A Boron Dipyrromethene (BODIPY)-Based CuII-Bipyridine Complex for Highly Selective NO Detection

Article abstract of DOI:10.1002/chem.201502191

A BODIPY-containing Cu^II-bipyridine complex for the simple selective fluorogenic detection of NO in air and in live cells is reported. The detection mechanism is based on NO-promoted Cu^II to Cu^I reduction, followed by demetallation of the complex, which results in the clearly enhanced emission of the boron dipyrromethene (BODIPY) unit.

Full text of DOI:10.1002/chem.201502191

DOI: 10.1002/chem.201502191
Communication
&
Fluorescence |Hot Paper|
II
A Boron Dipyrromethene (BODIPY)-Based Cu –Bipyridine Complex
for Highly Selective NO Detection
[
a, b]
[a, c, d]
[a, b]
L. Alberto Juµrez,
MµÇez,
Andrea Barba-Bon,
Ana M. Costero,*
Ramón Martínez-
[
a, c, d]
[a, c, d]
[a, b]
[a, b]
FØlix Sancenón,
Margarita Parra,
Pablo GaviÇa,
M.
[
a, e]
[a, e]
Carmen Terencio,
and M. JosØ Alcaraz
[
4]
hazard. Even though these methods offer certain benefits,
they also show some limitations which typically involve poor
specificity and the use of expensive experimental apparatus,
which restrict their application in practice. Recently, the devel-
opment of fluorogenic probes has gained growing interest as
II
Abstract: A BODIPY-containing Cu –bipyridine complex
for the simple selective fluorogenic detection of NO in air
and in live cells is reported. The detection mechanism is
II
I
based on NO-promoted Cu to Cu reduction, followed by
demetallation of the complex, which results in the clearly
enhanced emission of the boron dipyrromethene
[5]
an alternative to classical instrumental procedures. In this
context, fluorogenic probes are especially appealing because
they allow simple detection in situ or/and at site, usually with-
out any sample pre-treatment. Moreover, changes in emission
can be detected by simple equipment and it is a very sensitive
detection technique. Changes in emission properties can also
be detected by the naked eye, which makes this procedure
very appealing.
(
BODIPY) unit.
In recent years, environmental awareness has grown due to
[
1]
social dissatisfaction with the state of the environment. As
a result, more restrictive environmental laws have been intro-
duced. In this context, air pollution is one of the major prob-
lems in urban areas, whose main sources of pollutants are the
combustion processes of fossil fuels used in power plants, ve-
hicles, and other incineration processes. The main combustion-
Several probes for the fluorogenic detection of NO have
been reported. For instance, poorly fluorescent vicinal dia-
mines can be transformed into triazoles by NO, which results
[
6]
in a significant increase in fluorescence. Other sensing proto-
[
7]
generated air contaminants are nitrogen oxides (NO ), consid-
cols based on NO-induced ring closure, deamination reac-
x
[
8]
[4b]
ered primary pollutants of the atmosphere as they are respon-
sible for photochemical smog, acid rain, and ozone layer de-
tions, or NO-induced aromatization,
have been recently
studied. Metal-based probes that take advantage of the reac-
tivity of NO at the metal site have also been reported; for in-
[
2]
pletion. On the other hand, NO is a well-known bioactive
molecule that participates in a wide range of bioregulatory
II
stance, nitric oxide sensing has been accomplished with Co ,
[3]
II
II
II
II
[9]
and immune response processes.
Fe , Ru , Rh , and Cu complexes. However, some of these
probes display drawbacks, such as dependence on pH or the
tendency of certain dyes to form aggregates. Given the signifi-
cance of NO to human health and diseases, most probes have
been tested to monitor NO production in vivo, whereas very
few studies have been conducted into nitric oxide detection in
air.
For these reasons, intensive experimental research is being
conducted for NO monitoring, and several analytical tech-
niques, such as electrophoresis, electron paramagnetic reso-
nance (EPR) or GC-mass spectroscopies, chemiluminescence, or
electrochemical methods, have been developed to detect this
[
a] L. A. Juµrez, Dr. A. Barba-Bon, Prof. A. M. Costero, Prof. R. Martínez-MµÇez,
Dr. F. Sancenón, Prof. M. Parra, Dr. P. GaviÇa, Dr. M. C. Terencio,
Prof. M. J. Alcaraz
Centro de Reconocimiento Molecular y Desarrollo Tecnológico (IDM)
Universidad de Valencia-Universidad PolitØcnica de Valencia (Spain)
E-mail: Ana.Costero@uv.es
Following our interest in designing probes for the fluorogen-
[
10]
ic detection of gases, we focused on the potential use of
BODIPY for designing NO sensing probes. BODIPY is a well-
known fluorophore that possesses valuable optical characteris-
tics, such as absorption and fluorescence transitions in the visi-
ble spectral region with high molar absorption coefficients and
fluorescence quantum yields, good stability, and no depend-
[
b] L. A. Juµrez, Prof. A. M. Costero, Prof. M. Parra, Dr. P. GaviÇa
Departamento de Química Orgµnica. Universidad de Valencia.
Dr. Moliner, 50., 46100-Burjassot (Spain)
[
11]
ence on pH. Yet despite these advantageous BODIPY fluoro-
phore optical properties, as far as we know, no metal com-
plexes based on BODIPY derivatives have been reported for
fluorogenic NO detection.
[
c] Dr. A. Barba-Bon, Prof. R. Martínez-MµÇez, Dr. F. Sancenón
Departamento de Química. Universidad PolitØcnica de Valencia.
Camino de Vera s/n. 46022-Valencia (Spain)
[
d] Dr. A. Barba-Bon, Prof. R. Martínez-MµÇez, Dr. F. Sancenón
CIBER de Bioingeniería, biomateriales y nanomedicina (CIBER-BBN)
The design of our probe was based on two concepts. First, it
is known that the BODIPY derivatives that bear a 4-pyridine
moiety attached to the meso position induce fluorescence
quenching when the pyridine group is protonated, through
[
e] Dr. M. C. Terencio, Prof. M. J. Alcaraz
Departamento de Farmacología. Universidad de Valencia.
Vicente AndrØs EstellØs s/n. 46100-Burjassot, Valencia (Spain)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/chem.201502191.
[
12]
the formation of a weakly emissive charge-transfer state. We
Chem. Eur. J. 2015, 21, 15486 – 15490
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postulated that similar BODIPY-bipyridine derivatives that coor-
dinate metal cations would also be poorly emissive. On the
other hand, it has been well-established that the copper
II
Table 1. The UV/Vis and fluorescence data of 1 and 1-Cu .
[
a]
l
abs [nm]
log e
l
em [nm]
F
II
I
centre in Cu complexes undergoes reduction to Cu in the
presence of NO, followed by demetallation. Based on these
1
1-Cu
500.5
504.0
7.6
7.1
520.5
519.0
0.570
0.071
II
II
concepts, we prepared complex 1-Cu (see Scheme 1). This
[
a] Quantum yields were calculated using rhodamine B in ethanol as
complex is expected to be poorly fluorescent, whereas signifi-
cant fluorescence enhancement is expected to be selectively
observed in the presence of NO.
a standard (FEtOH =0.49, lexc =478 nm).
II
Scheme 1. Complex 1-Cu and schematic outline of the sensing paradigm.
À5
Figure 1. Fluorescence spectra of 1 (acetonitrile, 1.0”10 m) with increasing
amounts of Cu . Inset: molar ratio graphic.
The synthesis of BODIPY derivative 1 began with the prepa-
ration of bipyridine aldehyde 2 by the oxidation of 4,4’-dimeth-
II
[13]
yl-2,2’-bipyridine with selenium oxide.
Compound 1 was
readily obtained by the condensation of 2 with 2,4-dimethyl-
K=4.9Æ1.6 was determined. Additional experiments demon-
I
pyrrole following standard BODIPY synthesis procedures (see
strated that addition of Cu to the acetonitrile solutions of 1 in-
[
14]
1
13
Scheme 2). BODIPY 1 was characterised by H and C NMR
spectroscopy and MS (see the Supporting Information for de-
tails).
duced no changes in either the UV or emission spectra, which
suggests this cation’s poor coordination with the bipy unit. It
was also found that the emission quenching of 1, which was
II
similar to that found for 1 with Cu , was observed in the pres-
II
ence of diamagnetic cation Zn . This finding indicates that the
II
poor emission observed for the 1-Cu complex was not due to
the presence of a paramagnetic cation, but resulted from the
coordination of the metal with the pyridine moiety attached to
the meso position of the BODIPY fluorophore. Finally, the fluo-
rescence emission intensity of 1 was evaluated at different pH
values, with only minor changes in the pH range between 4–
Scheme 2. Synthesis of ligand 1. DDQ=2,3-dichloro-5,6-dicyano-1,4-benzo-
quinone; TEA=triethylamine.
1
2 (see the Supporting Information).
II
Complex 1-Cu was easily isolated by simply stirring 1 with
one equivalent of Cu(NO ) in EtOH/water for 2 h. The resulting
3
2
À4
Solutions of 1 (1.0”10 m in acetonitrile) showed an intense
yellow precipitate was recrystallised from EtOH/water, filtered,
II
absorption band at l=500.5 nm (log e=7.6) and an intense
emission band at l=520.5 nm (lexc =478 nm, F=0.570).
and dried. In order to evaluate the sensing ability of 1-Cu , the
water/acetonitrile (9:1 v/v) mixtures of the complex (1.0”
II
À5
Moreover, fluorescence quenching was observed when Cu (as
10 m) were exposed for 5 min to N atmospheres, which con-
2
nitrate salt) was added (see Table 1). This was in agreement
tained increasing quantities of NO, and the corresponding
emission spectra were recorded. The presence of NO greatly
II
with the coordination of the Cu cation to the bipyridine bind-
ing group. This metal complexation likely triggered an intra-
molecular charge-transfer process in the excited state from the
increased the fluorescence emission at 520.5 nm (lexc =
478 nm), which was attributed to the presence of free
1 (Figure 2). In fact, exactly the same emission band was ob-
served for the solutions of 1 in the water/acetonitrile (9:1 v/v)
II
BODIPY core to the bipy–Cu unit, and the resulting charge-
[12]
transfer state became weakly fluorescent.
II
II
The formation of 1:1 complexes between 1 and Cu was
mixtures. The fluorogenic sensing ability of probe 1-Cu was
confirmed through titration experiments in acetonitrile. The
also observed by the naked eye. In particular, a bright-green
II
II
stability constant for the formation of the corresponding 1-Cu
emission was clearly seen when the solutions of 1-Cu exposed
complex was calculated from fluorescence titration experi-
to NO were illuminated at 254 nm with a conventional UV
lamp (Figure 2).
[15]
ments (Figure 1) with the SPECFIT program. A value of log
Chem. Eur. J. 2015, 21, 15486 – 15490
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a dry atmosphere for 24 h to obtain the corresponding sensing
membrane.
In a typical assay, the membrane was placed into a container
that held NO (1 ppm). After 5 min, fluorescence clearly en-
hanced, as observed by the naked eye with a conventional
2
54 nm UV lamp (Figure 4). A LOD of 0.3 ppm to the naked
[
17]
eye after 10 min for sensing NO in air was determined.
II
Figure 2. Fluorescence spectra of complex 1-Cu (water/acetonitrile 9:1 v/v,
À5
1
8
.0”10 m) in the presence of increasing NO concentrations from 0 to
0 ppm after 5 min. Inset: the visual changes (lex =254 nm) observed for 1-
Cu before and after exposure to 1 ppm of NO.
II
II
Figure 4. Emission of a polyethylene oxide membrane of 1-Cu
(
lex =254 nm) (left) and after exposure to 1 ppm of NO in air for
From the titration studies, limits of detection (LOD) of
5 min (right).
3
ppm (from fluorescence) and 0.5 ppm (from UV/Vis) were cal-
culated by the 3Sb1/S procedure (Sb1 is the standard deviation
of the blank solution and S is the slope of the calibration
One important issue for designing probes for pollutant
gases is the role played by the potential interferents or false-
positive outcomes produced by other species. When bearing
[
16]
curve).
II
I
In addition, 1-Cu was recovered after the oxidation of Cu to
II
II
Cu induced by atmospheric oxygen. In particular, the regener-
this in mind, the potential reactivity of 1-Cu -containing mem-
II
II
ation of the 1-Cu probe was achieved after keeping the 1-Cu -
NO mixture in NO-free air. This process was repeated at least
five times with only minimal loss of fluorescence intensity
branes to other hazardous gases (i.e., NO , CO , H S, SO ) at
2
2
2
2
a concentration of up 100 ppm in air was also studied. The
probe was also tested in the presence of vapours of acetone,
hexane, chloroform, acetonitrile, and toluene, and up to a con-
centration of 100 ppm in air. No emission changes were ob-
served in the presence of any of these chemical species. Be-
sides, competitive experiments demonstrated that the mem-
brane was able to detect NO in a mixture that also contained
the aforementioned gases and vapours (see the Supporting In-
(
Figure 3).
II
formation for details). Such behaviour indicated that 1-Cu was
a suitable highly selective probe for detecting nitric oxide in
complex air samples.
Finally, we also assessed the ability of the probe to detect
NO release in cells (Figure 5). In order to further demonstrate
II
the biological application of BODIPY–copper complex 1-Cu ,
RAW 264.7 macrophages were stimulated for 18 h with Escheri-
[
18]
chia coli lipopolysaccharide to induce NO synthase (iNOS).
II
After washing cells with phosphate buffered saline, 1-Cu
(10 mm) was added and NO release was initiated by the addi-
tion of l-arginine as a substrate of iNOS. RAW 264.7 macro-
II
À5
Figure 3. The cycles of emission intensity of 1-Cu (1.0”10 m in acetoni-
trile) at lem =520.5 nm (lexc =500.5 nm) upon successive exposures to NO
(
5 min) and to a NO-free air atmosphere (24 h).
II
phages stimulated with only arginine or only 1-Cu showed
low fluorescence, whereas the addition of arginine in the pres-
II
ence of 1-Cu significantly increased fluorescence, which dem-
Encouraged by the sensing ability of BODIPY derivative 1-
onstrates this compound’s ability to detect NO released by
cells. Even though these results indicate that other compo-
nents of the complex cellular matrix did not interfere with the
detection results, further sensing experiments were carried out
II
Cu , we decided to move another step forward and we studied
the potential use of the probe for detecting NO in air. To this
II
end, a membrane that contained 1-Cu was designed. In a typi-
II
cal preparation, polyethylene oxide (Mw 400000 Dalton) was
with some possible biological competitors in Cu binding, such
II
slowly added to a solution of 1-Cu in dichloromethane. The
as cysteine, glutathione, or histidine. No significant increment
in fluorescence was observed in any case (see the Supporting
Information, Figure S14)
mixture was stirred until a highly viscous mixture was formed
and finally poured into a glass plate. The system was kept in
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Communication
II
was calculated. Probe 1-Cu was also suitable for NO detection
in live RAW 264.7 macrophages with no cytotoxic effects.
Acknowledgements
The authors thank the DGICYT and European FEDER funds
(
MAT2012-38429-C04-01 and MAT2012-38429-C04-02) and the
Generalitat Valenciana (PROMETEOII/2014/047) for support.
SCSIE (Universidad de Valencia) is gratefully acknowledged for
all the equipment employed.
Keywords: BODIPY · cells · fluorescence · NO detection ·
quimiodosimeter
Figure 5. NO detection in RAW 264.7 macrophages stimulated with lipopoly-
II
À5
saccharide (LPS). After cell washing, 1-Cu (1.0”10 m), dissolved in water/
acetonitrile 95:5 v/v (vehicle), was incubated for 30 min with cells, and Argi-
nine (Arg) 100 mm was added to induce NO release. Data are expressed as
meanÆSEM (n=8–12). ***p<0.001 compared to the LPS+vehicle-treated
cells. Dunnett’s t test for multiple comparisons.
[
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II
2
64.7 cells stimulated with LPS and the incubation of 1-Cu
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sion was clearly enhanced in the cells that contained NO. In
II
a parallel experiment, absence of the cytotoxicity of 1-Cu
2
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Figure 6. Fluorescence imaging of RAW 264.7 stimulated with LPS and incu-
bated for 30 min with 1-Cu (10 mm) in the presence or absence of Arginine
II
(
Arg) 100 mm.
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II
Probe 1-Cu contains a BODIPY fluorophore and a bipyridine
II
II
I
unit capable of coordinating Cu . Reduction of Cu to Cu medi-
ated by NO resulted in demetallation and a significant en-
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probe 1-Cu in the polyethylene oxide membranes was satis-
1
2
factorily used to monitor NO levels in air. Furthermore, the re-
II
sponse of probe 1-Cu to NO was selective in air and other
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hazardous gases, such as NO , CO , H S, SO , and vapours of
2
2
2
2
[
different solvents at concentrations of up 100 ppm were
unable to induce emission modulations. A LOD as low as
[
0
.3 ppm to the naked eye after 10 min for NO sensing in air
Chem. Eur. J. 2015, 21, 15486 – 15490
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ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Communication
[
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Received: June 4, 2015
Published online on August 3, 2015
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15490
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