Synthesis and application of a full water-soluble and red-emitting chemosensor based on phenoxazinium for copper(ii) ions

Article abstract of DOI:10.1002/cjoc.201200452

A phenoxazinium-based chemosensor (1) bearing di(2-picolyl)amine unit was successfully synthesized. The result shows that it is a red-emitting and full water-soluble chemosensor for the selective detection of Cu²⁺ in pure water. The fluorescence on-off mechanism was studied by ab initio calculations. To confirm the suitability of 1 for biological applications, it was employed in the fluorescence detection of the intracellular Cu²⁺ with cultured KB cells. The results indicated that 1 had good membrane permeability and could be useful for the fluorescence microscopic imaging. A phenoxazinium-based chemosensor (1) bearing di(2-picolyl)amine unit was successfully synthesized. The result shows it is a red-emitting and full water-soluble chemosensor for the selective detection of Cu²⁺ in pure water. The fluorescence on-off mechanism was studied by ab initio calculations. It was also indicated that 1 had good membrane permeability and could be useful for the fluorescence microscopic imaging. Copyright

Full text of DOI:10.1002/cjoc.201200452

FULL PAPER
DOI: 10.1002/cjoc.201200452
Synthesis and Application of a Full Water-Soluble and
Red-Emitting Chemosensor Based on Phenoxazinium
for Copper(II) Ions
Yan, Baolonga(闫保龙)
Sun, Rua(孙如)
Ge, Jianfeng*,a(葛健锋)
Xu, Yujieb(许玉杰)
Zhang, Qianqiana(张欠欠)
Yang, Xueboa(杨学波)
Lu, Jianmei*,a,c(路建美)
a Key Laboratory of Organic Synthesis of Jiangsu Province, College of Chemistry, Chemical Engineering and Ma-
terial Science, Soochow University, Suzhou, Jiangsu 215123, China
b School of Radiation Medicine and Protection, Medicine College of Soochow University, Suzhou, Jiangsu 215123,
China
c Key Laboratory of Functional Materials for Environment Protection of Suzhou City, Soochow University, Suzhou,
Jiangsu 215123, China
A phenoxazinium-based chemosensor (1) bearing di(2-picolyl)amine unit was successfully synthesized. The
＋
result shows that it is a red-emitting and full water-soluble chemosensor for the selective detection of Cu2 in pure
water. The fluorescence on-off mechanism was studied by ab initio calculations. To confirm the suitability of 1 for
＋
biological applications, it was employed in the fluorescence detection of the intracellular Cu2 with cultured KB
cells. The results indicated that 1 had good membrane permeability and could be useful for the fluorescence micro-
scopic imaging.
Keywords chemosensor, phenoxazinium, water-soluble, copper
Introduction
made to synthesize fluorescent chemosensors, such as
coumarin, rhodamine derivatives and so on, for selec-
＋
tive, sensitive detection of Cu2 .[4,5] Water-compatible
＋
Recently, fluorescent probes have become the hot
research topics. The long wavelength probes (650—900
nm) are highly preferable for applications in biological
systems due to their several advantages:[1] The
auto-fluorescence and photo-damage to living cells are
reduced; the low absorption and light scattering of bio-
logical samples facilitate deeper penetration of long
wavelength radiation into biological matrices, which
can help to obtain information from the inner regions of
tissues. Therefore, a pressing need exists to develop
fluorescent dyes satisfying the multiple criteria such as
long-wavelength excitation/emission, high selectivity
and live-cell permeability.[1b-1d]
fluorescent chemosensors for Cu2 -selective detection
were recently reported and used with some progress in
biological applications. However, most of them were
neither
long-wavelength
excitation/emission
probes,[4,5b-5f] nor full water soluble compounds.[4b-4e,5]
Hence, auto-fluorescence and photo-damage to living
cells would emerge in more complicated biological sys-
tem; and the membrane permeability would be a prob-
lem in vitro test. Therefore, development of new full
water soluble Cu2＋-selective probe with good mem-
brane permeability, which has long-wavelength excita-
tion or emission, is important and necessary.
Copper ions play an important role in various bio-
Symmetric and unsymmetric 3,7-bis(dialkylamino)-
phenoxazin-5-ium type derivatives contain a phenoxaz-
inium cation with absorption in the 640—650 nm region
and have corresponding emission bands between 650
and 670 nm.[6] It is also known that phenoxazinium type
compounds have good membrane permeability and
comparatively low toxicity according to previous in vi-
tro and in vivo assay results.[7] A red-emitting phenol-
xazinium based probe,[8a] 3-(diethylamino)-7-(1,4-di-
oxa-7,13-dithia-10-azacyclo-pentadecan-10-yl)phe-
logical processes ranging from bacteria to mammals.[2]
＋
The abnormal concentration of Cu2 can cause neu-
rodegenerative diseases such as Menkes disease, Wilson
disease, and Alzheimer's disease.[3] Long-term exposure
can cause liver or kidney damage. Even a short period
of exposure to a high level of copper can cause gastro-
intestinal disturbance. The US Environmental Protection
Agency has set the limit of copper in drinking water to
1.3 ppm (≈20 μmol/L). Considerable efforts have been
*
E-mail: lujm@suda.edu.cn (J. Lu), ge_jianfeng@hotmail.com (J. Ge); Tel.: 0086-0512-65880367; Fax: 0086-0512-65884717
Received May 8, 2012; accepted July 5, 2012; published online XXXX, 2012.
Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cjoc.201200452 or from the author.
Chin. J. Chem. 2012, XX, 1—6
© 2012 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
1

Yan et al.
FULL PAPER
noxa-zin-5-ium chloride, has been reported as a low in
OCH3), 4.81 (s, 4H, 2×NCH2), 6.48—6.13 (m, 3H, 3×
ArH), 7.07 (t, J＝8.2 Hz, 1H, ArH), 7.16 (td, J＝7.0,
1.6 Hz, 2H, 2×ArH), 7.27 (d, J＝7.5 Hz, 2H, 2×ArH),
7.62 (td, J＝7.7, 1.4 Hz, 2H, 2×ArH), 8.58 (d, J＝4.8
Hz, 2H, 2×ArH); 13C NMR (75 MHz, CDCl3) δ: 55.3
(OCH3), 57.5 (2×NCH2), 99.3 (ArCH), 102.4 (ArCH),
105.8 (ArCH), 121.0 (2×ArCH), 122.2 (2×ArCH),
130.3 (ArC), 137.1 (2×ArCH), 149.9 (3×ArCH),
159.0 (ArCH), 161.0 (ArCH); IR (neat) νmax: 2934,
2837 (CH2), 1731, 1505, 1176, 893, 819 (ArH), 1348
cost, and open to real-time monitoring probe for selec-
＋
tive ratiometric detection of Hg2 in pure water, and a
benzo[a]phenoxazinium-based red-emitting chemosen-
＋
sor[8b] for Zn2 in PBS buffer (pH＝7.4) and in cultured
KB cells has also been reported.
We assume that 3,7-bis(dialkylamino)phenoxazin-
5-ium derivatives appended with a di(2-picolyl) amine
＋
(DPA) unit will coordinate with Cu2 , and the absorp-
tion and emission properties will be modulated due to
the extended p-π conjugation between the nitrogen atom
and phenoxazinium. The fluorescent sensor (1) was de-
signed based on the above assumption. The synthesis
－
1
(Aryl-N, Tertiary amine), 1265, 1035 (Aryl-O-C) cm ;
HRMS (EI-TOF) calcd for C19H19N3O: 305.1528, found
305.1526.
and application of the red-emitting chemosensor based
3-Methoxy-4-nitroso-N,N-bis(2-picolyl) aniline (4)
To a stirred solution of 3 (1900.0 mg, 6.22 mmol) and 6
＋
on phenoxazinium for Cu2 in pure water and cultured
mol•L－ aqueous hydrochloride solution (25 mL) in an
1
KB cells are discussed as follows in details.
ice-water bath, a solution of NaNO2 (472.2 mg, 6.84
mmol) in 2 mL water was added dropwise in 10 min.
After the addition, the reaction was stirred for 1 h. Then,
aqueous NaOH solution (10%) was added until the so-
lution turned dark green and solid precipitated (pH≈10).
The mixture was extracted with CH2Cl2 (50 mL×3),
and combined organic phase was dried over anhydrous
MgSO4. The deep green solid, which was obtained by
evaporation, was purified by chromatography with silica
gel eluting with CH2Cl2. 4 (1768.0 mg, 85%) was ob-
tained. m.p. 99.2—100.1 ℃; 1H NMR (300 MHz,
CDCl3) δ: 4.00 (s, 3H, OCH3), 4.96 (s, 4H, 2×NCH2),
6.22 (d, J＝8.8 Hz, 1H, ArH), 6.40 (s, 1H, ArH), 6.56 (d,
J＝9.0 Hz, 1H, ArH), 7.39—7.11 (m, 4H, 4×ArH),
7.69 (t, J＝7.4 Hz, 2H, 2×ArH), 8.62 (s, 2H, 2×ArH);
13C NMR (75 MHz, CDCl3) δ: 56.2 (OCH3), 57.7 (2×
NCH2), 94.7 (ArCH), 104.7 (ArCH), 112.9 (ArCH),
121.0 (2×ArCH), 122.8 (2×ArCH), 137.2 (2×ArCH),
150.0 (2×ArCH), 156.2 (ArCH), 156.4 (ArCH), 157.1
(ArCH), 164.4 (ArCH); IR (neat) νmax: 2932, 2838
(CH2), 1728, 894, 818, (Ar-H) 1340 (Aryl-N, Tertiary
Experimental
General
Starting materials were purchased from TCI
(Shanghai) Development Co., Ltd. or Sinogharm
Chemical Reagent Co., Ltd. All analytic grade solvents
(A.R.) were obtained from commercial suppliers and
used directly. Flash chromatography was performed
with silica gel (300—400 mesh). Nuclear magnetic
resonance spectra were recorded on Varian Inova NMR
spectrometers (400 or 300 MHz). TMS was used as an
1
internal standard for H NMR and solvent peak used as
an internal standard for 13C NMR. High resolution mass
spectra were obtained on a GC-TOF or a Saturan2200
LC-MS instrument. UV/vis spectrum experiments were
recorded on a U-3900 spectrometer. Fluorescence meas-
urements were performed on a HORIBA JobinYvon's
fluorescence spectrofluorometers at room temperature.
Metaling points were determined on an X-4 microscope
electron thermal apparatus (Taike, China). All pH meas-
urements were made with a Model PHS-3C meter
(Jingke, China). Elementary analyses were conducted
on a Carlo Erba-MOD1106 elementary analysis appa-
ratus. IR spectra were recorded on a Nicolet 5200 FT-IR
instrument using solid samples dispersed in KBr pellets.
A Shimadzu LC-20A was used for HPLC analysis.
－
1
amine), 1253, 1047 (Aryl-O-C), 1515 (Aryl-NO) cm ;
HRMS (EI-TOF) calcd for C19H18N4O2: 334.1430,
found 334.1436.
3-(Diethylamino)-7-(di(2-picolyl)amino)phenoxa-
zin-5-ium chloride (1)
3-(Diethylamino)phenol
(395.3 mg, 2.39 mmol) and 90% i-PrOH (30 mL) were
stirred at 70 ℃ in a 100 mL two-neck bottle with dis-
tilling apparatus filled with N2. A suspended solution,
which was obtained by the mixture of 4 (800.0 mg, 2.39
Synthesis
3-Methoxy-N,N-bis(2-picolyl)aniline (3)
To a
1
solution of 2-chloromethyl -prydine hydrochloride (3.78
g, 23.0 mmol) in H2O (1 mL), 3-methoxyaniline (1.42 g,
11.5 mmol), 5 mol/L NaOH (5.8 mL), and hexadecy-
trimethylammonium chloride (40 mg) were added under
N2 protection. The mixture was stirred vigorously for 48
h at room temperature. The mixture was extracted with
CH2Cl2 (50 mL×3), and the extract was washed with
H2O and dried with MgSO4. After evaporation of the
solvent, the desired product 3 (1.2 g) was obtained as
yellow oil in 34.2% yield via column chromatography
(silica, from petroleum ether∶AcOEt (1∶1＝V/V) to
AcOEt). 1H NMR (400 MHz, CDCl3) δ: 3.68 (s, 3H,
mmol), 1 mol•L－ HCl (2.54 mL, 2.54 mmol), water (8
mL) and i-PrOH (30 mL), was injected with a syringe
into the above mixture in four portions during 1.5 h.
After 2 h at 70 ℃, the temperature rose to reflux. When
about 30 mL of the solvent was distilled out, 30 mL of
90% i-PrOH was added to the reaction mixture. This
procedure was repeated three times during 5 h. The
dark-blue solution was evaporated, and the residue was
purified by column chromatography with silica gel,
eluting with CH2Cl2∶MeOH from 15∶1 to 10∶1
(V/V). The deep-blue solution was evaporated. The
powder was dried in vacuum. Green solid 1 (290.4 mg,
2
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Chin. J. Chem. 2012, XX, 1—6

Synthesis and Application of a Full Water-Soluble and Red-Emitting Chemosensor
28%) with metallic luster was obtained, purity＝99.3 %;
m.p.＞200 ℃; 1H NMR (400 MHz, CD3OD) δ: 1.36 (t,
J＝7.1 Hz, 6H, 2×CH3), 3.98—3.66 (m, 4H, 2×
ArCH2), 5.22 (s, 4H, 2×NCH2Ar), 6.99 (s, 2H, 2×
ArH), 7.45—7.29 (m, 8H, 8×ArH), 7.83 (td, J＝7.8,
1.4 Hz, 2H, 2×ArH), 8.56 (d, J＝4.5 Hz, 2H, 2×ArH);
13C NMR (75 MHz, CD3OD) δ: 11.9 (2×CH3), 47.0 (2
×ArNCH2), 57.3 (2×NCH2Ar), 96.5 (ArCH), 97.7
(ArCH), 117.0 (ArCH), 119.1 (ArCH), 122.2 (2×
ArCH), 123.3 (2×ArCH), 133.6 (ArCH), 135.0 (ArCH),
136.8 (ArCH), 137.9 (2×ArCH), 148.7 (ArCH), 149.5
(2×ArCH), 150.0 (ArCH), 155.8 (ArCH), 157.6 (2×
ArCH); IR (neat) νmax: 2930, 2850 (CH2), 857, 820,
(ArH), 1337 (Aryl-N, Tertiary amine) cm－1; HRMS
(ESI＋) calcd for C28H28N5O＋: 450.2294, found [M－
Cl－]＋ 450.2276.
Figure 1 shows the changes in the absorption spec-
＋
trum of 1 as a function of Cu2 concentration in water
at room temperature. The UV-vis spectrum of 1 in water
is characterized by a very intense band centered at 632
－
－
1
1
nm (ε＝42000 mol •L•cm ). This is responsible for
the light blue color of the solution. During photometric
＋
titrations of 1 with Cu2 , a blue shift of the long-wave-
length absorption maximum from 632 nm to 582 nm
－
1
－
1
(ε＝18500 mol •L•cm ) (with an isosbestic point at
560 nm) was observed. This indicates the complexity of
＋
Cu2
with the DPA unit. As it is reported, the
difference in spatial configuration (Figure S1) and the
change in electric charge distribution, (Table 1) will
affect the frontier molecular orbitals,[4c] then the
absorption spectrum of phenoxazinium cation (1＋) and
＋
1＋•Cu2 . Decreases in the electron donating capability
of nitrogen, proven by density functional theory (DFT)
calculations with B3LYP at the level of 6-31G* for C,
H, N, O atoms and LANL2DZ for Cu atom, indicate the
changes of electric charge distribution (Table 1).
Cell incubation
KB cells were cultured in Roswell Park Memorial
Institute (RPMI-1640), supplemented with 10% calfse-
rum, 1% penicillin and 1% streptomycin at 37 ℃ in a
5∶95 CO2-air incubator. The cells were cultured for 2 d
then loaded onto a 35 mm diameter glass-bottomed
cover slip. An Nikon AY laser scanning microscopy
system equipped with a 630 nm laser head was applied
to confocal image KB cells stained with 1. The emission
was collected at 650—750 nm. All of the images were
gathered at the same confocal microscope settings and
processed with Nikon AY software.
0.8
0 equiv.
0.7
0.6
0.5
5 equiv.
0.4
0.3
0.2
0.1
0.0
Results and Discussion
3-(Diethylamino)-7-(di(2-picolyl)amino)phenoxazin-
5-iumchl-oride (1) was obtained by reacting 3-(diethyl-
amino)phenol with DPA derivative (4), which was syn-
thesized from 3-methoxyaniline with two steps (Scheme
1). Compound 1 exhibits good solubility in water and
polar solvents such as methanol and chloroform.
400
450
500
550
600
650
700
750
Wavelength/nm
Figure 1 Changes in the UV-vis spectra of 1 (20 μmol/L in
water) upon titration by CuCl2 from 5 to 100 μmol/L.
Scheme 1 Synthesis of phenoxazinium 1
＋
Table 1 Calculated NBO charges (electrons) of 1＋and 1＋•Cu2
N(1)
N(2)
O(1)
N(3)
1＋
1＋•Cu2
－0.381
－0.290
－0.334
－0.313
－0.438
－0.436
－0.354
－0.522
N
O
NH2
O
N
N
＋
i
3
2
The measured absorbance [1/(A－A0)] at 632 nm
＋
varied as a function of 1/[Cu2 ] in a linear relationship,
＋
indicating the 1∶1 stoichiometry between Cu2 and 1
N
ii
iii
O
N
N
(Figure 2). This is according to the linear Benesi-
Hildebr and expression.[9] On the basis of the 1∶1
stoichiometry and UV-vis titration data in Figure 1, the
ON
4
N
＋
association constant of 1 with Cu2 in water was found
－
1
to be (5.60±0.08)×104 mol •L. Job’s plot analysis[5d]
of the UV-vis titrations carried out in water revealed a
maximum of 50% mole fraction in accordance with the
proposed 1∶1 binding stoichiometry (Figure 3).
Et
N
O
N
N
+
Et
N
-
Cl
1
The fluorescence titration of 1 (20 μmol/L) in the
(i) 2-chloromethylpyridine hydrochloride, 34.2%; (ii) (1) NaNO2, HCl; (2)
NaOH, 85%; (iii) 3-(diethylamino)phenol, 28%.
＋
presence of different Cu2 concentrations was then
Chin. J. Chem. 2012, XX, 1—6
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3

Yan et al.
FULL PAPER
8x105
7x105
6x105
5x105
4x105
3x105
8x105
7x105
6x105
5x105
4x105
3x105
2x105
0 equiv.
5 equiv.
0
20 40 60 80 100
-1
Cu2+/(μmol L )
.
1x105
0
600 625 650 675 700 725 750 775 800
Wavelength/nm
Figure 4 Emission spectra of compound 1 in the presence of an
＋
increasing Cu2 concentration (from 5 to 100 μmol/L) in water.
The excitation wavelength was 560 nm with 5 nm slit widths. The
concentration of chemosensor 1 was 20 μmol/L. Inset: Fluores-
＋
cence intensity of 1 versus the Cu2 ×concentration at 673 nm.
＋
Figure 2 Benesi-Hilderbrand plot of 1 with Cu2
.
2.25x10-5
2.00x10-5
1.75x10-5
1.50x10-5
1.25x10-5
1.00x10-5
7.50x10-6
5.00x10-6
2.50x10-6
Figure 5 Frontier molecular orbitals of 1＋ and 1＋•Cu2 (a: 1＋;
＋
0.00
b: 1＋•Cu2＋).
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0
xCu2+
Table 2 The contributions of each electronic oscillator (orbital
＋
Figure 3 Job’s plot analysis of 1 and Cu2 in water; the total
＋
transition) to the lowest energy transition of 1＋ and 1＋•Cu2 (for
＋
molar concentration of 1 and Cu2 is 40 μmol/L. The absorbance
orbitals) in gas phase by CIS//TDDFT
was employed at 632 nm.
Structure Character
1＋
HOMO－1 → LUMO (7.0%, LLCT)
performed. In Figure 4, sensor 1 showed a strong fluo-
rescence emission at 673 nm. This emission intensity
HOMO → LUMO (93.0%, LLCT)
was gradually quenched upon increasing the concentra-
＋
1＋·Cu2
＋
tion of Cu2 from 5 to 100 μmol/L. At the employed
HOMO－12 → LUMO (6.6%, LMCT/LLCT)
HOMO－6 → LUMO (4.3%, LMCT/LLCT)
HOMO－4 → LUMO (3.0%, LMCT/LLCT)
HOMO →LUMO (84.3%, LMCT/LLCT)
HOMO → LUMO＋2 (1.8%, LLCT)
concentration of the probe (20 μmol/L), we can detect
＋
Cu2 in the range of 5—100 μmol/L (Figure 4 Inset).
The fluorescence quenching of the ligand may occur
through excitation energy transfer from the ligand to the
metal d-orbital and/or ligand to metal charge transfer
(LMCT) as reported.[10]
To demonstrate the fluorescence on-off mechanism,
the excited states of phenoxazinium cation (1＋) and
1＋•Cu2＋were obtained with the CIS method using
Hartree-Fock wave functions (Figure 5, Table 2). The
main mode of electron transition for 1＋ is from HOMO
to LUMO (93.0%). According to ligand to ligand charge
transfer (LLCT), the fluorescence intensity does not
weaken. For 1＋•Cu2 , the electronic oscillators corre-
＋
sponding to the transitions from HOMO(β) to LUMO(β)
are calculated to be 84.3%. The occupied orbital of
HOMO(β) is essentially localized on the phenoxazinium
ring, however, the LUMO(β) is occupied on N(3), N(4),
and N(5) atoms, Cu2 ＋ , and two water molecules.
Therefore, the main charge displacement takes place
4
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Chin. J. Chem. 2012, XX, 1—6

Synthesis and Application of a Full Water-Soluble and Red-Emitting Chemosensor
＋
from the phenoxazinium ring to Cu2 . In simple terms,
the best description of the emitting state is that of
1.0
LMCT. This provides a pathway for the nonradioactive
deactivation of the excited state, resulting in fluores-
cence intensity reduction and even fluorescence
quenching.[11,4c]
0.8
0.6
0.4
0.2
0.0
Control experiments were performed to evaluate the
＋
selectivity of the fluorescent probe 1 toward Cu2 ions
＋
＋
(Figure 6). The influence of the addition of Hg2 , Cd2 ,
＋
＋
＋
＋
＋
＋
＋
＋
＋
,
Zn2 , Ni2 Cu2 , Pb2 , Co2 , Fe3 , Fe2 , Ag , Mn2 ,
K＋, Na＋, Mg2 , and Ca2 on the emission properties of
＋
＋
1 was studied, respectively. The concentration of the
metal ions was 100 μmol/L except for K＋, Na＋, Mg2 ,
＋
2+
3+
K Na Ca Mg Zn Pb Ag Ba Fe Fe Ni Mn Hg Cd Co
＋
and Ca2 (100 mmol/L) because these ions may have a
Figure 7 Fluorescence response of 1 (20 μmol/L) containing
＋
＋
100 μmol/L Cu2 to the selected metal ions (100 μmol/L: Hg2
,
similar concentration in the real samples. Note that only
＋
＋
the additions of Cu2 and Fe3 ions lead to significant
＋
＋
＋
＋
＋
＋
＋
Cd2 , Zn2 , Ni2 , Pb2 , Co2 , Fe3 , Fe2 , Ag＋, Mn2＋; 100
＋
changes in the fluorescence of 1. In the case of Fe3 , the
mmol/L: K＋, Na＋, Mg2 , Ca2＋) in water. F1
indicates the
＋
＋
Cu
＋
fluorescence signals of 1 in the presence of Cu2 and F1
emission maximum was slightly quenched. The intro-
＋
＋
Cu
M
＋
duction of Fe2 results in about 10 nm blue-shift in the
＋
denotes the fluorescence signals of 1 in the presence of Cu2 and
competing ions. Excitation was at 560 nm and emission was at
673 nm.
fluorescence spectrum without changes in the emission
intensity .
＋
means 1 can be used to analyze intracellular Cu2 ions
in biological samples.
1.0x106
1 only, K+, Na+, Ca2+, Mg2+,
Zn2+, Pb2+, Ag+, Ba2+, Ni2+,
Mn2+, Hg2+, Cd2+, Co2+
Chemosensor 1 is also well-suited to fluorescence
imaging in living cells. Human oral epidermoid carci-
noma cells (KB cell) were incubated with 20 μmol/L 1
for 20 min and a strong fluorescence emission was ob-
served, as shown in Figure 8b, then washed with
physiological saline to remove excess 1. After it had
been treated with 5 equiv. of copper ions for 20 min,
intracellular fluorescence was almost completely sup-
pressed, as shown in Figure 8c. Therefore, 1 is
cell-permeable and can respond to copper ions within
living cells.
1+Fe2+
8.0x105
6.0x105
4.0x105
1+Fe3+
2.0x105
0.0
1+Cu2+
650
600
700
750
800
850
Wavelength/nm
Figure 6 Fluorescence spectra change of 1 (20 μmol/L) upon
＋
＋
＋
addition of different metal cations (100 μmol/L: Hg2 , Cd2 , Zn2
,
Ni2 , Cu2 , Pb2 , Co2 , Fe3 , Fe2 , Ag＋, Mn2＋; 100 mmol/L: K＋,
＋
＋
＋
＋
＋
＋
Na＋, Mg2 , Ca2＋) in water. Excitation was at 560 nm.
＋
＋
The interference of other cations to Cu2 is also car-
＋
ried out. As shown in Figure 7, the titration of Cu2 to
＋
Figure 8 (a) Bright-field of the KB cell; (b) the cells were in-
cubated with 1 (20 μmol/L) for 20 min then washed with physio-
logical saline; (c) the cells were then incubated with CuCl2 (100
μmol/L) for 20 min. Excitation was at 633 nm.
1 in the presence of the screened metal cations (Hg2 ,
＋
＋
＋
＋
＋
＋
＋
＋
Cd2 , Zn2 , Ni2 , Cu2 , Pb2 , Co2 , Fe3 , Fe2 , Ag＋,
＋
and Mn2 ) did not lead to significant changes in fluo-
rescence output compared with that in the absence of
other metal cations. The fluorescence of 1•Cu2＋has
slightly changes in the presence of a large excess of K＋,
Conclusions
＋
＋
Na＋, Mg2 , and Ca2 (100 mmol/L) (Figure 7), pre-
sumably resulting from the large ionic strength.[12]
The fluorescence intensity changes at 673 nm with
pH ranging from 5.90 to 8.10 were also discussed (Fig-
In conclusion, a selection of the suitable fluorophore
for ion probes from existing or potential drug molecules
was presented. A phenoxazinium-based chemosensor
bearing di(2-picolyl) amine unit (1) with long-wave-
length excitation and emission was used as a promising
analytical tool for fluorometric detecting copper ions in
ure S3). The fluorescence intensity of 1 was relatively
＋
unaffected by protons. With the introduction of Cu2 ,
the intensity of 1 was obviously quenched under acid
condition, and the differential of emission intensity
around pH ～7.4 did not have much effect, which
pure water. Notably, the selectivity of the probe for
＋
Cu2 over other metal ions is relatively high. The cor-
responding quenching mechanism was explained by ab
Chin. J. Chem. 2012, XX, 1—6
© 2012 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.cjc.wiley-vch.de
5

Yan et al.
FULL PAPER
Kim, K.-M.; Yoon, J. Sens. Actuator B: Chemical 2009, 137, 597; (g)
Yu, M. C.; Jin, Y. Q.; Qi, X.; Bao, L. J.; Qian, Z. S.; Zhu, C. Q.
Chin. J. Chem. 2010, 28, 1165.
initio calculations. In addition, 1 can be one of the most
＋
promising probes for the detection of Cu2 in living
cells due to its good membrane permeability.
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Z. X.; Qiao, L.; Li, C. Y.; Jian, L. X.; Shen, G. L.; Yu, R. Q. Anal.
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Acknowledgement
We are grateful to Professor Masataka Ihara (Hoshi
University, Japan) for the helpful discussions regarding
the synthesis of 1. This work was financially supported
by the National High Technology Research and
Development Program of China (863 Program,
SQ2009AA06XK1482331), the National Environmental
Community Project of China (200909044), the National
Natural Science Foundation of China (51273136) and
the Project funded by the Priority Academic Program
Development of Jiangsu Higher Education Institutions.
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Chin. J. Chem. 2012, XX, 1—6