A fast-response, highly sensitive and selective fluorescent probe for the ratiometric imaging of nitroxyl in living cells

Article abstract of DOI:10.1039/c4cc00980k

A fast-response, highly sensitive and selective fluorescent probe with the 2-(diphenylphosphino)benzoate moiety as a recognition receptor for the ratiometric imaging of nitroxyl in living cells was first developed. the Partner Organisations 2014.

Full text of DOI:10.1039/c4cc00980k

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A fast-response, highly sensitive and selective
fluorescent probe for the ratiometric imaging
of nitroxyl in living cells†
Cite this: Chem. Commun., 2014,
50, 6013
Received 6th February 2014,
Accepted 16th April 2014
Caiyun Liu,ab Huifang Wu,b Zuokai Wang,b Changxiang Shao,b Baocun Zhu*b and
Xiaoling Zhang*a
DOI: 10.1039/c4cc00980k
www.rsc.org/chemcomm
A fast-response, highly sensitive and selective fluorescent probe the same strategy.6g,h Despite advances in the development of
with the 2-(diphenylphosphino)benzoate moiety as a recognition fluorescent probes for HNO, the above-mentioned fluorescent
receptor for the ratiometric imaging of nitroxyl in living cells was probes might be prone to be disturbed in bioimaging applica-
first developed.
tions by abundant biological reductants including glutathione
(GSH) and ascorbate in living matrices.6 Additionally, as far as
Nitroxyl (HNO) is the one-electron reduced or protonated form we know, all of the current fluorescent probes respond to HNO
of the well-known signaling agent nitric oxide (NO), and can be with changes only in fluorescence intensity, which might be
formed directly from nitric oxide synthase under the appro- influenced in quantitative detection by many factors, such as
priate conditions.1 However, recent investigations demonstrate variabilities in probe distribution, excitation and emission
that HNO displays important biological roles with potential efficiency, the environment around the probe (pH, polarity,
pharmacological applications distinct from those of NO.2 temperature, etc.), and effective cell thickness in the optical
For example, HNO has been shown to possess the potential beam.7 So, ratiometric fluorescent probes for HNO are urgently
for therapeutic applications in a variety of diseases including needed because they can eliminate numerous ambiguities by self-
treatments for heart failure and alcohol abuse.3 Moreover, calibration of two emission bands. On the other hand, fast-
HNO reacts as an electrophile with thiols to resist superoxide response and sensitive fluorescent probes for HNO are also in
infraction in mammalian vascular systems.4 Unfortunately, studies high demand as HNO and NO may be able to interconvert in the
on the elucidation of HNO mechanisms in vivo and the identifi- presence of superoxide dismutase (SOD).8 Based on the above
cation of endogenous sources are hampered by a lack of reliable considerations, in this communication, we developed the first
detection methods.
fluorescent probe for the ratiometric imaging of HNO levels in
Among the various available methods, the fluorescence living systems, which features fast-response, high sensitivity, the
technique with the help of fluorescent probes is the preferred important properties of a large emission shift for enhancing
method for in situ visualization of biologically important resolution in the ratiometric bioimaging, and especially, excellent
species in living systems owing to its various advantages such selectivity even at high concentrations of GSH and ascorbate.
as high sensitivity, non-invasiveness, and high spatiotemporal
The 1,8-naphthalimide fluorophore containing an electron
resolution.5 Therefore, the development of fluorescent probes donor and an acceptor has been continually used in ratiometric
for the determination of HNO has attracted intense interest.5,6 fluorescent probes owing to its outstanding internal charge
Lippard et al. pioneered a series of fluorescent probes for transfer (ICT) structure and desirable photophysical properties,
HNO based on the design strategy of HNO-induced reduction such as a large Stokes shift and insensitivity to pH.9 Recently,
of Cu(II) to Cu(I).6a–f Subsequently, Yao et al. developed two we successfully developed a series of 4-hydroxynaphthalimide-
fluorescent probes for imaging HNO in living cells employing derived ratiometric fluorescent probes.10 In connection with our
continuing research, we herein describe the design and synthesis of
a Key Laboratory of Cluster Science of Ministry of Education, Beijing Key Laboratory
of Photoelectronic/Electrophotonic Conversion Materials, School of Chemistry,
Beijing Institute of Technology, Beijing 100081, China.
a simple 4-hydroxynaphthalimide-based ratiometric fluorescent
probe (Scheme 1, 1) employing the 2-(diphenylphosphino)benzoate
moiety as a recognition receptor for the detection of HNO. Probe 1
possesses a compromised ICT structure due to the opposite
electron-withdrawing carbonyl group of the 2-(diphenylphosphino)-
benzoate moiety. HNO-mediated cleavage of the 2-(diphenyl-
phosphino)benzoate moiety shows long-wavelength absorption
E-mail: zhangxl@bit.edu.cn; Fax: +86-10-88875298; Tel: +86-10-88875298
b School of Resources and Environment, University of Jinan, Jinan 250022, China.
E-mail: lcyzbc@163.com
† Electronic supplementary information (ESI) available: Synthesis procedures,
additional spectra and experimental details. See DOI: 10.1039/c4cc00980k
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of ratiometric fluorescent probes with a large emission shift. As
expected, upon addition of HNO (Angeli’s salt (AS), a commonly
employed HNO donor), the maximum emission peak exhibited
a 128 nm red shift, which makes probe 1 possess the potential
of enhancing resolution in the ratiometric bioimaging (Fig. 1).
At the same time, the maximum absorption peak underwent a red
shift to 454 nm accompanied by the color change of the solution
from colorless to yellow, and thus 1 can serve as a ‘‘naked-eye’’
probe for HNO (Fig. S1, ESI†). The remarkable changes in absorp-
tion and fluorescence spectra might be attributed to the cleavage
of the opposite electron-withdrawing carbonyl group of the
2-(diphenylphosphino)benzoate moiety and the coinstantaneous
production of stronger electron-push capacity of the oxygen
anion. Additionally, there was a good linearity between the
fluorescence intensity ratio, R (F546/F418), and the concentrations
of AS in the range of 2 to 35 mM with a detection limit of 0.5 mM12
(Fig. 1). These results demonstrated that probe 1 could detect
HNO qualitatively and quantitatively by the ratiometric fluores-
cence method with excellent sensitivity.5,6
Scheme 1 Proposed reaction mechanism of 1 and HNO.
and fluorescence of 4-hydroxy-1,8-naphthalimide owing to
the stronger electron donating ability of oxygen anions.11 The
detailed recognition mechanism was further confirmed by the
identification of resultant products 4-hydroxy-1,8-naphthalimide
and (diphenylphosphonyl)benzamide. The recognition mechanism
of probe 1 for HNO is shown in Scheme 1. Probe 1 was prepared
in a satisfactory yield from the corresponding 4-hydroxy-1,8-
naphthalimide and 2-(diphenylphosphino)benzoic acid. Detailed
procedures and characterization are described in the ESI.†
We firstly investigated the spectroscopic properties of probe
1 under physiological conditions (5 mM PBS, pH 7.4). In the
absence of HNO, probe 1 displays one major absorption band
centered at 350 nm with a corresponding blue-colored fluores-
cence maximum at 418 nm (F = 0.18) (Fig. 1 and Fig. S1, ESI†).
The relative blue shift of these absorption and emission spectra
compared to other 4-alkoxylnaphthalimide dyes is ascribed to
the introduction of opposite electron-withdrawing carbonyl
groups, and is of significant importance for the construction
Response time is a fundamental parameter for most
reaction-based probes, and the kinetic profile of the reaction
of probe 1 and HNO at room temperature was examined
(Fig. S2–S5, ESI†). Upon addition of 100 mM AS, the pseudo-first-
order rate constant was determined to be kobs = 0.2759 minꢀ1
.
With the various concentrations of AS (40, 50 and 100 mM), the
second-order rate constant for theꢀr3eaction of probe 1 and AS was
also calculated as k0 = 3.3 ꢁ 10 mMꢀ1 minꢀ1. These results
implied that our proposed probe would provide a rapid analytical
method for the detection of HNO.
Then we evaluated the selectivity of probe 1 towards HNO
under the same analytical conditions. As shown in Fig. 2, nearly
no fluorescence intensity changes were observed in the presence
of NO3ꢀ, NO2ꢀ, O2ꢀ, tert-butylhydroperoxide (TBHP), ascorbate,
H2O2, the tert-butoxy radical (ꢂOtBu), the hydroxyl radical (ꢂOH),
Fig. 1 Fluorescence responses of 1 (5 mM) toward different concentra-
tions of AS (final concentration: 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26,
28, 30, 35, 40, 45, 50, 70, and 100 mM); inset: the fluorescence intensity
ratio (F546/F418) of 1 vs. increasing concentrations of AS (final concen-
tration: 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, and 35 mM).
Each spectrum was acquired 20 min after AS addition at 25 1C.
Fig. 2 Fluorescence responses of 1 (5 mM) toward various biospecies
(50 mM except for notation). 1, NO3ꢀ and NO2ꢀ; 2, HNO; 3, O2ꢀ; 4, TBHP;
5, ascorbate (1 mM); 6, H2O2; 7, ꢂOtBu; 8, ꢂOH; 9, Cys; 10, GSH; and 11,
GSH (1 mM). Bars represent fluorescence intensity ratio F546/F418. Each
spectrum was acquired 20 min after various analytes addition at 25 1C.
6014 | Chem. Commun., 2014, 50, 6013--6016
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In conclusion, we have presented the design, synthesis and
properties of an ICT-based ratiometric fluorescent probe 1 for
HNO with the design platform of carbonyl protected 4-hydroxy-
naphthalimide. Probe 1 exhibits high HNO-selectivity even in
the presence of high concentration of GSH and ascorbate,
which is ascribed to the adoption of the 2-(diphenylphos-
phino)benzoate moiety. In addition, probe 1 displays a 104 nm
red-shift of absorption spectra and the color changes from
colorless to yellow upon addition of HNO, and thus can serve
as a ‘‘naked-eye’’ probe for HNO. Importantly, probe 1 can detect
HNO quantitatively by the ratiometric fluorescence method with
a 128 nm red-shifted emission with excellent sensitivity.
We highlight the simplicity of the design and synthesis of probe
1, and its combined properties, such as high specificity and
sensitivity, fast response, visual and ratiometric fluorescence
determination with a large red-shifted emission and ratiometric
bioimaging in living cells, and anticipate that this probe would
be of great benefit to biological researchers for investigating the
detailed function of HNO in living systems.
Fig. 3 Confocal fluorescence images of living RAW 264.7 macrophage
cells: RAW 264.7 macrophage cells incubated with probe 1 (5 mM) for
20 min: (b) blue channel, (c) orange channel, (d) ratio image generated
from (c) and (b), and (a) bright-field transmission image; RAW 264.7
macrophage cells incubated with probe 1 (5 mM) for 20 min were further
treated with 50 mM AS for another 10 min: (f) blue channel, (g) orange
channel, (h) ratio image generated from (g) and (f), and (e) bright-field
transmission image; RAW 264.7 macrophage cells incubated with probe 1
(5 mM) for 20 min were further treated with 100 mM AS for another 10 min:
(j) blue channel, (k) orange channel, (l) ratio image generated from (k) and
(j), and (i) bright-field transmission image. Incubation was performed at
37 1C under a humidified atmosphere containing 5% CO2.
We gratefully acknowledge financial support from the
National Nature Science Foundation of China (No. 21275018,
21107029 and 21203008), Outstanding Young Scientists Award
Fund of Shandong Province (BS2013HZ007), Postdoctoral
Science Foundation of China (2013M541953), Research Fund
for the Doctoral Program of Higher Education of China (RFDP)
cysteine (Cys) and GSH, which is ascribed to the adoption of a (No. 20121101110049) and the 111 Project (B07012).
HNO-specific receptor of the 2-(diphenylphosphino)benzoate
moiety. In addition, the effects of interference of the above-
mentioned other analytes on monitoring HNO were investigated
(Fig. S6, ESI†). Thus, these results demonstrated that probe 1
possesses high selectivity towards HNO even in the presence of
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6016 | Chem. Commun., 2014, 50, 6013--6016
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