A specific fluorescent probe for NO based on a new NO-binding group

Article abstract of DOI:10.1039/c4cc03540b

By incorporation of a specific NO-binding group, 2-amino-3′- dimethylaminobiphenyl, into a Bodipy dye, fluorescent probe 1 was constructed, which exhibited high selectivity for NO over other ROS/RNS as well as DHA, AA and MGO. the Partner Organisations 2014.

Full text of DOI:10.1039/c4cc03540b

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A specific fluorescent probe for NO based on a
new NO-binding group†
Xin Lv,a Yue Wang,a Song Zhang,b Yawei Liu,a Jian Zhanga and Wei Guo*a
Cite this: Chem. Commun., 2014,
50, 7499
Received 11th May 2014,
Accepted 22nd May 2014
DOI: 10.1039/c4cc03540b
www.rsc.org/chemcomm
By incorporation of a specific NO-binding group, 2-amino-30- which the off–on fluorescence response is achieved through sup-
dimethylaminobiphenyl, into a Bodipy dye, fluorescent probe 1 pression of the photoinduced electron transfer (PET) process.
was constructed, which exhibited high selectivity for NO over other Although in some cases they can provide useful information, the
ROS/RNS as well as DHA, AA and MGO.
basic chemical problems still remain. First, dehydroascorbic acid
(DHA) and ascorbic acid (AA) (micromolar to millimolar levels in
Nitric oxide (NO), produced by nitric oxide synthases, has been many cells9) were reported to be able to condense with the OPD
regarded as a ubiquitous signaling molecule, and plays key roles in group to turn on the fluorescence of such probes.10 Second, it was
various physiological as well as pathological processes. Endogenous recently reported that a OPD-based Bodipy fluorescent probe could
NO mediates multiple processes in the various physiological also detect methylglyoxal (MGO),11 a dicarbonyl metabolite pro-
systems, such as the cardiovascular system, immune system, duced by all living cells, under physiological conditions.12 Third,
and the central and peripheral nervous systems.1 Also, studies benzotriazoles are pH-sensitive, and their deprotonation at physio-
have shown that its deregulation correlates with the symptoms of logical pH results in an electron-rich triazolate, which may induce the
cancer, endothelial dysfunction, and neurodegenerative diseases.2 undesirable fluorescence quenching through the PET mechanism to
However, the mechanisms by which it performs its diverse lower the NO detection sensitivity.4d,f Therefore, further efforts to
biological roles still remain elusive. Therefore, an efficient method overcome these limitations are highly required.
for sensitively and selectively probing NO in biological systems is
highly required for better understanding of its origin, activities, cyano-a-naphthylamine fluorescent probe for NO (Fig. 1A).13 The
and biological functions. probe itself was nonfluorescent, but displayed the obvious fluores-
In 2010, Anslyn et al. exploited a 2-dimethlyaminophenyl-5-
Due to their simplicity, sensitivity, real-time imaging, and espe- cence off–on response upon NO treatment due to the NO-triggered
cially nondestructive detection, fluorescence techniques combined formation of a new fluorophore. Notably, the probe displayed
with microscopy have been regarded as a powerful tool for sensing excellent selectivity for NO over other reactive oxygen/nitrogen
and imaging targeted biomolecules in live cells or tissues. Given this, species (ROS/RNS) as well as DHA, and thus appears to be
a number of fluorescent NO probes have been reported to date3 by more advantageous than the conventional OPD-based ones
exploiting the specific reactions of NO with the o-phenylenediamine from the viewpoint of selectivity. To extend the method to a
(OPD) moiety,4,5 metal–ligand complexes,6 and others.7 Among these versatile design platform, in this work we present its modified
methods, OPD-based fluorescent probes, pioneered by Nagano’s
group, have shown the immense potential to visualize NO in vitro
and in vivo.4,5 Moreover, some of them are commercially available,8 and
have greatly promoted NO-related biological investigation. The corre-
sponding sensing mechanism is based on the irreversible reaction of
the OPD group with NO+ or N2O3 to give benzotriazole derivatives, by
a School of Chemistry and Chemical Engineering, Shanxi University,
Taiyuan 030006, China. E-mail: guow@sxu.edu.cn
b Shanxi Academy of Analytical Sciences, Taiyuan 030006, China
† Electronic supplementary information (ESI) available: Experimental proce-
dures, supplemental spectra, and the 1H-, 13C-NMR, and MS spectra. See DOI:
Fig. 1 The design strategies for fluorescent NO probes.
10.1039/c4cc03540b
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Fig. 2 Proposed sensing mechanism of probe 1 with NO.
Fig. 3 HRMS charts of probe 1 in the absence (A) and presence (B) of the
excess of DEAꢀNONOate in EtOH-PBS buffer (20 mM, pH 7.4, 1 : 1, v/v).
Inset: fluorescent images of probe 1 in the absence (A) and presence (B) of
the excess of DEAꢀNONOate in the same buffer.
version for constructing the fluorescent NO probe through integra-
tion of a NO-binding group, 2-amino-30-dimethylaminobiphenyl
(AD), and a selected fluorophore (Fig. 1B). We think that the strategy
may be more useful for the future design of fluorescent NO probes.
As a proof of concept, we designed probe 1 by attaching the
NO-binding group, AD into the widely used Bodipy dye14 (Fig. 2). In
fact, AD functions not only as a NO-binding group, but also as a
fluorescence quencher via PET to ensure the low background fluores-
cence. We reasoned that probe 1 is weakly fluorescent due to the PET
process from the electron-rich AD group to the excited Bodipy
fluorophore. Upon treatment with NO, diazo product 2 was expected
to be produced based on the reaction mechanism proposed by
Anslyn.13 Due to the absence of the electron-donating amino group
in 2, the above-mentioned PET process would be inhibited, thereby
leading to the fluorescence turn-on of the Bodipy fluorophore.
Probe 1 was synthesized according the route outlined in Scheme 1.
To anchor the AD group to the Bodipy core, the commercially avail-
able 4-bromo-3-nitro-benzaldehyde 5 was used as the starting material.
By use of the standard Bodipy synthetic procedures, intermediate 4
could be easily obtained. Suzuki coupling of 4 with 3-dimethylamino-
phenylboronic acid gave intermediate 3. The subsequent reduction of 3
with SnCl2 gave probe 1. The detailed experimental procedure and
characterization data are provided in the ESI.†
fluorescence turn-on response from a dark background (Fig. 3,
inset). The preliminary results are in accordance with our
proposed sensing mechanism.
Next, we performed the time-dependent fluorescence spectral
studies of probe 1 treated with different concentrations of
DEAꢀNONOate in EtOH-PBS buffer (20 mM, pH 7.4, 1 : 1, v/v).
The obtained results indicated that the reaction could be completed
within 30 min (Fig. 4A). With a time point of 30 min after addition of
DEAꢀNONOate, we performed the fluorescence titration studies of 1
for NO. As shown in Fig. 4B, probe 1 exhibited a weak emission
at 518 nm due to PET from the aniline unit to the excited Bodipy
fluorophore. Treatment with DEAꢀNONOate elicited an obvious
enhancement. The changes in the emission intensities became less
obvious when the amount of DEAꢀNONOate was more than 75 mM.
In this case, an approximately 5-fold fluorescence enhancement was
observed. Moreover, the fluorescence intensities of 1 at 518 nm
showed a good linear relationship with DEAꢀNONOate concentra-
tions from 0 to 15 mM (Fig. 4B, inset), and the detection limit was
estimated to be 30 nM based on S/N = 3. Thus, probe 1 is potentially
useful for monitoring NO in living cells.
Further, we evaluated the specificity of probe 1 for NO. We
screened a wide array of possible competitive species, including
H2O2, ClOꢁ, ꢂOH, O2ꢁ, 1O2, ONOOꢁ, NO2ꢁ, NO3ꢁ, DHA, AA, and
MGO, all of which are biologically related (Fig. 5A). In fact,
probe 1 was inert to most of these ROS and RNS, and only
1O2 and ONOOꢁ elicited a minor fluorescence enhancement of
1.4- and 1.5-folds, respectively. Notably, probe 1 did not show
With probe 1 in hand, we first tested the reaction of 1 with NO
in EtOH-PBS buffer (20 mM, pH 7.4, 1:1, v/v) at 37 1C by HRMS.
DEAꢀNONOate was used as the source of NO, which is commer-
cially available with a half time of 16 min. The free probe 1
exhibited a main peak at m/z 459.2565 (Fig. 3A), corresponding to
[1 + H+]+ (m/z, calcd: 459.2532). However, after incubation of the
probe with excess of DEAꢀNONOate, a new peak was observed at
m/z 470.2338 (Fig. 3B), which could be assigned to [2 + H+]+ (m/z,
calcd: 470.2328). Moreover, the reaction also elicited an obvious
Fig. 4 (A) Time-dependent fluorescence intensity changes of 1 (5 mM) upon
addition of varied concentrations of DEAꢀNONOate in EtOH-PBS buffer.
(B) Fluorescence spectra of probe 1 (5 mM) upon addition of DEAꢀNONOate
(0–75 mM) recorded after 30 min. Condition: EtOH-PBS buffer (20 mM,
pH 7.4, 1 : 1, v/v); 37 1C; lex = 480 nm; lem = 518 nm; slits: 5/5 nm.
Scheme 1 Synthesis of 1. (1) (i) 2,4-Dimethyl-pyrrole, TFA; (ii) DDQ and
(iii) Et3N, BF3-Et2O. (2) 3-Dimethylaminophenylboronic acid, Pd(PPh3)4,
K2CO3. (3) SnCl2, conc. HCl.
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In summary, we have presented a novel fluorescent probe
for NO by incorporation of a new NO-binding group, 2-amino-
30-dimethylaminobiphenyl, into a Bodipy dye. The probe can
selectively sense NO over ROS/RNS as well as AA/DHA/MGO
with a detection limit as low as 30 nM. The preliminary
cell imaging experiments indicate its potential to probe NO
chemistry in biological systems.
We acknowledge the National Natural Science Foundation
of China (NO. 21302114 and 21172137) and Scientific and
Technological Innovation Programs of Higher Education Insti-
tutions in Shanxi for financially supporting this work.
Fig. 5 (A) Fluorescence spectra of 1 (5 mM) treated with various species
1
ꢁ
(10 equiv. of NO, ONOOꢁ and O2; 100 equiv. of H2O2, ClOꢁ, ꢂOH, O2
,
NO2ꢁ, NO3ꢁ, DHA, AA, and MGO) in EtOH-PBS buffer (20 mM, pH 7.4, 1 : 1,
v/v). (B) Effects of pH on the fluorescence intensity of 1 (5 mM) in the
absence (’) or presence ( ) of DEAꢀNONOate (10 equiv.). lex = 480 nm,
lem = 518 nm, slits: 5/5 nm.
Notes and references
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DEAꢀNONOate in different pH environments. As shown in Fig. 5B,
probe 1 showed a stable and weak emission in the pH region of
4.5–8.0, and displayed the best response for NO in the pH region
of 4.5–7.5. Thus, probe 1 can function well under physiological
conditions. However, under alkaline conditions, the fluorescence
enhancement was inhibited, likely due to the fact that DEAꢀNON-
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Fig. 6 Fluorescent images of NO in HL-7702 cells using probe 1 (5 mM)
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Scale bar: 200 mm.
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