R.A. Steiner et al. / Journal of Inorganic Biochemistry 137 (2014) 1–11
3
Quantitative yield. 1H NMR (CD3OD, 400 MHz, 25 °C, δ from TMS): 8.17
(q, 2H), 8.06 (q, 2H), 7.54 (t, 1H), 7.46 (m, 1H), 7.36 (t, 1H), 6.92 (d,
2H) 6.85 (m, 1H). Selected IR bands: (cm−1) 1575 (νN = C), 1446 (m),
1095 (m), 717 (s).
on F2 with SHELXTL (Version 2008) [35]. All non-hydrogen atoms were
refined anisotropically. Hydrogen atoms on carbon were included in cal-
culated positions and were refined using a riding model. Crystal data and
refinement details are presented in Table 1 for complexes 1–4 while se-
lected bond distances and angles are listed in Tables 2 and 3 respectively.
The structure of 3∙CH3OH was found to be non-merohedrally twinned.
The two-component orientation matrix produced by CELL_NOW was
used to integrate the data which was subsequently scaled and absorption
corrected with TWINLABS (V2008/4) [35]. The initial solution was refined
with single component data for the stronger domain before final refine-
ment with data from both domains.
2.3.4. Synthesis of Cu(Q(oBt))(NO3)2(H2O)∙CH3OH (1∙CH3OH)
A batch of Q(oBt) (104 mg, 0.395 mmol) was suspended in MeOH.
Separately, Cu(NO3)2·3H2O (74 mg, 0.395 mmol) was dissolved in ~15
mL of MeOH. The copper solution was then added drop-wise to the ligand
suspension resulting in a dark green mixture. The reaction was refluxed
for 30 min and stirred for 10 h. After the solution was gravity filtered,
slow diffusion of diethylether (Et2O) resulted in the formation of
green crystalline 1 within 24 h. Yield: 156 mg (89%). Selected IR bands:
(cm−1) 1595 (νN = C), 1482 (s), 1268 (s), 761 (s). Electronic absorption
spectrum in MeOH: λmax (nm) (ε, M−1 cm−1) 211 (47003). Elemental
analysis (%): calc. for C17H16CuN4O8S: C, 40.80; H, 3.2; N, 11.3; S, 6.4.
Found: C, 40.7; H, 3.1; N, 11.3; S, 6.2.
2.5. DNA cleavage experiments
Chemical nuclease activity was monitored using agarose gel electro-
phoresis. SC-DNA was incubated for 30 min at 37 °C in 1.5 mL
microcentrifuge tubes containing incubation buffer in a total volume of
15 μL. Complex solutions in 100% dimethylformamide (DMF) were dilut-
ed in incubation buffer, resulting in a final DMF concentration of 20% or
less. The concentration of complexes was varied from 0 to 50 μM for 1
and 2, and from 0 to 80 μM for 3. The concentration of ascorbic acid
was twice the concentration of the complex (0–160 μM). When included,
DMSO was present at 10% (v/v) and L-histidine was present at 2 mM.
After incubation, samples were centrifuged at 12,000 ×g in a Mini-Spin
microcentrifuge (Eppendorf). A 10 μL sample of the supernatant was
removed and mixed with 2 μL of 6× loading dye, then loaded into a
well in a 1% (w/v) agarose gel prepared in gel running buffer. The gels
were run at 5–8 V/cm for 75 min in gel running buffer, stained in a
solution of 0.5 mg/mL EtBr for 30 min, then rinsed in DI water for
10 min before imaging with a UV transilluminator. The relative amounts
of DNA after cleavage experiments were determined by dividing the
fluorescence intensity of each band by the sum of the fluorescence inten-
sities of all bands in the same lane.
2.3.5. Synthesis of Cu(8OHQ(oBt))Cl2∙CH3OH (2∙CH3OH)
A batch of 8OHQ(oBt) (240 mg, 0.862 mmol) was suspended in 20 mL
MeOH. Separately, CuCl2·2H2O (147 mg, 0.862 mmol) was dissolved in a
minimum amount of MeOH, and was added drop-wise to the ligand
suspension. The dark orange solution was stirred for 10 h and then gravity
filtered. Slow diffusion of Et2O into the methanolic solution resulted in the
formation of dark orange crystalline 2 within 12 h. Yield: 161 mg (84%).
Selected IR bands: (cm−1) 3184 (νOH), 1574 (νN
C), 1342 (m),
=
1020 (s), 749 (s). Electronic absorption spectrum in MeOH: λmax (nm)
(ε, M−1 cm−1) 205 (44316). Elemental analysis (%): calc. for C17H14Cl2-
CuN2O2S: C, 45.9; H, 3.2; N, 6.3; S, 7.2. Found: C, 45.5; H, 2.9; N, 6.3; S, 7.2.
2.3.6. Synthesis of Cu(8OQ(oBt))Cl(CH3OH)∙CH3OH (3∙CH3OH)
A batch of CuCl2·2H2O (126 mg, 0.736 mmol) was dissolved in a
minimal amount of MeOH and then added drop-wise to freshly
prepared Na[8OQ(oBt)] (204 mg 0.736 mmol) in MeOH. The solution
immediately turned red and microcrystalline 3 precipitated from the
solution. After 10 h, the red brown solid was collected using gravity
filtration and dried under vacuum. Yield: 222 mg (70%). Selected IR
bands: (cm−1) 1589 (νN = C), 1455 (s), 1114 (m), 753 (s). Electronic
absorption spectrum in MeOH: λmax (nm) (ε, M−1 cm−1) 205 (32306).
Elemental analysis (%): calc. for C18H17ClCuN2O3S: C, 49.0; H, 3.9; N, 6.4;
S, 7.3. Found: C, 48.8; H, 3.7; N, 6.6; S, 7.7.
2.6. DNA binding studies
Samples of CT-DNA (12.5 μM) were incubated with ethidium
bromide (12.5 μM) and complex 1, 2 or 3 (0–80 μM) for 30 min at
room temperature in a total volume of 10 mL of gel running buffer.
Emission spectra were obtained with slit widths of 4 mm, an excitation
wavelength of 546 nm and emission wavelengths from 550 to 700 nm.
Values for fluorescence intensity were recorded at the λmax. Data were
analyzed by Stern–Volmer plots using the equation I0/I = Kq[Q] + 1
[36], where I0 is the fluorescence intensity of the CT-DNA alone, I is
the emission intensity of the CT-DNA plus complex, [Q] is the concentra-
tion of the complex, and Kq is the quenching constant. I0/I vs. [Q] was
plotted and the slope of the best-fit line was used to calculate Kq.
Binding constants were determined using the equation KEtBr [EtBr] =
2.3.7. Synthesis of [Cu(8OH1/2Q(oBt))(CH3OH)(NO3)]2(NO3) (4)
First, 8OHQ(oBt) (108 mg, 0.389 mmol) was suspended in 20 mL
MeOH. Separately, Cu(NO3)2·3H2O (73 mg, 0.389 mmol) was dissolved
in ~10 mL MeOH. The metal salt was then added drop-wise to the ligand
suspension. The deep red solution was stirred for 10 h and gravity
filtered. Slow diffusion of Et2O into the methanolic solution formed
orange crystalline 4 within 12 h. Yield: 271 mg (75%). Selected IR
bands: (cm−1) 3062 (νOH), 1567 (νN = C), 1275 (s), 1010 (m), 749 (s).
K
app [complex]50%, where KEtBr is the binding constant of EtBr to DNA
(1 × 107 M−1) [37], the concentration of ethidium bromide was
12.5 μM, and [complex]50% is the concentration of complex required to
reduce the emission intensity of EtBr by 50% calculated using the
Stern–Volmer plot.
Electronic absorption spectrum in MeOH: λmax (nm) (ε, M−1 cm−1
)
203 (46975). Elemental analysis (%): calc. for C34H27Cu2N7O13S2: C,
43.7; H, 2.9; N, 10.5; S, 6.9. Found: C, 43.2; H, 2.5; N, 10.5; S, 7.0.
2.4. X-ray data collection and structure solution and refinement
2.7. Cell viability assays
Suitable crystals for X-ray analysis of complexes 1–4 were obtained
by slow diffusion of Et2O into separate solutions of each compound dis-
solved in methanol: complex 1 was isolated as green needles, complex 2
was isolated as orange needles, complex 3 was isolated as red plates and
4 was isolated as orange plates. X-ray diffraction data were collected on
a Bruker APEX 2 CCD platform diffractometer (Mo Kα (λ = 0.71073 Å))
equipped with an Oxford liquid nitrogen cryostream. Crystals were
mounted in a nylon loop with Paratone-N cryoprotectant oil. The struc-
tures were solved using direct methods and standard difference map
techniques, and were refined by full-matrix least-squares procedures
For viability assays, cells were plated in 96 well plates at 1000 cells/
well and incubated for 24 h before adding copper compounds (concen-
tration range of 0–100 μM) or DMF, the solvent used to dissolve the
compounds. Each concentration was assayed in triplicate in each experi-
ment, and all experiments were repeated at least two times. Cell viability
was measured 72 h after addition of copper compounds by MTS (3-(4,5-
dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-
2H-tetrazolium) assay (Promega) according to the manufacturer's
instructions and color product measured at 490 nm using an Infinite
M200 Pro plate reader controlled by I-control 1.7 software (Tecan,