Article
Inorganic Chemistry, Vol. 49, No. 7, 2010 3263
Hc,Hc ), 8.54 (tt, 2H, Hb, Hb ), 9.17 (d, 2H, Ha, Ha ). 13C{1H}
NMR (CDCl3): δ(SiMe4) 174.032 (CdO); 148.332, 124.291,
138.330, 126.968, 155.894 (Ca, Cb, Cc, Cd, Ce of bipy); 66.480
(C0 of phenylmalonate).
For the fluorescence quenching experiment, the FS-DNA was
pretreated with ethidium bromide (EtBr) in the ratio
[DNA]/[EtBr] = 5 for 2 h at room temperature. The metal
complexes were then added to the DNA-EtBr system at varying
concentrations (1.25-40 μM), and their effect on the emission
intensity was measured. The concentration range was limited by
precipitation of the DNA at Pd(II) complexes > 55 μM. Under
these conditions, the fluorescence intensity of the respective
complexes, FS-DNA and EtBr, was very small and could be
ignored. The interaction of the respective complexes with DNA
in vitro was studied as described in the literature.36,37
A fluorescence Scatchard plot was created to study binding
constant determination. Essentially, the method is considered to
be the titration of a given amount of DNA-metal complexes
with increasing concentrations of EtBr (0.5-5 μM) and mea-
surement of the changes in fluorescence intensity. The binding
isotherms were obtained from the Scatchard equation:38 rb/c =
0
0
0
2.3.2. Synthesis of the [Pd(L3)(bipy)] 2H2O (3). The complex
3
was prepared by the same method used for the preparation of
complex 1 except using phenethylmalonate (0.2082 g) instead of
phenylmalonate. Yield: 53%. Anal. Calcd (%) for C21 H22 N2
O6 Pd1 (3): C, 49.96; H, 4.39; N, 5.55; Found (%): C, 49.69; H,
4.21; N, 5.45. IR(KBr): νmax/cm-1: 3418 (m), 3085 (w), 2925 (w),
1608 (s), 1452 (m), 1380 (s), 13 465 (w), 1163 (w), 966 (w), 773 (s),
723 (m), 701 (m), 487 (w). 1H NMR (300 MHz, DMSO-d6,
dppm): 2.72 (t, 2H, CH2CH2CHPh), 2.14 (m, 2H,
CH2CH2CHPh), 3.63 (t, 1H, CH2CH2CHPh), 7.83 (d, 2H,
0
0
Hd, Hd ), 8.26 (bm, 5H, Ph), 8.30 (tt, 2H, Hc,Hc ), 8.46 (tt, 2H,
Hb, Hb ), 9.15 (d, 2H, Ha, Ha ). 13C{1H} NMR (CDCl3):
δ(SiMe4) 174.925 (CdO); 148.390, 124.291, 142.085, 124.256,
156.019 (Ca, Cb, Cc, Cd, Ce of bipy); 33.687, 32.201, 58.413 (C1,
C2, C0 of phenethylmalonate).
0
0
K
app(n - rb), where rb is the number of molecules bound per
DNA nucleotide phosphate, c is the free drug concentration,
and Kapp is the apparent binding constant. A plot of experimen-
tally determined rb/c values versus rb yields a value for n, the
maximum number of binding sites per DNA base pair, from the
x intercept. rb can be determined by the equation rb = n - rE -
2.3.3. Synthesis of the [Pd(L4)(bipy)] 5H2O (4). The complex
3
was prepared by the same method used for the preparation of
complex 1, except phenylpropylmalonate (0.2220 g) was used
instead of phenylmalonate. Yield: 45%. Anal. Calcd (%) for
C22H30N2O9 Pd (4): C, 46.12; H, 5.28; N, 4.89. Found (%): C,
46.21; H, 5.24; N, 4.92. IR(KBr): νmax/cm-1: 3427 (m), 3108 (w),
2921 (w), 1602 (s), 1448 (s), 1373 (m), 1164 (m), 1037 (m), 762 (s),
720 (m), 699 (w), 650 (w). 1H NMR (300 MHz, DMSO-d6,
dppm): 1.71 (t, 2H, CH2CH2CH2CHPh), 1.34 (m, 2H,
CH2CH2CH2CHPh), 2.52 (m, 2H, CH2CH2CH2CHPh), 3.16
(t, 1H, CH2CH2CH2CHPh), 7.18 (bm, 5H, Ph), 7.79 (d, 2H, Hd,
rE/(KE cE),39 where cE is the concentration of free ethidium and
3
KE is a linear Scatchard constant dependent on the ratio of the
bound concentration of EtBr to the concentration of DNA, and
the KE value is obtained as the slope of the rE/CE versus rE linear
plot.38 Different DNA-metal complexes correspond to differ-
ent values of rf, where rf is the formal ratio of the complex to
nucleotide concentration with values ranging from 0 to 3.5. The
plot of rb/c versus rb gives the association constant (slope) and
the apparent binding site size (x intercept) for the agents.
2.7. Plasmid DNA Cleavage by Complexes. The plasmid
DNA, pBR322 (as a solution in Tris buffer), with a length of
436 bp was purchased from Biopolymers, Inc. and kept at -20 °C.
The sample was incubated at 310 K for an appropriate time, and
a loading buffer was applied (0.25% bromphenol blue, 50%
glycerol). The gel electrophoresis experiments were performed
0
0
0
Hd ), 8.32 (tt, 2H, Hc, Hc ), 8.59 (tt, 2H, Hb, Hb ), 9.17 (d, 2H, Ha,
13
1
0
Ha ). C{ H} NMR (CDCl3): δ(SiMe4) 175.303 (CdO);
148.369, 124.221, 142.065, 125.908, 155.964 (Ca, Cb, Cc, Cd,
Ce of bipy); 35.555, 30.368, 29.415, 59.507 (C1, C2, C3, C0 of
phenylpropylmalonate).
2.4. X-Ray Structure Determination. Single-crystal X-ray
data on 1, 2, 3, and 4 were collected at 293(2) K on a BRUKER
SMART 1000 CCD detector with graphite-monochromatized
˚
Mo KR radiation (λ = 0.71073 A) by using the ω-scan techni-
by incubation at 310 K for 5 min of 0.5 mg mL-1 pBR322 DNA
3
que. Lorentz polarization and absorption corrections were
applied. The structures were solved by direct methods and
refined with the full-matrix least-squares technique using the
SHELXS-97 and SHELXL-97 programs.34,35 Anisotropic ther-
mal parameters were assigned to all non-hydrogen atoms. The
hydrogen atoms were set in calculated positions and refined as
riding atoms with a common fixed isotropic thermal parameter.
Analytical expressions of neutral-atom scattering factors were
employed, and anomalous dispersion corrections were incorpo-
rated.
and a 20 μM metal complex in a loading buffer (0.25% brom-
phenol blue, 50% glycerol). Concentration of the metal complex
was also varied from 20 to 120 μM. After incubation, the
solution was then subjected to electrophoresis on 0.8% agarose
gel in a TAE buffer (40 mM Tris acetate/1 mMEDTA) at 120 V
for 120 min. Gels were stained with ethidium bromide and
visualized under a UV transilluminator and photographed using
a digital camera. In all cases, the background fluorescence was
subtracted. A correction factor of 1.42 was used for supercoiled
DNA (form I) assessment since the intercalation between EtBr
and form I DNA is relatively weak compared with that of nicked
(form II) and linear DNA (form III).40-43
2.5. Determination of Electronic Absorption Spectra. UV-
visible spectra were recorded on a Shimadzu UV-240. Tris-HCl
buffer (pH 7.0) and a buffer (10 mM) solution of complexes 1, 2,
3, and 4 were titrated with a concentrated fish sperm DNA (FS-
DNA) solution. The absorbance of the solution at 308 nm,
260 nm, and 260 nm was measured initially for complexes 1, 2, 3,
and 4 after each addition of FS-DNA.
2.8. Cell Line and Culture. The cell lines used in this experi-
ment were routinely maintained in a RPMI-1640 medium
supplemented with 10% (v/v) heat-inactivated fetal bovine
serum, 2 mmol/L of glutamine, 100 U/mL of penicillin, and
2.6. Fluorescence Spectra. A fluorescence survey was per-
formed on a Perkin-Elmer LS55 spectrofluorometer. For all
fluorescence measurements, the entrance and exit slits were
maintained at 10 and 10 nm, respectively. The sample was
excited at 526 nm and its emission observed at 602 nm. The
buffer used in the binding studies was 50 mM Tris-HCl, pH 7.4,
containing 10 mM NaCl. The sample was incubated for 4 h at
room temperature (20 °C) before spectral measurements.
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