Synthesis and characterization of a new fluorescent probe for reactive oxygen species

Article abstract of DOI:10.1039/b702618h

We report the development of a new fluorescence sensor for reactive oxygen species (ROS) based on a benzofurazan structure. The sensor, NBFhd, is initially non-fluorescent and reacts with peroxyl radicals by hydrogen transfer in an aqueous medium under neutral conditions to release the fluorescent N-methyl-4-amino-7-nitrobenzofurazan (NBF) and 1,4-benzoquinone. NBFhd shows excellent contrast and no interference in the region of cell autofluorescence and is a new tool to detect ROS in homogeneous and biological systems. This journal is The Royal Society of Chemistry.

Full text of DOI:10.1039/b702618h

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Synthesis and characterization of a new fluorescent probe for reactive
oxygen species†
Belinda Heyne, Chad Beddie and J. C. Scaiano*
Received 19th February 2007, Accepted 12th March 2007
First published as an Advance Article on the web 2nd April 2007
DOI: 10.1039/b702618h
We report the development of a new fluorescence sensor for reactive oxygen species (ROS) based on a
benzofurazan structure. The sensor, NBFhd, is initially non-fluorescent and reacts with peroxyl radicals
by hydrogen transfer in an aqueous medium under neutral conditions to release the fluorescent
N-methyl-4-amino-7-nitrobenzofurazan (NBF) and 1,4-benzoquinone. NBFhd shows excellent
contrast and no interference in the region of cell autofluorescence and is a new tool to detect ROS in
homogeneous and biological systems.
contrast, and fluorescence in a region of the visible spectrum that
minimizes interference by cell autofluorescence.
Introduction
Reactive oxygen species (ROS) formed during the metabolism of
oxygen are believed to be implicated in aging processes and nu-
Experimental
merous diseases such as atherosclerosis and cancer.1–6 Recently, in
order to gain insight into the exact implication of ROS in different
physiopathologies, research has focused on the characterization of
new sensors able to detect ROS in organisms.7–11
Synthesis of nitrobenzofurazan derivatives
All the chemicals were purchased from Aldrich and used as
received. The 1H and 13C NMR spectra were recorded on a Bruker-
Avance-400 spectrometer at 400 MHz. Samples were prepared in
methyl sulfoxide-d6 (99%) purchased from Aldrich. The chemical
shifts are quoted using the d scale and the coupling constants are
expressed in Hz. Mass spectra (EI) were recorded on a Kratos-
Concept-II instrument.
Because of its non-invasive character and its high sensitiv-
ity, fluorescence is frequently the preferred technique to detect
ROS in vitro as well as in vivo. Several fluorescence sensors
have been developed for this purpose; among them, 2-[6-(4ꢀ-
hydroxy)phenoxy-3H-xanthen-3-on-9-yl] benzoic acid (HPF) is
commercially available. This molecule, initially non-fluorescent,
is able to release fluorescein, a highly fluorescent compound, upon
interaction with ROS.8,12 In an earlier publication, we investigated
the mechanism of reaction of the HPF sensor.12 We demonstrated
the ability of HPF to react with peroxyl radicals and other
oxidizing agents. We also characterized the formation of the
corresponding phenoxyl radical as the initial oxidation product
and we proposed that the reaction was then followed by radical–
radical reactions and hydrolysis, the latter being responsible for
the release of the fluorescent moiety.
1. Synthesis of N-methyl-4-amino-7-nitrobenzofurazan (NBF).
The compound NBF was prepared according to the literature
procedure.13 Briefly, 1 g of 4-chloro-7-nitrobenzofurazan (5 mmol)
was dissolved in 30 ml of methanol, to which 7.5 mmol of
methy◦lamine hydrochloride was added. The mixture was refluxed
at 75 C under N2. Then 1.26 g (15 mmol) of sodium hydrogen
carbonate, dissolved in 20 ml of millipore water, was added
dropwise while stirring. While cooling, red crystals of the desired
1
compound precipitated. H NMR (DMSO-d6, 298 K): d = 3.05
The key characteristic of fluorescent sensors of this type
is the covalent binding between a fluorescent moiety and a
removable fluorescence quencher. In the case of HPF, these
functions are performed by moieties derived from fluorescein and
p-hydroxyquinone, respectively.
(s, 3H), 6.3 (d, J = 8.8 Hz, 1H), 8.51 (d, J = 8.8 Hz, 1H), 9.48
(s, 1H). 13C NMR (DMSO-d6, 298 K, decoupled): d = 30.59,
99.5,◦121.14,◦138.45, 144.49, 144.72, 146.26. Mp: 259 ◦C–261 ◦C
(264 C–265 C).13 EI found: 194 m/z.
In the present work, we have used a similar concept for the
design of a new fluorescent sensor for ROS. We synthesized a new
molecule, NBFhd, which contains a fluorescent compound, N-
methyl-4-amino-7-nitrobenzofurazan (NBF), tethered to a pheno-
lic structure that acts as a quencher and also as a hydrogen donor.
This new sensor has several key advantages over HPF, including a
simple high-yield and inexpensive synthesis, an exceptionally high
2. Synthesis of 4[N-methyl-N(4-hydroxyphenyl)amino]-7-nitro-
benzofurazan (NBFhd). The synthesis of NBFhd has never been
reported previously. 500 mg of 4-chloro-7-nitrobenzofurazan
(2.5 mmol) was dissolved in 20 ml of methanol, to which 1.29 g
(3.75 mmol) of 4-(methylamino)phenol sulfate was added. The
mixture was refluxed at 75 ◦C under N2 for 1 h. Then 0.76 g
(7.5 mmol) of sodium hydrogen carbonate, dissolved in 10 ml of
millipore water, was added dropwise under stirring. While cooling,
bright red crystals of the desired compound precipitated (668 mg,
University of Ottawa, Department of Chemistry, 10 Marie Curie, Ottawa,
ON, K1N 6N5, Canada. E-mail: tito@photo.chem.uottawa.ca
† Electronic supplementary information (ESI) available: NMR spectra,
crystal structures, ORTEP diagrams, crystallographic data for nitroben-
zofuran derivatives, and color representations of the calculated structures
from Fig. 6 and 7. See DOI: 10.1039/b702618h
1
93% yield). H NMR (DMSO-d6, 298 K): d = 3.81 (s, 3H), 6.21
(d, J = 8.8 Hz, 1H), 6.89 (d, J = 8.8 Hz, 2H), 7.24 (d, J = 8.8 Hz,
2H), 8.45 (d, J = 9.2 Hz, 1H), 9.97 (s, 1H). 13C NMR (DMSO-d6,
298 K, decoupled): d = 45.2, 104.64, 117.1, 121.58, 127.89,
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136.64, 137.32, 145.17, 145.18, 147.04, 157.79. Mp: 250 ◦C–
252 ◦C. The accurate mass was measured to be 286.0648 m/z for a
structure C13H10N4O4. X-ray quality crystals were grown by slow
evaporation of a dimethylformamide–dichloromethane solution.
The data and the structure of the molecule are shown in the ESI†.
The structure of NBFhd was unequivocally determined by
X-ray crystallography on a crystal obtained from a mixture
of dimethylformamide and dichloromethane (see ESI†). The
accurate mass of NBFhd gave 286.0648 g mol−1and is in agreement
with the proposed structure. 1H and 13C NMR spectra of NBFhd
are also consistent with the proposed structure and confirm the
purity of the sample (see ESI†).
Fluorescence measurements
The new probe, NBFhd, presents an absorption maximum
at 500 nm (Fig. 1) with a molar extinction coefficient of
22810 M−1 cm−1; NBFhd does not show any fluorescence within
our detection limit, thus offering excellent contrast for the
detection of ROS. The absence of fluorescence can be rationalized
on the basis of an electron transfer between the phenol moiety
and the excited NBF chromophore. Indeed, as reported on
the Stern–Volmer plot in Fig. 2, phenol is able to quench the
fluorescence of excited NBF. The fluorescence lifetime of NBF
was measured as 6.0 ns (data not shown), which gives a measured
quenching constant of 3.6 × 1010 M−1 s−1. This value is diffusion
controlled, which is not surprising since a similar value has already
been reported for fluorescence quenching of eosin by phenol.32
In phosphate buffer at pH 7 containing 1% DMSO, the two
molecules, NBF and NBFhd, obey the Beer–Lambert law between
at least 1 × 10−7 M and 5 × 10−5 M, indicating the absence of self-
association in the ground state.
Steady state emission spectra were recorded using a Photon Tech-
nology International fluorimeter. The excitation of the sensor was
set at 468 nm. When necessary, the temperature was maintained at
40 ◦C using a cuvette holder equipped for circulating thermostated
water.
Lifetime fluorescence measurements were performed on a
easylife LS (Photon Technology International). The excitation
was performed with a 440 nm pulsed LED and the emission was
measured at 525 nm.
Computational details
All calculations were performed using the Gaussian 03 suite of
programs.14 Optimized gas-phase geometries were obtained using
the Becke3 exchange functional,15 in combination with the Lee,
Yang, and Parr correlation functional,16 i.e. the B3LYP method,
as implemented in Gaussian 03. The 6–31+G(dꢀ,pꢀ) basis set17–23
was used for geometry optimizations and energy calculations.
Calculating the harmonic vibrational frequencies and noting the
number of imaginary frequencies confirmed the nature of all the
intermediates (NImag = 0). Solvent effects were approximated
with single-point calculations using B3LYP/6–31+G(dꢀ,pꢀ) on
gas-phase B3LYP/6–31+G(dꢀ,pꢀ) geometries using the IEF-PCM
method24–26 with the water solvent parameters. The atomic radii
for the IEF-PCM calculations were specified using the UAHF
keyword, while the free energy of solvation was generated using the
SCFVAC keyword. Mulliken27 atomic spin densities were obtained
from the gas-phase B3LYP/6–31+G(dꢀ,pꢀ) geometries.
Results and discussion
Our main interest was to develop a sensor able to release a
molecule with a fluorescence maximum in a region where cellular
fluorochromes such as flavin and NADPH do not show a signif-
icant autofluorescence.28 N-methyl-4-amino-7-nitrobenzofurazan
(NBF) proved to be an interesting candidate.13,29–31 This molecule
presents an absorption maximum at 468 nm and a fluorescence
maximum at 540 nm, well separated from cell autofluorescence.
The synthesis of NBFhd was based on a strategy similar to that
reported by Heberer et al.13 The high-yield one-pot synthesis is
outlined in Scheme 1.
Fig. 1 Absorption (left) and fluorescence (right) spectra of a 5 × 10−6
M
solution of NBF (thin line) and NBFhd (thick line) in phosphate buffer
solution (PBS) at pH 7.4. The excitation wavelength was set at 468 nm
and fluorescence spectra (right axis) for unexposed samples include a few
identifier points (᭹).
Fig. 2 Stern–Volmer plot of the quenching of fluorescence of a 5 × 10−6
M
Scheme 1 Synthesis of NBFhd.
NBF solution in methanol by phenol.
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The ability of the new fluorescent probe, NBFhd, to react
with reactive oxygen species was monitored by fluorescence
spectroscopy. As illustrated in Fig. 3, when peroxyl radicals
are generated by the thermal decomposition of 2,2ꢀ-azobis(2-
amidinopropane) dihydrochloride (AAPH) at 40 ◦C in aerated
solutions,33,34 an increase in fluorescence is observed with time
and was attributed to the release of the fluorescent NBF molecule
following radical attack on NBFhd; this was demonstrated by
HPLC with fluorescence detection (see ESI†). The decrease of
fluorescence observed in Fig. 3 after 120 min is likely due to peroxyl
radical attack on NBF.
concentration of TROLOX totally suppresses the release of NBF
suggests that the rate constant for radical attack on TROLOX is
significantly faster than that for NBFhd.
A GC/MS analysis of the products of the reaction between
NBFhd and peroxyl radicals indicate that 1,4-benzoquinone is a
product formed in this reaction (see ESI†). The results suggest
that the mechanism of reaction of the NBFhd sensor is similar
to that proposed for the commercially available probe, HPF.12
Peroxyl radicals are expected to abstract the hydrogen atom from
the phenolic moiety leading to the formation of the corresponding
phenoxyl radical, followed by reaction with a second peroxyl
radical and water as shown in Scheme 2. In aqueous solution
hydrogen atom transfer is likely mediated by electron transfer.35,36
Scheme 2 Proposed mechanism of reaction of the NBFhd sensor with
peroxyl radicals.
Density functional theory (DFT) calculations at the B3LYP/6–
31+G(dꢀ,pꢀ) level of theory indicate that hydrogen abstraction from
•
NBFhd by the model peroxyl radical OOCH3 is approximately
thermoneutral (Fig. 5).
Fig. 3 Evolution of the fluorescence intensity of an aerated NBFhd (5 ×
10−6 M) solution in PBS (pH 7.4) at 40 ◦C in the presence of AAPH (5 ×
10−3 M). The excitation wavelength was set at 468 nm. The inset represents
the evolution of the intensity at 540 nm.
The increase of fluorescence shown in Fig. 3 is completely
inhibited when TROLOX (a water soluble analog of Vitamin E
and an excellent hydrogen donor) is present in solution (Fig. 4),
indicating that the release of NBF from NBFhd is due to
its reaction with peroxyl radicals. The fact that an equimolar
Fig. 5 Calculated gas-phase and solvent corrected relative free energy of
hydrogen abstraction.
Analysis of the spin density of the NBFhd radical reveals that
the radical character is delocalized throughout the phenol moiety,
with the greatest spin densities located on the phenol oxygen and
on the carbon atoms that are ortho and para to the phenol oxygen
(Fig. 6), as expected.
Fig. 4 Effect of the addition of TROLOX, 5 × 10−6 M, on the release
of NBF from a NBFhd solution (5 × 10−6 M) in PBS (pH 7.4) at 40 ◦C
in presence of AAPH 5 × 10−3 M. Circles correspond to the solution in
the absence of TROLOX and the squares are the solution in presence of
TROLOX. The wavelength of excitation was set at 468 nm.
Fig. 6 Mulliken atomic spin densities of the NBFhd radical at the
B3LYP/6–31+G(dꢀ,pꢀ) level of theory. Spin densities between −0.05 and
0.05 are not shown. Unlabeled atoms are C (grey) and H (light grey).
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It is believed that the mechanism by which a second peroxyl
radical and water react with the NBFhd radical to release NBF and
1,4-benzoquinone involves attack at the para-position. However,
regardless of the mechanism by which NBF is released, the
NBFhd radical that was generated by hydrogen transfer, likely
will couple with other radicals, including peroxyl radicals. Using a
smaller model (Model 2, Fig. 7), we calculated the structures and
energies of isomers in which the peroxyl radical coupled to the
NBFhd radical in the ortho- and para-positions. These calculations
revealed that all of the conformers in which coupling occurs at the
ortho-positions are lower in energy than the isomers in the para-
position, which is likely because of the steric bulk of the NBF unit.
While the NBF released shows fluorescence emission in a region
where cell autofluorescence is weak, its fluorescence quantum yield
is modest in water (0.026 31). However, according to Uchiyama
et al.,31 the fluorescence quantum yield increases when the polarity
of the solvent decreases; this quantum yield was estimated to
be 0.23 in methanol, 0.31 in ethanol and 0.37 in propanol.
The polarity of those solvents is close to that found within
membranes.37 The polarity of NBF and NBFhd is also such
that its partition in biological media will favor phases with
intermediate polarity, rather than the aqueous phase. Therefore,
we can expect an increase of the NBF fluorescence quantum
yield within membranes and other biomaterials, providing an
interesting prospect for the use of this new fluorescent sensor in
biological applications.
The new fluorescent sensor, NBFhd, presents several advantages
compared to the available HPF probe. Indeed, its inexpensive
synthesis required only one step for a yield approaching 100%,
while according to the literature, the multistep synthesis of HPF
gave only 7% overall yield.8 In addition, the precursors are readily
available, the NBFhd system presents minimal interference with
cell autofluorescence and outstanding contrast between unreacted
and reacted probe.
Fig. 7 Gas-phase relative free energies of the ortho- and para-coupling
We have employed a computational chemistry approach to
establish if we could favor the formation of para-coupling products
by introducing steric bulk in the form of methyl groups at the
ortho-positions (Model 3, Fig. 6). The relative energies of the
isomers of the coupling products for the dimethyl system (Model
3) reveal that coupling to the ortho-positions is less favorable as
compared to the corresponding products for Model 2. In addition,
for the dimethyl complexes, the ortho- and para-coupling isomers
are closer in energy and less favorable energetically, which leads to
more para-coupling, and as a result may help minimize dead-end
pathways.
products for two models at the B3LYP/6–31+G(dꢀ,pꢀ) level of theory
•
compared to the gas-phase relative free energy of OC6R2H2NCH3Rꢀ +
•OOCH3 set to 0.0 kcal mol−1 (see ESI† for structures and computational
details).
The favorable energetics for formation of the ortho-coupling
product may serve as a side reaction that does not involve NBF
release. This result might explain why experimentally only 15 to
20% of NBF is released from the reaction of NBFhd with the
peroxyl radical.
The interaction of NBFhd with other reactive oxygen species
(ROS) was also investigated. As shown in Fig. 8, NBFhd is able
•
•
−
Conclusion
to react with OH and O2 to release NBF, while H2O2 seems
to have no effect on the fluorescent sensor. We note that only
qualitative comparisons can be made from these results since the
rate of production of ROS, as well as the rate constant of NBFhd
towards those ROS, are not known.
In summary, a new fluorescent sensor for reactive oxygen species
(ROS) detection has been developed. The initially non-fluorescent
NBFhd, a 4,7-disubstituted benzofurazan, reacts with peroxyl
radicals in phosphate buffer to release a strongly fluorescent com-
pound: NBF. The release of NBF is accompanied by the formation
of 1,4-benzoquinone, as demonstrated by chromatographic tech-
niques. NBFhd is a new tool to explore the involvement of reactive
oxygen species, in particular oxygen centered radicals, including
superoxide, for biological applications.
Acknowledgements
This work was generously supported by funding from the Natural
Sciences and Engineering Research Council of Canada, by the
Canadian Foundation for Innovation and by the Government of
Ontario. We would like also to thank Dr. Ilia Korobkov for his
help with the X-ray experiments.
Fig. 8 Fluorescence increase from a 5 × 10−6 M solution in PBS pH 7.4
upon reaction with •OH produced by a Fenton reaction [H2O2 1 mM
(white block), after addition of FeSO4 100 lM (grey block), after a second
addition of FeSO4 100 lM (black box)], H2O2 100 lM at 40 ◦C (white block
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