Highly selective fluorescent chemosensors for cadmium in water

Article abstract of DOI:10.1016/j.tet.2004.08.047

The design, synthesis and photophysical evaluation of two new chemosensors 1 and 2 is described for the selective detection of Cd(II) in water at pH 7.4. Both are based on the use of aromatic iminodiacetate receptors that connected to an anthracene fluoro

Full text of DOI:10.1016/j.tet.2004.08.047

Tetrahedron 60 (2004) 11239–11249
Highly selective fluorescent chemosensors for cadmium in water
b
a
Thorfinnur Gunnlaugsson,a, T. Clive Lee and Raman Parkesh
*
aDeparment of Chemistry, Trinity College Dublin, Dublin 2, Ireland
bDepartment of Anatomy, Royal College of Surgeons in Ireland, St. Stephen’s Green, Dublin 2, Ireland
Received 1 February 2004; revised 5 August 2004; accepted 19 August 2004
Available online 1 October 2004
Abstract—The design, synthesis and photophysical evaluation of two new chemosensors 1 and 2 is described for the selective detection of
Cd(II) in water at pH 7.4. Both are based on the use of aromatic iminodiacetate receptors that connected to an anthracene fluorophore by
covalent methyl spacers. These are highly water-soluble sensors where the fluorescence is ‘switched off’ between pH 3–11, due to
photoinduced electron transfer (PET) quenching of the anthracene excited state by the receptor. Upon protonation of the receptor, the
emission was however, ‘switched on’. From these changes pKas of 1.8 and 2.5 were determined for 1 and 2 respectively. Both showed good
selectivity for Cd(II) over competitive ions such as group II and Zn(II), Cu(II), Co(II). For 1, having a single receptor, only a weak monomer
anthracene emission was observed for the free sensor at pH 7.4 (HEPES buffer, 135 mM NaCl). Upon Zn(II) titration, a broad red shifted
emission occurred, centred at 468 nm. In the presence of Cd(II), a similar red shifted emission was also observed, however, this time centred
at 506 nm. In contrast to these results, the fluorescence of 2 in the presence of Zn(II) gave rise to typical monomeric anthracene emission, due
to suppression of PET, that is, the anthracene emission was ‘switched on’. Nevertheless, in the presence of Cd(II) a broad emission centred at
500 nm was observed, similar to that seen for 1. These ion induced long wavelength emission bands were assigned to the formation of charge-
transfer complexes (exciplexes) between the anthracene moieties and the ion-receptor complexes. Importantly, for both 1 and 2, a selective
detection of Cd(II) was possible, even in the presence of Zn(II).
q 2004 Elsevier Ltd. All rights reserved.
1. Introduction
satisfy the aforementioned criteria is thus currently of great
importance. In this paper we describe the design, synthesis
and the photophysical evaluation of two novel fluorescent
sensors, 1 and 2 for the selective sensing of Cd(II) under
physiological pH conditions.10
The development of luminescent sensors lies at the heart of
supramolecular chemistry.1 Several research groups have
recently reviewed work in this area.1–3 We have developed
various types of luminescent devices, some of which have
been developed as sensors for ions and molecules. These
include fluorescent4 and colorimetric5 sensors as well as
lanthanide luminescent6 chemosensors for cations such as
LiC, NaC, KC, Cu(II) and Zn(II) and anions such as FK,
AcOK, H2POK4 , pyrophosphate, carboxylates, and aromatic
carboxylates such as salicylic acid. The main driving force
for this work has been the increased use of chemosensors for
medical diagnostics,7 and in particular for critical care
analysis.8 During the course of our research we initiated an
investigation into developing luminescent sensors for metal
ions such as Cd(II), but only a few examples of sensors for
Cd(II) have been reported.9 Furthermore, those reported to
date for Cd(II) detection suffer from several drawbacks such
as low aqueous solubility, poor sensitivity and selectivity, as
well unsuitability for use in the physiological pH range. The
need for developing ideal chemosensors for Cd(II), that
Cadmium, an ion that poses an increasing environmental
and health risk, has a naturally low abundance in nature
(0.1–0.5 ppm).11 However, cadmium levels as high as
500 ppm have recently been reported to accumulate in
sedimentary rocks and marine phosphates and phosphor-
ites.12 One of the reasons for this is that Cd(II) is use in Ni–
Cd batteries, as well as in feritilsers.13 In the former,
Cd(OH)2 is used as one of the two principal electrode
materials.14 Even though LiC based batteries are now
becoming more common, the disposal of unused Ni–Cd
batteries is, and will be, a major environmental problem,
particularly since cadmium has profound biological effects
such as inducing renal dysfunction, reduced lung capacity
and emphysema.15 The tendency of this toxic ion to
accumulate in organs such as the kidneys, hippocampus,
thyroid and spleen, results in severe physiological effects.16
Although only about 0.4% of the total amount of ingested
cadmium is retained in the human body, recent studies
indicate that high toxic levels of Cd(II) in the kidney and
thyroid glands can occur, particularly in miners.17
Keywords: Chemosensors; Fluorescent; PET; Supramolecular chemistry.
* Corresponding author. Tel.: C35316083459; fax: C35316712628;
e-mail: gunnlaut@tcd.ie
0040–4020/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2004.08.047

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T. Gunnlaugsson et al. / Tetrahedron 60 (2004) 11239–11249
Furthermore, it has recently be suggested that Cd(II) affects
bone demineralisation by activating the osteoclast bone
cells.18 Moreover, cadmium is also thought to be a potential
carcinogen.19 All of these factors make the detection and
quantification of Cd(II) a vital area of research. With this in
mind we set out to develop the two chemosensors 1 and 2.
We designed these sensors on the fluorophore-spacer-
receptor and the receptor-spacer-fluorophore-spacer-recep-
tor models developed by de Silva et al. for PET sensors.20
Here we give a full account of our effort.10
2. Results and discussion
2.1. Synthesis and characterisation of 1 and 2
For the two chemosensors 1 and 2, we selected anthracene
as a fluorophore, and a simple aromatic iminodiacetate as
the receptor. The reasons for this were twofold. First, the
photophysical properties of anthracene in PET sensors is
well established.3,20 Secondly, the aniline based receptor
could be used under physiological pH conditions as the
nitrogen receptor moiety would only protonate under highly
acidic conditions.21 Moreover, this simple design would
overcome any competitive binding of any other physiologi-
cally important cations such as Mg2C and Ca2C 21
The use
.
of potassium salts of the carboxylates would impart high
water solubility to 1 and 2. In the case of 2, we predicted that
the presence of two receptor units would statistically
increase any PET rate quenching from the receptor to the
excited singlet state of the anthracene, resulting in effective
luminescent switching, where the emission would only be
switched on in the presence of Cd(II).
The synthesis of 1 and 2 is shown in Scheme 1. It began with
the synthesis of the iminodiester receptor 3, by reacting
aniline with ethyl bromoacetate, using potassium dihydro-
gen phosphate as a base in CH3CN in 89% yield. The two
sensors were made in two steps from this intermediate. For
1, this was achieved by Friedel–Crafts alkylation of 3 with
9-chloromethylanthracene (which was made in two steps
from 9-anthraldehyde by reduction followed by chlorin-
ation). This alkylation was very successful, giving the
desired product 4 in 70% yield using AlCl3 in CH3CN,
whereas using ZnBr2 as catalyst resulted in a low yield of
only 15%. The final product was purified using silica flash
column chromatography using ethyl acetate:hexane (2:3) as
eluant. In an analogous way, 2 was made by reacting 2 equiv
of 3 with 9,10-bischloromethylanthracene (made by
chloromethylation of anthracene in a single step),24
yielding the tetraester 5, in 72% yield, after purification
by flash column chromatography using ethyl acetate:hexane
(2:3) as eluant. The final products were obtained by
alkaline ester hydrolysis of 4 and 5 using aqueous KOH in
refluxing MeOH solution, yielding 1 and 2 in 92 and 90%
yields respectively after precipitation from the cold
solution.
Scheme 1. The synthesis of 1 and 2 from aniline. (i) CH3CN, KI, K2HPO4,
BrCH2CO2Et, reflux; (ii) 9-chloromethylanthracene, AlCl3, CHCl3, reflux;
(iii) 9,10-bischloromethylanthracene, KOH, H2O, MeOH, reflux.
6.47 ppm were observed for the phenyl protons. In
comparison, the H NMR of 1 showed two doublets for
1
the phenyl protons at 6.20 and 6.82 ppm, respectively. The
electrospray mass spectrum (ESMS) showed a peak at 476
1
mass units for MCHC (100%). In the H NMR of 5, the
phenyl protons appeared as doublets at 6.50 and 7.01 ppm
and a singlet was observed at 4.95 ppm for the two methyl
spacers. The symmetrical nature of the molecule was further
supported by the 13C NMR in which only 13 carbon
resonances were observed. Furthermore, the ESMS spec-
trum gave a peak at 733 mass units for MCHC. After
alkaline hydrolysis of 5 the 1H NMR spectrum of 2 (Fig. 1)
showed two double–doublet resonances for the anthracene
protons at 8.31–8.29 ppm and at 7.45–7.42 ppm whereas the
phenyl protons were observed as two doublets at 6.91 and
6.28 ppm. The two methyl spacers appeared as a singlet at
4.86 ppm and the two methyl iminodiester spacers as a
singlet at 4.86 ppm. The 13C NMR gave, as expected, rise to
only 11 resonances.
The structures of 1–4 were confirmed by the usual
spectroscopic methods (see the Section 4). In the H NMR
1
of 4, a singlet at 8.4 ppm and two multiplets at 8.21–8.24
and 8.02–8.05 ppm, respectively were attributed to the
anthracene protons, whereas two doublets at 6.98 and

T. Gunnlaugsson et al. / Tetrahedron 60 (2004) 11239–11249
11241
Figure 1. The 1H NMR of 2 (D2O, 400 MHz). The appropriate resonances have been assigned as a–f.
2.2. Ground and excited state evaluation of 1 and 2
In contrast to the above changes the fluorescence emission
of 1 and 2 was substantially changed upon protonation of the
amino moiety of the receptors (lexZ381 nm). For 1 in basic
solution (pHw12), the fluorescence was so minor that it can
be said that the emission was switched off. This is due to
PET quenching from the receptor to the excited state of the
fluorophore. However, upon titration with acid, a gradual
increase was observed for the anthracene emission, which
had three characteristic bands centred at 392, 415 and
439 nm and a weak shoulder at 466 nm respectively. This
fluorescence enhancement was approximately 60 fold. The
increase in fluorescence is due to the protonation of the
tertiary nitrogen atom of the receptor moiety, resulting in a
decrease in the reduction potential of the receptor, which is
typical PET behaviour. This prevents electron transfer from
the receptor to the excited state of the anthracene, thus
switching the emission on. Similar results were observed for
2 (lexZ385 nm), as seen in Figure 3. In basic solution, the
fluorescence of 2 was quenched, and in the pH range of
pHZ3.5–12 virtually no emission was observed. However,
at around pH 3.4, a broad band centered at 470 nm appeared
with an increase in the emission intensity (Fig. 3, insert).
Upon further acidification, between pHw3.3–1.0, the
normal anthracene emission with four main emission
bands at 405, 428 454 and 480 nm, respectively was
observed with concomitant increase in the fluorescence
intensity of ca. 600 fold.
The photophysical properties of the two chemosensors 1,
and 2 were evaluated in water and in pH 7.4 HEPES
buffered solution in the presence of 0.135 M of NaCl to
maintain constant ionic strength.
2.3. The effect of pH
We first evaluated the pH response of 1 and 2 in water. The
absorption spectra of 1 exhibited five main absorption bands
centred at 314, 337, 365, 385 and 402 nm (3Z17.1!
103 MK1cmK1). The absorption spectra of 1 as a function of
pH is shown in Figure 2. When an alkaline solution of 1 was
titrated with acid, no absorption shifts were seen in the
positions of the above mentioned bands. However, a small
decrease in the absorption intensity (!5%) was observed.
This was most likely due to the electronic effect observed
between the anthracene fluorophore and positively charged
aniline moiety upon protonation (through space). These
small effects are well known and signify that no significant
ground state interactions occur between the fluorophore and
the receptor, due to the presence of the covalent spacer. The
absorption spectra of 2 as a function of pH were recorded in
a similar manner. The absorption spectra of 2 showed four
absorption bands at 343, 362, 381 and 402 nm (3Z18.6!
103 MK1cmK1) respectively. The absorption spectra gen-
erated during the pH titration showed only a negligible
decrease in the intensity similar to that seen for 1 in
Figure 2. This shows that under these conditions only minor
ground state interactions take place between the receptors
and the anthracene moiety, confirming the insulating role of
the two methylene spacers.
We investigated these changes in some detail for 2. Our
studies revealed that from pH 12–3.5, the fluorescence of
the anthracene moiety is efficiently quenched via a PET
process from the receptors to the excited singlet state of the
anthracene. However, as the pH is decreased protonation of

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T. Gunnlaugsson et al. / Tetrahedron 60 (2004) 11239–11249
Figure 2. The changes in the absorption spectrum of 1 upon titration an alkaline solution of 1 with diluted acid.
the tertiary nitrogen of one receptor takes place, which
causes a weak charge transfer (CT)23 interaction between
the protonated receptor and the anthracene fluorophore. This
resulted in a charge transfer band at 470 nm. When the pH is
decreased further, both receptors become protonated. These
results in a repulsive interaction (RI), and to minimise it the
receptors move as far apart as possible in space. Conse-
quently, typical anthracene emission is observed in the pH
range of 3.3–1. We were unable to determine more than one
pKa from the changes, Figure 4. The reason for this is most
likely due to the fact that the changes in fluorescence
emission correspond to the protonation of only one of the
two receptors. Lippard et al. have reported similar
observations in the case of fluorescein based sensors for
Zn(II) ions, where the sensor also had two receptor moieties
that could be protonated.22
Plotting the changes in the fluorescence emission as a
function of pH resulted in a sigmoidal curve that changes
over two pH units for both sensors. This pH dependent
fluorescence behaviour is a typical ‘on-off’ PET sensor
characteristic, indicating a simple acid-base equilibrium and
one to one binding. The fluorescence intensity changes at
415 nm versus pH are shown for 2 in Figure 4. These, and
the changes at any other wavelength, can be used to
determine the pKa of the protonation of the nitrogen moiety.
Data fitting analysis of the emission intensity changes at
404, 428 and 454 nm respectively resulted in pKa values of
2.4, 2.3 and 2.8 providing an average pKa value of 2.5G0.1
for 2. Similarly for 1, the changes at 392, 415 and 439 nm
gave pKa values of 1.75, 1.74 and 2.0 (G0.1), providing an
average pKa of 1.83G0.1 for the protonation of the aniline
receptor in 1. This makes both 1 and 2 particularly attractive
chemosensors for both physiological and environmental
monitoring of Cd(II), for instance in highly acidic soils
Figure 3. The changes in the fluorescence emission spectra of 2 upon
acidification. Inserted are the changes observed between pH 3.4 and 12.
Figure 4. The plot of fluorescence intensity versus pH for 1 at 415 nm.
Fitting of these data points using non-linear regression gave the pKa.

T. Gunnlaugsson et al. / Tetrahedron 60 (2004) 11239–11249
11243
where the pH 3–4 can be reached. Similarly, the pH
[
[
[
[Zn(II)]
titrations of the two esters 4 and 5 switched the fluorescence
on for both of these molecules upon protonation of the
aniline amine.
20
10
0
2.4. The effect of group II and transition metal ions
The ability of 1 and 2 to recognise group II and various
transition metal ions was investigated at pH 7.4 in buffered
HEPES solution in the presence of 0.135 M of NaCl. We
first evaluated the ability of 1 and 2 to recognise group II
cations. The changes in the fluorescence emission spectra of
1 are shown in Figure 5 for titration with Ca2C. As can be
seen from this titration, the emission is very low in
comparison to the changes seen in the pH titration
(Fig. 3). The fluorescence is only minor and can be
considered as being switched off (identical experimental
settings were used for both measurements). Furthermore, no
Figure 6. The changes in the fluorescence emission spectra of 1 upon
titration with Zn(II).
significant changes occurred upon titration of 1 with Ca2C
.
In fact, even at high concentrations (0.01 M) of Mg2C or
Ca2C, no significant spectral changes were observed in
either the absorption or the fluorescence emission of the two
sensors. This indicates that the receptors in these sensors
were not coordinating significantly to these ions to prevent
quenching by the receptors.21
Figure 3. Here, however, the monomeric anthracene
emission was only slightly enhanced upon ion recognition,
with the concomitant formation of a new red shifted broad
emission band centred at 468 nm. Unlike that seen for the
protonation previously, the fluorescent enhancement was
much smaller for these changes. The affiliated changes in
the absorption spectra were also interesting, as at low and
medium concentration of Zn(II) the classical anthracene
absorption bands were observed at 335, 350, 365 and
389 nm. Furthermore, these were only slightly reduced in
intensity upon Zn(II) titration. Hence the classical PET
behaviour was observed. However, at higher Zn(II)
concentrations (w2 mM) dramatic shifts (DlZ14 nm)
were observed in the absorption bands, which now became
centred at 360, 376 and 404 nm, respectively. In conjunction
with the changes in the fluorescence emission spectra
(Fig. 6), one can deduce that these changes are due the
formation of an intramolecular charge-transfer complex23
(intramolecular exciplex) between the cation-bound accep-
tor and anthracene donor (see later) (Fig. 7).24
We next evaluated the response of 1 and 2 towards various
transition metal ions such as Co(II), Ni(II), Cu(II), Zn(II),
Cd(II) and Hg(II) (as their ClK, NO3K or ClO4K salts). Of
these, only Zn(II) and Cd(II) gave rise to any significant
changes in the fluorescence emission spectra and the
absorption spectra. For 1, the changes in the fluorescence
spectra (lexZ370 nm) upon titration with Zn(II) are shown
in Figure 6. We foresaw that the coordination of the receptor
(via the nitrogen lone pair and the two carboxylates) would,
in a similar way to the protonation of the amino moiety,
increase the oxidation potential of the receptor and prevent
PET quenching. However, as can be seen from Figure 6, the
monomeric anthracene emission was not switched on in an
analogous way as previously shown for the pH titration in
In comparison to these results we carried out an identical
titration using 2. The changes in the absorption spectra of 2
at low and high concentrations of Zn(II) are shown in
Figure 8. Here it is clear that no major changes took place
upon titration of 2 with Zn(II), with the three absorption
bands being shifted by only 2–3 nm. Hence, one can
conclude that in comparison to 1, there are no major ground
state interactions occurring between the fluorophore and the
receptor. The corresponding changes in the fluorescence
emission spectra are shown in Figure 9. They clearly
demonstrate that the monomeric emission of the anthra-
cence fluorophore is switched on, similar to that seen earlier
for the pH titration. This indicates that the Zn(II) ion was
able to coordinate to the carboxylates, as well as the aniline
nitrogen and hence increase the oxidation potential of the
receptors in a usual PET fashion. For these changes a
fluorescence enhancement factor of several hundreds was
observed. Most importantly, the Zn(II) recognition does not
lead to any charge transfer interactions as seen previously
for 1. Hence, 2 is behaving as a typical PET sensor for
Zn(II). A likely reason for this difference could be that the
Figure 5. The changes in the fluorescence emission of 1 at pH 7.4, in
0.135 M NaCl, upon titration with Ca2C, [Ca2C]Z0/0.01 M.

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T. Gunnlaugsson et al. / Tetrahedron 60 (2004) 11239–11249
Figure 9. The changes in the fluorescence emission spectra of 2 upon
titration with Zn(II).
Figure 7. The changes in the absorption spectra of 1 upon titration with
Zn(II).
affinity for Zn(II). We were unable to obtain a reliable
binding constant from the absorption spectrum of 1, as these
changes were too small to give accurate binding.
Zn(II) binding forces the receptor to further away from the
anthracene fluorophore possibly due to steric effecots.
However, we have not been able to obtain reliable
experimental proof for this.
In a similar manner we evaluated the affinity of 1 and 2 for
Cd(II). The absorption response of 1 to Cd(II) was quite
similar to that observed in the Zn(II) titration. Here, three
anthracene absorption bands were observed at 350, 365 and
389 nm, and a small decrease in the absorption intensity
was observed upon addition of Cd(II). However, at higher
concentrations of Cd(II), the absorption spectra was
gradually shifted to longer wavelengths with absorption
maxima at 360, 376 and 406 nm, with the formation of
isosbestic points at 370, 382 and 395 nm, respectively. As
explained earlier, these latter changes are probably due to
the charge transfer interaction occurring between the
anthracene and receptor-Cd(II) complex. From these
changes we were able to determine log b of 4.0 (G0.1).
We were able to evaluate the affinity of both 1 and 2 for
Zn(II) by plotting the relative intensity changes at 468 and
415 nm for 1 and 2 respectively, as a function of pZn
(pZnZKlog [Zn(II)]). For both, a sigmoidal curve was
observed, which switched on over two logarithmic units
between pZnw5–3. This can be seen in Figure 10 for 2.
Hence, these binding interactions can be determined to be
due to 1:1 binding between the two receptors of 2, and
Zn(II). This was indeed confirmed by using the Job plot
method. From these changes a binding constant log b of
3.8 (G0.1) was determined. Similarly, for 1, a log b of 3.8
(G0.1) was determined. Hence, both sensors have the same
Figure 10. The relative changes in the fluorescence emission of 2 at 415 nm
as a function of pZn.
Figure 8. The changes in the absorption spectra of 2 at low and high
concentration of Zn(II).

T. Gunnlaugsson et al. / Tetrahedron 60 (2004) 11239–11249
11245
Figure 11. The changes in the fluorescence emission spectra of 1 upon
titration with Cd(II).
Figure 13. The changes in the fluorescence emission of 2 upon titration
with Cd(II).
The corresponding fluorescence emission changes for 1 can
be seen in Figure 11. From these changes it is evident that
the fluorescence emission is shifted to the red upon Cd(II)
recognition, and that the monomeric emission is reduced.
Furthermore, in contrast to the changes seen for the Zn(II)
titration the emission is substantially more shifted, now
centred at 506 nm. Moreover, the fluorescence enhance-
ments are substantially greater than seen for the Zn(II)
titration. Again, we suggest that these changes are due to the
formation of a charge transfer complex between the
anthracene moiety and the bound receptors, in a similar
manner to that seen for the Zn(II) titration. As previously
demonstrated it was possible to evaluate the binding affinity
of 1 towards Cd(II), by plotting the changes at 506 nm as a
function of pCd which gave rise to a sigmoidal curve that
changed over two pCd units. Fitting these fluorescence
changes gave a log b of 4.2 (G0.1), indicating that selective
detection of Cd(II) over Zn(II) should be feasible. Same
results were observed when exciting at the isosbestic point.
The fluorescence response of 2 towards Cd(II) was similar
to that observed for 1. However, the absorption spectra were
somewhat different to that seen for the Zn(II) titration, as
seen in Figure 12. Here the recognition of the ion caused a
red shift of ca. 3 nm, with clear isosbestic points being
observed at 385 and 405 nm, respectively. From these
changes, we were able to determine a binding constant log b
of 4.1 (G0.2). In contrast to these results the fluorescence
emission was red shifted, as shown in Figure 13, with the
formation of a broad structure less red shifted band, centred
at ca. 500 nm As explained before, we believe that this is
due to the anthracene-Cd(II) charge transfer interaction, in a
similar manner to that seen for 1. The same trends and
spectral changes were observed upon excitation at the
isosbestic points.
Figure 12. The changes in the UV–Vis spectra of 2, upon titration with
Cd(II).
Figure 14. The changes in the fluorescence at 500 nm as a function of pCd.

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T. Gunnlaugsson et al. / Tetrahedron 60 (2004) 11239–11249
A plot of fluorescence changes at 500 nm for 2 against
pCd(II) resulted in a sigmoidal curve with two logarithmic
unit changes, indicating a simple 1:1 equilibrium, Figure 14.
From these changes a binding constant log b of 3.9 (G0.1)
was determined. These results demonstrate that both 1 and
2 can be considered as fluorescence chemosensors for
Cd(II), as both have a marginally better selectivity for Cd(II)
over that of Zn(II). The binding results are summarised in
Table 1.
selective fluorescent chemosensors for Cd(II), that can
operate under physiological pH conditions.
2.5. Competitive Cd(II) measurements of 1 and 2
To confirm the selectivity of 2 towards Cd(II) over Zn(II),
we performed a Cd(II) titration in the presence of excess
Zn(II). These results are summarised in Figure 15. A
1 mM solution of 2 was prepared in HEPES buffer at pH
7.4. Under this condition the emission can be said to be
switched off. This solution was adjusted to contain 3 mM
of Zn(II), which caused normal fluorescence emission to
be switched on. This Zn(II)-2 solution was then titrated
with Cd(II) in the same manner as described above. As
predicted the addition of Cd(II) induced a shift to longer
wavelength with the appearance of a new broad band at
500 nm, demonstrating that 2 is capable of selectively
signalling the presence of Cd(II) over Zn(II). Similar
effects were seen for 1, where the emission further red
shifted upon addition of Cd(II) to a solution of Zn(II).
These results clearly show the high selectivity that these
sensors have for Cd(II), even in the presence of highly
competitive ions such as Zn(II). Its possible that as the
binding constants for the two ions is so similar that this
selectivity is due to the formation of a more kinetically
stable complex in the case of Cd(II).
Table 1
Parameters
1
2
pKa
1.83 (G0.1)
—
3.8 (G0.1)
4.0 (G0.1)
4.2 (G0.1)
2.5 (G0.1)
—
3.8 (G0.1)
4.1 (G0.1)
3.9 (G0.1)
Log b Zn(II)abs
Log b Zn(II)flu
Log b Cd(II)abs
Log b Cd(II)flu
In summary, we have demonstrated efficient fluorescent
sensing of Cd(II). For 1, the sensing of both Zn(II) and
Cd(II) gives rise to red shifts in the fluorescence spectra,
with relatively little changes in the monomeric emission.
However, there is a significant difference between these red
shifts, for example, 468 versus 506 nm for Zn(II) and Cd(II)
respectively. However, the largest contrast is seen for 2,
where the Zn(II) gives rise to large enhancements in the
monomeric anthracene emission, but Cd(II) gives rise to the
formation of the charge transfer band at long wavelengths.
Consequently, 2 is a particularly attractive chemosensor for
Cd(II) as it gives rise to a very different spectral response to
different competitive ions. Furthermore, this difference was
clearly visible under UV-light (lexZ366 nm) as at pH 7.4
the emission is switched off, whereas in the presence of
Zn(II) and Cd(II) the sensors emits in the blue and green part
of the electronic spectrum respectively. When these
titrations were repeated using the two esters 4 and 5, no
fluorescence enhancement, or red shift emission was
observed, indicating that these molecules were unable to
coordinate to these ions. But what gives rise the large red
shifted emission upon ion recognition? As we have
indicated previously we believe that these are due to
charge-transfer interactions between the metal bound
receptor and the anthracene moiety. Similar results have
recently been reported by Yoon et al.9c and Czarnik et al.9h
utilising the anthracene fluorophore and aliphatic Cd(II)
based receptors. Both researchers suggested that their
anthracene–Cd(II) interactions were initially due to p-
complex formation, which then gave rise to the formation of
a s-complex. However, for both of these examples the
fluorescence emission was centred on 446 nm. This is a
significantly smaller red shift than observed for either 1 and
2 upon recognition of Cd(II). Although, we do not predict
that such s-complex interaction can occur for either 1 or 2,
solely due to steric effects, as we, unlike Yoon et al. and
Czarnik et al.9h use aromatic based receptors, the
formation of the long emitting emission bands for both
1 and 2 strongly suggest the formation of p-complex
interactions. Moreover, as the concentration of the sensor
used in the titration studies is very low (1 mM),
intermolecular exciplex, excimer and mixed excimer for-
mation can be effectively ruled out. To the best of our
knowledge, 1 and 2 are the first examples of highly
Figure 15. The titration of 2 with Cd(II) ([Cd(II)] 6 mM/2.0 mM) in the
presence of 2.0 mM of Zn(II) at pH 7.4.
3. Conclusion
Herein we have presented the results of two novel
anthracene based fluorescent chemosensors 1 and 2 for the
selective recognition of Cd(II). These were designed on the
PET principle, using an anthracene fluorophore, connected
to either one or two iminodiacetate receptors via a
methylene spacer. These sensors were easily synthesized
in two steps from the receptor 3 in a good yield.

T. Gunnlaugsson et al. / Tetrahedron 60 (2004) 11239–11249
11247
The sensors exhibited high pH stability and aqueous
solubility, where the fluorescence was switched off above
pH w3, enabling the use of these sensors in competitive pH
media such as in physiological pH. The evaluation of the
ground and excited state responses of these sensors
demonstrated their capability to detect Zn(II) and Cd(II)
and to discriminate these ions from group 2 and other
transition metal ions. Whereas the recognition of Cd(II) at
pH 7.4, gave rise to the formation of charge transfer
complexes (s-complexes) for both sensors (lmax ca. 506 and
500 nm for 1 and 2 respectively), the recognition of Zn(II)
only switched on the (monomeric) anthracene emission of 2,
while for 1 it was red shifted (lmaxZ468 nm). A
discrimination test performed to evaluate the selectivity
for Cd(II) in the presence of Zn(II) proved very successful,
as the monomeric anthracene emission was red shifted upon
detection of Cd(II). These results strongly support the
possible application of these chemosensors as Cd(II) sensors
in physiological samples. This is particularly the case for 2
which exhibits the unique feature of emitting in the blue
upon detection of Zn(II), whereas upon the detection of
Cd(II) it emits in the green. In this context further evaluation
of 2 is required to determine the degree of toxicity, cell
permeability and real time response. To the best of our
knowledge these are the first examples of such highly
selective Cd(II) fluorescent chemosensors.
evaporation of the solvent, crude product was subjected to
column chromatography using ethyl acetate/hexane (2:3) as
eluant to yield pure 4 (1.89 g, 70%) as light yellow thick
liquid. MpZ140–142 8C. MS (ESC) m/zZ456 (MH)C.
Anal. Calcd for C29H29NO4: C, 76.46; H, 6.42; N, 3.07.
1
Found: C, 76.18; H, 6.28; N, 3.07. H NMR (400 MHz,
CDCl3): d, 1.25 (t, 6H, JZ7.0 Hz, NH2CH2CO2CH2CH3),
4.07 (s, 4H, NCH2CO2CH2CH3), 4.18 (q, 4H, JZ7.0 Hz,
NCH2CO2CH2CH3), 4.92 (s, 2H, CH2), 6.47 (d, 2H
JZ8.5 Hz, Ar–H), 6.98 (d, 2H, JZ8.5 Hz, Ar–H), 7.45–
7.48 (m, 4H, Ar–H), 8.03–8.05 (m, 2H, Ar–H), 8.22–8.25
(m, 2H, Ar–H), 8.43 (s, 1H, Ar–H). 13C NMR (100 MHz,
CDCl3): d, 170.50, 145.68, 132.03, 130.12, 130.04, 131.24,
128.57, 128.45, 125.82, 125.26, 124.54, 124.40, 112.26,
60.53, 53.06, 32.02, 13.72. IR (nmax, NaCl, cmK1): 3345,
2977, 1895, 1931, 1854, 1765, 1676, 1615, 1568, 1521,
1480, 1447, 1386, 1355, 1334, 1274, 1177, 1065, 970, 879,
820, 786, 728, 691, 650, 635, 602, 570, 538, 517.
4.1.2. [(4-Anthracen-9-ylmethyl-phenyl)-ethoxycarbonyl-
methyl-amino]-acetic acid potassium salt (1). [(4-Anthra-
cen-9-ylmethyl-phenyl)-ethoxycarbonylmethyl-amino]-
acetic acid ethyl ester (4) (1 g, 2.19 mmol) was dissolved in
methanol (20 mL) while stirring. To this was added aqueous
KOH (1 mL, 3 M). The mixture was refluxed for 2 h. After
cooling to room temperature, the reaction mixture was kept
in the fridge, where the potassium salt precipitated. The
resulting solution was filtered and the precipitate dried in
vacuum to afford 1 as a pale yellow solid (0.96 g, 95%).
MpZ340 8C (decomp.). MS (ESC) m/zZ476 (MH)C.
Anal. Calcd for C25H19K2NO4.2H2O: C, 58.69; H, 4.53,
N, 2.74. Found: 57.80; H, 4.33; N, 2.56. 1H NMR
(400 MHz, D2O): d, 3.67 (s, 4H, NCH2CO2K), 4.65 (s,
2H, CH2), 6.20 (d, 2H, JZ8.5 Hz, Ar–H), 6.82 (d, 2H,
JZ8.53 Hz, Ar–H), 7.36–7.38 (m, 4H, Ar–H), 7.91 (d, 2H,
JZ5.5 Hz, Ar–H), 8.12 (d, 2H, JZ6.0 Hz, Ar–H), 8.30 (s,
1H, Ar–H). 13C NMR (100 MHz, D2O): d, 177.61, 144.74,
131.10, 129.26, 127.61, 126.89, 126.68, 126.55, 124.06,
123.95, 123.20, 122.57, 109.48, 53.54, 29.22. IR (nmax, KBr,
cmK1): 3400, 2877, 2844, 1964, 1915, 1723, 1660, 1500,
1445, 1336, 1256, 1186, 519, 535, 564, 601, 618, 656, 697,
758, 790, 819, 852, 1158, 1100, 987, 955, 903, 884.
4. Experimental
4.1. General
Starting materials were obtained from Sigma Aldrich, Strem
Chemicals and Fluka. Columns were run using silica gel 60
(230–400 mesh ASTM) or aluminum oxide (activated,
Neutral, Brockmann I STD grade 150 mesh). Solvents were
used at GPR grade unless otherwise stated. Infrared spectra
were recorded on a Mattson Genesis II FTIR spectrophoto-
meter equipped with a Gateway 2000 4DX2-66 workstation.
Oils were analysed using NaCl plates, solid samples were
1
dispersed in KBr and recorded as clear pressed discs. H
NMR spectra were recorded at 400 MHz using a Bruker
Spectrospin DPX-400 instrument. Tetramethylsilane (TMS)
was used as an internal reference standard, with chemical
shifts expressed in parts per million (ppm or d) downfield
from the standard. 13C NMR were recorded at 100 MHz
using a Bruker Spectrospin DPX-400 instrument. Mass
spectroscopy was carried out using HPLC grade solvents.
Mass spectra were determined by detection using Electro-
spray on a Micromass LCT spectrometer, using a Shimadzu
HPLC or Water’s 9360 to pump solvent. The whole system
was controlled by MassLynx 3.5 on a Compaq Deskpro
workstation.
4.1.3. [(4-{10-[4-(Bis-ethoxycarbonylmethyl-amino)-
benzyl]-anthracen-9-ylmethyl}-phenyl)-ethoxycarbonyl-
methyl-amino]-acetic acid ethyl ester (5). 9,10-Bischloro-
methyl anthracene (1.0 g, 3.63 mmol), phenyliminodi-
acetic acid diethyl ester (3) (1.93 g, 7.26 mmol) and AlCl3
(0.96 g, 7.26 mmol) were dissolved in dry CHCl3 (50 mL) at
K5 8C. The solution was refluxed under stirring overnight
(12 h). After the reaction was complete, (monitored by
TLC) the solution was cooled and washed with three
100 mL portions of water. The organic portion was dried
over MgSO4. After evaporation of the solvent, the crude
product was subjected to column chromatography and
yielded pure 5 (1.56 g, 58%) as light yellow solid. MpZ120–
122 8C. MS (ESC) m/zZ733 (MH)C. Anal. Calcd for
C44H48N2O8: C, 72.11; H, 6.60; N, 3.82. Found: C,
4.1.1. [(4-Anthracen-9-ylmethyl-phenyl)-ethoxycar-
bonyl-methyl-amino]-acetic acid ethyl ester (4). 9-Chloro-
methylanthracene (1.5 g, 6.64 mmol) and phenyliminodi-
acetic acid diethyl ester (3) (1.76 g, 6.64 mmol) and AlCl3
(0.91 g, 6.64 mmol) were dissolved in dry CHCl3 (50 mL) at
K5 8C. The solution was refluxed overnight. After the
reaction was complete (monitoring by TLC) the solution
was cooled and washed with three 100 mL portions of
water. The organic portion was dried over MgSO4. After
1
72.26; H, 6.45; N, 3.69. H NMR (400 MHz, CDCl3): d
1.27 (t, 12H, JZ7.6 Hz, NCH2CO2CH2CH3), 4.09 (s, 8H,
NCH2CO2CH2CH3), 4.19 (q, 8H, JZ7.0 Hz, NCH2CO2
CH2CH3), 4.95 (s, 4H, CH2), 6.50 (d, 4H, JZ9.0 Hz, Ar–H),
7.01 (d, 4H, JZ9.0 Hz, Ar–H), 7.44 (m, 4H, Ar–H), 8.27

11248
T. Gunnlaugsson et al. / Tetrahedron 60 (2004) 11239–11249
(m, 4H, Ar–H). 13C NMR (100 MHz, CDCl3, ppm): d,
170.52, 145.68, 131.35, 130.25, 130.00, 128.49, 125.19,
124.79, 112.29, 60.54, 53.07, 32.30, 13.74. IR (nmax, KBr,
cmK1): 3450, 2979, 1895, 1931, 1859, 1734, 1616, 1568,
1524, 1448, 1371, 1030, 969, 865, 812, 781, 763, 655, 601,
573, 543, 506.
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4.1.4. [(4-{10-[4-(Bis-ethoxycarbonylmethyl-amino)-
benzyl]-anthracen-9-ylmethyl}-phenyl)-ethoxycarbonyl-
methyl-amino]-acetic acid potassium salt (2). [(4-{10-[4-
(Bis-ethoxycarbonylmethyl-amino)-benzyl]-anthracen-9-
ylmethyl}-phenyl)-ethoxycarbonylmethyl-amino]-acetic
acid ethyl ester (5) (1 g, 1.36 mmol) was dissolved in
methanol (20 mL) while stirring. To this aqueous KOH
(2 mL, 3 M) was added. The mixture was refluxed for 2 h.
After cooling to room temperature the mixture was kept in
the fridge, where the potassium salt precipitated out. The
resulting solution was filtered and the precipitate dried in
vacuum to afford 2 as a pale yellow solid (0.95 g, 90%).
MpZ320 8C (decomp.). MS(ESC) m/zZ773 (MCH)C.
Anal. Calcd for C36H28K4N2O8.3H2O: C, 52.28; H, 4.14; N,
3.39. Found: C, 52.46; H, 4.02; N, 3.23. 1H NMR
(400 MHz, D2O): d, 3.70 (s, 8H, NCH2CO2K), 4.86 (s,
4H, CH2), 6.28 (d, 4H, JZ6.1 Hz, Ar–H), 6.91 (d, 4H,
JZ6.1 Hz, Ar–H), 7.42–7.45 (m, 4H, Ar–H), 8.29–8.31 (m,
4H, Ar–H). 13C NMR (100 MHz, D2O): d, 179.22, 146.34,
129.27, 128.41, 128.15, 125.23, 124.95, 111.11, 55.11,
45.30, 31.13. IR (nmax, KBr, cmK1): 3450, 3006, 2005,
1851, 1660, 1574, 1515, 1446, 1404, 1320, 1209, 1043,
1029, 976, 913, 820, 790, 746, 706, 601.
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We would like to thank TCD, RCSI and HRB for financial
support. We also thank Dr. Hazel M. Moncrieff and Dr. John
E. O’Brien for their help and discussion. We especially
thank Lisa J. Gillespie for her assistance in the preparation
of this manuscript.
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