G Model
BIOMAC-8426; No. of Pages11
ARTICLE IN PRESS
S. Sangeetha, M. Murali / International Journal of Biological Macromolecules xxx (2017) xxx–xxx
3
2.2. Synthesis of N-(2-quinolylmethylidene)aminophenol
[H(quamol)]
a constant concentration of the complex. The absorbance (A) of the
most red-shifted band of the complex was recorded after successive
additions of CT DNA.
A mixture of 2-quinolinecarboxaldehyde (1.57 g, 10 mmol) and
2-aminophenol (1.09 g, 10 mmol) was heated to reflux in anhy-
drous methanol (60 mL) for 4 h. After the solvent was removed
under reduced pressure, 30 mL n-hexane-CH2Cl2 (V:V = 1:1) was
then added, and the mixture was refluxed for another 0.5 h. The
reaction mixture was filtered and left to stand at −20 ◦C overnight
and bright yellow crystals were collected from the filtrate. Yield:
1.56 g, 63%. 1H NMR (300 MHz, dmso-d6) ı/ppm: 7.40 (d, 1H, 7.8,
Ha), 7.16 (t, 1H, 8.4, Hb), 6.88 (t, 1H, 6.5, Hc), 6.96 (d, 1H, 7.8, Hd),
Circular dichroic (CD) spectral experiments were done using a
cylindrical 0.1 cm path length quartz cell. Each CD spectrum was
collected after averaging over at least four accumulations using
a scan speed of 100 nm min−1 and 1 s response time. Machine
plus cuvette baselines were subtracted and the resultant spectrum
zeroed outside the absorption bands.
For emission intensity measurements, the 2% DMF/5 mM Tris-
HCl/50 mM NaCl buffer was used as a blank to make preliminary
adjustments. The excitation wavelength was fixed and the emis-
sion range was adjusted before measurements. DNA was pretreated
with ethidium bromide in the ratio [NP]:[EthBr] = 1:1 for 30 min at
27 ◦C. The metal complex was then added to this mixture and their
effect on the emission intensity was measured.
9.33 (s, 1H, OH), 8.90 (s, 1H, CH
N ), 8.56 (d, 1H, 7.6, H1), 8.48
(d, 1H, 7.6, H2), 8.11 (d, 1H, 8.2, H3), 7.67 (t, 1H, 7.8, H4), 7.82 (t, 1H,
7.6, H5) and 8.04 (d, 1H, 7.2, H6). Selected IR peaks (ꢁ, cm−1): 3471
b (ꢁO H), 3052 m (ꢁsC H), 2901 m (ꢁasC H), 1651 s (ꢁC
)
,
N
azomethine
1610 s (ꢁC
988 w (ꢁ
)
quinoline, 1491, 1448 s (ꢁC
)quinoline, 1281 s (ꢁPh O) and
Cyclic voltammetry (CV) and differential pulse voltammetry
(DPV) were performed in a CHI 620C electrochemical analyzer
at 25 0.2 ◦C. The working electrode was a glassy carbon disk
(0.0707 cm2) and the reference electrode, a saturated calomel elec-
trode. A platinum wire was used as the counter electrode. The
supporting electrolyte was 2% DMF/5 mM Tris-HCl/50 mM NaCl
buffer (pH 7.1). Solutions were deoxygenated by purging with
nitrogen gas for 15 min prior to measurements; during measure-
ments a stream of N2 gas was passed over them. The redox potential
E1/2 was calculated from the anodic (Epa) and cathodic (Epc) peak
potentials of CV traces as (Epa + Epc)/2 and also from the peak poten-
tial (Ep) of DPV response as Ep + ꢄE/2 (ꢄE is the pulse height).
N
C
). Anal. Cacld. For C16H12N2O: C, 77.40; H, 4.87; N,
CH
N
11.28. Found: C, 77.14; H, 4.98; N, 11.22%.
2.3. Synthesis of [Cu(quamol)Cl]·H2O 1
A methanol solution (15 mL) of CuCl2·2H2O (0.085 g, 0.5 mmol)
was added dropwise to a yellow solution of H(quamol) (0.124 g,
0.5 mmol) in methanol (15 mL) with stirring for 2 h. The greenish
brown solution obtained was filtered and the filtrate left to stand
at 20 ◦C for slow evaporation. The crystalline dark brownish green
solid formed after four days was collected by filtration. It is not suit-
able for X-ray structure determination. Yield: 0.108 g, 59%. Selected
IR peaks (ꢁ, cm−1): 3342 b (ꢁO
)water, 3070 m (ꢁsC H), 2930 m
H
(ꢁasC H), 1634 s (ꢁC
)azomethine, 1568 s (ꢁC
)quinoline, 1506, 1463 s
N
N
(ꢁC
446 (ꢁCu
)
quinoline, 1310 s (ꢁPh-O), 998 w (ꢁ
), 500 (ꢁCu )
,
C
CH N-
N
quinoline
)azomethine, 404 (ꢁCu O) and 316 (ꢁCu Cl). Anal. Cacld. For
N
2.5. Protein binding experiments
C
16H13N2O2ClCu: C, 52.75; H, 3.60; N, 7.69. Found: C, 53.10; H,
3.13; N, 7.29%. ꢂM in aqueous DMF at 25 ◦C: 8 ꢀ−1 cm2 mol−1
.
The stock solution of protein (1.0 × 10−4 mol L−1) was pre-
pared by dissolving the solid BSA in 0.05 M phosphate buffer at
pH 7.4 and stored at 0–4 ◦C in the dark for about a week and
then diluted to 1.0 × 10−6 mol L−1 using phosphate buffer (pH 7.4,
0.05 M) when used. The concentration of BSA was determined from
optical density measurements, using the value of molar absorptiv-
ity of 280 = 44720 M−1 cm−1 [20]. All fluorescence measurements
were performed using a 10 mm quartz cuvette at two different
temperatures (300 and 310 K).
ESI–MS in DMF solution: [Cu(quamol)Cl] displays a peak at
m/z 346.6 (calcd. 346.3). Electronic absorption spectrum in DMF
[ꢃmax/nm (εmax/dm3 mol−1 cm−1): 608 (255), 434sh, 414 (2320),
313 (16580), 268 (20980)]. EPR spectrum in polycrystalline solid
at RT: giso = 2.123. EPR spectrum in frozen DMF solution at 77 K:
g|| = 2.265, A|| = 153 × 10−4 cm−1, g = 2.066, g /A = 148 cm, G = 4.0.
⊥
|| ||
Quantitative analyses of the interaction between complex and
BSA were performed by fluorimetric titration (0.05 M phosphate
buffer, pH 7.4). A 3.0 mL portion of an aqueous solution of BSA was
titrated by successive additions of the complex. Titrations were
done manually by using an Eppendorf micro-pipette. For every
addition, the mixture solution was shaken and allowed to stand
for 20 min at the corresponding temperature (300 and 310 K) and
then the fluorescence intensities were measured with an excitation
wavelength of 280 nm and emission wavelengths in the interval
290–500 nm. No correction for the inner filter effect was applied
since complex represented very low absorbance (less than 0.1) at
excitation and emission wavelengths. The excitation and emission
slit width (each 5.0 nm), scan rate (fast) were constantly maintained
for all the experiments. In the meantime, the synchronous fluo-
rescence intensity of the mixed solution was measured by setting
the excitation and emission wavelength interval (ꢅꢃ) at 15 and
60 nm. The UV–vis absorption spectra of 1.0 M free BSA as well as
BSA/complex (equal molar ratio) in 0.5 M phosphate buffer of pH
7.4 were recorded from 200 to 500 nm. The far-UV CD measure-
ments of BSA (1.0 M) in the absence and presence of copper(II)
complex (1:0.1, 1:0.2, 1:0.3) were recorded from 200 to 260 nm in
0.05 M phosphate buffer (pH 7.4) at room temperature.
Solutions of DNA in the 5 mM Tris HCl/50 mM NaCl buffer gave
a ratio of UV absorbances at 260 and 280 nm, A260/A280, of 1.9
[26], indicating that the DNA was sufficiently free of protein. Con-
centrated stock solutions of DNA (13.5 mol dm3) were prepared in
buffer and sonicated for 25 cycles, where each cycle consisted of
30 s with 1 min intervals. The concentration of DNA in nucleotide
phosphate (NP) was determined by UV absorbance at 260 nm
after 1:100 dilutions. The extinction coefficient, ε260, was taken
as 6600 dm3 mol−1 cm−1. Stock solutions were stored at 4 ◦C and
used after no more than 4 days. Concentrated stock solutions of
copper(II) complex was prepared by dissolving in 2% DMF/5 mM
Tris-HCl/50 mM NaCl buffer at pH 7.1 and diluting suitably with the
corresponding buffer to required concentrations for all the experi-
ments. For absorption and emission spectral experiments, the DNA
solutions were pretreated with solutions of copper(II) complex to
ensure no change in concentrations of the copper(II) complex.
Absorption spectral titration experiments were performed by
maintaining a constant concentration of the complex and varying
the nucleic acid concentration. This was achieved by dissolving an
appropriate amount of the metal complex and DNA stock solutions
while maintaining the total volume constant (1 mL). This results in
a series of solutions with varying concentrations of DNA, but with
Sangeetha, Somasundaram
