230
Y.-Y. Qi et al. / European Journal of Medicinal Chemistry 154 (2018) 220e232
(0.020 g, 0.5 mmol) were mixed in aqueous solution (5 mL) with
stirring, followed by addition of Cu(ClO4)$6H2O (0.186 g, 0.5 mmol).
Then, methanol solution (20 mL) of HPBM (0.105 g, 0.5 mmol) was
added dropwise, and the pH value was adjusted to 4.86 with
perchloric acid solution. After being stirred for 1 h at 50 ꢂC, the
resulting solutions were filtered and left to evaporate at room
temperature to obtain the complexes.
KEB [EB] ¼ Kapp [complex]
(3)
where KEB ¼ 1.0 ꢁ 107 Mꢀ1, [EB] ¼ 8
m
M, and [complex] is the value
at 50% decrease of the fluorescence intensity of EB.
CD spectra of CT DNA (100 mM) with the addition of the com-
plexes ([complex]/[DNA] ratios of 0e1.0) were obtained in the
range of 220e320 nm at room temperature, using a 10 mm path
quartz cuvette. Each spectrum was obtained by averaging three
scans and subtracting the background signal of the buffer at
100 nm minꢀ1 scanning rate.
All cyclic voltammetric measurements were performed in a
single compartment cell with a glass carbon electrode as the
working electrode, a Pt wire as the auxiliary electrode and a satu-
rated calomel electrode (SCE) as the reference electrode, at
200 mV sꢀ1 scanning rate for the potential range from ꢀ0.6 to 0.6 v
in buffer solution (pH 7.2) containing 10 mM Tris-HCl/50 mM NaCl
as supporting electrolyte. The whole experiments were carried out
in a nitrogen atmosphere. The ratios of binding constants of the
reduced state Cu(I) and oxidation state Cu(II) were calculated using
the following equation (Eq. (3)) [30]:
4.3.1. [Cu(Gly-gly)(HPBM)(H2O)]ClO4·0.5H2O (1)
Yield: 72%. Anal. Calcd for C17H21N5O8.5ClCu: C, 38.49; H, 3.99; N,
13.20%. Found: C, 38.70; H, 3.71; N, 13.00%. FT-IR (KBr, cmꢀ1):
n
(ꢀOH) 3412; nas(ꢀNH2) 3091; ns(ꢀNH2) 2925; nas(eCOOe) 1610;
ns(eCOOe) 1395;
n
(C¼N) 1456;
n(CueO) 626; n(CueN) 420. UVevis.
(MeOH) lnm/nm, ε/(Mꢀ1 cmꢀ1): 317, 2.44 ꢁ 104; 665, 74.81. Molar
conductivity L (S cm2 molꢀ1) in methanol: 89.3. ESI-MS (m/z,
m
MeOH): 403.9 for [Cu(Gly-gly)(HPBM)]þ. ESR (MeOH): g ¼ 2.2784,
jj
g⊥ ¼ 2.0444.
4.3.2. [Cu(Gly-L-leu)(HPBM)(H2O)]ClO4 (2)
Yield: 75%. Anal. Calcd for C21H28N5O8ClCu: C, 43.67; H, 4.88; N,
12.13%. Found: C, 43.48; H, 4.59; N, 11.95%. FT-IR (KBr, cmꢀ1):
00
00
E
bꢀ E ¼ 0.059 log[KCu(I)/KCu(II)
]
(4)
f
n
(ꢀOH) 3437; nas(ꢀNH2) 3082; ns(ꢀNH2) 2960; nas(eCOOe) 1615;
ns(eCOOe) 1381;
n
(C¼N) 1450;
n
(CueO) 623;
n
(CueN) 436. UVevis.
00
00
where E b and E f are the condition potential of Cu(I)/Cu(II) of the
complexes alone or in combination with DNA, respectively. And
KCu(I) and KCu(II) are the corresponding binding constants of the
Cu(I) or Cu(II) complexes with DNA, respectively.
(MeOH) lnm/nm, ε/(Mꢀ1 cmꢀ1): 323, 2.53 ꢁ 104; 677, 64.45. Molar
conductivity L (S cm2 molꢀ1) in methanol: 97.8. ESI-MS (m/z,
m
MeOH): 459.0 for [Cu(Gly-L-leu)(HPBM)]þ. ESR (MeOH):
g ¼ 2.2570, g⊥ ¼ 2.0477.
jj
The CT DNA viscosity experiments in the absence and presence
of EB (standard) or complex 1/2 were conducted on Ostwald
Viscometer, immersed in a water bath maintained at 29 0.1 ꢂC.
4.4. DNA binding and cleavage experiments
The concentration of CT DNA was 200
mM, and the concentrations
Absorption titration experiments were done by maintaining the
concentration of the complexes constant and varying CT DNA
concentration. The absorption spectra were recorded in the range
of 225e500 nm. The base line was corrected by subtracting Tris
buffer, and CT DNA was placed to remove the absorbance of the
DNA itself. Every experiment was allowed to incubate for 8 min
before the spectra were recorded. From the spectroscopic titration
data, the binding constants (Kb) were calculated using the following
equation (Eq. (1)) [26]:
of EB and the complexes varied from 0 to 70
tervals. The flow times were measured with a digital stopwatch and
m
M with 10 M in-
m
each sample solution was tested three times to get an average time.
Data were presented as (
0.05, 0.1, 0.15, 0.20, 0.25, 0.30, 0.35), where h and h0 are the viscosity
h
/
h0
)
1/3 versus r (r ¼ [complex]/[DNA] ¼ 0,
of the CT DNA solutions in the absence and presence of the com-
plexes, respectively. The relative viscosity values ( ) were calcu-
lated based on the observed flow times of the DNA econtaining
solutions (t) corrected from the flow time of buffer alone (t0),
t0)/t0.
h
h
¼ (t-
[DNA]/(εa e εf) ¼ [DNA]/(εb e εf) þ 1/Kb(εb e εf)
(1)
The studies of the complexes docked into DNA were performed
using AutoDock Vina1.1.2 set of programs with the Lamarckian
Genmetic Algorithm (LGA) [57]. The crystal structure of DNA d(50-
G-Diu-TGCAAC-30) (PBD ID:454D) was downloaded from the Pro-
tein Data Bank, the water molecules were deleted from this struc-
ture before performing docking calculations. The molecular
structures of the complexes were sketched via Gaussian viewer. A
grid box (60, 60, 60) was selected to enclose the whole DNA
molecule, with a spacing of grid 0.375 Å, all other parameters were
kept at their default setting. The docking results were visualized
using PyMol software.
where [DNA] is the concentration of DNA in the base pairs. The
apparent absorption coefficient εa, εb and εf correspond to Aobs/CCu
,
the extinction coefficients of the complexes in the bound and free
forms, respectively. In plots of [DNA]/(εa e εf) versus [DNA], Kb is
given by the ratio of the slope (1/(εb e εf)) to the intercept (1/Kb(εb e
εf)).
The competitive experiments were carried out in the buffer (pH
7.2) by maintaining [DNA]/[EB] ¼ 1.25 and varying the concentra-
tion of the complexes. Each sample solution was scanned using an
excitation wavelength of 525 nm and emission wavelength set at
550e660 nm, and the mixtures were allowed to incubate for 8 min
before the spectra were recorded. The SterneVolmer constants
(KSV), a measure of the binding propensity of the complexes to DNA,
were calculated using the following equation (Eq. (2)) [30]:
The DNA cleavage was carried out using pBR322 plasmid DNA in
Tris buffer (pH 7.2) with the concentrations of the complexes
(5e20 mM) and ascorbic acid (50 mM) in a total volume of 10 mL. The
samples were incubated for 1 h at 37 ꢂC in the dark. A loading buffer
was added, and then electrophoresis was done at 100 V for 40 min
in standard Tris-boric acid-EDTA (TBE) buffer (pH 8.3). The ob-
tained gel bands were visualized and photographed using a Gel
Imaging System (BIO-RAD Laboratories-Segrate).
I0/I ¼ 1 þ Ksv [Q]
(2)
where I0 and I stand for the fluorescence intensities at 550e660 nm
in the absence and presence of the complexes, respectively. Ksv is
the linear StermeVolmer quenching constant and [Q] is the con-
centration of the complexes. Additionally, the apparent binding
constants (Kapp) were calculated using the equation (Eq. (3)) [31]:
In addition, the possible DNA cleavage mechanism of the com-
plexes was investigated in the presence of typical radical scaven-
gers such as hydroxyl radical scavengers (DMSO, ethanol and tert-
butyl), a superoxide anion radical scavenger (SOD) and a singlet
oxygen quencher (NaN3). Each sample was incubated for 1 h at