Inorganic Chemistry
Article
6
3
70.27. Anal. Calcd for PtC H N O Sb F ·2 H O: C, 20.37; H,
which time cells were precipitated with 25 μL ice-cold TCA (50% w/
v) and fixed for 60 min at 4 °C. Then the SRB assay was performed.
2
0
36 12 2·
2
12
2
21
.41; N, 14.25%. Found: C, 19.98; H, 3.27; N, 14.60.
trans-Pt(ipa) (9-EtG) (NO ) b4: White solid (35%). NMR
The optical density (OD) of each well was measured at 492 nm, using
BioTek’s PowerWave XS Absorbance Microplate Reader. Values were
corrected for background OD from wells only containing medium.
Cell Cycle Analysis. Cells were seeded in six well plates at a density
2
2
3 2
1
(
1
D O): δ( H, 300.13 MHz) 1.07 (d, J = 6.2 Hz 6H, CH -EtG),
.51(dd, J = 7.25 and 7.29 Hz 12H, CH ipa) 2.42 (sp J = 6.2 Hz 2H,
2
3
3‑
CH-ipa), 4.23 (q, J = 7.29 Hz 4H, CH -EtG), 5.56 (4H, NH ), 8.62 (s,
2
2
13
5
2
4
H, H -EtG). δ ( C, 125.76 MHz) 14.1 (CH -EtG), 22.3 (CH -ipa)
of 2.5−5 × 10 cells/well. After 24 h, the products were added to the
8
3
3
0.1 (CH ,9-EtG), 48.4 (CH-ipa), 114.0 (C1, 9-EtG), 156.9 (C2, 9-
respective well and incubated for an additional period of 24 h. Cells
were trypsinized, harvested, transferred to test tubes (12 × 75 mm),
and centrifuged at 1500 rpm for 10 min. The supernatant was
discarded, and the cell pellets were resuspended in 200 μL of cold PBS
and fixed by the addition of 1 mL ice-cold 70% EtOH. Fixed cells were
incubated overnight at −20 °C, after which time were centrifuged at
1500 rpm for 10 min. The cell pellets were resuspended in 500 μL of
PBS, and 5 μL of DNase-free RNase solution (10 mg/mL) was added.
The mixture was incubated at 37 °C for 30 min. Finally, 5 μL of PI
(0.5 mg/mL) was added. Flow cytometric determination of DNA
content (20 000 cells/sample) was analyzed on a LRSII Flow
2
EtG), 154.6 (C4, 9-EtG), 151.0 (C6, 9-EtG), 141.3 (C8, 9-EtG) δ
1
95
(
Pt, 64.53 MHz, Na PtCl ) −2491.0 ppm. MS (EI) m/z:
2
6
2+
[
Pt(ipa)(9-EtG)] = 431 [M ].
trans-Pt(ipa) (9-EtG) (SbF ) b4·SbF : White solid (80%). NMR
2
2
6
2
6
1
(
D O): δ( H, 300.13 MHz) 1.07 (d, J = 6.4 Hz 6H, CH -EtG),
2
3
1.51(dd, J = 7.35 and 7.26 Hz 12H, CH ipa) 2.42 (sp, J = 6.4 Hz 2H,
3‑
CH-ipa), 4.23 (q, J = 7.35 Hz 4H, CH -EtG), 5.56 (bs, 4H, NH ), 8.62
2
2
13
(
s, 2H, H -EtG). δ( C, 125.76 MHz) 14.1 (CH -EtG), 22.3 (CH -
8
3
3
ipa) 40.1 (CH ,9-EtG), 48.4 (CH-ipa), 114.0 (C1, 9-EtG), 156.9 (C2,
2
9-EtG), 154.6 (C4, 9-EtG), 151.0 (C6, 9-EtG), 141.3 (C8, 9-EtG).
1
95
δ( Pt, 64.53 MHz, Na PtCl ) −2491.0 ppm. MS (EI) m/z:
Cytometer (Becton Dickinson, San Jose,
́
CA, U.S.A.). The fractions of
2
6
2
+
[
Pt(ipa)(9-EtG)] = 431 [M ]. Anal. Calcd for PtC H N O Sb F ·
20 36 12 2· 2 12
the cells in G /G , S, and G /M phase were analyzed using FACS Diva
0
1
2
H O: C, 20.68; H, 3.29 ; N, 14.47%. Found: C, 20.39; H, 3.27; N,
́
6.0 (BD Software San Jose, CA, U.S.A.) software.
2
1
4.69.
Mono- and Bis-Adducts of 1 and 4 with 9-EtG. Samples of
complex 1 and 4 were prepared by mixing 0.5 mL of complex solution
50 mg, 0.09 mmol) in D O:acetone-d (1:2) with 9-EtG (4 mg, 22.5
Annexin V Binding. Cells were seeded in six well plates at a density
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of 0.25−0.5 × 10 cells. After 24 h, the test drug was added, and the
cells were incubated for period of 24 h. Then, cells were trypsinized,
harvested, transferred to test tubes (12 × 75 mm), and centrifuged at
400 g for 10 min. The supernatant was discarded, and the cells pellets
were resuspended in 100 μL of ice-cold 1x Binding Buffer (0.1 m
Hepes/NaOH (pH 7.4) 1.4 M NaCl, 25 mM CaCl2).
(
2
6
−3
x10 mmol) into a NMR tube. The tube was maintained at 37 °C
1
13
under slight stirring during the entire experiment (72 h). H, C, 2D
HSQC], and 1 Pt NMR spectra were acquired at various times to
95
[
monitor the reactivity.
ESI MS Analysis. ESI-MS spectra of modified amino acids samples
were recorded on an QSTAR (Applied Biosystems) mass
spectrometer operating in positive ion mode. Experimental parameters
were set as follows: ion spray voltage, 5.5 kV; declustering potential, 50
V; focusing potential 210 V, ion source gas, 10 psi; acquisition
window, m/z 50−2000; and flow rate, 10 μL/min with a solution of
methanol.
The HPLC purified synthetic oligonucleotide 5′-d-
(TAATTAAGCATAATAT)-3′ was purchased from Microsynth
(Balgach, Switzerland). For ESI-MS analysis of the oligonucleotide,
samples contained a platinum complex:oligonucleotide ratio of 3:1
(75:25 μM) in 10 mM ammonium acetate (pH 7.4) and were
incubated for 24 h at 37 °C. ESI-MS spectra were recorded on an
Ultima II q-TOF mass spectrometer (Waters, Manchester, U.K.) fitted
with a standard Z-spray ion source operating in negative ion mode.
Experimental parameters were set as follows: capillary voltage, 3 kV;
sample cone, 50 V; source temperature, 80 °C; desolvation
temperature, 200 °C; acquisition window, m/z 500−1500 in 1 s. A
volume of 5 μL of sample (20 μM) was introduced into the mass
spectrometer by infusion at a flow rate of 20 μL/min with a solution of
Reactivity with Biomolecules. A mixture of 150 mg (0.26 mmol) of
complex 4 and 258 mg (1.58 mmol) or 47.4 mg (0.29 mmol) of N-
acetyl-L-cysteine (Pt:N-AcCys ratio 1:1.1 or 1:6, respectively) in 2 mL
1
13
1
13
195
of acetone-d was prepared. H, C, 2D [ H, C] HSQC, and Pt
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NMR spectra were recorded at various times (2, 4, 12, and 24 h).
To afford the metathesis of the iodide ligands and force the
formation of N-acetyl-L-cysteine adducts, an additional sample was
prepared as follows: a solution of AgNO (16.5 mg, 0.097 mmol) in
3
0
.2 mL of D O was added to a solution of complex 4 (50 mg, 0.088
2
mmol) in acetone-d and stirred at room temperature in the dark for
6
1
0 min. The mixture was filtered and mixed with a freshly prepared
solution of N-acetyl-L-cysteine (15.82 mg, 0.097 mmol) in 0.2 mL of
acetone-d6. Then H, C, 2D [ H, C] HSQC, and Pt NMR
spectra were acquired after 4 and 24 h.
1
13
1
13
195
UV−Visible Absorption Spectrophotometry. The absorption
spectra of 1−6 in the UV−visible region were recorded on a Perkin-
−
4
Elmer Lambda 20 Bio spectrophotometer. Solutions of 1−6 (10 M)
MiliQ water were monitored, collecting the electronic spectra over 24
h at room temperature.
Cells, Culture, and Plating. The human solid tumor cell lines HBL-
00, HeLa, SW1573, T-47D, and WiDr were used in this study. These
cell lines were a kind gift from Prof. G. J. Peters (VU Medical Center2,
Amsterdam, The Netherlands). Cells were maintained in 25 cm
culture flasks in RPMI 1640 supplemented with 5% heat inactivated
ACN/H O 50:50 (v:v). External calibration was carried out with a
2
1
solution of phosphoric acid at 0.01% in 50% acetonitrile. Data
acquisition and analysis were carried out using the MassLynx 4.1
software bundle (Waters).
ESI-MS spectra of 9-EtG samples were recorded by direct
introduction (5 μL/min) into an Orbitrap high-resolution mass
spectrometer (Thermo, San Jose, CA) equipped with a conventional
ESI source. The working conditions were as follows: spray voltage 5
kV, capillary voltage 35 V, and capillary temperature 275 °C. Sheath
gas was set at 8 au. For spectra acquisition, a nominal resolution (at m/
z 400) of 100 000 and Xcalibur 2.0 software (Thermo) were used.
Horse heart cytochrome c was purchased from Sigma (Code
C7752). Metal complexes/cyt c adducts were prepared in tetramethyl
ammonium acetate buffer (TMAA) (pH 7.4) with a protein
fetal calf serum and 2 mM L-glutamine in a 37 °C, 5% CO , 95%
2
humidified air incubator. Exponentially growing cells were trypsinized
and resuspended in an antibiotic-containing medium (100 units
penicillin G and 0.1 mg of streptomycin per mL). Single cell
suspensions displaying >97% viability by trypan blue dye exclusion
were subsequently counted. After counting, dilutions were made to
give the appropriate cell densities for inoculation onto 96 well
microtiter plates. Cells were inoculated in a volume of 100 μL per well
at densities of 10 000 (SW1573 and HBL-100), 15 000 (HeLa and T-
7D), and 20 000 (WiDr) cells per well, on the basis of their doubling
times.
Chemosensitivity Testing. Compounds were initially dissolved in
DMSO at 400 times the desired final maximum test concentration.
Control cells were exposed to an equivalent concentration of DMSO
−4
4
concentration of 10 M and platinum to protein ratio of 3:1. The
reaction mixtures were incubated at 37 °C for different time intervals
over 48 h. Samples were extensively ultrafiltered using Centricon YM-
3 (Amicon Bioseparations, Millipore Corporation) in order to remove
the unbound platinum complex. After a 20 fold dilution with HCOOH
0.1%, ESI-MS spectra were recorded by an Orbitrap high-resolution
mass spectrometer as detailed above. The working conditions were as
follows: spray voltage 3.1 kV, capillary voltage 45 V, and capillary
(
0.25% v/v, negative control). Each agent was tested in triplicate at
different dilutions in the range of 1−100 μM. The drug treatment was
started on day 1 after plating. Drug incubation times were 48 h, after
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dx.doi.org/10.1021/ic202036c | Inorg. Chem. 2012, 51, 1717−1726