A rhodamine-based chemosensor that works in the biological system

Article abstract of DOI:10.1021/ol800976d

(Figure Presented) A new rhodamine-based reversible chemosensor (L ₁) is reported, which could bind Hg²⁺ and Cu²⁺ in aqueous methanol solution with detectable change in color. Cu²⁺ and Hg²⁺ ions responded differently toward the fluorescence output signals on binding to L1. L1 could also be used as a selective probe for monitoring Hg²⁺ adsorbed on bacteria using an optical microscope.

Full text of DOI:10.1021/ol800976d

ORGANIC
LETTERS
2008
Vol. 10, No. 14
3013-3016
A Rhodamine-Based Chemosensor that
Works in the Biological System
Moorthy Suresh, Anupama Shrivastav, Sandhya Mishra, E. Suresh, and
Amitava Das*
Central Salt and Marine Chemicals Research Institute (CSIR),
BhaVnagar 364002, Gujarat, India
amitaVa@csmcri.org
Received April 28, 2008
ABSTRACT
A new rhodamine-based reversible chemosensor (L1) is reported, which could bind Hg2+ and Cu2+ in aqueous methanol solution with detectable
change in color. Cu2+ and Hg2+ ions responded differently toward the fluorescence output signals on binding to L1. L1 could also be used as
a selective probe for monitoring Hg2+ adsorbed on bacteria using an optical microscope.
Mercury is an unsafe toxin that has posed a great threat to
our environment.1 Oxidation of mercury vapor in atmosphere
to water-soluble Hg2+ ions and its consequent metabolism
by aquatic microbes produces methyl mercury, which bio-
accumulates through the food chain.2 This is expected to
have a severe effect on human health and the environment.
The best way to detect Hg2+ that has gone into the food
chain or contaminated the environment is to monitor the
extent of mercury present in microorganisms such as bacteria,
which survive in waste water or effluents. Recently, Chang
and co-workers reported a fluorescent probe for the detection
of mercury in fish, where the appearance of the new
fluorescence could be detected by confocal laser microscopy.3
In the past few years, a number of fluorescent chemosensors
for the selective detection of Hg2+ ions have been reported.4
However, examples for the detection of mercury in biological
systems are extremely rare.5 Limiting factors for design of
such a sensor molecule are low solubility in water, cross-
sensitivity toward other metal ions, and spectral/optical
sensitivity in physiological conditions. Generally, Hg2+ is
known to cause fluorescence quenching of the fluorophores
via the spin-orbit coupling effect,6 and this is reflected in
the turn-off fluorescence response reported in most instances.7
Very recently, few rhodamine-based fluorescent probes are
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10.1021/ol800976d CCC: $40.75
Published on Web 06/14/2008
2008 American Chemical Society

reported which show a selective turn-on response to Hg2+,8
although reports on the use of such probe molecules for
detection of Hg2+ uptake by live microorganisms are rare.5
Fluorescent probes, which show fluorescence enhancement
on binding to the cation of interest, are preferred as sensors
as these allow a lower detection limit and high-speed spatial
resolution via microscopic imaging.3,9
Information), and this was unequivocally corroborated on
the basis of the single-crystal X-ray analysis (Figure 1).13
Rhodamine-based dyes are known for their excellent
spectroscopic properties with a large molar extinction coef-
ficient (ε) and high fluorescence quantum yield (Φ). Earlier,
rhodamine-based spirolactam (fluorescence “off” state) was
employed as a molecular scaffold to design chemosensors
for selective recognition of Cu2+ and Pb2+10,11 as coordina-
tion of these metal ions induced spirolactam ring opening
(fluorescence “on” state) and thereby allowed detection
through an enhancement in fluorescence intensity.12
Here, we report a new rhodamine-based spirolactam
derivative (L1) as a chemosensor for Hg2+ and Cu2+, when
binding phenomena could be probed through binding-induced
changes in an electronic spectral pattern. Further, binding
of these metal ions to L1 caused color changes, which could
also be detected by the naked eye. Interestingly, binding of
only Hg2+ to L1 caused significant fluorescence enhancement
in an aqueous-methanol mixture. In this mixed solvent
media, two different modes of binding for Hg2+ and Cu2+
to L1 were observed. Cu2+ formed a 1:1 complex (CuL1),
whereas Hg2+ formed a 2:1 complex (Hg(L1)2). The newly
synthesized rhodamine 6G derivative (L1) (Scheme 1) was
prepared in high yield (see Supporting Information).
Figure 1. ORTEP diagram of the compound L1 (40% probability
level for the thermal ellipsoids).
This compound (L1) remained colorless in water-methanol
(1:1, v/v) solution at pH 7.0. This indicates that the
spirolactam form of L1 predominantly existed under this
condition. The 13C NMR spectrum was recorded for L1. A
characteristic peak for the C7-atom appeared near 66 ppm
and confirmed this proposition.14 Spectrophotometric titra-
tions for L1 with varying pH revealed that L1 retained
the spirocyclic form within the pH range of 5.0-13.0 (see
Supporting Information). Below pH 5.0, the fluorescence
intensity tended to increase with a further decrease in the
pH of the solution, which signified the spirolactam ring
openingsas the acyclic form of rhodamine derivatives are
known to be strongly fluorescent.
Scheme 1. Synthetic Route of L1
Electronic spectra of L1 (20 µM), recorded in the water/
methanol (1:1, v/v) mixed solvent at neutral pH, exhibited a
very weak band above 530 nm, which could be attributed to
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J. Am. Chem. Soc. 2007, 129, 10322–10323. (b) Sasaki, E.; Kojima, H.;
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7386–7387.
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W.; Yoon, J. J. Am. Chem. Soc. 2005, 127, 10107–10111.
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2037. (b) Tanaka, M.; Kamada, K.; Ando, H.; Kitagaki, T.; Shibutani, Y.;
Kimura, K. J. Org. Chem. 2000, 65, 4342–4347. (c) Winkler, J. D.; Bowen,
C. M.; Michelet, V. J. Am. Chem. Soc. 1998, 120, 3237–3242. (d) Tanaka,
M.; Nakumara, M.; Salhin, M. A. A.; Ikeda, T.; Kamada, K.; Ando, H.;
Shibutani, Y.; Kimura, K. J. Org. Chem. 2001, 66, 1533–1537. (e) Hung,
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F.; Yi, T.; Huang, C. Chem. Commun. 2008, DOI: 10.1039/b801503a.
(13) Crystal data for the compound: CCDC no. 685033; molecular
formula: C34H31N4O4, M ) 559.63, crystal size: 0.40 × 0.36 × 0.20 mm3,
Triclinic, space group P-1 with a ) 9.132(3) Å, b ) 11.574(4) Å, c )
13.473(4) Å, R ) 92.904 (5)°, ꢀ ) 92.889(5)°, γ ) 97.025(5)°, V )
1409.2(8) Å3, Z ) 2, Dcalcd ) 1.319 g/cm, T ) 100(2) K, F(000) ) 590,
Absorption coefficient ) 0.088 mm-1, λ ) 0.71073 Å, 11 469 reflections
were collected, 6158 observed reflections with (I g 2σ(I)), R(int) ) 0.0633.
R1) 0.0780, wR2 ) 0.1987, goodness of fit on F2 ) 1.057. The largest
difference peak and hole: 0.867 and -0.411 eÅ-3, respectively.
(14) Anthoni, U.; Christophersen, C.; Nielsen, P.; Puschl, A.; Schaum-
burg, K. Struct. Chem. 1995, 3, 161–165.
The proposed molecular structure and its purity were
confirmed by various spectroscopic analyses (see Supporting
(7) (a) Moon, S.-Y.; Cha, N. R.; Kim, Y. H.; Chang, S.-K. J. Org. Chem.
2004, 69, 181–183. (b) Moon, S.-Y.; Yoon, N. J.; Park, S. M.; Chang, S.-
K. J. Org. Chem. 2005, 70, 2394–2397. (c) Chae, M.-Y.; Czarnik, A. W.
J. Am. Chem. Soc. 1992, 114, 9704–9705. (d) Prodi, L.; Bargossi, C.;
Montalti, M.; Zaccheroni, N.; Su, N.; Bradshow, J. S.; Izatt, R. M.; Savage,
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969
.
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Chem. Soc. 2003, 125, 14270–14271. (c) Descalzo, A.; Martinez-Manez,
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.
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Org. Lett., Vol. 10, No. 14, 2008

the presence of a trace (∼0.22%) amount of the ring-opened
form of L1. On addition of Hg2+ and Cu2+, a new absorption
band appeared at 534 and 528 nm, respectively (Figure 2).
constant values reported here are an average of at least five
independent experimental results in each case. Presumably,
the larger covalent radius (1.49 × 10-10 m) for Hg2+ as
compared to the Cu2+ (1.17 × 10-10 m) accounts for the
difference in the coordination behavior of L1.
On addition and gradual increase of [Hg2+] to the
nonfluorescent aqueous methanol solution (1:1, v/v) of L1,
a significant enhancement in fluorescence intensity at 554
nm was observed following excitation at 500 nm, and the
emission quantum yield (Φ) was found to be 0.82 relative
to rhodamine 6G. No such change was observed on addition
of Cu2+. The quenching of the fluorescence of the open ring
form of L1 by Cu2+ could be explained based on the well-
known paramagnetic effect of the d9 Cu(II) system.16
Fluorescence intensity of L1 (10 µM) upon addition of Hg2+
(0-8 mol equiv) was found to enhance by 90 fold at 554
nm (Figure 4). Various alkali, alkaline earth metal ions,
Figure 2
. Absorption spectra of L1 (20 µM) in water-methanol
(1:1, v/v) at pH 7.0 (a) upon addition of 0-35 mol equiv of Cu2+
.
Inset: Job’s plot that indicates the 1:1 stoichiometry for complex
formation. (b) Upon addition of 0-8 mol equiv of Hg2+. Inset:
Job’s plot that indicates 2:1 stoichiometry for complex formation.
([L1] + [Cu2+] or [Hg2+] ) 100 µM).
This enhancement in absorbance clearly suggests the forma-
tion of the delocalized xanthane moiety of the rhodamine
group, associated with a distinct color change from colorless
to pink. Other metal ions, such as Co2+, Fe2+, Ni2+, Zn2+,
Pb2+, Cd2+, Mg2+, Ca2+, Ba2+, Li+, K+, and Na+ did not
show any significant color and spectral change under
identical conditions (see Supporting Information).
2+
An association constant (KaHg ) of 8.0 × 105 ( 0.1 M-2
Figure 4. Change in fluorescence spectra of L1 (10 µM) upon
L2 was evaluated assuming a 2:1 stoichiometry for [Hg(L1)2]
addition of Hg2+ (0-8 equiv) in water-methanol (1:1, v/v) at pH
7. λext at 500 nm. Inset: Emission intensity of L1 (2 × 10-5 M) at
554 nm as a function of [Hg2+] in ppb level.
(Figures 2 and 3) from the nonlinear fitting of the titration
and transition metal ions (Co2+, Ni2+, Cu2+, Fe2+, Cd2+
,
Zn2+, and Pb2+) did not show any significant fluorescence
enhancement at 554 nm even upon addition of 25 mol
equiv of respective metal ions. Relative fluorescence
enhancement of L1 in the absence and presence of various
other metal ions and thereby its selectivity for Hg2+ is
shown in Figure 5.
Figure 3. .
Proposed binding mode of L1 with Hg2+ and Cu2+
curve. This binding stoichiometry was also confirmed from
Job’s plots.15
Spectrophotometric titrations with Cu2+ revealed a 1:1
complex formation (CuL1) (Figures 2 and 3), and the
2+
association constant (KaCu ) of 1.68 × 105 ( 0.012 M-1
L
was evaluated. This stoichiometry was also confirmed by
ESI-MS mass spectra (see Supporting Information). The
peak at m/z ) 623.3 corresponding to CuL1 was observed,
whereas L1 without Cu2+ exhibited peaks only at m/z )
561.5, which corresponds to [L1 + H]+. The association
Figure 5. Fluorescence spectra of L1 (8 µM) in water-methanol
(1:1, v/v) at pH 7 with respective metal cations (33 equiv). λext at
500 nm.
Competitive recognition of Hg2+ in the presence of various
other metal ions, in even higher concentration, was also
(15) Vosburgh, W. C.; Copper, G. R. J. Am. Chem. Soc. 1941, 63, 437–
442.
Org. Lett., Vol. 10, No. 14, 2008
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studied, which revealed that Hg2+ present in 33 mol equiv
could be detected even in the presence of 33 mol equiv of
other metal ions like Na+, K+, Ba2+, Mg2+, Ca2+, Co2+, Ni2+,
Cu2+, Fe2+, Cd2+, Zn2+, and Pb2+ (see Supporting Informa-
tion). The lower detection limit for Hg2+, using this
chemosensor (L1), was also evaluated. The fluorescence
titration profile of L1 (20 µM) with Hg2+ demonstrates that
Hg2+ could be detected at the parts per billion level.17 Please
note that the signal-to-noise ratio for this specific concentra-
tion was at least three.
cell wall of the bacteria. Adsorption of the Hg2+ ion on the
cell wall was also confirmed by SEM images (Figure 7)
Consequently, it was of great interest to investigate the
reversibility of the system. I- is known to have a strong
binding affinity for Hg2+,4i,5d and upon addition of aqueous
methanol solution of 5 equiv of KI to a solution mixture of
L1 (20 µM) and Hg2+ (8 µM), color changed from pink to
colorless. Simultaneously, about 95% of the fluorescence
intensity gets quenched (see Supporting Information) signify-
ing decomplexation of Hg2+ by I- and followed by a
spirolactam ring closure reaction (see Supporting Informa-
tion). Thus, L1 can be classified as a reversible chemosensor
for Hg2+.
Figure 7. SEM images of Pseudomonas putida (a) before and (b)
after being exposed to Hg2+ and then to L1.
recorded for Pseudomonas putida, before and after exposing
the live cells to the Hg2+ ion and then to L1.
The difference in the relative contrasts in the SEM images
of the sample surfaces signifies the nonhomogeneous chemi-
cal nature of the sample surface. Relatively brighter cell
exterior for Gram -Ve bacteria indicates the accumulation
of higher charges on the extracellular surface.
The Hg2+ ion is one of the most potent pollutants present
in various effluents. There are several microorganisms which
exist in soil and various effluents and are known to have a
high affinity for adsorption of the Hg2+ ion.
In conclusion, we have synthesized a new rhodamine-
based chemosensor L1 that displayed 1:2 complex formation
with Hg2+ and 1:1 complex formation with Cu2+ in aqueous
solution. Complex formation processes could be monitored
by the spectral changes, as well as through color changes,
which could be detected by the naked eye. Binding to Hg2+
caused a significant enhancement in the observed fluores-
cence at 554 nm. This could also be visualized in the dark
when irradiated with 365 nm of light. Remarkably, high
selectivity for the Hg2+ ion in aqueous solution could be
demonstrated when it was probed using the fluorescence
emission mode. Further, this sensor molecule could detect
the Hg2+ ion adsorbed on the cell surface of the microorgan-
ism such as Pseudomonas putida and thus could be useful
for determining the amount of Hg2+ that could enter in the
food chain affecting human beings along with phytoplanktons
and zooplanktons.
Pseudomonas putida, a Gram -Ve bacteria is one such
bacteria.18,19 To evaluate the application potential of this
newly developed chemosensor for Hg2+, we have used this
bacterial cell for detection of Hg2+ adsorbed when exposed
to the Hg2+ solution. Pseudomonas putida was cultured in
the King’s B (KB) medium (Peptone 20 g, glycerol 15 g,
K2HPO4 1.5 g, MgSO4·7H2O 1.5 g, distilled water 1000 mL,
pH 7.2). The cells were harvested and vortexed for making
the homogeneous suspension in sterile distilled water.
These bacterial cells were exposed to Hg2+ (10.0 µM) in
water–ethanol (7:3, v/v) for 10 min at 25 °C and then
successively exposed to L1 (20.0 µM) under the same
conditions and monitored through a light microscope (AXIO
IMAGER, Carl Zeiss; 100×). Microscopic images revealed
that, after treatment with Hg2+ and L1 the color of bacterial
Acknowledgment. CSIR and DST (India) have supported
this work. M.S. (JRF) and A.S. (SRF) acknowledge CSIR
for a Research Fellowship. S.M. and A.D. thank Dr. P.K.
Ghosh (CSMCRI, Bhavnagar) for his keen interest in this
work.
Supporting Information Available: Synthetic details,
characterization data for the compound L1, and selected
spectroscopic data of L1. This material is available free of
charge via the Internet at http://pubs.acs.org.
Figure 6
.
Light microscopy images (100×) of (a) blank cells of
Pseudomonas putida, (b) bacteria cells exposed to Hg2+ solution
(10 µM), and (c) cells exposed to aqueous solution of Hg2+ (10
µM) and then to a water–ethanol (7/3, v/v) solution of L1 (20
µM).
OL800976D
(16) Kim, J. S.; Quang, D. T. Chem. ReV. 2007, 9, 3780–3799.
(17) The EPA standard for the maximum allowable amount of Hg2+ in
drinking water is 2 ppb.
(18) Beveridge, T. J. Biotechnol. Bioeng. 1986, 16, 127.
(19) Ledin, M.; Pedersen, K.; Allard, B. Water, Air, Soil Pollut. 1997,
93, 367–381.
cells changed to pink (Figure 6). These results demonstrate
that L1 could be used for detecting Hg2+ adsorbed on the
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