Visible-near-infrared and fluorescent copper sensors based on julolidine conjugates: Selective detection and fluorescence imaging in living cells

Article abstract of DOI:10.1002/chem.201101906

We present novel Schiff base ligands julolidine-carbonohydrazone 1 and julolidine-thiocarbonohydrazone 2 for selective detection of Cu²⁺ in aqueous medium. The planar julolidine-based ligands can sense Cu²⁺ colorimetrically with characteristic absorbance in the near-infrared (NIR, 700-1000 nm) region. Employing molecular probes 1 and 2 for detection of Cu ²⁺ not only allowed detection by the naked eye, but also detection of varying micromolar concentrations of Cu²⁺ due to the appearance of distinct coloration. Moreover, Cu²⁺ selectively quenches the fluorescence of julolidine-thiocarbonohydrazone 2 among all other metal ions, which increases the sensitivity of the probe. Furthermore, quenched fluorescence of the ligand 2 in the presence of Cu²⁺ was restored by adjusting the complexation ability of the ligand. Hence, by treatment with ethylenediaminetetraacetic acid (EDTA), thus enabling reversibility and dual-check signaling, julolidine-thiocarbonohydrazone (2) can be used as a fluorescent molecular probe for the sensitive detection of Cu²⁺ in biological systems. The ligands 1 and 2 can be utilized to monitor Cu ²⁺ in aqueous solution over a wide pH range. We have investigated the structural, electronic, and optical properties of the ligands using ab initio density functional theory (DFT) combined with time-dependent density functional theory (TDDFT) calculations. The observed absorption band in the NIR region is attributed to the formation of a charge-transfer complex between Cu²⁺ and the ligand. The fluorescence-quenching behavior can be accounted for primarily due to the excited-state ligand 2 to metal (Cu²⁺) charge-transfer (LMCT) processes. Thus, experimentally observed characteristic NIR and fluorescence optical responses of the ligands upon binding to Cu ²⁺ are well supported by the theoretical calculations. Subsequently, we have employed julolidine-thiocarbonohydrazone 2 for reversible fluorescence sensing of intracellular Cu²⁺ in cultured HEK293T cells. Copyright

Full text of DOI:10.1002/chem.201101906

DOI: 10.1002/chem.201101906
Visible–Near-Infrared and Fluorescent Copper Sensors Based on Julolidine
Conjugates: Selective Detection and Fluorescence Imaging in Living Cells
Debabrata Maity,[a] Arun K. Manna,[b] D. Karthigeyan,[c] Tapas K. Kundu,[c]
Swapan K. Pati,[b] and T. Govindaraju*[a]
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Chem. Eur. J. 2011, 17, 11152 – 11161

FULL PAPER
Abstract: We present novel Schiff base
ligands julolidine–carbonohydrazone 1
and julolidine–thiocarbonohydrazone 2
for selective detection of Cu2+ in aque-
ous medium. The planar julolidine-
based ligands can sense Cu2+ colori-
metrically with characteristic absorb-
ance in the near-infrared (NIR, 700–
1000 nm) region. Employing molecular
probes 1 and 2 for detection of Cu2+
not only allowed detection by the
naked eye, but also detection of vary-
ing micromolar concentrations of Cu2+
restored by adjusting the complexation
ability of the ligand. Hence, by treat-
ment with ethylenediaminetetraacetic
acid (EDTA), thus enabling reversibili-
ty and dual-check signaling, julolidine–
thiocarbonohydrazone (2) can be used
as a fluorescent molecular probe for
the sensitive detection of Cu2+ in bio-
logical systems. The ligands 1 and 2 can
be utilized to monitor Cu2+ in aqueous
solution over a wide pH range. We
have investigated the structural, elec-
tronic, and optical properties of the lig-
(TDDFT) calculations. The observed
absorption band in the NIR region is
attributed to the formation of a charge-
transfer complex between Cu2+ and
the ligand. The fluorescence-quenching
behavior can be accounted for primari-
ly due to the excited-state ligand 2 to
metal (Cu2+) charge-transfer (LMCT)
processes. Thus, experimentally ob-
served characteristic NIR and fluores-
cence optical responses of the ligands
upon binding to Cu2+ are well support-
ed by the theoretical calculations. Sub-
sequently, we have employed juloli-
dine–thiocarbonohydrazone 2 for re-
versible fluorescence sensing of intra-
cellular Cu2+ in cultured HEK293T
cells.
due to the appearance of distinct col
ation. Moreover, Cu2+ selectively
quenches the fluorescence of juloli-
dine–thiocarbonohydrazone among
or-
ACHTUNGTRENNaUNG nds using ab initio density functional
theory (DFT) combined with time-de-
pendent density functional theory
T
2
all other metal ions, which increases
the sensitivity of the probe. Further-
more, quenched fluorescence of the
ligand 2 in the presence of Cu2+ was
Keywords: bioimaging · chemosen-
sors
· copper · fluorescence ·
visible–near infrared
Introduction
plemented through human diet in regular amounts for ab-
sorption.[4] The adult human body contains between 1.4–
2.1 mg of copper per kilogram of body weight under normal
conditions. Copper-dependent enzymes act as catalysts to
help a number of body functions to provide energy for bio-
chemical reactions, transform melanin for pigmentation of
the skin, assist the formation of cross-links in collagen and
elastin, and thereby maintain and repair connective tissues.
This is especially important for the heart and arteries[5] and
research suggests that copper deficiency is one of the factors
leading to an increased risk of developing coronary heart
disease.[6] In general most human diets contain enough
copper (2–5 mgdayꢀ1) to prevent a deficiency, but not suffi-
cient to cause toxicity. The World Health Organization
(WHO) and the Food and Agricultural Administration
(FAA) reports suggest that the population mean intake of
copper should not exceed 10–12 mgdayꢀ1 for adults. Copper
in excessive amounts can be toxic and cause oxidative stress
and disorders associated with neurodegenerative diseases in-
cluding Alzheimerꢁs, Parkinsonꢁs, Menkes, Wilsonꢁs, and
prion diseases.[7,8] Although protein and organically bound
copper appears to be less toxic, free solvated Cu2+ is partic-
ularly damaging, since it catalyses the formation of reactive
organic species (ROS), including radical and nonradical spe-
cies, which can also trigger oxidative damage to proteins, nu-
cleic acids, and lipids.[9] The common nutritional deficiencies
of zinc, manganese, and other trace minerals also facilitate
the accumulation of very high levels of copper.
Molecular sensors have been developed for selective recog-
nition of different species on the basis of host–guest interac-
tions making use of hydrogen bonding, electrostatic force,
metal–ligand coordination, and hydrophobic and van der
Waals interactions.[1] In recent years, the development of
novel colorimetric and fluorescent sensors of biologically
active metal ions have been actively investigated because of
their potential applications in life sciences, medicine,
chemistry, and biotechnology.[2] The design and synthesis of
highly selective sensors for metal ions, such as mercury,
lead, iron, zinc, and copper, is particularly important, since
these metal ions can have detrimental effects on the envi-
ronment and human health.[3] Copper is one of a relatively
small group of trace metal nutrients that are essential to sus-
tain normal human health. However, copper has to be sup-
[a] D. Maity, Dr. T. Govindaraju
Bioorganic Chemistry Laboratory, New Chemistry Unit
Jawaharlal Nehru Centre for Advanced Scientific Research
Jakkur, Bangalore-560064 (India)
Fax : (+91)80-22082627
E-mail: tgraju@jncasr.ac.in
[b] A. K. Manna, Prof. S. K. Pati
Theoretical Sciences Unit
Jawaharlal Nehru Centre for Advanced Scientific Research
Jakkur, Bangalore-560064 (India)
[c] D. Karthigeyan, Prof. T. K. Kundu
Molecular Biology and Genetics Unit
Jawaharlal Nehru Centre for Advanced Scientific Research
Jakkur, Bangalore-560064 (India)
In the past few years, many colorimetric and fluorescent
chemosensors have been reported for sensing copper ions
with absorbance and emission in the visible region.[10–18] Al-
though chemosensors with visible (absorbance/emission) re-
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201101906.
Chem. Eur. J. 2011, 17, 11152 – 11161
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T. Govindaraju et al.
sponses have important roles in various research endeavors,
molecular probes with near-infrared (NIR, 700–1000 nm)
optical responses are particularly gaining special interest in
recent years.[19] Unlike UV/Vis radiation, NIR radiation can
penetrate much deeper into the sample, since there is no or
limited absorption and scattering of NIR radiation. Addi-
tionally, there is no interference of autofluorescence gener-
ated from the chromophores and macromolecules present in
the analytic samples. This phenomenon enables the assess-
ment of molecular and physiological events in several layers
deep inside the analyte samples and tissues.[20] There have
been few reports on UV/Vis–NIR sensing of cation and
anions.[21–24] Recently, we have reported Schiff base conju-
gates of urea/thiourea and salicylaldehyde as colorimetric
chemosensors with characteristic NIR response for selective
detection of copper.[25] Designing novel and efficient NIR
chemosensors necessitates the need for understanding de-
tailed molecular mechanism involved in the origin of such
interesting phenomenon. The studies aimed at understand-
ing the underlying mechanism and origin of NIR responses
are scarce and hence theoretical calculations would be of
great help to understand the microscopic details of such
changes in the photophysical characteristics. Along with visi-
ble and NIR absorbance signals, a fluorescence response
would add a new dimension to the sensing of metal ions
over the methods involving just one kind of optical re-
sponse. Additionally, the fluorescence chemosensors are
known for their sensitivity, specificity, and real-time moni-
toring with fast response time.[26] Thus, designing a colori-
metric chemosensor that combines the advantage of the
characteristics of an NIR optical response with the sensitivi-
ty of fluorescence is of prime importance. In accordance
with this concept we have designed two novel compounds
julolidine–carbonohydrazone 1 and julolidine–thiocarbono-
hydrazone 2 for selective detection of Cu2+ by conjugating
hydroxyjulolidinal with carbohydrazide and thiocarbohydra-
zide (Scheme 1). We have demonstrated their ability to
detect Cu2+ colorimetrically with characteristic absorbance
in the NIR region. Moreover julolidine–thiocarbonohydra-
zone 2 was used for the detection of Cu2+ fluorometrically,
since it overall increases the sensitivity of the probe. Fur-
thermore, we have demonstrated the utility of 2 for reversi-
ble bioimaging of Cu2+ in cultured living cells. The experi-
mentally observed characteristic NIR optical response of lig-
Scheme 1. Schiff base ligands synthesized by conjugating various alde-
hydes with carbohydrazide (1, 3, 5, and 7) and thiocarbohydrazide (2, 4,
6, and 8).
tered. The precipitate was washed with ethanol and dried
under vacuum to obtain Schiff base ligands julolidine–car-
bonohydrazone 1 and julolidine–thiocarbonohydrazone 2,
respectively, in quantitative yield. Similarly, ligands 3–8
were prepared by condensing different salicylaldehyde de-
rivatives with electron-donating and withdrawing functional
groups with carbohydrazide or thiocarbohydrazide in good
yields (Scheme 1). All the synthesized Schiff base ligands
(1–8) were characterized by NMR spectroscopy, mass spec-
troscopy, and elemental analysis.
Absorbance study of Schiff base ligands with Cu2+ and
other metal ions: We studied photophysical properties of
different Schiff base ligands carrying electron-withdrawing
and electron-donating functional groups. First, the photo-
physical properties of julolidinal-based Schiff base ligands 1
and 2 were investigated by monitoring the absorption spec-
tral behavior upon addition of several metal ions such as
Li+, Na+, Ba2+, Sr2+, Mg2+, Al3+, Ca2+, Mn2+, Fe2+, Co2+
,
Ni2+, Zn2+, Ag+, Cd2+, Hg2+, Pb2+, and Cu2+ in an aqueous
buffer medium (50 mm, 2-(4-(2-hydroxyethyl)-1-piperazin-
AHCTUNGTRENNUNG
A
ACHTUNGTRENNUNG
visible (460–600 nm) and NIR (700–1100 nm) regions in the
presence of Cu2+ as shown in Figures 1 and 2. The presence
of other metal ions did not lead to the appearance of any
such visible and NIR absorbance of ligands 1 and 2 (Fig-
ures 1 and 2). In contrast, other Schiff base ligands (3–8) did
not exhibit any characteristic optical response in the pres-
ence of Cu2+ (see Figure S1 in the Supporting Information).
Julolidine–carbonohydrazone 1 shows an absorption band
centered around 380 nm, which remains unchanged upon
Results and Discussion
Synthesis of Schiff base ligands: We have synthesized a
series of Schiff base ligands by a one-step condensation re-
action. A solution of 8-hydroxyjulolidinal in ethanol was
added slowly to a solution of carbohydrazide or thiocarbo-
hydrazide in water. The reaction mixture was heated to
reflux for 24 h with constant stirring. The reaction mixture
was cooled to room temperature and the precipitate was fil-
addition of 50.0 equivalents of Li+, Na+, Ba2+, Sr2+, Mg2+
,
Al3+, Ca2+, Mn2+, Fe2+, Ag+, Cd2+, and Pb2+. With the ad-
dition of 50.0 equivalents of Co2+, Ni2+, Zn2+, and Hg2+ the
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Visible–Near-Infrared and Fluorescent Copper Sensors
FULL PAPER
from 930 to 825 nm and that remains unchanged upon fur-
ther addition of Cu2+. The final solution color of ligand 1 is
aqua colored. The complete colorimetric titration of ligand
1 with sequential addition of Cu2+ is shown in Figure 3.
Figure 1. UV/Vis absorption spectra of julolidine–carbonohydrazone 1
(10.0 mm) and on addition of salts (50.0 equiv) of Li+, Na+, Ba2+, Sr2+
,
,
Mg2+, Al3+, Ca2+, Mn2+, Fe2+, Co2+, Ni2+, Zn2+, Ag+, Cd2+, Hg2+, Pb2+
and Cu2+ in aqueous medium (50 mm HEPES/CH3CN, 6:4, v/v; pH 7.2).
Figure 3. UV/Vis absorption spectra of julolidine–carbonohydrazone 1
(10.0 mm) on addition of different concentrations of Cu2+ (0, 2.5, 5, 10,
15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, and 100 mm)
in aqueous medium (50 mm HEPES/CH3CN, 6:4, v/v; pH 7.2). Inset: Ab-
sorbance at 825 nm as a function of [Cu2+].
Cu2+ can be detected by an NIR response at least down to
20 mm when 10 mm of 1 is employed in an aqueous medium
(50 mm, HEPES/CH3CN, 6:4, v/v; pH 7.2). An absorbance
peak in the NIR region would be useful for sensing Cu2+ in
systems that contains unwanted interference of endogenous
chromophores in the visible region. Ligand 2 (10 mm) exhib-
ited an absorption band in the visible region (lmax =570 nm)
accompanied by a well-distinguished NIR absorption band
around 820 nm as shown in Figure 2. During sequential titra-
tion, two absorption bands appeared at 570 and 980 nm and
correspond to the visible and NIR regions, respectively,
after addition of 1.0 equivalent of Cu2+ (Figure 4). At this
concentration of added Cu2+ the color of the solution of
Figure 2. UV/Vis absorption spectra of julolidine–thiocarbonohydrazone
2 (10.0 mm) and on addition of salts (50.0 equiv) of Li+, Na+, Ba2+, Sr2+
,
,
Mg2+, Al3+, Ca2+, Mn2+, Fe2+, Co2+, Ni2+, Zn2+, Ag+, Cd2+, Hg2+, Pb2+
and Cu2+ in aqueous medium (50 mm HEPES/CH3CN, 6:4, v/v; pH 7.2).
absorbance intensity decreases and is slightly red shifted to
different extents as shown in Figure 1. At a similar concen-
tration of Cu2+ (50.0 equiv), ligand 1 showed characteristic
absorbance in the visible (495 nm) and NIR (823 nm) re-
gions. During sequential titration, the absorption spectra of
ligand 1 in the visible region gradually red shifted to 412 nm
with the addition of 1.5 equivalents of Cu2+ and the color-
less solution turns to light green. Increasing the concentra-
tion of Cu2+ in the solution of ligand 1 from 1.5 to
2.0 equivalents leads to a change in the solution color from
light green to light purple. A new absorption band (lmax
=
570 nm) appeared in the visible region accompanied by a
distinguishable NIR band with lmax centered at 930 nm. The
absorbance at 570 nm reaches a maximum upon addition of
6.0 equivalents of Cu2+ with an extinction coefficient (e) of
2ꢂ104 mꢀ1 cmꢀ1 and then decreases with further addition of
Cu2+. The absorbance in the NIR region reaches a maxi-
mum (e=5.2ꢂ104 mꢀ1 cmꢀ1) upon addition of a total of
8.0 equivalents of Cu2+ with a gradual blue shift of lmax
Figure 4. UV/Vis absorption spectra of julolidine–thiocarbonohydrazone
2 (10.0 mm) on addition of different concentrations of Cu2+ (0, 5, 10, 20,
30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190,
and 200 mm) in aqueous medium (50 mm HEPES/CH3CN, 6:4, v/v;
pH 7.2). Inset: Absorbance at 825 nm as a function of [Cu2+].
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T. Govindaraju et al.
ligand 2 changes from greenish to light purple. The absorb-
ance at 570 and 980 nm is enhanced by increasing the con-
centration of added Cu2+ from 1.5 to 2.0 equivalents and the
purple-colored solution turns to light violet. The absorbance
at 570 nm reaches the maximum at 5.0 equivalents of Cu2+
with an extinction coefficient (e) of 2ꢂ104 mꢀ1 cmꢀ1 and a
consequent increase in the intensity of the NIR absorption
band was observed. At this composition of ligand 2 and
Cu2+ the solution color changes from light violet to blue.
Further addition of Cu2+ resulted in a gradual decrease in
the absorbance at 570 nm as shown in Figure 4, whereas the
absorbance in the NIR region increases and the band was
gradually blue shifted to 820 nm and reaches a maximum
with e=2.5ꢂ104 mꢀ1 cmꢀ1 upon overall addition of
15.0 equivalents of Cu2+ (Figure 4). The final solution color
of ligand 2 becomes greenish aqua. By employing 10 mm
concentration of ligand 2, Cu2+ can be detected by an NIR
response at least down to 10 mm in an aqueous medium.
Apart from selective detection of Cu2+ using ligands 1 and
2, different concentrations of Cu2+ can also be detected
(Figure 5). The specific micromolar concentrations of added
Stoichiometry of binding: The stoichiometry of binding of
Cu2+ to ligands 1 and 2 were studied by various analytical
techniques. Jobꢁs plots from UV/Vis absorbance data show
1:2 stoichiometric complexation between ligands 1 and 2
with Cu2+ (Figures S3 and S4 in the Supporting Informa-
tion). This data was further supported by mass spectrometry
analysis. The addition of Cu2+ to ligands 1 and 2 resulted in
deprotonation of the hydroxyl functional groups of the julo-
lidine moieties followed by complex formation. MALDI/
TOF-MS shows the formation of a complex between depro-
tonated ligands and two copper ions: [C27H32Cu2N6O3 +Na+
ꢀ2H+]: m/z: 635.17; calcd for C27H32Cu2N6O3 +Na+: 637.1
and [C27H32Cu2N6O2S+Na+ꢀ3H+]: m/z: 650.07; calcd for
C27H31Cu2N6O2S+Na+, 653.07 (Figures S13 and S14 in the
Supporting Information).
Response parameter and binding constant: The response pa-
rameter (a) which is defined as the ratio of free ligand con-
centration to the initial concentration of ligand is plotted as
a function of Cu2+ concentration (see Figures S5 and S6 in
the Supporting Information). This plot can serve as the cali-
bration curve for the detection of Cu2+. The association con-
stant (logKa) of ligands 1 and 2 with Cu2+ was calculated as
13.52 and 14.36 mꢀ1, respectively, as determined from Liꢁs
equations.
pH dependence study: The influence of pH on the absorb-
ance of ligands 1 and 2 upon complexation with Cu2+ was
studied in aqueous medium (50 mm HEPES/CH3CN, 6:4, v/
v; Figures S7 and S8 in the Supporting Information).[27] Cu2+
can be clearly detected from the visible and NIR absorbance
measurements by using ligands 1 and 2 over a pH range of
2–11. Therefore ligands 1 and 2 can be used for the environ-
mental monitoring and biological detection of copper in
most of commonly encountered pH ranges.
Figure 5. Detectable colorimetric change with increasing concentration of
Cu2+ to the solution of a) julolidine–carbonohydrazone 1 (10.0 mm) and
b) julolidine–thiocarbonohydrazone
2 (10.0 mm) in aqueous medium
(50 mm HEPES/CH3CN, 6:4, v/v; pH 7.2). The values indicated on the
vials correspond to [Cu2+].
Fluorometric detection of Cu2+ using Julolidine–thiocarbo-
nohydrazone 2: Fluorometric behavior of both the ligands 1
and 2 was studied upon addition of 20.0 equivalents of Li+,
Cu2+ gave distinct coloration to aqueous solutions of ligands
1 and 2 (Figure 5a and 5b, respectively). These colorimetric
changes correspond to well-distinguishable visible absorb-
ance spectra with a characteristic NIR signature in Figure 3
and Figure 4.
Na+, Ba2+, Sr2+, Mg2+, Al3+, Ca2+, Mn2+, Fe2+, Co2+, Ni2+
,
Zn2+, Ag+, Cd2+, Hg2+, Pb2+, and Cu2+ in aqueous medium
(50 mm HEPES/CH3CN, 6:4, v/v; pH 7.2). Ligand 1 did not
show any specific changes in the fluorescence emission in
the presence of Cu2+ and other metal ions used upon excita-
tion at 402 nm (see Figure S9 in the Supporting Informa-
tion). On the other hand, ligand 2 shows strong fluorescence
emission around 535 nm upon excitation at 430 nm. The
fluorescence intensity around 535 nm was quenched in the
presence of only Cu2+ with 430 nm excitation, whereas no
significant changes were observed in the fluorescence emis-
sion of ligand 2 in the presence of other metal ions under
similar conditions (Figure 6a). The quenched fluorescence
of ligand 2 was restored upon treating the ligand 2–2Cu2+
complex with ethylenediaminetetraacetic acid (EDTA;
Inset: Figure 6a). Fluorescence emission of ligand 2 with se-
quential addition of increasing concentrations (0 to 50 mm)
Competitive binding experiment: The selectivity of ligands 1
and 2 as Vis–NIR chemosensors for Cu2+ was studied in the
presence of various competing metal ions. For this purpose
ligands 1 and 2 were treated with a mixture of 5.0 equiva-
lents of Cu2+ and 10.0 equivalents of all other metal ions.
Data shown in Figure S2 (see the Supporting Information)
confirms that there is no interference for the detection of
Cu2+ in the presence of all other metal ions tested. The
aqua color of the solutions of ligands 1 and 2 persist even in
the presence of other metal ions in excess. Thus, ligands 1
and 2 can serve as highly selective colorimetric, as well as
NIR, sensors for Cu2+ in the presence of most competing
metal ions.
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Visible–Near-Infrared and Fluorescent Copper Sensors
FULL PAPER
perimentally observed photophysical characteristics of lig-
AHCTUNGTRENNUNG
ands 1 and 2 upon complexation with Cu2+, we have investi-
gated the structural, electronic, and optical properties using
ab initio density functional theory (DFT) combined with
time-dependent density functional theory (TDDFT) calcula-
tions as implemented in the Gaussian 03 package.[28] We
have adopted the hybrid B3LYP[29] exchange and correlation
functional using an effective core potential with the
LANL2DZ[30] basis set for transition-metal Cu and the 6-
31 gACTHNUGRTENUNG(d,p) basis set for all the other elements in the calcula-
tions. Jobꢁs plots and mass spectrometry analysis showed
that ligands 1 and 2 form complexes with Cu2+ in a 1:2 stoi-
chiometric ratio. Consequently, we have considered two
magnetic Cu2+ (Szt =1/2) complexes with 1 and 2 for the
DFT calculations. To find out the minimum energy magnetic
ground state, unrestricted DFT calculations were performed
considering both the high-spin (Szt =1) and low-spin (Szt =0)
states within the broken symmetry (BS) approach.[31] We
have also conducted vibrational energy calculations to con-
firm the local energy minimum structures. All of the DFT
optimized geometries are shown in Figure 7.
Our calculated results show that the two magnetic Cu2+
centers are coupled with ferromagnetic (FM) spin align-
ments when forming complexes with both the ligands 1 and
2. The FM state is stabilized over the antiferromagnetic
(AFM) state by 5.61 kcalmolꢀ1 for the ligand 1+2Cu2+
complex and 3.58 kcalmolꢀ1 for the ligand 2+2Cu2+ com-
plex (see Table S1 in the Supporting Information). To focus
on the strength of the magnetic coupling constant (J), we
make use of the simple 1D Heisenberg Hamiltonian for the
interaction of two spins[32] and have obtained the FM cou-
pling constants for both the complexes using the energy of
Figure 6. a) Fluorescence spectra of julolidine–thiocarbonohydrazone 2
(10.0 mm) and on addition of salts (20.0 equiv) of Li+, Na+, Ba2+, Sr2+
,
,
Mg2+, Al3+, Ca2+, Mn2+, Fe2+, Co2+, Ni2+, Zn2+, Ag+, Cd2+, Hg2+, Pb2+
and Cu2+ in aqueous medium (50 mm HEPES/CH3CN, 6:4, v/v; pH 7.2).
Inset: Fluorescence spectra of ligand 2 (10.0 mm), [2+Cu2+] and [2+
Cu2+ +EDTA] (10.0 mm). b) Fluorescence spectra of julolidine–thiocar-
bonohydrazone 2 (10.0 mm) on addition of different concentrations of
Cu2+ (0, 1, 2, 3, 5, 10, 20, 30, 40, 50, and 60 mm) in aqueous medium.
Inset: Intensity at 535 nm as a function of [Cu2+].
low-spin states calculated within
a BS approach (see
of Cu2+ is shown in Figure 6b.
This data shows that ligand 2
can easily detect copper ions at
least down to 1.0 mm in aqueous
medium. The reason behind the
fluorescence quenching of
2
upon complexation with Cu2+
was understood by theoretical
calculations as discussed in the
Theoretical
Study
section.
Ligand 2 can be used for detec-
tion of Cu2+ by fluorescence
“on–off” phenomena in a wide
pH (2–11) range in aqueous
medium (see Figure S10 in the
Supporting Information).
Theoretical Study
To understand the molecular
Figure 7. The optimized structures of julolidine–carbonohydrazone 1, julolidine–thiocarbonohydrazone 2 and
mechanism underlying the ex- their Cu2+ complexes. The numbers in each structure show selected important bond-length parameters in ꢃ.
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11157

T. Govindaraju et al.
Table S1 in the Supporting Information). As can be seen
from Table S1 (in the Supporting Information), the relative-
ly higher J value of 1+2Cu2+ complex results from the
strong super exchange interaction between the two magnetic
Cu2+ ions mediated by the oxygen bridge as opposed to the
sulfur bridge in 2+2Cu2+ complex. The spin density distri-
bution (See Figure S11 in the Supporting Information) for
both complexes also confirms the FM spin alignments of the
two copper ions. It should be noted that a significant
amount of spin density is distributed over the delocalized
conjugated p orbital in both complexes. To obtain an insight
on the charge-density profile and bonding aspects, we have
performed calculations of the natural bond orbital (NBO)
and natural electronic configuration. A significant amount
of charge transfer from ligand 1 and 2 to the Cu2+ center
was observed (see Table S1 in the Supporting Information).
This excess transferred electronic charge was found to be
mainly localized on the d orbital of the two Cu2+ centers.
The experimentally observed peaks in the visible and
NIR regions of the absorption spectra upon complexation
with Cu2+ were studied by using TDDFT computations on
ground-state optimized geometries of free ligands 1 and 2,
as well as their Cu2+ complexes. As shown in Figure 8, the
TDDFT-computed excitation energies for free ligands (1
and 2) are in quantitative agreements with the experimental
transition energies (Table S1 in the Supporting Information).
From an analysis of frontier molecular orbitals (FMO) the
lowest energy transition found consists of mainly promoting
the electron from the delocalized highest occupied molecu-
lar orbital (HOMO) to the lowest unoccupied molecular or-
bital (LUMO) delocalized over the entire molecule (see Fig-
ure S12 in the Supporting Information). Interestingly, the
observed shift in absorption peak position towards near-IR
in forming a charge-transfer complex between Cu2+ and lig-
tal findings (Figure 8). The relevant FMOs responsible for
these transitions are shown in Figure S12 (in the Supporting
Information). The b-spin orbital contributes mainly to the
observed new transition of these Cu2+ complexes. Here, it
should be pointed out that the observed red shifting in tran-
sition energy upon Cu2+ chelating compared only qualita-
tively with the experimental results. This may be due to the
fact that we have considered the complexation of two Cu2+
ions with each ligand (1 and 2) neglecting any coordination
from water molecules. To compare and contrast the effect of
water coordination on photophysical properties, we consider
two water molecules coordinating with Cu2+ in the ligand 2–
2Cu2+ complex (Figure 7).[33] Interestingly, the shifting of
the UV/Vis spectral peak position is less in comparison to
the water-free coordination complex (Figure 8, bottom
panel), which results in good agreement with the experimen-
tally observed shift in peak position.
To investigate the mechanism (energy transfer and/or
charge transfer process) of fluorescence-quenching behavior
upon chelation with Cu2+, we have performed excited-state
geometry optimization and subsequent single-point transi-
tion-energy calculations employing the TDDFT method as
implemented in the Gaussian suite of programs for com-
pound 2 in the presence of Cu2+ and two explicit water mol-
ecules. From an analysis of FMOs corresponding to the
lowest energy transition the fluorescence quenching by Cu2+
could be rationalized in terms of the occupancy of FMOs.
As shown in Figure 9, the HOMO (b)!LUMO+1 (b),
HOMOꢀ1 (b)!LUMO+1 (b), and HOMOꢀ2 (b)!
LUMO+1 (b) electronic excitations are found to be rele-
vant for the lowest energy fluorescence process showing pre-
dominantly ligand-to-metal charge transfer (LMCT) and
their contributions to the lowest energy excitation are 24,
18, and 9%, respectively. The transferred charge is mainly
localized on the two Cu2+ centers, as well as on its nearby
atoms. Note that the calculated values are comparatively
small and strongly dependent on the method and basis sets
used for their computations. Depending on this, the extent
of excited-state charge transfer may significantly alter and
cause the fluorescence quenching. These excitations corre-
spond to the charge transfer from the excited state of ligand
ACHTUNGTRENNUNGands 1 and 2 corroborates qualitatively with the experimen-
Figure 8. The calculated absorption spectra for 1 (i), 2 (iii) and their Cu2+
complexes: ii) 1+2Cu2+, iv) 2+2Cu2+, and v) 2+2Cu2+ +2H2O. The in-
dices a (H!L), b (H-9(b)!L(b)), and c (H(b)!L(b)) and a*
(H!L),
Figure 9. The relevant frontier molecular orbitals (FMOs) of the 2+
2Cu2+ +2H2O complex corresponding to the excited-state charge-transfer
process. The symbols H and L represent the highest occupied molecular
orbital and lowest unoccupied molecular orbital, respectively.
11158
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Chem. Eur. J. 2011, 17, 11152 – 11161

Visible–Near-Infrared and Fluorescent Copper Sensors
FULL PAPER
2 to the Cu2+ center (LMCT) and thus provide a pathway
for the nonradiative deactivation of the excited state. Thus,
fluorescence-quenching behavior can be accounted for pri-
mented with EDTA (10 mm) in DMEM without FBS for
10 min at 378C to recover the fluorescence. Since the cells
stained with ligand 2 show a clear-cut cytoplasmic localiza-
tion these studies clearly suggested that ligand 2 is cell-per-
meable and can respond to copper ions within living cells.
marily due to the excited-state ligand (2) to metal (Cu2+
)
charge transfer (LMCT) processes.
Bioimaging
Conclusion
Subsequent experiments proved the ability of julolidine–
thio
carbonohydrazone (2) to track Cu2+ levels in living cells
We have established a design strategy to develop new Vis–
NIR chemosensors for Cu2+ detection. Julolidine–carbono-
hydrazone 1 and julolidine–thiocarbonohydrazone 2 can in-
dependently detect Cu2+ with high selectivity in the pres-
ence of other metal ions with a characteristic NIR signature
in aqueous medium. Ligands 1 and 2 can be used for detec-
ACHTUNGTRENNUNG
by using fluorescence microscopy. HEK293T cells were
grown to 50% confluency in a 30 mm dish with Dulbeccoꢁs
modified eagle medium (DMEM) and 10% fetal bovine
serum (FBS) at 378C and 5% CO2. The cells were washed
thrice with phosphate-buffered saline (PBS) and stained
with ligand 2 (10 mm) in the growth media without FBS for
10 min. The adherent cells were washed thrice with PBS to
remove excess stain. Fluorescence emission was observed in
the optical window at 450–650 nm as shown in Figure 10.
The stained cells were subsequently supplemented with Cu-
tion with the naked eye of different concentrations of Cu2+
,
since they exhibit distinct colors in aqueous solution and
therefore one can easily assess the rough concentration
levels of Cu2+ in analyte samples. In addition, fluorescent
julolidine–thiocarbonohydrazone 2 selectively senses Cu2+
by fluorescence quenching in the presence of all other metal
ions under aqueous conditions. The quenched fluorescence
emission of ligand 2 can be restored by the addition of
EDTA. We have further demonstrated the utility of ligand 2
as a reversible biosensor by employing it for living-cell
imaging of Cu2+ in HEK293T cells. Results obtained from
theoretical calculations corroborate the experimental find-
ings and provide us with a detailed microscopic understand-
ing of the observed NIR and fluorescence properties of lig-
ACHTUNGTRENNUNG(ClO4)2·6H2O (10 mm) in DMEM without FBS at 378C and
5% CO2 for 10 min and then intracellular fluorescence was
almost completely suppressed. Finally, the excess Cu-
ACHTUNGTRENNUNG(ClO4)2·6H2O was washed off with PBS and then supple-
AHCTUNGTRENNUNG
ands 1 and 2 upon Cu2+ chelation.
Acknowledgements
We thank Prof. C. N. R. Rao, FRS for constant support, JNCASR and
the Department of Science and Technology (DST), India for financial
support and CSIR, New Delhi, India for awarding a JRF to D.M. and
A.K.M.
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Published online: September 1, 2011
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