Benzo[a]phenoxazinium-based red-emitting chemosensor for zinc ions in biological media

Article abstract of DOI:10.1021/ol2008022

A benzo[a]phenoxazinium-based chemosensor bearing an N,N-di(2-picolyl) ethylenediamine unit was successfully synthesized. It is a long-wave emission and fully water-soluble fluorescent sensor with good membrane permeability for the selective detection of Zn²⁺.

Full text of DOI:10.1021/ol2008022

ORGANIC
LETTERS
2011
Vol. 13, No. 10
2710–2713
Benzo[a]phenoxazinium-Based
Red-Emitting Chemosensor for
Zinc Ions in Biological Media
Xue-Bo Yang,† Bai-Xia Yang,‡ Jian-Feng Ge,*,† Yu-Jie Xu,‡ Qing-Feng Xu,† Jie Liang,†
and Jian-Mei Lu*,†
Key Laboratory of Organic Synthesis of Jiangsu Province, College of Chemistry,
Chemical Engineering and Material Science, Soochow University, 199 Ren’Ai Road,
Suzhou 215123, China, and School of Radiation Medicine and Public Health, Soochow
University, 199 Ren’Ai Road, Suzhou 215123, China.
lujm@suda.edu.cn; ge_jianfeng@hotmail.com
Received March 26, 2011
ABSTRACT
A benzo[a]phenoxazinium-based chemosensor bearing an N,N-di(2-picolyl)ethylenediamine unit was successfully synthesized. It is a long-wave
emission and fully water-soluble fluorescent sensor with good membrane permeability for the selective detection of Zn2þ
.
Zinc is the second most abundant transition metal in the
human body, since it is mostly trapped within proteins as a
structural or catalytic cofactor,1 and there is a pool of Zn2þ
which is loosely bound or chelatable in the brain.2 It is also
known that disorder of Zn2þ metabolism is closely asso-
ciated with many severe neurological diseases, including
Alzheimer’s disease (AD),3 cerebral ischemia,4 and
epilepsy.5 Therefore, the measurement of Zn2þ is impor-
tant in monitoring biological processes. For this purpose,
several fluorescent Zn2þ sensors have been documented.6aꢀm
However, improvements are needed to overcome several
limitations when they were applied to detect zinc in biological
samples. First, most of reported sensors need to be excited by
UV light,6aꢀi which can cause damage to living cells. Second,
their fluorescence lies in the visible region,6 which cannot
penetrate deep enough into human tissue. The sensor with
long-wave absorption or emission region (650ꢀ900 nm)
attracted much attention, since autofluorescence and photo-
damage to living cells are reduced in this region.6n,o However,
(6) (a) Maruyama, S.; Kikuchi, K.; Hirano, T.; Urano, Y.; Nagano,
T. J. Am. Chem. Soc. 2002, 124, 10650. (b) Xu, Z.; Han, S. J.; Lee, C.;
Yoon, J.; Spring, D. R. Chem. Commun. 2010, 46, 1679. (c) Hanaoka, K.;
Muramatsu, Y.; Urano, Y.; Terai, T.; Nagano, T. Chem.;Eur. J. 2010,
16, 568. (d) Chen, H.; Gao, W.; Zhu, M.; Gao, H.; Xue, J.; Li, Y. Chem.
Commun. 2010, 46, 8389. (e) Xue, L.; Liu, C.; Jiang, H. Chem. Commun.
2009, 1061. (f) Aoki, S.; Sakurama, K.; Matsuo, N.; Yamada, Y.;
Takasawa, R.; Tanuma, S.-i.; Shiro, M.; Takeda, K.; Kimura, E.
Chem.;Eur. J. 2006, 12, 9066. (g) Komatsu, K.; Kikuchi, K.; Kojima,
H.; Urano, Y.; Nagano, T. J. Am. Chem. Soc. 2005, 127, 10197. (h)
Felton, C. E.; Harding, L. P.; Jones, J. E.; Kariuki, B. M.; Pope, S. J. A.;
Rice, C. R. Chem. Commun. 2008, 6185. (i) Zhang, L.; Dong, S.; Zhu, L.
Chem. Commun. 2007, 1891. (j) Komatsu, K.; Urano, Y.; Kojima, H.;
Nagano, T. J. Am. Chem. Soc. 2007, 129, 13447. (k) Wong, B. A.;
Friedle, S.; Lippard, S. J. Inorg. Chem. 2009, 48, 7009. (l) Qian, F.;
Zhang, C.; Zhang, Y.; He, W.; Gao, X.; Hu, P.; Guo, Z. J. Am. Chem.
Soc. 2009, 131, 1460. (m) Zhou, X.; Yu, B.; Guo, Y.; Tang, X.; Zhang,
H.; Liu, W. Inorg. Chem. 2010, 49, 4002. (n) Guo, Z.; Zhu, W.; Zhu, M.;
Wu, X.; Tian, H. Chem.;Eur. J. 2010, 16, 14424. (o) Peng, X.; Wu, T.;
Fan, J.; Wang, J.; Zhang, S.; Song, F.; Sun, S. Angew. Chem., Int. Ed.
2011, 50, 4180.
† Key Laboratory of Organic Synthesis of Jiangsu Province.
‡ School of Radiation Medicine and Public Health.
(1) Berg, J. M.; Shi, Y. Science 1996, 271, 1081.
(2) Frederickson, C. J. Int. Rev. Neurobiol. 1989, 31, 145.
(3) Bush, A. I.; Pettingell, W. H.; Malthaup, G.; Paradis, M.;
Vonsattel, J. P.; Gusella, J. F.; Beyreuther, K.; Masters, C. L.; Tanzi,
R. E. Science 1994, 265, 1464.
(4) Koh, J. Y.; Suh, S. W.; Gwag, B. J.; He, Y. Y.; Hsu, C. Y.; Choi,
D. W. Science 1996, 272, 1013.
(5) Frederickson, C. J.; Hernandez, M. D.; McGinty, J. F. Brain Res.
1989, 480, 317.
r
10.1021/ol2008022
Published on Web 04/26/2011
2011 American Chemical Society

cases of Zn2þ sensors in the long-wave fluorescent emission
are still less common.7 Only two of them, which worked in
organic cosolvent required conditions, could be used in
imaging in biological systems.7a,b And there is no report on
a full water-soluble long-wave fluorescence probe for ima-
ging zinc in biological samples. In this regard, it is highly
desirable to develop Zn2þ-selective full water-soluble long-
wave fluorescent sensors to match harmless imaging and
visualization of Zn2þ in living cells.
Scheme 1. Synthesis of Benzo[a]phenoxazinium 1
Benzo[a]phenoxazinium derivatives have absorption
bands in the 580ꢀ6108 or 629ꢀ632 nm9 region, and the
corresponding emissionbandsare between 650 and 670 nm
in water.8,9 They have good membrane permeability and
comparativelylow toxicityaccording toinvitro and invivo
assay results,10a,b and a red-emitting phenoxazinium based
probe, 3-(diethylamino)-7-(1,4-dioxa-7,13-dithia-10-aza-
cyclopentadecan-10-yl)phenoxazin-5-ium chloride, has
been reported as a low-cost and real-time monitoring
probe for selective ratiometric detection of Hg2þ in pure
water.10c Therfore, we have focused on the synthesis of a
new sensor based on the benzo[a]phenoxazinium skeleton
and investigated its chemodosimetric properties that can
provide sensitive measurement of the Zn2þ in a 100%
aqueous environment. Compound 1 exhibits an absorp-
tionpeakat 582 nm and hasthe maximal emission bands at
656 nm with a high quantum yield in PBS buffer. More
importantly, the chloride salt shows an excellent solubility
and intense fluorescence, even in aqueous media. In addi-
tion, for demonstration of its application in biological
samples, the application of 1 to cultured KB human oral
epidermoid carcinoma cell (KB cell) were implemented
and discussed.
the light blue color of the solution. During photometric
titrations of 1 with Zn2þ, the absorption wavelength at 582
nm was slightly changed, and there was gradually decrease
of absorption at 623 nm. These may be induced by
the changes of the molecular orbitals resulting of the
complexation of Zn2þ with the N,N-di(2-picolyl)ethyl-
enediamine (DPEN) unit.
As depicted in Scheme 1, the benzo[a]phenoxazinium
derivative (1) was prepared in 65% yield from the reaction
of 5-(ethylamino)-4-methyl-2-nitrosophenol (2) with
naphthalene derivative (3) in acidic ethanol solution. The
identities of all synthetic compounds were fully confirmed
1
by H NMR, 13C NMR, and mass spectroscopy. All
photochemical experiments were carried out in PBS buffer
at pH 7.4 without any organic cosolvent.
Figure 1 shows the changes in the absorption spectrum
of 1 as a function of Zn2þ concentration at room tempera-
ture. The UVꢀvis spectrum of 1 is characterized by two
Figure 1. Changes in the UVꢀvis spectra of 1 (20 ꢁ 10ꢀ6 M in
PBS buffer, pH = 7.4) upon titration by ZnCl2 from 1.5 ꢁ 10ꢀ6
M to 25 ꢁ 10ꢀ6 M.
absorption maxima at 582 nm (ε = 35000 Mꢀ1 cmꢀ1) and
3
623 nm (ε = 25000 Mꢀ1 cmꢀ1). These are responsible for
3
According to the linear BenesiꢀHildebrand expr-
ession,11 the measured absorbance [1/(A ꢀ A0)] at 623
nm shows a linear relationship with a change of 1/[Zn2þ
]
(7) (a) Lu, X.; Zhu, W.; Xie, Y.; Li, X.; Gao, Y.; Li, F.; Tian, H.
Chem.;Eur. J. 2010, 16, 8355. (b) Tang, B.; Huang, H.; Xu, K.; Tong,
L.; Yang, G.; Liu, X.; An, L. Chem. Commun. 2006, 3609. (c) Atilgan, S.;
Ozdemir, T.; Akkaya, E. U. Org. Lett. 2008, 10, 4065. (d) Hung, C.-H.;
Chang, G.-F.; Kumar, A.; Lin, G.-F.; Luo, L.-Y.; Ching, W.-M.;
Wei-Guang Diau, E. Chem. Commun. 2008, 978. (e) Kiyose, K.; Kojima,
H.; Urano, Y.; Nagano, T. J. Am. Chem. Soc. 2006, 128, 6548.
(8) (a) Frade, V. H. J.; Gonc-alves, M. S. T.; Moura, J. C. V. P.
Tetrahedron Lett. 2006, 47, 8567. (b) Lee, M. H.; Lee, S. W.; Kim, S. H.;
Kang, C.; Kim, J. S. Org. Lett. 2009, 11, 2101.
(9) Jose, J.; Ueno, Y.; Burgess, K. Chem.;Eur. J. 2009, 15, 418.
(10) (a) Ge, J. F.; Arai, C.; Yang, M.; Md., A. B.; Lu, J.; Ismail,
N. S. M.; Wittlin, S.; Kaiser, M.; Brun, R.; Charman, S. A.; Nguyen, T.;
Morizzi, J.; Itoh, I.; Ihara, M. ACS Med. Chem. Lett. 2010, 1, 360. (b)
Ihara, M. Heterocycles 201110.3987/REV-11-706. (c) Yang, X. B.; Ge,
J. F.; Xu, Q. F.; Zhu, X. L.; Li, N. J.; Gu, H. W.; Lu, J. M. Tetrahedron Lett.
2011, 52, 2492.
(R = 0.994), indicating the 1:1 stoichiometry between
Zn2þ and 1 (Figure 2 and Figure S3, Supporting Infor-
mation). The job plot analysis12 of the UVꢀvis titrations
carried out in water revealed a maximum of 50% mole
fraction in accordance with the proposed 1:1 binding
stoichiometry (Figure S1, Supporting Information). On
the basis of the 1:1 stoichiometry and UVꢀvis titration
(11) Zhu, M.; Yuan, M. J.; Liu, X. F.; Xu, J. L.; Lv, J.; Huang, C. S.;
Liu, H. B.; Li, Y. L.; Wang, S.; Zhu, D. B. Org. Lett. 2008, 10, 1481.
(12) Zhou, Y.; Wang, F.; Kim, Y.; Kim, S.-J.; Yoon, J. Org. Lett.
2009, 11, 4442.
Org. Lett., Vol. 13, No. 10, 2011
2711

Figure 2. BenesiꢀHilderbrand plot of 1 with Zn2þ
.
Figure 4. Fluorescence response of 1 (20 ꢁ 10ꢀ6 M) containing
20 ꢁ 10ꢀ6 M Zn2þ to the selected metal ions (20 ꢁ 10ꢀ6 M: Cd2þ
Cu2þ, Pb2þ, Fe3þ, Agþ, Ba2þ, Mn2þ; 100 ꢁ 10ꢀ3 M: Kþ, Naþ,
Mg2þ, Ca2þ) in PBS buffer (pH = 7.4). F1þZn indicates the fluo-
rescence signals of 1 in the presence of Zn2þ and F1þZnþM den-
otes the fluorescence signals of 1 in the presence of Zn2þ and
competing ions. Excitation was at 579 nm and emission was at
656 nm.
,
data in Figure 1, the association constant of 1 with Zn2þ in
water was found to be 7.35 ( 0.90 ꢁ 104 Mꢀ1
.
The fluorescence titration of 1 (20 ꢁ 10ꢀ6 M) in the pre-
sence of different Zn2þ concentrations was also performed. As
shown in Figure 3, sensor 1 showed a fluorescence emission at
656 nm (Φf = 0.15). Upon increasing the concentration of
Zn2þ to 25 ꢁ 10ꢀ6 M, the emission intensity at 656 nm gradu-
ally increased (Φf = 0.28, Table S1, Supporting Information).
The fluorescent probe 1 (20 ꢁ 10ꢀ6 M) can detect the con-
centration of Zn2þ from 1.5 ꢁ 10ꢀ6 to 20 ꢁ 10ꢀ6 M (Figure 3
Inset) in PBS buffer (pH = 7.4). At the employed concentra-
tion of the probe (20 ꢁ 10ꢀ6 M), the limit of detection for Zn2þ
is 1.5 ꢁ 10ꢀ6 M (Figure 3 inset). The excitation and emission
spectra are shown in Figure S4, Supporting Information.
The titration of Zn2þ to 1 in the presence of the screened
metal cations (Kþ, Naþ, Mg2þ, Pb2þ, Agþ, Ba2þ, and
Mn2þ) did not lead to any changes to the fluorescence
output. Cd2þ and Ca2þ enhanced the fluorescence inten-
sity, and some paramagnetic metal ions, such as Fe3þ and
Cu2þ, slightly quench the fluorescence.
Figure 5. The fluorescence intensity of compound 1 (20 ꢁ 10ꢀ6
M) in the absence (9) and in the presence (1) of Zn2þ (24 ꢁ 10ꢀ6
M) at different pH values in a phosphate buffer (100 ꢁ 10ꢀ3 M);
Figure 3. Emission spectra of compound 1 in the presence of an
increasing Zn2þ concentration (from 1.5 ꢁ 10ꢀ6 to 25 ꢁ 10ꢀ6 M)
in PBS buffer (pH = 7.4). The excitation wavelength was 579 nm
with 5 nm slit widths. The concentration of chemosensor 1 was
20 ꢁ 10ꢀ6 M. Inset: Fluorescence intensity of 1 versus the Zn2þ
concentration at 656 nm.
λ
ex = 579 nm, λem = 656 nm.
The fluorescence intensity changes at 656 nm at a pH
ranging from 5.90 to 7.70 are shown in Figure 5. The
fluorescence intensity of 1 was slight enhanced with the
increase of pH value, and the situation was opposite on the
mixed solution of 1 and Zn2þ. The differentials of emission
intensity around pH ∼7.4 did not have a large variation,
which means 1 can be used to analysize intracellular Zn2þ
ions in biological samples.
To evaluate the selectivity of the fluorescent probe 1
toward Zn2þ ions, competition experiments were per-
formed. As shown in Figure 4, only some of the other
cations interfere with the fluorimetric analysis of Zn2þ
.
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Org. Lett., Vol. 13, No. 10, 2011

In addition, the MTT assay was used to investigate the
cytotoxicity of 1 to the KB cell (Figures S5, Supporting
Information). The cell viability is about 89ꢀ109%, which
was obtained by the percentage of KB cell viability remain-
ing after cell treatment with 1 (untreated cells were con-
sidered to have 100% survival) between 10 ꢁ 10ꢀ6 and
30 ꢁ 10ꢀ6 M, and the damage of MTT for the cells will be
reduced by the protecting from compound 1 at 20 ꢁ 10ꢀ6
M concentration. The result indicated the probe 1 has no
obvious cytotoxicity in the concentration region, and 1 can
be a suitable fluorescence chemosensing probe for the
detection of Zn2þ in biological systems.
In conclusion, this study presents an example of the
selection of a suitable fluorophore for zinc ion probes
from existing or potential drug molecules. A benzo[a]-
phenoxazinium-based chemosensor bearing an N,N-di-
(2-picolyl)ethylenediamine unit (1) was used as a promis-
ing analytical tool for detecting fluorometric zinc ions with
long-wave emission. Specifically, this probe is one of the
few red-emitting fluorescent probes that allows for the
selective detection of zinc ions in PBS buffer with no
organic cosolvent required. Notably, the selectivity of the
probe for Zn2þ over other metal ions is extremely high. In
the sense of long-wave excitation/emission, water solubi-
lity, low cytotoxicity, and membrane permeability nature,
1 can be one of the most promising probes for the detection
of Zn2þ in water and in living cells.
Figure 6. Confocal fluorescence images in KB cells. Top (a, b):
Cells incubated with 1 (20 ꢁ 10ꢀ6 M) for 0.5 h. Bottom (c, d):
Cells incubated with ZnCl2 (100 ꢁ 10ꢀ6 M) for 0.5 h and washed
with PBS, then 1 (20 ꢁ 10ꢀ6 M) for 0.5 h. Emission was collected
at 520 ꢀ 620 nm upon excitation at 514 nm. Bright field (a and c)
fluorescence (b and d).
The application of 1 on the cultured cells was checked.
Bright-field measurements confirmed that the cells after
treatment with Zn2þ and 1 were viable throughout the
imaging experiments (Figure 6a and 6c). Under selective
Acknowledgment. We are grateful to Professor Masataka
Ihara (Hoshi University, Japan) for the helpful discussion
regarding the synthesis of 1. The project is financially sup-
ported by the National Environmental Community Project
of China (200909044), Natural Science Fund (BK2009113),
and Natural Science Fund for Colleges and Universities
(08KJB430013) in Jiangsu Province.
excitation of 1, staining KB cells with a 20 ꢁ 10ꢀ6
M
solution of 1 for 30 min at 37 °C led to very weak
intracellular fluorescence (Figure 6b). After the cells were
supplemented with ZnCl2 (20 ꢁ 10ꢀ6 M) for 30 min at
37 °C and washed with PBS, then loaded with 1 under the
same conditions, a significant increase in the fluorescence
from the intracellular areawas observed (Figure6d). These
results suggested that 1 has good membrane permeability
Supporting Information Available. Synthesis; charac-
terization of 1; KB cell culture MTT assay. This material
is available free of charge via the Internet at http://pubs.
acs.org
and can be used to monitor changes in intracellular Zn2þ
.
It can be useful in clarifying the intracellular concentration
of Zn2þ in biological systems.
Org. Lett., Vol. 13, No. 10, 2011
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