Development and applications of fluorescent indicators for Mg2+ and Zn2+

Article abstract of DOI:10.1021/jp110305k

In a study of the spectroscopic behavior of two Schiff base derivatives, salicylaldehyde salicylhydrazone (1) and salicylaldehyde benzoylhydrazone (2), Schiff base 1 has high selectivity for Zn²⁺ ion not only in abiotic systems but also in living cells. The ion selectivity of 1 for Zn²⁺ can be switched for Mg²⁺ by swapping the solvent from ethanol-water to DMF (N,N-dimethylformamide)-water mixtures. Imine 2 is a good fluorescent probe for Zn²⁺ in ethanol-water media. Many other ions tested, such as Li⁺, Na⁺, Al³⁺, K⁺, Ca ²⁺, Cr³⁺, Mn²⁺, Fe³⁺, Co ²⁺, Ni²⁺, Cu²⁺, Ag⁺, Cd ²⁺, Sn²⁺, Ba²⁺, Hg²⁺, and Pb ²⁺, failed to induce any spectral change in various solvents. The selectivity mechanism of 1 and 2 for metal ions is based on a combinational effect of proton transfer (ESPT), C=N isomerization, and chelation-enhanced fluorescence (CHEF). The coordination modes of the complexes were investigated.

Full text of DOI:10.1021/jp110305k

ARTICLE
pubs.acs.org/JPCA
Development and Applications of Fluorescent Indicators
for Mg2þ and Zn2þ
Lina Wang, Wenwu Qin, Xiaoliang Tang, Wei Dou, and Weisheng Liu*
Key Laboratory of Nonferrous Metals Chemistry and Resources Utilization of Gansu Province and State Key Laboratory of Applied
Organic Chemistry, College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou 730000, China
S Supporting Information
b
ABSTRACT:
In a study of the spectroscopic behavior of two Schiff base derivatives, salicylaldehyde salicylhydrazone (1) and salicylaldehyde
benzoylhydrazone (2), Schiff base 1 has high selectivity for Zn2þ ion not only in abiotic systems but also in living cells. The ion
selectivity of 1 for Zn2þ can be switched for Mg2þ by swapping the solvent from ethanol-water to DMF (N,N-
dimethylformamide)-water mixtures. Imine 2 is a good fluorescent probe for Zn2þ in ethanol-water media. Many other ions
tested, such as Liþ, Naþ, Al3þ, Kþ, Ca2þ, Cr3þ, Mn2þ, Fe3þ, Co2þ, Ni2þ, Cu2þ, Agþ, Cd2þ, Sn2þ, Ba2þ, Hg2þ, and Pb2þ, failed to
induce any spectral change in various solvents. The selectivity mechanism of 1 and 2 for metal ions is based on a combinational effect
of proton transfer (ESPT), CdN isomerization, and chelation-enhanced fluorescence (CHEF). The coordination modes of the
complexes were investigated.
’ INTRODUCTION
A fluorescent probe is a molecular system for which the physico-
chemical properties change upon interaction with a chemical species
in such a way as to produce a detectable fluorescent signal.11 A number
of signaling mechanisms have been developed and widely applied for
the optical detection of different species, such as photoinduced
electron/energy transfer (PET),12 metal-ligand charge transfer
(MLCT),13 intramolecular charge transfer (ICT),14 excimer/exciplex
formation,15 excited-state intra/intermolecular proton transfer
(ESPT),16 and CdN isomerization. Schiff bases (imines) are known
to be good ligands for metal ions.17,18 Some Schiff base-metal
complexes have antitumor properties,19 antioxidative activities,20 and
attractive electronic and photophysical properties.21 In addition, Schiff
base derivatives incorporating a fluorescent moiety are appealing tools
for optical sensing of metal ions. In this paper, we synthesized three
Schiff bases 1 (salicylaldehyde salicylhydrazone), 2 (salicylaldehyde
benzoylhydrazone), and 3 (benzaldehyde salicylhydrazone) . They
were prepared in a manner similar to that of a reported procedure
(Scheme 1).22 Imines are poorly fluorescent, in part due to isomer-
ization of the CdN double bond in the excited state23 and in part due
to ESPT involving the phenolic protons of the salicylic amide
moiety.24 We reasonably expect that the ESPT and CdN isomeriza-
tion may be inhibited upon the complexation with certain metal ions.
Fluorescent probes for metal ions have found widespread use not
only in environmental monitoring but also in biological studies.1
The development of highly sensitive fluorescent probes for metal
ion analysis is an active research field.2 Fluorescence offers signifi-
cant advantages over other methods for metal ion detection and
measurement because of its generally nondestructive character, high
sensitivity, and instantaneous response.3 Zinc and magnesium are
indispensable for mediating many enzyme-catalyzed reactions in
humans.4 These elements play important roles in a wide variety of
physiological and pathological processes.5,6 Zinc is widespread in
biology and there is much interest in its detection In vivo where
its concentration ranges from 10-9 mol dm-3 in the cytoplasm to
10-2 mol dm-3 in some vesicles.7 Magnesium is found in the bone
in macronutrient quantities, and it plays a key role in bone remodeling
and skeletal development.8 Fluorescent indicators for the determi-
nation of Mg2þ or Zn2þ are attractive targets in molecular design
and synthesis. It is a challenge to design selective Zn2þ probes
because the chemical properties of Zn2þ are so similar to those of
Cd2þ. In recent years, progress in the area of chemosensors has
contributed significantly to the development of a variety of Zn2þ
probes based on quinoline, anthracene, BODIPY, and fluorescein
as fluorophores.9,10 Most of them are excellent Zn2þ sensors.
Unfortunately, some Zn2þ sensors have disadvantages such as
poor water-solubility or complicated synthesis methods. However,
few Mg2þ sensors have been reported up to now.
Received: October 28, 2010
Revised:
January 18, 2011
Published: February 17, 2011
r
2011 American Chemical Society
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Scheme 1. Synthesis of Compounds 1, 2, and 3a
5.237 mL of salicylaldehyde (6.075 g, 49.8 mmol) was slowly
dropped into the above solution. The reaction mixture was refluxed
for 5 h during which an ivory white precipitate appeared. The
resulting precipitate was filtered and washed three times with 15
mL of ethanol. After being dried over anhydrous sodium sulfate in
vacuum, 12.111 g (95% yield) of salicylaldehyde salicylhydrazone (1)
was obtained: mp 269 °C. 1H NMR (300 MHz, DMSO-d6) δ 12.01
(s, 1H), 11.76 (s, 1H), 11.18 (s, 1H), 8.66 (s, 1H), 7.87 (d, 1H,
J = 7.8 Hz), 7.55 (d, 1H, J = 7.8 Hz), 7.44 (t, 1H, J = 7.8 Hz), 7.30
(t, 1H, J = 6.6 Hz), 6.89-6.98 (m, 4H) ppm. 13C NMR (75 MHz,
DMSO-d6) δ 164.5, 159.0, 157.5, 149.0, 134.0, 131.7, 129.5, 128.6,
119.4, 119.1, 118.7, 117.3, 116.5, 115.6, 40.4, 40.1, 39.8, 39.5, 39.2,
39.0, 38.7 ppm. HRMS (ESI) m/z calcd for C14H12N2O3, 256.0848;
found 257.0921 (M þ 1).
a Reagents and conditions: (i) ethanol, reflux, 5 h, 75%.
The spectroscopic behavior of Schiff bases shows that 1 is an excellent
chemosensor for Mg2þ and Zn2þ in various organic-water mixtures.
Especially, 1 can be used for Zn2þ monitoring in living cells. Schiff
base 2 is also a good fluorescent probe for Zn2þ in ethanol-water.
Synthesis of Compound 2. Salicylaldehyde benzoylhydra-
zone (2) was prepared via a synthetic method analogous to that
for 1 and was obtained as an ivory white solid (11.4 g, 95% yield):
mp 172 °C. 1H NMR (300 MHz, DMSO-d6) δ 12.10 (s, 1H),
11.28 (s, 1H), 8.63 (s, 1H), 7.93 (d, 2H, J = 6.6 Hz), 7.50-7.60
(m, 4H), 7.30 (t, 1H, J = 7.8 Hz), 6.91 (t, 2H, J = 7.5 Hz) ppm.
13C NMR (75 MHz, DMSO-d6) δ 162.9, 157.5, 148.3, 132.8,
131.4, 129.6, 128.6, 127.7, 119.4, 118.7, 116.5, 40.3, 40.1, 39.8,
39.5, 39.2, 40.0, 38.7 ppm. HRMS (ESI) m/z calcd for
C14H12N2O2, 240.0899; found 241.0972 (M þ 1).
’ EXPERIMENTAL SECTION
Materials. All the materials for the syntheses were purchased
from commercial suppliers and used without further purification.
All of the solvents used were of analytical reagent grade. Water
was deionized. The solutions of the metal ions were prepared
from their perchlorate salts, except for Cr3þ and Sn2þ, which
were used as chloride salts, and Kþ, which was available as KNO3.
Instrumentation. The pH of the solutions was measured with a
SPM-10A acidity meter (Shanghai, China). 1Hand13CNMRspectra
were taken on a Varian Mercury-300 spectrometer with tetramethyl-
silane (TMS, 0.00 ppm) as an internal standard and DMSO-d6 as
solvent. Chemical shift multiplicities are reported as s = singlet, d =
doublet, t = triplet, q = quartet, and m = multiplet. High resolution
mass data (HRMS) were determined on a Bruker Daltonics APEXII
47e FT-ICR spectrometer. ESI-MS spectra were determined on a
Bruker Daltonics Esquire 6000 spectrometer. Melting points were
determined on a Kofler apparatus. Absorption spectra were measured
on a Varian UV-Cary100 spectrophotometer using quartz cells of
1.0 cm path length. Fluorescence spectra measurements were perfor-
med on a Hitachi F-4500 spectrofluorimeter and a Shimadzu RF-540
spectrofluorophotometer equipped with quartz cuvettes of 1 cm path
length with a xenon lamp as the excitation source. An excitation and
emission slit of 2.5 or 5.0 nm were used for the measurements in the
solid state. Quantum yields were determined by an absolute method
using an integrating sphere based upon that originally developed by
de Mello25 et al. Experiments were conducted on an FLS920 from
Edinburgh Instruments. The quantum yields were also determined
according to the following equation: Φu = Φs (FuAsnu2/FsAuns2),
where Φu is the quantum yield, F is the integrated area under the
corrected emission spectra, A is the absorbance at the excitation
wavelength, n is the refractive index of the solution, and the subscripts
u and s refer to the unknown and the standard, respectively. Acridine
orange (λex = 400 nm, Φf = 0.20) was used as the fluorescence
standard.26 All spectra were recorded at room temperature except for
fluorescence microscope images. The fluorescence microscope ex-
periment was carried out with a Nikon TE2000-5.
Synthesis of Compound 3. Benzaldehyde salicylhydrazone (3)
was prepared via a synthetic method analogous to that for 1 and was
obtained as an ivory white solid (11.4 g, 95% yield): mp 231 °C. 1H
NMR (300 MHz, DMSO-d6) δ11.86 (s, 2H), 8.48 (s, 1H), 7.90 (d,
1 H, J = 7.5 Hz), 7.75 (q, 2H, J = 3.2 Hz), 7.42-7.47 (m, 4H),
6.94-6.70 (m, 2H) ppm. 13C NMR (75 MHz, DMSO-d6) δ 164.8,
159.0, 148.7, 134.1, 133.8, 130.3, 128.9, 128.6, 117.2, 119.0, 117.3,
115.9, 40.3, 40.1, 39.8, 39.5, 39.2, 40.0, 38.7 ppm. HRMS (ESI) m/z
calcd for C14H12N2O2, 240.0899; found 241.0969 (M þ 1).
Synthesis of Complexes. Zn(ClO4)2 (0.1 mmol) was added
to an ethanol solution containing 1 or 2 (0.1 mmol). The
complexes were obtained by stirring the mixtures for 5 h.
1-Zn2þ: ESI-MS m/z calcd for C28H22N4O6Zn, 574.1, found
m/z 575.1 (M þ 1). 2-Zn2þ: ESI-MS m/z calcd for
C28H22N4O4Zn, 542.2, found m/z 543.1 (M þ 1).
Cell Incubation and Imaging. Human bladder cancer cell line
EJ was obtained from Laboratory Center for Medical Science of
Lanzhou University. Cells were cultured in RPMI 1640 (Gibco, New
York, NY) supplemented with 10% fetal bovine serum (FBS, Rongye
Biotech, China) and penicillin (100 U/mL)/streptomycin (100 mg/L)
in an atmosphere of 5% CO2 at 37 °C. One day before imaging, cells
were seeded in 96-well flat-bottomed plates. Immediately before the
experiments, cells were incubated with the probe 1 (50 μM) for 0.5 h
in RPMI 1640 at 37 °C under 5% CO2 and then washed with
phosphate-buffered saline (PBS) three times before incubating with
Zn(ClO4)2. Next, stained cells were incubated with 50 μM Zn-
(ClO4)2 for another 0.5 h, cells were rinsed with PBS three times
again, and then intracelluar fluorescence imaging was performed
under a Nikon Eclipse TE2000-5 inverted fluorescence microscope
with a 20ꢀ objective lens (excited with blue light). The EJ cells that
were only incubated with 1(50μM) for 0.5 h at 37 °Cunder5%CO2
were a control.
Synthesis of Compound 1. The synthesis of salicylaldehyde
salicylhydrazone (1) was carried out as outlined in Scheme 1. The
hydrazide salicylate 4 was obtained by refluxing the ester (8.5 mL, 66
mmol) and hydrazine hydrate (18 mL, 320 mmol) in absolute
ethanol(20mL) ina100mLone-necked bottle for 8 h (7.573 g, 75%
yield). In a 200 mL three-necked bottle, compound 4 (7.573 g, 49.8
mmol) was dissolved in 30 mL of absolute ethanol. The mixture was
refluxed at 80 °C until the dissolution of hydrazide salicylate. Next,
’ RESULT AND DISCUSSION
The electronic absorption spectra properties of 1were investigated
in N,N-dimethylformamide (DMF) or ethanol. The effects of Mg2þ
on the absorption spectra of 1 in DMF are displayed in Figure S1
(Supporting Information). Because DMF absorbs strongly below
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Scheme 2. ESPT and Distribution of the Absorption Bands to Compound 1
270 nm, it is difficult to observe the absorption bands of 1 at
wavelengths lower than 270 nm. The absorption spectrum of 1in ion-
free solution presents two important transitions with intense absorp-
tion bands at 299 and 335 nm, respectively. As Scheme 2 describes,
there is an equilibrium between the two configurations (Normal and
Tautomer) due to ESPT. The band at 335 nm (332 nm in Scheme 2)
is assigned to the transition of OdC-CdC-(NH) in Tautomer.
The band at 299 nm (288 nm in Scheme 2) is attributed to the
azomethine CdN bond, and the band at 239 nm which is assigned to
CdO derived from salicyloyl hydrazone does not emerge from the
DMF spectrum. After the addition of Mg2þ, the absorbance of
compound 1at 299 nm increases gradually; however, the peak at 335 nm
starts to disappear. The spectral change indicated that the addition of
Mg2þ ion induced the decrease of tautomer. We speculated that the
nitrogen of azomethine took part in the coordination with Mg2þ and the
formation of the 1-Mg2þ complex inhibited the ESPT (Scheme 2).
Moreover, two new bands appeared at 387 and 410 nm, which are
certainly caused by a new, larger conjugated system (1-Mg2þ com-
plex). We also investigated the electronic absorption spectral properties
of ligands 2 (R1 = H, R2 = OH, Scheme 1) and 3 (R1 = OH, R2 = H,
Scheme 1). The main difference between compounds 1-3 is that they
haveone or twoOH(orH) group(s) atthe2,20 (ortho) positions. For
compounds 2 and 3, in which one of the hydroxyl groups of compound
1 is missing, there are no new peaks centered at 400 nm after the
addition of Mg2þ ion. We speculated that the hydroxyl groups in
compound 1 (i.e., OH of the salicylic amide moiety and OH of the
salicylaldimine part: R1 = R2 = OH in Scheme 1) were required for
nitrogen of azomethine was involved in the formation of the 1-Zn2þ
complex. In addition, the transition of CdO of salicyloyl hydrazone at
239 nm had a blue shift with slight increase in absorption. This
indicates that the CdO particpates in the coordination. A new peak at
397 nm was observed with the addition of Zn2þ, caused by the
formation of the 1-Zn2þ complex. We investigated the electronic
absorption spectra of 2and 3to determine which OH of compound 1
was involved in the coordination to Zn2þ. The spectra of 2 (in which
the hydroxyl group of the salicylic amide moiety in compound 1 is
missing) showed a change similar to that of 1 after the addition of
Zn2þ (Figure S3, Supporting Information), which suggested that the
hydroxyl group of the salicylic amide moiety (R1 = OH) in 1 was not
essential for the coordination to the Zn2þ ion. No change was
observed in the spectra of compound 3(R1 = OH, R2 =H,Scheme1)
after the injection of Zn2þ ions indicated the participation of R2 =OH
in the complexation of 1 with Zn2þ (Scheme 2). A similar coordina-
tion mode occurs between 2 and the Zn2þ ion.
The changes in the fluorescence emission spectra of 1 at pH ∼ 7
induced by addition of Mg2þ in DMF-H2O (9:1, v/v, λex
=
410 nm) is portrayed in Figure 1. The fluorescent signal of 1 at
453 nm increases significantly with an increase in the concentration
of Mg2þ in DMF-H2O solution. There is a 6-fold increase of the
fluorescence quantum yield Φf of 1 in the presence of Mg2þ [from
0.044 in ion-free DMF-H2O to 0.26 at 30 μM Mg2þ]. In ethanol-
H2O (9:1, v/v), the maximum of the fluorescence emission band of 1
shifts from 453 nm in ion-free solution to 488 nm in the presence of
Zn2þ and is accompanied by a 6-fold increase in Φf (Figure 2) [Φf
increases from 0.05 for ion-free 1 to 0.31 in the presence of 1 equiv of
the selectivity for Mg2þ
.
The absorption spectra of 1in the absence and presence of Zn2þ in
ethanol are depicted in Figure S2 (Supporting Information). In ion-
free solution, there are three key peaks at 239 nm, 288 nm, and 332
nm, respectively. They are assigned in Scheme 2. The absorbance of
compound 1at 288 had a red shift (from 288 nm to 297 nm), and the
peak at 332 nm disappeared gradually. The disappearance of the
absorption band at 332 nm indicated that the formation of 1-Zn2þ
complex inhibited ESPT and led to the large decrease of tautomer.
Because the peak at 288 nm had a red shift, we speculated that the
Zn2þ]. Other ions tested, such as Liþ, Naþ, Al3þ, Kþ, Ca2þ, Cr3þ
,
Mn2þ, Fe3þ, Co2þ, Ni2þ, Cu2þ, Agþ, Cd2þ, Sn2þ, Ba2þ, Hg2þ, and
Pb2þ, gave no distinct change in the spectrum of 1 in both DMF-
H2O and ethanol-H2O. This indicated that 1 could serve as an
excellent fluorescent probe for Mg2þ and Zn2þ by switching
solvents. The significant increase of fluorescence can be explained
as follows: Imine 1 is poorly fluorescent in part due to isomerization
of the CdN double bond in the excited state and in part due to
excited-state proton transfer (ESPT) involving the phenolic protons
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Scheme 3. CdN Isomerization
Figure 1. Fluorescence emission spectra of 1 (30 μM) in the absence
(ion-free) and presence of 1 equiv of various metal ions in DMF-H2O
(9:1, v/v) solution. λex = 410 nm.
Figure 3. Fluorescence emission spectra of 2 (30 μM) in the absence
(ion-free) and presence of 1 equiv of different metal ions in ethanol-
H2O (9:1, v/v) solution. λex = 390 nm.
Moreover, with coordination to the metal ion, an extended π-
electroconjugation system was formed. At the same time, a selective
chelation-enhanced fluorescence(CHEF) effect was observed, which
was a significant enhancement in the fluorescence intensity of Schiff
base 2. No change in the fluorescence spectra could be found after the
addition of other ions to the solution of 2. This interesting feature
reveals that 2 can serve as an excellent selective fluorescent chemo-
sensor for Zn2þ. Thefluorescence spectra of 1, 2, and3indicated that
the CONHNdCH function, which is present in all three compounds
(1-3), is not sufficient to warrant selectivity for a specific metal ion.
Indeed, the o-hydroxy groups play decisive roles in the selectivity. For
the complex between 1 and Mg2þ in DMF-H2O solution, the two
hydroxyl groups (i.e., OH of the salicylic amide moiety and OH of the
salicylaldimine part: R1 = R2 = OH in Scheme 1) are needed for
coordination of 1 to Mg2þ. The selectivity of 1 and 2 for Zn2þ
requires the OH of the salicylaldimine moiety, indicating the
participation of R2 = OH in the complexation of Zn2þ. However,
the hydroxyl group of the salicylic amide moiety is not necessary for
the coordination of Zn2þ to ligand.
The fluorescence titration experiments were performed by mixing
various amounts of metal ions with 1. Figures 4 and 5 show the
fluorescence emission changes of 1 after the addition of various
concentrations of Mg2þ and Zn2þ, respectively, in 9:1 (v/v) organic/
aqueous solvent. For 1-Mg2þ, the fluorescence intensity enhanced
gradually along with the changes in concentration of Mg2þ ions. It did
not reach a maximum until the concentration of ions achieved 1
equiv. For 1-Zn2þ, the fluorescence intensity increased obviously
until the concentration of Zn2þ achieved 0.5 equiv. The ground-state
dissociation constants Kd of the 1-Mg2þ and 1-Zn2þ complexes
were determined by direct fluorimetric titration as a function of
Figure 2. Fluorescence emission spectra of 1 (30 μM) in the absence
(ion-free) and presence of 1 equiv of various metal ions in ethanol-H2O
(9:1, v/v) solution, λex = 390 nm.
of the salicylic amide moiety. Upon stable chelation with cartain
metal ions, the CdN isomerization is inhibited (Scheme 3); ad-
ditionally, the coordination of 1 with the metal ion prevents ESPT
(Scheme 2), leading to fluorescence enhancement. Moreover, the
reaction of a metal ion with a chelating agent induces rigidity in the
resulting molecule and tends to produce a large CHEF effect which
induces the large enhancement of fluorescence.
We also investigated the effect of Zn2þ and Mg2þ on the
fluorescence properties of compounds 2 and 3. Compound 3
exhibited no spectral change from any metal ion in any of the solvents
tested (methanol, ethanol, acetone, acetonitrile, chloroform, DMF,
and DMSO). Mg2þ did not induce any spectral change of 2 in the
above solvents. However, 2 is a good indicator for Zn2þ in ethanol-
H2O (9:1, v/v). The change of fluorescence emission spectra of 2
after the addition of Zn2þ ions is shown in Figure 3. In the absence of
Zn2þ, the fluorescence emission peak of 2 was very weak due to
isomerization of the CdN double bond and ESPT effect. Upon
addition of Zn2þ to the solution of 2, there was a significant
enhancement in the fluorescence emission band with a red shift,
reaching a maximum at 495 nm. This enhancement of fluorescence
intensity was due to inhibition of the CdN isomerization and ESPT
effect after the injection of Zn2þ into the solution mixture of 2.
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Figure 4. Fluorescence spectra of 1 (30 μM) in the absence and
Figure 6. Fluorescence spectra of 2 (30 μM) in the absence and presence
of Zn(ClO4)2 (0-15 μM) in 10% aqueous ethanol. λex = 390 nm.
presence of Mg(ClO4)2 (0-30 μM) in 10% aqueous DMF. λex
=
410 nm.
Nonlinear curve fitting of eq 1 to the fluorescence data Frecorded as a
function of [Zn2þ] yields ground-state dissociation constants Kd and
n of the 2-Zn2þ complex as 42.38 ( 3.20 μM and 0.5 (Figure 6
inset), respectively. Job’s plots confirm the value of n (Figure S6,
Supporting Information), which indicates that the coordination mode
of probe 2 with Zn2þ is also 2:1 stoichiometry.
Job’s plots suggested that a 1:1 stoichiometry is the most likely
binding mode between 1 and Mg2þ. The dominating peak in ESI
mass spectra of 1-Mg2þ (Figure S16, Supporting Information) was
in agreement on a 1:1 stoichiometry. The peak at m/z = 352.1
(calcd =352.1) corresponding to [MgL1(DMF)]þ (L1 = 1) was
observed. Thus, according to the above spectral data and some
structures of Mg2þ complexes,29 we presented the probable binding
mode between 1 and Mg2þ as shown in Figure 1 inset. DMF
molecules were involved in the coordination of Schiffbase 1to Mg2þ
,
and some hydrogen bonding with the remaining OH group in Schiff
base 1. Maybe there was another bonding mode: Each Mg2þ center is
coordinated to a chelating ligand 1 and the OH of a neighboring
[1-Mg2þ] unit. Note that the peaks at m/z = 269.0 (calcd =
269.0) and m/z = 279.1 (calcd =279.1) were attributed to
[Mg(DMF)2(ClO4)]þ and [MgL1 þ H]þ (L1 = 1), respectively.
Perhaps the coordination modes between 1 and Mg were diversified.
As demonstrated for complexes 1-Zn2þ and 2-Zn2þ, 1 and 2 can
serve as good fluorescent probes for Zn2þ ion; however, 3 has no
selectivity for Zn2þ. On the basis of the structures of 1, 2, and 3, we
estimated the configuration of the 1-Zn2þ complex as displayed in
Figure 2 inset. More direct evidence of this binding mode was
obtained from the ESI mass spectra of 1-Zn2þ (Figure S17,
Supporting Information). A peak at m/z = 575.2 (calcd =575.1)
corresponding to [Zn(L1)2 þ H]þ (L1 = 1) was observed. This
binding mode was also supported by Job’s plots. Job’s plots also
indicated a 2:1 stoichiometry between 2 and Zn2þ. The complex
2-Zn2þ exhibited peak at m/z = 543.2 (calcd =543.1), which
corresponds to [Zn(L2)2 þ H]þ (Figure S18, Supporting Informa-
tion, L2 = 2). Figure 3 inset shows the proposed structure of the
2-Zn2þ complex. However, other metal ions fail to form such a rigid
framework, probably because of several combined influences coop-
erating to achieve the unique selectivity for the Mg2þ and Zn2þ ion,
such as the suitable coordination geometry conformation of the
bischelating Schiff base receptor, the appropriate ion radius and
sufficient binding energy of the Mg2þ and Zn2þ, and the suitable
coordination ability of the specific solvent.
Figure 5. Fluorescence spectra of 1 (30 μM) in the absence and presence
of Zn(ClO4)2 (0-15 μM) in 10% aqueous ethanol . λex = 390 nm.
[M2þ] (M2þ - Mg2þ or Zn2þ). Nonlinear curve fitting of eq 127
n
Fmax½Mꢁ þ FminKd
F ¼
ð1Þ
n
Kd þ ½Mꢁ
to the fluorescence data F recorded as a function of [M2þ] yields
values of Kd, the fluorescence signals Fmin and Fmax at minimal and
maximal [M2þ], respectively (corresponding to the free and M2þ
bound forms of the probe, respectively), and n (the number of M2þ
bound per probe). The ground-state dissociation constants Kd of the
1-Mg2þ complex was found to be 33.84 (3.19 μM(Figure4inset).
Fitting eq 1 to the steady-state fluorescence data F with n, Kd, Fmin
,
and Fmax as freely adjustable parameters always gave values of n close
to 1; n was kept fixed at 1 in the final curve fittings, which indicated
that 1 formed a 1:1 complex with Mg2þ. This binding mode was also
supported by the data of Job’s plots evaluated from the fluorescence
spectra of 1 and Mg2þ with a total concentration of 100 μM (Figure
S4, Supporting Information).28 The ground-state dissociation con-
stants Kd of the 1-Zn2þ complex were found to be 35.87 (3.59 μM.
Job’s plots (Figure S5, Supporting Information) and fitting eq 1 to the
steady-state fluorescence data F (Figure 5 inset) gave values of n close
to 0.5, which indicated a 2:1 stoichiometry between 1 and Zn2þ
.
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Figure 9. Fluorescence emission spectra of 1 (30 μM) in various mixing
ratios of DMF-water solution (%, v/v). λex = 410 nm.
Figure 7. Metal-ion response for 1 (30 μM) in the absence (F0) and
presence (F) of metal ions in DMF-water (9/1, v/v). One equivalent of
NaF was used for masking Fe3þ, and 1 equiv of Na2S2O3 was used for
masking Cu2þ and Co2þ
.
Figure 10. Fluorescence emission spectra of 1 (30 μM) in various
mixing ratios of ethanol-water solution (%, v/v). λex = 390 nm.
Except for ethanol, methanol, and acetone, the change of the
fluorescence spectra after Zn2þ was added to 1 was not significant
enough to be worth considering in other solvents. Zn2þ changed the
spectrum of 1 in methanol and acetone; however, the enhancement
of fluorescence intensity of 1 upon the addition of Zn2þ in methanol
and acetone was less than that in ethanol. The selectivity of 1 for
Zn2þ in ethanol is better than that in any other solvent. In addition,
the fluorescence intensity changed as a function of the ratio of
organic solvent to water. The fluorescence intensity is quenched
gradually by increasing the amount of water.
The fluorescence change that occurred upon addition of water
to a DMF solution containing 1 and Mg2þ ion was investigated.
The fluorescence intensity of a solution containing 1 and Mg2þ
ion declined gradually with the increase in the concentration of
water (Figure 9). In water-free DMF solution containing 1 and
Mg2þ, there is obvious blue fluorescence. The fluorescence
became very weak when the content of water reached 30% in
solution (keeping the concentration of 1 and Mg2þ constant).
Figure 10 shows the change in emission spectra of ethanol
solution containing 1 and Zn2þ with the increase of water. A
similar change occurred in the fluorescence spectra of the 2-
Zn2þ complex in ethanol (Figure S8, Supporting Information).
Figure 8. Metal-ion response for 1 (30 μM) in the absence (F0) and
presence (F) of metal ions in ethanol-water (9/1, v/v). One equivalent
of NaF was used for masking Fe3þ, and 1 equiv of Na2S2O3 was used for
masking Cu2þ and Co2þ
.
It is known that achieving a high selectivity for the analyte of
interest over a complex background of potentially competing species
is a challenging task in sensor development. Figure 7 and Figure 8
illustrate the fluorescence response of 1 to Mg2þ and Zn2þ in the
presence of other metal ions, respectively. Except for Fe3þ, Co2þ,
and Cu2þ, a background of most competing metal ions does not
interfere with the detection by 1of Mg2þ in DMF-H2OandofZn2þ
in ethanol-H2O. However, the addition of 1 equiv of masking agent
NaF to the solution of 1 containing Fe3þ and Mg2þ in DMF-H2O
or Fe3þ and Zn2þ in ethanol-H2O weakens the influence of Fe3þ 30
.
Analogously, Na2S2O3 was used to weaken the interference of Cu2þ
and Co2þ.
Next, we investigated whether the selectivity of 1 for metal ions
could be produced in other solvents (Figure S7, Supporting
Information). The addition of Mg2þ to 1 in other solvents, such
as methanol, ethanol, acetonitrile, acetone, chloroform, and dimethyl
sulfoxide (DMSO), induced negligible fluorescence enhancement.
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Figure 13. Fluorescence microscope images of human bladder cancer
cell line EJ. EJ cells loaded with probe 1 (50 μM) for 30 min under a 20ꢀ
objective lens (left). Fluorescence image of 1-stained EJ cells exposed to
50 μM Zn2þ for 30 min under a 20ꢀ objective lens (right). Incubation
was performed at 37 °C.
intensity after the addition of 10% water to the ethanol solution of 2is
shown in Figure S9 (Supporting Information).
Subsequent experiments probed the ability of 1 to track Zn2þ
levels in living cells using fluorescence microscopy. To determine the
cell permeability of 1, human bladder cancer cell line EJ was
incubated with 1. EJ cells incubated with 50 μM 1 for up to 30
min at 37 °C show negligible intracellular fluorescence. In contrast,
1-stained cells exposed to 50 μM Zn2þ for 30 min at 37 °C display
enhanced cytosolic fluorescence (Figure 13). Further control experi-
ments without ligand 1 or Zn2þ give no fluorescence over back-
ground levels. Taken together, it clearly demonstrated that the probe
is membrane-permeable, and the fluorescence change in the human
bladder cancer cell line EJ was really due to the synchronous
appearance of 1 and Zn2þ. The result confirmed that the probe 1
can be used to image intracellular Zn2þ in living cells. It should
therefore be potentially useful to study the content or bioactivity of
Zn2þ in living cells.
Figure 11. Fluorescence intensity of DMF solution of 1 (30 μM) with
addition of various 10% (%, v/v) water samples.
’ CONCLUSIONS
In conclusion, we synthesized two Schiffbase fluorescent indicators
1and 2.1is an excellent indicator with solvent-dependent, ion-tunable
selectivity for Mg2þ and Zn2þ. The presence of o-hydroxy groups and
the nature of the solvent play crucial roles in the selectivity for metal
ions and the switch of selectivity from Mg2þ to Zn2þ. In addition, 1 is
membrane-permeable and can also be used for Zn2þ monitoring in
living cells. Schiff base 2 has selectivity to Zn2þ ion in various solvents.
Many other ions tested, such as Liþ, Naþ, Al3þ, Kþ, Ca2þ, Cr3þ,
Mn2þ, Fe3þ, Co2þ, Ni2þ, Cu2þ, Agþ, Cd2þ, Sn2þ, Ba2þ, Hg2þ, and
Pb2þ, have no influence on the spectrum of 1 or 2. The luminescence
mechanism is as follows: The formation of a rigid framework between
Schiff bases and Mg2þ or Zn2þ inhibits the CdN isomerization and
ESPT, leading to fluorescence enhancement. Simultaneously, the
chelation between metal ion and ligand causes a large CHEF effect,
which induces the increase of fluorescence intensity.
Figure 12. Fluorescence intensity of ethanol solution of 1 (30 μM) with
addition of various 10% (%, v/v) water samples.
Generally, one of the most important and useful applications for a
fluorescent probe is the detection of metal ions in water samples.
Herein, we choose several water samples from different water
sources: water from Yellow River, tap water, mineral water, pure
water, distilled water, ultrapure water, and HEPES-buffered distilled
water. Probe 1 is sensitive enough to detect the concentrations of
Mg2þ in these water samples. The change of fluorescence intensity
after the addition of 10% water to the DMF solution of 1 is shown in
Figure 11. The fluorescence intensity of 1 upon the addition of 10%
Yellow River and tap water increased significantly, which indicates
that the content of Mg2þ in both water samples is high. Figure 11
also shows the concentration of Mg2þ ion in mineral water is
medium, and in other water samples is lower. Figure 12 displays the
change of flurescence intensity after the addition of 10% water to the
ethanol solution of 1. The enhancement of fluorescence with the
addition of Yellow River and tap water indicates the existence of
Zn2þ ions in these water samples. Figure 12 shows that the
concentration of Zn2þ ions in mineral water is lower than that in
Yellow River and tap water, which suggests that the amount of Zn2þ
ion in other samples is very small. The change of fluorescence
’ ASSOCIATED CONTENT
Supporting Information. 1H NMR spectrum, 13C NMR
S
b
spectrum, and MS spectra for the compounds and selected
spectroscopic data. This material is available free of charge via
the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*Tel: þ86-0931-8915151. Fax: þ86-0931-8912582. E-mail:
liuws@lzu.edu.cn.
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’ ACKNOWLEDGMENT
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This study was supported by the NSFC (Grant Nos.
20771048, 20931003) and the Fundamental Research Funds
for the Central Universities (lzujbky-2009-k06).
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