ORGANIC

LETTERS

New Fluorescent Rhodamine Hydrazone 2006

Vol. 8, No. 13
Chemosensor for Cu(II) with High 2863-2866
Selectivity and Sensitivity
Yu Xiang, Aijun Tong,* Peiyuan Jin, and Yong Ju

Department of Chemistry, Tsinghua UniVersity, Beijing 100084, P. R. China

[email protected]

Received April 28, 2006

ABSTRACT

A new fluorescent probe, salicylaldehyde rhodamine B hydrazone (1), was synthesized and displayed selective Cu(II)-amplified absorbance
and fluorescence emission above 500 nm in neutral buffered media. Upon the addition of Cu(II), the spirolactam ring of 1 was opened and a
1:1 metal−ligand complex was formed. The detection of Cu(II) by 1 at a lower micromolar level was successful even in buffered water.

The development of artificial receptors for the sensing and enhancement response; moreover, the selectivity for Cu(II)
recognition of environmentally and biologically important over other ions, such as Fe(III) and Pb(II), is not very
ionic species, especially transition-metal ions, is currently satisfactory for some of the probes. To overcome these
of great interest.1 Because copper is a widely used pollutant disadvantages, fluoroionophores which show a selective
and an essential trace element in biological systems, much response to Cu(II) by a copper-amplified fluorescence
attention has been drawn to the design of fluorescent probes emission have been well developed in recent years.4 Among
for the detection of copper ions due to the nondestructive, these sensors, unfortunately, those that can be applied in
quick, and sensitive advantages of emission signals.2 Most aqueous solutions at neutral pH are still rare mainly because
of the classic and early-reported cation sensors, however, of the strong hydration ability of Cu(II) in water. Actually,
generally undergo fluorescence quenching upon the binding sensors of this kind are always considered to be much more
of Cu(II),3 which is not as sensitive as a fluorescence attractive and efficient in the respect that most copper-
containing samples are near-neutral aqueous systems.1,2
(1) For reviews, see: (a) de Silva, A. P.; Gunaratne, H. Q. N.;
Gunnlaugsson, T.; Huxley, A. J. M.; McCoy, C. P.; Rademacher, J. T.;
On the other hand, fluorophores of long-wavelength
Rice, T. E. Chem. ReV. 1997, 97, 1515. (b) Valeur, B.; Leray, I. Coord. emissions are often preferred to serve as the reporting group
Chem. ReV. 2000, 205, 3. (c) Rurack, K. Spectrochim. Acta 2001, 57A, for analyte to avoid the influence of background fluorescence
2161. (d) Amendola, V.; Fabbrizzi, L.; Forti, F.; Licchelli, M.; Mangano,
C.; Pallavicini, P.; Poggi, A.; Sacchi, D.; Taglieti, A. Coord. Chem. ReV.
2006, 250, 273. (4) (a) Wu, Q.; Anslyn, E. V. J. Am. Chem. Soc. 2004, 126, 14682. (b)
(2) Krämer, R. Angew. Chem., Int. Ed. 1998, 37, 772. Gunnlaugsson, T.; Leonard, J. P.; Murray, N. S. Org. Lett. 2004, 6, 1557.
(3) (a) Breuer, W.; Epsztejn, S.; Millgram, P.; Cabantchik, I. Z. Am. J. (c) Mokhir, A.; Krämer, R. Chem. Commun. 2005, 2244. (d) Royzen, M.;
Physiol. 1995, 268, C1354. (b) McCall, K. A.; Fierke, C. A. Anal. Biochem. Dai, Z.; Canary, J. W. J. Am. Chem. Soc. 2005, 127, 1612. (e) Zheng, L.;
2000, 284, 307. (c) Chavez-Crooker, P.; Garrido, N.; Ahearn, G. A. J. Exp. Miller, E. W.; Pralle, A.; Isacoff, E. Y.; Chang, C. J. J. Am. Chem. Soc.
Biol. 2001, 204, 1433. 2006, 128, 10.

10.1021/ol0610340 CCC: $33.50 © 2006 American Chemical Society

Published on Web 06/03/2006
(<400 nm). The introduction of the rhodamine moiety to range immediately as a result of the Cu(II)-induced ring
construct probes of the “off-on” type is a reliable method opening of the spirolactam form. We also found that a
because of the well-known spirolactam (fluorescence “off”) solution of 10 µM 1 in 50% (v/v) buffered (10 mM Tris-
to ring-opened amide (fluorescence “on”) equilibrium (Figure HCl, pH ) 7.0) water/CH3CN could display an obvious
1) of rhodamine derivatives.6 We have successfully designed purple color in the presence of Cu(II) at the micromolar level,
and other ions of our interest showed little interference
(Figure 2). These results suggested that 1 could serve as a

Figure 1. Chemical structures of compound 1, 1-Cu(II),5 and 2.

a fluorescence chemosensor for Fe(III) utilizing rhodamine

B as the fluorophore in our previous work.6d Herein, we
describe a new rhodamine-based chemosensor 1 (Figure 1),
which shows a reversible, selective, and sensitive fluores- Figure 2. Pictures of 10 µM 1 as a selective naked-eye chemosen-
sor for Cu(II) in 50% (v/v) water/CH3CN (10 mM Tris-HCl, pH
cence enhancement response to Cu(II) in neutral buffered ) 7.0). Top (from left to right): 0, 2.5, 5.0, 10.0 µM Cu(II). Bottom
media. (from left to right): 50 µM Fe(III), Fe(II), Zn(II), Pb(II), Hg(II).
Compound 1 was facilely synthesized from rhodamine B
by a two-step reaction (Supporting Information, S-Figure 1).
This Shiff base was stable in neutral water solutions for at “naked-eye” chemosensor selective for Cu(II) in neutral
least 2 days. It was designed to chelate with metal ions via buffered media.
its carbonyl O, imino N, and phenol O atoms.7 The The absorption spectra of 1 (10 µM) in 50% (v/v) water/
spirolactam moiety of the rhodamine group acted as a signal CH3CN (10 mM Tris-HCl buffer, pH ) 7.0) exhibited only
switcher, which was envisioned to turn on when the cation a very weak band above 500 nm, which was ascribed to the
was bound. A solution of 1 in Tris-HCl buffer (5 mM, pH trace ring-opened form of molecules of 1. Upon the addition
) 7.0) or organic media is colorless and weakly fluorescent, of up to 5 equiv of Cu(II), the absorbance was significantly
indicating that the spirolactam form of 1 exists predomi- enhanced (> 500-fold) and a new peak at 558 nm was
nantly. The characteristic peak of the 9-carbon of 1 near 66 observed (Figure 3a), suggesting the clear formation of the
ppm in the 13C NMR spectrum (Supporting Information, ring-opened amide form of 1. Other metal ions had little
S-Figure 2d) also supports this consideration.8 Besides, no interference. Only Fe(II) displayed a 65-fold enhancement
obvious characteristic color or fluorescence of rhodamine at the same concentration, but a much longer response time
could be observed for 1 between pH 5.0 and 10.0 (Supporting was required (Supporting Information, S-Figure 4a-d). The
Information, S-Figure 3), suggesting that 1 is insensitive to nonlinear fitting of the titration curve assumed a 1:1
pH and that the spirolactam form is still preferred in this stoichiometry for the 1-Cu(II) complex (Figure 1) with an
range. As we expected, addition of Cu(II) to a solution of 1 association constant Ka value of far more than 106,9 showing
in either CH3CN or water caused a significant enhancement the high affinity of 1 to Cu(II). This binding mode was also
of absorbance and fluorescence intensity in the 500-650 nm supported by the data of Job’s plots10 evaluated from the
absorption spectra of 1 and Cu(II) with a total concentration
(5) The copper ion may be chelated by the counteranion or solvent
oxygen to satisfy the need for four-coordination. They are omitted for clarity. of 3.3 µM (Supporting Information, S-Figure 5). A more
(6) For rhodamine-based chemosensors for metal ions, see: (a) Dujols, direct evidence was obtained by comparing the ESI mass
V.; Ford, F.; Czarnik, A. W. J. Am. Chem. Soc. 1997, 119, 7386. (b) Yang,
Y.-K.; Yook, K.-J.; Tae, J. J. Am. Chem. Soc. 2005, 127, 16760. (c) Kwon, spectra of 1 and 1-Cu(II) (Figure 4). The unique peak at
J. Y.; Jang, Y. J.; Lee, Y. J.; Kim, K. M.; Seo, M. S.; Nam, W.; Yoon, J. m/z ) 622.5 (calcd ) 622.3) corresponding to [1 + Cu-
J. Am. Chem. Soc. 2005, 127, 10107. (d) Xiang, Y.; Tong, A. Org. Lett. H]+ was clearly observed when 1.2 equiv of Cu(II) was
2006, 8, 1549.
(7) Lee, P. F.; Yang, C.-T.; Fan, D.; Vittal, J. J.; Ranford, J. D.
Polyhedron 2003, 22, 2781. (9) (a) Connors, K. A. Binding Constants-The Measurement of Molecular
(8) Anthoni, U.; Christophersen, C.; Nielsen, P.; Puschl, A.; Schaumburg, Complex Stability; John Wiley & Sons: New York, 1987; Chapter 4.
K. Struct. Chem. 1995, 3, 161. (10) Vosburgh, W. C.; Cooper, G. R. J. Am. Chem. Soc. 1941, 63, 437.

2864 Org. Lett., Vol. 8, No. 13, 2006

 Figure 4. ESI mass spectra (positive) of 1 (10 µM) in the absence
and presence of Cu(II) (1.2 equiv). Right: calculated (top) and
observed (bottom) isotopic patterns for the [Cu(1-H)]+ cation,
indicating the formation of a 1:1 metal-ligand complex.

The high enhancement factor (>500) of absorbance of 1

upon the binding of Cu(II) and the large association constant
(Ka > 106) of the 1-Cu(II) complex implied the possibility
of copper sensing at a very low concentration level by 1.
Indeed, the absorbance of 1 (10 µM) at 558 nm was found
to increase linearly with the concentration of Cu(II) in the
range of 25 nM-3.3 µM (Supporting Information, S-Figure
Figure 3. (a) Absorption spectra of 1 (10 µM) in Tris-HCl (10 7a-d). However, signals acquired from absorption spectra
mM, pH ) 7.0) buffer containing 50% (v/v) water/CH3CN in the
presence of different amounts of Cu(II). Inset: absorbance at 558 are generally not as efficient as those evaluated from
nm as a function of Cu(II) concentration, indicating a 1:1 metal- fluorescence spectra. The fluorescence titration12 was also
ligand ratio. (b) Fluorescence spectra of 1 (10 µM) under the same carried out using 10 µM 1 in 50% (v/v) buffered water/CH3-
conditions. Excitation was performed at 520 nm. Inset: fluorescence CN at pH 7.0. Although the emission spectra of 1 underwent
enhancement factor (F - F0)/F0 at 583 nm as a function of Cu(II) a red shift from 576 to 583 nm upon the addition of 5 equiv
concentration.
of Cu(II), the fluorescence intensity at the new peak (583
nm) had only a 3.8-fold enhancement (Figure 3b), which
was much weaker compared to that of absorbance. Neverthe-
added to 1, whereas 1 without Cu(II) exhibited peaks only
less, the selective sensing of Cu(II) at the 100 nM level was
at m/z ) 561.4 (calcd ) 561.3) and 583.3 (calcd ) 583.3)
still available when using 1.0 µM 1 under the same
which corresponded to [1 + H]+ and [1 + Na]+, respectively.
conditions (Supporting Information, S-Figure 8).
To achieve this 1:1 stoichiometry, carbonyl O, imino N, and
To get a practical application view, the fluorescence
phenol O atoms of 1 are the most possible binding sites for
sensing behavior of 1 for Cu(II) in buffered water solution
Cu(II).7 In fact, the phenol group of 1 which undergoes
(with no more than 2% CH3CN) was also investigated. The
deprotonation during the binding plays an indispensable role
titration was performed using 10 µM solutions of 1 in Tris-
in the affinity of 1 to Cu(II) because the contrast compound
HCl (5 mM, pH ) 7.0) aqueous buffer (Figure 5). A 9.4-
2 (no phenol group compared with 1, Figure 1) displayed
fold enhancement of fluorescence and a continuous red shift
nearly no response to Cu(II) in either absorption or ESI mass
of the emission peak from 573 to 585 nm were observed
spectra when treated with excess (10 equiv) Cu(II) (Sup-
upon the addition of 20 equiv of Cu(II) compared to that of
porting Information, S-Figure 6a-c). It was also confirmed
only 1 in solution. The nonlinear fitting of the titration curve
that the response of 1 to Cu(II) was reversible rather than a
and the data of Job’s plots from absorption spectra (Sup-
cation-catalyzed reaction:11 (i) the color and fluorescence of
porting Information, S-Figures 9 and 10) also assumed a 1:1
1-Cu(II) disappeared instantly upon the addition of EDTA,
stoichiometry for the 1-Cu(II) complex with an association
whereas excess Cu(II) would recover the signal; (ii) the TLC
constant around Ka ) 69 110. The response was proved to
results and the mass spectra data of a buffered solution
be revisable, and the selectivity of 1 to Cu(II) over other
containing 10 µM 1 and 12 µM Cu(II) after standing at room
cations was still satisfactory. We found that alkali and
temperature for 2 days indicated no other chemical species
alkaline earth metal ions had hardly any effect on the
except for 1 and 1-Cu(II).
(12) All the fluoresence spectra were measured by excitation at 520 nm
(11) For sensors of this type, see ref 6a,b. to obtain a full view of emissions from 530 to 700 nm.

Org. Lett., Vol. 8, No. 13, 2006 2865

 absorption and fluorescence signals.5c,d In the case of 1,
binding of Cu(II) does open the spirolactam ring, but at the
same time, the fluorescence of the ring-opened amide form
is probably partially quenched by Cu(II). The quenching
mechanism may be similar to that of some Cu(II) probes
displaying fluorescence quenching for the paramagnetic
nature of the copper ion.1,13 From a sensitivity viewpoint, it
is preferable to inhibit this quenching effect to generate a
more notable fluorescence enhancement. Chemical modifica-
tion on the phenol group may be a good improvement,14 but
the affinity of 1 to Cu(II) could probably decrease for the
important role of the phenol moiety. Works related to this
topic are now under our investigation.
In conclusion, we have synthesized a new rhodamine-
based fluoroionophore 1, which displayed a reversible
Figure 5. Fluorescence spectra of 1 (10 µM) in Tris-HCl (5 mM, absorption and fluorescence enhancement response to Cu(II)
pH ) 7.0) aqueous buffer solutions in the presence of different via a 1:1 binding mode. Its selectivity toward Cu(II) is very
amounts of Cu(II). Excitation was performed at 520 nm. Inset:
high because little interference was observed for other
fluorescence enhancement factor at 585 nm as a function of Cu(II)
concentration. commonly coexistent metal ions. Furthermore, the sensitivity
of 1 for Cu(II) can be lower than 25 nM in 50% (v/v)
buffered water/CH3CN by the absorption spectra method (still
fluorescence spectra of 1 (10 µM) in buffered water even at at the 0.1 µM level for the fluorescence method under this
the millimolar level. Other transition-metal ions, such as condition). Even in neutral buffered aqueous solutions, the
Fe(III), Co(II), Ni(II), Zn(II), Mn(II), Ag(I), Cd(II), and fluorescent sensing of Cu(II) at a lower micromolar level
Hg(II), gave a much weaker response compared to Cu(II) at was successful.
the same concentration (100 µM) (Supporting Information,
Acknowledgment. The authors are grateful for the
S-Figure 11), and the fluorescence signal of 1-Cu(II) in the
financial support from the National Natural Science Founda-
presence or absence of these contrast ions also exhibited only
tion of China, No. 20375021.
a mild difference (Supporting Information, S-Figure 12).
Furthermore, the detection of Cu(II) from 0.25 to 2.0 µM Supporting Information Available: Experimental pro-
was successful when utilizing 1.0 µM 1 in 1 mM Tris-HCl cedures, characterization data for the compounds described,
aqueous buffers at pH 7.0 (Supporting Information, S-Figure and selected spectroscopic data of 1 and 2. This material is
13), indicating the high sensitivity of 1 for Cu(II) even in available free of charge via the Internet at http://pubs.acs.org.
aqueous media.
OL0610340
With careful investigation, one may note that when Cu(II)
was added to 1, the enhancement of absorbance was much (13) (a) Varnes, A. W.; Dodson, R. B.; Wehry, E. L. J. Am. Chem. Soc.
more significant than that of fluorescence intensity. However, 1972, 94, 946. (b) Torrado, A.; Walkup, G. K.; Imperiali, B. J. Am. Chem.
Soc. 1998, 120, 609. (c) Li, Y.; Yang, C. M. Chem. Commun. 2003, 2884.
the ring opening of the spirolactam form of rhodamine (14) Wen, Z.-C.; Yang, R.; He, H.; Jiang, Y.-B. Chem. Commun. 2006,
derivatives generally results in comparable amplifications of 106.

2866 Org. Lett., Vol. 8, No. 13, 2006