M. H.-W. Lam, Y.-W. Lam et al.
as received unless stated otherwise. Solvents used for synthesis were of
analytical grade. THF was distilled from sodium/benzophenone prior to
use. 2-Phenyl-6-(1H-pyrazol-3-yl)pyridine was prepared according to the
published procedures.[12]
clometalated platinum(II) complex, the emission peak intensities were
plotted as a function of the incident power at 700 nm.
Two-photon cross-section measurements: The theoretical framework and
experimental protocol for the two-photon cross-section measurement
have been outlined by Webb and Xu.[25] The two-photon excitation
(TPE) ratios of the reference and sample systems were given by Equa-
tion (1):
Compound HL2: Freshly distilled THF (75 mL) was added to a mixture
of 2-phenyl-6-(1H-pyrazol-3-yl)pyridine (1.12 g, 5 mmol) and NaH (60%
by weight dispersed in mineral oil) (0.24 g, 6 mmol). The mixture was
stirred for 15 min at room temperature. 1,5-Dibromopentane (2.31 g,
10 mmol) was then added and the mixture was heated to reflux for 24 h.
After concentration under reduced pressure, the resulting crude solid
was chromatographically purified by using 4:1 (v/v) petroleum ether/di-
ethyl ether as the eluent to give a white crystalline solid (1.46 g, 78%).
1H NMR (300 MHz, CDCl3): d=8.09–8.14 (m, 2H; phenyl CH), 7.92 (dd,
J=0.9, 8.1 Hz, 1H; pyridyl CH), 7.78 (t, J=7.8 Hz, 1H; pyridyl CH),
7.65 (dd, J=0.9, 7.5 Hz, 1H; pyridyl CH), 7.38–7.52 (m, 4H; phenyl
CH+pyrazolyl CH), 7.05 (d, J=2.1 Hz, 1H; pyrazolyl CH), 4.20 (t, J=
6.9 Hz, 2H; aliphatic CH2), 3.40 (t, J=6.9 Hz, 2H; aliphatic CH2), 1.85–
2.02 (m, 4H; aliphatic CH2), 1.44–1.56 (m, 2H; aliphatic CH2); IR (KBr):
n˜ =3062, 1590 cmꢀ1 (aromatic C=C); ESIMS: 371 [M+1]+.
sS2 ꢀS
CRnSFSðlÞ
ð1Þ
¼
sR2 ꢀR CSnRFRðlÞ
in which f is the quantum yield, C is the concentration, n the refractive
index, and F(l) is the integrated photoluminescent spectrum. The super-
scripts S and R represent the sample and reference, respectively. In our
measurements, we have ensured that the excitation flux and the excita-
tion wavelengths are the same for both the sample and the reference.
The two-photon absorption cross-section s2 of the cyclometalated plati-
num(II) complex [Pt(L3)Cl]+ was determined by using Rhodamine 6G as
a reference.
Compound HL3: A mixture of 2-(1-(5-bromopentyl)-1H-pyrazol-3-yl)-6-
phenylpyridine (1.11 g, 3 mmol) and PPh3 (1.57 g, 6 mmol) in acetonitrile
(50 mL) was heated to reflux with stirring for 72 h. After reaction, an
aqueous solution (25 mL) of KCl (8.95 g, 120 mmol) was added. After
concentration under reduced pressure, the resulting crude solid was chro-
matographically purified by using 2:1 (v/v) dichloromethane/methanol as
the eluent to give a white solid (1.27 g, 72%). 1H NMR (300 MHz,
CDCl3): d=8.06–8.12 (m, 2H; phenyl CH), 7.37–7.83 (m, 22H; phenyl
CH+pyridyl CH+pyrazolyl CH), 6.87–6.89 (d, J=6.9 Hz, 1H; pyrazolyl
CH), 4.15 (t, J=6.9 Hz, 2H; aliphatic CH2), 3.00–3.11 (m, 2H; aliphatic
CH2), 1.87–2.00 (m, 2H; aliphatic CH2), 1.56–1.67 (m, 2H; aliphatic
CH2), 1.48–1.57 (m, 2H; aliphatic CH2); IR (KBr): n˜ =3062, 1589 cmꢀ1
(aromatic C=C); ESIMS: 553 [MꢀCl]+.
Cell culture: HeLa and 3T3 cells were cultured in Dulbeccoꢂs Modified
Eagle Medium (DMEM) from Gibco, supplemented with 10% foetal
bovine serum (FBS) in the presence of antibiotic-antimycotic solution
(Gibco). The cells were cultured in a humidified chamber with 5% CO2
at 378C.
Immunofluorescence: For immunofluorescence experiments, HeLa cells
were grown on 18 mm cover slips (no.1; Corning) and fixed with cold
methanol for 10 min, washed three times with phosphate-buffered saline
(PBS). Immunofluorescence staining was carried out as described previ-
ously.[26] Antibodies used were anti-fibrillarin polyclonal (dilution 1:100,
Santa Cruz) and anti-FUS/TLS monoclonal (dilution 1:100, Santa Cruz),
and TRITC conjugated secondary antibodies (Sigma). After immuno-
fluoresence, cells were incubated with [Pt(L3)Cl]+ at 1 mm for 15 min. In
some experiments, prior to mounting on slides, cover slips were incubat-
ed with 4 mm Hochest 33342 (Sigma) in water for 5 min to counterstain
DNA. Coverslips were mounted in 50% glycerol and sealed with nail
polish.
ACHTUNGTRENNUNG
[Pt(L3)Cl][PF6]: A mixture of the free ligand HL3 (0.35 g, 0.5 mmol) and
K2PtCl4 (0.21 g, 0.5 mmol) in degassed glacial acetic acid (15 mL) was
heated at reflux for 12 h to give a yellow solution. Addition of an aque-
ous solution (15 mL) of KPF6 (0.92 g, 5 mmol) caused precipitation of a
yellow solid. The precipitate was collected by filtration and washed with
water and diethyl ether (0.38 g, 82%). 1H NMR (300 MHz, CDCl3): d=
7.92–7.93 (d, J=2.8 Hz, 1H; pyrazolyl CH), 7.83–7.90 (m, 5H; phenyl+
pyridyl CH), 7.69–7.79 (m, 12H; phenyl CH), 7.45–7.47 (dd, J=0.8, 8 Hz,
1H; pyridyl CH), 7.37–7.41 (m, 2H; phenyl+pyridyl CH), 7.25–7.29 (dt,
J=1.33, 7.47 Hz, 1H; phenyl CH), 7.15–7.19 (dt, J=1.33, 7.47 Hz, 1H;
phenyl CH), 6.78 (d, J=2.8 Hz, 1H; pyrazolyl CH), 4.68 (t, J=7.4 Hz,
2H; aliphatic CH2), 3.25–3.33 (m, 2H; aliphatic CH2), 2.13–2.15 (m, 2H;
aliphatic CH2), 1.81–1.80 (m, 4H; aliphatic CH2); IR (KBr): n˜ =3062,
1584 cmꢀ1 (aromatic C=C); ESIMS: 782 [MꢀPF6]+.
MTT cell viability assay: HeLa cells were plated in triplicates into 96-
well tissue culture plates. After 24 h, cells were treated with [Pt(L3)Cl]+
at concentrations of 0, 0.1, 0.5 and 1 mm for 15 min. Cells were then
washed with DMEM and replaced with new medium and incubated for
1, 2 and 4 h. The cells were then incubated with 0.5 mgmLꢀ1 of 3-(4,5-di-
methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Sigma) for 30 min
before being lysed with 1-isopropanol. Absorption was measured at
570 nm by using a microplate reader (PowerWave XS, BioTek). For com-
parison of cell viability between the treatments, a relative viability was
calculated by dividing all the absorbance readings with those of cells not
treated with [Pt(L3)Cl]+.
Physical measurements and instrumentation: 1H NMR spectra were re-
corded by using a Varian YH300 300 MHz NMR spectrometer. Electro-
spray (ESI) mass spectra were measured by using a PE SCIEX API 365
LC/MS/MS system. UV/Vis spectra were measured on a Hewlett Packard
8452A UV/Vis diode array spectrophotometer. Emission spectra were re-
corded by using a Horiba FluoroMax-3 spectrofluorimetric with a 5 nm
slit width and 0.5 s integration time. Emission lifetime measurements
were performed by using a SPEX FluoroLog 3-TCSPC spectrofluorime-
ter in the Fast MCS mode with a NanoLED N-370 as the laser source for
excitation. Emission quantum yields were measured by the method of
RNase treatment experiment: To investigate whether the cellular staining
of [Pt(L3)Cl]+ was dependent on RNA integrity, HeLa cells were grown
on cover slips, fixed with cold methanol for 10 min, were then washed
thrice with PBS. The cells were treated with 100 mgmLꢀ1 ribonuclease A
from bovine pancreas (Sigma) for 30 min prior labelling with 1 mm of
[Pt(L3)Cl]+ in PBS for 15 min. Cells were mounted in 50% glycerol and
sealed with nail polish.
Histology: An adult (10 weeks old) male BALB/c mouse (obtained by
the Lab Animal Unit, Chinese University of Hong Kong) was sacrificed
by cervical dislocation. The mouse was maintained and handled by proce-
dures in compliance to the Prevention of Cruelty of Animals Ordinance
(Cap. 169) of Hong Kong. The heart, kidney, ileum and kidney were dis-
sected and rinsed in PBS. The organs were snap-frozen in liquid nitrogen,
embedded in tissue freezing medium (Jung) and sectioned on a cryostat
(Jung CM 1500, Leica Instruments). The sections were dried on glass
slides before being fixed in cold methanol (10 min, RT). After fixation,
the sections were labelled with 1 mm of [Pt(L3)Cl]+ in PBS for 30 min.
Hoechst 33342 (4 mm, 15 min) was used to counterstain DNA. Sections
were mounted in 50% glycerol and sealed with nail polish.
Demas and Crosby[23] with [Ru
ACHTUNGTRNENUG(bpy)3]ACHTUNGTREN[NUGN (PF6)2] in degassed acetonitrile as
the standard (fr =0.062). Sample and standard solutions were degassed
with at least three freeze–pump–thaw cycles.
Two-photon-induced emission measurements: The 700 nm pump source
was from the fundamental of a femtosecond mode-locked Ti:sapphire
laser system (output beam ꢂ150 fs duration and 1 kHz repetition rate).
The laser was focused to a spot size of ꢂ50 mm through an f=10 cm lens
onto the sample. The emitting light was collected with a backscattering
configuration into a 0.5 m spectrograph and detected by a liquid-nitro-
gen-cooled CCD detector. A power meter was used to monitor the uni-
form excitation.[24] To further confirm the two-photon process of the cy-
3948
ꢁ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2010, 16, 3942 – 3950
Koo, Chi-Kin
