X-Cite 120 metal
halide lamp (EXFP). Dierential interference contrast (DIC) and
uorescence images were obtained using an oil immersion 63×
objective lens with exposure times ranging from 50 ms to 2 s. The
microscope was operated with the Volocity 6.01 software (Improvi-
sion), and images were analyzed with the Volocity 6.01 software. All
uorescent images were deconvoluted and background-corrected.
Images were measured before and after addition of Angelis salt or
NO-releasing agent. The image before addition of Angelis salt or NO-
releasing agent was taken as the background level.
RESULTS AND DISCUSSION
Design and Synthesis of Benzoresorun-Based
Sensors. An ongoing goal of our laboratory is the development
of uorescent sensors for NO and HNO. We found that an
eective strategy for designing such species is to couple a metal-
Inorganic Chemistry Article
dx.doi.org/10.1021/ic302793w | Inorg. Chem. 2013, 52, 328532943287
binding site with a uorophore.
50
In the o state of the
sensor, a paramagnetic ion in the metal-binding site quenches
the emission of the uorescent reporter by photoinduced
electron transfer (PET). Upon reaction with NO, the
paramagnetic ion is reduced, displaced, or both, thus
eliminating the PET quenching pathway and triggering an
emission turn-on response.
32,35,5154
Our most successful NO
sensors, which operate nicely in cellular environments, contain
a 2-methyl-8-aminoquinoline metal-binding site and either a
uorescein (FL series) or a seminaphthouorescein (SNFL
series) dye as uorescent reporters.
2730
Cu
II
is used as the
paramagnetic uorescence quencher. The methyl group in the
2 position of the aminoquinoline gives rise to only moderate
Cu
II
-binding a nities, characterized by K
d
values of approx-
imately 1.5 μM. In this study, we sought to change both the
uorescent reporter and metal-binding site. Benzoresorun,
which emits in the red, was chosen as the uorophore, and in
addition to our standard 2-methyl-8-aminoq uinoline Cu
II
-
binding site, we investigated 8-aminoquinoline and 2-
(methylamino)pyridine.
The overall synthetic strategy for preparing the desired
benzoresorun-based sensors is shown in Scheme 2. This
synthetic approach is general and therefore oers a viable
pathway to a wide variety of monofunctionalized benzoresor-
un dyes. Following a similar procedure for the synthesis of
naphthophenoxazones,
55
the reaction of 2 -methyl-4-nitro-
resorcinol
37
and 1,3-dihydroxynaphthalene aorded 1, which
is readily puried in high yield by formation of the acetyl ester
derivative 2. The molecular structure of this compound, as
determined by single-crystal X-ray diraction, is presented in
Figure S1 in the SI. To enable functionalization of the
benzoresorun moiety, we sought to selectively brominate the
benzylic position. The bromination of compound 2 was carried
out with 1,3-dibromo-5,5-dimethylhydantoin and VAZO88,
yielding dibrominated species 3 in high yield. The position of
the two bromine atoms was unambiguously veried by single-
crystal X-ray analysis (Figure S2 in the SI). The formation of 3
from 2 was monitored by
1
H NMR spectroscopy, revealing that
bromination occurs rst on the quinoid position, thus
preventing isolation of a singly brominated species (Figure S3
in the SI). Because the exocyclic nitrogen atoms of amino-
quinolines are only weak nucleophiles, the direct S
N
2 reaction
on bromide of 3 with 8-aminoquinolines was not a viable
pathway to the target molecule. An alternative approach of
reductive amination was therefore sought, involving oxidation
of dibromide 3 to aldehyde 4. This aldehyde is a versatile
intermediate that may be used for functionalization of the dyes
with a variety of metal-binding sites, thus allowing ne-tuning
of the metal-binding properties and the electronic properties of
the sensors. Three dierent sensors with varying metal-binding
sites were synthesized by reductive amination. In all cases, a
Schi-base adduct was initially formed by reaction of aldehyde
4 with 2-methyl-8-aminoquinoline, 8-aminoquinoline, or 2-
(aminomethyl)pyridine followed by reduction with sodium
cyanoborohydride to aord the benzoresorun-based dyes
BRNO1, BRNO2, and BRNO3, respectively, in modest yield.
For the more nucleophilic 2-(aminomethyl)pyridine, direct
substitution of the benzylic bromine in 3 could be achieved in
methanol with potassium carbonate as the base, aording
BRNO3 with yield similar to that of the two-step route.
Photophysical Properties. The photophysical properties
of BRNO13 were investigated in 50 mM PIPES buer (100
mM KCl, pH 7) to reect physiological conditions. The results
are summarized in Table 1. At pH 7, BRNO1 and BRNO2
exhibit a very broad band in their absorption spectra, with a
maximum at 470 nm and a lower-energy shoulder at 570 nm.
Scheme 2. Synthetic Scheme for the Synthesis of Benzoresorun-Based Sensors BRNO13
Inorganic Chemistry Article
dx.doi.org/10.1021/ic302793w | Inorg. Chem. 2013, 52, 328532943288
The extinction coecients for the 470 nm maximum are only
5590 M
1
cm
1
for BRNO1 and 4390 M
1
cm
1
for BRNO2.
In contrast, the absorption spectrum of the 2-(aminomethyl)-
pyridine derivative BRNO3 is marked by a lower-energy band
centered at 563 nm of greater intensity (ε
563
= 11900 M
1
cm
1
; Figure 1). Upon excitation of BRNO1 and BRNO2 at
570 nm, a weak emission band (ϕ
BRNO1
= 0.055 ± 0.002;
ϕ
BRNO2
= 0.048 ± 0.004) is observed at 625 nm. These low
luminescence quantum yields are similar to those reported for
previously described NO sensors FL1 (ϕ = 0.077 ± 0.002)
27
and SNFL1 (ϕ = 0.027 ± 0.004)
30
and suggest a common
quenching eect caused by the nitrogen lone pair of the amine
in the metal-binding group. BRNO3, on the other hand,
exhibits stronger emission. Upon excitation at 563 nm, an
emission band at 625 nm is observed, and the photo-
luminescence quantum yield is 0.25. The signicantly higher
quantum yield and extinctio n coecient of BRNO3 in
comparison to the other derivatives demonstrate that the
metal-binding site can modify the electronic properties of the
ground and excited states of the dye. This observation suggests
that modifying the metal-binding sites of metal-based sensors is
a viable strategy for adjusting not only the binding anity but
also the photophysical properties of the system.
As for the uorescein-based FL dyes, the absorbance and
emission spectra of BRNO13 are sensitive to changes in pH
because of the proton-accepting properties of the metal-binding
sites and the dye itself. The pH dependence of the absorbance
and emission properties of the metal-free dyes was therefore
investi gated. Under basic conditions (pH 11), the three
compounds all display a strong broad absorbance feature
between 550 and 610 nm. This band, presumably arising from
the deprotonated sensors, is more intense than those observed
at pH 7. At low pH values, the absorption decays with
concomitant formation of a weaker feature centered at 470 nm
(Figure 1). The emission properties of the uorophores also
vary with pH. Maximum emission intensity was observed
between pH 7.5 and 9.5 for BRNO1 and BRNO2, whereas for
BRNO3, maximum emission occurs between pH 7.0 and 9.0.
The slightly dierent ranges for the maximum emission
intensity of the aminoquinoline-based (BRNO1 and BRNO2)
and (aminomethyl)pyridine-based (BRNO3) sen sors most
likely reect disparate pK
a
values associated with the dierent
metal-binding sites. Analysis of the integrated emission data as a
function of pH returned apparent pK
a
values of 6.5 and 9.6 for
BRNO1, 6.5 and 9.7 for BRNO2, and 5.9 and 9.6 for BRNO3
(Figure 2 and Table 1). These values are signicantly higher
than the pK
a
values reported for SNFL1 (4.9, 6.3, and 7.5),
30
indicating that the benzoresorun confers a higher degree of
basicitity to the molecules. The pH-depe ndent emission
properties of the compounds reveal that all dyes described
here exhibit high uorescence under physiological conditions,
with less than a 10% change in uorescence within 2 pH units
from pH 7.
DFT Calculations. To gain insight into the pH-dependent
photophysical properties of BRNO13, DFT calculations were
employed. Because BRNO1 and BRNO2 contain similar
aminoquinoline metal-binding sites, DFT calculations were
only carried out for BRNO1 and BRNO3 to compare how their
dierent metal-binding groups aect the properties of the
sensors. The geometries of BRNO1 and BRNO3, in both
Table 1. Photophysical Properties of the BRNO Derivatives
BRNO1 BRNO2 BRNO3
absorption
λ
max
(nm),
ε (M
1
cm
1
)
470,
5590 ± 300
470,
4390 ± 570
470, 5030 ± 630
570,
2280 ± 230
570,
2160 ± 210
563,
11900 ± 1100
emission
λ
max
(nm),
Φ (%)
623, 5.5 ± 0.2 623, 4.8 ± 0.4 625, 25 ± 4
brightness
(Φε,M
1
cm
1
) 125 103 2974
acid/base constants
pK
a,1
6.46 ± 0.05 6.49 ± 0.03 5.93 ± 0.05
pK
a,2
9.57 ± 0.14 9.75 ± 0.15 10.29 ± 0.37
Figure 1. UVvis absorption spectra of BRNO1 (top), BRNO2
(center), and BRNO3 (bottom) at dierent pH values.
Figure 2. Fluorescence spectrum of BRNO3 at dierent pH values
(left) and the pH dependence of the uorescence intensity (right).
Inorganic Chemistry Article
dx.doi.org/10.1021/ic302793w | Inorg. Chem. 2013, 52, 328532943289
neutral protonated and anionic deprotonated forms, were
optimized in the gas phase at the B3LYP/6-31g(d,p) level of
theory. Using these geometries, TDDFT calculations with an
implicit water solvation model were employed to simulate the
UVvis absorbance spectra and to investigate the nature of the
corresponding excited states. For the deprotonated anionic
form of BRNO1, a transition (S
2
) having a large oscillator
strength (f = 0.736) is predicted at 528 nm. This calculated
transition energy is 0.12 eV (30 nm) greater than that of the
experimentally observed absorbance, which has a maximum at
560 nm (Figure S4 in the SI). An EDDM depicting the charge
redistribution in this excited state is shown in Figure 3, where
green lobes correspond to holes and purple lobes to electrons.
On the basis of the EDDM, the excited state is assigned to a
mixed transition with components of both benzoresorun
ππ* and aminoquinoline-to-benzoresorun ligand-to-ligand
charge transfer (LLCT) character. The mixture of LLCT into
this excited state may give rise to the low observed emission
quantum yield. The S
1
state of this deprotonated anion occurs
at 616 nm with a low oscillator strength (f = 0.015). On the
basis of its EDDM, the nature of the S
1
state is identical with
that of the allowed S
2
state (Figure 3). For the neutral
protonated form of BRNO1, the main allowed transition (S
2
; f
= 0.531) blue shifts relative to that of the deprotonated form,
appearing at 471 nm. This calculation is in good agreement
with the experimentally observed transition centered at 470 nm
for BRNO1 measured at pH 3 in aqueous solutions (Figure S5
in the SI), The EDDM of this excited state reveals it to be
primarily benzoresorun ππ* in character. The S
1
excited
state of the protonated species exhibits a low oscillator strength
(f = 0.0003) and is therefore not expected to be an allowed
transition. The EDDM (Figure 3) of the S
1
state indicates that
it corresponds to an aminoquinoline-to-benzoresorun LLCT.
This dark charge-transfer excited state provides a plausible
nonradiative decay pathway for the ππ* S
2
state. This
hypothesis is consistent with the signicant decrease in
emission intensity of BRNO1 observed upon adjustment of
the solution pH from 11 to 3 (Figure 2). The S
1
excited state of
the deprotonated anionic form of BRNO3, which contains an
(aminomethyl)pyridine metal-binding site, is computed to
occur at 529 nm, with a large oscillator strength (f = 0.8182).
This calculated value is 0.23 eV higher in energy than that
measured experimentally from the absorbance spectrum of
BRNO3 in pH 11 aqueous solution (Figure S6 in the SI). The
EDDM of this excited state (Figure 4) reveals it to be a
benzoresorun ππ* transition. The pure ππ* character of
the lowest-energy transition for BRNO3 is in contrast to the
lowest-energy excited state of BRNO1, which is characterized
in part as a LLCT. The lack of this charge transfer, which is
expected to favor nonradiative emission, in the S
1
state of
BRNO3 is consistent with the higher photoluminescent
quantum yield of this sensor (Φ = 25%) compared to those
of BRNO1. The large oscillator strength (f = 0.5469) of the S
1
state suggests that this transition is allowed, whereas the smaller
oscillator strength (f = 0.0639) of S
2
indicates a smaller degree
of allowed character. The S
1
and S
2
excited states of the neutral,
protonated form of BRNO3 are separated by only 0.028 eV.
The computed absorbance maximum of 469 nm is in good
agreement with the value of 470 nm experimentally determined
for BRNO3 at pH 3 in aqueous solution (Figure S7 in the SI).
The nature of the S
1
and S
2
excited states is given by their
EDDMs, shown in Figure 4. The S
1
state is primarily a
benzoresorun ππ* transition, whereas the S
2
state originates
from charge transfer from the amine lone pair to the
benzoresorun π* orbital (nπ*). Of these two excited states,
only the S
1
state is expected to be emissive because it involves
only the uorescent benzoresorun unit. The small energy
dierence (0.058 eV) between the emissive S
1
and dark S
2
states renders these two states thermally accessible at room
temperature ( k
B
T = 0.0256 eV at 25 °C). Therefore, the
signicant emission quenching at low pH of BRNO3 (Figure 2)
arises from thermal population of the dark S
2
state. At higher
pH values, where BRNO3 is largely deprotonated, the
nonemissive S
2
state (Figure 4) lies 0.52 eV above the S
1
state and is not accessible at room temperature.
Metal-Binding Properties. Metal ions are endogenous to
biol ogical systems and can potentially inter act with and
inuence the emissive properties of metal-based sensors. We
therefore investigated the emission response of BRNO13in
the presence of several such metal ions. Upon addition of 1000
equiv of alkali or alkaline-earth metals, no signicant change in
uorescence intensity was observed for all three dyes (Figure
5). When late-rst-row transition metals were added, a small
decrease of uorescence intensity was observed. The largest
Figure 3. EDDMs of the two lowest-energy singlet excited states of
BRNO1 in both the anionic deprotonated (left) and neutral
protonated (right) forms. Green lobes represent holes, whereas purple
lobes are electrons.
Figure 4. EDDMs of the two lowest-energy singlet excited states of
BRNO3 in both the anionic deprotonated (left) and neutral
protonated (right) forms. Green lobes represent holes, whereas purple
lobes are electrons.
Inorganic Chemistry Article
dx.doi.org/10.1021/ic302793w | Inorg. Chem. 2013, 52, 328532943290
decrease in emission intensity occurred following addition of
Cu
2+
or Fe
3+
. Addition of the diamagnetic metal ions, Zn
2+
and
Cd
2+
, induced small increases in the emission of BRNO1 and
BRNO2, whereas BRNO3 displayed a slight decrease in
emission. The small response of the BRNO sensors to Zn
2+
is somewhat surprising because analogous monotopic uo-
rescein-based derivatives (QZ1) exhibit a 42-fold turn-on upon
Zn
2+
addition.
56
For uorescein-based zinc sensors, the zinc ion
serves to lower the energy of the nitrogen lone pairs in the
metal-binding sites, thereby inhibiting them from quenching
the uorescence by a PET mechanism.
57
The DFT calculations
presented above are consistent with the inability of the BRNO
sensors to turn-on with Zn
2+
. For BRNO1 and BRNO2, the
eect of zinc coordination on the energy of the the nitrogen
lone pair would have little impact on quenching charge transfer
from the aromatic π orbitals of the quinoline that is predicted
for t he S
1
and S
2
excited states. For BRNO3 in the
deprotonated anionic form, the nπ* transition is signicantly
higher in energy than the emissive benzoresorun ππ* and
therefore unable to quench emission, even in the metal-free
form. The lack of a turn-on response upon interaction with
Zn
2+
is an advantageous property of these sensors over rst-
generation analogues because no false-positive response of the
sensors to highly abundant endogenous Zn
2+
will occur.
Previously reported NO and HNO sensors utilize the Cu
2+
/
Cu
+
redox couple to modulate the uorescence response. In the
2+ oxidation state, the paramagnetic copper ion quenches the
emission of the uorophore.
27,33
Upon reduction of the copper
ion to the diamagnetic 1+ oxidation state by NO or HNO, the
emission is restored. To investigate the viability of this sensing
mechanism for the BRNO compounds, more detailed metal-
binding and photophysical studies were carried out in the
presence of Cu
2+
. Titration of the sensors with CuCl
2
revealed
a 1:1 binding stoichiometry (Figure S8 in the SI). Addition of 1
equiv of CuCl
2
resulted in a decrease in uorescence intensity
(Figure S8 in the SI), from which the Cu
2+
dissociation
constants (K
d
) were determined by emission titrations as 4470
± 50 (BRNO1), 400 ± 60 (BRNO2), and 18 ± 2nM
(BRNO3) (Figures S9 and S10 in the SI). These values are
consistent with the nature of the dierent metal-binding sites in
the three sensors. The chelating groups of BRNO1 and
BRNO2 dier only by the presence, in BRNO1, of a methyl
group ortho to the nitrogen atom of the quinoline ring in
BRNO2. This methyl group presumably destabilizes Cu
2+
binding due to steric crowding. This destabilization is reected
by a dissociation constant of BRNO1 that is 1 order of
magnitude higher than that of BRNO2. BRNO3, with its 2-
(aminomethyl)pyridine group, binds Cu
2+
most eectively. The
dissociation constant of the Cu
2+
-BRNO3 complex is more
than 1 order of magnitude smaller than that of BRNO2. The
larger Cu
2+
anity of BRNO3 may arise from the greater
exibility as well as the higher basicity of the secondary
nitrogen atom of the 2-(aminomethyl)pyridine unit by
comparison to the more rigid aminoquinoline metal-binding
sites in the other two sensors.
Reactivity with ROS and RNS. The response of our
copper-based sensors (CuBRNO13), assembled by treatment
of BRNO13 with 1 equiv of CuCl
2
under anaerobic
conditions, to dierent RNS and ROS was investigated. The
CuBRNO probes were treated with 500 equiv of the RNS or
ROS, and the emission response was recorded after 60 min
(Figure 6). The reaction with the oxidizing agents sodium
hypochlorite and hydrogen peroxide induced no signicant
change in emission for any of the three sensors. Peroxynitrite, a
strong oxidant, decreases the emission, probably owing to the
formation of nonuorescent oxidized resazurin derivatives.
Unexpectedly, the reaction with NO only led to 2.6- (BRNO1),
1.5- (BRNO2), and 1.7-fold (BRNO3) uorescence increases.
A similar response was observed when the NO-releasing SNAP
was added. Addition of Angelis salt, Na
2
N
2
O
3
, an HNO source,
generated a larger turn-on response, with a 4.8-fold increase in
uorescence intensity for CuBRNO1. Angelis salt decomposes
in aqueous solutions to release HNO and nitrite ions.
58
To
verify that nitrite was not causing the increase in emission, this
ion was added to the probes. No increase in uorescence was
observed, proving that HNO rather than NO
2
gives rise to the
turn-on response. On the basis of emission, these probes are
more eective for sensing HNO than NO. Furthermore, they
exhibit selectivity over other ROS and RNS tested here.
Because Fe
3+
, like Cu
2+
, quenches the emission of the BRNO
sensors, we investigated the response of the Fe
III
BRNO
complexes to ROS and RNS. These studies revealed little
Figure 5. Fluorescence response (F/F
0
) of the BRNO sensors upon
addition of 1000 equiv of metal ions.
Figure 6. Comparison of the selectivity for RNS/ROS for
[CuBRNO1]
+
, [CuBRNO2]
+
, and [CuBRNO3]
+
in 50 mM PIPES
buer (100 mM KCl, pH 7, 37 °C, 60 min) after addition of 500 equiv
of RNS/ROS. Excitation wavelength: 570 nm, BRNO1 and BRNO2;
563 nm, BRNO3. integrated uorescence emission from 590 to 800
nm.
Inorganic Chemistry Article
dx.doi.org/10.1021/ic302793w | Inorg. Chem. 2013, 52, 328532943291
utility of the Fe
III
BRNO system for sensing RNS and ROS
(Figure S11 in the SI).
Mechanism of HNO and NO Sensing. We previously
reported mechanistic investigations for a related NO-sensing
probe, CuFL1, which utilizes a 2-methyl-8-aminoquinoline
Cu
2+
-binding site like that in BRNO1 but with uorescein as
the uorophore.
35
The reaction of CuFL1 with NO reduces
copper from the 2+ to 1+ oxidation state, forming an N-
nitrosated uorescein derivative, FL1-NO, in the process. In
contrast to FL1 and CuFL1, FL1-NO is highly uorescent. The
sensing mechanism of our previously reported HNO sensor,
CuBOT1, has also been investigated.
33
Although this
compound does not react with NO, HN O reduces the
emission-quenching paramagnetic Cu
II
ion and induces a
turn-on response. As in these previous studies, we sought to
investigate the mechanisms by which CuBRNO1 responds to
both NO and HNO.
The reaction of BRNO1 with 1 equiv of CuCl
2
decreases the
emission intensity to one-fth of its original value, most likely
due to quenching eect of the photoexcited state by the
paramagnetic Cu
II
ion. An ESI-MS spectrum of freshly prepared
CuBRNO1 shows the molecular ion peak of [Cu
II
BRNO]
+
at
m/z 573.1 (calcd m/z 573.0). Addition of NO or SNAP to
CuBRNO1 induces a 2.6-fold emission turn-on, and the ESI-
MS spectrum revealed the presence of a molecular ion peak at
m/z 538.9, corresponding to the N-nitrosated species BRNO1-
NO (calcd m/z 539.0 for [BRNO1 + NO H]
+
). Reaction of
NOBF
4
with BRNO1 in acetonitrile aorded BRNO1-NO, as
evidenced by the same molecular ion peak in the ESI-MS
spectrum. Treatment of BRNO1 with NO gas in the absence of
copper, on the other hand, gave no reaction, thereby
demonstrating the importance of the Cu
II
ion as an electron
acceptor. An aliquot from the reaction mixture of NOBF
4
and
BRNO1 in MeCN was diluted into PIPES buer after 1 h, and
the emission spectrum was recorded. Unexpectedly, a 1.8-fold
decrease in uorescence intensity compared to that of BRNO1
was observed (Figure S12 in the SI). Therefore, in contrast to
FL1-NO and FL1, BRNO1-NO is less emissive than BRNO1.
The relative order of emission intensity is BRNO1 > BRNO1-
NO > CuBRNO1. The low observed turn-on response induced
upon trea tment of CuBRNO1 with NO is therefore a
consequence of the intrinsically low emission intensity of
BRNO1-NO. This result is not predicted by TDDFT
calculations. The lowest-energy singlet excited state of
BRNO1-NO is a benzoresorun ππ* transition with a large
oscillator strength of 0.701 (Figure S13 in the SI). The lack of
charge-transfer character in this excited state suggests that it
should be highly emissive, or at least more so than BRNO1.
Therefore, additional nonradiative decay pathways must
operate in BRNO1-NO. Decay by internal conversion, perhaps
through the newly introduced NNO vibrational mode, is one
possible explanation.
As discussed above, treatment of CuBRNO1 with Angelis
salt as an HNO source induces a 4.8-fold increase in emission
after 2 min (Figure S14 in the SI). This 4.8-fold turn-on
eectively corresponds to restoration of the uorescence of
copper-free BRNO1 because removal of copper from
CuBRNO1 with ethylenediaminetetraacetic acid generates the
same response. After the 4.8-fold turn-on, subsequent additions
of Angelis salt produced no further increases in emission
intensity. Additionally, the emission properties of copper-free
BRNO1 do not change in response to Angelis salt. By analogy
to the mechanism of HNO sensing for CuBOT1,
33
we
hypothesized that HNO reduces the paramagnetic Cu
II
center
in CuBRNO1 to restore the emission of BRNO1. To
investigate this possibility, the reaction of CuBRNO1 with
Angelis salt was monitored by EPR spectroscopy. For
CuBRNO1, an axial signal is observed in the EPR spectrum
due to the S =
1
/
2
Cu
II
ion (Figure S15 in the SI). Addition of
Angelis salt to this solution led to the disappearance of this
signal (Figure S15 in the SI). This observation is consistent
with reduction of Cu
II
by HNO to form a diamagnetic, EPR-
silent Cu
I
ion. Although this EPR study veries that Cu
II
is
reduced by HNO, it does not establish whether the newly
formed Cu
I
ion remains bound and whether or not this ion can
aect the photophysical properties of the sensor. An ESI-MS
spectrum of the reduced EPR solution revealed only the
presence of free BRNO1, suggesting that reduction of copper
leads to its dissociation. A 1:1 mixture of the Cu
I
source
[Cu(CH
3
CN)
4
]PF
6
and BRNO1 in acetonitrile, however, does
show evidence for the formation of a Cu
I
complex judging by
the observation of a molecular ion peak corresponding to
[CuBRNO + H]
+
in the ESI-MS spectrum (m/z 575.9; calcd
m/z 576.0). The uorescence spectrum of this mixture in
aqueous buer does not show Cu
I
-induced changes in the
emission intensity of BRNO1. Therefore, the Cu
I
ion either
does not bind strongly to BRNO1 or, if it does, it has little
eect on the emission intensity, as for other diamagnetic metal
ions tested (Figure 5).
Because the Cu
II
/Cu
I
redox couple is crucial for mediating
the detection of HNO in this system, the electrochemical
properties of CuBRNO13 were investigated by cyclic
voltammetry in acetonitrile. Quasi-reversible reduction features
occur at 90, 150, and 10 mV vs Fc/Fc
+
for CuBRNO13,
respectively (Figure S16 in the SI). These values are close to
that of the Cu
II
/Cu
I
couple of CuCl
2
under the same conditions
(130 mV vs Fc/Fc
+
). The reduction potential corresponding to
the HNO/NO couple is 0.36 V in water.
59
In acetonitrile,
conditions similar to those used for our copper complexes, NO
is reduced irreversibly with an onset potential of approximately
1 V versus the Fc/Fc
+
couple. Therefore, all three sensors are
thermodynamically capable of oxidizing HNO to NO. To
conrm the formation of NO gas, EI-MS measurements were
carried out on the head space of a reaction mixture of
CuBRNO1 and Angelis salt. These measurements conrmed
the formation of NO gas (Figure S17 in the SI), consistent with
our hypothesis that HNO reduces the CuBRNO sensors.
NO and HNO Detection in Living Cells. Fluorescence
imaging studies were carried out to investigate whether the
CuBRNO sensors can detect NO and HNO in living cells, as
they do in cuvettes. Only CuBRNO1 and CuBRNO3 were
used for these studies because they contain signicantly
dierent metal-binding sites and were therefore expected to
exhibit dierent intracellular properties. Human cervical cancer
cells, HeLa, and murine macrophage cells, Raw 264.7, were
investigated. The Raw 264.7 cells allow for stimulation of
endogenous NO production by iNOS following treatment with
LPS and INF-γ.
60
Treatment of both cell types with 2.5 μM
CuBRNO1 or CuBRNO3 resulted only in negligible emission
in the absence of an analyte. In contrast, treatment of both cell
lines with 500 equiv of Angelis salt in the presence of
CuBRNO1 or CuBRNO3 produced a distinct enhancement in
emission after 15 min of incubation (Figure 7). In all cases, the
observed increase in emission was comparable to that found
when similar experiments were performed in a cuvette,
demonstrating the suitability of the sensors to detect HNO
Inorganic Chemistry Article
dx.doi.org/10.1021/ic302793w | Inorg. Chem. 2013, 52, 328532943292
in live cells. To assess their ability to detect intracellular NO, we
induced NO production by iNOS in Raw 264.7 cells by adding
LPS and INF-γ .
60
Raw 264.7 macrophages generate micromolar
concentrations of NO following treatment with endotoxins and
cytokines.
61
Accordingly, these cells are ideal for studying the
response of NO probes to endogenously produced NO. After
stimulation of NO production, 1.4- and 1.2-fold increases in
emission intensity were observed for CuBRNO1 and
CuBRNO3, respectively (Figure 7). As a control experiment,
Raw 264.7 macrophages were treated with 500 equiv of GSNO.
This experiment provides a direct comparison of the turn-on
response of CuBRNO1 and CuBRNO2 induced by endoge-
nous and exogenous NO sources within the same cell line. In
agreement with our ndings for endogenously produced NO,
treatment of cells containing our sensors with GSNO resulted
in a similar, small enhancement of emission for both sensors
(Figure 7). Representative cell images are presented in Figures
8 and S18 in the SI. Colocalization experiments, performed
with the sensor in the presence of Angelis salt, indicated that
both CuBRNO1 and CuBRNO3 localize to the endoplasmic
reticulum (ER). The spatial emission overlap between the
BRNO sensors and the ER tracker dye is marked by a Pearson
correlation coecient
62
of 0.61 for CuBRNO1 and 0.74 for
CuBRNO3 (Figures S19 and S20 and Table S13 in the SI).
This observation is in good agreement with literature reports
that resorun dyes localize in the ER.
63
In addition to the ER, a
signicant degree of membrane staining was observed.
CONCLUSION
We have presented three novel benzoresorun-based NO and
HNO uorescent sensors. The use of benzoresorun as the
uorophore gives rise to emission at 625 nm, a region favorable
for biological imaging studies. In their copper-free forms,
BRNO1 and BRNO2 exhibit only weak uorescence. BRNO3,
which contains a 2-(aminomethyl)pyridine metal-binding site,
has a signicantly higher photoluminescent quantum yield
owing to the absence of low-energy charge-transfer excited
states. In the Cu
II
-bound form, these complexes serve as
selective sensors for NO and HNO over other ROS and RNS
with modest increases in emission intensity. The observation
that higher turn-on responses for these sensors occur for HNO
rather than NO is an unexpected result based on the structural
similarities of the metal- and NO-reacting sites with respect to
those in previously reported NO sensors in the FL and SNFL
series. These results suggest that, in addition to the chemistry
occurring at the metal-binding site, the energy levels of the
uorophore unit also play a signicant role in modulating the
emission response and must be considered carefully. Moreover,
they indicate that a secondary amine in the copper-binding site
is necessary but not sucient for selectively sensing NO,
whereas the best HNO sensor is obtained by blocking this
secondary amine and further tuning the electronic properties.
Mechanistic studies revealed that Cu
II
reduction is necessary for
both HNO or NO turn-on. The uorescence turn-on observed
with the Cu
II
BRNO probes reveals that the redox potentials of
the complexes are largely inuenced by the uorescent dye,
which participates in metal coordination, and are only slightly
inuenced by the dierent amine chelating moieties. The metal-
binding anity inuences uorescence turn-on after addition of
NO or HNO. There is a lower uorescence turn-on for both
HNO and NO with tighter binding of Cu
II
. Despite the small
turn-on response to NO, these sensors are quite eective at
detecting NO and HNO in both the cuvette and living cells.
ASSOCIATED CONTENT
*
S
Supporting Information
NMR data of all compounds, as well as crystallographic data of
compounds 2 and 3, additional DFT results, Cu
II
-binding
studies, RNS/ROS studies with [FeBRNO]
2+
, time dependency
study of HNO turn-on, EPR data, cyclic voltammetry data,
headspace EI-MS data, and intracellular localization experi-
ments. This material is available free of charge via the Internet
at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*E-mail: lippard@mit.edu.
Notes
The authors declare no competing nancial interest.
Figure 7. Quantied uorescence turn-on in cells after treatment with
Angelis salt as well as NO-releasing GSNO and stimulation of iNOS
by LPS/INF- γ in the presence of in situ generated [CuBRNO1]
+
and
[CuBRNO3]
+
.
Figure 8. Fluorescence imaging of HNO in HeLa and Raw 264.7 cells.
For each set, the top image corresponds to treatment of cells with the
uorescent probe. The bottom image corresponds to cells treated with
Angelis salt. (left) DIC images. (right) Fluorescence images. Scale bar
=25μm.
Inorganic Chemistry Article
dx.doi.org/10.1021/ic302793w | Inorg. Chem. 2013, 52, 328532943293
ACKNOWLEDGMENTS
This work was supported the National Science Foundation
(Grant CHE-0611944 to S.J.L.). Instrumentation in the MIT
DCIF is maintained with funding from NIH Grant
1S10RR13886-01. U.-P.A. thanks the Alexander von Humboldt
Foundation for a postdoctoral fellowship. J.J.W. is a grateful
recipient of a David H. Koch Graduate Fellowship. Daniel J.
Graham and Dr. Amit Majumdar are thanked for assistance
with headspace EI-MS experiments and X-ray crystallography,
respectively.
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