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A simple fluorescent receptor selective for Mg2+†
Yingying Ma,a Hong Liu,b Shaopu Liua and Rui Yang*a
Received 12th February 2012, Accepted 2nd March 2012
DOI: 10.1039/c2an35200a
Published on 06 March 2012 on http://pubs.rsc.org | doi:10.1039/C2AN35200A

A structurally simple fluorescent receptor (receptor 1) has been Experimental

synthesized and was found to show a dramatic enhancement in its
Reagents and instruments
fluorescence emission upon complexation with Mg2+. This was maybe
contributed to by the inhibition of the C]N isomerization in the 4-phenylsemicarbazide was purchased from commercial supplier
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excited state. The experimental results show that the receptor was
selective and sensitive towards Mg2+ in the presence of competing
ions, with a low detection limit of 3.5
109 mol L1 (3s).
Introduction
Mg2+ is the second abundant intracellular cation1 and plays essen-
tial roles in many biological processes.2 Designing receptors to be
highly selective for Mg2+ remains a challenging subject in supra-
molecular chemistry. Fluorescent receptors, owing to their high
detection sensitivity and intrinsic operation simplicity, have great
potential for practical applications in Mg2+ recognition. The
majority of the available receptors bear structural and conforma-
tional merits that match well with the shape and size of Mg2+,
thereby fluorophores modified by crown ethers,3 azacrown ethers,4
polyethers5 and calixarene derivatives6 that could form a rigid or
flexible cavity around Mg2+ have been used widely. However, these
receptors are usually synthetically demanding, furthermore limited
in distinguishing Mg2+ from Ca2+. Thus far few examples providing
selectivity in Mg2+ recognition have been reported3a,4c,d,5b–d,f,6,7 due to
the similar chemical properties of Ca2+ and Mg2+. Herein, we report
a structurally simple compound, 1-(2-hydroxybenzylidene)-4-phe-
nylsemicarbazide (receptor 1, Scheme 1), as a selective fluorescent
receptor for Mg2+. A 2-hydroxybenzaldehyde acylhydrazone moiety
was chosen as the binding unit due to its good coordination
capacity for metal ions.8 Receptor 1 was synthesized via a simple
one-step reaction (Scheme 1) and it could discriminate Mg2+ from
Ca2+ using fluorescence spectroscopy, with the low detection limit
of 3.5
109 mol L1. The selectivity and sensitivity of receptor 1
towards Mg2+ suggested its potential use in the detection of trace
amounts of Mg2+.
(J&K Technology Co., Ltd.), other reagents were purchased from
commercial suppliers (Sinopharm Reagent Co., Ltd). The solutions
of metal ions were prepared from their perchlorate salts. All chem-
icals expect acetonitrile (ACN) used in this work were of analytical
grade and were used without further purification. ACN was HPLC
grade and was used with further purification, without fluorescence
impurity. Distilled water was used as solvent.
Fluorescence spectra measurements were performed on a Hitachi
F-2500 spectrofluorophotometer (Hitachi Company, Japan) equip-
ped with a xenon discharge lamp and using a 1 cm quartz cell.
Fluorescence quantum yields were measured using quinine sulfate as
the standard (0.55 in 0.50 mol L1 H2SO4).9 Absorption spectra
measurements were performed on a UV-8500 spectrophotometer
(Shimadzu Corporation, China). 1H NMR and 13C NMR were
acquired in DMSO-d6 on Bruker 300 MHz NMR spectrometer using
TMS as an internal standard. HRMS were obtained on a Micromass
LCT spectrometer by injection of methanol solution of the sample.
IR spectra measurements were obtained on a PENFOR27 Fourier
transform infrared spectroscopy (BRUKER, Germany).
Computational details
The geometry optimizations were performed in vacuum using the
hybrid density functional Becke-3-Lee-Yang-Parr (B3LYP) potential
in combination with a 6-31G* basis set for the H, C, N, O atoms and
a LANL2DZ effective core potential basis set for Mg atom, as
implemented in GAUSSIAN 2003 software package, which is often
used to estimate the geometry optimization of receptor with metal
interaction.

a
Key Laboratory on Luminescence and Real-Time Analysis, Ministry of
Education, School of Chemistry and Chemical Engineering, Southwest
University, Chongqing 400715, P.R. China. E-mail: ruiyang@swu.edu.cn
b
Department of Chemistry, Bijie University, Bijie 551700, Guizhou
Province, P.R. China
† Electronic supplementary information (ESI) available: Absorption and
fluorescence spectra, 2D COSY spectrum and IR spectra of receptor 1.
See DOI: 10.1039/c2an35200a Scheme 1 Synthesis of receptors 1 and 2.

This journal is ª The Royal Society of Chemistry 2012 Analyst, 2012, 137, 2313–2317 | 2313
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Synthesis of receptors 1 and 2

Receptors 1 and 2 were synthesized by refluxing in ethanol equimolar

quantities of 4-phenylsemicarbazide and 2-hydroxybenzaldehyde and
2-methoxybenzaldehyde, respectively. The crude products were
recrystallized from ethanol and characterized by 1H NMR, 13C NMR
and HRMS.

Receptor 1. 1H NMR (300 MHz, DMSO-d6), d (ppm): 10.63 (s,

1H), 10.08 (s, 1H), 8.87 (s, 1H), 8.26 (s, 1H), 7.95 (d, J ¼ 7.5 Hz, 1H),
7.63 (d, J ¼ 7.9 Hz, 2H), 7.27 (d, J ¼ 7.0 Hz, 2H), 7.20 (d, J ¼ 7.5 Hz,
1H), 7.00 (t, J ¼ 7.2 Hz, 1H), 6.86 (t, J ¼ 8.4 Hz, 2H). 13C NMR
(75 MHz, DMSO-d6) d (ppm): 156.09, 152.94, 139.18, 130.59, 128.47,
127.08, 122.38, 120.37, 119.69, 119.23, 116.01. HRMS (ESI): found
Published on 06 March 2012 on http://pubs.rsc.org | doi:10.1039/C2AN35200A

for [receptor 1 + H]+ 256.1086; calcd for [receptor 1 + H]+: 256.1081.

Receptor 2. 1H NMR (300 MHz, DMSO-d6), d (ppm): 10.72

(s, 1H), 8.86 (s, 1H), 8.30 (s, 1H), 8.18 (d, J ¼ 7.5 Hz, 1H), 7.65 (d, J ¼
7.8 Hz, 2H), 7.38 (t, J ¼ 7.6 Hz, 1H), 7.29 (t, J ¼ 7.5 Hz, 2H), 7.07 (d,
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J ¼ 8.3 Hz, 1H), 7.01 (t, J ¼ 7.3 Hz, 2H), 3.84 (s, 3H). 13C NMR
(75 MHz, DMSO-d6) d (ppm): 157.29, 153.07, 139.10, 136.25, 130.83,
128.41, 126.13, 122.41, 120.52, 119.85, 111.54, 55.59. HRMS (ESI):
found for [receptor 2 + H]+ 270.1243; calcd for [receptor 2 + H]+:
270.1237.

Optical responses of receptor 1 to Mg2+

The absorption spectra of receptor 1 in ACN exhibited three bands
peaked at 282, 290 and 318 nm, with respective molar absorption Fig. 1 Absorption (a) and fluorescence (b) spectra of receptor 1
coefficients of 2.41
104, 2.33
104, 1.54
104 M1 cm1, indicative (1.0
105 mol L1) in ACN in the presence of increasing concentration
of the (p, p*) transition character, respectively (Fig. 1a). When Mg2+ of Mg2+ (0.0–4.0
105 mol L1).
was added, the absorbance band at about 282 nm increased slightly,
and the band at 318 nm decreased, while a new peak appeared Mg2+ (212) was the strongest, showing an approximately 3.5 times
at longer wavelength (from 350 to 400 nm) (Fig. 1a). Three clear larger value than that of Ca2+. In contrast, scarce responses were
isosbestic points at 270, 298 and 333 nm were observed, indicating the observed in the presence of Na+, K+ and Ba2+. Apparently, the results
formation of a well-defined 1–Mg2+ complex in the ground state. indicate that receptor 1 could easily distinguish Mg2+ from other
Fig. 1b shows the changes in the fluorescence emission spectra of cations by means of fluorescence spectroscopy. In fact, the complex
receptor 1 as a function of the Mg2+ concentrations. Receptor 1 in 1–Mg2+ could be readily distinguished by the naked eye under a UV
ACN emitted extremely weak fluorescence, with a fluorescence lamp as shown in Scheme 2 and Fig. S1†.
quantum yield (F) of 0.0015. This was indicative of the C]N To test whether the other cations could interrupt the response of
isomerization in the excited state.10 In the presence of Mg2+, however, receptor 1 towards Mg2+, the fluorescence emission spectra of 1–Mg2+
a significant enhancement of the emission band at 436 nm occurred, in the presence of other competing metal ions were investigated as
by an enhancement of up to 212 fold (Fig. 2) and with F reaching
0.262. This was contributed to by the inhibited C]N isomerization11
of receptor 1 arising from the Mg2+ binding (Scheme 2). Fig. 1b also
shows that the fluorescence intensity of receptor 1 increased notice-
ably when the concentration of Mg2+ was 2.0
107 mol L1 in the
solution of receptor 1 with the concentration of 1.0
105 mol L1,
which indicates that the receptor was highly sensitive towards Mg2+.

Selectivity and sensitivity

To examine the selectivity of receptor 1 towards Mg2+, the related
alkali and alkaline earth cations such as Ca2+, K+, Na+ and Ba2+ were
introduced to investigate their influence on the fluorescence intensity
of receptor 1. Meanwhile, the binding constants between receptor 1
and cations were calculated by the non-linear fit12 and are listed in
Table S1 (See the ESI†). Fig. 2 shows the fluorescence intensity
enhancement ratio (I/I0) of receptor 1 in the presence of various Fig. 2 Fluorescence intensity of receptor 1 (1.0
105 mol L1) in the
cations mentioned above. It was obvious that the largest value for presence of different metal ions (2.0
105 mol L1).

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Scheme 2 Proposed mechanism for the fluorescence changes of receptor 1 upon addition of Mg2+.
Published on 06 March 2012 on http://pubs.rsc.org | doi:10.1039/C2AN35200A
shown in Fig. 3. The fluorescence intensity of 1–Mg2+ showed a slight
change, when Ca2+, Na+, K+ and Ba2+ were added, respectively,
suggesting the selectivity of receptor 1 towards Mg2+.
The relationship between the fluorescence intensity I and concen-
tration of Mg2+ in mM (c) was as I ¼ 136.9c  5.7. The linear range of
the method was found to be 2.0
107 to 1.0
106 mol L1 Mg2+
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with a correlation coefficient of R2 ¼ 0.9981. The detection limit was
found to be 3.5
109 mol L1, which indicates that the receptor was
highly sensitive towards Mg2+.
Fluorescence spectra of receptor 1 response to Mg2+ in ACN–H2O
solutions
The response of receptor 1 to Mg2+ in ACN–H2O solutions were
investigated preliminarily. In ACN–H2O (9 : 1, v/v), the fluorescence
of receptor 1 emitted extremely weak fluorescence, while an
enhancement of emission was observed at 448 nm when Mg2+ was
added to the solution of receptor 1 (Fig. S2†). Meanwhile, the fluo-
rescence of receptor 1 was hardly changed by Ca2+, Ba2+, Na+ and K+,
which suggested receptor 1 could be employed as a selective fluo-
rescent receptor in aqueous solution for Mg2+ (Fig. S3†).
Binding mode and mechanism
In order to explore the structural contribution of 2-hydroxy group in
1–Mg2+ binding, the involved proton was substituted by a methyl
group, producing 1-(2-methoxybenzylidene)-4-phenylsemicarbazide
Fig. 3 Fluorescence intensity of 1–Mg2+ in the presence of other
competing metal ions. The concentration of receptor 1 was 1.0
105
mol L1, while that of Mg2+ and other cations were 2.0
105 mol L1,
respectively.
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(receptor 2) (Scheme 1). In adding Mg2+ to receptor 2, there were
almost no any changes in the absorption spectra and the fluorescence
emission of receptor 2 (Fig. S4†), indicating there were no 2–Mg2+
complexes formed, whether in ground state or excited state. This is
probably because of the more steric hindrance of 2–OCH3 group in
Mg2+ binding to receptor 2. These results demonstrate that the
2-hydroxy group of receptor 1 plays a very important role in the
specific recognition of Mg2+.
A 1 : 1 stoichiometry of Mg2+ binding to receptor 1 was made
evident from Job plots. In exploring the binding model between
receptor 1 and Mg2+, density functional theory (DFT) calculations
were done using B3LYP/6-31G* level (GAUSSIAN03). The opti-
mized configuration showed that one Mg2+ was suitably at the
coordination center of receptor 1 via its carbonyl O, imino N and
2-hydroxy O atoms (Fig. 4). The average bond length of Mg–O was
reported as 1.95 A (both for Mg–O (31) and Mg–O (15)), and those
of Mg–N (18) was reported as 2.05 A,  respectively. Evidently, the
2+
coordination of Mg and imino N (18) atom disrupted the C]N
isomerization in free receptor 1, which led to the obvious fluorescence
enhancement.
The interaction of receptor 1 with Mg2+ was further investigated by
1
H NMR titrations in DMSO-d6 (DMSO-d6 was chosen as the
solvent because of the bad solubility for receptor 1 in CD3CN-d3). As
seen in Fig. 5, two –NH (Ha and Hb) resonances of receptor 1 were at
8.87 and 10.63 ppm, –N]C–H (Hc) was at 8.26 ppm, and –OH (Hd)
proton was at 10.08 ppm, respectively (signals of active hydrogen
protons were assigned by referring to the 2D COSY spectrum
(Fig. S5†)). In the presence of up to 1.0 equiv. Mg2+, an obvious
downfield shift of the hydroxy proton Hd from 10.08 to 10.13 ppm
was observed. This reflected the deshielding effect of the metal ion
binding,13 as a result of the coordination of Mg2+ to 2-hydroxy O
atom. Interestingly, upon addition of 0.5 equiv. of Mg2+, the proton
Hc experienced a downfield shift from 8.26 to 8.28 ppm, after which,
it moved upfield shift to 8.27 ppm. This was possibly due to the
Fig. 4 Conformation of 1–Mg2+ optimized by density functional theory
calculations.
Analyst, 2012, 137, 2313–2317 | 2315

Published on 06 March 2012 on http://pubs.rsc.org | doi:10.1039/C2AN35200A
1
Fig. 5 Partial H NMR spectra of receptor 1 in DMSO-d6 in the pres-
ence of increasing equivalents of Mg2+. The concentration of receptor 1
was 0.24 mol L1 and equivalents of Mg2+ were indicated in the figure.
competition between the deshielding effect of the metal ion chelating
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and the C]N–metal p–p orbital interaction through space13b,14
(Scheme 3). Meanwhile, the signals of Ha and Hb experienced a tiny
upfield to 8.86 and 10.59 ppm, respectively, indicating the involve-
ment of the carbonyl O atom in binding Mg2+.13a,15
Further evidence for the binding mode between receptor 1 and
Mg2+ was provided by IR experiment. In the IR spectrum of receptor
1 (Fig. S6†), the peaks at 1649 and 1608 cm1 ascribed to the C]O
and C]N stretching, respectively, were found, upon Mg2+ binding,
to shift to lower wavenumbers of 1624 and 1566 cm1, respectively,
indicating carbonyl O and imino N atoms complexation with Mg2+.16
The IR peak at 1273 cm1 ascribed to the C–O (phenolic O)
stretching had a small higher wavenumber shift and became much
weaker, indicating phenolic O atom coordination with Mg2+.8a
Computational data, 1H NMR spectra and IR spectra testified
receptor 1 complexation with Mg2+ via its carbonyl O, imino N and
2-hydroxy O atoms, which resulted in the increase in fluorescence
emission of receptor 1. Receptor 1 contained an unbridged C]N
structure and the C]N isomerization was the predominant decay
process in its excited state10 which induced weak fluorescence
emission of receptor 1. However, by binding to Mg2+, the C]N
isomerization was inhibited, which led to the increase in fluorescence
emission of receptor 1.
Conclusion
In summary, a simple yet selective and sensitive fluorescent receptor
for Mg2+ was designed and synthesized. The receptor 1 shows a linear
range of 2.0
107 to 1.0
106 mol L1 and low detection limit of
3.5
109 mol L1 Mg2+. The C]N isomerization mechanism was
utilized effectively to detect Mg2+ selectively, nevertheless those
Scheme 3 Chelation of receptor 1 with Mg2+ showing the C]N–metal
p–p orbital interaction.
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successful Mg2+ fluorescent receptors were mainly based on the
fluorescence signaling mechanisms of photoinduced electron transfer
(PET)3b,4a,c,5a,7 and intramolecular charge transfer (ICT).3,5a–c,f,6 Upon
binding with Mg2+, the C]N isomerization was eliminated and the
fluorescence was turned from ‘‘off’’ to ‘‘on’’. The 2-hydroxy group
played a crucial role in the specific recognition of Mg2+ and the C]N
bond should be inside of the cycle formed with metal ion. The
conclusion reported here provides a new strategy for constructing
turn-on fluorescent receptors for alkaline earth cations.
Acknowledgements
This work was supported by the Fundamental Research Funds for
the Central Universities (XBJK2009c179) and the Doctoral Foun-
dation of Southwest University (SWUB2008044).
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