The specific detection of Cu(II) using an AIE-active alanine ester

Article abstract of DOI:10.1016/j.cclet.2013.05.014

Cu(II) detection is important because it plays crucial role in several biological processes and ecological systems. Fluorescent techniques have attracted more and more attention in Cu(II) detection. In this report, we contribute a novel strategy to use fluorescence spectroscopy for Cu(II) specific detection. The specificity relies on the fact that, of the many metal cations, only Cu(II) can catalyze the hydrolyzation of α-amino acid ester. The novelty originates from the unique aggregation-induced emission (AIE) property of the fluorescent label. We designed a model α-amino acid ester (TPE-Ala) constructed with alanine and tetraphenylethene-functionalized methanol (TPE-methanol). In comparison with the precursor TPE-Ala, TPE-methanol has lower solubility and is easy to form aggregates in water, thereby displaying a higher fluorescent response. Thus, the Cu(II) catalyzed hydrolyzation can be monitored by recording the fluorescence enhancement and fluorescent detection Cu(II) is rationally achieved.

Full text of DOI:10.1016/j.cclet.2013.05.014

Chinese Chemical Letters 24 (2013) 668–672
Contents lists available at SciVerse ScienceDirect
Chinese Chemical Letters
journal homepage: www.elsevier.com/locate/cclet
Original article
The specific detection of Cu(II) using an AIE-active alanine ester
a,b,
Shuang Zhang a, Ji-Ming Yan a, An-Jun Qin a, Jing-Zhi Sun a, , Ben-Zhong Tang
*
*
a MoE Key Laboratory of Macromolecule Synthesis and Functionalization, Department of Polymer Science and Engineering, Zhejiang University, Hangzhou 310027, China
b Department of Chemistry, Institute of Molecular Functional Materials, the Institute for Advanced Study (IAS), The Hong Kong University of Science & Technology, Hong Kong, China
A R T I C L E I N F O
A B S T R A C T
Article history:
Cu(II) detection is important because it plays crucial role in several biological processes and ecological
systems. Fluorescent techniques have attracted more and more attention in Cu(II) detection. In this
report, we contribute a novel strategy to use fluorescence spectroscopy for Cu(II) specific detection. The
specificity relies on the fact that, of the many metal cations, only Cu(II) can catalyze the hydrolyzation of
Received 25 February 2013
Received in revised form 11 April 2013
Accepted 17 April 2013
Available online 10 June 2013
a
-amino acid ester. The novelty originates from the unique aggregation-induced emission (AIE) property
of the fluorescent label. We designed a model -amino acid ester (TPE-Ala) constructed with alanine and
a
Keywords:
tetraphenylethene-functionalized methanol (TPE-methanol). In comparison with the precursor TPE-Ala,
TPE-methanol has lower solubility and is easy to form aggregates in water, thereby displaying a higher
fluorescent response. Thus, the Cu(II) catalyzed hydrolyzation can be monitored by recording the
fluorescence enhancement and fluorescent detection Cu(II) is rationally achieved.
ß 2013 Jing-Zhi Sun and Ben-Zhong Tang. Published by Elsevier B.V. on behalf of Chinese Chemical
Society. All rights reserved.
Aggregation-induced emission
Fluorescence enhancement
Cu(II) catalyzed
1. Introduction
and homocysteine (Hcy). For example, we used aldehyde function-
alized silole, tetraphenylethene (TPE) and carbazole derivatization
Copper is an essential element in the ecosphere and the third
abundant element after ferrum and zinc in our bodies. The divalent
copper cation, i.e. Cu(II), participates a series of biological processes
and the change in Cu(II) concentration can be considered as a clue
of degenerated diseases, such as Menkes syndrome, Alzheimer’s
disease, Prion disease and amyotrophic lateralizing sclerosis [1–5].
As a result, the detection of Cu(II) is important in both fields of
fundamental research and practical application.
Due to the high sensitivity, ease of manipulation, and visual
observation, fluorescent detection has become an attractive
technique with many instructive research reports on the fluorescent
detection of Cu(II) [6–14]. Most fluorescent detection methods take
advantage of the coordination capacity of Cu(II) to amino and/or
oxygen moieties. The coordination may result in the variation of
fluorescence features including emission intensity, color and life
time, thus the fluorescent detection of Cu(II) is achieved.
to detect Cys over other amino acids and glucose [21–23]. The
specially designed probe can selectively react with Cys and Hcy to
form thiazinane and thiazolidine derivatives in the presence of
diverse amino acids to protect Cys, and glucose. The discrimination
relies on the reaction-dependent fluorophore aggregation, or the
solubility of adducts of the probe molecule and analytes. This
strategy is intrinsically a fluorescent titration, which combines the
high sensitivity of fluorescence spectroscopy and the reliability of
precipitate titration methodology.
Herein, we report our efforts to expand this strategy to Cu(II)
detection. It was found that Cu(II) could catalyze the hydrolyzing of
a
-amino acid ester in aqueous solution at room temperature to
produce -amino acid and corresponding alcohol [24,25]. This
a
commonplace, but simple reaction, triggered the idea of adapting
the AIE mechanism to detect Cu(II). The basic concept is shown in
Scheme 1. Given that the solubility of the AIE-labeled a-amino acid
Aggregation-induced emission (AIE) is a unique fluorescent
phenomenon [15] and it has been found versatile applications in
chemical and biological detections [16–19]. According to AIE
mechanism [16,19,20], the fluorescence turn-on/turn-off process
can be observed if the reaction between the substrate and the
resultant moleculescanalterthe aggregationbehavior(solubility)of
the fluorescent species. These observations triggered the attempt of
using AIE luminogens as fluorescent probes to detect cysteine (Cys)
ester is higher than that of AIE-labeled aliphatic alcohol (hydro-
lyzed resultant), the Cu(II)-catalyzed hydrolyzing process must be
accompanied by the increase of fluorescence intensity, thus Cu(II)
can be detected by fluorescence change.
2. Experimental
2.1. Materials and instruments
Protected
a-alanine, 4,6-dimethyl-2-aminopyridine (DMAP),
piperidine, N,N0-dicyclohexylcarbodiene (DCC), N,N0-dicyclohex-
* Corresponding authors.
E-mail addresses: sunjz@zju.edu.cn (J.-Z. Sun), tangbenz@ust.hk (B.-Z. Tang).
ylcarbodiene were purchased from Aldrich Chemical Co.
1001-8417/$ – see front matter ß 2013 Jing-Zhi Sun and Ben-Zhong Tang. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
http://dx.doi.org/10.1016/j.cclet.2013.05.014

[
S. Zhang et al. / Chinese Chemical Letters 24 (2013) 668–672
669
H2N
O
Cu2+
OH
2+
Cu
O
O
NH2
O
O
NH2
OH
TPE-Ala
TPE-methanol
Relative hydrophilic
Less aggregation
Lower fluorescence
More hydrophobic
Easier aggregation
Higher fluorescence
Scheme 1. Cu(II) catalyzed hydrolyzation of an AIE-labeled
a-amino acid ester and expected fluorescence change.
Dichloromethane, ethyl acetate, and petroleum ether (60–90 8C)
were purchased from Huadong Medicine Co., Ltd. Copper nitrate,
sodium nitrate, potassium nitrate, calcium nitrate, ferric nitrate,
cobalt nitrate, magnesium nitrate, nickel nitrate, zinc nitrate, and
chromium nitrate were purchased obtained from Sinopharm
Chemical Reagent Co., Ltd. They were all analytical grade reagents
and used as received. All other chemicals and regents were
commercially available and used without further purification.
Tris–HCl buffer solutions were prepared as standard procedure.
UV–visible (UV–vis) absorption spectra were measured on a Varian
CARY 100 Bio UV–vis spectrophotometer. Fluorescence spectra
were recorded on a Perkin–Elmer LS 55 spectrofluorometer. The
1H NMR data were measured on a Mercury plus 300 MHz NMR
spectrometer using tetramethylsilane (TMS) as internal standard.
obtained in a yield of about 70%. Characterization data: 1H NMR
(300 MHz, CDCl3): 7.80–7.68 (d, 2H, J = 6.4 Hz), 7.60 (d, 2H,
J = 6.2 Hz), 7.40 (m, 2H), 7.31 (m, 2H), 7.15–6.92 (m, 19H), 5.42 (d,
1H, J = 9.1 Hz), 5.10 (m, 2H), 4.40 (m, 3H), 4.23 (m, 1H), 1.42 (d, 3H,
J = 5.2 Hz). The 1H NMR spectrum can be found in the Supporting
information as Fig. S1. Elementary analysis, calculated for
d
C45H35NO4 (%): C 82.70, H 5.36, N 2.14; found (%): C 81.96, H
5.89, N 2.22.
2.3. Synthesis of the target TPE-labeled
a-amino acid ester (TPE-Ala)
Into a baked 20 mL double-necked flask was added 200 mg
(0.30 mmol) of the protected TPE-functionalized methanol and
8 mL of dichloromethane. The solution was cooled with an ice bath
to 0–5 8C, and 2 mL piperidine was introduced into the cooled
solution under stirring. The mixture was kept at 0–5 8C and stirred
for 2 h then stirred at room temperature for 2 h. The solvent was
removed by rotatory evaporation and the residual was extracted
twice with ethyl acetate and water. The obtained solid was
recrystallized with absolute alcohol and the target resultant TPE-
Ala was obtained in a yield of 83%. Characterization data (see Fig.
2.2. Synthesis of the protected TPE-labeled
a-amino acid ester
The synthetic route to the target molecule is shown in Scheme
-Alanine was chosen to be the prototype of -amino acids due
2.
a
a
to the simplicity in its chemical structure. TPE was used as the AIE
label for its easiness in preparation and functionalization. The
synthesis of TPE-functionalized methanol was reported elsewhere
and we used it directly as reported in this work [26]. Into a baked
50 mL double-necked flask was added 300 mg (0.83 mmol) of TPE-
functionalized methanol, protected alanine (310 mg, 0.99 mmol),
DCC (340 mg, 1.66 mmol), 4,6-dimethylaminopyridine (DMAP)
(200 mg, 1.66 mmol), and DCM (15 mL). The mixture was stirring
at room temperature for 12 h. The resultants were filtered and the
filtrate was rotatory-evaporated. The residual solid was purified on
a silica gel column with ethyl acetate/petroleum (1:1, v/v) as
eluent. The resultant was recrystallized using absolute alcohol as
S2 in Supporting information): 1H NMR (300 MHz, DMSO-d6):
d
8.78–8.62 (s, 2H), 7.20–6.93 (m, 19H), 5.14–5.11 (s, 2H), 4.10–4.02
(m, 1H), 1.43–1.38 (d, 3H, J = 5.5 Hz). Elementary analysis,
calculated for C30H26NO2 (%): C 83.33, H 6.02, N 3.24; found (%):
C 82.89, H 6.41, N 3.21.
2.4. Fluorescent detection of Cu(II) and other cations
The appropriate amount of copper nitrate was dissolved in 4 mL
pure water or 10 mmol/L Tris–HCl buffer solution with proper pH
value, then a suitable amount of TPE-Ala was added under vigorous
solvent and the protected TPE-labeled
a-amino acid ester was
[(Scheme_2)TD$FIG]
O
OH
(i)
(ii)
+
NHFmoc
O
O
O
O
OH
NHFmoc
NH2
TPE-Ala
TPE-methanol
O
ProtectedTPE-Ala
(i) DCC, DMAP/DCM, reflux
(ii) piperidine, DCM, 0ºC
Fmoc=
O
CH2
Scheme 2. Synthetic route to the target TPE-labeled
a-amino acid ester (TPE-Ala).

670
S. Zhang et al. / Chinese Chemical
2
Letters 24 (2013) 668–672
600
stirring to dissolve TPE-Ala in the aqueous solution as soon as
possible. The upper limit of the solubility of TPE-Ala in pure water is
about 4
mmol/L and this concentration was determined as an
500
400
300
200
100
0
optimizedconcentrationinordertogetbetterfluorescentresponses.
The mixture was stirred at room temperature for 2 h in the presence
of 100
mmol/L Cu(II) cations and underwent the necessary fluores-
cent measurement. Other metal nitrates were similarly treated and
the fluorescent measurement procedures were identical to the case
of copper nitrate. The purpose of using relative high concentration of
metal cations is to achieve faster fluorescent detection. As revealed
by Fig. S3 (in Supporting information), at low Cu(II) concentration,
the fluorescence enhancement requires a longer time, which is
unfavorable for practical detection.
3. Results and discussion
It can be seen from Scheme 1 that TPE-Ala ester is constructed by
a hydrophobic TPE moiety and a hydrophilic alanine moiety. Upon
Cu(II) catalyzed hydrolyzation, the hydrophilic alanine and the
hydrophobic TPE moieties separate from each other, with the
resulted TPE-methanol having a higher hydrophobicity than TPE-
Ala. As a result, TPE-methanol molecules are easier to form
aggregates in aqueous solution and exhibit stronger fluorescence.
Based onthe fluorescenceenhancement, the presenceof Cu(II) inthe
aqueous solution is confirmed. Since TPE-methanol and TPE-Ala
have the identical fluorophore (TPE) and the emission peak appears
at around 460 nm, the change in fluorescence intensity depends on
the relative solubility of TPE-methanol and TPE-Ala. Due to the large
hydrophobic TPE group, TPE-Ala has a low solubility in water. When
theconcentrationishigherthan4
form in aqueous solution at room temperature. Fortunately, TPE-
methanol is insoluble in water. Thus we can observe the
fluorescence enhancement. As shown in Fig. 1, when 100
Cu(II) (from copper nitrate) was added to a 4 mol/L TPE-Ala
aqueous solution, an observed fluorescence increase was recorded
after 2 h. Without the addition of copper nitrate, the fluorescence
intensity of TPE-Ala aqueous solution remained unchanged. It is
noted that extending the reaction time can lead to higher
fluorescenceintensity,butfortheultimateapplicationoffluorescent
detection, the prolonged response time is inappropriate. Therefore,
we use 2 h as a unified time in all detection experiments.
pH 4.2
pH 7.2
mol/L TPE-Ala aqueous solution
pH 6.8
pH 5.2
Fig. 2. Variation of fluorescence intensity of 4
m
with (filled column) and without Cu(II) (open column) in Tris–HCl (0.01 mol/L)
buffer at different pH values. ex = 321 nm; concentration Cu(II) = 100 mol/L.
l
m
According to the literature, Cu(II) can effectively catalyze the
hydrolyzation reaction of -amino acid esters at a proper pH
a
window [25]. To explore the pH value at which Cu(II) can properly
catalyze the hydrolyzation of TPE-Ala, we tried the fluorescence
responses of TPE-Ala to Cu(II) at different pH values, which were
tuned by using Tris–HCl buffer solution. Considering that Tris–HCl
has efficient pH tuning capacity in the pH region of 3 to 9, we
selected four typical pH values of 4.2, 5.2, 6.8, and 7.2. The
experimental data are summarized in Fig. S4. The data of peak
fluorescence intensity (at 460 nm) are extracted to organize the
histogram in Fig. 2. At pH 4.2, TPE-Ala itself shows weak
fluorescence. When the pH is adjusted to 5.2, the solubility of
TPE-Ala in buffer solution decreases and the solution exhibits
stronger fluorescence than at pH 4.2. Addition of Cu(II) to the
solution leads to an observed increase in fluorescence intensity.
Further increasing the pH value to 6.8, the fluorescence intensity of
the TPE-Ala aqueous solution becomes even stronger in compari-
son with the case of pH 5.2. After addition of Cu(II) to the solution,
an over 200% enhancement in fluorescence intensity was recorded.
mmol/L,aggregatesofTPE-Alawill
mmol/L
m
[(Fig._1)TD$FIG]
[F(ig._3)TD$FIG]
500
400
300
200
100
800
600
400
200
TPE-Ala
TPE-Ala + Cu(II)
TPE-Ala +50 mol/L Cu2+
TPE-Ala +40 mol/L Cu2+
TPE-Ala +30 mol/L Cu2+
TPE-Ala +20 mol/L Cu2+
TPE-Ala +10 mol/L Cu2+
TPE-Ala + 4 mol/L Cu2+
TPE-Ala + 1 mol/L Cu2+
TPE-Ala
0
400
450
500
Wavelength (nm)
550
600
0
400
450
500
550
600
Wavelength (nm)
Fig. 1. Fluorescent spectra of 4
containing 100
mol/L Cu2+ (after stirring for 2 h; reaction temperature = 25 8C;
excitation wavelength ( ex) = 321 nm).
mmol/L TPE-Ala aqueous solution and its mixture
Fig. 3. Effect of Cu(II) concentration on the fluorescent intensity change of 4
TPE-Ala aqueous solution. Solution pH = 6.8, ex = 321 nm.
m mol/L
m
l
l

[
S. Zhang et al. / Chinese Chemical Letters 24 (2013) 668–672
671
Fig. 4. (A) Fluorescence responses of 4
in comparison with the control sample (without any metal ion, used as baseline). Fluorescence measurements were carried out in pH 6.8 Tris–HCl buffer solution at room
temperature and ex = 321 nm.
mmol/L TPE-Ala to 100 mmol/L different metal cations. (B) The relative change of fluorescence in the presence of different metal cations
l
The observed pH dependent fluorescence change can be ascribed to
the protonation effect of the amino group. At lower pH, the
protonation increased the solubility of TPE-Ala in aqueous
solution, which reduced the fluorescence emission. Meanwhile,
the protonation reduced the affinity of amino group to Cu(II), thus
weakened the Cu(II) catalyzed hydrolyzation of TPE-Ala, thus
addition of Cu(II) into the buffer solution at pH 4.2 showed limited
fluorescence enhancement. At low pH, the addition of Cu(II) to the
solution has a weak negative effect on the fluorescence change,
since Cu(II) has some fluorescence quenching effect.
To clarify the origin of the observed fluorescence enhancement,
thin layer chromatograph (TLC) was used to monitor the process of
the reaction. In the resultant mixtures of TPE-Ala and Cu(II) at pH
5.2 and 6.8, we found that a component has the same Rf value as the
standard TPE-methanol when hexane, hexane/acetic ether mixture
and dichloromethane were used as developing solvents. Therefore,
we conclude that the fluorescence enhancement is associated with
the production of TPE-methanol. At pH 7.2, the fluorescence
intensity of the TPE-Ala aqueous solution is higher than in aqueous
acid solutions because the solubility of TPE-Ala becomes lower in
neutral and basic aqueous solutions. We did not try the fluorescent
response in higher pH values considering that at basic conditions
the ester can be hydrolyzed directly by hydroxyl group and it is
impossible to clearly demonstrate the catalyzing effect of Cu(II).
Based on the above experimental results, we established 6.8 as
the optimized pH value to do the successive fluorescent detection
experiments. The effect of Cu(II) concentration on the fluorescent
fluorescence response of TPE-Ala in pH 6.8 buffer solution with
the experimental results summarized in Fig. 4A and 4B. If the peak
fluorescence intensity of 4
solution is set as the base, the data in Fig. 4B clearly exhibit the
following trends. Firstly, at the concentration of 100 mol/L, metal
mmol/L TPE-Ala in pH 6.8 buffer
m
cations in IA and IIA groups, such as Na(I), K(I), Mg(II), and Ca(II),
show very weak influence on the fluorescence of TPE-Ala.
Secondly, the divalent transition metal cations in the same period
as copper, including Co(II), Ni(II) and Zn(II), also have little effect on
the fluorescence change. Thirdly, it is found that the trivalent metal
cations, such as Fe(III) and Cr(III), display a negative effect on the
fluorescence intensity. This effect can be explained as following:
both Fe(III) and Cr(III) have strong hydrolyzation capacities and
100
m
mol/L Fe(III) and Cr(III) can lead to the acidification of the
mol/L
aqueous solutions. As a result, the intrinsic pH value of 100
m
Fe(III) and Cr(III) aqueous solutions is evidently lower than 6.8, and
the decrease of fluorescence intensity is expected, just as the
situation observed in Fig. 2. As a pertinent proof, we recently took
the advantage of their pronounced hydrolyzation properties to
specifically detect trivalent cations by monitoring the fluorescent
changes of the pyridinyl-functionalized TPE derivative [27]. Finally
and most importantly, among all the tested metal cations, TPE-Ala
shows a large and positive fluorescent response only with Cu(II).
This result provides a solid support of the specific detection of
Cu(II) by fluorescent methodology.
4. Conclusion
intensity of 4
mmol/L TPE-Ala aqueous solution (pH 6.8) is shown
in Fig. 3. It should be pointed out that there is fluctuation of
fluorescence intensity between different patches. The fluctuation
comes from the fact that the hydrolyzation resultant TPE-methanol
is easy to form aggregates, and the size and number of the
aggregates are influential on the fluorescence intensity. The data in
Fig. 3, which are extracted from a typical patch of fluorescent
detection indicate that the fluorescence enhancement is propor-
We have explored the feasibility of detecting Cu(II) in aqueous
mediaby a fluorescenttechniqueby usingana-amino acid esterasa
model compound (TPE-Ala). The hydrolyzation of TPE-Ala in
aqueous solution can be catalyzed by Cu(II) at room temperature
and TPE-methanol is derived. The more hydrophobic TPE-methanol
shows higher ability toform aggregates in aqueous media. Thanks to
the unique AIE activity of TPE moiety, the aggregation of the
generated TPE-methanol molecules can be monitored as evident by
the fluorescence enhancement of the system. Thus in the presenceof
tional to Cu(II) concentration. At around 100
mmol/L Cu(II), the
fluorescence enhancement shows a decreasing trend (see Fig. S5),
which may be associated with the fluorescent quenching effect of
Cu(II), thereaction solution isvalidated. A concentrationof4
mmol/L
high concentration Cu(II), thus we set 100
Cu(II) concentration.
The specification is one of the crucial factors of practical
fluorescence detection. Accordingly, we studied the influence of a
m
mol/L as the maximum
TPE-Ala, pH 6.8 in aqueous solution, and 2 h reaction time are the
optimized detection condition. Under these conditions, the fluores-
cence enhancement is proportional to Cu(II) concentration from
1
mmol/L to 100
mmol/L. At the concentration of 100 mmol/L, or the
series of metal cations (concentration: 100
mmol/L) on the
upper limit concentration for Cu(II), metal cations including

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S. Zhang et al. / Chinese Chemical Letters 24 (2013) 668–672
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National Natural Science Foundation of China (No. 51273175);
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