Fe(III)- and Hg(II)-selective dual channel fluorescence of a rhodamine-azacrown ether conjugate

Article abstract of DOI:10.1016/j.tetlet.2008.04.102

A rhodamine-azacrown ether conjugate (1) demonstrates Fe(III)-selective green fluorescence, while showing Hg(II)-selective orange fluorescence. This is the first example of rhodamine-based fluorescent probe that shows dual channel fluorescence for two different metal cations.

Full text of DOI:10.1016/j.tetlet.2008.04.102

Available online at www.sciencedirect.com
Tetrahedron Letters 49 (2008) 4178–4181
Fe(III)- and Hg(II)-selective dual channel fluorescence
of a rhodamine–azacrown ether conjugate
Xuan Zhang, Yasuhiro Shiraishi *, Takayuki Hirai
Research Center for Solar Energy Chemistry, and Division of Chemical Engineering, Graduate School of Engineering Science,
Osaka University, Toyonaka 560-8531, Japan
Received 7 March 2008; revised 12 April 2008; accepted 17 April 2008
Available online 22 April 2008
Abstract
A rhodamine–azacrown ether conjugate (1) demonstrates Fe(III)-selective green fluorescence, while showing Hg(II)-selective orange
fluorescence. This is the first example of rhodamine-based fluorescent probe that shows dual channel fluorescence for two different metal
cations.
Ó 2008 Elsevier Ltd. All rights reserved.
Fluorometric detection of ionic species has attracted a
great deal of attention.1 Of particular interest is the develop-
ment of fluorescent probes for heavy and transition metal
cations, such as Hg2+ and Fe3+, due to their biological
and environmental importance.2 Hg2+ is one of the most
hazardous components in the environment,3 and Fe3+ plays
a pivotal role in many biochemical processes in a cellular
level.4 A number of fluorescent probes for the detection of
Hg2+ and Fe3+ have been proposed so far.5,6 However,
most of these probes show fluorescence quenching (turn-
off) response,5 and fluorescent probes that show fluores-
cence enhancement (turn-on) response are still rare.6
Rhodamine is a dye used extensively as a fluorescent
labeling reagent due to its excellent photophysical proper-
ties, such as long absorption and emission wavelengths
elongated to visible region, high absorption coefficient,
and high fluorescence quantum yield.7 Recently, various
rhodamine-based turn-on fluorescent probes for Hg2+ or
Fe3+ have been proposed.8,9 The cation sensing mechanism
of these probes is based on the change in structure between
the spirocyclic and open-cycle forms. Without cations,
these probes exist in a non-emissive spirocyclic form.
Addition of metal cation leads to spirocycle opening via
a reversible coordination or an irreversible chemical reac-
tion with the probe, resulting in an appearance of orange
fluorescence (550–650 nm). These probes show this single
channel fluorescence against Hg2+ or Fe3+ and, hence,
can detect either Hg2+ or Fe3+
.
Herein, we report that a new rhodamine derivative (1)
containing an aza-18-crown-6 moiety (Scheme 1, synthe-
sis10) behaves as a dual channel fluorescent probe for
Hg2+ and Fe3+. Compound 1 shows Hg2+-selective
ordinary orange fluorescence (550–650 nm), while showing
POCl3
COCl
COOH
Cl
reflux, 18 h
N
O
N
N
O
N
RB HCl
Br
H2N
TEAa, CH3CN
rt, 24 h
O
37%
O
N
O
O
O
N
O
Br
N
aza-18-crown-6
N
O
DIEAb, CH3CN
reflux, 24 h
20%
N
N
O
O
N
1
2
*
Scheme 1. Synthesis of the probe 1. (a) Triethylamine. (b) N,N-
Corresponding author. Tel.: +81 6 6850 6271; fax: +81 6 6850 6273.
Diisopropylethylamine.
E-mail address: shiraish@cheng.es.osaka-u.ac.jp (Y. Shiraishi).
0040-4039/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2008.04.102

X. Zhang et al. / Tetrahedron Letters 49 (2008) 4178–4181
4179
Fe3+-selective green fluorescence (490–600 nm). To the best
of our knowledge, this is the first example of the rhoda-
mine-based fluorescent probe that shows dual channel fluo-
rescence for two different metal cations.
Figure 1 shows fluorescence spectra (kex = 480 nm) of 1
(5 lM) measured in CH3CN with respective metal cations
(90 equiv). Without cations, 1 is non-fluorescent. Addition
of Hg2+, however, leads to an appearance of orange fluo-
rescence at 578 nm (fluorescence enhancement: 45-fold,
Fig. S110). In contrast, addition of Fe3+ creates a remark-
ably enhanced green fluorescence at 525 nm (fluorescence
enhancement: 378-fold, Fig. S110). These data clearly indi-
cate that probe 1 detects Hg2+ and Fe3+ with two different
fluorescence channels.
a
12 equiv
10
200
9
200
100
0
8
Hg2+
100
0
7
6
5
4
λ
em = 578 nm
0
5
10
15
20
3
[Hg2+] / [1]
1
550
600
650
700
Wavelength / nm
90 equiv
b
Figure 2 shows the results of fluorescence titration of 1
with Hg2+ and Fe3+. Hg2+ addition (Fig. 2a) leads to a
monotonous increase in the orange fluorescence (578 nm),
600
400
200
0
60
50
40
600
400
200
0
where the increase is saturated with 12 equiv of Hg2+ 11
.
Fe3+
This emission behavior is similar to that for early-reported
rhodamine-based Hg2+ probes.8 As shown in Figure 2b,
addition of <10 equiv of Fe3+ creates similar emission at
ca. 575 nm. Further Fe3+ addition, however, leads to
blue-shift of the emission, along with a drastic intensity
increase, where the emission color changes from pale
orange to green (Fig. S210). The blue-shift and enhance-
ment of the emission stop upon the addition of 90 equiv
of Fe3+ (Fig. 2b).11 It must be noted that the Fe3+-selective
‘green’ emission is the first example among the rhodamine-
based probes.9
30
24
λem = 525 nm
20
0
30
60
90
120
[Fe3+] / [1]
10
4
500
550
600
650
Wavelength / nm
Fig. 2. Fluorescence titration of 1 (5 lM) in CH3CN with (a) Hg2+
(kex = 510 nm) and (b) Fe3+ (kex = 480 nm). Intensity change (inset).
As shown in Figure 3, without cations, 1 scarcely shows
an absorption at 500–600 nm, indicating that 1 exists as a
spirocycle-closed form.8,9 This is confirmed by a distinctive
spirocycle carbon shift at 64.81 ppm in the 13C NMR spec-
trum of 1.10 Hg2+ addition leads to an appearance of
strong 556 nm absorption, along with a clear color change
from colorless to pink, as is observed for ordinary rhoda-
mine-based probes.8,9 Fe2+, Cu2+, and Pb2+ show minor
absorption increase, whereas other metal cations show neg-
ligible increase. In contrast, Fe3+ shows a blue-shifted
absorption at 502 nm.
Fe3+
1·Fe3+
1
1·Hg2+
Fig. 3. Absorption spectra of 1 (5 lM) measured in CH3CN with
respective metal cations (90 equiv). Change in solution color (inset).
600
400
200
0
Figure 4 shows the results of absorption titration of 1.
Hg2+ addition leads to monotonous increase in the
556 nm absorption, which is saturated upon the addition
of 12 equiv of Hg2+. With <10 equiv of Fe3+, 556 nm
absorption also increases. However, with >10 equiv of
Fe3+, the absorption decreases and the 502 nm absorption
then increases. This increase is saturated with >90 equiv of
1 only,
Cu2+, Fe2+, Pb2+, Zn2+, Cd2+,
Co2+, Ni2+, Cr3+, Mn2+, Ca2+,
Ag+, Li+, Na+, K+
Hg2+
Fe3+
.
500
550
600
650
700
Excitation spectra of 1 with Hg2+, monitored at 580 nm
(orange emission), appear at 559 nm (Fig. S310), which are
similar to the absorption spectra (Fig. 4a). With <10 equiv
of Fe3+, similar excitation spectra appear at 559 nm (Fig.
Wavelength / nm
Fig. 1. Fluorescence spectra of 1 (5 lM) measured in CH3CN with
90 equiv of various metal cations (kex = 480 nm). Change in fluorescence
color (inset).

4180
X. Zhang et al. / Tetrahedron Letters 49 (2008) 4178–4181
a
a
600
10
8
12 equiv
0.4
0.2
0.0
0.4
0.2
4
Fe3+
400
Hg2+
8
7
20
556 nm
200
2
0.0
90 equiv
0
5
10
15
20
4
1
[Hg2+] / [1]
0
400
450
500
550
600
450
500
550
600
650
700
Wavelength / nm
Wavelength / nm
0.2
0.1
0.0
b
0.2
0.1
0.0
10 equiv
b
90 equiv
1200
800
400
0
556 nm
Fe3+
Fe3+
502 nm
24
20
90 equiv
0
30
60
[Fe3+] / [1]
90
120
10
8
400
450
500
550
600
450
500
550
600
650
700
Wavelength / nm
Wavelength / nm
Fig. 5. Excitation spectra of 1 (5 lM) in CH3CN with Fe3+ monitored at
(a) 580 nm and (b) 530 nm.
Fig. 4. Absorption titration of 1 (5 lM) in CH3CN with (a) Hg2+ and (b)
Fe3+. Change in absorbance (inset).
5a). However, with >10 equiv of Fe3+, this band decreases
and a blue-shifted band appears at 450–550 nm; with 90
equiv of Fe3+, only the 505 nm band remains. The appear-
ance of the blue-shifted band is more apparent when the
spectra are monitored at 530 nm (green emission; Fig.
5b): excitation band blue-shifts continuously upon Fe3+
addition. These suggest that the respective orange and
green emissions originate from different ground state
species.
Hill analysis of the fluorescence titration data (578 nm)
obtained with Hg2+ (Fig. S410) provides a Hill coefficient
n = 2.0 with association constant log Ka = 8.7, indicative
of the formation of 1Á(Hg2+)2 complex.12 In contrast, anal-
ysis of the titration data obtained with Fe3+ (525 nm; Fig-
ure S410) provides an unresolved coefficient n = 3.3 (log
Ka = 12.6). This indicates that 1 associates with Fe3+ in a
complicated stoichiometry.
electron density of these moieties, indicating that 1 actually
coordinates with Hg2+ or Fe3+ 9d,13
In addition, upon
.
addition of Hg2+ or Fe3+, CH3 proton of 1 (1.12 ppm)
decreases, and new protons appear downfield, indicating
that the coordination of 1 with Hg2+ or Fe3+ leads to spi-
rocycle opening.9d Addition of ethylenediamine to the solu-
tion of 1 containing either Hg2+ or Fe3+ leads to the
disappearance of both absorption and emission spectra,
indicating that 1 coordinates reversibly with these cat-
ions.8,9,13 The emission behaviors of 1 are therefore
explained by the ordinary spirocycle opening mecha-
nism:8,9,13 coordination of metal cations with the amide
carbonyl and azacrown ether moieties of 1 leads to the for-
mation of spirocycle-opened emitting species (orange emit-
ter). The formation of the ‘green’ emitter of 1 upon Fe3+
addition involves a different mechanism in addition to the
spirocycle opening mechanism.
IR analysis of 1 in CH3CN (Fig. S510) reveals that both
amide carbonyl (C@O) and ether (C–O) absorptions of 1 at
1684.5 and 1120.5 cmÀ1 shift to lower frequency upon the
addition of Hg2+ (1632.9 and 1100.2 cmÀ1) and Fe3+
(1635.8 and 1101.6 cmÀ1). This indicates that both car-
bonyl and azacrown ether moiety are involved in metal cat-
ion coordination.9d,13 1H NMR titration in CD3CN (Fig.
S610) shows that both aromatic and azacrown ether pro-
tons of 1 shift downfield and become broader upon the
addition of Fe3+ or Hg2+. This is due to the decrease in
Recently, we found that a rhodamine derivative contain-
ing an ethylenediamine-N,N-diacetic acid moiety shows
blue-shifted absorption and emission spectra upon Cu2+
addition.14 Inherent aggregation properties of rhodamine
in solution15 and the spectral data imply that the emitting
species for the blue-shifted emission is an ‘aggregate’ of
multiple molecules formed by coordination association
with multiple Cu2+ ions. The absorption and emission
behaviors of the rhodamine derivative are similar to those
of 1 with Fe3+; therefore, the green emission of 1 upon

X. Zhang et al. / Tetrahedron Letters 49 (2008) 4178–4181
4181
Fe3+ addition may probably be explained by Fe3+-induced
aggregation mechanism. This assumption is supported by
some spectral data: Hill analysis of the fluorescence titra-
tion data shows unresolved stoichiometry (n = 3.3; Fig.
S410); absorption spectra do not show clear isosbestic point
(Fig. 4b). These indicate that 1 aggregates via coordination
association with multiple Fe3+ ions. The appearances of
the blue-shifted excitation spectra (Fig. 5) clearly indicate
that the green emission is formed via direct photoexcitation
of the ground state aggregates. In addition, saturation of
the green emission increase after Fe3+ addition (296 K)
requires >5 h, while the orange emission increase after
Hg2+ addition saturates relatively faster (<1 h) ( Fig.
S710). As reported,16 rhodamine aggregation is enhanced
at higher temperature due to the decrease in solvation
interaction. The green emission increase after Fe3+ addi-
tion occurs rapidly at higher temperature (Fig. S710). These
findings clearly indicate that the aggregation interaction is
involved in the formation of the green emitter.
3. Boening, D. W. Chemosphere 2000, 40, 1335–1351.
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In conclusion, we found that a new rhodamine deriva-
tive (1) containing an azacrown ether moiety shows Fe3+
-
and Hg2+-selective dual channel fluorescence in CH3CN.17
This is the first rhodamine-based probe showing dual chan-
nel fluorescence for different metal cations. Although the
detailed mechanism for the Fe3+-selective green emission
is unclear, the results presented here may contribute to
the design of more useful rhodamine-based fluorescent
probes for heavy and transition metal cations.
7. Lakowicz, J. R. Principles of Fluorescence Spectroscopy, 3rd ed.;
Springer: New York, 2006; pp 67–69.
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Hwang, I.-C.; Kim, K. S.; Kim, J. S. Org. Lett. 2007, 9, 907–910; (d)
Lee, M. H.; Wu, J.-S.; Lee, J. W.; Jung, J. H.; Kim, J. S. Org. Lett.
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Acknowledgements
This work is partly supported by Grants-in-Aid for Sci-
entific Research (No. 19760536) from the Ministry of Edu-
cation, Culture, Sports, Science and Technology, Japan
(MEXT). We are grateful to the Division of Chemical
Engineering for the Lend-Lease Laboratory System.
9. For Fe3+: (a) Xiang, Y.; Tong, A.-J. Org. Lett. 2006, 8, 1549–1552; (b)
Zhang, M.; Gao, Y.-H.; Li, M.-Y.; Yu, M.-X.; Li, F.-Y.; Li, L.; Zhu,
M.-W.; Zhang, J.-P.; Yi, T.; Huang, C.-H. Tetrahedron Lett. 2007, 48,
3709–3712; (c) Bae, S.; Tae, J. Tetrahedron Lett. 2007, 48, 5389–5392;
(d) Zhang, X.; Shiraishi, Y.; Hirai, T. Tetrahedron Lett. 2007, 48,
5455–5459; (e) Mao, J.; Wang, L.-N.; Dou, W.; Tang, X.-L.; Yan, Y.;
Liu, W.-S. Org. Lett. 2007, 9, 4567–4570.
Supplementary data
10. See the Supplementary Data.
11. Fluorescence quantum yields of 1 with 12 equiv of Hg2+ and 90 equiv
of Fe3+ are determined to be 0.15 and 0.49, respectively, based on
fluorescein standard (UF = 0.85 in 0.1 M NaOH): Parker, C. A.;
Rees, W. T. Analyst 1960, 85, 587–600.
Supplementary data (materials, synthesis, methods, and
Figures) associated with this article can be found, in the
online version, at doi:10.1016/j.tetlet.2008.04.102.
12. Hennrich, G.; Rurack, K.; Spieles, M. Eur. J. Org. Chem. 2006, 516–
521.
References and notes
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with Fe3+ or Hg2+ is completely quenched by an addition of 5% water
to CH3CN.