Dicarboxylated ethynylarenes as buffer-dependent chemosensors for Cd(II), Pb(II), and Zn(II)

Article abstract of DOI:10.1016/j.tetlet.2013.07.111

Two dicarboxylated ethynylarenes were prepared efficiently from condensation of 1,3-bis(3-aminophenylethynyl)benzene with 2 equiv of either succinic anhydride or glutaric anhydride. These compounds behave as fluorescent chemosensors selective for Cd(II), Pb(II), and Zn(II) cations under buffered aqueous conditions, with analyte binding observed as bathochromically shifted, intensified fluorescence. It was noteworthy that the fluorescence responses varied significantly with buffer identity. A conformational restriction mechanism involving reversible interactions between the fluorophore, metal cation, and buffer itself is proposed.

Full text of DOI:10.1016/j.tetlet.2013.07.111

Tetrahedron Letters 54 (2013) 5366–5369
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Dicarboxylated ethynylarenes as buffer-dependent chemosensors
for Cd(II), Pb(II), and Zn(II)
⇑
James T. Fletcher , Brent S. Bruck, Douglas E. Deever
Department of Chemistry, Creighton University, 2500 California Plaza, Omaha, NE 68178, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Two dicarboxylated ethynylarenes were prepared efficiently from condensation of 1,3-bis(3-aminophen-
ylethynyl)benzene with 2 equiv of either succinic anhydride or glutaric anhydride. These compounds
behave as fluorescent chemosensors selective for Cd(II), Pb(II), and Zn(II) cations under buffered aqueous
conditions, with analyte binding observed as bathochromically shifted, intensified fluorescence. It was
noteworthy that the fluorescence responses varied significantly with buffer identity. A conformational
restriction mechanism involving reversible interactions between the fluorophore, metal cation, and buf-
fer itself is proposed.
Received 18 June 2013
Revised 17 July 2013
Accepted 21 July 2013
Available online 27 July 2013
Keywords:
Sensor
Fluorescent
Ratiometric
Cation detection
Ethynylarene
Ó 2013 Elsevier Ltd. All rights reserved.
Because the majority of transition metal cations cannot be
observed directly by spectroscopic measurement, the indirect
detection of such analytes through the use of fluorescence chemo-
sensors constitutes an ongoing area of research.1 As the most abun-
dant transition metal in the body, Zn(II) is an important target,2 as
are Cd(II) and Pb(II) due to their strong toxicity and persistence as
environmental pollutants.3 Selective detection of such analytes in
aqueous environments is challenging.4
NH2
NH2
NH2
Pd(PPh3)4
CuI
I
I
+
2
tetrahydrofurhan
triethylamine
500C, 24 h
1, (78%)
O
O
O
O
O
O
2
2
Arene-based sensors operating via conformational restriction
mechanisms can be used to detect a variety of cationic,5,6 anionic,7
and small molecule7c,8 analytes. Ethynylarenes are also attractive
templates for constructing such fluorescent chemosensors9 due
to their efficient and modular construction, well-defined optoelec-
CH2Cl2
r.t. 24 h
CH2Cl2
r.t. 24 h
O
O
O
OH
HO
O
OH HO
tronic properties, and rotationally mobile p
-systems.10 Conforma-
H
H
H
H
N
O
O
N
N
O
O
N
tional restriction sensing relies on analyte binding at a peripheral
recognition site leading to rigidification of the covalently con-
nected fluorophore unit.6a,11 This results in an intensification of
fluorescence signal relative to the analyte unbound state. If coplan-
arity between the fluorophore’s arene units is simultaneously per-
turbed, hypsochromic or bathochromic shifts in emission can also
be observed.6
The goal of this investigation was to develop a new family of
fluorescence chemosensors comprised of ethynylarene fluorophore
units and carboxylic acid containing analyte recognition units.
These molecules were designed to operate via a conformational
restriction mechanism. In the absence of analyte the arene
3, (98%)
Scheme 1. Preparation of ethynylarene sensors.
2, (95%)
subunits can freely rotate at the alkyne bonds, while cooperative
binding of divalent cation analytes between sensor’s peripheral
carboxylate groups causes rigidification and increased coplanarity
in the ethynylarene fluorophore.
⇑
Scheme 1 summarizes the simple preparation of the two dicarb-
oxylated ethynylarenes used in this investigation. Sonogashira
Corresponding author. Tel.: +1 402 280 2273; fax: +1 402 280 5737.
E-mail address: jamesfletcher@creighton.edu (J.T. Fletcher).
0040-4039/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetlet.2013.07.111

J. T. Fletcher et al. / Tetrahedron Letters 54 (2013) 5366–5369
5367
Figure 1. Changes in fluorescence emission of 2 (50 lM) upon titration with Cd(II), Pb(II), and Zn(II) with TRIS (pH 7.6) buffer.
Figure 2. Changes in fluorescence emission of 3 (50 lM) upon titration with Cd(II), Pb(II), and Zn(II) with TRIS (pH 7.6) buffer.
coupling12 between 1,3-diiodobenzene and two equivalents of
meta-ethynylanaline produced diamine 1 as previously reported.13
Condensation of 1 with two equivalents either succinic or glutaric
anhydride resulted in dicarboxylated ethynylarenes 2 and 3,
respectively. These reactions were conducted in CH2Cl2 solvent,
and as each reaction progressed the carboxylated products precip-
itated from solution. Reaction progress was monitored by HPLC,
where condensation products displayed longer retention times
than reactant 1 (Supplementary data). Reaction completion was
also assessed by 1H NMR in CD3OD, where aromatic region signals
of 2 and 3 displayed significant downfield shifts relative to reactant
1. Each of these products was soluble in aqueous buffer at pH 7.6
and upon excitation at kmax = 300 nm displayed fluorescence emis-
sion at kmax = 350 nm.
attractive template to modularly incorporate a diversity of periph-
eral analyte binding units for future chemosensor development.
At this stage of the study, the hypothesized mechanism for ana-
lyte binding was cooperative intramolecular chelation between
each carboxylate unit of the sensor and the divalent cation analyte.
Unexpectedly, when TRIS buffer was replaced with phosphate dur-
ing subsequent control studies, no ‘turn-on’ signals were observed
despite the identical pH 7.6 conditions of the assays (Supplemen-
tary data). Because TRIS has been shown to perturb the results of
metal binding assays by coordinating metal cations,15 a survey of
buffers was conducted to evaluate the influence of buffer identity
on chemosensor performance.
Figure 4 summarizes the commercially available buffers and
control compounds selected for this survey. These can be organized
into three families: the trihydroxy-containing TRIS family, the
morpholine-containing MOPS family and the piperazine-contain-
ing HEPES family. Buffers were selected so that the impact of any
minor structural differences on sensor performance could be iden-
tified. The small organic molecules triethanolamine (TEA), N-meth-
ylmorpholine (NMM), and dimethylpiperazine (DMP) were also
employed as buffers in this study to gain additional insight on
structure–property relationships.
Aqueous solutions of 2 and 3 were prepared using each of these
buffers adjusted to pH 7.6 with HCl or NaOH. High throughput
screening was then performed against the same panel of cations
as the initial TRIS study. Varying the buffer identity did not lead
to any significant ‘turn-on’ signals for analytes beyond Cd(II),
Pb(II), and Zn(II), but there was surprising variation in sensor out-
put observed for 2 (Fig. 5) and 3 (Supplementary data).
The modest size difference in the carboxylate chains of 2 and 3
significantly impacted signal output for the buffers surveyed, with
2 displaying generally weaker signal strengths than 3. A notable
feature of this system is that analyte selectivity can be tuned sim-
ply by changing buffer identity. For example, while 2 + TRIS de-
tected each of Cd(II), Pb(II), and Zn(II), 2 + TAPS was selective for
Cd(II) and 2 + DMP was selective for Zn(II).
High-throughput fluorescence screening1c,14 was used to exam-
ine whether these compounds would display the predicted spec-
troscopic responses upon exposure to cationic analytes in
aqueous solution. TRIS buffered aqueous 100
of 2 and 3 were mixed with varying ratios of 100–500
metal chloride salt solutions in 96 well plates. The resulting solu-
tions of 50 M sensor mixed with 50–250 M metal cations were
lM stock solutions
lM aqueous
l
l
analyzed for fluorescence changes induced by increasing metal
concentrations. Among the twelve metal cation analytes studied,
only Cd(II), Pb(II), and Zn(II) produced bathochromic shifts in fluo-
rescence output when mixed with 2 (Fig. 1) or 3 (Fig. 2). Concur-
rent signal intensification varied significantly among metal/
sensor combinations. These observations are consistent with a con-
formational restriction mechanism of signal generation. Enabled
by the 30–40 nm shift in emission wavelength upon analyte bind-
ing, a ratiometric comparison of emission intensities (390/340 nm)
was used to define the ‘turn-on’ sensor response (Fig. 3).
In comparing the results of 2 and 3 it is evident that the single
methylene unit difference between succinic and glutaric units sig-
nificantly impacts signal generation. Compound 2 shows a greatly
enhanced signal strength for Zn(II) relative to 3, while 3 shows a
strong selectivity toward Pb(II). The significant bathochromic re-
sponses observed for 2 and 3 enabling ratiometric interpretation
of data positions the 1,3-bis(arylethynyl)benzene motif as an
Because 2 and 3 are each inactive when using phosphate or cit-
ric acid buffers, amine functionality appears necessary for ‘turn-on’

5368
J. T. Fletcher et al. / Tetrahedron Letters 54 (2013) 5366–5369
Figure 3. Ratiometric analysis for assays of 2 and 3 with 1–5 equiv of metal cation
analytes, showing ‘turn-on’ responses for Cd(II), Pb(II), and Zn(II).
HO
HO
HO
HO
HO
HO
SO3H
NH2
N
H
SO3H
N
H
HO
TRIS
HO
HO
TAPS
TES
HO
O
OH
O
HO
OH
OH
HO
OH
N
N
H
HO
OH
HO
O
O
OH
Triethanolamine
Tricine
Citric Acid
SO3H
N
SO3H
N
N
O
O
O
Figure 5. Ratiometric sensing output for 2, comparing impact of buffer identity.
MOPS
MES
NMM
SO3H
SO3H
N
N
N
‘turn-on’ responses. In comparing TES with TAPS and MES with
MOPS, longer spacing between ammonium and sulfonate groups
in the buffer correlates to stronger ‘turn-on’ signals for most ana-
lytes. These observations collectively indicate that electrostatic
repulsion between buffer sulfonate and sensor carboxylate groups
destabilize analyte binding, and results in an interesting diversity
of ‘turn-on’ sensing patterns toward Cd(II), Pb(II), and Zn(II) at
the concentrations studied.
In the absence of analyte there were no observed differences in
the fluorescence emission of 2 and 3 among the buffers studied. So
while the data generated from this buffer survey suggests that a
direct interaction between sensor and buffer is necessary for
‘turn-on’ signal generation, these two constituents alone are un-
able to generate fluorescence changes. ‘Turn-on’ signals were only
observed when metal cation analytes were present, supporting a
three-component binding event that leads to conformational
N
N
N
HO
HO3S
HEPES
PIPES
Figure 4. Identities of buffers surveyed.
DMP
signal generation. It is proposed that the amine containing buffers
interact electrostatically with the sensor carboxylate units via their
ammonium groups present at pH 7.6, supported by the observation
that all sensors failed to operate at either high (pH 10) or low (pH
3) pH levels. Optimal ‘turn-on’ signals were observed for
buffer:sensor ratios of 1000:1 or 500:1 but were lost at ratios of
100:1 or less. Hence, this weak electrostatic interaction driving
buffer/sensor binding in these assays is likely enforced by the large
statistical excess of buffer relative to sensor.
The importance of this electrostatic interaction between buffer
and sensor is also supported by the inactivity of tricene buffered
sensors, where buffer carboxylate groups could compete with sen-
sor carboxylate groups for ammonium affinity. In contrast, buffers
possessing sulfonate groups were able to generate ‘turn-on’ sig-
nals. Responses by buffers with either one or zero sulfonate groups
(such as MOPS vs NMM and HEPES vs DMP) were nearly identical,
while the buffer with two sulfonate groups (PIPES) showed no
restriction and increased p-system coplanarity in 2 and 3. It is pro-
posed that the hydroxy, ether, and amine functionalities present in
the TRIS, MOPS, and HEPES families of sensors are essential for
metal analyte binding, but additional study is needed to precisely
define these collective binding interactions.
To identify the detection limits afforded by
2 and 3 in
various buffers, serial dilution assays were performed on those

J. T. Fletcher et al. / Tetrahedron Letters 54 (2013) 5366–5369
5369
Institute for General Medical Science (NIGMS) (5P20GM103427),
a component of the National Institutes of Health (NIH) and its con-
tents are the sole responsibility of the authors and do not necessar-
ily represent the official views of NIGMS or NIH.
HO
H
O
NH3
M+2
+
O
H
_
_
O
O
O
O
O
O
O
M+2
_ O
Supplementary data
_
H
H
H
H
N
O
O
N
N
O
O
N
Supplementary data (details of synthetic procedures and char-
acterization of 1, 2, and 3, fluorescence titration assay results,
and table of detection limits) associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/
j.tetlet.2013.07.111.
Figure 6. Illustrations of binding mechanisms for 2. Left: initially hypothesized
binary interaction between sensor and metal. Right: possible ternary interaction
between sensor, metal, and buffer (TRIS example shown).
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sensor/buffer/metal combinations showing strong ‘turn-on’ signals
(Supplementary data). The reversibility of analyte binding is
supported by the design of these dilution experiments where
pre-saturated ‘turn-on’ signals were lost upon dilution. The metal
concentration at which the bathochromic shift became absent
was defined as the lower detection limit for each system. Buffer
identity impacted binding affinity up to two orders of magnitude,
and 3 generally showed 10-fold stronger binding affinity than 2
for analogous buffers. Several buffer–metal combinations for 2
and 3 continued to generate ‘turn-on’ signals at the 1 lM lower
detection limit of the assay, establishing the ability of this chemo-
sensing motif to detect aqueous Cd(II), Pb(II), and Zn(II) at biolog-
ically relevant concentrations.
It is proposed that the ‘turn-on’ fluorescence chemosensing re-
sponses of 2 and 3 for micromolar Cd(II), Pb(II), and Zn(II) analytes
in aqueous solution involves the direct participation of buffer mol-
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study serves as a cautionary example that buffers themselves can
significantly perturb fluorescence output, and that an evaluation
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Future work will aim to more precisely define the noncovalent
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This publication was made possible by grants from the National
Center for Research Resources (5P20RR016469) and the National