Synthesis and biological studies of some gold(I) complexes containing functionalised alkynes

Article abstract of DOI:10.1039/b911234k

The propargyl ethers 7-chloro-(4-propargyloxy)quinoline, 1-propargyloxynaphthalene and 2-propargyloxybenzophenone react with [AuCl(PPh₃)] in the presence of KOH to give the gold(I) alkynyl complexes [Au(CCOCH₂Ar)(PPh₃)] in good yields. The compounds were fully characterised by spectroscopic methods and were subsequently examined for their biological activity against four tumour cell lines as well as their activity against Plasmodium falciparum, the parasite responsible for malaria. The compounds show antiproliferative effects in human cancer cells with IC₅₀ values ranging from 0.4-12 μM. The Royal Society of Chemistry 2009.

Full text of DOI:10.1039/b911234k

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PAPER
www.rsc.org/dalton | Dalton Transactions
Synthesis and biological studies of some gold(I) complexes containing
functionalised alkynes
Esther Schuh,a Seied M. Valiahdi,b Michael A. Jakupec,b Bernhard K. Keppler,b Peter Chibac and
Fabian Mohr*a
Received 10th June 2009, Accepted 17th September 2009
First published as an Advance Article on the web 8th October
DOI: 10.1039/b911234k
The propargyl ethers 7-chloro-(4-propargyloxy)quinoline, 1-propargyloxynaphthalene and
2-propargyloxybenzophenone react with [AuCl(PPh3)] in the presence of KOH to give the gold(I)
≡
alkynyl complexes [Au(C COCH2Ar)(PPh3)] in good yields. The compounds were fully characterised
by spectroscopic methods and were subsequently examined for their biological activity against four
tumour cell lines as well as their activity against Plasmodium falciparum, the parasite responsible for
malaria. The compounds show antiproliferative effects in human cancer cells with IC50 values ranging
from 0.4–12 mM.
Introduction
Results and discussion
Cancer and malaria are both diseases responsible for a significant
number of deaths world-wide thus creating a huge economic and
social burden.1-3 Given that within the next century the number of
people affected by these diseases will increase,4 the development
of a cure is an important field of chemical and biomedical
research. Cisplatinum, cis-[PtCl2(NH3)2], has been in clinical use
for the treatment of various cancers for more than 30 years.
However, despite the success of this compound, it has significant
drawbacks including high toxicity (resulting in severe side-effects
for the patient) as well as poor solubility and development of
cellular resistance.5 In the case of malaria, the most active and
also most used drug Chloroquine is today almost completely
ineffective, due to cell resistance. Research groups all over the
world have therefore developed new approaches in the design of
novel metal-based anti-cancer and anti-malaria drugs.6-10 The use
of gold containing medicines for the treatment of various ailments
has a long history; today there are several anti-arthritis drugs
containing gold(I) thiolate complexes in clinical use. Recently,
the anti-tumour activity of gold(I) and gold(III) complexes have
also been recognised.9,11 Navarro et al. reported the anti-malaria
activity of some cationic gold(I) phosphine complexes containing
chloroquine.12,13 These complexes showed a significant increase of
anti-malaria activity against Plasmodium parasites compared to
that of Chloroquine itself. Given our interest in metal complexes
with biological activity,14-17 we wished to prepare some gold
complexes containing derivatives of known anti-malaria drugs,
in which the gold atom is bound tightly to the ligand through a
strong Au-C bond. The preparation and biological studies of these
complexes is presented herein.
The propargyl ethers 7-chloro-(4-propargyloxy)quinoline,
1-propargyloxynaphthalene and 2-propargyloxybenzophenone
react with [AuCl(PPh3)] in the presence of KOH to afford the
colourless, air- and moisture-stable gold(I) alkynyl complexes
≡
[Au(C COCH2Ar)(PPh3)] (1-3) in good yields (Scheme 1).
Scheme 1
Complexes 1-3 were fully characterised by spectroscopic tech-
niques including 1D 1H, 13C and 31P NMR spectroscopy as well as
2D (1H-13C HSQC and HMBC) NMR techniques. Unfortunately,
we were unable to grow crystals suitable for X-ray diffraction,
since solutions of the complexes decomposed and deposited
metallic gold after a few days. The spectral data is nevertheless
1
fully consistent with the proposed structures. The 31P{ H} NMR
spectra of complexes 1-3 show singlet resonances with chemical
shifts typically observed for Ph3PAu complexes. Deprotonation
of the propargyl ethers is evident from the H NMR spectra of
1-3: the triplet resonance of the acetylenic proton has disappeared
and the OCH2 signal has collapsed to a singlet. Further evidence
of deprotonation is seen in the IR spectra of the complexes, in
1
aFachbereich C, Anorganische Chemie Bergische Universita¨t Wuppertal,
Gaußstr. 20, D-42929, Wuppertal, Germany. E-mail: fmohr@uni-wuppertal.
de; Fax: +49 202 439 3053; Tel: +49 202 439 3641
bInstitute of Inorganic Chemistry, University of Vienna, 1090, Vienna,
Austria
≡
cInstitute of Medical Chemistry, Medical University of Vienna, 1090, Vienna,
Austria
which the C C-H stretching frequency has disappeared. The most
interesting feature of the 13C NMR spectra of complexes 1-3 is the
Dalton Trans., 2009, 10841–10845 | 10841
This journal is
The Royal Society of Chemistry 2009
©

Table 1 In vitro anti-malaria activity of complexes 1-3 and chloroquine
in two strains of Plasmodium falciparum
Table 2 In vitro cytotoxicity of complexes 1-3 and cisplatin in four human
cancer cell lines
IC50 [mM]
IC50 [mM]a
Compound
3D7
K1
Compound
CH1
SK-OV-3 HeLa
SW480
1
14.8
7.2
14.5
0.01
14.8
19.5
23.6
0.3
1
1.5 0.3
0.6 0.2
0.4 0.1
0.16 0.03
10
12
7.4 0.9
1.9 0.3
4
6
4.6 0.2
6.0 2.3
4.1 0.6
0.37 0.06
9.3 1.0
2
2
10
2
3
3
4.5 0.7
3.5 0.3
Chloroquine
cist-[PtCl2(NH3)2]
a 50% inhibitory concentration in the assay (96 h exposure). Values are
means standard deviations of at least three independent experiments.
≡
location of the resonances from the C CAu unit. The signal of the
carbon atom attached directly to gold appears as a doublet at ca.
131 ppm with coupling constants of approximately 142 and 129 Hz
for complexes 1 and 3, respectively. The corresponding signal of
colon cancer cell line SW480. On the other hand, the resistance
mechanisms of SK-OV-3, an ovarian cancer cell line with intrinsic
resistance to classic platinum drugs probably related to its distinct
genomic aberrations (tetraploidy, double minutes),20 seem to affect
sensitivity to compounds 1-3, too. Comparison of the results for
complexes 1 and 2 shows that the presence of a chloride substituent
at the 7-position and nitrogen at position 4 of the naphthyl
ring seems to have no significant effect on their cytotoxicity. In
general, the similarity of the cytotoxic potencies seems to suggest
that the observed activity is governed to a higher degree by the
(triphenylphosphine)gold(I)propargyl moiety common to all three
compounds rather than by the variable part of the structure. The
mechanism of action of anti-tumour active gold complexes is
to date not well understood. Mechanistic considerations have
shifted in recent years from DNA to mitochondrial targets21
and in particular thioredoxin reductase,22 a redox enzyme with
a selenocysteine residue suitable as a binding site for gold.
However, it remains unclear whether the effects reported for
gold(I) phosphine complexes can be generalized to our gold
compounds.
complex 2 could not be observed. These values are very similar to
18
≡
those reported by Schmidbaur et al. for [Au(C CH){P(p-tol)3}].
Similarly, in all three complexes the carbon signal of the acetylenic
carbon not attached to gold could be observed at ca. 100 ppm. In
this case, the P-C coupling constants ranged from 0 to 26 Hz.
Similar observations of such variations in P-C coupling constants
have been observed in gold alkynyl complexes by us and others
previously.18,19
The in vitro anti-malaria activity of the three complexes was
examined in two different strains of plasmodium falciparum
cultures. The results (Table 1) show that these complexes have
low activity against the malaria parasite strains 3D7 (chloroquine
sensitive) and K1 (chloroquine resistant). IC50 values for growth
inhibition of the blood stages of P. falciparum were between 7.2 and
23 mM. A collateral resistance with chloroquine was not observed.
The IC50 values of the complexes were several orders of magnitude
higher than that observed for chloroquine itself (0.01 and 0.3 mM
for the 3D7 and K1 strain, respectively).
These results were unexpected given that Navarro et al.12,13
observed that cationic chloroquine gold derivatives show higher
(or at least similar) anti-malaria activity than free chloroquine.
It may, however, simply be that the weak nitrogen-gold bond in
their complex is broken under physiological conditions, allowing
release of free chloroquine. The AuPPh3 moiety might thus
simply help to bring the molecule to the site of action within
the cell. In our system, the gold-carbon bond is expected to be
considerably stronger than a gold-nitrogen bond, which means
that hydrolysis of the complexes under physiological conditions
is unlikely. The very low anti-malaria activity of these complexes
might then be explained by the fact that the molecule can simply
not reach the site of action. Given these disappointing results,
we wished to investigate if complexes 1-3 showed any other
in vitro bioactivity. These compounds were therefore screened
in four human tumour cell lines. To the best of our knowledge,
these results are amongst the first such studies carried out on
mononuclear gold(I) compounds. The results, shown in Fig. 1 and
Table 2, were much more encouraging, yielding IC50 values in the
high nanomolar (cell line CH1) to low micromolar range (all other
cell lines).
At the moment, the major achievement of this study is to have
demonstrated for the first time that mononuclear organometallic
gold(I) complexes can indeed show antiproliferative activity in
human cancer cells in vitro in a reasonable range of concentrations.
Further studies of this well known class of compounds are
therefore clearly warranted and ongoing in our laboratories.
Experimental
General
1
1H, 13C and 31P{ H} NMR spectra were recorded on a 400 MHz
Bruker ARX spectrometer. Chemical shifts are quoted relative to
external SiMe4 (1H, 13C) and 85% H3PO4 (31P). The splitting of
1
proton resonances in the reported H NMR spectra are defined
as s = singlet, d = doublet, t = triplet and m = multiplet; coupling
constants are reported in Hz. IR spectra were run as KBr pellets
on a Bruker Tensor 27 instrument. Elemental analyses were per-
formed by staff of the microanalytical laboratory of the University
of Wuppertal. All Reactions were carried out under an atmosphere
of dry dinitrogen using standard Schlenk techniques. All chemicals
and solvents (anhydrous quality) were sourced commercially and
used as received. 7-Chloro-(4-propargyloxy)quinoline, as well as
[AuCl(PPh3)] were prepared as described in the literature.23,24
Out of the three complexes, compound 3 shows the highest
activity in all four cell lines tested, but differences in the cytotoxic
potencies of these compounds are not very pronounced and mainly
within the ranges of variation. In particular, complex 3 gives
IC50 values of similar magnitude to those of cisplatin in the
broadly chemosensitive ovarian cancer cell line CH1 and in the
10842 | Dalton Trans., 2009, 10841–10845
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The Royal Society of Chemistry 2009
©

Fig. 1 Concentration-effect curves of complexes 1-3, obtained by the MTT assay (96 h exposure) in four human cancer cell lines: CH1 (A), SK-OV-3
(B), HeLa (C) and SW480 (D).
≡
Preparation of propargyl ethers
78.0 (C CH), 113.3 (C3), 121.5 (C5), 128.1 (C3¢, C5¢), 129.6 (C2),
129.7 (C1¢), 129.8 (C2¢, C6¢), 131.6 (C6), 132.8 (C4¢), 137.6 (C4),
To a solution of the appropriate phenol in acetone (60 mL) was
added K2CO3 (3 eq.) followed by propargyl bromide (1.2 eq.).
The mixture was heated to reflux for ca. 18 h. The suspension
was subsequently filtered and the filtrate was taken to dryness in
vacuum. The resulting solid was purified by flash chromatography
(SiO2, CH2Cl2) to give the propargyl ethers as oils in high yields.
-1
=
≡
155.1 (C1), 195.9 (C O). IR (cm CH2Cl2) 3302 n(C CH), 2124
n(C C), 1711 n(C O).
≡
=
Preparation of the gold(I) alkynyl complexes
≡
[Au(C COCH2Ar)(PPh3)]
To a solution of the propargyl ether (0.34 mmol) in EtOH
and MeOH (1:1 10 mL) was added a solution of 2 M KOH
in MeOH (0.2 mL, 0.4 mmol). After stirring the mixture for
ca. 5 min, solid [AuCl(PPh3)] (0.100 g, 0.20 mmol) was added
and the mixture was stirred at 50 ◦C for 1 h. The resulting
solution was cooled and concentrated in vacuum to a volume
of ca. 5 mL. Addition of hexane led to the precipitation of the
complexes. Complete removal of KCl and excess base was achieved
by dissolving the crude material in CH2Cl2, filtration through
Celite and precipitation of the complexes by addition of hexane.
Using this procedure the following compounds were prepared:
1-Propargyloxynaphthalene
This was prepared as described above from 1-naphthol (2.0 g,
14 mmol), K2CO3 (5.9 g, 42 mmol) and propargyl bromide
1
(2.2 mL, 20 mmol). Yield: 2.2 g (87%) red-brown oil. H NMR
◦
≡
(400 MHz, CDCl3, 25 C): d 2.56 (t, J = 2.3 Hz, 1 H, C CH),
4.90 (d, J = 2.2 Hz, 2 H, OCH2), 6.96 (d, J = 7.5 Hz, 1 H H2),
7.41 (t, J = 7.9 Hz, 1 H, H3), 7.49-7.54 (m, 3 H, H4, 6, 7), 7.83
(m, 1 H, H5), 8.31 (m, 1 H, H8). 13C NMR (100 MHz, CDCl3,
◦
≡
≡
25 C): d 56.1 (OCH2), 75.5 (C CH), 78.6 (C CH), 105.5 (C2),
121.2 (C4), 121.9 (C8), 125.4 (C7), 125.7 (C4a), 125.5 (C3), 126.4
(C6), 127.4 (C5), 134.5 (C8a), 153.3 (C1). IR (cm-1 CH2Cl2) 3302
1
≡
[Au(C COCH2Ar)(PPh3)] (Ar = 7-chloro-(4-propargyloxy)-
≡
≡
n(C CH), 2124 n(C C) .
quinoline) 1. Yield: 0.087 g (87%) colourless solid. Found: C,
52.9; H, 3.3; N, 1.85; Calc. for C30H22AuClNOP: C, 53.3; H, 3.3;
N, 2.1%. 1H NMR (400 MHz, CDCl3, 25 ◦C): d 4.89 (s, 2H, OCH2),
6.26 (d, J = 7.8 Hz, 1 H, H3), 7.31 (dd, 3J = 8.6 Hz, 4J = 1.7 Hz,
1 H, H6), 7.44–7.52 (m, 15H, PPh3), 7.65 (s, 1 H, H8), 7.92 (d,
J = 7.8 Hz, 1 H, H5), 8.37 (d, J = 8.6 Hz, 1 H, H2). 13C-NMR
2-Propargyloxybenzophenone
This was prepared as described above from 2-hydroxy-
benzophenone (1.0 g, 5 mmol), K2CO3 (2.1 g, 15 mmol) and
propargyl bromide (0.8 mL, 8 mmol). Yield: 1.1 g (89.7%) brown
oil. 1H NMR (400 MHz, CDCl3, 25 ◦C): d 2.43 (t, J = 2.3 Hz, 1
◦
(101 MHz, CDCl3, 25 C): d 43.8 (OCH2), 93.7 (d, J = 26.8Hz,
≡
≡
H, C CH), 4.59 (d, J = 2.3 Hz, 2H, OCH2), 7.09 (t, J = 7.6 Hz,
C C-Au), 110.6 (C3), 115.6 (C8), 124.3 (C6), 125.6 (C4a), 128.6
1 H, H5), 7.14 (d, J = 8.2 Hz, 1 H H3), 7.38-7.43 (m, 3 H, H3¢, 4,
5¢), 7.47 (td, J = 8.2 Hz, J = 1.7 Hz, 1 H, H6), 7.54 (t, J = 7.3 Hz,
(C5), 129.2 (d, J = 11.6Hz, m-PPh3), 129.4 (d, J = 56.5Hz, ipso-
≡
PPh3), 131.5 (d, J = 141.9 Hz, C CAu), 131.7 (d, J = 1.7Hz,
1 H H4¢), 7.81 (dd, J = 7.1 Hz, J = 1.0 Hz, 2 H, H2¢, 6¢). 13C
p-PPh3), 134.2 (d, J = 13.7 Hz, o-PPh3), 138.5 (CCl), 140.6 (C8a),
◦
≡
NMR (100 MHz, CDCl3, 25 C): d 56.1 (OCH2), 75.7 (C CH),
142.4 (C2), 177.7 (C-O). Atom numbering as shown in Scheme 1.
This journal is
The Royal Society of Chemistry 2009
Dalton Trans., 2009, 10841–10845 | 10843
©

◦
1
31P{ H} NMR (162 MHz, CDCl3, 25 C): d 42.98. IR (cm-1, KBr
from culture flasks by trypsinization and seeded in 100 mL aliquots
into 96-well microculture plates (Iwaki/Asahi Technoglass) in
the following densities, in order to ensure exponential growth of
untreated controls throughout the experiment: 1.5 ¥ 103 (CH1,
HeLa), 2.5 ¥ 103 (SW480) and 3.5 ¥ 103 (SK-OV-3) viable cells
per well. Cells were allowed to settle and resume exponential
growth in drug-free complete culture medium for 24 h, followed
by the addition of dilutions of the test compounds in 100 mL/well
complete culture medium. For this purpose, the poorly water-
soluble compounds were dissolved in dmso first and then diluted
in the medium such that the effective dmso content did not
exceed 0.3%. After exposure for 96 hours, medium was replaced
by 100 mL/well RPMI 1640 medium (supplemented with 10%
heat-inactivated fetal bovine serum and 4 mM L-glutamine)
plus 20 mL/well solution of MTT in phosphate-buffered saline
(5 mg/mL) (all purchased from Sigma-Aldrich). After incubation
for 4 h, medium/MTT mixtures were removed, and the formazan
product formed by vital cells was dissolved in dmso (150 mL/well).
Optical densities at 550 nm were measured with a microplate
reader (Tecan Spectra Classic), using a reference wavelength of
690 nm to correct for unspecific absorption. The quantity of
vital cells was expressed as percentage of untreated controls,
and 50% inhibitory concentrations (IC50) were calculated from
concentration-effect curves by interpolation. Evaluation is based
on means from at least three independent experiments, each
comprising three replicates per concentration level.
≡
disk) 2115 n(C C).
≡
[Au(C COCH2Ar)(PPh3)] (Ar = 1-propargyloxy-naphthalene)
2. This was prepared as described above, except that the mixture
was refluxed for one hour. Yield: 0.078 g (60%) colourless solid.
Found: C, 58.1; H, 3.6; Calc. for ◦C31H24AuOP: C, 58.1; H, 3.8%.
1H NMR (400 MHz, CDCl3, 25 C): d 5.05 (s, 2H, OCH2), 7.05
(dd, 3J = 6.9 Hz, 4J = 1.6 Hz, 1 H, H2), 7.36-7.54 (m, 19H, H3,
3
4
H4, H6, H7, PPh3), 7.73 (dd, J = 7.1 Hz, J = 2.1 Hz, 1 H,
H5), 8.34 (dd, J = 6.9 Hz, J = 2.4 Hz, 1 H, H8). 13C-NMR
3
4
◦
≡
(101 MHz, CDCl3, 25 C): d 57.3 (OCH2), 98.2 (C CAu), 105.5
(C2), 120.3 (C3), 122.6 (C8), 124.9 (C-naph), 125.9 (C-naph), 125.9
(C8a.), 126.1 (C-naph), 127.2 (C5), 129.1 (d, J = 11.1Hz, m-PPh3),
130.0 (d, J = 52.5Hz, ipso-PPh3), 131.4 (d, J = 2.1 Hz, p-PPh3),
≡
134.2 (d, J = 14.0 Hz, o-PPh3), 134.5 (C4a.), 154.06 (C1), C CAu
not observed. Atom numbering as shown in Scheme 1. 31P{ H}
1
NMR (162 MHz, CDCl3, 25 ◦C): d 43.3. IR (cm-1, KBr disk) 2134
≡
n(C C).
≡
[Au(C COCH2Ar)(PPh3)] (Ar
=
2-propargyloxy-benzo-
phenone) 3. This was prepared as described above, except that
the mixture was stirred at room temperature over night. Yield:
0.129 g (92%) colourless solid. Found: C, 58.8; H, 3.5; Calc. for
1
C34H26AuO2P: C, 58.8; H, 3.8%. H NMR (400 MHz, CDCl3,
25 ◦C): d 4.75 (s, 2H, OCH2), 7.03 (t, J = 7.4 Hz, 1 H, H5),
7.36-7.53 (m, 21H, H3, H3¢, H4, H4¢, H5¢, H6, PPh3), 7.84 (d,
J = 7.0 Hz, 2 H, H2¢, H6¢). 13C-NMR (101 MHz, CDCl3, 25 ◦C):
≡
d 57.4 (OCH2), 97.7 (d, J = 2.2 Hz, C CAu), 113.8 (C3), 120.7
Malaria studies
(C5), 128,1 (C3¢, 5¢), 128.7 (d, J = 57.2 Hz, ipso-PPh3), 129.2
(d, J = 11.3 Hz, m-PPh3), 129,3 (C2.), 129.6 (C4), 129.9 (C1¢.),
129.9 (C2¢, 6¢), 131.6 (d, J = 2.2 Hz, p-PPh3), 131.8 (C6), 132.0
Continuous culture of laboratory strains of P. falciparum Protozoa
were grown in RPMI 1640 medium supplemented with gentamycin
and 7.5% sodium bicarbonate and 10% human serum at a
hematokrit of 5% of blood group 0 rhesus positive red blood
cells following the protocol for continuous culture by Jensen
and Trager.25,26 The parasites were cultured i◦n cell culture flasks
incubated in a tissue culture incubator at 37 C in a gas mixture
composed of 5% CO2, 5% O2, and 90% N2. The culture medium
was changed every 24 to 48 hours. Fresh erythrocytes were added
whenever the parasite density reached more than 1%. Cultures
with a parasitemia of 6-10% were used for growth assays.
≡
(d, J = 129.0 Hz, C CAu), 132.7 (C4¢), 134.3 (d, J = 13.8Hz,
=
o-PPh3), 156.1 (C1), 196.4 (C O). Atom numberi◦ng as shown in
Scheme 1. 31P{ H} NMR (162 MHz, CDCl3, 25 C): d 43.2. IR
1
-1
≡
=
(cm , KBr disk) 2152 n(C C), 1700 n(C O).
Cell lines and culture conditions
CH1 and SK-OV-3 cells (both ovarian carcinoma, human) were
kindly provided by Lloyd R. Kelland (CRC Centre for Cancer
Therapeutics, Institute of Cancer Research, Sutton, U.K.) and
Evelyn Dittrich (Department of Medicine I, Medical University
of Vienna, Austria), respectively. HeLa cells (cervical carcinoma,
human) and SW480 cells (colon carcinoma, human) were kindly
provided by Thomas Czerny (Institute Of Genetics, University
of Veterinary Medicine Vienna, Austria) and Brigitte Marian
(Institute of Cancer Research, Medical University of Vienna,
Austria), respectively. Cells were grown in 75 cm2 culture flasks
(Iwaki/Asahi Technoglass) as adherent monolayer cultures in
complete culture medium, i.e., Minimal Essential Medium (MEM)
supplemented with 10% heat-inactivated fetal bovine serum, 1 mM
sodium pyruvate, 4 mM L-glutamine, and 1% non-essential amino
acids (100¥) (Sigma-Aldrich) without antibiotics. Cultures were
Drug screening and drug sensitivity testing
A highly sensitive HRP2 ELISA was employed for drug sensitivity
assays. Stock solutions of the compounds as well as of chloro-
quine were prepared as previously described.27 Chloroquine was
dissolved in 70% ethanol and gold complexes were dissolved in
DMSO (final concentration of solvent not to exceed 0.1%). The
resulting stock solutions were diluted with RPMI 1640 to achieve
the desired final concentrations.
Sample preparation
After synchronization the samples from continuous culture were
diluted with RPMI 1640 containing 10% serum to obtain a
hematocrit of 1.5%, and with uninfected red blood cells (blood
group 0) to obtain 0.05% parasitemia. 200 ml of the cell medium
mixture were added to each well. The plates were then incubated
at 37 ◦C for 72 hours in a gas mixture containing 5% CO2, 5% O2,
and 90% N2.
◦
maintained at 37 C in a humidified atmosphere containing 5%
CO2 and 95% air.
Cytotoxicity in cancer cell lines
Cytotoxicity in the cell lines mentioned above was determined by a
colorimetric microculture assay (MTT assay). Cells were harvested
10844 | Dalton Trans., 2009, 10841–10845
This journal is
The Royal Society of Chemistry 2009
©

HRP2 double-site antigen capture ELISA28
Notes and references
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High binding 96-well ELISA plates (Costarꢀ 3590, Corning
Inc., NY) were coated with 100 ml/well of a 1.0 mg/ml solu-
tion of anti-HRP2 IgM antibody solution (MPFM-55A, Dunn
Labortechnik, Asbach, Germany) in phosphate buffered saline
(PBS). Subsequently plates were sealed and incubated overnight
◦
at 4 C. The supernatant was discarded and plates saturated for
2 hours with 200 ml/well of a 2% solution of bovine serum albumin
(BSA, Sigma-Aldrich, A9647) in PBS. The supernatant was again
discarded and plates were washed 3 times with 200ml/well of
R
PBS/Tween washing solution (0.5% Tweenꢀ20 in PBS). The
plates were then sealed and stored at or below -20 ◦C.
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an equivalent of approximately 0.05% starting parasitemia. The
ELISA plates were then incubated with the samples for one hour
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the diluted conjugate, the plates were incubated for another hour
at room temperature and washed three times with PBS/Tween
solution.
100 ml of 3,3¢,5,5¢-tetramethylbenzidine (TMB) were added to
each well and the plates were incubated in the dark for 5 to
10 min. The reaction was stopped by addition of 50 ml of 1 M
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at 450 nm.
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Acknowledgements
FM gratefully acknowledges generous funding from the Fonds der
Chemischen Industrie as well as the University of Wuppertal for
this project.
This journal is
The Royal Society of Chemistry 2009
Dalton Trans., 2009, 10841–10845 | 10845
©