Imaging intracellular zinc by using a glyoxal bis(4-methyl-4-phenyl-3- thiosemicarbazone) ligand

Article abstract of DOI:10.1002/ejic.201300324

The ligand glyoxal bis(4-methyl-4-phenyl-3-thiosemicarbazone) (GTSCH ₂) is shown to be a selective fluorescence "turn-on" sensor for zinc ions (Zn²⁺). This sensor is easy to synthesize, exhibits excellent sensitivity and selectivity

Full text of DOI:10.1002/ejic.201300324

FULL PAPER
DOI:10.1002/ejic.201300324
Imaging Intracellular Zinc by Using a Glyoxal Bis(4-
methyl-4-phenyl-3-thiosemicarbazone) Ligand
Duraippandi Palanimuthu,[a] Sridevi Vijay Shinde,[b] Disha Dayal,[b]
Kumaravel Somasundaram,*[b] and Ashoka G. Samuelson*[a]
Keywords: Zinc / Thiosemicarbazones / Imaging agents / Fluorescence / Sensors
The ligand glyoxal bis(4-methyl-4-phenyl-3-thiosemicarb-
azone) (GTSCH2) is shown to be a selective fluorescence
“turn-on” sensor for zinc ions (Zn2+). This sensor is easy to
synthesize, exhibits excellent sensitivity and selectivity
towards Zn2+ over other physiologically relevant cations, and
has sub-nanomolar binding affinity. It displays maximum
fluorescence response to Zn2+ when the metal/ligand ratio is
1:1 and displays stable fluorescence over a broad pH range.
The potential of GTSCH2 to image Zn2+ inside the cell was
demonstrated in MCF-7 cells (human breast cancer cell line)
by using flow cytometry and confocal fluorescence micro-
scopy. Cell viability studies reveal that the probe is biocom-
patible and suitable for cellular applications.
Histochemistry- and radioactivity-based methods have
been extensively employed to monitor the concentration of
Zn2+ in cells, but inherent problems in these methods have
motivated chemists and biologists to find alternative tools
for better visualization with good optical resolution.[7] Con-
focal fluorescence microscopy is well suited and has been
widely used in recent times for in situ measurements of
cellular events such as cellular uptake, localization, traffick-
ing, and efflux processes in real time with spatial and tem-
poral resolution.[8]
Introduction
Zinc is an essential nutrient for all three domains of life
that include archaea, bacteria, and eukaryota.[1] Unlike
many other transition-metal ions, redox-inactive Zn2+
serves as a cofactor to bring about various catalytic trans-
formations in all six classes of enzymes owing to its excel-
lent Lewis acid properties.[2] It is also the second most
abundant transition-metal ion after iron in the human body
and plays a pivotal role in the regulation of various meta-
bolic processes, transcription factors, immune functions, ion
channels, and neurotransmission.[3] The concentration of
zinc in cells is in the millimolar range, but the concentration
of the free and loosely bound forms of Zn2+ in the cytosol
can vary from the picomolar to micromolar range.[4]
Whereas the intracellular concentration of Zn2+ is tightly
controlled by regulating proteins, some cell types, in par-
ticular neuronal and pancreatic beta cells, accumulate high
concentrations of free Zn2+ for a special function such as
neurotransmission or insulin secretion.[5] Upsetting Zn2+
homeostasis leads to various diseases and disorders such as
cancer, diabetes, and many forms of neurodegeneration.[6]
To detect Zn2+ in a living system, various cell-permeable
fluorescent chemosensors having binding affinities for Zn2+
in the millimolar to femtomolar range have been devel-
oped.[9] Most of them utilize dyes such as cyanine,[10]
fluorescein,[11] and rhodamine[12] as fluorophores. Gen-
erally, these molecules are functionalized and attached to
various groups that would be responsible for binding Zn2+
selectively. Whereas most fluorescent sensors have nano-
molar to micromolar binding affinities for Zn2+ [13]
a few
,
of them have sub-nanomolar binding affinity for zinc.
These utilize 2-picolylamine-[14] and quinoline-based[15] che-
lating ligands as receptors because of their high binding af-
finity for Zn2+. The Zn2+ binding group quenches the
fluorescence of the dye in its apo form by a through-space
photoinduced electron-transfer (PeT) process.[16] However,
the fluorescence intensity is enhanced if Zn2+ binds to the
receptor part, which makes it a turn-on fluorescence sensor.
Though these chemosensors exhibit excellent quantum
yields upon complexation with Zn2+, the emission spectra
of these complexes overlap with the fluorescence of the free
ligand as a result of a small Stokes shift.[17] In addition,
these sensors may not be useful for the quantitative deter-
mination of Zn2+ owing to variations in excitation and
emission intensity in local cellular environments, photo-
[a] Department of Inorganic and Physical Chemistry,
Indian Institute of Science,
Bangalore 560012, India
Fax: +91-80-2360-1552
E-mail: ashoka@ipc.iisc.ernet.in
Homepage: http://ipc.iisc.ernet.in/~ashoka/
[b] Department of Microbiology and Cell Biology,
Indian Institute of Science,
Bangalore 560012, India
Fax: +91-80-2360-2697
E-mail: skumar@mcbl.iisc.ernet.in
Homepage: http://mcbl.iisc.ernet.in/KumarS/
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejic.201300324.
Eur. J. Inorg. Chem. 2013, 3542–3549
3542
© 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.eurjic.org
FULL PAPER
bleaching, and uneven dye loading.[18] Some of these diffi- amined by UV/Vis spectroscopy in an aqueous 3-morphol-
culties can be overcome by carrying out a ratiometric mea- inopropanesulfonic acid (MOPS) buffer (MOPS, 50 mm;
surement of the emission intensities of the metal-free ligand NaCl, 100 mm; pH = 7.3) containing 30% DMSO (v/v) at
and the complexed ligand. Because the ratio between the physiological pH. As shown in Figure 2, the UV/Vis spec-
two emission intensities is not affected by cellular environ- trum of GTSCH2 exhibits an intense band in the UV region
ments, it permits quantitation.[19] The requirement for UV with a maximum at 336 nm (ε = 4.0ϫ104 m–1cm–1). When
excitation by ratiometric sensors and special instrumen- titrated with increasing concentrations of Zn2+ (0–
tation required to monitor the multiphoton excitation and 1.4 equiv.), a new band appears in the visible region cen-
emission limit their usage in cellular studies.[20]
tered at 458 nm (ε = 1.6ϫ104 m–1cm–1 at 1:1 ratio) with a
Thus, developing sensors based on different ligand sys- concomitant decrease in the absorption band at 336 nm (ε
tems having sub-nanomolar binding affinity for Zn2+ and = 1.0ϫ104 m–1cm–1 at 1:1 ratio), which is attributed to a
having better selectivity for Zn2+ over other physiologically ligand-to-metal charge-transfer (LMCT) transition (Fig-
abundant metal ions is of great interest. More importantly, ure 2). Appearance of a distinct isosbestic point at 380 nm
if the fluorescent dye is integrated with the Zn2+ receptor indicates the formation of single species. Spectral changes
and if it has a large Stokes shift upon binding zinc, it would indicating the formation of the zinc complex were com-
be even better. Such a system was realized by Dilworth et pleted within 5 min. A plot of the absorbance at 458 nm
al. who showed the fluorescence of zinc–bis(thiosemicarb- (A458) versus [Zn2+]/[GTSCH2] resulted in a linear increase
azone) complexes and their distribution in various cancer in the absorbance until a 1:1 ratio of GTSCH2/Zn2+ was
cell lines by using confocal fluorescence microscopy.[21] Re- attained, and further incremental additions of Zn2+ did not
cently, Wedd and co-workers reported a water-soluble bis- show any enhancement in the absorption (Figure 2). Job’s
(thiosemicarbazone), which is an absorption-based chemo- plot analysis further confirmed the 1:1 binding of GTSCH2
sensor for Zn2+ with a high binding affinity of 5.9 nm for and Zn2+ in solution (Figure S2, Supporting Information).
zinc.[22] In general, bis(thiosemicarbazones) are easy to syn-
thesize, have good chelating ability with transition-metal
ions, and are cell-permeable.[23] We have studied a series of
bis(thiosemicarbazone) ligands and their zinc and copper
complexes. Among them glyoxal bis(4-methyl-4-phenyl-3-
thiosemicarbazone) (GTSCH2) forms a novel trimeric zinc
complex [Zn(GTSC)]3 that has been used as a live cell-im-
aging agent,[24] whereas the corresponding copper com-
plexes have been utilized as anticancer agents.[25]
Here, we show the utility of the GTSCH2 ligand (Fig-
ure 1) for imaging intracellular Zn2+. The GTSCH2 probe
exhibited excitation and emission in the visible region upon
binding Zn2+. It is selective towards Zn2+ even in the pres-
ence of biologically abundant metal ions and has a binding
affinity in the sub-nanomolar range. It is cell-permeable,
exerts minimal cytotoxicity, and is capable of imaging intra-
cellular zinc.
Figure 2. UV/Vis spectra of GTSCH2 (50 μm) in aqueous buffer
containing MOPS (MOPS, 50 mm; NaCl, 100 mm; pH = 7.3) and
DMSO (30% v/v) in the presence of various concentrations of Zn2+
ranging from 0 to 70 μm. Inset plot of A458 versus [Zn2+]/[GTSCH2]
shows the 1:1 binding nature.
The fluorescence spectra of GTSCH2 (50 μm) in the ab-
sence of and in the presence of Zn2+, are shown in Figure 3
following excitation at 420 nm. Virtually no fluorescence
was exhibited by the ligand, but it showed an emission band
in the green region of the visible spectrum centered at
560 nm with a large Stokes shift of 102 nm upon complex-
ation with Zn2+ (Figure 3). The observed fluorescence in-
tensity increases with increasing concentration of Zn2+ until
1 equiv. is reached. Excitation at 380 nm produced a similar
emission profile. Thus, fluorescence-based binding analysis
Figure 1. Structure of the glyoxal bis(4-methyl-4-phenyl-3-thio-
semicarbazone) (GTSCH2) ligand.
Results and Discussion
The ligand, GTSCH2, was prepared according to a litera- is consistent with the UV/Vis absorption experiment. No-
ture procedure, as shown in Scheme S1 (Supporting Infor- tably, the addition of an equimolar amount of Zn2+ to a
mation).[24] The Zn2+–GTSCH2 binding behavior was ex- solution of GTSCH2 induced a 5.5-fold enhancement in the
Eur. J. Inorg. Chem. 2013, 3542–3549
3543
© 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.eurjic.org
FULL PAPER
fluorescence. The fluorescence quantum yield for the Zn2+
–
GTSCH2 (1:1) mixture in DMSO was estimated to be
2.0ϫ10–3 by using [Ru(bpy)3](PF6)2 (Φf = 42ϫ10–3 in
water, bpy = 2,2Ј-bipyridyl) as a standard.[26] However, the
fluorescence intensity was quenched by 25% in aqueous
phosphate-buffered saline (PBS) solution containing 30%
(v/v) of DMSO at pH = 7.3.
Figure 4. High-resolution mass spectrum of a solution containing
GTSCH2 (50 μm) and zinc nitrate (50 μm) in DMSO. The solution
was diluted with methanol prior to spectral acquisition. Inset
shows the measured spectrum (red line) of the major species and
the calculated isotopic distribution profile (black line).
The fluorescence sensitivity of GTSCH2 for Zn2+ and
other biologically available metal ions was investigated by
titration with various metal ions, and the results are shown
in Figure 5a and b (blue bar). This fluorescence response
study of GTSCH2 (50 μm) with Zn2+ (1 equiv.) exhibited
“turn-on” fluorescence, whereas other divalent transition-
metal ions including Cu2+, Ni2+, Co2+, Fe2+, and Mn2+ did
not induce fluorescence even at concentrations that were
10-fold higher (0.5 mm). When the experiment was carried
Figure 3. Fluorescence spectra of GTSCH2 (50 μm) in DMSO and
changes observed upon addition of a zinc nitrate solution (0–
80 μm) in DMSO. Inset plot showing the 1:1 stoichiometric binding
nature, as monitored by fluorescence intensity at 560 nm.
The nature of the species present in solution was probed out with ubiquitous intracellular metal ions such as K+,
with soft ionization HRMS (ESI–QTOF). The Zn2+ Na+, Ca2+, and Mg2+, which exist at very high concentra-
–
GTSCH2 (1:1 mixture) in DMSO was diluted with meth- tions inside the cell, no significant fluorescence was ob-
anol for mass spectrometry. Mass spectral analysis in the served, even at concentrations that were 100-fold higher
positive ion mode showed a major peak at m/z (%) = (5 mm; Figure 5a and b, blue bar).
447.0395 (100), which was assigned to the Zn(GTSC)
Metal-ion selectivity was also examined to probe if
monomer (Figure 4). Additionally, the measured isotopic GTSCH2 could be used as a selective sensor for Zn2+ in the
pattern matched the simulated spectrum of the monomer. presence of other competitive cations found in biological
We observed trace amounts of dimeric (ca. 2%) and tri- systems. Emission spectra were measured for a 1:1 mixture
meric (ca. 0.3%) species in the mass spectrum. This sug- of GTSCH2 and Zn2+ in the presence of other metal ions.
gested that the ligand forms a 1:1 complex with Zn2+ very The prominent fluorescence enhancement observed upon
readily. Signature peaks for GTSCH2 were not observed in mixing GTSCH2 and Zn2+ remained unchanged, even in
the spectrum, which indicates the complete conversion of the presence of a 100-fold excess of metal ions such as K+,
the ligand into the zinc complex. It also suggests that in Na+, Ca2+, and Mg2+ (Figure 5b, red bar). This confirms
solutions of methanol, the major species is the monomer. the excellent selectivity of GTSCH2 for Zn2+ over other
It is likely that the polar methanol solvent shifts the equilib- abundant cations in the cell. Notably, the fluorescence of
rium in favor of the monomer.
the zinc complex was completely quenched in the presence
The binding affinity (Kd) of GTSCH2 for Zn2+ was esti- of metal ions such as Cu2+, Ni2+, and Co2+ when used in
mated by using competitive binding with ethylene glycol- 10-fold excess. It is interesting to note that the fluorescence
bis(2-aminoethylether)-N,N,NЈ,NЈ-tetraacetic acid (EGTA), of GTSCH2 in the presence of Zn2+ is not quenched by
a suitable competitor having a known dissociation constant other first-row transition-metal ions such as Mn2+ and Fe2+
of 1 nm to Zn2+ at pH = 7.3.[27] The binding event was probably as a result of poor binding affinity (Figure 5b, red
monitored by UV/Vis spectroscopy (Figure S3, Supporting bar).
Information). On the basis of the occupancy of Zn2+ in
To understand the effect of pH on the fluorescence inten-
both GTSCH2 and EGTA, the Kd value was estimated to sity of GTSCH2 in the absence of and in the presence of
be 0.53 nm in aqueous MOPS (MOPS, 50 mm; NaCl, Zn2+ (1 equiv.), the emission spectra were measured as a
100 mm; pH = 7.3) buffer containing 30% (v/v) DMSO function of pH (varied from 3 to 11.2) with gradual ad-
(Table S4, Supporting Information). This sub-nanomolar dition of NaOH. As shown in Figure 6, the ligand itself
binding affinity makes the probe suitable for cellular appli- does not display significant changes in its fluorescence pro-
cations.
file in both acidic and basic media. Upon addition of Zn2+
Eur. J. Inorg. Chem. 2013, 3542–3549
3544
© 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.eurjic.org
FULL PAPER
Figure 6. The pH-dependent fluorescence response of GTSCH2
(50 μm) in the absence and in the presence of Zn2+ (1 equiv.) in
universal pH buffer containing 30% DMSO (v/v). The emission
spectra were recorded following excitation at 420 nm, and the
fluorescence intensity at 550 nm was plotted against pH increasing
from 3.0 to 11.2.
meter after excitation at 488 nm. As expected, GTSCH2-
treated cells showed no significant increase in fluorescence
relative to that shown by vehicle-treated cells at all the con-
centrations tested (Figure 7a, b). In a separate experiment,
the concentration of zinc inside the cell was elevated by sup-
plementing cells with zinc (100 μm) along with pyrithione
(20 μm), a zinc-specific ionophore that facilitates the trans-
port of zinc ions across the cell membrane, at room tem-
perature for 30 min. After incubation, the cells were washed
thoroughly with fresh PBS buffer to remove extracellular
Zn2+ and then incubated with GTSCH2 (10–40 μm) for an
additional 30 min. The resulting histogram showed a 6.3-
fold increase (at 10 μm of GTSCH2) in the cellular fluores-
cence relative to that shown by cells that were treated with
GTSCH2 alone. This is in good agreement with the fluores-
cence enhancement obtained from zinc titration studies
(Figure 3). However, the intensity did not significantly de-
crease/increase with the addition of increasing amounts of
GTSCH2, and this indicates that 10 μm is sufficient to ob-
tain maximal fluorescence. Further incubation of cells
with N,N,NЈ,NЈ-tetrakis(2-pyridylmethyl)ethylenediamine
(TPEN, 100 μm), a cell-permeable and high-affinity zinc
chelator (Kd = 92 fm), for 10 min resulted in a 3.3-fold
Figure 5. (a) Fluorescence spectra of GTSCH2 (50 μm) in the pres-
ence of various metal ions of interest at different concentrations in
DMSO following excitation at 420 nm (0.5 mm of Cu2+, Ni2+
,
Co2+, Fe2+, and Mn2+; 5 mm of Ca2+, Mg2+, K+, and Na+).
(b) Metal-ion sensitivity and selectivity for GTSCH2. Blue bars
represent the fluorescence sensitivity of GTSCH2 (50 μm) to vari-
ous metal ions (quantified from Figure 5a). Red bars represent the
fluorescence response measured after the addition of Zn2+ (50 μm)
to the indicated metal ion–GTSCH2 (10:1 for transition metal ions
and 100:1 for alkali and alkaline earth metal ions) mixture. The
response was quantified as F/F0, for which the integrated emission
(500–750 nm) of a 1:1 mixture of Zn2+ and GTSCH2 (50 μm) in the
absence (F0) and in the presence of other metal ions (F).
(1 equiv.) to GTSCH2, an approximate sevenfold enhance- quenching (at 10 μm of GTSCH2) of cellular fluorescence,
ment in fluorescence was observed at pH = 5.0–7.8. How- possibly by trans-chelation of Zn2+ from Zn(GTSC) to
ever, the fluorescence intensity decreased dramatically when TPEN.[29] These results show that GTSCH2 forms a zinc
the pH dropped below 5.0 probably owing to the instability complex by utilizing free zinc in the cells.
of the complex. The excellent fluorescence response of
The potential of GTSCH2 to enter the cell and image
GTSCH2 with Zn2+ in the pH range from 5.0 to 7.8 sug- intracellular zinc was examined on MCF-7 cells by using
gests that the probe may be suitable for imaging zinc in confocal fluorescence microscopy following excitation at
both normal cells and slightly acidic cancer cells.[28]
488 nm. The cells were treated with GTSCH2 (10 μm) at
To test whether GTSCH2 has the ability to form a zinc 37 °C for 30 min with and without pretreatment with exter-
complex within the cell, MCF-7 (human breast cancer) cells nal zinc (100 μm). As shown in Figure 8e–h, treatment of
suspended in PBS buffer (pH = 7.4) were incubated either GTSCH2 (10 μm) did not increase the green fluorescence
with vehicle [1% DMSO (v/v) in PBS] or with increasing relative to that of vehicle-treated MCF-7 cells (Figure 8a–
concentrations of GTSCH2 (10–40 μm) at room tempera- d). In contrast, the addition of GTSCH2 (10 μm) to cells
ture for 30 min in the presence of or in the absence of added having transiently increased amounts of Zn2+ [pretreated
zinc. Cell fluorescence was quantified by using a flow cyto- with zinc acetate (100 μm) and pyrithione (20 μm) for
Eur. J. Inorg. Chem. 2013, 3542–3549
3545
© 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.eurjic.org
FULL PAPER
Figure 8. Confocal fluorescence imaging of MCF-7 cells treated
with GTSCH2 in the presence of or in the absence of Zn2+. Treat-
ment of MCF-7 cells with 1% DMSO in phenol red-free DMEM
media (a–d) or 10 μm GTSCH2 (e–h) at 37 °C for 30 min. Cells
supplemented with zinc acetate/pyrithione (5:1) for 30 min and
then treated with GTSCH2 (10 μm) at 37 °C for 30 min (i–l). After
addition of TPEN (100 μm) to the cells containing GTSCH2 and
Zn2+ at 37 °C for 30 min (m–p). After all treatments, cells were
fixed by using 4% paraformaldehyde, and images were captured
immediately in fluorescence and brightfield modes by using a Zeiss
confocal fluorescence microscope at 63ϫ magnification. Cells were
treated with the nucleus stain propidium iodide (shown in red: b,
c, f, g, j, k, n, and o).
Figure 7. (a) MCF-7 cells treated with 1% DMSO (black line),
GTSCH2 (10 μm) before (blue line) and after treatment with zinc
acetate (100 μm) along with pyrithione (20 μm) for 30 min (green
line). Addition of TPEN (100 μm) to the cells that were preincu-
bated with zinc acetate (100 μm) along with pyrithione (20 μm) for
30 min followed by GTSCH2 (10 μm) for 30 min (red line). The cell
fluorescence was measured by using a flow cytometer. (b) Histo-
gram showing mean fluorescence intensity (MFI) from cells that
were treated with 1% DMSO (black bar) or different concentra-
tions of GTSCH2 (10, 20, 30, and 40 μm) (blue bar) or zinc acetate
(100 μm) along with pyrithione (20 μm) followed by GTSCH2 (10,
20, 30, and 40 μm) (green bar). MFI after addition of TPEN
(100 μm) to the cells containing GTSCH2 and Zn2+ (red bar).
viability graphs are shown in Figure 9. The IC50 values, cor-
responding to the cytotoxic activity, obtained from cell via-
bility graphs are shown in Table 1. The IC50 values obtained
for GTSCH2 are similar to those of its zinc complex in the
HaCaT cell line, whereas the IC50 values were found to be
higher (Ͼ 1.6-fold) than those of the zinc complex in the
HepG2 and MCF-7 cell lines. Moreover, treatment of cells
with 10 μm of GTSCH2, the concentration used for im-
aging, did not affect the cell viability significantly, as 90–
99% of the cells were viable in all the cell lines tested, even
after 48 h. Overall, the cell viability studies confirmed that
GTSCH2 is biocompatible even for long periods (48 h) and
at high concentrations.
30 min] significantly enhanced cellular fluorescence (Fig-
ure 8i–l), and the observed fluorescence was quenched by
63% with subsequent addition of TPEN (100 μm; Fig-
ure 8m–p). Co-staining of cells with propidium iodide (PI),
a nuclear stain, clearly revealed that the probe was localized
in the cytoplasm of the cell. All these results confirm that
the probe is cell-permeable and that it is capable of moni-
toring intracellular zinc. Control experiments with the vehi-
cle [1% DMSO (v/v) in phenol-red-free Dulbecco’s Modi-
fied Eagle’s Medium (DMEM)] or zinc acetate along with
pyrithione-treated cells in the absence of GTSCH2 did not
show any fluorescence, which points to the fact that the
observed fluorescence is due to the formation of an intracel-
lular zinc complex with GTSCH2.
Table 1. IC50 values[a] as evaluated from an MTT assay in a panel
of three cancer cell lines.
Cell line
IC50 value [μm]
[Zn(GTSC)]3
Given that imaging probes should be ideally nontoxic,
we evaluated the viability of human-derived breast cancer
cell line MCF-7, hepatoma cancer cell line HepG2, and im-
mortalized keratinocyte cell line HaCaT by MTT assay af-
ter exposure to GTSCH2 for 48 h. The corresponding zinc
GTSCH2
MCF-7
HepG2
HaCaT
Ͼ50.0
53.6Ϯ3.5
47.5Ϯ3.5
23.3Ϯ6.0
32.5Ϯ1.5
47.0Ϯ1.4
[a] Data are presented as the mean of three independent experi-
complex [Zn(GTSC)]3 was included for comparison. Cell ments.
Eur. J. Inorg. Chem. 2013, 3542–3549
3546
© 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.eurjic.org
FULL PAPER
Experimental Section
Materials and Methods: The synthesis and characterization of the
GTSCH2 ligand is given in our earlier work.[24] [Ru(bpy)3](PF6)2
(bpy = 2,2-bipyridine) and TPEN were synthesized and recrys-
tallized according to literature procedures.[30] MOPS was purchased
from SRL, Mumbai, India. EGTA and 2-pyridine N-oxide
(pyrithione) were procured from Aldrich. Mass spectra were re-
corded with an Agilent 6538 High accurate-QTOF-LC–MS instru-
ment. All the UV/Vis experiments were carried out with a Perkin–
Elmer Lambda 35 instrument. Fluorescence measurements were
carried out with a Horiba Jobin Yvon Fluoromax-4 spectro-
fluorimeter. All solutions and buffers were prepared by using puri-
fied water from Milli-Q Biocel made by Millipore. For studying the
effect of pH, a stock solution of universal buffer (Britton-Robinson
buffer) containing 0.04 m of boric acid, phosphoric acid, and acetic
acid was prepared according to a literature procedure.[31] The solu-
tion was adjusted to the pH range from 3.0 to 11.2 by adding incre-
mental amounts of NaOH (0.2 m). DMEM media, penicillin, and
streptomycin were obtained from Sigma (USA). Sterile 96-well,
flat-bottomed tissue-culture plates and other plastic ware were pur-
chased from Tarsons (India). Fetal calf serum (FCS) and 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
used for growth inhibition assays were obtained from Sigma Chem-
ical Co. (India).
Flow Cytometry: MCF-7 cells were cultured in DMEM media,
trypsinized, washed with PBS (pH = 7.4), and centrifuged. The
resulting pellet was resuspended in PBS buffer (pH = 7.4, 0.5 mL)
in a FACS tube. Stock solutions of GTSCH2, zinc acetate, pyri-
thione, and TPEN were prepared in DMSO (HPLC grade). In the
first set of experiments, GTSCH2 (10, 20, 30, and 40 μm) was added
to the cells and incubated at room temperature for 30 min. In the
second set, cells were pretreated with zinc acetate (100 μm) and pyr-
ithione (20 μm). After 30 min of incubation at room temperature,
the cells were pelleted by centrifuge, washed with PBS buffer, and
resuspended in fresh PBS buffer (0.5 mL). GTSCH2 (10, 20, 30,
and 40 μm) was then added, and the cells were incubated at room
temperature for an additional 30 min. In the third set of experi-
ments, the same conditions used for the second set were used.
TPEN was then added, and the cells were incubated for another
10 min. In a control experiment, DMSO [1% (v/v) in PBS] was
added, and the cells were incubated for 30 min. Finally, the samples
were subjected to flow cytometry by using Beckman Coulter CyAn
flow cytometer analysis, and the fluorescence was acquired in the
FL1 channel for 10000 counts. The forward- and side-scattering
parameters were adjusted to keep the background fluorescence to
a minimum.
Figure 9. Effect of increasing concentrations of GTSCH2 and
[Zn(GTSC)]3 (in zinc) on the cell viability of (a) MCF-7,
(b) HepG2, and (c) HaCaT cells after 48 h of treatment. Values are
expressed as the mean of triplicates Ϯ the standard deviations of
one experiment.
Confocal Fluorescence Imaging: Fluorescence imaging was carried
out in MCF-7 cells with and without added Zn2+ by using a Zeiss
LSM 510 META confocal fluorescence microscope. The cells were
grown in DMEM medium in a 6-well dish containing circular cover
Conclusions
We have shown for the first time that the bis(thiosemicar- slips. The next day, the medium was replaced by phenol-red-free
DMEM medium (pH = 7.4) containing 10% FCS, penicillin, and
streptomycin. The GTSCH2 ligand (10 μm) and DMSO [1% (v/v)]
were added to separate wells, and the cells were incubated at 37 °C
for 30 min. In a third well, zinc acetate (100 μm) along with pyri-
thione (20 μm) was added. After incubation at 37 °C for 30 min, the
cells were washed with fresh medium and incubated with GTSCH2
(10 μm) at 37 °C for 30 min. The fourth well was given the same
treatment as the third but washed with medium, and then TPEN
(100 μm) was added, and the cells were incubated at 37 °C for
30 min. After various treatments, the spent medium was removed,
and the cells were fixed by using 4% paraformaldehyde. Cells were
bazone) ligand GTSCH2 can be used as a zinc-specific sen-
sor. The GTSCH2 probe forms a complex with Zn2+ rapidly
and selectively. The potential of GTSCH2 in imaging intra-
cellular zinc was demonstrated successfully in the MCF-7
human breast cancer cell line by using flow cytometry and
confocal fluorescence microscopy, in which the probe was
found to be localized in the cytoplasm. The probe was also
found to be noncytotoxic to a range of cell lines. All these
properties make GTSCH2 an excellent ligand that is highly
suitable for imaging Zn2+ in biological systems.
Eur. J. Inorg. Chem. 2013, 3542–3549
3547
© 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.eurjic.org
FULL PAPER
then treated with RNAse (200 μgmL–1 in PBS) for 15 min followed
by propidium iodide (1 μgmL–1 in PBS) for 15 min. Finally, the
coverslips were removed and mounted on glass slides, and fluores-
cence images were captured immediately at an excitation of 488 nm
for Zn–GTSCH2 (green) and 543 nm for PI (red). The correspond-
ing brightfield images were also captured.
Curr. Opin. Chem. Biol. 2004, 8, 182–191; e) E. Tomat, S. J.
Lippard, Curr. Opin. Chem. Biol. 2010, 14, 225–230; f) M. D.
Pluth, E. Tomat, S. J. Lippard, Annu. Rev. Biochem. 2011, 80,
333–355; g) K. Komatsu, K. Kikuchi, H. Kojima, Y. Urano, T.
Nagano, J. Am. Chem. Soc. 2005, 127, 10197–10204; h) M. M.
Henary, Y. Wu, C. J. Fahrni, Chem. Eur. J. 2004, 10, 3015–
3025; i) R. A. Bozym, R. B. Thompson, A. K. Stoddard, C. A.
Fierke, ACS Chem. Biol. 2006, 1, 103–111; j) Y. Hitomi, C. E.
Outten, T. V. O’Halloran, J. Am. Chem. Soc. 2001, 123, 8614–
8615.
a) B. Tang, H. Huang, K. Xu, L. Tong, G. Yang, X. Liu, L. An,
Chem. Commun. 2006, 3609–3611; b) K. Kiyose, H. Kojima, Y.
Urano, T. Nagano, J. Am. Chem. Soc. 2006, 128, 6548–6549.
a) G. K. Walkup, S. C. Burdette, S. J. Lippard, R. Y. Tsien, J.
Am. Chem. Soc. 2000, 122, 5644–5645; b) S. C. Burdette, G. K.
Walkup, B. Spingler, R. Y. Tsien, S. J. Lippard, J. Am. Chem.
Soc. 2001, 123, 7831–7841; c) C. J. Chang, E. M. Nolan, J. Ja-
worski, S. C. Burdette, M. Sheng, S. J. Lippard, Chem. Biol.
2004, 11, 203–210; d) E. M. Nolan, J. W. Ryu, J. Jaworski, R. P.
Feazell, M. Sheng, S. J. Lippard, J. Am. Chem. Soc. 2006, 128,
15517–15528; e) E. M. Nolan, S. J. Lippard, Inorg. Chem. 2004,
43, 8310–8317.
MTT Assay: MTT assay was carried out for GTSCH2 and its zinc
complex on the human breast cancer cell line MCF-7, hepatoma
cancer cell line HepG2, and immortalized keratinocyte cell line
HaCaT to measure cell viability. An appropriate number of cells
was seeded onto a 96-well plate. After 24 h, varying concentrations
of either GTSCH2 or [Zn(GTSC)]3 were added, and the cells were
incubated in a 37 °C incubator for 48 h. After 45 h of incubation,
MTT was added, and the absorbance was read 3 h later on a micro-
plate reader (Molecular devices M5e) at 550 nm. A graph of con-
centration versus percentage cell viability gave the IC50 value, a
concentration at which 50% cell death occurred.
[10]
[11]
Supporting Information (see footnote on the first page of this arti-
cle): Synthetic scheme, Job’s plot, and binding affinity data.
[12]
a) S. L. Sensi, D. Ton-That, J. H. Weiss, A. Rothe, K. R. Gee,
Cell Calcium 2003, 34, 281–284; b) S. L. Sensi, D. Ton-That,
P. G. Sullivan, E. A. Jonas, K. R. Gee, L. K. Kaczmarek, J. H.
Weiss, Proc. Natl. Acad. Sci. USA 2003, 100, 6157–6162; c) P.
Du, S. J. Lippard, Inorg. Chem. 2010, 49, 10753–10755.
a) E. L. Que, D. W. Domaille, C. J. Chang, Chem. Rev. 2008,
108, 1517–1549; b) B. Daniela, J. A. Horowitz, S. J. Lippard, J.
Am. Chem. Soc. 2011, 133, 4101–4114; c) Z. Liu, W. He, Z.
Guo, Chem. Soc. Rev. 2013, 42, 1568–1600.
a) J. E. Kwon, S. Lee, Y. You, K.-H. Baek, K. Ohkubo, J. Cho,
S. Fukuzumi, I. Shin, S. Y. Park, W. Nam, Inorg. Chem. 2012,
51, 8760–8774; b) Z. Xu, J. Yoon, D. R. Spring, Chem. Soc.
Rev. 2010, 39, 1996–2006; c) B. A. Wong, S. Friedle, S. J. Lip-
pard, Inorg. Chem. 2009, 48, 7009–7011.
Acknowledgments
D. P. acknowledges the Council of Scientific and Industrial Re-
search (CSIR), New Delhi, for a senior research fellowship. D. D.
thanks the Indian Institute of Science (IISc) for a centenary post-
doctoral fellowship. K. S. and A. G. S. thank the Department of
Science and Technology (DST), New Delhi, for a research grant.
K. S. also thanks the Department of Science and Technology for a
J. C. Bose fellowship. We would like to thank the Indian Institute
of Science for the imaging facility, the Centre for Imaging Research
in Science & Engineering (IRIS), and Ms. Meenakshi for assisting
us with the the confocal fluorescence microscopy experiments.
[13]
[14]
[15]
a) E. M. Nolan, J. Jaworski, K. I. Okamoto, Y. Hayashi, M.
Sheng, S. J. Lippard, J. Am. Chem. Soc. 2005, 127, 16812–
16823; b) Y. Mikata, A. Yamashita, A. Kawamura, H. Konno,
Y. Miyamoto, S. Tamotsu, Dalton Trans. 2009, 21, 3800–3806;
c) Y. Zhang, X. Guo, W. Si, L. Jia, X. Qian, Org. Lett. 2008,
10, 473–476; d) H.-H. Wang, Q. Gan, X.-J. Wang, L. Xue, S.-
H. Liu, H. Jiang, Org. Lett. 2007, 9, 4995–4998.
[1] a) S. J. Lippard, J. M. Berg, Principles of Bioinorganic Chemis-
try, University Science Books, Mill Valley, California, USA,
1994, pp. 139–175; b) C. Andreini, L. Banci, I. Bertini, A. Ros-
ato, J. Proteome Res. 2006, 5, 3173–3178.
[2] a) J. E. Coleman, Curr. Opin. Chem. Biol. 1998, 2, 222–234;
b) J. M. Berg, Y. Shi, Science 1996, 271, 1081–1085; c) T. V.
O’Halloran, Science 1993, 261, 715–724.
[3] a) J. E. Coleman, Annu. Rev. Biochem. 1992, 61, 897–946; b)
B. L. Vallee, K. H. Falchuk, Physiol. Rev. 1993, 73, 79–118; c)
T. Fukada, S. Yamasaki, K. Nishida, M. Murakami, T. Hirano,
J. Biol. Inorg. Chem. 2011, 16, 1123–1134.
[4] C. E. Outten, T. V. O’Halloran, Science 2001, 292, 2488–2492.
[5] a) K. Toth, Annu. Rev. Nutr. 2011, 31, 139–153; b) N. Wijesek-
ara, F. Chimienti, M. B. Wheeler, Diabetes Obes. Metab. 2009,
11, 402–414.
[6] a) R. B. Franklin, L. C. Costello, J. Cell. Biochem. 2009, 106,
750–757; b) A. B. Chausmer, J. Am. Coll. Nutr. 1998, 17, 109–
115; c) C. L. Pao, A. S. Prasad, Nutr. Res. 1988, 8, 889–897; d)
M. P. Cuajungco, G. J. Lees, Neurobiol. Dis. 1997, 4, 137–169;
e) V. Tougu, P. Palumaa, Coord. Chem. Rev. 2012, 256, 2219–
2224.
[16]
[17]
P. Jiang, Z. Guo, Coord. Chem. Rev. 2004, 248, 205–229.
a) X. Peng, F. Song, E. Lu, Y. Wang, W. Zhou, J. Fan, Y. Gao,
J. Am. Chem. Soc. 2005, 127, 4170–4171; b) K. Hanaoka, K.
Kikuchi, H. Kojima, Y. Urano, T. Nagano, J. Am. Chem. Soc.
2004, 126, 12470–12476.
a) E. Tomat, S. J. Lippard, Inorg. Chem. 2010, 49, 9113–9115;
b) Z. Wang, M. A. Palacios, G. Zyryanov, P. Anzenbacher,
Chem. Eur. J. 2008, 14, 8540–8546.
a) P. Carol, S. Sreejith, A. Ajayaghosh, Chem. Asian J. 2007, 2,
338–348; b) K. Komatsu, Y. Urano, H. Kojima, T. Nagano,
J. Am. Chem. Soc. 2007, 129, 13447–13454; c) C. J. Chang, J.
Jaworski, E. M. Nolan, M. Sheng, S. J. Lippard, Proc. Natl.
Acad. Sci. USA 2004, 101, 1129–1134.
G. J. Brakenhoff, M. Muller, R. I. Ghauharali, J. Microsc.
1996, 183, 140–144.
A. R. Cowley, J. Davis, J. R. Dilworth, P. S. Donnelly, R. Dob-
son, A. Nightingale, J. M. Peach, B. Shore, D. Kerr, L. Seym-
our, Chem. Commun. 2005, 845–847.
G. Buncic, P. S. Donnelly, B. M. Paterson, J. M. White, M.
Zimmermann, Z. Xiao, A. G. Wedd, Inorg. Chem. 2010, 49,
3071–3073.
a) H. Beraldo, D. Gambino, Mini-Rev. Med. Chem. 2004, 4,
31–39; b) B. M. Paterson, P. S. Donnelly, Chem. Soc. Rev. 2011,
40, 3005–3018.
[18]
[19]
[20]
[21]
[7] C. J. Frederickson, G. A. Howell, The Neurobiology of Zinc,
Alan R. Liss, New York, 1984.
[8] a) D. W. Domaille, E. L. Que, C. J. Chang, Nat. Chem. Biol.
2008, 4, 168–175; b) R. McRae, P. Bagchi, S. Sumalekshmy,
C. J. Fahrni, Chem. Rev. 2009, 109, 4780–4827; c) J. W. Licht-
man, J. A. Conchello, Nat. Methods 2005, 2, 910–919.
[9] a) N. C. Lim, H. C. Freake, C. Bruckner, Chem. Eur. J. 2005,
11, 38–49; b) E. M. Nolan, S. J. Lippard, Acc. Chem. Res. 2009,
42, 193–203; c) K. Dhara, S. Karan, J. Ratha, P. Roy, G. Chan-
dra, M. Manassero, B. Mallik, P. Banerjee, Chem. Asian J.
2007, 2, 1091–1100; d) K. Kikuchi, K. Komatsu, T. Nagano,
[22]
[23]
[24]
D. Dayal, D. Palanimuthu, S. V. Shinde, K. Somasundaram,
A. G. Samuelson, J. Biol. Inorg. Chem. 2011, 16, 621–632.
Eur. J. Inorg. Chem. 2013, 3542–3549
3548
© 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.eurjic.org
FULL PAPER
[25] D. Palanimuthu, S. V. Shinde, K. Somasundaram, A. G. Sam-
uelson, J. Med. Chem. 2013, 56, 722–734.
b) A. E. Martell, R. J. Motekaitis, Determination and Use of
Stability Constants, VCH, Weinheim, 1988.
[26] a) J. V. Caspar, T. J. Meyer, J. Am. Chem. Soc. 1983, 105, 5583–
5590; b) S. F. Forgues, D. J. Lavabre, J. Chem. Educ. 1999, 76,
1260–1264.
[27] A. E. Martell, R. M. Smith (Eds.), Critical Stability Constants,
vol. 1 (Amino Acids), Plenum Press, New York, 1974, pp. 469.
[28] J. R. Griffiths, Br. J. Cancer 1991, 64, 425–427.
[29] a) A. E. Martell, R. M. Smith, NIST Standard Reference Data-
base 46: NIST Critical Stability Constants of Metal Complexes,
US Department of Commerce, Gaithersburg, 1998, vol. 5.0;
[30]
a) K. W. Lee, J. D. Slinker, A. A. Gorodetsky, S. Flores-Torres,
H. D. Abruna, P. L. Houston, G. G. Malliaras, Phys. Chem.
Chem. Phys. 2003, 5, 2706–2709; b) N. Hirayama, S. Iimuro,
K. Kubono, H. Kokusen, T. Honjo, Talanta 1996, 43, 621–626.
[31] H. T. K. Britton, R. A. Robinson, J. Chem. Soc. 1931, 1456–
1462.
Received: March 9, 2013
Published Online: June 4, 2013
Eur. J. Inorg. Chem. 2013, 3542–3549
3549
© 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim