Naphthalimide appended rhodamine derivative: Through bond energy transfer for sensing of Hg2+ ions

Article abstract of DOI:10.1021/ol2001073

A naphthalimide appended rhodamine based fluorescent chemosensor '1' is synthesized which undergoes through bond energy transfer in the presence of Hg²⁺ ions in mixed aqueous media.

Full text of DOI:10.1021/ol2001073

ORGANIC
LETTERS
2011
Vol. 13, No. 6
1422–1425
Naphthalimide Appended Rhodamine
Derivative: Through Bond Energy
Transfer for Sensing of Hg2þ Ions
Manoj Kumar,*,† Naresh Kumar,† Vandana Bhalla,† Hardev Singh,†
Parduman Raj Sharma,‡ and Tandeep Kaur‡
Department of Chemistry, UGC-Center for Advance Studies-1, Guru Nanak Dev
University, Amritsar, Punjab, India, and Department of Cancer Pharmacology,
Indian Institute of Integrative Medicine, Canal Road, Jammu-180001, India
mksharmaa@yahoo.co.in
Received January 13, 2011
ABSTRACT
A naphthalimide appended rhodamine based fluorescent chemosensor ‘1’ is synthesized which undergoes through bond energy transfer in the
presence of Hg2þ ions in mixed aqueous media.
Mercury is one of the most significant cations among
various heavy and soft cations because of its toxic effects.1
Mercury contamination occurs through a variety of nat-
ural and anthropogenic sources including oceanic and
volcanic emissions, gold mining, and combustion of fossil
fuels.2 The biological targets and toxicity profile of mer-
cury species depend on their chemical composition.3 The
exposure to mercury, even at very low concentration, leads
to digestive, kidney, and especially neurological diseases4
as mercury can easily pass through the biological
membranes. Thus, keeping in view the role played by
mercury in day-to-day life, simple and rapid sensing of
mercury5 in biological and environmental systems is very
important. Fluorescence signaling is one of the first choices
due to its high detection sensitivity and simplicity which
translates molecular recognition into tangible fluorescence
signals.6 In most of the fluorescent sensors the cation
binding involves photophysical changes such as photoin-
duced electron transfer (PET),7 photoinduced charge
transfer (PCT),8 formation of monomer/excimer,9 and
energy transfer,10 and more recently fluorescence reso-
nance energy transfer (FRET) where the excitation energy
† Guru Nanak Dev University.
‡ Indian Institute of Integrative Medicine.
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r
10.1021/ol2001073
Published on Web 02/16/2011
2011 American Chemical Society

from one fluorophore, the donor, is transferred to another
fluorophore, the acceptor, without emission of a photon.11
However, there is a problem in many biochemical experi-
ments which involve irradiation of different fluorescent
labels with a single excitation source. The dye which is to
emit at a longer wavelength absorbs at the excitation source
less effectively and hence results in loss in fluorescence
intensity. This is an important issue in those cases where
detection of low levels of fluorescence is involved. Fluores-
cence resonance energy transfer (FRET) provides a solution
to some extent. However, the number of FRET based
systems is less, as these systems require that donor emission
must overlap with the acceptor absorption.11 On the other
hand, through bond energy transfer (TBET) is theoretically
not subjected to the requirement of spectral overlapbetween
the donor emission and acceptor absorption and is expected
to have large Stokes shifts and emission shifts.12 These
spectral benefits are very important for the use of fluorescent
dyes in chemistry, biology, medicine, and material science.
In TBET systems the donor and acceptor are joined by a
conjugated spacer which prevents them from becoming flat
and conjugated. These types of systems absorb at a wave-
length characteristic of a donor then emit via a receptor.
Recently, Burgees et al.12a,b have developed excellent
(TBET) systems based on rhodamine and fluorescein for
use in biotechnology, but no such systems for fluorogenic
sensing of metal ions have been developed so far.
Suzuki-Miyaura cross coupling of boronic ester 313
with 214 catalyzed by Pd(II) furnished compound 4
(Scheme 1) in 70% yield (Supporting Information pp
Scheme 1. Synthesis of 1
S5-S8, S23-S25). The reaction of compound 4 with
rhodamine acid chloride 5 formed from the reaction of
rhodamine and phosphorus oxychloride gave the desired
compound 1 in 55% yields. The structure of compound 1
was confirmed from its spectroscopic and analytical data
(Supporting Information pp S16-S18).
In the present investigation, we have designed and
synthesized a naphthalimide appended rhodamine based
chemosensor where through bond energy transfer has been
used for the selective sensing of Hg2þ ions in mixed
aqueousmedia. Theattachmentofanaphthalimidemoiety
with rhodamine through a conjugated spacer like benzene
exhibits the phenomenon of through bond energy transfer
in the presence of mercury ions. To the best of our knowl-
edge, this is the first report where a TBET is observed
between naphthalimide and rhodamine moieties in the
presence of Hg2þ ions.
The binding behavior of compound 1 was studied
toward different metal cations (Hg2þ, Fe2þ, Fe3þ, Pb2þ
,
Cd2þ, Cu2þ, Zn2þ, Ni2þ, Agþ, Co2þ, Mg2þ, Liþ, Naþ, and
Kþ) as their perchlorate salts by UV-vis and fluorescence
spectroscopy. The absorption spectrum of compound 1 in
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Figure 1. UV-vis spectra of 1 (5 μM) in THF/H2O (9.5:0.5, v/v)
buffered with HEPES, in the presence of Hg2þ ions (100 equiv).
Inset showing the change in color before and after the addition
of Hg2þ ions.
THF/H2O (9.5: 0.5, v/v) shows two absorption bands at
320 and 363 nm (Figure 1) due to the naphthalimide
moiety, but there was no band corresponding to the
rhodamine moiety. However, on addition of Hg2þ ions
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Tetrahedron Lett. 2009, 50, 2649.
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Org. Lett., Vol. 13, No. 6, 2011
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(0-100 equiv), the intensity of the band at 363 nm
increases and a new absorption band appears at 565 nm
along with a color change from colorless to pink. The
formation of a new band at 565 nm is due to the opening of
the spirolactam ring of the rhodamine moiety. Thus, in the
presence of mercury ions, compound 1 shows an absorp-
tion spectrum characteristic of both the donor and accep-
tor components. Under the same conditions as those used
above for compound 1, we also carried out UV-vis studies
of model compound 6 (naphthalimide donor; Supporting
Information pp S5-S8, S26-S28) and rhodamine accep-
tor 715 (Supporting Information pp S5-S8) with Hg2þ
ions independently and found that the combined behavior
was the same (Supporting Information p S15) as was
observed with compound 1 in which two moieties are
attached to each other through a conjugated spacer. This
indicates that naphthalimide and rhodamine moieties in
compound 1 are interacting with Hg2þ ions independent of
each other. In other words, there are no electronic inter-
actions between these in the ground state in the presence of
Hg2þ ions, and thus compound 1 behaves like a cassette12
and not as a planar totally conjugated dye.
Figure 3. Hg2þ induced TBET OFF-ON. Inset showing the
fluorescence of ring opened rhodamine B (a) and receptor 1 (b).
operating through bonds, i.e., via the congugated linker
which allows energy transfer from donor to acceptor
through bonds. However, the energy transfer was not
100% because some of the flourescence leaks from the
naphthalimide donor rather being transferred to the ac-
ceptor. Under the same conditions as those used above for
compound 1 we also carried out fluorescence studies of an
equimolar mixture of naphthalimide donor 6 and rhoda-
mine acceptor (ring opened form of rhodamine B) and
found that no visible quenching of 6 and no enhancement
in the fluorescenceemission of the rhodamine acceptor was
observed when the mixture was excited at the naphthali-
mide absorption band, i.e., at 360 nm (Supporting Infor-
mation p S13), which clearly indicates that there is no
intermolecular energy transfer between the naphthalimide
donor and rhodamine acceptor in the mixture. Thus, the
advantage of the TBET system for energy transfer is
obvious. Further, for practical applications it is very
important that the fluorescence intensity of the acceptor
in the cassette is greater than that of the acceptor without
the donor when it is excited at the donor absorption
wavelength. The fluorescence enhancement factor for
compound 1 is 407-fold compared to the ring opened form
of rhodamine B when excited at 360 nm (Supporting
Information p S14). This enhancement factor is far higher
when compared with other FRET based systems.16 More-
over the fluorescence of compound 1 is much brighter than
that of the ring opened form of rhodamine B (inset of
Figure 3).
The fluorescence spectrum of compound 1 in THF/H2O
(9.5:0.5, v/v), in the absence of mercury ions, exhibited a
very weak emission at 472 nm attributed to the naphtha-
limide moiety when excited at 360 nm (Figure 2). The weak
Figure 2. Fluorescence spectra of 1 (5 μM) in response to the
presence of Hg2þ ions (350 equiv) in THF/H2O (9.5:0.5, v/v)
buffered with HEPES, pH = 7.0; λex = 360 nm. Inset showing
the fluorescence before and after the addition of Hg2þ ions.
We also tested the fluorescence response of 1 to other
metal ions such as Fe2þ, Fe3þ, Pb2þ, Cd2þ, Cu2þ, Zn2þ
,
Ni2þ, Agþ, Co2þ, Mg2þ, Liþ, Naþ, and Kþ, in mixed
aqueous media (THF/H2O; 9.5:0.5); however, no signifi-
cant variation in the fluorescence spectra of 1 (Figure 4A)
was observed with any other metal ion except Fe2þ and
Fe3þ (Supporting Information p S10) which also induce
similar fluorescence emission but to a small extent. To
check the practical ability of compound 1 as a Hg2þ
selective fluorescent sensor, we carried out competitive
experiments in the presence of Hg2þ at 350 equiv mixed
fluorescence emission of receptor 1 is due to the photo-
induced electron transfer (PET) from nitrogen atom of
the spirolactam ring to the photoexcited naphthalimide
moiety. Upon addition of Hg2þ ions (0-10 equiv) to the
solution of 1 in mixed aqueous media (THF/H2O, 9.5:0.5)
an emission band characteristic of the acceptor component
appears at 578 nm. This fluorescence enhancement at 578
nm is attributed to the opening of the spirolactam ring of
rhodamine to an amide form (Figure 3). The mode of
energy transfer in receptor 1 is a very fast mechanism
(16) (a) Su, J.; Tian, H.; Chen, K. Dyes Pigm. 1996, 31, 69. (b) Tian,
H.; Tang, Y.; Chen, K. Dyes Pigm. 1994, 26, 159. (c) Lee, L. G.;
Spurgeon, S. L.; Rosenblum, B. U.S. Patent 7,169,939, 2007.
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Org. Lett., Vol. 13, No. 6, 2011

Figure 5. Fluorescence and brightfield images of PC3 cells lines.
(a) Green fluorescence images of cells treated with probe 1
(1.0 μM) only for 20 min at 37 °C. (b) Brightfield images of (a).
(c) Overlay image of (a) and (b). (d) Red fluorescence images of
cells upon treatment with probe 1 (1.0 μM) and then Hg(ClO4)2
(10.0 μM) for 20 min. (e) Brightfield images of (d). (f) Overlay
image of (d) and (e); λex = 488 nm.
Figure 4. Fluorescence response of 1 (5 μM) to various cations
(350 equiv) in THF/H2O (9.5:0.5, v/v) buffered with HEPES,
pH = 7.0; λex = 360 nm. Bars represent the emission intensity
ratio (I - I0/I0) ꢀ 100 (I0 = initial fluorescence intensity at
578 nm; I = final fluorescence intensity at 578 nm after the
addition of Hg2þ ions). The black bars represent the addition of
individual metal ions while the gray bars represent the change in
the emission that occurs upon the subsequent addition of Hg2þ
(350 equiv) to the above solution.
cancer (PC3) cell lines. The prostate cancer (PC3) cell
lines were incubated with receptor 1 (1.0 μM in THF/
H2O (9.5:0.5, v/v) buffered with HEPES, pH = 7.0) in
an RPMI-1640 medium for 20 min at 37 °C and washed
with phosphate buffered saline (PBS) buffer (pH 7.4) to
remove excess of receptor 1. Microscope images showed a
weak green intracellular fluorescence which indicated that
compound 1 is cell permeable (Figure 5a). The cells were
then treated with mercury perchlorate (10.0 μM) in the
RPMI-1640 medium and incubated again for 20 min at
37 °C and washed with PBS buffer. After treatment with
Hg2þ ions the color of the cells changed to red which
clearly indicates the quenching of green emission with the
appearance of red emission (Figure 5d). These results
suggest that 1 is an effective intracellular Hg2þ imaging
agent with the change in fluorescence emission from green
tored, attributedtotheworking of theTBET phenomenon
within the cells.
with Fe2þ, Fe3þ, Pb2þ, Cd2þ, Cu2þ, Zn2þ, Ni2þ, Agþ,
Co2þ, Mg2þ, Liþ, Naþ, and Kþ (350 equiv each). As
shown in Figure 4B no significant variation in the fluores-
cence emission was observed by comparison with or with-
out the other metal ions. It was found that 1 has a detection
limit of 2 ꢀ 10-6 mol L-1 for Hg2þ which is sufficiently low
for the detection of the submillimolar concentration range
of Hg2þ ions found in many chemical systems. The fluo-
rescence quantum yield17 (Φfs) of compound 1 in the free
and Hg2þ-bound state was found to be 0.06 and 0.54
respectively. Fitting the changes in the fluorescence spectra
of compound 1 with Hg2þ ions using the nonlinear regres-
sion analysis program SPECFIT18 gave a good fit and
demonstrated that a 1:1 stoichiometry (host/guest) was the
most stable species in the solution with a binding constant
log β1 = 4.85. The method of continuous variation (Job’s
plot) (Supporting Information p S9) was alsousedtoprove
the 1:1 stoichiometry.19 We also carried out a reversibility
experiment which proved that binding of Hg2þ ions to
compound 1 was reversible. In the presence of KI, the
iodide ions because of the strong affinity for Hg2þ ions
form a complex with it, which results in the decomplexa-
tion of the receptor Hg2þ complex. On further addition of
Hg2þ ions, the fluorescence intensity was revived again
indicating the reversible behavior of the chemosensors 1
for the Hg2þ ions (Supporting Information p S12).
In conclusion, we synthesized naphthalimide appended
rhodaminebased fluorescent chemosensor1 which showed
through bond energy transfer in the presence of Hg2þ ions
in mixed aqueous solution. Chemosensor 1 can also be
used as a fluorescent probe for imaging Hg2þ ions in PC3
cell lines which will help in the understanding of biological
processes at the molecular level.
Acknowledgment. We are thankful to CSIR (New Delhi)
(ref. No. 01 (2326)/09/EMR-II) for financial support. We
are also thankful to reviewers for their valuable suggestions.
The potential biological application of the receptor was
evaluated for in vitro detection of Hg2þ ions in prostate
Supporting Information Available. Experimental data
and synthetic details of compound 1 and 4 are given in the
Supporting Information. This material is available free of
charge via the Internet at http://pubs.acs.org.
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