Inorganic Chemistry
Article
source (the excitation wavelength was 380 nm), an integrating sphere,
and a red-sensitive multichannel photodetector (Hamamatsu
Photonics, PMA-12).
4H2O) and CCDC-987780 (K2.75H1.25-2-3EtOH·7H2O) contain
supplementary crystallographic data for this paper. These data can
be obtained free of charge from the Cambridge Crystallographic Data
Preparation of K4[{Re6(μ3-S)8}(BTA)6]·3.5EtOH·4H2O (K4-1-
3.5EtOH·4H2O) and K2.75H1.25[{Re6(μ3-Se)8}(BTA)6]·3EtOH·7H2O
(K2.75H1.25-2-3EtOH·7H2O). Synthetic procedures for both com-
pounds were the same. For the preparation of K4-1-3.5EtOH·4H2O,
K4[{Re6(μ3-S)8}(OH)6]·8H2O (200 mg, 0.113 mmol) and 1H-
benzotriazole (200 mg, 1.679 mmol) were heated in a sealed glass
tube at 150 °C for 48 h. After the reaction mixture was cooled, it was
dissolved in 100 mL of hot ethanol and filtered. The solution was
evaporated at 70 °C until the formation of an orange crystalline
precipitate. The precipitate was filtered and dried in air. Yield: 253 mg
(91%). The compound K2.75H1.25-2-3EtOH·7H2O was prepared in the
same way: K4[{Re6(μ3-Se)8}(OH)6]·8H2O (200 mg, 0.093 mmol) was
heated with 1H-benzotriazole (200 mg, 1.679 mmol). Yield: 230 mg
(88%). Anal. Calcd for C43H53K4N18O7.5Re6S8 (K4-1-3.5EtOH·4H2O):
C, 20.9; H, 2.2; N, 10.2; S, 10.4. Found: C, 20.2; H, 1.8; N, 10.5; S,
10.6. Anal. Calcd for C42H57.25K2.75N18O10Re6Se8 (K2.75H1.25-2-3EtOH·
7H2O): C, 17.8; H, 2.0; N, 8.9. Found: C, 17.9; H, 1.9; N, 8.8. EDS
shows the following: K:Re:S ratio of 4.2:6:8.1 for
C43H53K4N18O7.5Re6S8 (1); K:Re:Se ratio of 2.6:6:7.9 for
C42H57.25K2.75N18O10Re6Se8 (2). UV−vis in MeCN, λmax, nm (ε,
mol−1 dm3 cm−1): 330 (sh, 27850), 297 (sh, 44800), 279 (sh, 59150),
273 (sh, 62700), 250 (sh, 66600), 211 (205000) for 1; 340 (sh,
15750), 310 (sh, 28150), 282 (sh, 63250), 276 (64500), 258 (sh,
72700), 251 (74950), 211 (sh, 200550) for 2. UV−vis in 0.1 M
aqueous solution of KOH, λmax, nm (ε, mol−1 dm3 cm−1): 330 (sh,
25450), 297 (sh, 42500), 279 (sh, 60100), 274 (61950) for 1; 340 (sh,
13050), 310 (sh, 24950), 282 (sh, 66050), 274 (70950) for 2. The IR
spectra (400−4000 cm−1) of compounds 1 and 2 in KBr pellets show
all of the peaks expected for benzotriazole ligands. Crystals suitable for
X-ray structure determination were separated from the precipitate
formed in an ethanol solution during evaporation.
Preparation of [{Re6(μ3-S)8}(1H-BTA)4(BTA)2] (H4-1) and
[{Re6(μ3-Se)8}(1H-BTA)4(BTA)2] (H4-2). The compounds were
quantitatively precipitated as orange powders by acidifying the
aqueous solution of K4-1-3.5EtOH·4H2O or K2.75H1.25-2-3EtOH·
7H2O, respectively, with 0.1 M HCl until the pH was equal to ∼2. The
powders were separated by centrifugation and then dried in an oven at
110 °C for 1 h. Anal. Calcd for C36H28N18Re6S8 (H4-1): C, 20.7; H,
1.4; N, 12.1; S, 12.3. Found: C, 21.1; H, 1.6; N, 12.5; S, 11.8. Anal.
Calcd for C36H28N18Re6Se8 (H4-2): C, 17.6; H, 1.1; N, 10.2. Found:
C, 18.0; H, 1.4; N, 10.5. EDS shows the absence of potassium and
chlorine in samples of H4-1 and H4-2.
Titration. H4-1 (130 mg, 0.0625 mmol) or H4-2 (154 mg, 0.0625
mmol) was added to 30.0 mL of degassed water. The titration
measurements were performed with an 0.084 M aqueous solution of
KOH using a Hanna HI 9024 pH meter. The pH meter was calibrated
at pH 9.18 and 6.86 by using standard titrants: aqueous solutions of
Na2B4O7·10H2O and KH2PO4, respectively.
Cell Culture. Human larynx carcinoma cell line (Hep2) was
purchased from the State Research Center of Virology and
Biotechnology VECTOR and cultured in Eagle’s Minimum Essential
Medium (EMEM, pH ∼7.4) supplemented with a 10% fetal bovine
serum under a humidified atmosphere (5% CO2 plus 95% air) at 37
°C.
Confocal Microscopy. Hep2 cells were seeded on slides (1.5 ×
105 cells/slide) and incubated overnight at 37 °C under a 5% CO2
atmosphere. The medium was then replaced with a fresh medium
containing rhenium cluster complexes (12.5, 25, or 50 μM) and
incubated for 24 h. Finally, the cells were washed twice with phosphate
buffered saline (PBS), fixed in 4% paraformaldehyde, and visualized by
using a Zeiss LSM 510 confocal microscope (Carl Zeiss Inc., Jena,
Germany) equipped with a laser diode (405 nm) for fluorescence and
with a 100× λ-blu corrected oil immersion objective. The images were
obtained and analyzed with ZEN 2009 software. Each experiment was
repeated three times on separate days.
Flow Cytometry. The cells (106 cells/mL) were incubated
overnight at 37 °C under a 5% CO2 atmosphere, and the cellular
uptake of rhenium clusters was measured by incubating the cells with a
given concentration of rhenium cluster complexes (12.5, 25, or 50
μM) for 24 h. The cells were washed twice with PBS, harvested by
trypsinization, and then suspended in PBS containing a 0.1% bovine
serum albumin and NaN3. The accumulation of rhenium clusters in
the cells was analyzed by using a FACSCanto II (Becton Dickinson).
The cells incubated in the absence of rhenium clusters were also used
as a control. All of the data were the mean fluorescence obtained from
a population of 10000 cells. The experiments were carried out in
triplicate and expressed as the percentage of viable cells compared to
the control group.
Cell Proliferation/Viability. The percentage of cell proliferation/
viability was measured using the 3-[4,5-dimethylthiazol-2-yl]-2,5-
diphenyltetrazolium bromide (MTT) colorimetric assay. The Hep2
cells were seeded into 96-well plates at 5 × 104 cells/well in a medium
containing K4-1-3.5EtOH·4H2O or K2.75H1.25-2-3EtOH·7H2O with
concentrations from 3.125 to 400 μM and then incubated for 72 h
under a 5% CO2 atmosphere. The effect of the cluster complexes on
the cell proliferation/viability was also determined using the MTT
assay. Ten microliters of the MTT solution with a concentration of 5
mg/mL was added to each well, and the plates were incubated for 4 h
and then solubilized with a dimethyl sulfoxide solution. The optical
density was measured with a plate reader (Sunrise, TECAN) at 620
nm. The experiment was repeated three times on separate days.
Singlet Oxygen Generation. The singlet oxygen generation was
investigated as follows: a solution of K4-1-3.5EtOH·4H2O (5 mg,
0.002 mmol) or K2.75H1.25-2-3EtOH·7H2O (6 mg, 0.002 mmol) and
2,3-diphenyl-p-dioxene9 (48 mg, 0.2 mmol) in 1 mL of acetone-d6 in a
conventional NMR tube was saturated with oxygen for 10 min and
then irradiated with a DRSh-500 mercury lamp with a filtered light
wavelength longer than 400 nm for 2 h. 1H NMR (200 MHz) spectra
were collected on a Bruker Avance 200 NMR spectrometer before and
after irradiation of the samples.
Single-Crystal Diffraction. The single-crystal X-ray diffraction
data were collected using graphite-monochromated Mo Kα radiation
(λ = 0.71073 Å) at 150(2) K on a Bruker APEX DUO diffractometer
(K4-1-3.5EtOH·4H2O) and a Bruker Nonius X8 APEX diffractometer
(K2.75H1.25-2-3EtOH·7H2O) equipped with a 4K CCD area detector.
The φ-scan technique was employed to measure the intensities.
Absorption corrections were applied empirically using the SADABS
program.7 The structures were solved by the direct methods of
difference Fourier synthesis and then refined by the full-matrix least-
squares method using the SHELXTL package.8 The atomic thermal
parameters for non-hydrogen atoms were refined anisotropically. The
positions of hydrogen atoms of the benzotriazole ligands and ethanol
molecules of crystallization (except for the hydroxyl groups) were
calculated in accordance with their geometric conditions and refined
using the rigid-body approximation (riding model). The hydrogen
atoms for crystallization water molecules and the hydroxyl groups of
ethanol were not localized. More experimental details are given in
Table 1. Table S1 in Supporting Information gives selected interatomic
distances and angles for 1 and 2. CCDC-987781 (K4-1-3.5EtOH·
RESULTS AND DISCUSSION
■
Synthesis and Structure. The hexahydroxo cluster
complexes K4[{Re6(μ3-Q)8}(OH)6]·8H2O (Q = S, Se) were
found to be convenient parent compounds for ligand
substitution reactions. It was demonstrated that the OH−
groups could be substituted under various conditions by
different inorganic and organic ligands, such as halides,6,10
cyanide,1n azide,1p carboxylates,1j−l,5 and pyridine deriva-
tives.1k,11 In fact, the possibility to controllably modify the
ligand environment of the cluster complexes is a handy tool for
C
dx.doi.org/10.1021/ic500553v | Inorg. Chem. XXXX, XXX, XXX−XXX