A novel zinc bis(thiosemicarbazone) complex for live cell imaging

Article abstract of DOI:10.1007/s00775-011-0764-0

Fluorescent zinc complexes have recently attracted a lot of interest owing to their vast applications in cellular imaging. We report the synthesis as well as physical, chemical and biological studies of a novel zinc glyoxalbis(4-methyl-4-phenyl-3-thiosemi

Full text of DOI:10.1007/s00775-011-0764-0

J Biol Inorg Chem (2011) 16:621–632
DOI 10.1007/s00775-011-0764-0
ORIGINAL PAPER
A novel zinc bis(thiosemicarbazone) complex for live cell imaging
Disha Dayal
Sridevi Vijay Shinde
Ashoka G. Samuelson
•
Duraippandi Palanimuthu
•
•
Kumaravel Somasundaram
•
Received: 27 October 2010 / Accepted: 29 January 2011 / Published online: 8 March 2011
Ó SBIC 2011
Abstract Fluorescent zinc complexes have recently
attracted a lot of interest owing to their vast applications in
cellular imaging. We report the synthesis as well as
physical, chemical and biological studies of a novel zinc
glyoxalbis(4-methyl-4-phenyl-3-thiosemicarbazone), [Zn
complex can potentially serve as a biocompatible fluores-
cent imaging agent for live cells.
Keywords Bis(thiosemicarbazone) ꢀ Imaging agent ꢀ
Cell viability ꢀ Breast cancer ꢀ Zinc
(
GTSC)] , complex. As compared with the well-studied
3
zinc biacetylbis(4-methyl-3-thiosemicarbazone), Zn(ATSM),
complex, which was used as a reference, [Zn(GTSC)] had
3
Introduction
2
.5-fold higher fluorescence. When cellular fluorescence
was measured using flow cytometry, we observed that
Zn(GTSC)] had 3.4-fold to 12-fold higher fluorescence
There is widespread interest in developing novel imaging
agents that can be effectively and reliably used in many
disease conditions, including cancer, therapy and treatment
monitoring [1–4]. Several reports have demonstrated the use
of small organic fluorophores for imaging, but these dyes
have been found to have serious side effects. For example,
commercial dyes such as rhodamine 123 and fluorescein
isothiocyanate can cause cytotoxicity and even lung fibrosis
in rodents. They also appear to produce symptoms such as
nausea and vomiting [5–8]. The use of metal complexes for
live cell imaging, however, remains a less explored area.
[
3
than Zn(ATSM) in various cell lines (n = 9) of different
tissue origin. Confocal fluorescence microscopy results
showed that [Zn(GTSC)]3 appeared to have a nuclear
localization within 30 min of addition to MCF7 cells.
Moreover, [Zn(GTSC)]3 showed minimal cytotoxicity
compared with Zn(ATSM), suggesting that [Zn(GTSC)]3
may be less deleterious to cells when used as an imaging
agent. Our data suggest that the novel [Zn(GTSC)]3
6
4
Although several radioactive copper ( Cu) bis(thiosemi-
carbazone) complexes have been tested for imaging and
treatment, the corresponding zinc-based complexes are
excellent alternatives for fluorescent imaging [9–25]. Not
only are zinc complexes readily available, they are also
generally less cytotoxic to cells and are conveniently mon-
itored using simple fluorescence techniques.
D. Dayal and D. Palanimuthu contributed equally.
Electronic supplementary material The online version of this
article (doi:10.1007/s00775-011-0764-0) contains supplementary
material, which is available to authorized users.
D. Dayal ꢀ S. V. Shinde ꢀ K. Somasundaram (&)
Department of Microbiology and Cell Biology,
Indian Institute of Science,
Bangalore 560012, India
e-mail: skumar@mcbl.iisc.ernet.in
Recently, Cowley et al. [26] reported an intrinsically
fluorescent zinc biacetylbis(4-methyl-3-thiosemicarba-
zone), Zn(ATSM), complex whose cellular uptake and
distribution in live cells could be monitored by confocal
fluorescence microscopy. Zn(ATSM) was shown to accu-
mulate in significant concentrations at the nucleus of PC-3
D. Palanimuthu ꢀ A. G. Samuelson (&)
Department of Inorganic and Physical Chemistry,
Indian Institute of Science,
Bangalore 560012, India
e-mail: ashoka@ipc.iisc.ernet.in
(prostate cancer) cells, whereas in the IGROV (ovarian
cancer) cell line, the uptake is greater in acidic subcellular
123

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J Biol Inorg Chem (2011) 16:621–632
organelle lysosomes [22]. Selective cellular localization
can prove valuable for understanding various mechanisms
of action during therapeutic studies. Analogous zinc com-
plexes such as zinc acenaphthenequinonebis(4-allyl-3-
thiosemicarbazone) have also shown cellular fluorescence.
However, their use as imaging agents is likely to be limited
by their cytotoxicity. The latter complex has been shown to
be as cytotoxic (LC50 12.5 lM) as cisplatin in the MCF-7
cell line [27, 28]. Thus, there is a need to develop new
imaging agents which exhibit high fluorescence in cells,
low background fluorescence and low cellular toxicity. We
report here a novel trimeric zinc complex formed by a
glyoxalbis(4-methyl-4-phenyl-3-thiosemicarbazone) (GTS
dissolved completely. The clear solution was cooled to room
temperature and glyoxal (0.317 g, 2.2 mmol) was added as a
40% aqueous solution. The color changed from colorless to
yellow. Addition of one drop of concentrated H SO brought
2
4
about the immediate precipitation of the product, which was
collected by filtration. The residue was washed repeatedly
with ethanol and dried in air for 12 h to give GTSCH as a
2
1
light-yellow solid. Yield: 54%. H NMR (400 MHz,
CDCl ): d = 3.72 (s, 3H, NCH ), 7.39 (s, 1H, CH),
3
3
7.21–7.24 (dd, 2H, Ph), 7.45 (m, 1H, Ph) 7.50–7.53 (m, 2H,
Ph), 8.60 (s, 1H, NH). Electrospray ionization (ESI) mass
?
spectroemetry (MS) calcd for [M?Na ] : m/z: 407.11;
?
?
found: m/z (%): 407.60 (100) [M?Na ] , ESI(-): calcd for
?
?
[M–H ] : m/z: 383.11; found: m/z (%): 383.67 [M–H ]
-
? -
CH ) ligand. The complex shows good fluorescence
2
response and low cytotoxicity, making it a promising agent
for live cell imaging.
(100); elemental analysis calcd (%) for C H S N :
18 20 2 6
C 55.76, H 5.33, N 20.80, S 16.22; found: C 56.10, H 5.47,
-
1
N 21.22, S 16.19; IR (KBr): mmax (cm ) 3,230 (N–H), 1,522
C=N), 1,320 (thioamide); UV–vis (dimethyl sulfoxide,
(
-
1
-1
Materials and methods
DMSO): kmax(e) = 354 nm (37,300 M cm ).
All the reagents and solvents were obtained from com-
mercial sources and used without further purification.
Benzilbis(4-pyrrolidinyl-3-thiosemicarbazone)
(
Amines and CS used in the study were purified by stan-
2
To an ethanolic solution of benzil (0.5 g, 2.4 mmol) and
4-pyrrolidinyl-3-thiosemicarbazide (0.7 g, 4.8 mmol) were
added a few drops of concentrated H SO . The resulting
dard methods prior to experiments.) NMR spectra were
recorded with a Bruker AMX 400 spectrometer with tet-
ramethylsilane as the internal standard. IR spectra were
recorded in the solid state using KBr pellets with a Perk-
inElmer Spectrum One spectrometer. All UV–vis experi-
ments were carried out with a PerkinElmer Lambda 35
instrument and the fluorescence measurements were car-
ried out with an Horiba Jobin Yvon Fluoromax-4 spec-
trofluorimeter. A standard solution of zinc was purchased
from Fluka Analytical and the amount of zinc in cells was
estimated using a PerkinElmer Optima 2000 DV induc-
tively coupled plasma optical emission spectroscopy (ICP-
OES) instrument. All reagents used for cell culture study
and DNA binding experiments were obtained from Sigma.
2
4
reaction mixture was refluxed for 4 h. The solid product
was isolated and washed with ethanol. This crude product
was further recrystallized from a 1:1 mixture of chloroform
and ethanol to give benzilbis(4-pyrrolidinyl-3-thiosemi-
1
carbazone) (BTSCH ) as a yellow crystals. Yield: 61%. H
2
NMR (400 MHz, CDCl3): d = 1.62 (quintet, 2H,
NCH CH ), 1.73 (quintet, 2H, NCH CH ), 2.01 (quintet,
2H, NCH CH ), 2.07 (quintet, 2H, NCH CH ), 2.96 (t, 2H,
2
2
2
2
2
2
2
2
NCH ), 3.70 (t, 2H, NCH ), 3.90 (t, 2H, NCH ), 4.08 (bs,
2
2
2
2H, NCH ), 7.29-7.58 (m, 10H, Ph), 8.59, 8.77 (s, 2H, NH,
2
1
3
exchanges with D O). C NMR (100.6 MHz, d -DMSO):
2
6
d = 24.17, 24.90 (NCH CH ), 49.8, 50.3 (NCH ) 127.45,
2
2
2
127.69, 128.26, 128.71, 130.45, 131.71, 134.18 (Ph),
Synthesis and characterization
of bis(thiosemicarbazone) ligands
and their zinc complexes
144.55 (C=N), 176.10 (C=S); ESI–MS calcd for
?
?
[M?Na ] : m/z: 487.17; found: m/z (%): 487.87 (100)
?
?
[M?Na ] ; elemental analysis: calcd for C H S N : C
2
4 28 2 6
6
2.04, H 6.07, N, 17.64; found: C 61.77, H 6.25, N 17.91;
-
1
Biacetylbis(4-methyl-3-thiosemicarbazone)
IR (KBr): mmax (cm ) 3,175 (N–H), 1,580 (C=N), 1,448
thioamide); UV–vis (DMSO): kmax(e) = 302 nm
(29,500 M cm ).
(
-
1
-1
The ligand biacetylbis(4-methyl-3-thiosemicarbazone)
(
ATSMH ) was prepared following the literature procedure
2
and was characterized by spectroscopic techniques [20].
Zinc biacetylbis(4-methyl-3-thiosemicarbazone)
Glyoxalbis(4-methyl-4-phenyl-3-thiosemicarbazone)
The Zn(ATSM) complex was prepared from zinc acetate
(0.09 g, 0.410 mmol) and an ethanolic solution (10 mL) of
An ethanolic solution (30 mL) of 4-methyl-4-phenylthio-
semicarbazide (0.8 g, 4.4 mmol) was heated to 60 °C and
stirred until the 4-methyl-4-phenylthiosemicarbazide
ATSMH (0.1 g, 0.384 mmol) following the literature pro-
2
cedure [20]. After the addition of zinc acetate, the color
changed from light yellow to intense yellow and the solution
1
23

J Biol Inorg Chem (2011) 16:621–632
623
was refluxed for 4 h. The yellow precipitate formed was
collected by filtration, washed with ethanol followed by
1
diethyl ether and dried in vacuo. Yield: 56%. H NMR
Crystal structure determination
X-ray crystallographic studies were carried out using a
Bruker CCD diffractometer with graphite-monochroma-
tized MoKa radiation (k = 0.71073) controlled by a Pen-
tium-based PC running the SMART (version 5.05, 1998;
Bruker AXS single crystal diffractometer; Bruker, Madi-
son, WI, USA) software package. Single crystals were
mounted on glass fibers at room temperature, and data were
collected at room temperature (20 °C). The structures were
solved by direct methods (SIR-92) followed by the full-
(
400 MHz, d -DMSO): d = 2.19 (s, 3H, CH –C=N), 2.81
6
3
1
s, 3H, CH –N), 7.21 (bs, 1H, NH); C NMR (100.6 MHz,
3
(
3
d -DMSO): d = 14.79 (CH ), 30.15 (NCH ), 146.15 (C=N),
1
6
3
3
-
1
76.62 (C = S); IR (KBr): mmax (cm ) 3,335 (NH), 1,525
(
C=N), 1,372 (thioamide); UV–vis (DMSO): kmax(e) = 313,
-
1
35 nm (11,700, 11,900 M cm ).
-1
4
Zinc glyoxalbis(4-methyl-4-phenyl-3-thiosemicarbazone)
2
matrix least-squares procedure of F for all reflections
(SHELXL-97). All non-hydrogen atoms were refined by
Zinc glyoxalbis(4-methyl-4-phenyl-3-thiosemicarbazone),
anisotropic displacement parameters and hydrogen atoms
were fixed at idealized positions [29, 30].
[
Zn(GTSC)] , was prepared using GTSCH2 (0.14 g,
3
0
.36 mmol) and zinc nitrate (0.1 g, 0.36 mmol) in ethanol
(
10 mL) using the procedure used to prepare the
Zn(ATSM) complex. About 50 mg of the complex was
isolated as an orange solid. The single crystal used for the
Cell culture
X-ray diffraction study was grown in chloroform. Yield:
1
All cell lines were grown in Dulbecco’s modified Eagle
medium (DMEM) supplemented with 10% fetal calf serum
(FCS), penicillin and streptomycin. Cell cultures were
3
1%. H NMR (400 MHz, CDCl ): d = 3.59 (s, 3H,
3
NCH ), 7.29–7.40 (m, 3H, Ph), 7.50–7.53 (m, 2H, Ph), 8.30
3
1
3
(
s, 1H, CH). C NMR (100.6 MHz, CDCl ): d = 41.21
maintained at 37 °C and 5% CO
in an incubator.
2
3
(
NCH ), 127.29, 127.86, 129.12, 146.18 (Ph), 146.88,
3
1
44.55 (C=N), 176.10 (C=S); ESI–MS calcd for
Estimation of zinc in cells by ICP-OES
?
M?H ] : m/z: 447.30: found: m/z (%): 447.33 [M?H ]
?
? ?
[
(
100); elemental analysis: calcd for C H S N Zn: C
18 18 2 6
MCF-7 cells were treated with [Zn(GTSC)] for 1 h at
3
4
1
8.27, H 4.05, N 18.76, S 14.32; found: C 48.08, H 4.29, N
-
37 °C and harvested using a cell scraper. The cell sus-
pension was centrifuged at 2,000 rpm for 5 min at 4 °C.
The cell pellet so obtained was washed with phosphate-
buffered saline (PBS) and an aliquot was taken for protein
analysis. The suspension was again centrifuged at
1
8.19, S 13.71; IR (KBr): mmax (cm ) 1,437 (C=N), 1,367
(
thioamide); UV–vis (DMSO): kmax(e) = 339, 475 nm
-
21,900, 10,900 M cm ).
1
-1
(
2
,000 rpm for 5 min at 4 °C and the cell pellet was col-
Zinc benzilbis(4-pyrrolidinyl-3-thiosemicarbazone)
lected. Dry cell pellets were then lysed using one freeze–
thaw cycle and 1% HNO solution. The final volume was
3
Zinc
Zn(BTSC)(DMSO)], was prepared following the procedure
used for preparing Zn(ATSM) except that zinc nitrate
0.068 g, 0.228 mmol) in ethanol (5 mL) was used with
BTSCH (0.099 g, 0.213 mmol) dissolved in chloroform
benzilbis(4-pyrrolidinyl-3-thiosemicarbazone),
made up to 10 mL and used for analysis with the ICP-OES
instrument. The instrument was calibrated for zinc using
solutions containing 10, 50, 100, 500 and 1,000 ppb zinc.
The concentration of zinc obtained was normalized to
cellular protein concentration (measured using Bradford
protein assay) [31, 32].
[
(
2
(
5 mL). The product, [Zn(BTSC)], was isolated as a bright-
orange solid. Recrystallization of [Zn(BTSC)] from DMSO
gave single crystals of [Zn(BTSC)DMSO] suitable for X-ray
1
diffraction studies. Yield: 36%. H NMR (400 MHz, d -
Confocal fluorescence imaging
6
DMSO): d = 1.78 (s, 4H, NCH CH ), 3.60 (s, 4H, NCH ),
2
2
2
1
3
7
.13–7.15 (3H, m, Ph), 7.28 (2H, d, JH, H = 7.2, Ph).
NMR (100.6 MHz, d -DMSO): d = 25.35 (NCH CH ),
C
Fluorescence imaging studies were done using a Leica
confocal fluorescence microscope (TCS SP5) in HCX APO
L 20.0 9 1.00 water-objective mode. Cells were grown in
appropriate media in six-well dishes containing circular
cover slips. On the day of the experiment, the medium was
replaced with phenol red free DMEM (pH 7.4) containing
10% FCS, penicillin and streptomycin. The cover slip was
placed on the imaging chamber, zinc complex/ligand
(1.5 mL in 1% DMSO in phenol red free DMEM) was
6
2
2
4
9.5, 50.20 (NCH ) 127.91, 128.74, 131.15, 134.57 (Ph),
2
?
45.01 (C=N), 176.48 (C=S); ESI–MS calcd for [M?H ] :
?
1
?
m/z: 527.10: found: m/z (%): 527.0 [M?H ] (100); ele-
?
mental analysis: calcd for C H S N Zn: C 54.59, H 4.96,
24 26 2 6
N 15.91; found: C 53.80, H 4.85, N 15.43; IR (KBr): m
max
-
1
cm ) 1,437 (C=N), 1,367 (thioamide); UV–vis (DMSO):
(
-
1
-1
kmax(e) = 333, 490 nm (10,500, 11,100 M cm ).
123

6
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J Biol Inorg Chem (2011) 16:621–632
added and fluorescence was recorded using 488-nm exci-
tation and emission was collected in the 500–600-nm
region. Quantification of microscopy images was done
using ImageJ (NIH) [33].
Flow cytometry
Cell pellets were collected following trypsinization from
exponentially growing cultures and washed with PBS (pH
7
.4). The pellet was then resuspended in 1 mL PBS buffer
(
pH 7.4), zinc complexes were added and fluorescence was
recorded in the FL1 channel of a Beckman Coulter CyAn
flow cytometer. The mean fluorescence intensity was rep-
resented as an average of 10,000 cells gated according to
forward and side scatter.
3
-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
Fig. 1 Molecular structures of bis(thiosemicarbazone) ligands and
their zinc complexes
bromide assay
3
-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
Results and discussion
mide (MTT) assay was carried out to measure cell viabil-
ity. An appropriate number of cells were seeded onto the
wells of a 96-well plate. After 24 h, various concentrations
of the zinc complexes were added and incubation was
carried out for 48 h in a 37 °C incubator. After 45 h
incubation, MTT was added and the absorbance at 550 nm
was read 3 h later on a microplate reader (Molecular
Devices). A graph of the concentration versus percentage
cell viability was plotted and the concentration at which
Synthesis and characterization
The bis(thiosemicarbazone) ligands and their zinc com-
plexes were synthesized (Fig. 1) by following known
protocols with minor modifications (Scheme S1). The
1
ligands and zinc complexes were well characterized by H
1
3
NMR spectroscopy,
C NMR spectroscopy, UV–vis
spectroscopy, IR spectroscopy, CHN analysis, ESI–MS
and fluorescence spectroscopic techniques. The crystal
structures of BTSCH2 ligand and [Zn(GTSC)]3 and
5
0% cell death occurred was used as the IC50 value.
[Zn(BTSC)(DMSO)] complexes were characterized using
DNA binding constant estimation by ethidium bromide
displacement assay
single-crystal X-ray crystallography (Figs. S2, S3, S4,
1
Table S5).
The versatile coordinating behaviour of bis(thiosemi-
carbazone) ligands and the structural flexibility of zinc
leads to the formation of a variety of zinc bis(thiosemi-
carbazone) complexes with different coordination numbers
and geometries. The formation of monomers, dimers and
polymeric zinc bis(thiosemicarbazone) complexes in both
square pyramidal and tetrahedral geometry around zinc
have been reported [14, 34–38]. However, our crystallo-
The DNA binding experiments were done in aqueous
solutions of sodium perchlorate (400 mM) and the zinc
complexes dissolved using minimum amounts of DMSO.
The purity of calf thymus DNA was evaluated from the
ratio of the absorbance at 260 and 280 nm, which was
found to be more than 1.8, and the concentration was
calculated from the absorbance at 260 nm. DNA was added
to ethidium bromide (1.3 lM) solution until the fluores-
cence intensity at 602 nm saturated (kex = 560 nm). Sub-
sequently, quenching was observed in the presence of
increasing amounts of zinc complexes. The apparent
binding constant (Kapp) was calculated from the equation
graphic analysis of the zinc complex of GTSCH revealed
2
the formation of an unusual trimeric [Zn(GTSC)] com-
3
plex. This trimeric structure resembles a trigonal prism
with each face capped by a ligand. Each zinc is coordinated
to two ligands through sulphur and nitrogen ligation, thus
forming a ZnS N framework where zinc is in a distorted
[
EtBr] 9 KErBr = [complex]50% 9 Kapp
,
where [EtBr]
2
2
refers the concentration of ethidium bromide and [com-
plex]50% is the concentration required for 50% quenching
of the DNA–EtBr adduct. The reported values are the
average of two independent experiments.
1
The Cambridge Crystallographic Data Centre (CCDC) numbers for
Zn(GTSC)] , [Zn(BTSC)(DMSO)] and BTSCH are CCDC 776332,
CCDC 776330 and CCDC 776331.
[
3
2
1
23

J Biol Inorg Chem (2011) 16:621–632
625
Fig. 3B and C. The studies were also repeated using 5%
DMSO in PBS (Fig. 3D) and 1% DMSO in water (Fig. S6)
to ascertain the fluorescence response in aqueous media. It
was observed that in aqueous media the fluorescence
intensity was significantly quenched. The quantum yields
for [Zn(GTSC)] , [Zn(BTSC)(DMSO)] and Zn(ATSM)
3
complexes were estimated in DMSO (0.02 mM) using
[
Ru(bpy) ](PF ) (bpy is bipyridyl) as a standard [40–42].
3 6 2
-
3
The observed values for [Zn(GTSC)] (/ = 3.0 9 10
)
3
-
3
and [Zn(BTSC)(DMSO)] (/ = 2.1 9 10 ) were 2.5 and
.7 times higher, respectively, than the value for
1
-
3
Zn(ATSM) (/ = 1.2 9 10 ) under identical conditions.
Photophysical properties of all the synthesized zinc com-
plexes are listed in Table 1. The stock solutions of
[Zn(GTSC)] and [Zn(BTSC)(DMSO)] were found to be
3
stable for at least 10 days in DMSO. Emission spectra
recorded with excitation at 488 nm showed that
Fig. 2 Crystal structure of the trimeric [Zn(GTSC)]
3
complex.
˚
Hydrogen atoms are omitted for clarity. Selected bond lengths (A):
Zn(2)–S(3), 2.264(16); Zn(2)–S(2), 2.255(16); Zn(2)–N(4), 2.085(4);
Zn(2)–N(6), 2.071(4); S(3)–C(26), 1.724(6); S(2)–C(11), 1.745(5);
N(5)–C(11), 1.325(6); N(8)–C(26), 1.326(5); N(4)–N(5), 1.366(5);
N(6)–N(8), 1.362(5)
[Zn(GTSC)]3 and [Zn(BTSC)(DMSO)] exhibit fluores-
cence with maxima at 565 and 575 nm, respectively, and
have better a fluorescence response than Zn(ATSM).
Interestingly, the commonly available excitation wave-
length 488 nm is close to the absorption maxima of both
complexes, making them more convenient cellular imaging
agents. The [Zn(BTSC)(DMSO)] complex showed a much
tetrahedral geometry (Fig. 2). [Zn(GTSC)] is structurally
3
similar to the previously reported zinc (1,2-ethanediyl-
iden)-bis(S-methylhydrazonecarbodithionate) complex [39].
In the case of [Zn(BTSC)(DMSO)], the complex is a
monomer and DMSO is coordinated to zinc as a fifth
ligand, resulting in a distorted square pyramidal structure.
The absence of bulky groups on the diketone and the
presence of methyl and phenyl groups on the nitrogen of
thiosemicarbazide enforce an s-trans conformation for the
lower fluorescence quantum yield than [Zn(GTSC)] and
3
was not investigated further. The unique trimeric nature of
[Zn(GTSC)]3 is presumably responsible for the greater
fluorescence of this complex.
In vitro cellular uptake studies by flow cytometry
We then investigated the ability of [Zn(GTSC)] to enter
3
GTSC ligand in [Zn(GTSC)] . In most complexes, the s-cis
3
cells and fluoresce in an intracellular environment. To
optimize the concentration and time required for the
conformation is preferred as shown by the [Zn(BTSC)
(
DMSO)] complex, resulting in a distorted square pyramidal
maximum cellular uptake of [Zn(GTSC)] , MCF-7 breast
3
geometry. Our examination of bond lengths in the ligands
and in zinc complexes revealed elongation of the C=S bond
and shortening of the adjacent C–N bond length relative to
the free ligand, indicating extensive delocalization and
formation of the thiolate form in zinc complexes.
cancer cells were treated with various concentrations of
[Zn(GTSC)] , were incubated for either 30 or 60 min and
3
fluorescence was measured by flow cytometry. A dose-
dependent cellular uptake of [Zn(GTSC)] was observed
3
(Fig. 4A). There was no significant difference in uptake at
30 or 60 min of incubation, suggesting that maximum
UV–vis and fluorescence studies
uptake of the complex occurs within 30 min of the treat-
ment (Fig. 4B). Furthermore, the dose-dependent increase
in intracellular zinc concentration following treatment with
UV–vis absorption spectra were recorded for the com-
plexes in DMSO. [Zn(GTSC)] and [Zn(BTSC)(DMSO)]
3
[Zn(GTSC)] (Fig. 4C), as measured by ICP-OES, pro-
3
complexes showed a characteristic absorption band cen-
tered at 475 and 490 nm, respectively, whereas Zn(ATSM)
showed a band centered at 435 nm. These lower-energy
vided evidence for entry of [Zn(GTSC)] into cells. On the
3
basis of these experimental results, 10 lM [Zn(GTSC)]3
and 30 min incubation were taken as the ideal concentra-
tion and time for internalization and were used for all
further experiments.
bands in [Zn(GTSC)] and [Zn(BTSC)(DMSO)] can be
3
attributed to charge transfer transitions from sulphur to zinc
(
Fig. 3A).
To compare the uptake of [Zn(GTSC)] with that of the
3
Fluorescence spectra of complexes dissolved in DMSO
10 lM in zinc) were recorded and the results are shown in
previously known Zn(ATSM) complex, MCF-7 and normal
(
keratinocyte HaCat cells were treated with either 10 lM
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J Biol Inorg Chem (2011) 16:621–632
Fig. 3 UV–vis spectra (A) and
fluorescence spectra at
k
k
ex = 420 nm (B) and at
ex = 488 nm (C) for solutions
of zinc complexes (10 lM in
zinc) in dimethyl sulfoxide
(
DMSO). Fluorescence spectra
of the [Zn(GTSC)] complex in
% DMSO in phosphate-
3
5
buffered saline (pH 7.4) (D)
Table 1 Photophysical properties of the zinc complexes
a
b
c
Complexes
Absorbance kmax (nm)
Emission
max (nm)
Quantum yield
-3
/ 9 10
Optical brightness
-1
4
-1
-1
-1
(e 9 /, M cm )
(
e 9 10 M cm
)
k
Zn(ATSM)
313 (1.17), 435 (1.19)
333 (1.05), 490 (1.11)
339 (2.19), 475 (1.09)
526
563
541
1.2
2.1
3.0
12.0
7.2
[
[
a
b
c
Zn(BTSC)(DMSO)]
Zn(GTSC)]
3
23.6
See Fig. 1 for the structures
Emission spectra were measured in dimethyl sulfoxide solvent with 420-nm excitation and 5-nm slit width
-3
3 6 2
The quantum yield was calculated by using [Ru(bpy) ](PF ) ] (bpy is bipyridyl) (/ = 42 9 10 ) as a reference
[
Zn(GTSC)] or 10 lM Zn(ATSM) for 30 min and fluores-
Cellular localization by confocal fluorescence imaging
To visualize the cellular localization and cellular retention
3
cence was measured using flow cytometry. In MCF-7 cells,
[
(
Zn(GTSC)] showed a 12-fold higher fluorescence response
3
45.5 ± 1.1) than Zn(ATSM) (3.8 ± 0.2) (Fig. 5A). When
time of [Zn(GTSC)]
10 lM [Zn(GTSC)]
monitored immediately and followed up to 30 min by con-
3
, MCF-7 cells were treated with either
HaCat cells were treated with [Zn(GTSC)] , 6.5-fold higher
3
3
or 10 lM Zn(ATSM) and uptake was
fluorescence was observed (18.6 ± 1.1 for [Zn(GTSC)] vs.
3
2
.8 ± 0.1 for Zn(ATSM)) (Fig. 5B). In another breast can-
focal fluorescence microscopy. As shown in Fig. 6A and B,
cer cell line, MDA-MB-231, [Zn(GTSC)] showed 3.4-fold
3
[Zn(GTSC)] localizes on the cell membrane/cytoplasm
3
higher fluorescence (17.2 ± 1.3 for [Zn(GTSC)]3 vs.
of MCF-7 cells initially and then becomes internalized into
the nucleus. In agreement with flow cytometry data,
5
.1 ± 0.4 for Zn(ATSM)). Under our experimental condi-
tions, no increase above the background level was seen when
either MCF-7 or HaCat cells were treated with Zn(ATSM)
[Zn(GTSC)] showed a higher fluorescence response than
3
Zn(ATSM) (Fig. 6C, D) in MCF-7 cells. Figure 6E shows
the quantification of cellular fluorescence from images taken
(
Fig. 5). These data show that [Zn(GTSC)] has higher
3
fluorescence than Zn(ATSM) inside the cells as well.
from 2 to 30 min of [Zn(GTSC)] incubation. It appears that
3
1
23

J Biol Inorg Chem (2011) 16:621–632
627
Fig. 4 A MCF-7 cells were
treated with different
concentrations of [Zn(GTSC)]
for 30 min and fluorescence was
recorded using a flow
3
cytometer. B Flow cytometry
quantification for MCF-7 cells
treated with different
concentrations of [Zn(GTSC)]
for two different time points,
3
3
0 and 60 min. C Elemental
analysis was carried out using
inductively coupled plasma
optical emission spectroscopy
and intracellular zinc
concentration was estimated in
MCF-7 cells treated with either
2
5 or 50 lM (in zinc)
[
fluorescence intensity
Zn(GTSC)] . MFI mean
3
Fig. 5 A MCF-7 and B HaCat
cells were incubated with either
[
Zn(GTSC)]
3
or Zn(ATSM)
(
both 10 lM in zinc) for 30 min
and fluorescence was recorded
using a flow cytometer
cytoplasmic fluorescence decreases and nuclear fluores-
cence increases within the first 30 min. However, the overall
cellular fluorescence remains constant for more than 60 min
15 min, and cellular morphology was also retained. This
fact is evident from the overlaid fluorescence and bright
field images, as shown in Fig. 7B. No fluorescence was
observed from the cells with an addition of an equal
amount of DMSO under identical conditions (Fig. 7A).
(
Fig. 6F), suggesting that the complex does not efflux from
the cell for at least 1 h. To ensure that the cellular viability
is not affected by [Zn(GTSC)] treatment, we carried out
3
This suggests that [Zn(GTSC)] enters the cell and is able
3
MTT-based cell viability assay following treatment with
to exhibit stable fluorescence in the intracellular envi-
ronment. To show further that the zinc complex is
essential for the intracellular fluorescence, a confocal
fluorescence imaging experiment was performed in live
MCF-7 cells treated with [Zn(GTSC)] or GTSCH ligand
[
Zn(GTSC)] for 15, 30, 45 or 60 min. We also examined the
3
cell morphology for any signs of stress following
Zn(GTSC)] treatment. We observed that neither the cell
[
3
morphology (Fig. S7a–e) nor the cell viability (Fig. S7f) was
3
2
affected by [Zn(GTSC)] treatment, suggesting that the
3
(10 lM) and the fluorescence was monitored for 30 min.
We observed significant fluorescence from the cells
complex is non-toxic. To investigate whether our complex
enters the cell, we carried out co-localization measurement
of the fluorescence and bright field images following treat-
treated with [Zn(GTSC)] (Fig. 7D). The observed intra-
3
cellular fluorescence was quenched by the addition of an
0
0
ment offixed MCF-7 cells with [Zn(GTSC)] . When the cells
3
excess (100 lM) of N,N,N ,N -tetrakis(2-pyridylmethyl)
ethylenediamine, a cell-permeable, non-fluorescent and
high-affinity zinc chelator (Fig. 7F). It is apparent that the
were treated with [Zn(GTSC)] , intense intracellular fluo-
3
rescence was observed at the cytoplasmic region after
123

6
28
J Biol Inorg Chem (2011) 16:621–632
Fig. 6 Representative images
of MCF-7 cells following
treatment with [Zn(GTSC)]
A, B) or Zn(ATSM) (10 lM in
3
(
zinc) (C, D) recorded using a
confocal fluorescence
microscope. E MCF-7 cells
were treated with 10 lM
[
3
Zn(GTSC)] for 60 min.
Images were recorded using a
confocal microscope after every
2
min, generating a total of 30
images. For quantification, ten
cells were selected per image
and fluorescence was measured
using ImageJ (NIH). The
average of the fluorescence of
ten cells is shown in the graph.
F MCF-7 cells were treated with
[
3
Zn(GTSC)] (10 lM in zinc)
and images were recorded over
a period of 30 min using a
confocal fluorescence
microscope. The fluorescence
intensity of ten cells and that of
corresponding nuclei were
quantified using ImageJ. The
latter was subtracted from the
former to get the fluorescence
intensity of the cytoplasm. The
graph represents the change in
fluorescence of the cytoplasm
and the nucleus over 30 min and
error bars represent the
standard deviation (SD) from
ten different cells
intracellular fluorescence was quenched by the process of
the nucleus of HaCat cells as well. In another breast cancer
cell line, MDA-MB-231, nuclear localization was visible
trans chelation. Very faint fluorescence was observed for
the cells treated with GTSCH ligand alone under similar
only at lower concentrations of [Zn(GTSC)] (Fig. S8).
3
2
conditions (Fig. 7E). A control experiment with untreated
MCF-7 cells showed little fluorescence (Fig. 7C). All
these results confirm that internalization of the trimeric
This suggests that [Zn(GTSC)] is cell-permeable and can
3
fluoresce in an intracellular environment of cancer or
normal cells. Since the fluorescence of the complex is
partially quenched in aqueous media, and the intracellular
environment is mostly aqueous, we hypothesize that the
observed intracellular fluorescence is due to the binding of
zinc complex [Zn(GTSC)] is essential for the observed
3
intracellular fluorescence.
In HaCat cells as well, [Zn(GTSC)] showed higher
3
fluorescence than Zn(ATSM) (Fig. 8). As observed in the
[Zn(GTSC)] with hydrophobic membranes of intracellular
3
case of MCF-7 cells, [Zn(GTSC)] appears to localize in
3
organelles.
1
23

J Biol Inorg Chem (2011) 16:621–632
629
Fig. 7 A Autofluorescence of fixed MCF-7 cells. B MCF-7 cells
were treated with [Zn(GTSC)]
of live MCF-7 cells was recorded without any fixation. D Live MCF-7
cells were treated with [Zn(GTSC)] (10 lM) in Dulbecco’s modified
3
3
(10 lM in zinc) in an eight-chamber
slide. After 15 min, spent medium was removed and cells were fixed
using 4% paraformaldehyde and fluorescence images were recorded
using a Zeiss confocal fluorescence microscope (940 magnification)
with 488-nm excitation. Simultaneously, bright field images were
recorded and the two images were overlaid to visualize co-localiza-
tion. For both A and B, a the fluorescence image, b the bright field
image and c the co-localization of images a and b. C Autofluorescence
Eagle medium and fluorescence was recorded after 30 min incubation
with a Leica confocal fluorescence microscope (920 magnification).
E Live MCF-7 cells were treated with GTSCH
and fluorescence was recorded. F Live MCF-7 cells were treated as
2
ligand (10 lM) alone
0
0
in D and subsequently quencher N,N,N ,N -tetrakis(2-pyridylmethyl)
ethylenediamine (TPEN) (100 lM) was added; fluorescence was
recorded after 5 min incubation with TPEN
123

6
30
J Biol Inorg Chem (2011) 16:621–632
Fig. 8 Representative images
of HaCat cells following
3
treatment with [Zn(GTSC)] or
Zn(ATSM) (10 lM in zinc)
recorded using a confocal
fluorescence microscope
Table 2 Cytotoxicity of [Zn(GTSC)]
represented as IC50 (lM)
3
and Zn(ATSM) complexes
Table 3 Summary of mean fluorescence intensity (in arbitrary units)
in different cancer cell lines following treatment with [Zn(GTSC)]
3
or
Zn(ATSM) (10 lM in zinc)
Complex
HaCat
MCF-7
MDA-MB-231
Cells
Origin
Zn(ATSM)
[Zn(GTSC)]
3
Zn(ATSM)
24.5 ± 5.5
47.0 ± 1.4
6.5 ± 3.6
7.7 ± 1.2
MCF-7
Breast
3.8 ± 0.24
5.1 ± 0.43
2.8 ± 0.06
4.2 ± 0.10
5.8 ± 0.10
3.2 ± 0.03
3.9 ± 0.11
3.2 ± 0.09
2.7 ± 0.04
45.5 ± 1.10
17.2 ± 1.32
18.6 ± 1.12
16.5 ± 0.57
42.5 ± 0.78
27.5 ± 1.38
27.9 ± 3.67
33.4 ± 0.96
21.1 ± 0.84
[
Zn(GTSC)]
3
23.3 ± 6.0
13.0 ± 5.0
MDA-MB-231
a
HaCat
Breast
Each value is the average of three independent experiments
Keratinocytes
Hepatocellular
Kidney
HepG2
2
93T
Cell viability studies
C33A
LN18
U251
U87
Cervical
Glioma
To ascertain the effect of these complexes on cellular
viability after prolonged treatment, we performed MTT
Glioma
Glioma
assay following 48 h treatment with both [Zn(GTSC)] and
3
Each data set represents the average of measurements on three
independent samples
Zn(ATSM). The IC50 value obtained for [Zn(GTSC)] was
3
nearly 1.7-fold to 3.6-fold higher in cells than that for
Zn(ATSM) even after 48 h, showing that the complex is
relatively non-toxic (Table 2; Fig. S9). Moreover, the
concentration at which maximum biological fluorescence
was observed (10 lM) is below the IC50 value for
a
Immortalized human keratinocytes
lower toxicity of [Zn(GTSC)] compared with Zn(ATSM),
3
we measured the binding ability of [Zn(GTSC)]3 and
Zn(ATSM) for DNA, a possible target for these complexes,
by ethidium bromide displacement assay. We observed that
[
Zn(GTSC)] and cells had 60–80% viability.
3
We also found [Zn(GTSC)] to be less toxic (1.7 and 3.3
3
the apparent DNA binding constant of [Zn(GTSC)]
3
4
-1
(4.6 ± 1.4 9 10 M ) was nearly twofold lower than that
times) to normal cells (immortalized human keratinocyte
cells—HaCat) than to MCF-7 and MDA-MB-231 breast
cancer cells (Table 2). In addition, IC50 was twofold higher
for [Zn(GTSC)]3 than for Zn(ATSM) in HaCat cells
4
-1
of Zn(ATSM) (8.3 ± 0.1 9 10 M ), suggesting that it
may cause less DNA damage and hence low cytotoxicity.
The possibility of the ligand leaching out from the metal
complex, inside the cell, is excluded as these ligands
(
Table 2). To investigate the possible reason behind the
1
23

J Biol Inorg Chem (2011) 16:621–632
631
Fig. 9 A Fluorescence of
Zn(GTSC)]
(blue curve) in comparison with
autofluorescence (black curve)
A
HaCat
MCF-7
MDA-MB-231
[
3
(10 lM in zinc)
7
42
1000
500
375
250
556
371
7
5
2
50
00
and fluorescence of Zn(ATSM)
(
185
0
50
0
125
0
10
10 lM) (red curve) in nine
0
1
2
3
4
1
2
0
1
10
2
10
3
10
4
10
1
0
10
10
10
10
0
3
4
1
0
10
10
10
10
different cancer cell lines of
different tissue origin.
B Quantification of A. Error
bars represents the SD from
three independent samples
HepG2
293T
C33A
5
4
2
1
95
46
97
406
304
203
661
495
330
48
0
101
0
165
0
0
1
2
10
3
4
10
0
1
10
2
10
3
4
0
1
10
2
10
3
10
4
10
1
0
10
10
10
10
10
10
LN18
U251
U87
5
65
23
82
576
432
288
556
417
278
4
2
1
41
0
144
0
139
0
0
0
1
10
2
10
3
10
4
10
0
1
10
2
10
3
10
4
10
0
1
10
2
10
3
10
4
10
1
10
10
Fluorescence Intensity
B
Auto fluorescence
Zn(ATSM)
5
4
4
3
3
2
2
1
1
0
5
0
5
0
5
0
5
0
5
0
[Zn(GTSC)]3
possess lower cytotoxicity than their zinc complexes (data
synthesize and exhibits enhanced fluorescence owing to its
unique trimeric structure. It is significantly better than the
other zinc complexes owing to its low cytotoxicity. Since
the fluorescence of [Zn(GTSC)]3 is quenched in the
aqueous phase, background fluorescence is minimal.
not shown). Thus, [Zn(GTSC)] appears to be safer and
3
biocompatible, which bodes well for its use as an imaging
agent for cancer cells and normal cells.
Quantification of complex uptake in different cell lines
[Zn(GTSC)] appears to be stable in an intracellular envi-
3
ronment and fluoresces for a long time (more than 1 h)
without being quenched in the hydrophobic regions of the
cell. It shows high fluorescence in several cell lines, and in
some it shows nuclear localization. Additionally, it is rel-
atively non-toxic to two breast cancer cell lines and a
normal cell line tested in this study. These features make it
an excellent agent for live cell imaging.
One of the desired qualities of an imaging agent is that it
should be able to fluoresce in various types of tissues. To
determine the utility of [Zn(GTSC)] in cancers of different
3
tissue origin, we tested its fluorescence by flow cytometry
in nine cancer cell lines of six different tissue types. In all
cell lines, we observed that [Zn(GTSC)] had higher fluo-
3
rescence than Zn(ATSM) (Table 3; Fig. 9).
Acknowledgments D.D. thanks the Indian Instutite of Science
(
IISc) for an IISc Centenary Postdoctoral Fellowship. D.P.
acknowledges the Council of Scientific & Industrial Research for a
Senior Research Fellowship. K.S. and A.G.S. thank the Department of
Science and Technology (New Delhi) for a research grant. We thank
IRIS, IISc and Pushpa Janardhan for assistance with the confocal
fluorescence microscopy experiments.
Conclusion
In conclusion, we have shown that the novel trimeric zinc
complex [Zn(GTSC)] is a good imaging agent, is easy to
3
123

6
32
J Biol Inorg Chem (2011) 16:621–632
References
21. Dearling JLJ, Blower PJ (1998) Chem Commun 2531–2532
2
2. Lim SC, Price KA, Chong SF, Paterson BM, Caragounis A,
Barnham KJ, Crouch PJ, Peach JM, Dilworth JR, White AR,
Donnelly PS (2010) J Biol Inorg Chem 15:225–235
1
2
. Cai W, Rao J, Gambhir SS, Chen X (2006) Mol Cancer Ther
:2624–2633
. Caroll L, Bejot R, Hueting R, King R, Bonnitcha P, Bayly S,
Christlieb M, Dilworth JR, Gee AD, Declerck J, Gouverneour V
5
2
3. Pascu SI, Waghorn PA, Kennedy BWC, Arrowsmith RL, Bayly
SR, Dilworth JR, Christlieb M, Tyrrell RM, Churchill GC,
Aigbirhio FI (2010) Chem Asian J 5:506–519
(2010) Chem Commun 46:4052–4054
2
2
2
4. You Y, Tomat E, Hwang K, Atanasijevic T, Nam W, Jasanoff
AP, Lippard SJ (2010) Chem Commun 46:4139–4141
5. Gao Y, Wu J, Li Y, Sun P, Zhou H, Yang J, Zhang S, Jin B, Tian
Y (2009) J Am Chem Soc 131:5208–5213
6. Cowley AR, Davis J, Dilworth JR, Donnelly PS, Dobson R,
Nightingale A, Peach JM, Shore B, Kerr D, Seymour L (2005)
Chem Commun 845–847
7. Pascu SI, Waghorn PA, Conry TD, Betts HM, Dilworth JR,
Churchill GC, Pokrovska T, Christlieb M, Aigbirhio FI, Warren
JE (2007) Dalton Trans 43:4988–4997
8. Pascu SI, Waghorn PA, Conry TD, Lin B, Betts HM, Dilworth
JR, Sim RB, Churchill GC, Aigbirhio FI, Warren JE (2008)
Dalton Trans 16:2107–2110
9. Walker N, Stuart D (1983) Acta Crystallogr Sect A 39:158–166
0. Altomare A, Cascarano G, Giacovazzo C, Gualardi A (1993)
J Appl Crystallogr 26:343–350
1. Donnelly PS, Caragounis A, Du T, Laughton KM, Volitakis I,
Cherny RA, Sharples RA, Hill AF, Li QX, Masters CL, Barnham
KJ, White AR (2008) J Biol Chem 283:4568–4577
2. Deng Z, Dailey LA, Soukup J, Stonehuerner J, Richards JD,
Callaghan KD, Yang F, Ghio AJ (2009) Biometals 22:803–815
3. Rasband WS (1997–2004) ImageJ. National Institutes of Health,
Bethesda. http://rsb.info.nih.gov/ij/
4. Christlieb M, Struthers HSR, Bonnitcha PD, Cowley AR, Dil-
worth JR (2007) Dalton Trans 5043–5054
5. Al-Karawi AJM, Clegg W, Harrington RW, Henderson RA
3
4
5
6
7
. Ferreira MF, Martins AF, Martins JA, Ferreira PM, Toth E,
Geraldes CFGC (2009) Chem Commun 42:6475–6477
. Craig P, Benjamin MS, Elizabeth NJ, Pal R, Parker D (2009) Acc
Chem Res 42:925–937
. Castro DJ, Saxton RE, Fetterman HR, Castro DJ, Ward PH
(1992) J Clin Laser Med Surg 10:83–90
. Lampidis TJ, Bernal SD, Summerhayes IC, Chen LB (1983)
Cancer Res 43:716–720
2
2
. Roberts SN, Howie SE, Wallace WA, Brown DM, Lamb D,
Ramage EA, Donaldson K (1995) J Pathol 176:309–318
. Rumbaut RE, Sial AJ (1999) Microcirculation 6:205–213
. Lewis JS, Laforest R, Buettner TL, Song SK, Fujibayashi Y,
Connett JM, Welch MJ (2001) Proc Natl Acad Sci USA
8
9
2
3
9
8:1206–1211
0. Kraker A, Krezoski S, Schneider J, Minkel D, Petering DH
1985) J Biol Chem 260:13710–13718
1. Fujibayashi Y, Taniuchi H, Yonekura Y, Ohtani H, Konishi J,
Yokoyama A (1997) J Nucl Med 38:1155–1160
2. Vavere AL, Lewis JS (2007) Dalton Trans 4893–4902
3. Xiao Z, Donnelly PS, Zimmermann M, Wedd AG (2008) Inorg
Chem 47:4338–4347
4. Holland JP, Aigbirhio FI, Betts HM, Bonnitcha PD, Burke P,
Christlieb M, Churchill GC, Cowley AR, Dilworth JR, Donnelly
PS, Green JC, Peach JM, Vasudevan SR, Warren JE (2007) Inorg
Chem 46:465–485
1
1
(
3
1
1
3
3
3
3
3
3
3
3
1
1
1
5. Katherine AP, Aphrodite C, Paterson BM, Filiz G, Volitakis I,
Masters CL, Barnham KJ, Donnelly PS, Crouch PJ, White AR
(
2009) Dalton Trans 564–570
6. Zhong X, Yi J, Sun J, Wei HL, Liu WS, Yu KB (2006) Eur J Med
Chem 41:1090–1092
7. Buncic G, Donnelly PS, Paterson BM, White JM, Zimmermann
M, Xiao Z, Wedd AG (2010) Inorg Chem 49:3071–3073
8. Torres EL, Mendiola MA, Pastor CJ, Perez BS (2004) Inorg
Chem 43:5222–5230
(2009) J Med Chem 52:6606–6620
6. Holland JP, Barnard PJ, Collison D, Dilworth JR, Edge R, Green
JC, Heslop JM, McInnes EJL, Salzmann CG, Thompson AL
(
2008) Eur J Inorg Chem 22:3549–3560
7. Beraldo H, Boyd LP, West D (1998) Transition Met Chem
3:67–71
8. Cowley AR, Dilworth JR, Donnelly PS, Labisbal E, Sousa A
2002) J Am Chem Soc 124:5270–5271
9. Holland JP, Fisher V, Hickin JA, Peach JM (2010) Eur J Inorg
Chem 48–58
0. Holland JP, Barnard PJ, Collison D, Dilworth JR, Edge R, Green
JC, McInnes JL (2008) Chem Eur J 14:5890–5907
1
1
1
2
2
9. Hennig C, Hallmeier KH, Zahn G, Tschwatschal F, Hennig H
(
1999) Inorg Chem 38:38–43
0. Caspar JV, Meyer TJ (1983) J Am Chem Soc 105:5583–5590
1. Williams ATR, Winfield SA, Miller JN (1983) Analyst
(
4
4
108:1067–1071
2. Forgues SF, Lavabre D (1999) J Chem Educ 76:1260–1264
4
1
23