A new bis(rhodamine)-based fluorescent chemosensor for Fe3+

Article abstract of DOI:10.1007/s10895-011-1022-0

A new bis(rhodamine)-based fluorescent probe 4 was synthesized, and it exhibited high selectivity for Fe³⁺ over other commonly coexistent metal ions in both 50% ethanol and Tris-HCl buffer. Upon the addition of Fe ³⁺, the spirocyclic ring of 4 was opened and a significant enhancement of visible color and fluorescence in the range of 500-600 nm was observed. The Author(s) 2011.

Full text of DOI:10.1007/s10895-011-1022-0

J Fluoresc (2012) 22:789–794
DOI 10.1007/s10895-011-1022-0
RAPID COMMUNICATION
A New bis(rhodamine)-Based Fluorescent Chemosensor
for Fe3+
Xiaopo Chen & Huijie Hong & Rui Han & Di Zhang &
Yong Ye & Yu-fen Zhao
Received: 17 September 2011 /Accepted: 29 November 2011 /Published online: 7 December 2011
#
The Author(s) 2011. This article is published with open access at Springerlink.com
Abstract A new bis(rhodamine)-based fluorescent probe 4
was synthesized, and it exhibited high selectivity for Fe3+
over other commonly coexistent metal ions in both 50%
ethanol and Tris–HCl buffer. Upon the addition of Fe3+,
the spirocyclic ring of 4 was opened and a significant
enhancement of visible color and fluorescence in the range
of 500–600 nm was observed.
of great interest [1–5]. Numerous excellent works focus
on the selective and sensitive detection of transition
metal ions; e.g., detection of Cu2+, Pb2+, Zn2+, and
Hg2+ have been reported [6–15]. The trivalent form of
iron is an essential element in man. It provides the
oxygen-carrying capacity of heme and acts as a cofactor
in many enzymatic reactions involved in the mitochon-
drial respiratory chain [16–18]. However, the develop-
ment of new fluorescent Fe3+ indicators, especially
those that exhibit selective Fe3+-amplified emission
[19–22], is still a challenge. Therefore, there is an
urgent need to develop chemical sensors that are capa-
ble of detecting the presence of iron in environmental
and biological samples at a physiological pH value. On
the other hand, rhodamine derivatives are nonfluorescent
and colorless, whereas ring-opening of the corresponding
spirolactam gives rise to strong fluorescence emission
and a pink color [23–28]. Recently, some bis(rhoda-
mine)-based fluorescent probe were reported for the
detection of Fe3+ metal ions [29–31]. In this paper, we
report three new rhodamine-based turn-on fluorescent
sensors 2–4 (Scheme 1) for Fe3+. Sensor 4 shows very
high sensitivity and selectivity toward Fe3+ over other
metal ions.
3+
.
.
Keywords Fluorescent probe Rhodamine Fe
Introduction
During the recent two decades, the design and synthesis
of compounds for sensing environmentally and biologi-
cally relevant important ionic species, particularly for
heavy metal and transition metal cations, is currently
Electronic supplementary material The online version of this article
(doi:10.1007/s10895-011-1022-0) contains supplementary material,
which is available to authorized users.
:
:
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X. Chen H. Hong R. Han D. Zhang Y. Ye (*) Y.-f. Zhao
Phosphorus Chemical Engineering Research Center of Henan
Province, Department of Chemistry, Zhengzhou University,
Zhengzhou 450052, China
e-mail: yeyong03@tsinghua.org.cn
Experimental Section
Y.-f. Zhao
Key Lab Chem Biol, Fujian Prov Coll Chem & Chem Engn,
Xiamen Univ,
Xiamen 361005, China
All the materials for synthesis were purchased from com-
mercial suppliers and used without further purification. The
solutions of metal ions were prepared from their nitrate salts,
except for FeCl2, CrCl3 and MnCl2. The use of Fe(NO3)3
and FeCl3 yielded nearly the same results. Tris–HCl buffer
solutions (pH07.15) were prepared using 0.01 M Tris,
0.1 M KNO3 and proper amount of HCl (about 0.01 M).
:
Y. Ye Y.-f. Zhao
Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical
Biology(Ministry of Education), Department of Chemistry,
Tsinghua University,
Beijing 100084, China

790
J Fluoresc (2012) 22:789–794
Scheme 1 Synthetic route of title compounds
Rhodamine B derivative (1) was synthesized according to
the literature [1–5]. A Hitachi F-4500 spectrofluorimeter
was used for fluorescence measurements. The absorption
spectra were recorded with a Techcomp UV-8500 spectro-
photometer (Shanghai, China). NMR spectra were measured
on a Bruker AMX-400 spectrometer at 400 MHz in CDCl3.
Elemental analyses were carried out with a Flash EA 1112
instrument. Mass spectra were acquired in positive ion mode
using a Bruker ESQUIRE 3000 ion trap spectrometer
equipped with a gas nebulizer probe, capable of analyzing
ions up to m/z 6000.
6.46 (s, 1H), 6.41 (d, 2H, J03 Hz), 6.28 (d, 2H, J03 Hz), 3.47
(m, 2H), 3.23–3.37 (m, 8H), 3.23 (m,2H),1.16–1.19 (m, 12H);
13C NMR (100 MHz, CDCl3, δ ppm): 170.3, 166.9, 153.9,
153.3, 148.9, 134.3, 132.8, 131.1, 130.3, 128.4, 128.1, 127.1,
123.9, 122.8, 108.3, 104.5, 97.7, 65.9, 44.3, 41.9, 39.9, 12.6;
ESI-MS: m/z 589.2 [M+H] +; Element analysis (%): found: C
75.50, H 6.88, N 9.54, calcd: C 75.48, H 6.85, N 9.52.
Synthesis of 3
Isophthalaldehyde (0.166 g,1.24 mmol) was slowly added
into the solution of 1 (1.20 g, 2.48 mmol)in 30 mL MeOH
under argon. After the addition three drops of AcOH and
some 4 Å molecular sieve, the mixture was refluxed for 8 h.
NaBH4 (0.469 g, 12.4 mmol) was added in three portion
at 0 °C. Then the reaction was stirred for 12 h at room
temperature before quenching with HCl (5 mL) and
adjusted to pH 7 by NaOH aqueous solution. After removal
of the solvent, the residue was extracted with HCCl3 (3×
15 mL). The combined organic layers were washed with brine
and dried over NaSO4. Removal of the solvents in vacuo and
purification by silica gel column chromatography with
Synthesis of 2
Benzoyl chloride (0.140 g, 1 mmol) was slowly added into
the mixture of 1 (0.484 g, 1 mmol) and triethylamine
(0.5 mL) in 20 mL CH2Cl2 at 0 °C. After the addition, the
mixture was stirred at room temperature for 5 h. The solvent
was removed and the residue was purified by silica gel column
chromatography with EtOAc/PE (4/1, v/v) as eluent to afford
1
4,yield 98.5%; H NMR (400 MHz,CDCl3, δ ppm):8.33 (s,
1H), 7.96 (m,3H), 7.47 (m, 5H), 7.10 (s, 1H), 6.48 (s, 1H),
Fig. 1 Color change of the
neutral buffer solution of 4 in the
presence of metal ions

J Fluoresc (2012) 22:789–794
791
CH2Cl2/MeOH (20/1, v/v) yielded the desired product 3 as
1
solid. yield 85.7%, M.p: 121–123 °C. H NMR (400 MHz,
CDCl3, δ ppm):7.89 (m, 2H), 7.42 (m, 4H), 7.06 (m, 5H), 6.38
(m, 8H), 6.22 (m, 4H), 3.56 (br, 4H), 3.29–3.35 (m, 20H),
2.45(br, 4H), 1.14–1.17 (t, 24H, J06.8 Hz); 13C NMR
(100 MHz, CDCl3, δ ppm):168.7, 153.6, 153.2, 148.7,
139.4, 132.4, 130.9, 128.6, 128.2, 128, 126.8, 123.7, 122.7,
108.1, 105.3, 97.7, 65.1, 52.9, 47.5, 44.3, 39.9, 12.6;
ESI-MS: m/z 1071.7 [M+H] +; Element analysis (%): found: C
76.26, H 7.21, N 10.43, calcd: C 76.23, H 7.34, N 10.46
Fig. 3 Changes of fluorescence intensity (F/F0) of compound 4
(10 μM) upon the addition of different metal ions in ethanol–water
solution. F and F0 are the fluorescence intensities of 4 with and without
adding the interference
Synthesis of 4
m-Phthaloyl chloride (0.17 g, 0.843 mmol) was slowly
added into the mixture of 1 (0.918 g, 1.897 mmol) and
triethylamine(1 ml) in 10 mL CH2Cl2 at 0 °C. After the
addition, the mixture was stirred at room temperature for
5 h. The solvent was removed and the residue was purified
by silica gel column chromatography with EtOAc/PE (2/1,
v/v) as eluent to afford 4,yield 95.8%, M.p : 166–168 °C; 1H
NMR (400 MHz,CDCl3, δ ppm):8.39 (s, 1H), 8.21 (s, 1H),
8.02 (s, 1H), 8.0 (s, 1H), 7.95 (m, 2H), 7.53 (t, 1H, J08 Hz),
7.45 (m, 4H), 7.1 (m, 2H), 6.47 (d, 4H, J08.8 Hz), 6.39 (m,
4H), 6.29 (d, 2H, J02.4 Hz), 6.27 (d, 2H, J02.4 Hz), 3.46
(m, 4H), 3.28–3.44 (m, 16H), 3.26 (m, 4H), 1.16 (t, 24H, J0
6.8 Hz); 13C NMR (100 MHz, CDCl3, δ ppm):170.2, 166.4,
153.8, 153.2, 148.9, 134.6, 132.7, 130.4, 129.7, 128.7,
128.4, 128.1, 126.1, 123.8, 123, 108.3, 104.6, 97.7, 65.3,
44.3, 41.5, 40, 12.6; ESI-MS: m/z 1098.6 [M+H] +, m/z
1121.6 [M+Na]+; Element analysis (%): found: C 74.26, H
6.76, N 10.21, calcd: C 74.29, H 6.78, N 10.19.
literature and obtained as light orange crystals. As shown
in Scheme 1, compounds 2 and 4 were prepared in high
yield by reacting 1 with benzoyl chloride and m-phthaloyl
chloride in the presence of triethylamine respectively. Com-
pound 3 was synthesized in two steps: production of
Schiff’s bases from 1 and isophthalaldehyde, followed by
reduction by NaBH4 in MeOH. Their structures have been
confirmed using 1H NMR, 13C NMR, ESI mass spectrometry,
and elemental analysis (see Supporting Information). Al-
though 4 is a derivative of rhodamine B, it forms a nearly
colorless solution in either Tris–HCl aqueous buffer (pH 3–9)
or absolute ethanol, indicating that the spirocyclic form exists
predominantly. The characteristic peak near 65.0 ppm (9-
carbon) in the 13C NMR spectrum of 4 also supports this
consideration [32]. Besides, neither the color nor the fluores-
cence (excited at 530 nm) characteristics of rhodamine could
be observed for 4 between pH 3.0 and 9.0 in water, suggesting
that the spirocyclic form was still preferred in this range
(S-Figure 10 and 11).
Results and Discussion
The free probes 2–3 are also colorless and nonfluorescent
because they are both in “ring-closed” states. After addition
of Fe3+ to 2–3 solutions, the complexation induced the three
Compound 1 was facilely synthesized from rhodamine B
and ethylenediamine according to procedures in the
Fig. 2 Fluorescence intensity of 4 (10 μM) and 16 eq of various other
metal ions in ethanol–water (1: 1,v/v) Solution Ex:548 nm, Em:
575 nm slit: 5
Fig. 4 Changes in the absorption spectra of 4 in the presence of
different metal ions in ethanol in ethanol-water

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J Fluoresc (2012) 22:789–794
Fig. 7 Fluorescence intensities of 4 (80 μM) with gradual addition of
Fig. 5 Change in the absorbance at 560 nm of 4 (10 μm) in presence
of 16 eq of various different metal ions in ethanol–water (1 1,v/v)
different amounts of (from bottom 0–8 eq Fe3+
)
The fluorescence enhancement effects of various metal
ions on 4 were investigated under excitation at 548nm
(Figs. 2 and 3). In the absence of metal ions, 4 exhibited a
very weak fluorescence peak near 580 nm, which was
probably the emission of trace open-ring molecules of 4.
When Fe3+ was introduced to a 10 μM solution of 4 in
ethanol-water, a bit red shift (~5 nm) and obvious enhance-
ment of fluorescence spectra were observed, whereas other
ions of interest displayed much weaker response. In ethanol-
water, 4 (10 μM) exhibited a 117-fold enhancement of
fluorescence intensity at peak wavelength λmax0575 nm in
the presence of 16 equiv Fe3+. A mild fluorescence enhance-
ment factors (FEF) were also detected for Cr3+ (40-fold), Fe2+
(6-fold), and Li+, Pb2+, Ag+, K+, Co2+, Ni2+, Na+, Zn2+, Ca2+,
Ba2+, Mn2+, Mg2+, Cd2+, Cu2+ or Hg2+ showed nearly no
response (Fig. 2). 4 displays better selectivity and sensitivity
than 2 and 3 maybe due to it has more amides to binding
the Fe3+.
probes to their “ring-open” states, thus leading to evident
color change (from colorless to brilliant pink) and emission
of a strong fluorescence. The fluorescence enhancement of 2
to Fe3+, Cr3+ and Fe2+ is as high as 33, 20 and 6-fold,
respectively(S-Figure 6). The fluorescence enhancement of
3 (10 uM) to Fe3+ (16 equiv) is as high as 53-fold (S-
Figure 8). The colorimetric and fluorometric responses be-
tween the probes and Fe3+ can also be conveniently detected
by the naked eye (S-Figure 9).
Interestingly, the addition of Fe3+ into the colorless sol-
utions (in both neutral buffer and ethanol) of 4 also gener-
ated a purple color and orange fluorescence rapidly, while
other ions, such as Li+, Pb2+, Ag+, K+, Co2+, Ni2+, Na+, Zn2+,
Ca2+, Ba2+, Mn2+, Mg2+, Cd2+, Cu2+ and Hg2+, gave no
visible change except for Cr3+ and Fe2+, which caused a mild
effect compared to Fe3+ in ethanol-water (1:1). This interest-
ing feature reveals that 4 can serve as a selective “naked-eye”
chemosensor for Fe3+ (Fig. 1).
Fig. 8 Fluorescence intensity of 4 (10 μM) to Fe3+ in ethanol–water
(1: 1,v/v) solution. (1) baseline: 10 μM 4 only; (2): 10 μM 4 with 16 eq
3-
Fig. 6 Plots according to the method for continuous variations, indi-
cating the 1:2 stoichiometry for 4 -Fe3+(the total concentration of 4 and
Fe3+ is 100 μm). XFe3þ ¼ CFe3þ=CFe3þ þ C4
Fe3+;(3): 4 with16 eq Fe3+ and then addition of 64 eq PO4 (K salt);
(4): 4 with 16 eq Fe3+ and 64 eq PO43−, then addition of 16 eq Fe3+
excitation wavelength:543 nm, slit: 5
;

J Fluoresc (2012) 22:789–794
793
Figure 4 shows the absorption spectra of 4 in the pres-
ence of various metal ions in ethanol-water. When no metal
ion was added to the solution of 4 (10 μM), almost no
absorption above 570 nm could be observed, whereas a
significant enhancement of the characteristic absorption of
rhodamine B emerged soon after Fe3+ was injected into the
solution. There was a large enhancement factor (178-fold) of
absorbance at λmax0560 nm upon the addition of 16 equiv
of Fe3+. A mild increase of absorbance at 560 nm was also
detected when the same amount (160 μM) of Cr3+ (causing
61-fold absorption enhancement) was added due to their low
binding affinity to 4. Other cations of interest gave no
response (Fig. 5).
as Hg2+, Pb2+, Co2+, Ni2+, Zn2+, Ca2+, Ba2+, Mn2+, Mg2+,
Cd2+, Cu2+, Na+, Ag+, K+ or Li+. The colorimetric and
fluorescent response to Fe3+ can be conveniently detected
even by the naked eye, which provides a facile method for
visual detection of Fe3+. The main limitation of this probe is
probably its moderate binding capacity to Fe3+ in aqueous
media, which hinders its usefulness in biochemical applica-
tions. However, its selectivity is excellent, and the detection
of Fe3+ at 10−5 M level is still available. The modification of
4 to develop new fluorescent probes for Fe3+ with stronger
binding ability is now under investigation.
Acknowledgments This work was financially supported by the Nation-
al Science Foundation of China (Nos. 20972143, 20732004 and 20972130)
The Fe3+ binding stoichiometry of 4 can be determined
from Fe3+ titration and the Job plot [33]. Detailed kinetic
analyses and the mechanisms are discussed later. The “ac-
tivity Job plots [34, 35]” with a maximum at XFe~0.36 are
best fitted to Fe3+:4 0 2:1 (Fig. 6), indicating that the
complex (Fe3+)2–4 is the predominant active species.
To further investigate the binding stoichiometry of 4 and
Fe3+ ion, a fluorescence titration experiment was carried out.
An increase of fluorescence intensity of 4 could be observed
with gradual addition of Fe3+ ion. The Fig. 7 indicates that a
1:2 stoichiometry is most possible for the binding mode of
Fe3+ and 4 in ethanol-water (1:1). The stability constant (K)
of 4 with Fe3+ ion was calculated according to the 1:2 model
(K02.25×104). The moderate stability constant of the 4-Fe3+
complex is mainly because the need of Fe3+ for six-
coordination is not satisfied, and moreover, the strong hydra-
tion ability of iron in water. As with many reported rodamine-
based spirolactam chemosensors, the Fe3+ induced fluores-
cence enhancement of chemosensor 4 is most likely the result
of the spiro ring-opening mechanism. That is, the chelation of
Fe3+ with the oxygen atoms of the amide groups of 4 results in
the formation of the open-ring form.
Open Access This article is distributed under the terms of the Crea-
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