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N. Chowdhury et al. / Journal of Photochemistry and Photobiology B: Biology 130 (2014) 188–198
ice cooled condition. The reaction was stirred at 35 °C for 4–
5 h. After completion of the reaction, the reaction mixture
was diluted with DCM and washed with water. Evaporation
of the organic phase gave an oily residue, which was purified
by column chromatography using as eluent a mixture of 25%
EtOAc/hexane to afford compound 10a in moderate yield
(0.16 g, 56%). Compound 10b was also prepared by using sim-
ilar procedure.
2.5.4. Photoinduced DNA cleavage of quinoline hydroperoxide 10b by
adding different additives
The photoinduced DNA cleavage experiments were done by
adding different additives like D2O (4
(50 mM) in sodium phosphate buffer solution containing quinoline
hydroperoxide 10b (30 M) in 10% acetonitrile solution, upon irra-
lL), DMSO (4 lL), KI
l
diation of UV light (P350 nm) under aerobic condition at room
temperature for 10 min. The sample buffer containing bromophe-
nol and glycerol was added to the reaction mixture and loaded
on 1% agarose gel. After addition of the tank buffer (1X TAE), the
electrophoresis apparatus was attached to a power supply. The
gel was visualized by GEL LOGIC 200 Imaging System.
2.4. Preparative photolysis of quinoxaline and quinoline
hydroperoxides 5b and 10b
Photolysis of quinoxaline and quinoline hydroperoxides 5b and
10b (200 mg, 0.7 mM) in 80 mL argon saturated acetonitrile solu-
tion were carried out using 125 W medium pressure Hg lamp as
light source (P350 nm) and 0.1 M CuSO4 solution as UV cut-off
filter. The reactions were monitored by TLC at regular interval of
time. After completion of photolysis, solvent was removed under
reduced pressure and photoproducts were isolated by flash column
chromatography using 20%EtOAc/hexane as an eluent. The ester
compounds 11 and 12 were isolated as major photoproducts along
with minor amounts of 3b and 8b.
2.6. DNA-binding studies of quinoline hydroperoxide 10b
2.6.1. Electronic absorption titration of quinoline hydroperoxide 10b
Absorption titration experiments were performed in 3 cm
quartz cuvette by taking absorption in between 300 and –400 nm.
3 mL of 50 lM quinoline hydroperoxide 10b in ethanolic solution
was titrated with increasing concentration of ct-DNA (0–100
lM)
in phosphate buffer (10 mM, pH 7.0) containing 50 mM NaCl
2.6.2. Fluorimetric titration of quinoline hydroperoxide 10b
Fluorimetric titrations were performed in a 3 cm quartz cuvette
using an excitation wavelength of 350 nm having emission max-
ima at 400 nm. The titration experiments were performed with
2.5. DNA cleavage experiments
fixed concentrations of quinoline hydroperoxide 10b (50
ethanolic solution, with gradually increasing the concentration of
ct-DNA (0–100 M) in phosphate buffer (10 mM, pH 7.0) contain-
ing 50 mM NaCl. A quantitative estimation of quenching experi-
ments in term of the binding constant calculation is obtained
from Stern–Volmer equation
lM) in
2.5.1. Procedure for DNA cleaving ability of quinoline hydroperoxide
10b at different concentrations
l
The reaction mixtures (20
pBR322 DNA stock solution (form I, 62.5
phate buffer (10 mM, pH 7.0), quinoline hydroperoxide 10b at dif-
ferent concentrations (10–50 M) in 10% acetonitrile solution
individually in eppendrof were irradiated with UV light
lL) containing supercoiled circular
l
g/mL) in sodium phos-
l
logðF0 ꢀ FÞ=F ¼ log Kb þ n log½DNAꢁ
ð1Þ
a
(P350 nm) under aerobic conditions at room temperature for
10 min. The sample buffer containing bromophenol and glycerol
was added to the reaction mixture and loaded on 1% agarose gel.
After addition of the tank buffer (1X TAE), the electrophoresis
apparatus was attached to a power supply. The gel was visualized
by GEL LOGIC 200 Imaging System.
where Kb and n are the binding constant and the number of
binding sites respectively, [DNA] is the concentration of ct-DNA
in base pairs, F0 and F are the fluorescence intensities of quinoline
hydroperoxide 10b in absence and presence of DNA respectively.
From the linear plot of log (F0 ꢀ F)/F vs log DNA, the binding con-
stant (Kb) value was calculated.
2.6.3. Ethidium bromide (EB) displacement assay of quinoline
hydroperoxide 10b
2.5.2. General procedure for photoinduced DNA cleavage by
quinoxaline and quinoline 5a–b and 10a–b
The DNA cleavage experiments were carried out containing
pBR322 DNA stock solution in sodium phosphate buffer (10 mM,
Fluorimetric titrations were performed in a 3 cm quartz cuvette
using an excitation wavelength of 480 nm. 3 mL of 20
lM ct-DNA
was saturated with 2 M of ethidium bromide (EB) solution in
l
pH 7.0), hydroperoxides 5a–b and 10a–b (30 lM) in 10% acetoni-
10 mM phosphate buffer pH 7.0 containing 50 mM NaCl. The EB-
ct-DNA solution was then titrated by successive addition of hydro-
trile solution individually in a eppendrof upon irradiation of UV
light (P350 nm) for 10 min. The sample buffer containing bromo-
phenol and glycerol was added to the reaction mixture and loaded
on 1% agarose gel. After addition of the tank buffer (1X TAE), the
electrophoresis apparatus was attached to a power supply. The
gel was visualized by GEL LOGIC 200 Imaging System.
peroxide 10b to reach a final concentration of ꢂ35
lM. Emission
spectra were recorded from 500 nm to 750 nm. The spectra were
analyzed according to the classical Stern–Volmer equation
F0=F ¼ 1 þ Ksv½Qꢁ
ð2Þ
where F0 and F are the fluorescence intensities of ethidium bromide
in absence and presence of hydroperoxide 10b respectively, Ksv is
the linear Stern–Volmer quenching constant, [Q] is the concentra-
tion of the hydroperoxide 10b.
2.5.3. Photoinduced DNA cleavage of quinoline hydroperoxide 10b in
different concentration of NaN3
The photoinduced DNA cleavage experiments were carried out
in presence of increasing amount of NaN3 in the sodium phosphate
buffer solution containing quinoline hydroperoxide 10b (30
l
M) in
2.7. Docking studies of quinoline hydroperoxide 10b
10% acetonitrile solution, upon irradiation of UV light (P350 nm,)
under aerobic condition at room temperature for 10 min. The sam-
ple buffer containing bromophenol and glycerol was added to the
reaction mixture and loaded on 1% agarose gel. After addition of
the tank buffer (1X TAE), the electrophoresis apparatus was at-
tached to a power supply. The gel was visualized by GEL LOGIC
200 Imaging System.
The B-DNA crystal structure used for the docking studies was
obtained from the Protein Data Bank (PDB ID: 453D) [27]. The
DNA file was prepared for docking by removing water molecules
and adding polar hydrogen atoms with Gasteiger charges. The 3D
structure of the ligands was generated in Sybyl 6.92 (Tripos Inc.,
St. Louis, USA) and its energy-minimized conformation was