Coumarin-TPA derivative: A reaction-based ratiometric fluorescent probe for Cu(I)

Article abstract of DOI:10.1016/j.tetlet.2013.08.046

A coumarin-based reactive probe 1 is reported for the highly selective detection of Cu⁺. The benzylic ether (C-O) bond of probe 1 can be cleaved selectively by the reaction with Cu⁺ under a physiological reducing environment, resulting in a fluorescent change. The maximum emission peak exhibited a red shift from 410 nm to 472 nm, and a remarkable enhancement of emission intensity ratios (I₄₇₂/I₄₁₀) from 0.26 to 13.82 was observed.

Full text of DOI:10.1016/j.tetlet.2013.08.046

Tetrahedron Letters 54 (2013) 5771–5774
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Coumarin–TPA derivative: a reaction-based ratiometric
fluorescent probe for Cu(I)
⇑
⇑
Kang-Kang Yu, Kun Li , Ji-Ting Hou, Xiao-Qi Yu
Key Laboratory of Green Chemistry and Technology (Ministry of Education), College of Chemistry, Sichuan University, Chengdu 610064, China
a r t i c l e i n f o
a b s t r a c t
A coumarin-based reactive probe 1 is reported for the highly selective detection of Cu+. The benzylic ether
(C–O) bond of probe 1 can be cleaved selectively by the reaction with Cu+ under a physiological reducing
environment, resulting in a fluorescent change. The maximum emission peak exhibited a red shift from
410 nm to 472 nm, and a remarkable enhancement of emission intensity ratios (I472/I410) from 0.26 to
13.82 was observed.
Article history:
Received 7 May 2013
Revised 29 July 2013
Accepted 15 August 2013
Available online 22 August 2013
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
Coumarin
Reactive probe
Ratiometric probe
Copper sensor
As one of the most abundant soft transition metal elements,
copper plays a very important role in the human body. A variety
of enzymes, such as tyrosinase,1 need copper to serve as an indis-
pensable cofactor due to its redox-active properties. Additionally,
copper is closely connected with structural elements in the human
skeleton and collagen tissue, as well as functional determinants of
the endocrine and nervous systems. On the other hand, when
in vivo concentrations of copper exceed the normal range, the re-
dox active metal becomes a biological hazard via the generation
of reactive oxygen species (ROS), which can cause disruptions in
cellular metabolism.2,3 Alterations in copper concentration at
cellular homeostasis are related to serious neurodegenerative
diseases, such as Wilson and Alzheimer diseases, Menkes syn-
drome, familial amyotrophic lateral sclerosis, and prion diseases.4
In consideration of the strict concentration-dependent balance
for copper between toxicity and biological, it is of great importance
to develop an exact and convenient method for the detection of
copper in environmental and biological samples.
In recent years, more and more metal-responsive fluorescent
sensors have been constructed. This method provides a convenient
and effective approach for detecting metals because of its relative
low cost, high sensitivity, and logistical simplicity.5 Despite the
large success in the development of specific fluorescent probes
for other cations, copper-selective probes are relatively rare and
the research of copper-selective probes is particularly challenging
for the dominant oxidation state in the biological system (+1).
Cu+ has a paramagnetic character and is thus a potent free radical
Scheme 1. Synthesis of probes.
quencher in all coordination environments.6 Most reported cop-
per-specific fluorescent probes are selective for Cu2+, with only
limited examples indicative of Cu+. More importantly, a large part
of these probes relies on fluorescence intensity-based measure-
ments.7 Fluorescence intensity-based detection methods are typi-
cally not appropriate for complex real-world samples where
measuring initial probe concentration and overall excitation and
emission intensity are complicated with other environmental
factors.8 Compared to intensity-based sensors, ratiometric fluores-
cence sensors show obvious advantages. They allow for the simul-
taneous recording of two measurable signals in the presence and/
or absence of the analyte and can, in principle, allow for accurate
and quantitative readouts.9 Accordingly, it is absolutely essential
⇑
Corresponding authors. Tel./fax: +86 28 85415886.
E-mail addresses: kli@scu.edu.cn (K. Li), xqyu@scu.edu.cn (X.-Q. Yu).
0040-4039/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetlet.2013.08.046

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K.-K. Yu et al. / Tetrahedron Letters 54 (2013) 5771–5774
Figure 1. (A) Cation selectivity (kex = 360 nm) of probe 1 (10 lM) in PBS buffer (25 mM, pH = 7.20). (B) The bars represent the ratiometric fluorescence intensity [I472/I410] of
the mixture, from 1 to 20: probe 1 only, Li+, Na+, K+, Ca2+, Mg2+, Ba2+, Pb2+, Al3+, Cr3+, Cd2+, Fe2+, Fe3+, Ag+, Hg2+, Ni2+, Zn2+, Cu2+, Co2+, and Cu+.
Scheme 2. The supposed mechanism of probe 1 with Cu+.

K.-K. Yu et al. / Tetrahedron Letters 54 (2013) 5771–5774
5773
Figure 2. (A) Cation selectivity (kex = 360 nm) of probe 4 (10
From 1 to 19: probe 4 only, Li+, Na+, K+, Ca2+, Mg2+, Ba2+, Pb2+, Al3+, Cr3+, Fe2+, Fe3+, Ag+, Hg2+, Ni2+, Zn2+, Cu2+, Co2+, and Cu+.
l
M) in PBS buffer (25 mM, pH 7.20). (B) The bars represent the fluorescence intensity at 410 nm of the [1-Cu+].
to design a novel ratiometric fluorescence sensor for Cu+. Herein,
we present a reaction-based ratiometric probe 1 for Cu+ under a
physiological reducing environment.
The emission intensity ratios (I472/I410) of Cu2+ and Cu+ are 0.42
and 13.82, respectively. Obviously, probe 1 is capable of discrimi-
nating between Cu+ and Cu2+ and other metals. Additionally, the
significant red-shift indicated that the benzylic ether (C–O) linkage
indeed was cleaved, accompanied by the release of the coumarin
moiety. Notably, the cleavage of C–O bond only requires approxi-
mately 20 min (Fig. S3); we use a 30 min mixing time to ensure
a complete reaction. In order to provide support for the proposed
mechanism, various ESI-MS spectra of the reaction between probe
1 and a Cu+ solution were collected at different times (Scheme 2).
In the absence of Cu+, the peak at m/z 544.1 was recognized as cor-
responding to probe 1 (Fig. S4, Supplementary data); after Cu+ was
added, peaks m/z 554.75 and 602.72, corresponding to [1-Cu+] and
[1-Cu+-H2O] intermediates, respectively, were found immediately
(Fig. S5). After 30 min the peak for probe 1 and [1-Cu+] disappeared
and a new peak at m/z 396.1 was detected. This new peak corre-
sponds to the complex between Cu2+ and TPA (Fig. S6). These re-
sults provide strong evidence for the Cu+ catalyzed oxidative
cleavage of the benzylic C–O bond of probe 1 in the presence of
O2. From these results, we infer that Cu+ first coordinates with
probe 1 and water. This complex is then oxidized by O2 resulting
eventually in cleavage of the C–O bond and formation of a stabi-
lized Cu2+–TPA complex.3a,13
Coumarin was selected as the fundamental platform, because it
is known to be a strongly fluorescent compound, and it is relatively
easy to synthesize a large variety of structural varients.10 Couma-
rin-based probes are widely used in various biological assays.11
Recently, Taki et al. reported a reaction-based turn-on sensing of
Cu+ by using a tetradentate ligand tris[(2-pyridyl)-methyl]amine
(TPA).1a Inspired by this work, probe 1 was prepared by combining
the coumarin platform (compound 2) with a tetradentate moiety
through an oxidatively-cleavable benzyl ether bond (Scheme 1).
Compound 2 and probe 1 exhibited native blue-green and violet
fluorescence, respectively. Probe 1 was designed so that in the
presence of Cu+ the reaction between the probe and the redox-ac-
tive cation would selectively remove TPA from probe 1 by oxida-
tively cleaving the benzylic ether bond, resulting in the release of
the bright blue-green coumarin fluorophore with a resultant
increase in the quantum yield12 and a large Stokes shift in the
observed fluorescence emission.
The target compound (probe 1) could be synthesized easily via a
two-step nucleophilic substitution with a moderate overall yield of
54%. Meanwhile, pyridylmethylene ether 4 was also synthesized to
serve as a control compound. The probes and intermediates all
were characterized by 1H NMR, 13C NMR, and MS (Supplementary
data).
The fluorescence responses of probe 1 to a range of physiologi-
cally and environmentally relevant metal cations were measured
in an aqueous buffer solution (25 mM PBS, pH 7.20), containing
2 mM glutathione (GSH) for maintaining the dominant oxidation
state of Cu+. As exhibited in Figure 1, the addition of 10 equiv of
Li+, Na+, K+, Ca2+, Mg2+, Ba2+, Pb2+, Al3+, Cr3+, Cd2+, Fe2+, Fe3+, Ag+,
Hg2+, Ni2+, Zn2+, Cu2+, and Co2+, did not cause any significant change
in the fluorescence spectra of probe 1, while Zn2+, Cd2+ could lead to
a moderate fluorescence enhancement at 410 nm. However, upon
addition of 10 equiv Cu+, a large red shift of probe 1 from 410 to
472 nm (with a corresponding color change from violet to blue-
green under a 365 nm UV lamp, Fig. S1) was observed, in addition,
Furthermore, to verify that cleavage of the benzylic C–O bond
only occurs after the [1-Cu+] complex is generated, the fluorescent
response of compound 4 was investigated under the same condi-
tions as with probe 1. As shown in Figure 2, the emission intensity
of 4 showed no obvious change upon the addition of the tested
ions, including Cu+. These results indicate the importance of the
TPA moiety. With the replacement of TPA by pyridine, Cu+ could
not effectively complex with 4, and the C–O bond of the ether
could not be broken, resulting in the maintenance of the fluores-
cence. That is to say, the benzylic ether (C–O) linkage is cleaved
only after Cu+ selectivity coordinates with the tetradentate moiety
of probe 1.
The fluorescence titration of probe 1 (10 l
M) toward Cu+ was
conducted in a buffered solution (25 mM PBS, pH 7.20) in the pres-
ence of 2 mM glutathione (GSH). With increasing concentrations of
Cu+, the fluorescence of probe 1 at 410 nm decreased gradually,
a remarkable enhancement in emission intensity ratios (I472/I410
from 0.26 to 13.82 was found. Meanwhile, the response of probe 1
(10
M) to 10 equiv of Cu+ in the presence of 10 equiv of different
)
accompanied by the appearance of
a
new peak at 472 nm
l
(Fig. 3). Additionally, the emission intensity ratios (I472/I410) in-
competing metal ions was also investigated (Fig. S2). The results
indicate that Zn2+, Ni2+ and Cd2+ greatly reduced the ability of probe
1 to detect Cu+ due to the strong coordination abilities of these
competing cations for the TPA moiety.
creased from 0.23 to 7.66 when 5 l
M Cu+ was added and the detec-
tion limit of probe 1 was calculated to 2.29 Â 10À7 M14 (Fig. S7).
Moreover, the effect of pH on the cleavage of the benzylic ether
(C–O) linkage was investigated and summarized in Figure 4.

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K.-K. Yu et al. / Tetrahedron Letters 54 (2013) 5771–5774
with a detecting limit of 2.29 Â 10À7 M. ESI-MS results indicated
that the oxidative properties of Cu+ are responsible for the mecha-
nism, which resulted in cleavage of the benzylic ether (C–O) bond.
This unique chemosensor is an important and valuable new ad-
vance for the ratiometric detection of Cu+.
Acknowledgments
This work was financially supported by the National Program
on Key Basic Research Project of China (973 Program,
2012CB720603 and 2013CB328900) and the National Science
Foundation of China (Nos. 21232005 and 21001077). We also
thank Analytical & Testing Center of Sichuan University for NMR
analysis. K.L. thanks Professor Jason J. Chruma (Sichuan University)
for assistance with document preparation.
Supplementary data
Figure 3. Fluorescence spectra (kex = 360 nm) of probe 1 (10 lM) in PBS buffer
(25 mM, pH 7.20) upon addition of Cu+ (0–5
lM). Inset: Ratiometric fluorescence
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.tetlet.2013.08.046.
intensity [I472/I410] as a function of Cu+.
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Figure 4. Ratiometric fluorescence intensity [I471/I410] as Cu+ (100
lM) was added
in different pH of probe 1 (10
lM, 25 mM PBS buffer). Black: probe 1 only; red:
probe 1 with Cu+ (2 nm GSH).
Throughout the biologically relevant pH range of 4.50–10.50, probe
1 could detect Cu+ by the mechanism we mentioned (Scheme 2).
Accordingly, there is no need to worry about interference from
pH effects in the detection of Cu+ with probe 1, except possibly
for strongly basic conditions (pH >10.50) which may result in
hydrolytic ring-opening of the coumarin moiety.
In conclusion, a reaction-based ratiometric chemosensor is re-
ported for the selective detection of Cu+ in aqueous solutions.
Probe 1 exhibited high selectivity toward Cu+ over other metals
14. Shortreed, M.; Kopelman, R.; Kuhn, M.; Hoyland, B. Anal. Chem. 1996, 68, 1414.