Highly selective colorimetric naked-eye Cu(II) detection using an azobenzene chemosensor

Article abstract of DOI:10.1021/ol0498951

Colorimetric azobenzene based chemosensors 1 and 2 were designed for detection of transition-metal ions such as Cu(II) under physiological pH conditions. The internal charge transfer (ICT) sensors are highly colored, absorbing in the green. For 1, the Cu(

Full text of DOI:10.1021/ol0498951

ORGANIC
LETTERS
2004
Vol. 6, No. 10
1557-1560
Highly Selective Colorimetric Naked-Eye
Cu(II) Detection Using an Azobenzene
Chemosensor
Thorfinnur Gunnlaugsson,* Joseph P. Leonard, and Niamh S. Murray
Department of Chemistry, Trinity College Dublin, Dublin 2, Ireland
gunnlaut@tcd.ie
Received January 16, 2004
ABSTRACT
Colorimetric azobenzene based chemosensors 1 and 2 were designed for detection of transition-metal ions such as Cu(II) under physiological
pH conditions. The internal charge transfer (ICT) sensors are highly colored, absorbing in the green. For 1, the Cu(II) recognition gives rise
to red-to-yellow color changes that are visible to the naked-eye and reversible upon addition of EDTA, whereas for 2, which lacks the aromatic
o-methoxy chelating group, no such changes were observed.
The recognition of ions and molecules is an essential part
of supramolecular chemistry.1 Over the years, many excellent
examples of luminescent-based devices, such as chemo-
sensors for various analytes, have been prepared and studied
for application in physiology and medical diagnostics.1-3
Such devices can give rise to real time, noninvasive, and
on-line monitoring in vitro or in vivo. We are interested in
this field and have developed fluorescent4 and lanthanide
luminescent Eu(II) and Tb(III) cyclen complexes as chemo-
sensors,5 switches,6 and logic gate mimics.7 Recently, we
have also focused our efforts on developing novel colori-
metric sensors for ions,8 nucleic acids,9 and oligonucleotides,9
where the aim is to develop simple-to-use, naked-eye
diagnostic tools (such as dipsticks), for the recognition of
essential electrolytes and molecules in serum for critical care
analysis.10 Herein we describe the synthesis and the spec-
(4) (a) Gunnlaugsson, T.; Kruger, P. E.; Lee, T. C.; Parkesh,R.; Pfeffer,
F. M.; Hussey, M. G. Tetrahedron Lett. 2003, 44, 6575. (b) Gunnlaugsson,
T.; Davis, A. P.; Glynn, M. Org. Lett. 2002, 4, 2449. (c) Gunnlaugsson,
T.; Bichell, B.; Nolan, C. Tetrahedron Lett. 2002, 43, 4989. (d) Gunnlaugs-
son, T.; Davis, A. P.; Glynn, M. Chem. Commun. 2001, 2556.
(5) (a) Gunnlaugsson, T.; Harte, A. J.; Leonard, J. P.; Senechal, K. J.
Am. Chem. Soc. 2003, 125, 12062. (b) Gunnlaugsson, T.; Harte, A. J.;
Leonard, J. P.; Nieuwenhuyzen, M. Supramol. Chem. 2003, 15, 505. (c)
Gunnlaugsson, T.; Harte, A. J.; Leonard, J. P.; Nieuwenhuyzen, M. Chem.
Commun. 2002, 2134.
(6) (a) Gunnlaugsson, T.; Leonard, J. P. Chem. Commun. 2003, 2424.
(b) Gunnlaugsson, T.; MacDo´naill, D. A.; Parker, D. Chem. Commun. 2000,
93. (c) Gunnlaugsson, T. Tetrahedron Lett. 2001, 42, 8901.
(7) Gunnlaugsson, T.; MacDo´naill, D. A.; Parker, D. J. Am. Chem. Soc.
2001, 123, 12866.
(8) (a) Gunnlaugsson, T.; Leonard, J. P. J. Chem. Soc., Perkin Trans. 2
2002, 1980. (b) Gunnlaugsson, T.; Nieuwenhuyzen, M.; Richard L.; Thoss,
V. J. Chem. Soc., Perkin Trans. 2 2002, 141. (c) Gunnlaugsson, T.;
Nieuwenhuyzen, M.; Richard L.; Thoss, V. Tetrahedron Lett. 2001, 42,
4725.
(9) Gunnlaugsson, T.; Kelly, J. M.; Nieuwenhuyzen, M.; O’Brien, A.
M. K. Tetrahedron Lett. 2003, 44, 8571.
(1) (a) de Silva, A. P.; McCaughan, B.; McKinney, B. O. F.; Querol,
M. Dalton Trans. 2003, 1902. (b) Anslyn, E. V. Angew. Chem., Int. Ed.
2001, 40, 3119. (c) Amendola, V.; Fabbrizzi, L.; Mangano, C.; Pallavicini,
P. Acc. Chem. Res. 2001, 34, 488. (d) Fabbrizzi, L.; Licchelli, M.;
Pallavicini, P. Acc. Chem. Res. 1999, 32, 846. (e) Czarnik, A. W. Acc.
Chem. Res. 1994, 27, 302.
(2) (a) de Silva, A. P.; Fox, D. B.; Huxley, A. J. M.; Moody, T. S. Coord.
Chem. ReV. 2000, 205, 41. (b) de Silva, A. P.; Gunaratne, H. Q. N.;
Gunnlaugsson, T.; Huxley, A. J. M.; McCoy, C. P.; Rademacher, J. T.;
Rice, T. E. Chem. ReV. 1997, 97, 1515.
(3) (a) Spichiger-Keller, U. S. Chemical Sensors and Biosensors for
Medical and Biological Applications; Wiley-VCH: Weinheim; Germany,
1998. (b) Czarnik, A. W. Fluorescent Chemosensors for Ion and Molecular
Recognition; American Chemical Society: Washington, DC, 1993.
10.1021/ol0498951 CCC: $27.50 © 2004 American Chemical Society
Published on Web 04/20/2004

troscopic evaluation of the two colorimetric chemosensors
1 and 2, which were developed for the detection of transition-
metal ions such as Cu(II) in competitive physiological pH
conditions.
Scheme 1. Synthesis of 1 and 2 from the Amines Receptors 5
and 6, Respectively
Both sensors are based on the well-known azobenzene
structure, where one of the aromatic rings is an integrated
part of the ion receptor. In these cases, simple phenyl
iminodiacetate receptors were chosen as the electron donors
and a nitro benzene moiety as the electron acceptor. In the
case of 1, the use of o-methoxy functionality gives rise to
an extra chelation site. The use of the o-methoxy group11 in
1, gives rise to high selectivity and sensitivity of this sensor
for Cu(II) under physiological pH conditions. Here, the Cu(II)
detection gives rise to large changes in the absorption spectra
(form red to yellow), which are clearly visible to the naked
eye. However, for 2, no transition-metal ion sensing was
observed. To the best of our knowledge, 1 is the first example
of such a highly selective and sensitive Cu(II) colorimetric
azobenzene-based chemosensor.
The synthesis of 1 and 2, Scheme 1, began with receptors
5 and 6, formed form o-anisidine (3) and aniline (4),
respectively, by alkylation using 2 equivalent of either ethyl
bromoacetate (for 5) or methyl boromoacetate (for 6) in the
presence of potassium dihydrogen phosphate in CH3CN or
DMF. Both were formed in ca. 90% yield. The azobenzene
esters 7 and 8 were made using standard diazonium
chemistry. For 1, this was achieved by using p-nitroaniline
that was diazonated using NaNO2 in a 1:1 mixture of THF/
H2O (20 mL), in the presence of (1 mL) HCl (12 M) at 0
°C. This mixture was then added at 0 °C to a 1:1 mixture of
THF/H2O and 5 and stirred for 2 h, followed by stirring at
room temperature for 12 h. The resulting dark-red aqueous
solution was reduced in volume and washed with CHCl3.
The organic solution was then further washed with water
and dried over MgSO4 and the solvent removed under
reduced pressure to give 7 as red oil. This crude material
was purified using flash silica column chromatography (70:
30 hexane/EtOAc) to produce 7 in a pure form as a red solid
in 15% yield. The final chemosensor 1 was then formed by
hydrolysis of 7 under alkaline conditions in a refluxing
mixture of aqueous KOH in EtOH in 95% yield. Similarly,
8 was formed in 10% yield and 2 in 90% yield using identical
conditions.
Both compounds were highly soluble in water. Initially
the pKa of 1 and 2 were determined in water in the presence
of 0.1 M tetramethylammonium chloride (to maintain
constant ionic strength), by observing the changes in the
UV-Vis spectra upon titration of an alkaline solution of
either 1 or 2 with diluted HCl solution. Figure 2 shows the
changes for 1. In alkaline solution (at pH 11.7), 1 had a strong
absorption band centered (λmax) at 509 nm (log ꢀ ) 4.35
cm-1 M-1) and a second band at ca. 289 nm. The former
was assigned to the internal charge-transfer character, ICT,
of the chromophore due to the push-pull effect of the
electron-donating amine and the electron-withdrawing nitro
group. As can be seen from these changes the absorption
Both chemosensors and intermediates were characterized
using conventional methods (see the Supporting Information).
Figure 1 shows the 1H NMR of 1 in D2O, where the
o-methoxy group appeared as a singlet at 3.69 ppm, the
methylene spacer of the iminodiacetate appeared as a singlet
at 3.89 ppm, and five sets of the aromatic protons appeared
at 8.19, 7.67, 7.42, 7.27, and 6.56 ppm, respectively. Similar
results were observed for 2.
(10) Fluorescent sensors for Na+ and K+ have been developed recently
for critical care analysis: (a) He, H.; Mortellaro, M. A.; Leiner, M. J. P.;
Fraatz, R. J.; Tusa, J. K. J. Am. Chem. Soc. 2003, 125, 1468. (b) He, H.;
Mortellaro, M. A.; Leiner, M. J. P.; Young, S. T.; Fraatz, R. J.; Tusa, J. K.
Anal. Chem. 2003, 75, 549.
(11) (a) An example of Cd(II) sensing: Gunnlaugsson, T.; Lee, C. T.;
Parkesh, R. Org. Lett. 2003, 5, 4065. (b) Example of Zn(II) sensing:
Gunnlaugsson, T.; Lee, C. T.; Parkesh, R. Org. Biomol. Chem. 2003, 1,
3265.
1
Figure 1. Partial H NMR of 1 in D2O (400 MHz).
1558
Org. Lett., Vol. 6, No. 10, 2004

based aryl iminodiacetate receptors in fluorescent PET
sensors, where we were able to selectively detect ions such
as Zn(II) and Cd(II) over other competitive ions such as
Ni(II), Fe(II), and Cu(II) under physiological pH conditions.11
However, we also established that this selectivity was
highly dependent on the nature of the fluorophore used.11
The changes in the absorption spectra of 1 in buffered
water at pH 7.4 are shown in Figure 4, where it is clear that
Figure 2. Changes in the UV-vis spectra of 1 upon pH titration
of an alkaline solution of 1 with acid.
maxima gradually decreased in intensity upon addition of
acid. These changes correspond to the protonation of the
amino moiety, reducing the ICT character of the molecule.
By plotting the changes at 509 nm as a function of pH a
sigmoidal curve was observed where the major changes
occurred over ca. two pH units. These changes were fully
reversible, as the addition of strong base to this acidic
solution reversed these effects. As can be seen from Figure
3, only minor changes occurred above ca. pH 5.5. From these
Figure 4. Changes in the UV-vis spectra of 1 at pH 7.4 (20 mM
HEPES, 135 mM NaCl) upon titration with CuCl2.
the absorption of 1 is highly affected by the presence of
Cu(II). Here, the absorption maxima at 509 nm gradually
reduced in intensity with the formation of a new absorption
band at ca. 325 nm and with the formation of an isosbestic
point at 401 nm. We propose that this is due to the
coordination of the Cu(II) to the two carboxylates, the amino
moiety and the o-methoxy group. This reduces the ability
of the amino moiety to participate in ICT in the same manner
as for the protonation of the amino moiety discussed earlier.
However, the role of the o-methoxy group is vital here, as
upon its coordination to the metal ion, it forces the amine of
the receptor to become deconjugated from the aromatic
receptor, inhibiting the ICT. Even though we do not have
direct 1H NMR or X-ray crystal structure evidence for this,
we have previously observed such phenomena in related 15-
crown-5 and 18-crown-6 based receptor, for the sensing of
Na+ and K+ respectively.12 Unlike that seen for the proto-
nation event (Figure 2), a clear isosbestic point is observed
in Figure 4.
Figure 3. Changes in the UV-vis spectra of 1 and 2 at 509 and
491 nm, respectively, upon titration of an alkaline solution of these
sensors with diluted HCl.
More importantly, the Cu(II) sensing and the concomitant
absorption changes were clearly visible to the naked eye, as
can be seen in the graphical abstract, where the red solution
of 1 became yellow upon titration with Cu(II). Moreover,
these changes are also fully reversible as the addition of
EDTA reversed the color changes, Figure 5. When these
titrations were repeated using Cd(II) or Zn(II), the absorption
spectra was also affected in a manner similar to that shown
above. By plotting the changes at λmax as a function of pM,
changes a pKa of 4.2 ((0.1) was determined. This clearly
demonstrates that 1 can be used in physiological environment
where the pH >5. Similar results were observed for 2, Figure
3.
We next investigated the affinity of both 1 and 2 for a
series of transition and group II metal ions, at pH 7.4 (20
mM HEPES) and in the presence of 135 mM NaCl to
maintain constant ionic strength. For the latter, only minor
changes were seen at very high concentrations for Mg(II)
or Ca(II). We have previously demonstrated the use of simple
(12) Gunnlaugsson, T.; Leonard, J. P. J. Chem. Soc., Perkin Trans. 1
2002, 1954.
Org. Lett., Vol. 6, No. 10, 2004
1559

no affect upon either the sensitivity or the selectivity of these
titration’s (see the Supporting Information). Importantly, all
three transition ions modulate the absorption over 2 pM units,
which is indicative of a simple equilibrium and one to one
binding. From the binding isotherms in Figure 6, we were
able to determine the binding constants (log â) as log â )
5.0 ((0.1), 3.4 ((0.1), and 3.0 ((0.1) for Cu(II), Zn(II),
and Cd(II), respectively, for 1. From these changes it can
be concluded that 1 is a good naked-eye sensor for the
selective detection of Cu(II) in competitive media. In
comparison to these result the changes in the absorption
spectra of 2 (λmax ) 491 nm) were very different.
As stated above, the absorption spectrum was blue shifted
upon pH titration. However, when 2 was titrated at pH 7.4
in HEPES buffer and in the presence of 135 mM of NaCl,
using the above ions, no significant spectral changes were
observed. However, at relatively high concentrations of Zn(II)
and Cu(II) precipitation occurred (See SI). This is quite
extraordinary, as the only difference between the two sensors
is the presence of the o-methoxy group. Hence, the selectivity
and the sensitivity of 1 is dependent on the presence of the
o-methoxy group.
Figure 5. UV-vis spectra of 1 before and after addition of Cu(II)
and reversed changes upon addition EDTA.
where pM ) -log[M] where M is the cations used, the
sensitivity and the selectivity of these ion recognitions can
be evaluated. From Figure 6, it is apparent that both Zn(II)
and Cd(II) modulate the absorption spectra.
In summary, we have developed a highly selective
azobenzene based colorimetric chemosensor 1, for the
detection of Cu(II) under physiological conditions. The
recognition of the ion gave rise to major color changes from
red to yellow that was clearly visible to the naked eye. Such
Cu(II) selective colorimetric chemosensors could be of great
importance in medical diagnostics such as serum studies. We
are currently working toward the development of such a
device by incorporating 1 into soft material such as hydro-
gels.
Acknowledgment. We thank TCD and Enterprise Ireland
for financial support. We also thank Dr. John E. O’Brien
for NMR and Donal F. O’Shea (University College Dublin)
for helpful discussions.
Figure 6. Changes in the UV-vis spectra of 1 at 509 nm in water
at pH 7.4 (20 mM HEPES, 135 mM NaCl) upon titration of several
cations.
Supporting Information Available: Synthesis of 1, 2,
7, and 8; the UV-Vis spectral changes upon titration of 1
and 2 with solutions of Zn(II), Cd(II), Ca(II), and Mg(II)l
the absorption vs pH profile for 2. This material is available
free of charge via the Internet at http://pubs.acs.org.
However, these changes occur at much higher concentra-
tions than that of Cu(II). Studies using different anions had
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