Detection of Nitric Oxide and Nitroxyl with Benzoresorufin-Based Fluorescent Sensors
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 Benzoresoruﬁn-Based
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
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.
Reactive nitrogen (RNS) and oxygen species (ROS) have
numerous biological consequences.
Among these species,
nitric oxide (NO), initially identiﬁed as an endothelial-derived
has a broad variety of biological regulatory
and signaling functions.
Biological NO is generated by
L-arginine to L-citruline by a class of enzymes
known as nitric oxide synthases (NOS).
Among its many
functions, NO plays important roles in the control of smooth
muscle relaxation and vasodilation,
platelet aggregation in
vascular endothelial cells,
of the immune response by macrophages.
Recently, it was
suggested that nitroxyl (HNO), the one-electron-reduced
congener of NO, is formed by NOS via oxidative degradation
Some recent in vitro studies using HNO-
releasing molecules demonstrated that HNO increases the
contractility of heart cells,
leads to vasorelaxation in muscle
and decreases platelet aggregation.
these ﬁndings suggest that HNO also plays a pivotal role in
In order to advance our insight into the physiological and
pathological roles of HNO and NO, our group has focused on
designing ﬂuorescent sensors that selectively respond to these
small molecules and aﬀord both sp atial and tem poral
information regarding the natural occurrence of these species
at the cellular level. Various NO sensors, including o-
diaminoﬂ uore scein, o-diaminonaphthalene, o-diam inocya-
luminescent lanthanide complexes,
detect NO in the presence of
oxygen. Sensors for HNO include metalloporphyrins,
In contrast to these reagents, copper-based
ﬂuorescent probes like CuFL1,
developed by our group (Scheme 1) and others
direct, selective, and fast detection of NO both in vitro and in
vivo. We have also recently described a selective HNO sensor,
The emission intensity of these copper-based
sensors is modulated by the oxidation state of the copper ion,
which, in turn, can be modiﬁed 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
ions that dissociate from the
The copper complex CuBOT1, on the other
hand, reacts selectively with HNO, leading to reduction of the
ion and ﬂuorescence enhancement.
diﬀerent 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
© 2013 American Chemical Society 3285 dx.doi.org/10.1021/ic302793w | Inorg. Chem. 2013, 52, 3285−3294
former, which is the preferred site of attack on CuFL by NO.
A limitation of both ﬂuorescein- 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
In our continuing eﬀorts to design improved sensors for NO
and HNO, we have functionalized a benzoresoruﬁn ﬂuorophore
with a Cu-binding site that contains a secondary amine, and we
investigated its ability to detect ROS and RNS. Resoruﬁn dyes
emit at wavelengths >600 nm with Stokes shifts up to 60 nm.
Accordingly, sensors based on resoruﬁn 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 benzoresoruﬁn-based sensors are
described herein. As with our previous systems, the Cu
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 benzoresoruﬁn-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
atmosphere using standard Schlenk techniques.
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
H signals from the deuterated
solvent and are reported in parts per million (ppm). 2-Methyl-4-
nitrosoresorcinol was synthesized by a literature procedure.
compounds were obtained from commercial vendors and used without
further puriﬁcation. 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). UV−vis spectra were recorded with a Varian
Cary 1E spectrometer at 25 °C. Fluorescence spectra were obtained on
a Quanta Master 4 L-format scanning spectroﬂuorimeter (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 (0.015−0.040 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 ﬁltration, 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 puriﬁcation. ESI-MS. Calcd for
: 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 ﬁltration 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
H NMR (CDCl
, 300 MHz, ppm): δ 8.71−8.67
(1H, m), 8.30−8.27 (1H, m), 7.78−7.73 (2H, m), 7.68 (1H, d,
Hz), 7.07 (1H, d,
J = 9 Hz), 6.46 (1H, s), 2.39 (3H, s), 2.27 (3H, s).
C NMR (CDCl
, 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
Found: 320.2. Mp: 234−235 °C.
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
atmosphere. Acetic acid (700 μL) was added, and the solution was
heated at 60 °C until
H NMR spectroscopy revealed full conversion
to the ﬁnal molecule (6−10 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
aﬀorded an orange solid, which was washed with diethyl ether/pentane
(1:1). The solid was collected by ﬁltration and dried under vacuum to
aﬀord 3.6 g (92%) of an orange solid.
H NMR (CDCl
, 300 MHz,
ppm): δ 8.71−8.69 (1H, m), 8.38−8.35 (1H, m), 7.86−7.76 (3H, m),
7.26−7.23 (1H, m), 4.73 (2H, s), 2.46 (3H, s). ESI-MS. Calcd for
: 497.9. Found: 497.8. Mp: 221 °C.
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 ﬁltration, washed with water, and dried under vacuum.
The crude material was puriﬁed 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 aﬀord 382 mg (29%) of a dark-orange solid.
H NMR (CDCl
MHz, ppm): δ 12.10 (1H, s), 10.71 (1H, s), 8.70−8.67 (1H, m),
8.40−8.37 (1H, m), 8.01 (1H, d,
J = 9 Hz), 7.85−7.74 (2H, m), 7.02
J = 9 Hz). ESI-MS. Calcd for [C
Found: 369.9. Mp: 254 °C.
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 ﬁltered
oﬀ and washed with copious amounts of water and then dichloro-
methane/hexane (10 mL, 1:1). Subsequently, the solid was dried
under vacuum to aﬀord 14.2 mg (41%) of a dark-red compound.
OD, 500 MHz, ppm): δ 12.33 (1H, s), 9.41 (1H, d,
Hz), 9.03 (1H, d,
J = 6 Hz), 8.95−8.91 (1H,m), 8.71−8.68 (1H, m),
8.64−8.61 (1H, m), 8.55 (1H, d,
J = 6 Hz), 8.20 (1H, d,
J = 6 Hz),
8.14−8.11 (1H, m), 7.95 (1H, d,
J = 6 Hz), 7.90 (1H, d,
J = 6 Hz),
7.87 (1H, d,
J = 6 Hz), 5.59 (2H, s), 3.46 (3H, s). ESI-HRMS. Calcd
: 510.0453. Found: 510.0439. Mp: 184 °C
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 ﬁltered oﬀ and washed with water. Drying under
vacuum aﬀorded 10.6 mg (39%) of a dark-purple solid.
, 500 MHz, ppm): δ 8.70 (1H, d,
J = 6 Hz), 8.57 (1H, d,
= 6 Hz), 8.22 (1H, d,
J = 6 Hz), 8.18 (1H, d,
J = 6 Hz), 7.83−7.80
(1H, m), 7.72−7.69 (1H, m), 7.61 (1H, d,
J = 6 Hz), 7.48−7.44 (1H,
m), 7.36−7.33 (1H, m), 7.17 (1H, d,
J = 6 Hz), 7.05 (1H, d,
Inorganic Chemistry Article
dx.doi.org/10.1021/ic302793w | Inorg. Chem. 2013, 52, 3285−32943286
Hz), 6.83 (1H, d,
J = 6 Hz), 4.73 (2H, s), 2.62 (3H, s). ESI-HRMS.
Calcd for [C
: 496.0297. Found: 496.0293. Mp:
256 °C (dec).
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 ﬁltered oﬀ, washed with water (10
mL), and dried under vacuum to aﬀord 21.4 mg (43%) of a purple
H NMR (CD
OD, 500 MHz, ppm): δ 8.72 (1H, d,
J = 6 Hz),
8.41 (1H, d,
J = 6 Hz), 8.34 (1H, d,
J = 6 Hz), 7.78−7.73 (2H, m),
7.67−7.63 (1H, m), 7.60 (1H, d,
J = 6 Hz), 7.51 (1H, d,
J = 6 Hz),
7.25−7.24 (1H, m), 6.79 (1H, d,
J = 6 Hz), 4.23 (2H, s), 4.03 (2H,
s). ESI-HRMS. Calcd for [C
: 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
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 Angeli’s salt. The cell was connected to a He gas-ﬂow 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
electrode in acetonitrile. The solvent contained n-Bu
) (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
through the solutions for 5 min prior to the measurements, and
the solutions were kept under N
for the duration of the study. All data
were referenced to the Fc/Fc
couple as an internal standard (E
+405 mV vs Ag/AgNO
X-ray Data Collection and Structure Solution Reﬁnement.
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 ﬁber loop, and
placed in a cold, gaseous N
stream on a Bruker APEX CCD X-ray
diﬀractometer performing φ and ω scans at 100(2) K. Diﬀraction
intensities were measured using graphite-monochromated Mo Kα
radiation (λ = 0.71073 Å). Data collection, indexing, initial cell
reﬁnements, frame integration, and ﬁ nal cell r eﬁnements were
accomplished with the program APEX2.
were applied using the program SADABS.
The structure was solved
by direct methods using SHELXS
and reﬁned against F
on all data
by full-matrix least squares with SHELXL-97
reﬁnement strategies. Crystallographic data collection and reﬁnement
parameters are presented in Tables S1 and S2 in the Supporting
Density Functional Theory (DFT) Calculations. All calculations
were performed with the Gaussian 03 program package
Geometry optimizations were carried out in the
gas phase using the 6-31g(d,p) basis set.
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 eﬀects were
modeled with the conductor-like polarizable continuum model for
Electron-density diﬀerence maps (EDDMs) and calculated
UV−vis absorbance spectra were generated with the program
Tables S3−S12 in the SI contain the coordinates of
optimized structures and a summary of the lowest-energy singlet
Spectroscopic Materials and Methods. Piperazine-N,N′-bis(2-
ethanesulfonic acid) (PIPES; Calbiochem) and potassium chloride
(99.999%, Aldrich) were used to prepare buﬀered solutions (50 mM
PIPES, 100 mM KCl, pH 7.0) in deionized water with resistivity ≥18
, obtained using a Milli-Q water puriﬁcation system. Nitric
oxide (NO) was purchased from Airgas and puriﬁed as previously
S-Nitroso-N-acetylpenicillamine (SNAP), S-nitroso-L-
glutathione (GSNO), sodium peroxynitrite, and Angeli’s salt were
purchased from Cayman Chemical and stored at −80 °C when not in
use. NO and the other RNS were injected into buﬀered solutions via a
gastight syringe. CuCl
O (99+%, Alfa Aesar) was used to prepare
7 mM CuCl
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 BRNO1−3 were determined in 50 mM
PIPES buﬀer (100 mM KCl, pH 7) using resoruﬁn(λ
= 585 nm, λ
= 572 nm, and Φ = 0.74
) as the reference in water (pH 9.5).
Cell Culture. HeLa cells and Raw 264.7 murine macrophages were
cultured in Dulbecco’s modiﬁed Eagle medium (DMEM; Cellgro,
MediaTek, Inc.), supplemented with 10% fetal bovine serum (FBS;
HyClone), 1% penicillin−streptomycin, 1% sodium pyruvate (Cellgro,
MediaTek, Inc.), 1% MEM nonessential amino acids (Sigma), and 1%
L-glutamine. For imaging studies, cells were grown to conﬂuence,
passaged, and plated onto poly-
D-lysine-coated plates. The plates,
containing 2 mL of DMEM, were incubated at 37 °C with 5% CO
at least 12 h. The media were removed, the cells were washed with 5
mL of PBS buﬀer, and solutions of the ﬂuorescent probes in 2 mL of
fresh DMEM were added. For all cell studies, the Cu
generated in situ by combining a stock solution of the ﬂuorescent
sensor and CuCl
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 495−4950 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 Angeli’s 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 Angeli’s salt.
Prior to ﬂuorescence 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 epiﬂuorescence microscope equipped
with an EM-CCD camera (Hamamatsu) and an X-Cite 120 metal
halide lamp (EXFP). Diﬀerential interference contrast (DIC) and
ﬂuorescence 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
ﬂuorescent images were deconvoluted and background-corrected.
Images were measured before and after addition of Angeli’s salt or
NO-releasing agent. The image before addition of Angeli’s salt or NO-
releasing agent was taken as the background level.
RESULTS AND DISCUSSION
Design and Synthesis of Benzoresoruﬁn-Based
Sensors. An ongoing goal of our laboratory is the development
of ﬂuorescent sensors for NO and HNO. We found that an
eﬀective strategy for designing such species is to couple a metal-
Inorganic Chemistry Article
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binding site with a ﬂuorophore.
In the “oﬀ” state of the
sensor, a paramagnetic ion in the metal-binding site quenches
the emission of the ﬂuorescent 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.
Our most successful NO
sensors, which operate nicely in cellular environments, contain
a 2-methyl-8-aminoquinoline metal-binding site and either a
ﬂuorescein (FL series) or a seminaphthoﬂuorescein (SNFL
series) dye as ﬂuorescent reporters.
is used as the
paramagnetic ﬂuorescence quencher. The methyl group in the
2 position of the aminoquinoline gives rise to only moderate
-binding a ﬃnities, characterized by K
values of approx-
imately 1.5 μM. In this study, we sought to change both the
ﬂuorescent reporter and metal-binding site. Benzoresoruﬁn,
which emits in the red, was chosen as the ﬂuorophore, and in
addition to our standard 2-methyl-8-aminoq uinoline Cu
binding site, we investigated 8-aminoquinoline and 2-
The overall synthetic strategy for preparing the desired
benzoresoruﬁn-based sensors is shown in Scheme 2. This
synthetic approach is general and therefore oﬀers a viable
pathway to a wide variety of monofunctionalized benzoresor-
uﬁn dyes. Following a similar procedure for the synthesis of
the reaction of 2 -methyl-4-nitro-
and 1,3-dihydroxynaphthalene aﬀorded 1, which
is readily puriﬁed in high yield by formation of the acetyl ester
derivative 2. The molecular structure of this compound, as
determined by single-crystal X-ray diﬀraction, is presented in
Figure S1 in the SI. To enable functionalization of the
benzoresoruﬁn 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 veriﬁed by single-
crystal X-ray analysis (Figure S2 in the SI). The formation of 3
from 2 was monitored by
H NMR spectroscopy, revealing that
bromination occurs ﬁrst 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
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 ﬁne-tuning
of the metal-binding properties and the electronic properties of
the sensors. Three diﬀerent sensors with varying metal-binding
sites were synthesized by reductive amination. In all cases, a
Schiﬀ-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 aﬀord the benzoresoruﬁn-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, aﬀording
BRNO3 with yield similar to that of the two-step route.
Photophysical Properties. The photophysical properties
of BRNO1−3 were investigated in 50 mM PIPES buﬀer (100
mM KCl, pH 7) to reﬂect 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 Benzoresoruﬁn-Based Sensors BRNO1−3
Inorganic Chemistry Article
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The extinction coeﬃcients for the 470 nm maximum are only
for BRNO1 and 4390 M
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
= 11900 M
; Figure 1). Upon excitation of BRNO1 and BRNO2 at
570 nm, a weak emission band (ϕ
= 0.055 ± 0.002;
= 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)
and SNFL1 (ϕ = 0.027 ± 0.004)
and suggest a common
quenching eﬀect 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 signiﬁcantly higher
quantum yield and extinctio n coeﬃcient 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 aﬃnity but
also the photophysical properties of the system.
As for the ﬂuorescein-based FL dyes, the absorbance and
emission spectra of BRNO1−3 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 ﬂuorophores 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 diﬀerent ranges for the maximum emission
intensity of the aminoquinoline-based (BRNO1 and BRNO2)
and (aminomethyl)pyridine-based (BRNO3) sen sors most
likely reﬂect disparate pK
values associated with the diﬀerent
metal-binding sites. Analysis of the integrated emission data as a
function of pH returned apparent pK
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 signiﬁcantly higher
than the pK
values reported for SNFL1 (4.9, 6.3, and 7.5),
indicating that the benzoresoruﬁn 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 ﬂuorescence under physiological conditions,
with less than a 10% change in ﬂuorescence within 2 pH units
from pH 7.
DFT Calculations. To gain insight into the pH-dependent
photophysical properties of BRNO1−3, 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
diﬀerent metal-binding groups aﬀect the properties of the
sensors. The geometries of BRNO1 and BRNO3, in both
Table 1. Photophysical Properties of the BRNO Derivatives
BRNO1 BRNO2 BRNO3
5590 ± 300
4390 ± 570
470, 5030 ± 630
2280 ± 230
2160 ± 210
11900 ± 1100
623, 5.5 ± 0.2 623, 4.8 ± 0.4 625, 25 ± 4
) 125 103 2974
6.46 ± 0.05 6.49 ± 0.03 5.93 ± 0.05
9.57 ± 0.14 9.75 ± 0.15 10.29 ± 0.37
Figure 1. UV−vis absorption spectra of BRNO1 (top), BRNO2
(center), and BRNO3 (bottom) at diﬀerent pH values.
Figure 2. Fluorescence spectrum of BRNO3 at diﬀerent pH values
(left) and the pH dependence of the ﬂuorescence intensity (right).
Inorganic Chemistry Article
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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
UV−vis absorbance spectra and to investigate the nature of the
corresponding excited states. For the deprotonated anionic
form of BRNO1, a transition (S
) 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 benzoresoruﬁn
π−π* and aminoquinoline-to-benzoresoruﬁn 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
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
state is identical with
that of the allowed S
state (Figure 3). For the neutral
protonated form of BRNO1, the main allowed transition (S
= 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 benzoresoruﬁn π−π* in character. The S
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
state indicates that
it corresponds to an aminoquinoline-to-benzoresoruﬁn LLCT.
This dark charge-transfer excited state provides a plausible
nonradiative decay pathway for the π−π* S
hypothesis is consistent with the signiﬁcant decrease in
emission intensity of BRNO1 observed upon adjustment of
the solution pH from 11 to 3 (Figure 2). The S
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
benzoresoruﬁn π−π* transition. The pure π−π* 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
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
state suggests that this transition is allowed, whereas the smaller
oscillator strength (f = 0.0639) of S
indicates a smaller degree
of allowed character. The S
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
excited states is given by their
EDDMs, shown in Figure 4. The S
state is primarily a
benzoresoruﬁn π−π* transition, whereas the S
from charge transfer from the amine lone pair to the
benzoresoruﬁn π* orbital (n−π*). Of these two excited states,
only the S
state is expected to be emissive because it involves
only the ﬂuorescent benzoresoruﬁn unit. The small energy
diﬀerence (0.058 eV) between the emissive S
and dark S
states renders these two states thermally accessible at room
temperature ( k
T = 0.0256 eV at 25 °C). Therefore, the
signiﬁcant emission quenching at low pH of BRNO3 (Figure 2)
arises from thermal population of the dark S
state. At higher
pH values, where BRNO3 is largely deprotonated, the
state (Figure 4) lies 0.52 eV above the S
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
inﬂuence the emissive properties of metal-based sensors. We
therefore investigated the emission response of BRNO1−3in
the presence of several such metal ions. Upon addition of 1000
equiv of alkali or alkaline-earth metals, no signiﬁcant change in
ﬂuorescence intensity was observed for all three dyes (Figure
5). When late-ﬁrst-row transition metals were added, a small
decrease of ﬂuorescence 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
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decrease in emission intensity occurred following addition of
. Addition of the diamagnetic metal ions, Zn
, 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
is somewhat surprising because analogous monotopic ﬂuo-
rescein-based derivatives (QZ1) exhibit a 42-fold turn-on upon
For ﬂuorescein-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 ﬂuorescence by a PET mechanism.
The DFT calculations
presented above are consistent with the inability of the BRNO
sensors to turn-on with Zn
. For BRNO1 and BRNO2, the
eﬀect 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
excited states. For BRNO3 in the
deprotonated anionic form, the n−π* transition is signiﬁcantly
higher in energy than the emissive benzoresoruﬁn π−π* and
therefore unable to quench emission, even in the metal-free
form. The lack of a turn-on response upon interaction with
is an advantageous property of these sensors over ﬁrst-
generation analogues because no false-positive response of the
sensors to highly abundant endogenous Zn
Previously reported NO and HNO sensors utilize the Cu
redox couple to modulate the ﬂuorescence response. In the
2+ oxidation state, the paramagnetic copper ion quenches the
emission of the ﬂuorophore.
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
. Titration of the sensors with CuCl
a 1:1 binding stoichiometry (Figure S8 in the SI). Addition of 1
equiv of CuCl
resulted in a decrease in ﬂuorescence intensity
(Figure S8 in the SI), from which the Cu
) 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 diﬀerent metal-binding sites in
the three sensors. The chelating groups of BRNO1 and
BRNO2 diﬀer 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
binding due to steric crowding. This destabilization is reﬂected
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
most eﬀectively. The
dissociation constant of the Cu
-BRNO3 complex is more
than 1 order of magnitude smaller than that of BRNO2. The
aﬃnity of BRNO3 may arise from the greater
ﬂexibility 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 (CuBRNO1−3), assembled by treatment
of BRNO1−3 with 1 equiv of CuCl
conditions, to diﬀerent 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 signiﬁcant
change in emission for any of the three sensors. Peroxynitrite, a
strong oxidant, decreases the emission, probably owing to the
formation of nonﬂuorescent oxidized resazurin derivatives.
Unexpectedly, the reaction with NO only led to 2.6- (BRNO1),
1.5- (BRNO2), and 1.7-fold (BRNO3) ﬂuorescence increases.
A similar response was observed when the NO-releasing SNAP
was added. Addition of Angeli’s salt, Na
, an HNO source,
generated a larger turn-on response, with a 4.8-fold increase in
ﬂuorescence intensity for CuBRNO1. Angeli’s salt decomposes
in aqueous solutions to release HNO and nitrite ions.
verify that nitrite was not causing the increase in emission, this
ion was added to the probes. No increase in ﬂuorescence was
observed, proving that HNO rather than NO
gives rise to the
turn-on response. On the basis of emission, these probes are
more eﬀective for sensing HNO than NO. Furthermore, they
exhibit selectivity over other ROS and RNS tested here.
, like Cu
, quenches the emission of the BRNO
sensors, we investigated the response of the Fe
complexes to ROS and RNS. These studies revealed little
Figure 5. Fluorescence response (F/F
) of the BRNO sensors upon
addition of 1000 equiv of metal ions.
Figure 6. Comparison of the selectivity for RNS/ROS for
, and [CuBRNO3]
in 50 mM PIPES
buﬀer (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 ﬂuorescence emission from 590 to 800
Inorganic Chemistry Article
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utility of the Fe
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
-binding site like that in BRNO1 but with ﬂuorescein as
The reaction of CuFL1 with NO reduces
copper from the 2+ to 1+ oxidation state, forming an N-
nitrosated ﬂuorescein derivative, FL1-NO, in the process. In
contrast to FL1 and CuFL1, FL1-NO is highly ﬂuorescent. The
sensing mechanism of our previously reported HNO sensor,
CuBOT1, has also been investigated.
compound does not react with NO, HN O reduces the
emission-quenching paramagnetic Cu
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
emission intensity to one-ﬁfth of its original value, most likely
due to quenching eﬀect of the photoexcited state by the
ion. An ESI-MS spectrum of freshly prepared
CuBRNO1 shows the molecular ion peak of [Cu
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
with BRNO1 in acetonitrile aﬀorded 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
ion as an electron
acceptor. An aliquot from the reaction mixture of NOBF
BRNO1 in MeCN was diluted into PIPES buﬀer after 1 h, and
the emission spectrum was recorded. Unexpectedly, a 1.8-fold
decrease in ﬂuorescence 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 benzoresoruﬁn π−π* 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
As discussed above, treatment of CuBRNO1 with Angeli’s
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
eﬀectively corresponds to restoration of the ﬂuorescence 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 Angeli’s salt produced no further increases in emission
intensity. Additionally, the emission properties of copper-free
BRNO1 do not change in response to Angeli’s salt. By analogy
to the mechanism of HNO sensing for CuBOT1,
hypothesized that HNO reduces the paramagnetic Cu
in CuBRNO1 to restore the emission of BRNO1. To
investigate this possibility, the reaction of CuBRNO1 with
Angeli’s salt was monitored by EPR spectroscopy. For
CuBRNO1, an axial signal is observed in the EPR spectrum
due to the S =
ion (Figure S15 in the SI). Addition of
Angeli’s salt to this solution led to the disappearance of this
signal (Figure S15 in the SI). This observation is consistent
with reduction of Cu
by HNO to form a diamagnetic, EPR-
ion. Although this EPR study veriﬁes that Cu
reduced by HNO, it does not establish whether the newly
ion remains bound and whether or not this ion can
aﬀect 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
and BRNO1 in acetonitrile, however, does
show evidence for the formation of a Cu
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 ﬂuorescence spectrum of this mixture in
aqueous buﬀer does not show Cu
-induced changes in the
emission intensity of BRNO1. Therefore, the Cu
does not bind strongly to BRNO1 or, if it does, it has little
eﬀect on the emission intensity, as for other diamagnetic metal
ions tested (Figure 5).
Because the Cu
redox couple is crucial for mediating
the detection of HNO in this system, the electrochemical
properties of CuBRNO1−3 were investigated by cyclic
voltammetry in acetonitrile. Quasi-reversible reduction features
occur at 90, 150, and 10 mV vs Fc/Fc
respectively (Figure S16 in the SI). These values are close to
that of the Cu
couple of CuCl
under the same conditions
(130 mV vs Fc/Fc
). The reduction potential corresponding to
the HNO/NO couple is −0.36 V in water.
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
conﬁrm the formation of NO gas, EI-MS measurements were
carried out on the head space of a reaction mixture of
CuBRNO1 and Angeli’s salt. These measurements conﬁrmed
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 signiﬁcantly
diﬀerent metal-binding sites and were therefore expected to
exhibit diﬀerent 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.
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 Angeli’ s 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
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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 .
Raw 264.7 macrophages generate micromolar
concentrations of NO following treatment with endotoxins and
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 ﬁndings 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 Angeli’s 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
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 resoruﬁn dyes localize in the ER.
In addition to the ER, a
signiﬁcant degree of membrane staining was observed.
We have presented three novel benzoresoruﬁn-based NO and
HNO ﬂuorescent sensors. The use of benzoresoruﬁn as the
ﬂuorophore 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 ﬂuorescence. BRNO3,
which contains a 2-(aminomethyl)pyridine metal-binding site,
has a signiﬁcantly higher photoluminescent quantum yield
owing to the absence of low-energy charge-transfer excited
states. In the Cu
-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
ﬂuorophore unit also play a signiﬁcant 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 suﬃcient 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
reduction is necessary for
both HNO or NO turn-on. The ﬂuorescence turn-on observed
with the Cu
BRNO probes reveals that the redox potentials of
the complexes are largely inﬂuenced by the ﬂuorescent dye,
which participates in metal coordination, and are only slightly
inﬂuenced by the diﬀerent amine chelating moieties. The metal-
binding aﬃnity inﬂuences ﬂuorescence turn-on after addition of
NO or HNO. There is a lower ﬂuorescence turn-on for both
HNO and NO with tighter binding of Cu
. Despite the small
turn-on response to NO, these sensors are quite eﬀective at
detecting NO and HNO in both the cuvette and living cells.
NMR data of all compounds, as well as crystallographic data of
compounds 2 and 3, additional DFT results, Cu
studies, RNS/ROS studies with [FeBRNO]
, 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
The authors declare no competing ﬁnancial interest.
Figure 7. Quantiﬁed ﬂuorescence turn-on in cells after treatment with
Angeli’s salt as well as NO-releasing GSNO and stimulation of iNOS
by LPS/INF- γ in the presence of in situ generated [CuBRNO1]
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
ﬂuorescent probe. The bottom image corresponds to cells treated with
Angeli’s salt. (left) DIC images. (right) Fluorescence images. Scale bar
Inorganic Chemistry Article
dx.doi.org/10.1021/ic302793w | Inorg. Chem. 2013, 52, 3285−32943293
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,
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