Highly selective fluorescence turn-on chemodosimeter based on rhodamine for nanomolar detection of copper ions

Article abstract of DOI:10.1021/ol203186b

A highly selective fluorescent chemodosimeter based on rhodamine is synthesized which undergoes Cu²⁺ driven hydrolysis in aqueous media to produce fluorescence turn-on changes with a detection limit up to the nanomolar range.

Full text of DOI:10.1021/ol203186b

ORGANIC
LETTERS
2012
Vol. 14, No. 1
406–409
Highly Selective Fluorescence Turn-on
Chemodosimeter Based on Rhodamine
for Nanomolar Detection of Copper Ions
Manoj Kumar,*,† Naresh Kumar,† Vandana Bhalla,† Parduman Raj Sharma,‡ and
Tandeep Kaur‡
Department of Chemistry, UGC Sponsored Centre for Advance Studies-1,
Guru Nanak Dev University, Amritsar-143005, Punjab, India, and Department
of Cancer Pharmacology, Indian Institute of Integrative Medicine, Canal Road,
Jammu-180001, India
*mksharmaa@yahoo.co.in
Received November 29, 2011
ABSTRACT
A highly selective fluorescent chemodosimeter based on rhodamine is synthesized which undergoes Cu2þ driven hydrolysis in aqueous media to
produce fluorescence turn-on changes with a detection limit up to the nanomolar range.
Copper is an essential soft transition metal ion that plays
a crucial role in environmental, biological, and chemical
systems. Copper, being both useful and cytotoxic, is a vital
trace element for the activities of enzymes because of its
redox-active nature.1 Copper deficiency may lead to he-
matological manifestations2 and a wide variety of neuro-
logical problems.3 An excess amount of copper in the body
causes gastrointestinal disturbance and damage to the liver
and kidneys.4 Thus, in view of the significance of copper
ions, simple and rapid sensing of copper ions in biological5
and environmentalsystemsisveryimportant. Copperions,
being of a catalytic nature, promoted the hydrolysis of
amides and esters, reactions including oxidations, dethioa-
cetalizations, rearrangements, and oxidative cyclizations.6
The method of copper induced reactions has been an
excellent approach for the development of selective che-
modosimeters for these ions. For example, Han and co-
workers6e and Li et al.6k reported efficient fluorescent
chemodosimeters which catalytically hydrolyzed in the
presence of Cu2þ ions. However, the reported examples
of Cu2þ mediated hydrolysis to induce fluorescence en-
hancement generally involve the generation of a single
fluorescent species and required a prolonged reaction time
to give the desired fluorescence change. The development
of chemodosimeters which undergo Cu2þ mediated hydro-
lysis to generate more than one fluorescent species is still a
† Guru Nanak Dev University.
‡ Indian Institute of Integrative Medicine.
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r
10.1021/ol203186b
Published on Web 12/15/2011
2011 American Chemical Society

challenge, yet this may open a new approach for the con-
struction of fluorescent chemodosimeters for desired ana-
lytical objectives.
Scheme 1. Synthesis of Compound 2
Our research work involves the design, synthesis, and
evaluation of fluorogenic receptors for selective sensing of
soft metal ions as well as anions and evaluation of their
switching behavior.7 In continuation of this work, we have
now designed and synthesized chemodosimeter 2 which
undergoes Cu2þ driven hydrolysis in mixed aqueous
media. The acylhydrazide moiety is introduced in receptor
2 due to its good binding affinity for transition metal
ions.6a,8 The catalytic hydrolysis of acylhydrazide moiety
in chemosensor 2 is selectively induced by Cu2þ ions to
produce irreversible fluorescence turn-on changes at 534
and 575 nm ascribed to the highly fluorescent hydrazide
derivative (HZD) exhibiting a twisted intramolecular charge
transfer phenomenon and a ring opened form of the
rhodamine, respectively. To the best of our knowledge,
this is the first report where catalytic hydrolysis of a recep-
tor in the presence of Cu2þ ions produces fluorescence
turn-on changes at two different wavelengths. Interest-
ingly, the change in fluorescence emission is observed im-
mediately after the addition of Cu2þ ions which in turn
indicates the highly reactive nature of Cu2þ promoted
hydrolysis of 2. Furthermore, biological application of
the chemodosimeter 2 is evaluated for in vitro detection
of Cu2þ ions in prostate cancer (PC3) cell lines.
Cu2þ ions (0ꢀ40 μM) in CH3CN/H2O (8:2, v/v) when
excited at 540 nm due to the ring opening of the spirolac-
tam form of the rhodamine moiety (Figure 1B). These
results indicate that either Cu2þ ions are interacting with
the dimethylaminovinylbenzene/rhodamine moieties inde-
pendent of each other or some other process is operating in
this system. To gain insight into the whole process, we
studied the fluorescence behavior of 2 with Cu2þ ions in
acetonitrile. Free 2 in acetonitrile exhibits very weak
charge transfer emission at 508 nm with a shoulder at
430 nm attributed to the locally excited emission when
excited at λex = 380 nm (inset of Figure 2A). The addition
The reduction of compound 17d with NaBH4 gave
desired compound 2 in 65% yield (Scheme 1, see Support-
ing Information (SI) p S5). The structure of compound 2
was confirmed by its spectroscopic and analytical data (see
SI p S15ꢀ17). The absorption spectrum of receptor 2
(5 μM) in CH3CN/H2O (8:2, v/v) shows two absorption
bands at 238 and 275 nm with a shoulder at 300 nm (see SI
p S6). On addition of Cu2þ ions (0ꢀ36 equiv) the bands at
238 and 275 nm show significant enhancement with the
simultaneous appearance of two new absorption bands at
394 and 556 nm. The addition of other transition and alkali
metal ions did not alter the absorption spectrum of recep-
tor 2 (see SI p S6). From the absorbance spectra of receptor
2 in the presence of Cu2þ ions, we expected that the system
might exhibit the phenomenon of resonance energy trans-
fer during the fluorescence studies. However, the 2-Cu2þ
system in aqueous media did not undergo any energy
transfer. The fluorescence spectrum of receptor 2 (1 μM)
did not exhibit any emission when excited at 380 nm in
CH3CN/H2O (8:2, v/v; Figure 1A). Upon addition of
increasing amounts of Cu2þ ions (0ꢀ22 μM) a remark-
able enhancement in emission intensity was observed at
534 nm with the appearance of green colored fluorescence
(Figure 1A). On the other hand, receptor 2 undergoes
fluorescence enhancement at 575 nm in the presence of
Figure 1. Fluorescence spectra of 2 (1 μM) in the presence of
Cu2þ ions in CH3CN/H2O (8:2, v/v) buffered with HEPES, pH
= 7.0: (A) in the presence of Cu2þ ions (0ꢀ22 μM), λex
=
380 nm; (B) in the presence of Cu2þ ions (0ꢀ40 μM), λex = 540 nm.
Insets show the fluorescence of (a) free 2 and (b) 2 þ Cu2þ ions.
of 0ꢀ10 μM of Cu2þ ions results in the fluorescence
enhancement at 508 nm along with appearance of a new
emission band at 586 nm (Figure 2A). Further additions of
Cu2þ ions (11ꢀ24 μM) result in the diminishing of emis-
sion at 508 nm while the emission at 586 nm undergoes
significant fluorescence enhancement (Figure 2B). These
results indicate that there is a resonance energy transfer
mechanism operating between the dimethylaminovinyl-
benzene and rhodamine moieties on addition of Cu2þ ions
in acetonitrile. The binding of Cu2þ with an acylhydrazide
moiety first induces an enhanced charge transfer from the
nitrogen atom of the dimethylamino moiety to the acylhy-
drazide moiety which results in the enhancement at 508 nm
corresponding to the donor emission (dimethylaminovinyl-
benzene). As the concentration of Cu2þ ions increases, the
(7) (a) Kumar, M.; Dhir, A.; Bhalla, V. Org. Lett. 2009, 11, 2567. (b)
Kumar, M.; Kumar, R.; Bhalla, V. Chem. Commun. 2009, 7384. (c)
Kumar, M.; Dhir, A.; Bhalla, V. Chem. Commun. 2010, 46, 6744. (d)
Kumar, M.; Kumar, N.; Bhalla, V. Tetrahedron Lett. 2011, 52, 4333.
(8) (a) Katiyar, S. S.; Tandon, S. N. Talanta 1964, 11, 892. (b)
Johnson, D. K.; Murphy, T. B.; Rose, N. J.; Goodwin, W. H.; Pickart,
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Vittal, J. J.; Ranford, J. D. Polyhedron 2003, 22, 2781.
Org. Lett., Vol. 14, No. 1, 2012
407

phenomenon of resonance energy transfer occurs which
involves energy transfer from the dimethylaminovinyl-
benzene moiety to the rhodamine moiety. No significant
change in the fluorescence behavior was observed in the
presence of other metal ions (see SI p S7). In fitting the
changes in the fluorescence spectra of 2 with Cu2þ ions in
acetonitrile, the nonlinear regression analysis program
SPECFIT9 gave a good fit and demonstrated that a 2:1
stoichiometry (host:guest) was the most stable species in
the solution with a binding constant (log β) = 10.30 with
( 0.07 error. The method of continuous variation10 (Job’s
plot) was also used to prove the 2:1 stoichiometry (see SI
p S13). The absorbance spectrum of 2 in acetonitrile (see SI
p S7) is same as that observed in the aqueous media.
However, the addition of Cu2þ ions results in the appear-
ance of new bands at 256, 354, 394, and 560 nm with a
simultaneous decrease of absorbance at 238, 275, and 300
nm (see SI p S7).
The complexation of DETA with Cu2þ ions did not affect
the highly fluorescent products generated by the Cu2þ ion
promoted hydrolysis of receptor 2, and thus the reaction of
2 and Cu2þ ions in aqueous solution is irreversible in
nature. The reaction of 2 with Cu2þ was carried out in
1
CD3CNꢀD2O (9:1, v/v) for 5 min, and then a H NMR
spectrum of the resulting solution (see SI p S18) was
recorded after eliminating the Cu2þ ions by Chelex resin.
The 1H NMR spectrum shows the appearance of a singlet
at 10.16 ppm corresponding to the hydroxyl group of ring
opened rhodamine and a broad multiplet at 1.75ꢀ1.80 ppm
corresponding to the amino protons of hydrazide deriva-
tive (HZD). This clearly indicates that receptor 2 was
catalytically hydrolyzed in the presence of Cu2þ ions
generating highly fluorescent products, a hydrazide deri-
vative (HZD) and ring-opened form of rhodamine
(Figure 3). The twisted intramolecular charge transfer
(TICT) state is responsible for the emission observed at
534 nm which is confirmed by observing the fluorescence
emission of receptor 2 with Cu2þ ions by varying the polarity
The UVꢀvis and fluorescence behavior of 2-Cu2þ in
acetonitrile is different from that in the mixed aqueous
media. The use of water as a cosolvent along with acetoni-
trile does not influence the emission pattern of receptor 2.
However, the addition of Cu2þ ions promoted the hydro-
Figure 2. Fluorescence spectra of 2 (1 μM) in the presence of
Cu2þ ions: (A) in the presence of 0ꢀ10 μM and (B) in the
presence of 11ꢀ24 μM of Cu2þ ions. Inset shows the fluores-
cence spectrum of free 2, in CH3CN, λex = 380 nm.
Figure 3. Cu2þ induced catalytic hydrolysis of 2.
lysis of receptor 2 rather thanthe resonance energy transfer
phenomenon which results in the generation of a highly
fluorescent hydrazide derivative (HZD) and a ring opened
form of rhodamine (Figure 3). Further, to confirm Cu2þ
mediated hydrolysis, we carried out reversibility experi-
ments in both acetonitrile and acetonitrile/water systems.
The addition of diethylenetriamine (DETA) to the 2-Cu2þ
complex in acetonitrile restored the fluorescence signal of 2
to its original level (see SI p S8). Further addition of Cu2þ
ions to the same solution gives the outcome of the 2-Cu2þ
complex indicating the reversible behavior of 2 in acetoni-
trile. On the other hand, addition of DETA to the aqueous
solution containing 2 and Cu2þ ions did not alter the
emission pattern of the 2-Cu2þ system (see SI p S9).
The fluorescence emissions at 534 and 575 nm remained
the same as was observed in the absence of DETA.
of the solvent system (see SI p S10). As we increase the
water content of the solvent system CH3CN/H2O (9:1, 8:2,
and 1:1; v/v), there is a continuous red shift of the emission
band confirming the twisted intramolecular charge trans-
fer nature of its emissive state.11 The kinetics of the
fluorescence enhancement at 534 nm by Cu2þ mediated
hydrolysis of 2 was also studied by time-dependent fluor-
escence spectroscopy (Figure 4). The maximum fluores-
cence emission enhancement at 534 nm was observed
within 5 min after the additions of different concentrations
of Cu2þ ions which clearly indicate the highly reactive
nature of the Cu2þ mediated hydrolysis of 2.
To check the practical ability of receptor 2as a Cu2þ selec-
tive fluorescent chemodosimeter, we carried out competitive
(9) Gampp, H.; Maeder, M.; Meyer, C. J.; Zuberbulher, A. D.
Talanta 1985, 32, 95.
(10) Job, P. Ann. Chim. 1928, 9, 113.
(11) (a) Grabowski, Z. R.; Rotkiewicz, K. Chem. Rev. 2003, 103,
3899. (b) Bangal, P. R.; Panja, S.; Chakravorti J. Photochem. Photobiol.
A: Chem. 2001, 139, 5.
408
Org. Lett., Vol. 14, No. 1, 2012

Figure 4. Variation of fluorescence of receptor 2 (1 μM) at
534 nm in CH3CN/H2O (8:2, v/v) buffered with HEPES, pH
= 7.0, λex = 380 nm in the presence of different concentrations
of Cu2þ ions (I/Io; Io = initial fluorescence intensity at 534 nm; I
= fluorescence intensity after the addition of Cu2þ ions at 534
nm); the fluorescence were recorded after every 1 min interval.
Figure 5. Fluorescence and brightfield images of PC3 cell lines.
(a and b) Fluorescence images of cells in the green and red
channels respectively treated with probe 2 (5 μM) for 20 min at
37 °C. (c) Brightfield image of (a) and (b). (d) Overlay image of
(a) and (b). (e and f) Fluorescence images of cells in the green and
red channels respectively upon treatment with probe 2 (5 μM)
and then Cu(ClO4)2 (30 μM) for 20 min at 37 °C. (g) Brightfield
image of (e) and (f). (h) Overlay image of (e) and (f), λex
=
experiments at two different emissions, in the presence of
Cu2þ mixed with different metal ions (see SI p S12). No
significant variation in the emission was observed by
comparison with or without the other metal ions. Further,
the fluorescence enhancement factor for receptor 2 at 534
and 575 nm in the presence of Cu2þ ions is increased 32-
and 27-fold, respectively. The fluorescence quantum
488 nm; fluorescence images are recorded at both green (520 (
20 nm) and red channels (570 ( 20 nm).
appearance of green and red fluorescence is attributed to
the hydrazide derivative and ring-opened form of rhoda-
mine produced by Cu2þ ion mediated hydrolysis of 2.
These results suggest that receptor 2 is cell permeable
and an effective intracellular Cu2þ ion imaging agent with
turn-on green and red colored fluorescence emissions.
In conclusion, we synthesized fluorescent chemodosi-
meter 2 which undergoes Cu2þ ion promoted hydrolysis to
produce fluorescence turn-on changes at two different
wavelengths. Further, receptor 2 can also be used as a
fluorescent probe for intracellular imaging of Cu2þ ions
with tunable emission (green and red) which will help in the
understanding of biological processes at the molecular level.
yield12 (see SI p S3) of the 2-Cu2þ complex is 0.20 (at λem
=
534 nm) and 0.16 (at λem = 575 nm) as compared to that of
free 2 (0.03 and 0.01, respectively). The detection limit of 2
for Cu2þ ions at 534 nm was found tobe 20 ꢁ 10ꢀ9 mol Lꢀ1
(see SI p S14) which is sufficiently low and within the
acceptable limit of the US EPA for the maximum allow-
able limit of Cu2þ ions (1.3 ppm) in drinking water.
The potential biological application of the chemodosi-
meter 2 was evaluated for in vitro detection of Cu2þ ions in
prostate cancer (PC3) cell lines (see SI p S4). The prostate
cancer (PC3) cell lines were incubated with receptor 2
(5 μM) 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 receptor 2. Microscopic images
showed no intracellular fluorescence which indicated that
receptor 2 is nonemissive in nature (Figure 5a and b).
However, after treatment with Cu2þ ions (30 μM), the cells
pretreated with receptor 2 (5 μM) show fluorescence in
both the green and red channels (Figure 5e and f). The
Acknowledgment. We are thankful to CSIR (New Delhi)
for financial support (Ref. No. CSIR Scheme 01 (2167)07/
EMR-II) N. K. is thankful to CSIR (New Delhi) for junior
research fellowship and Guru Nanak Dev University for
providing the research facility.
Supporting Information Available. Experimental data
and synthetic detail of compound 2. This material is
available free of charge via the Internet at http://pubs.
acs.org.
(12) Demas, J. N.; Crosby, G. A. J. Phys. Chem. 1971, 75, 991.
Org. Lett., Vol. 14, No. 1, 2012
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