A new triphenylamine fluorescent dye for sensing of cyanide anion in living cell

Article abstract of DOI:10.1016/j.tetlet.2013.07.011

A triphenylamine-based fluorescent probe 1 with larger Stokes shift (～141 nm) has been synthesized, which can make an efficient nucleophilic addition by reacting with CN^- in water. Upon addition of CN ^-, the red fluorescence of 1 was distinctly quenched. Importantly, 1 exhibits a highly selective response toward CN^- over other anions in water and a high sensitivity (detection limit ≤ 11 nM). Moreover, it has potential application to track CN^- levels in living cells by confocal laser scanning microscopy (CLSM).

Full text of DOI:10.1016/j.tetlet.2013.07.011

Tetrahedron Letters 54 (2013) 4942–4944
Contents lists available at SciVerse ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
A new triphenylamine fluorescent dye for sensing of cyanide anion
in living cell
a,
Yi Qu a,b, Bin Jin a, Yi Liu b, Yongquan Wu b, Lin Yang a, Junchen Wu a, , Jianli Hua
⇑
⇑
a Key Laboratory for Advanced Materials and Institute of Fine Chemicals, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, China
b Department of Chemistry & Laboratory of Advanced Materials, Fudan University, Shanghai 20043, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A triphenylamine-based fluorescent probe 1 with larger Stokes shift (ꢀ141 nm) has been synthesized,
which can make an efficient nucleophilic addition by reacting with CNÀ in water. Upon addition of
CNÀ, the red fluorescence of 1 was distinctly quenched. Importantly, 1 exhibits a highly selective
response toward CNÀ over other anions in water and a high sensitivity (detection limit 6 11 nM). More-
over, it has potential application to track CNÀ levels in living cells by confocal laser scanning microscopy
(CLSM).
Received 9 April 2013
Revised 27 May 2013
Accepted 2 July 2013
Available online 9 July 2013
Keywords:
Cyanide anion
Ó 2013 Elsevier Ltd. All rights reserved.
Fluorescent sensor
Triphenylamine–pyridine
Living cell
Cyanide ion, as a highly toxic anion, has drawn considerable
attention for its detection and determination.1 The high toxicity
of cyanide lies in its ability to bind with the ferric iron in cyto-
chrome oxidase and inhibit oxygen utilization by cells.2 According
to the guidelines of the World Health Organization (WHO), the
was used for larger Stokes shift13 in which the excitation energy
of the energy-donor is transferred to the energy-acceptor. On the
other hand, two signal moieties in one sensory unit make the big-
ger molecular weight and larger scale.
Therefore, we are interested in developing a specific fluorescent
sensor with a larger Stokes shift for sensitive CNÀ detection in
water and living cells. Our idea is to use the nucleophilic addition
reaction of CNÀ with a dicyanovinyl group as the strategy for the
design of a probe to track CNÀ levels in living cells with high sen-
sitivity. Triphenylamine was used as a fluorophore whose strong
donating property can induce an effective redshift of the maximum
emission peak with the connection of a smaller electron-with-
drawing group. For the purpose, the ICT-based sensor 1 was de-
signed and synthesized (Scheme 1 and Fig. S1). Compound 1
contains two pyridine groups and a dicyanovinyl group which is
connected by a triphenylamine.14 Pyridine-attached groups can
not only improve the water-solubility of 1 but also serve as an elec-
tron-withdrawing group. More importantly, a dicyanovinyl-intro-
duced group plays a vital role as a cyanide reactive site.15 As
shown in Figure 1A, 1 indicated two strong absorption bands at
ca. 360 and 465 nm in water, which were ascribed to the ICT pro-
cess. The obvious red emission at 612 nm was found as shown in
Figure 1B with a Stokes shift larger than 140 nm indicating that a
full emission spectrum can be obtained by choosing maximum
absorbance as excitation wavelength and a small overlap between
absorption and emission spectra. Upon the addition of CNÀ, a solu-
tion of 1 can result in a color change from orange to colorless
accompanying a gradual decrease in absorption intensity. The re-
sult indicated the generation of stabilized anionic species 1-CN
acceptable level of CNÀ is lower than 1.9
and 20
l
M in drinking water
l
M is considered as its lethal level.3 By now, the detection
limit of previous studies can only reach the micromolar concentra-
tion, which is higher than the physiologically lethal level
(ꢀ20
l
M).4 Moreover, in the presence of other anions, such as fluo-
ride and acetate, the selectivity of CNÀ in water is still a great
challenge.5
More recently, there has been increasing interest in designing
the chemodosimeter for the sensing of cyanide with the purpose
of minimizing the interference of other anions, and this usually
has been developed in different solvent systems by the nucleo-
philic addition of CNÀ to pyrylium,6 pyrazolone,7,8 tri-fluoroaceto-
phenone,9 benzil derivatives,10 Meldrum’s acid11, etc.12 However,
many achievements have been mainly focused on the design of
recognized groups, which always attach on the commercial fluoro-
phores with short excited/emission wavelength. For example, cou-
marin was an excellent fluorophore with brightness emission
around 490 nm, but small Stokes shift make obvious overlap be-
tween absorption and emission spectra which is a big puzzle for
selecting an optimized excitation wavelength. Among several
strategies, Föter resonance energy-transfer (FRET) mechanism
⇑
Corresponding authors. Tel.: +86 21 64250940; fax: +86 21 64252758.
E-mail addresses: jcwu@ecust.edu.cn (J. Wu), jlhua@ecust.edu.cn (J. Hua).
0040-4039/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetlet.2013.07.011

Y. Qu et al. / Tetrahedron Letters 54 (2013) 4942–4944
4943
HOMO
LUMO
LUMO+1
1
Scheme 1. Structures of 1 and 1-CN.
1-CN
which can be confirmed by 1H NMR, 13C NMR, HRMS, and IR spec-
tra (Figs. S2–S5).
Figure 2. Electron density maps of the frontier molecular orbitals of 1 and 1-CN.
Fluorescent properties of sensor 1 were also studied by adding
CNÀ to a pure water solution of 1 (2.5
lM) at room temperature. As
shown in Figure 1B, the emission 1 at 612 nm (kex = 465 nm) de-
creased rapidly upon the addition of CNÀ. When 2.4 equiv CNÀ
was added to the solution of 1 (Uf = 0.16, pH 7, Fig. S1), a fivefold
(F0/F) decrease in fluorescence intensity at 612 nm was observed.
These facts indicate that 1 can serve as a ‘naked-eye’ sensor for
CNÀ.
This is further supported through TD-DFT calculations on 1 and
1-CN compounds. The molecular orbitals of these compounds are
presented in Figure 2. The HOMO orbital of 1 is localized on the en-
tire molecule including the donor and acceptor. The LUMO orbital
is concentrated on the acceptor side of dicyano-vinyl and LUMO+1
is on the pyridine groups, thus resulting in a charge transfer from
triphenylamine to dicyano-vinyl and pyridine. Interestingly, the
electron density of 1-CN compound is significantly contributed to
the cyanation of the anionic species at the HOMO level, but the
LUMO state stretches out to the pyridine moiety. However, the
electron density of the phenyl group connected with the cyanine
anion is low. ICT process in sensor 1 is also weak. Furthermore,
we also calculated the lowest excitation energies of 1 and 1-CN
(Table 1). Calculation results indicated the absorbance at short
wavelength became stronger than that at long wavelength in 1-
CN, which is close to the experimental results (Table 1). Although
the computational absorption spectra underestimated the experi-
mental absorption maxima in general as shown in Table 2, analo-
gous spectroscopic behaviors were reproduced qualitatively.
Moreover, absorption band at 471 nm in 1-CN is close to the exper-
imental results (465 nm).
To better evaluate the cyanide-selective nature of 1, fluores-
cence changes caused by the addition of other anions, including
FÀ, ClÀ, BrÀ, IÀ, CO32À, HCOÀ3 , SO23À, H2POÀ, HPO2À, PO43À, SCNÀ,
4
4
NOÀ2 , S2O8À, AcOÀ, S2À and N3À were investigated. A pure water solu-
tion of 1 (2.5 lM), containing 40 equiv of each of these anions,
were maintained for 20 min at room temperature and then sub-
jected to fluorescence analysis. As shown in Figure 3, anions other
Table 1
Calculated TD-DFT (MPW1K) excitation energies for the lowest transition (eV, nm),
oscillator strengths (f), and composition in terms of molecular orbital contributions,
and experimental absorption maxima
State
Compositiona
E (nm)
f
Exp. (nm)c
1
S1
S2
S1
S2
94% H ? L
88% H ? L+1
98% H ? L
414.15
309.95
471.62b
419.19b
1.2040
0.7433
0.0083
0.0152
465
360
465
360
1-CN
83% H ? L+1
a
b
c
H = HOMO, L = LUMO, L+1 = LUMO+1.
TD-DFT calculation in vacuo.
In H2O.
Figure 1. (A) Absorption titration of 1 (2.5
1.2, 1.6, 2.0, 2.4, 2.8, 3.2, 3.6, 4.0, 4.4, 4.8, and 5.2
l
M) with the addition of CNÀ (0, 0.4, 0.8,
M) in water at room temperature
Figure 3. Various anions selectivity profiles of 1 (2.5
lM) in the presence of various
l
anions in water (pH 7): (black bars) emission ratios of (F0 À F)/F0 at 612 nm in the
(inset: the color change images of 1). (B) Emission titration of 1 (kex = 465 nm) with
presence of 40 equiv of FÀ, ClÀ, BrÀ, IÀ, CO23À, HCO3À, SO23À, H2POÀ, HPO2À, PO34À
,
4 4
addition of CNÀ (0, 0.4, 0.8, 1.2, 1.6, 2.0, 2.4, 2.8, 3.2, 3.6, 4.0, 4.4, 4.8, and 5.2
lM) in
SCNÀ, NO2À, S2OÀ8 , AcOÀ, S2À and NÀ3 ; (blue bars) fluorescence intensity in the
presence of various anions, followed by 4.0 equiv of CNÀ.
water at room temperature (inset: the fluorescent change images of 1).

4944
Y. Qu et al. / Tetrahedron Letters 54 (2013) 4942–4944
be more than 90%. Even at a much higher concentration of 1
(100 M), cell viabilities were still over 60% (Fig. S8). Hence, probe
l
1 has very low cytotoxicity.
In conclusion, we have demonstrated that 1 can be used as a
highly selective and sensitive probe for detecting the nanomolar
level of CNÀ in water. Furthermore, 1 is taken up by cells and can
thus be used for the cell image by CLSM. As 1 does not possess
any general cytotoxicity it might be of interest to use for a specific
bioimaging in vitro and in vivo. We are currently working on devel-
oping a turn-on fluorescent probe for detecting picomolar concen-
tration of CN in water.
Acknowledgements
Figure 4. (A–H) CLSM images (A) HeLa cells incubated with Hoechst 33258 for
15 min at 37 °C. (B) is then stained with 10
(D) are bright-field and overlay images of (A), (B) and (C), respectively. (E) HeLa cells
incubated with Hoechst 33258 for 15 min at 37 °C. (F) is stained with 10 M 1 in
PBS for 15 min at 37 °C, then further treated with 40
M CNÀ at 37 °C for 2 h. (G)
lM 1 in PBS for 15 min at 37 °C. (C) and
This work was supported by NSFC/China (2116110444 and
l
21172073),
National
Basic
Research
973
Program
l
(2013CB733700) and the Fundamental Research Funds for the
Central Universities (WJ0913001).
and (H) are bright-field and overlay images of (E), (F), and (G), respectively (Hoechst
33258: kex = 405 nm, kem = 460 30 nm; 1: kex = 488 nm, kem = 590 50 nm).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.tetlet.2013.07.
011.
References and notes
1. Kulig, K. W. Cyanide Toxicity; U.S. Department of Health and Human Services:
Atlanta, GA, 1991.
2. (a) Yang, R.; Wu, W.; Wang, W.; Li, Z.; Qin, J. Macromol. Chem. Phys. 2010, 211,
18–26; (b) Dong, S.; Ou, D.; Qin, J.; Li, Z. J. Polym. Sci. Part A 2011, 49, 3314–
3327; (c) Lou, X.; Zhang, Y.; Li, S.; Ou, D.; Wan, Z.; Qin, J.; Li, Z. Polym. Chem.
2012, 3, 1446–1452; (d) Chen, X.; Nam, S.-W.; Kim, G.-H.; Song, N.; Jeong, Y.;
Shin, I.; Kim, S. K.; Kim, J.; Park, S.; Yoon, J. Chem. Commun. 2010, 8953–8955.
3. World Health Organization Guidelines for DrinkingWater Quality; World Health
Organization: Geneva, 1996.
4. (a) Dai, Z.; Boon, E. M. J. Am. Chem. Soc. 2010, 132, 11496–11503; (b) Kim, Y.;
Huh, H. S.; Lee, M. H.; Lenov, I. L.; Zhao, H. Y.; Gabbaï, F. P. Chem. Eur. J. 2011, 17,
2057–2062.
Figure 5. Cell viability values (%) estimated by MTT proliferation test at different
concentrations of 1. HeLa cells were cultured in the presence of 1 at 37 °C for 24 h.
5. (a) Ballesteros-Garrido, R.; Abarca, B.; Ballesteros, R.; Arellano, C. R.; Leroux, F.
R.; Colobert, F.; García-España, E. New J. Chem. 2009, 33, 2102–2106; (b) Tang,
Y. H.; Qu, Y.; Song, Z.; He, X. P.; Xie, J.; Hua, J. L.; Chen, G. R. Org. Biomol. Chem.
2012, 10, 555–560.
6. Ros-Lis, J. V.; Martínez-Máñez, R.; Soto, J. Chem. Commun. 2002, 2248–2249.
7. Lou, B.; Chen, Z. Q.; Bian, Z. Q.; Huang, C. H. New J. Chem. 2010, 34, 132–136.
8. Liu, J. L.; Liu, Y.; Liu, Q.; Li, C. H.; Sun, L. N.; Li, F. Y. J. Am. Chem. Soc. 2011, 133,
15276–15279.
than CNÀ do not lead to significant changes in the fluorescence
intensity. As a result, the selectivity profile for CNÀ over other an-
ions is remarkably high. In addition, titration spectrum shows the
detection limit of 1 for CNÀ was estimated to be 11 nM (Fig. S6). It
should be noted that 1 can also be applied for detecting the level of
9. Niu, H. T.; Su, D. D.; Jiang, X. L.; Yang, W. Z.; Yin, Z. M.; He, J. Q.; Cheng, J. P. Org.
Biomol. Chem. 2008, 6, 3038–3040.
10. Sessler, J. L.; Cho, D. G. Org. Lett. 2008, 10, 73–75.
CNÀ in drinking water by the requirement of WHO (1.9
lM).
Furthermore, the application of 1 in the monitoring of intracel-
11. Li, H.; Wen, Z.; Jin, L.; Kan, Y.; Yin, B. Chem. Commun. 2012, 11659–11661.
12. (a) Hong, S. J.; Yoo, J.; Kim, S. H.; Kim, J. S.; Yoon, J.; Lee, C. H. Chem. Commun.
2009, 189–191; (b) Qian, G.; Li, X. Z.; Wang, Z. Y. J. Mater. Chem. 2009, 19, 522–
530; (c) Lou, X. D.; Zeng, Q.; Zhang, Y.; Wan, Z. M.; Qin, J. G.; Li, Z. J. Mater. Chem.
2012, 22, 5581–5586; (d) Lou, X. D.; Qiang, L.; Qin, J. G.; Li, Z. ACS Appl. Mater.
Interfaces 2012, 4, 2133–2138; (e) Lou, X. D.; Zhang, Y.; Qin, J. G.; Li, Z. Chem.
Eur. J. 2011, 17, 9691–9696; (f) Cheng, X. H.; Tang, R. L.; Jia, H. Z.; Feng, J.; Qin, J.
G.; Li, Z. ACS Appl. Mater. Interfaces 2012, 4, 4387–4392.
13. (a) Yuan, L.; Lin, W.; Cao, Z.; Wang, J.; Chen, B. Chem. Eur. J. 2012, 18, 1247–
1255; (b) Yuan, L.; Lin, W.; Xie, Y.; Chen, B.; Song, J. Chem. Eur. J. 2012, 18,
2700–2706; (c) Yuan, L.; Lin, W.; Chen, B.; Xie, Y. Org. Lett. 2012, 14, 432–435.
14. (a) Sumalekshmy, S.; Fahrni, C. J. Chem. Mater. 2011, 23, 483–500; (b) Bhaskar,
A.; Ramakrishna, G.; Twieg, R. J.; Goodson, T. J. Phys. Chem. C 2007, 111, 14607–
14611; (c) Cheng, X.; Li, Q.; Qin, J.; Li, Z. ACS Appl. Mater. Interfaces 2010, 2,
1066–1072.
lular CNÀ concentrations was investigated by confocal laser scan-
ning microscopy (CLSM). As shown in Figure 4B,
a red
luminescence was observed inside the HeLa cells. Overlay of lumi-
nescence and brightfield images demonstrated that the red lumi-
nescence was evident in the cytoplasm over the nucleus and
membrane, which was also confirmed by xz cross-sectional image
(Fig. 4D and Fig. S7). When the HeLa cells were again incubated
with 40 l
M CNÀ at 37 °C for 2 h, the red luminescence was dis-
tinctly decreased (Fig. 4F) while the blue luminescence was slightly
enhanced (Fig. 4H). Finally, these results indicate 1 can be taken
advantage of tracking CNÀ levels in living cells.
The toxicity of 1 for Hela cells was measured by using a stan-
dard MTT assay (Fig. 5). After Hela cells were cultured with 1
(40 lM) in PBS at 37 °C for 24 h, cell viabilities were estimated to
15. (a) Liu, Z.; Wang, X.; Yang, Z.; He, W. J. Org. Chem. 2011, 76, 10286–10290; (b)
Deng, L.; Wu, W.; Guo, H.; Zhao, J.; Ji, S.; Zhang, X.; Yuan, X.; Zhang, C. J. Org.
Chem. 2011, 76, 9294–9304.