Ratiometric detection of Cr3+ and Hg2+ by a naphthalimide-rhodamine based fluorescent probe

Article abstract of DOI:10.1021/ic202073q

Newly synthesized rhodamine derivatives, L₁ and L₂, are found to bind specifically to Hg²⁺ or Cr³⁺ in presence of large excess of other competing ions with associated changes in their optical and fluorescence spectral behavior. These spectral changes are significant enough in the visible region of the spectrum and thus, allow the visual detection. For L₁, the detection limit is even lower than the permissible [Cr³⁺] or [Hg²⁺] in drinking water as per standard U.S. EPA norms; while the receptor, L2 could be used as a ratiometric sensor for detection of Cr³⁺ and Hg²⁺ based on the resonance energy transfer (RET) process involving the donor naphthalimide and the acceptor Cr³⁺/Hg²⁺-bound xanthene fragment. Studies reveal that these two reagents could be used for recognition and sensing of Hg²⁺/Cr³⁺. Further, confocal laser microscopic studies confirmed that the reagent L₂ could also be used as an imaging probe for detection of uptake of these ions in A431 cells.

Full text of DOI:10.1021/ic202073q

Article
pubs.acs.org/IC
Ratiometric Detection of Cr3+ and Hg2+ by a Naphthalimide-
Rhodamine Based Fluorescent Probe
Prasenjit Mahato,† Sukdeb Saha,† E. Suresh,† Rosa Di Liddo,‡ Pier Paolo Parnigotto,‡
Maria Teresa Conconi,‡ Manoj K. Kesharwani,† Bishwajit Ganguly,*,† and Amitava Das*,†
†Central Salt and Marine Chemicals Research Institute (CSIR), Bhavnagar -364002, Gujarat, India
‡Interdepartmental Research and Service Centre for Biology and Regenerative Medicine-Department of Pharmaceutical Sciences,
University of Padua, Via Marzolo 5, 35131 Padua, Italy
S
* Supporting Information
ABSTRACT: Newly synthesized rhodamine derivatives, L1 and L2, are found to bind specifically to Hg2+ or Cr3+ in presence of
large excess of other competing ions with associated changes in their optical and fluorescence spectral behavior. These spectral
changes are significant enough in the visible region of the spectrum and thus, allow the visual detection. For L1, the detection
limit is even lower than the permissible [Cr3+] or [Hg2+] in drinking water as per standard U.S. EPA norms; while the receptor,
L2 could be used as a ratiometric sensor for detection of Cr3+ and Hg2+ based on the resonance energy transfer (RET) process
involving the donor naphthalimide and the acceptor Cr3+/Hg2+-bound xanthene fragment. Studies reveal that these two reagents
could be used for recognition and sensing of Hg2+/Cr3+. Further, confocal laser microscopic studies confirmed that the reagent L2
could also be used as an imaging probe for detection of uptake of these ions in A431 cells.
chemosensors for Hg2+ and Cr3+ in various media is of
considerable importance. In general, traditional analytical
INTRODUCTION
Development of chemosensors for sensing and recognition of
■
techniques like atomic absorption/emission spectroscopy or
environmentally and biologically important heavy and tran-
sition metal ions, for example, Hg2+, Pb2+, Cu2+, and Cr3+, have
inductively coupled plasma mass spectrometry are costly and
attracted considerable attention of current researchers.1,2
time-consuming methods, which are not convenient for “in-the-
Among the heavy and transition metal ions, Hg2+ and Cr3+
field” applications. Reports are there for electrochemical
sensors for detection of these two ions.9 However, among
appear ubiquitous because of various industrial and natural
sources. Apart from that Hg2+ that is being released in the
various methodologies that have been adopted for sensing of a
specific analyte, fluorescence based sensors have been most
environment along with the effluent, atmospheric oxidation of
popular for achieving higher sensitivity, rapid and reversible
mercury vapor also leads to the generation of water-soluble
Hg2+ ions that deposit onto land or into water. Hg2+ is
detection, and possible application in imaging studies for
diagnostic applications.10 Again Cr3+ is known to quench the
assimilated and converted by microorganisms to methylmer-
cury, a potent neurotoxin.3 Bioaccumulation of ionic mercury
luminescence of a fluorophore because of its paramagnetic
property and Hg2+ quench due to effective spin−orbit coupling
mechanism, bound to the receptor functionality of a sensor, and
this accounts for the most fluorescence off/quenching-based
sensors reported in the literature.11 However, the preference is
for the receptor with fluorescence on response because of the
obvious ease in the detection process.
and methylmercury allows this toxin to get into the food chain.4
As an environmental contaminant, chromium is found
mostly in Cr(VI) form and its bacterial reduction to Cr3+ is
considered as one of the promising strategies for bioremedia-
tion.5 However, a recent study reveals that soluble Cr3+ at pH
6−8 can be found transiently in significant concentrations and
has a deleterious effect on microorganisms, like Shewanella sp.
MR-4.6 It is also proposed that Cr3+ ion, present in the
cytoplasm, binds nonspecifically to DNA and other cellular
components and inhibits transcription and possibly DNA
replication.7 Chromium deficiency is known to influence
adversely the metabolism of glucose and lipids and cause
maturity-onset diabetes, cardiovascular diseases, and nervous
system disorders.8 Thus, development of sensitive and selective
Among various photoinduced processes that are commonly
involved in the signaling or response phenomena of
luminescence based chemosensors, the resonance energy
transfer (RET)12 based process is preferred for designing an
appropriate probe molecule over single dye-based probe
Received: September 23, 2011
Published: January 11, 2012
© 2012 American Chemical Society
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(100%) (M++H+), calc. for C34H30N4O4 is 558.6. Elemental Analysis
data: Calc. C, 73.10; H, 5.41; N, 10.03; Expt. C, 73.3; H, 5.39; N, 9.96.
Synthesis of Intermediate Compound A. A 200 mg portion
(0.44 mmol) of rhodamine B hydrazide and 121.5 mg of 4-bromo-1,8-
naphthalic anhydride (0.44 mmol) were taken in 10 mL of glacial
acetic acid to make a suspension. The suspension was heated to 110
°C for 24 h. Then it was cooled to room temperature and added to 40
mL of water. Then 1 M NaOH solution was added dropwise for
neutralization to pH 8. A violet colored precipitate appeared which
was filtered under vacuum in a G-3 crucible. Then the product was
dried in vacuum desiccator. Isolated yield of the compound A (yield
was calculated based on the starting compounds) was 95% (297 mg,
0.42 mmol). 1H NMR (500 MHz, CDCl3, Si(CH3)4, J (Hz), δ
(ppm)): 8.53 (1H, d, J = 8.5, H11), 8.3 (1H, d, J = 7, H12), 8.07 (1H, d,
J = 7, H14), 8.03 (1H, d, J = 7.5, H9), 7.94 (1H, d, J = 8, H10), 7.76−
7.67 (2H, m, H7,8), 7.65 (1H, t, H13), 7.31 (1H, d, J = 7.5, H6), 6.68
(2H, d, J = 9, H5), 6.38 (2H, d, J = 8, H4), 6.07 (2H, br, H3), 3.32−
3.27 (8H, m, H2), 1.09 (12H, t, H1). ESI-MS (+ve mode): m/z; 717.77
(7%) (M++H+), calc. for C40H35BrN4O4 is 716.2. Elemental Analysis
data: Calc. C, 67.13; H, 4.93; N, 7.83; Expt. C, 67.3; H, 4.97; N, 7.8.
Synthesis of L2. A 200 mg portion (0.28 mmol) of compound A
was added to 10 mL of (excess) pyrrolidine and heated to reflux under
inert atmosphere for 18 h with continuous stirring. The reaction
mixture was cooled to room temperature and added to ice-cold water.
A yellow colored precipitate appeared, which was filtered through a G-
3 crucible. The product was washed several times with cold water and
then dried under vacuum desiccator. Isolated yield of the compound
L2 (yield was calculated based on the starting compounds) was 92%
molecules, as the RET based process is independent of the
concentration of a single fluorescent dye and one can quantify
the analyte concentration by using the ratio of intensities of the
well resolved fluorescence peaks with reasonable intensities at
two different wavelengths for analyte-free and analyte bound
probe.13 However, despite many advantages, examples of RET
based turn-on fluorogenic sensors for Hg2+ and more specifically
for Cr3+ in aqueous solution are not common in the literature.14
Earlier reports on Cr3+ sensors, more than one metal ion (e.g.,
15
either Cu2+, Zn2+, Cd2+ and Al3+ or Ni2+ and Cd2+16) interfere
with the detection processes. Keeping this in mind we have
developed a new RET-based rhodamine derivative that is
capable of reporting the Cr3+ binding process through a RET-
based turn-on fluorescence response, while Hg2+ ion only
interferes with the detection process. To understand the
binding process and response phenomena well, we have also
synthesized another naphthalimide derivative (L3) as a control.
The change in spirocycle to open-ring form of the rhodamine
fragment in L1 and L2 results in the remarkable enhancement of
absorption or emission intensities, and these offer us the
possibility of studying the Cr3+ or Hg2+ recognition process
through the switch-on optical responsea criterion that is
important for developing an in-field detection reagent.
EXPERIMENTAL SECTION
■
1
(181.5 mg, 0.26 mmol). H NMR (500 MHz, CD2Cl2, Si(CH3)4, J
Rhodamine 6G, Rhodamine B, phthalic anhydride, 4-bromo-1,8-
naphthalic anhydride, hydrazine hydrate, pyrrolidine, Hg(ClO4)2,
Cu(ClO4)2, Zn(ClO4)2, Ni(ClO4)2, Fe(ClO4)2, Pb(ClO4)2, Cd-
(ClO4)2, Cr(ClO4)3, Ca(ClO4)2, Co(ClO4)2, NaClO4, KClO4, Mg-
(ClO4)2, CsClO4, Ba(ClO4)2, Sr(ClO4)2, AgClO4 were purchased
from Sigma-Aldrich (U.S.A.). All the other reagents used were
procured from S. D. fine chemicals, India. Acetonitrile, Methanol (AR;
Merck, India), Ethanol (Spectrosol; Spectrochem, India) was used as a
solvent. HPLC grade water (Merck, India) was used for experiments
and spectral studies. ESI-MS measurements were carried out on
Waters QTof-Micro instrument. Microanalysis (C, H, N) was
performed using a Perkin-Elmer 4100 elemental analyzer. FTIR
spectra were recorded as KBr pellets using a Perkin-Elmer Spectra GX
2000 spectrometer. 1H and 13C NMR spectra were recorded on
Bruker 500 MHz FT NMR (model: Avance-DPX 500) and Bruker
200 MHz FT NMR (model: Avance-DPX 200). Electronic spectra
were recorded with a Shimadzu UV-3101 PC/Varian Cary 500 Scan
UV−vis−NIR Spectrophotometer. Fluorescence spectra recorded with
a HORIBA JOBIN YVON spectrophotometer. Time resolved
emission studies measurements were carried out with an Edinburgh
OB920 spectrofluorimeter that works on Time Correlated Single
Photon Counting (TCSPC) technique.
(Hz), δ (ppm)): 8.54 (1H, d, J = 8.5, H28), 8.17 (1H, d, J = 7.5, H24),
8.05 (1H, d, J = 9, H26), 8.01 (1H, d, J = 7.5, H23) 7.69−7.61 (2H, m,
H21, 22), 7.41 (1H, t, H27), 7.26 (1H, d, J = 7.5, H25), 6.71−6.69 (3H,
m, J = 9, 2H19, 1H20), 6.37 (2H, d, J = 9, H18), 6.11(2H, dd, J 1= 7.3, J
1= 2, H17), 3.72 (4H, br, H29), 3.32−3.29 (8H, m, H16), 2.07−2.05
(4H, m, H30), 1.1(12H, t, H15) 13C NMR (DMSO-d6, 125 MHz,
Si(CH3)4, δ (ppm)): 164.0, 161.6, 160.34, 154.34, 152.97, 148.78,
133.56, 131.06, 130.39, 129.55, 125.11, 123.55, 122.17, 121.57, 108.8,
108.23, 105.52, 97.34, 79.53, 67.41, 53.35, 44.22, 25.99, 12.7. ESI-MS
(+ve mode): m/z; 706.09 (100%) (M++H+), calc. for C44H43N5O4 is
705.3. Elemental Analysis data: Calc. C, 74.87; H, 6.14; N, 9.92; Expt.
C, 74.7; H, 6.2; N, 10.0.
Synthesis of the Intermediate B. A 750 mg portion (2.71
mmol) of 4-bromo naphthalic anhydride was added to 50 mL of
ethanol and heated to reflux. To this suspension 132 μL (2.71 mmol)
of hydrazine hydrate was added dropwise and heated to reflux for
overnight; a yellow colored precipitate appeared. The reaction mixture
was cooled to room temperature and filtered through G-4 crucible.
The residue was washed several times with ethanol and then with
slight ether. Isolated yield of the intermediate B (yield was calculated
based on the starting compounds) was 91% (717 mg, 2.46 mmol). 1H
NMR (200 MHz, DMSO-d6, Si(CH3)4, J (Hz), δ (ppm)): 8.55−8.47
(2H, m, H33,35), 8.39 (1H, d, J = 7.8, H32), 8.17 (1H, d, J = 7.8, H31),
7.96 (1H, t, H34), 5.78 (2H, s, H36). ESI-MS (+ve mode): m/z; 315.1
(100%) (M++Na+), calc. for C12H7BrN2O2 is 292. Elemental Analysis
data: Calc. C, 49.51; H, 2.42; N, 9.62; Expt. C, 49.3; H, 2.4; N, 9.7.
Synthesis of Intermediate C. Compound C was synthesized
following a procedure similar to that for synthesis of L2 using the
intermediate B instead of the intermediate A. Isolated yield of the
intermediate B (yield was calculated based on the starting
Synthetic Methodology. Rhodamine 6G hydrazide and Rhod-
amine B hydrazide were prepared following a literature method.17
Synthesis of L1. A 300 mg portion (0.7 mmol) of rhodamine-6G
hydrazide was dissolved in 50 mL of dry acetonitrile by heating, with
continuous stirring under N2 atmosphere. Then 150 mg (0.77 mmol)
of phthalic anhydride was added to the solution and then heated to
reflux for 48 h. After that the reaction mixture was cooled to room
temperature and evaporated to dryness. Then to the reaction mixture
30 mL of ethanol was added and stirred for about 3 h; a precipitate
appeared which was filtered through G-4 crucible. The residue was
washed several times with ethanol. Isolated yield of the compound L1
(yield was calculated based on the starting compounds) was 55% (215
mg, 0.38 mmol). 1H NMR (500 MHz, DMSO-d6, Si(CH3)4, J (Hz), δ
(ppm)): 7.97 (1H, d, J = 7.5, Hj), 7.85 (4H, m, Hk,l,m,n), 7.75 (1H, t,
Hi), 7.68 (1H, t, Hh), 7.21 (1H, d, J = 7.5, Hg), 6.35 (2H, s, Hf), 6.1
(2H, s, Hd), 5.07 (2H, s, Hc), 3.07−3.05 (4H, m, Hb), 1.91 (6He, s,
He), 1.18 (6H, t, Ha). 13C NMR (DMSO-d6, 125 MHz, Si(CH3)4, δ
(ppm)): 163.75, 163.54, 151.77, 150.98, 147.88, 135.59, 134.31,
129.82, 129.09, 128.74, 128.04, 124.55, 123.99, 123.06, 117.47, 103.4,
94.96, 66.74, 37.43, 17.15, 14.09. ESI-MS (+ve mode): m/z; 559.24
1
compounds) was 89% (430 mg, 1.6 mmol). H NMR (200 MHz,
DMSO-d6, Si(CH3)4, J (Hz), δ (ppm)): 8.75 (1H, J = 8.4, d, H37), 8.5
(1H, d, J = 7.2, H39), 8.3 (1H, d, J = 8.8, H41), 7.59 (1H, t, H38), 6.87
(1H, d, J = 8.8, H40) 3.83 (4H, t, H42), 2.12 (4H, t, H43). ESI-MS (+ve
mode): m/z; 322.46 (100%) (M++H2O+Na+), calc. for C16H15N3O2 is
281.31. Elemental Analysis data: Calc. C, 68.31; H, 5.37; N, 14.94;
Expt. C, 68.1; H, 5.3; N, 14.9.
Synthesis of Compound L3. Compound L3 was synthesized by
using the procedure as L1; while the intermediate C was used instead
of rhodamine-6G hydrazide for the reaction. Isolated yield for L3
(yield was calculated based on the starting compounds) was 87.5%
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Scheme 1. (a−c) Methodologies Adopted for the Synthesis of Necessary Intermediates (A, B, and C) and Final Receptor
Molecules Like L1, L2, and L3, and (d) X-ray Single Crystal Structure for L1 and L2
1
(512 mg, 1.24 mmol). H NMR (200 MHz, DMSO-d6, Si(CH3)4, J
(Hz), δ (ppm)): 8.65 (1H, J = 8.6, d, H49), 8.58 (1H, d, J = 7.2, H51),
8.41(1H, d, J = 8.4, H53), 7.97 (2H, d, J = 3.4, H45, 48), 7.83 (2H, d, J =
3.4, H46, 47), 7.55 (1H, t, H50), 6.8 (1H, d, J = 8.8, H52), 3.81 (4H, br,
H54), 2.12 (4H, br, H55). ESI-MS (+ve mode): m/z; 434.4 (100%)
(M+ + Na+) calc. for C24H17N3O4 = 411.4. Elemental Analysis data:
Calc. C, 70.07; H, 4.16; N, 10.21; Expt. C, 70.3; H, 4.2; N, 10.2.
Biological Study. The efficacy of L2 as a sensor of Hg2+ and Cr3+
ions was studied in living human epidermoid A431 cells, as epithelia
are the tissues mainly exposed to a risk of damage from an excess of
Cr3+ and Hg2+.3a The organic anion transporters are involved in the
uptake of chromium18 and mercury,19 and they are nearly ubiquitously
expressed in barrier epithelia.19 Moreover, the overexpression of EGF
receptors enhances the uptake of heavy metals upregulating the metal
transporter proteins.19 Sumalekshmy et al.20 report that A431 cells
present a rapid internalization of metal-ion indicators in living cells and
are useful for in vitro testing. Confocal imaging experiments and flow
cytometry were used to detect the emission of fluorescence in cells
treated with L2 sensor after the exposure to Cr3+ or Hg2+. In this work,
A431 cells were obtained from EACC (Porton Down, Wilts, U.K.) and
were cultured in Dulbecco’s modified Eagle’s medium (DMEM,
Gibco, Carlsbad, CA), supplemented with 10% fetal bovine serum
(FBS, Gibco) and 1% antibiotic solution (Gibco).
at room temperature, atmospheric atmosphere, for 5 min, and in dark.
Data were acquired using a Leica TCS SP5 microscope (Leica
Microsystems, Mannheim, Germany) with excitation light at 488 nm
and filter cube N2.1 (bandpass 515−560 nm). As the activity of the L2
sensor was defined only by detecting an increased red fluorescence in
samples with respect to the controls, the microscopic observation was
performed without using any incubation and monitoring device
suitable for a time-lapse study.
Cytometry. Treated samples and negative controls were washed
twice with PBS and then mechanically reduced to a single cell
suspension. All samples were kept in the incubator at 37 °C until
analysis and then loaded on Moflo High speed cytometer (Beckman
Coulter, Brea, CA, U.S.A.). The excitation of samples was performed
at 488 argon laser, and the emission light was detected at 580 30
nm. For the analysis, 2 × 104 cells were acquired by Summit 4.3
software, and results were expressed as mean fluorescence intensity
(MFI)
SD of samples with respect to controls. The statistical
analysis was performed using t-student test.
Calculations for the Binding Constants Using Spectropho-
tometric Titration Data. The following equation was used for the
nonlinear least-squares analysis21 to determine the association
constant, as well as the binding stoichiometry for the formation of
the respective complex, [Hg2+·L] and [Cr3+·L] (where L = L1 and L2),
One day before testing, cells were trypsinized and seeded (5 × 103
n
n
cells/cm2) on tissue culture plates (Falcon BD Biosciences, San Jose,
́
A = (A + A K C )/(1 + K C
)
(1)
0
lim n M
n M
CA) with glass or polystyrene surface for confocal and cytometrical
analysis, respectively. The cells were incubated with 40 μM
Hg(NO3)2·H2O in DMEM or 50 μM Cr(NO3)3·9H2O for 1 h at 37
°C, washed with PBS to remove the remaining mercury or chromium
ions, and then incubated with 10 μM L2 in PBS for 30 min at 37 °C. In
parallel, populations incubated only with Hg(NO3)2·H2O or Cr-
(NO3)3·9H2O solutions for 1 h or with only L2 solution for 30 min
were prepared as controls. To have statistical significance, each sample
was prepared in triplicate.
where A0, A, and Alim are the respective absorbance of free L, L present
in the form of [Hg2+·L]/[Cr3+·L] in the complex, and L in presence of
excess amounts of Hg2+ or Cr3+ ions where the absorbance reaches a
limiting value. CM is the metal ion concentration, Kn is the binding
constant, and n is the stoichiometry of the complex formed between
the ligand and metal ion.
Calculations for the Binding Constants Using Emission
Titration Data. The following equation was used for the nonlinear
least-squares analysis21 to determine the association constant, as well
as the binding stoichiometry for the formation of the respective
complex, [Hg2+·L] and [Cr3+·L] (where L = L1 and L2),
Confocal Microscopy. After treatment, all samples were kept in
the incubator at 37 °C, 95% humidity, 0.5% CO2 until the analysis.
Confocal imaging experiments of living cells were performed in PBS,
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Figure 1. (A) Absorbance spectra of L1 (5 μM) with varying [Cr3+] (0−1.34 mM) in CH3CN-aq.HEPES buffer (1 mM, pH 7.2; 1:1, v/v). Inset:
Jobs plot between L1 and Cr3+. (B) Absorbance spectra of L1 (5.84 μM) with varying [Hg2+] (0−0.51 mM) in CH3CN-aq.HEPES buffer (1 mM, pH
7.2; 1:1, v/v). Inset: Jobs plot between L1 and Hg2+. (C) Image of change of color of L1 in presence of 50 mol equivalent of Hg2+ and Cr3+.
Figure 2. (A) Emission spectra of L1 (5 μM) with varying [Cr3+] (0−1.34 mM) in CH3CN-aq.HEPES buffer (1 mM, pH 7.2; 1:1, v/v). Inset: plot
of emission intensity vs [Cr3+]. (B) Emission spectra of L1 (5 μM) with varying [Hg2+] (0−0.43 mM) in CH3CN-aq.HEPES buffer (1 mM, pH 7.2;
1:1, v/v). Inset: plot of emission intensity vs [Hg2+]. (c) Image of change of fluorescence of L1 in presence of 50 mol equiv of Hg2+ and Cr3+.
Parr (B3LYP)22 exchange functional and mixed basis set combinations
that were given as general basis set input to Gaussian 09 (Gen
keyword). The LANL2DZ basis sets were used for Hg2+, whereas 6-
31G* were used for other atoms.23 All calculations were performed
using Gaussian 09 program.24
n
n
F = (F + F K C )/(1 + K C
)
(2)
0
lim n M
n M
where, F0, F, and Flim are the respective emission intensity of free L, L
present in the form of [Hg2+·L]/[Cr3+·L] in the complex, and L in
presence of excess amounts of Hg2+ or Cr3+ ions where the emission
intensity reaches a limiting value. CM is the metal ion concentration, Kn
is the binding constant, and n is the stoichiometry of the complex
formed between the ligand and metal ion.
RESULTS AND DISCUSSION
■
Evaluation of Different Parameters for FRET Process. The
Forster distance R was calculated using the expression shown in eq 3,
In the present study, we have carefully chosen rhodamine and
1,8-naphthalimide derivatives as the two fluorophores in
synthesizing the receptor molecule L2; as absorption spectra
of the Cr3+/Hg2+ ion bound open spirocycle form of the
rhodamine derivative develops a significant spectral overlap
with the emission spectra of the N,N-dialkylamine-naphthali-
mide derivative and offers the possibility of a RET process. For
free L2 FRET in its spirolactam form is otherwise suppressed,
and only the yellow emission of the 1,8-naphthalimide
derivative is observed upon excitation at 455 nm, the λmax
(absorption) for analogous derivative L3 (Scheme 1). Binding
of L2 to Cr3+/Hg2+ induces a RET process and results in an
intense rhodamine-based red emission, which was confirmed by
comparing results with analogous rhodamine derivative L1.
Details about the synthesis of L1, L2, and L3 are discussed above
and their characterization data are presented in the Supporting
Information, Figures S1−S8. Proposed molecular structures of
L1 and L2 were also confirmed by single crystal X-ray analysis
(Scheme 1 and Supporting Information, Figures S9−S10).
UV−vis spectra recorded for L1 (CH3CN-1.0 mM aq.HEPES
buffer, pH = 7.2; 1:1, v/v) shows an absorption maximum at
300 nm, which was predominantly due to intraligand π−π*
charge transfer (CT) transition. The binding ability of L1 was
checked with perchlorate salts of Hg2+, Cr3+, Cu2+, Pb2+, Zn2+,
Co2+, Ni2+, Fe2+, Ca2+, Cd2+, Mg2+, K+, Na+, Sr2+, Cs+, Ba2+, and
Ag+ in CH3CN-aq.HEPES buffer (1 mM, pH 7.2; 1:1, v/v). A
significant change in electronic spectral pattern was observed
only with Hg2+ and Cr3+, among all these metal ions used
(Supporting Information, Figure S11). A new absorption band
around 531 nm appeared with detectable change in solution
̈
0
−4
2 1/6
R = 0.211[(J)Q(n )(κ )]
(3)
0
where, n is the refractive index of the medium in between donor and
acceptor and was taken approximately to be equal to 1.4. κ2 is the
dipole orientation factor. Depending upon the relative orientation of
donor and acceptor, the value ranges from 0−4, and it is often
assumed to be 2/3. Q is the fluorescence quantum yield of the donor
in the absence of acceptor. J is the spectral overlap integral between
the emission spectrum of the donor and the absorption spectrum of
the acceptor and is shown in the following eq 4,
4
J = f (λ) ε(λ)λ dλ
∫
D
(4)
where f D(λ) is the normalized emission of the donor and ε(λ) is the
molar absorption coefficient (M−1 cm−1) of the donor.
Energy transfer efficiency (ΦET) was evaluated using the expression
shown in eq 5,
Φ
= 1 − (F′ /F )
D D
where F′D and FD denote the donor fluorescence intensity with and
without an acceptor, respectively.
(5)
ET
Energy transfer rate constant (KET) was calculated using eq 6,
Φ
= K /(1/τ + K )
ET ET
where τD denotes the fluorescence lifetime of the donor fragment in
(6)
ET
D
the absence of acceptor.
COMPUTATIONAL METHODS
■
All geometries for L1 and Hg2+·L1 were optimized by density
functional theory (DFT) calculations using the Becke-3-Lee−Yang−
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color from colorless to bright pink (Figure 1). The binding
A binding stoichiometry of 1:1 was confirmed from the Job’s
plot for binding of Hg2+ or Cr3+ to L1 (inset of Figure 1). This
1:1 stoichiometry was further confirmed from results the ESI-
MS studies (Supporting Information, Figure S17). FTIR
spectra of L1 revealed that the peak at 1724 cm−1, the
characteristic stretching frequency for the COAmide bond of the
rhodamine unit, shifted to 1610 cm−1 and 1611 cm−1 on
coordination to the Hg2+ and Cr3+ ions, respectively, in
presence of 1.5 equiv of the respective metal ion (Supporting
Information, Figure S18). Such shift in the stretching frequency
of COAmide bond of the rhodamine unit on binding to a metal
ion is reported earlier.26 Simultaneously, the stretching
frequency band at 1744 cm−1, corresponding to the carbonyl
group of the phthalimide moiety, was shifted to 1646 and 1641
cm−1 in presence of 1.5 equiv of Hg2+ and Cr3+ ions,
respectively. These appreciable shifts support the coordination
of the O>CO of the rhodamine and phthalimide units to the
Hg2+ or Cr3+ center and the possible binding mode for L1 to
Hg2+ or Cr3+ ion is shown in Scheme 2. The generation of the
affinity for these two respective metal ions towards L1 was
2+
evaluated from spectrophotometric titration (KHg = 3.13
0.08 × 105 M−1 and KCr = 1.38 0.04 × 105 M−1 at 25 °C);
3+
while 1:1 binding stoichiometry was evaluated using nonlinear
regression analysis (Figure 1, Supporting Information, Figure
S12).21 Subsequently, an intense emission band appeared at
max
λems = 557 nm on excitation at λext = 500 nm, which was
earlier absent for pure L1 (Figure 2). Thus a switch-on
luminescence response was observed at 557 nm for Cr3+and
Hg2+; while analogous experiment with other cations did not
show any such enhancement (Supporting Information, Figure
S11). Switch on responses for the absorption spectral band at
531 nm and the luminescence band at ∼557 nm on binding to
Hg2+ or Cr3+ suggest opening of the spirolactam ring in L1 on
metal ion coordination. Association constant values, calculated
from emission titration (Figure 2), for binding of L1 with Hg2+
and Cr3+ were found to be 3.07 0.3 × 105 M−1 (KHg2+) and
1.28 0.08 × 105 M−1 (KCr3+), respectively. These values are
very close to the respective binding constant for Hg2+ and Cr3+,
obtained from electronic spectral titrations. Further, spectral
studies revealed that a lower detection limit for Hg2+ and Cr3+
were 0.35 and 0.14 ppb (Supporting Information, Figures S13−
S14) (for signal-to-noise ratio of 3:1), respectively, where the
U.S. EPA limit for Hg2+ and Cr3+ (Cr3+ and Cr6+) are 2 and 100
ppb respectively.25
Scheme 2. Schematic Presentation Showing the Possible
Metal Ion Binding Mode of L1
Further L1 could be used as selective chemosensor for Cr3+,
while experiments were performed in presence of excess KI. On
addition of the aqueous solution of KI to the pink colored
solution of Hg2+·L1, spectral bands (absorption band with λMax
acyclic rhodamine form of L1, as shown in Scheme 2, was also
confirmed by the electronic and fluorescence spectral studies
(vide supra). However, the most convincing proof in favor of
the ring-opening of the spirolactam form of L1 on coordination
to Hg2+ ion was established from 13C NMR studies. Results of
the 13C NMR studies revealed that the signal at 66.74 ppm for
tertiary carbon of the spirolactam ring of L1 disappeared upon
addition of Hg2+ (Supporting Information, Figure S19).
at 532 nm and emission band with λMax at ∼557 nm for λExt
=
500 nm), as well as the color of the solution disappeared
(Figure 3). Again on addition of Cr3+ to this solution mixture
Spectral studies using a freshly prepared solution of
compound L3 in identical mixed solvent medium (CH3CN-
1.0 mM aq.HEPES buffer, pH 7.2; 1:1, v/v) revealed that the
emission spectra for L3 (N,N-dialkylamine-naphthalimide
derivative) had a significant overlap with absorption spectra
of rhodamine B (Figure 4),14f and this led us to develop a new
Figure 3. Absorption spectra of (A) L1 (17.9 μM), (B) L1 in presence
of Hg2+ (0.2 mM), (C) L1 in presence of Hg2+ (0.2 mM) and KI (0.5
mM), (D) L1 in presence of Hg2+ (0.2 mM), KI (0.5 mM) and Cr3+
(0.36 mM) in CH3CN-aq.HEPES buffer (0.01 M, pH 7.2; 1:1, v/v).
having KI, absorbance band at 530 nm and emission band at
557 nm reappeared (Figure 3, Supporting Information, Figure
S15). Simultaneously, the pink color of the solution was also
restored. Preferential binding of the iodide ion to the Hg2+ ion
led to the formation of HgI2 and the regeneration of the cyclic
lactam form of the reagent, a process which is well documented
for demonstrating the reversible binding of rhodamine
derivatives to Hg2+.10m However, the absorption and emission
spectral bands, as well the solution color, were restored on
coordination of the Cr3+ to L1. Further experiments reveal that
the absorbance spectra for Cr3+·L1 and its solution color
remained unchanged on addition of excess of iodide ion in the
form of KI (Supporting Information, Figure S16). Thus, in
presence of excess of KI, reagent L1 could be used for selective
recognition of Cr3+ from all other metal ions.
Figure 4. Overlap (shown with cyan shade) between emission and
absorption spectra of the donor and acceptor, respectively.
receptor, L2 with an aim for developing a reagent for
ratiometric sensing of Hg2+/Cr3+.
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The spectral properties of L2 in CH3CN-1.0 mM aq.HEPES
buffer (1 mM, pH 7.2; 1:1, v/v) were recorded. L2 displayed
absorption bands at 455 nm and on excitation at this
wavelength a yellow fluorescence centered at 533 nm was
observed; while L3 showed an absorption band at 462 nm
(Supporting Information, Figure S20). Thus, the absorption
spectrum for L2 was found to be the linear combination of
spectra for L1 and L3 with little blue shift for the L3-based
absorption band (Supporting Information, Figure S20). Thus
the emission at 533 nm, following excitation at 455 nm, is
attributed to an intramolecular charge transfer (ICT) process
associated with 1,8-naphthalimide chromophore;27 while the
rhodamine moiety retains its spirolactam form. In presence of
Cr3+/Hg2+, the switch on response at 561 nm for electronic
spectra and at 583 nm for luminescence spectra accounts for a
visually detectable change in solution color and luminescence
because of the opening of the spirolactam ring and generation
of the delocalized xanthene moiety.10h It will not be
unreasonable to presume that the similar binding motif for
Hg2+·L2 or Cr3+·L2 formation, as it was proposed for the other
reagent L1 with analogous structure, would prevail for the
reagent L2. Results of the different spectral studies support this
presumption. FTIR studies revealed that the characteristic
stretching frequency for the COAmide of the rhodamine moiety
at 1693 cm−1 was shifted to 1588 and 1589 cm−1 in presence of
1.5 equiv of Hg2+ and Cr3+, respectively. Whereas the stretching
frequency band at 1729 cm−1 for the CONaphthalimide was shifted
to 1644 and 1643 cm−1 in presence of 1.5 equiv of Hg2+ and
Cr3+ (Supporting Information, Figure S18), respectively.
Significant shifts in the stretching frequencies of both CO
groups of naphthalimide and rhodamine moieties of L2 support
the proposed binding mode similar to that of L1. The
coordination site of L2 for chelation with Hg2+ was also
NMR signal at 66.85 ppm for tertiary carbon of the spirolactam
ring of L2 upon addition of Hg2+ confirmed the opening of the
spirolactam ring and coordination through O>CO of the
rhodamine moiety (Supporting Information, Figure S21).
Chelation of Hg2+ through two O>CO atoms of L2, as discussed
above, is expected to deplete the electron density in the
aromatic rings of the xanthene and 1,8-naphthalimide moieties,
and this was reflected in the appreciable downfield shifts of the
associated aromatic protons (Figure 5). These shifts were more
pronounced for H17, H18, and H19 protons (Δδ = 1.1, 0.8, and
0.6 ppm, respectively), which revealed opening of the
spirolactam ring on coordination to Hg2+ with associated
charge transfer in the aromatic rings of the xanthene moiety.
Downfield shift of H25, H26, H27, and H28 (Δδ = 0.13, 0.05,
0.12, and 0.11 ppm, respectively) of the naphthalimide moiety
indicates the involvement of its carbonyl oxygen in Hg2+
binding.
Spectral (Electronic and emission) responses for L2 toward
Hg2+ and Cr3+ in presence of excess of KI were similar as it was
observed for L1 (Supporting Information, Figure S22), which
confirmed that this reagent also could be used for delineating
Cr3+ from Hg2+ in mixed solvent medium (CH3CN-1.0 mM
aq.HEPES buffer,1:1 v/v) pH 7.2).
The binding of Cr3+/Hg2+ ion induces opening of the
spirolactam ring in L2 with an associated switch on UV−vis
spectral response in the range 515−585 nm, which has a
significant spectral overlap with the emission spectrum of the
N,N-dialkylamine-naphthalimide fragment and makes non-
radiative transfer of excitation energy between donor
naphthalimide to acceptor xanthene moiety feasible and
initiates an intramolecular FRET process. Thus, with increasing
[Cr3+] or [Hg2+], the [Cr3+·L2] or [Hg2+·L2] increases with
associated increase in the absorbance intensity at 561 nm
1
confirmed by H NMR and 13C NMR spectra (Figure 5 and
(Figure 6) and emission at 583 nm (for λExt = 561 nm).
3+
Supporting Information, Figure S21). Disappearance of 13C
Respective binding constant values for two metal ions (KCr
=
(1.22 0.07) × 105 and KHg = (1.01 0.05) × 105 M−1 at 25
°C) were evaluated from the absorbance titration using λMon
2+
=
560 nm. Binding stoichiometry for the respective complex was
evaluated using nonlinear regression analysis and was found to
be for 1:1 for both cases.21 On excitation at 455 nm, a steady
decrease in emission intensity at 533 nm (characteristic for the
naphthalimide moiety) was observed along with a concomitant
increase in the intensity of the new fluorescence band at 583
nm (characteristic for spirocycle opening moiety) (Figure 7). A
well-defined iso-emissive point appeared at 548 nm.
This also resulted in a visually detectable change in solution
fluorescence (Figure 7). The binding constant values for two
metal ions (KCr = (1.12 0.01) × 105 and KHg = (1.09
0.02) × 105 M−1 at 25 °C) were evaluated from the emission
titrations using λMon = 583 nm, and these values are comparable
with those obtained from absorption titrations. The ratio of
emission intensities for rhodamine moiety to 1,8-naphthalimide
fragment at 583 and 533 nm (I583/I533), respectively, varied
from 0.47 to 11.72, which correspond to a 25.1 fold
enhancement in emission intensity in presence of Hg2+; while
this ratio for Cr3+ varied from 0.47 to 10.67 and resulted a
22.75 fold enhancement (Figure 7 and Supporting Information,
Figure S23) in emission intensity.
3+
2+
For both metal ions, 1:1 complex formation was established
from Job’s plot and thus, corroborated our earlier observations
on electronic spectral titration, as well from ESI-MS spectra
(Supporting Information, Figure S24−S25). The noninterfering
absorption bands with significant wavelength shift and the
1
Figure 5. Partial H NMR spectra for L2 (1 mM) in presence of
varying [Hg2+] (0−0.0057 M) in CDCl3:CD3OD = 1:1(v/v).
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Figure 6. Electronic spectra of (A) L2 (6.7 μM) with varying [Cr3+] (0−2.5 mM), inset: least square plot of absorbance vs [Cr3+]; (B) of L2 (6.7
μM) with varying [Hg2+] (0−3.2 mM), inset: least square plot of absorbance vs [Hg2+]. All studies were carried out in CH3CN-aq.HEPES buffer (1
mM, pH 7.2; 1:1, v/v) medium.
The proposed binding mode of the reagent L1 to Hg2+ was
also investigated with density functional theory (DFT)
calculations. All geometries were fully optimized using the
B3LYP method and general basis set (Gen keyword).22 The
carbon, nitrogen, oxygen, and hydrogen atoms were calculated
with 6-31G* basis set,23 whereas Hg2+ was calculated with the
LANL2DZ basis set. The B3LYP optimized geometry of L1 is
similar to the structure obtained from crystal study (Scheme 1d
and Figure 8). Since ESI-Mass spectra has suggested the
coordination of two water molecules to Hg2+ complexed with
Figure 7. Fluorescence spectra of (A) L2 (6.96 μM) with varying
[Cr3+] (0−0.92 mM), and (B) of L2 (7.4 μM) with varying [Hg2+]
L1 (Supporting Information, Figure S17), therefore, calcu-
(0−0.1 mM), inset: image of change of fluorescence of L2 in presence
lations have been performed with two coordinated water
of 50 mol equiv of Hg2+ and Cr3+. All studies were carried out in
molecules. The optimized geometry suggests a distorted
CH3CN-aq.HEPES buffer (1 mM, pH 7.2; 1:1, v/v) medium.
tetrahedral geometry around the Hg2+ ion (Figure 8). Studies
revealed that [Hg2+·L1]Spirolactam is 277.3 kcal/mol more stable
than separated reactants (L1 + 2H2O + Hg2+). In this
complexed structure, Hg2+ binds with the spirolactam and
phthalimide carbonyl oxygens of L1. The additional calculation
was performed for optimizing the geometry for the Hg2+·L1
having the xanthene moiety. The calculated complex Hg2+·L1
was found to be more stable by 19.3 kcal/mol than that of
[Hg2+·L1]Spirolactam. Further, optimized structure for Hg2+·L1
reveals that Hg2+-OII distance (2.175 Å) is shorter than the
Hg2+-OI distance (2.331 Å). This signifies a stronger binding
through the COII of the xanthene moiety than the COI moiety
of the phthalimide fragment. Thus, the calculated results
corroborate the proposed binding mode (Scheme 2) for
Hg2+·L1, and it will be reasonable to presume that similar
binding mode will prevail for reagent L2.
possibility to probe the binding of Cr3+/Hg2+ in mixed aqueous
organic medium at two emission maxima make the receptor L2
a unique ratiometric probe.
The singlet−singlet excitation energy-transfer efficiency
(ΦET) and rate constant for the energy-transfer process (kET)
between donor and acceptor were evaluated from steady state
and time-resolved fluorescence data. The value for ΦET and kET
was found to be 50% and 2.33 × 108 s−1, respectively; while the
Forster critical distance (R ) was calculated as 64.5 Å.
̈
0
Competitive binding of L1 and L2 to Cr3+/Hg2+ were
established in presence of 10 mol equiv of other metal ions like
Cu2+, Pb2+, Zn2+, Co2+, Ni2+, Fe2+, Ca2+, Cd2+, Mg2+, K+, Na+,
Sr2+, Cs+, Ba2+, and Ag+ is discussed in Supporting Information
(Supporting Information, Figure S26). Reversible binding of
Cr3+/Hg2+ to L1 and L2 was also established through spectral
studies in presence of 3 mol equiv of Na2EDTA (Supporting
Information, Figure S27).
Owing to its favorable properties, L2 sensor should be suited
for fluorescence imaging in living cells. We evaluated the
applicability of L2 as a probe of Hg2+ and Cr3+ by confocal
Figure 8. B3LYP optimized geometries and important bond distances (Å) of rhodamine derivative L1 and its complexes with Hg2+ ion. Binding
energy (EB) of complexes is also given. For clarity hydrogens are omitted (yellow = C; red = O; blue = N; light gray = Hg).
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microscope (Leica TCS SP5) and cytometer (Moflo High
Speed cytometer) on A431 cells treated with 50 μM Cr(NO3)3
or 40 μM Hg(NO3)2 for 1 h at 37 °C, and then with 10 μM L2
solution for 30 min at 37 °C.
As shown in Figure 9, a significant fluorescence emission
from the intracellular region was observed (Figure 9 B, C),
suggesting a subcellular distribution of Cr3+ and Hg2+ in the
cytoplasm. In contrast, when the cells were treated with only 10
μM L2, 50 μM Cr(NO3)3 or 40 μM Hg(NO3)2 a negligible
Model compounds L1 and L3 were synthesized to confirm
the binding mode and the unambiguous assignment of the
response processes. Probe molecule, like L2, which works in
physiological conditions is preferred for use either as a
colorimetric staining agent or as a reagent for imaging studies
with biological and environmental samples. Moreover, when
used on epithelial cells like A431, the reagent L2 could detect
successfully the cellular uptake of Cr3+ or Hg2+ ion.
ASSOCIATED CONTENT
* Supporting Information
■
S
Characterization, spectroscopic data and X-ray crystallographic
data in CIF format of L1 (CCDC 829120) and L2 (CCDC
829121). Cartesian coordinates of all optimized geometries.
This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Phone: +91 278 2567760 [672] (A.D.). Fax: +91 278
2656762 (A.D.). E-mail: amitava@csmcri.org (A.D.).
■
Figure 9. Confocal microscopic images of A431 cells (A) loaded with
10 μM L2, (B) 50 μM Cr3+ and 10 μM L2, (C) 40 μM Hg2+ and 10
μM L2.
intracellular fluorescence was detected (Figure 9A). The above
results indicated that L2 was cell permeable and the marked
enhancement of the intracellular red fluorescence confirmed
the binding of L2 with Cr3+ and Hg2+ within A431 cells (Figure
9). Consistent with morphological studies, the cytometrical
analysis by a Moflo High speed cytometer (Beckman Coulter)
(Figure 10) detected a significant increase of fluorescent cells
induced by L2 in samples treated respectively with Cr3+
ACKNOWLEDGMENTS
■
A.D. acknowledges DST and CSIR for financial support. P.M.
and S.S. acknowledge CSIR, and M.K.K. acknowledge UGC for
research fellowship. R.D.L., P.P.P., and M.T.C. thank Saint
Luca Hospital-ULSS 18 placed in Trecenta (Rovigo)-Italy for
providing laboratory facilities.
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■
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