1
88
N. Raman et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 127 (2014) 185–195
ꢁ
ꢁ1
1
375
Calc. for C28
.27%; Co, 8.52%. Found: C, 48.58%; H, 5.22%; N, 12.11%; S, 9.25%;
t
sy(COO ), (
D
t
6
= 46); 530 (M@O), 437 (M@N) cm ; Anal.
b
range of 30–180 lM. The equilibrium binding constant (K ) values
for the interaction of the complexes with DNA were obtained from
the absorption spectral titration data using the following equation:
H36CoN
O
7
S
2
: C, 48.62%; H, 5.25%; N, 12.15%; S,
9
ꢁ3
ꢁ1
ꢁ1
2
Co, 8.48%. MS: m/z 692.
BM) 4.86; kmax in DMSO 773, 627 and 339 nm.
K
m
ꢂ 10 (O mol cm ) 10.93;
l
eff
½
DNAꢃ=ð Þ ¼ ½DNA=ð
e
a
ꢁ
e
f
e
b
ꢁ
e
f
Þꢃ þ 1=K
b
ð
e
b
ꢁ
e
f
Þ
ð1Þ
(
1
[
ZnL (Met)
2
] (3) Yield: 80%. M.Wt: 698, m.p: 188 °C; FT-IR (KBr
where e the extinction coefficient observed for the charge transfer
a
disc): 3319
t
(NH
2
), 1648
t
D
(C@O), 1614
= 54); 535 (M@O), 454 (M@N)
) d: 6.8–7.5 (phenyl multiplet), 9.3 (s,
), 3.4 (s, N@CH ); [3.9 (t, CH), 2.1 (dd,
) 2.3 (s, SACH ) (Met protons)] ppm; C NMR
): d = 116.0–138.1 (aromatic C), 14.8 (ACACH ), 40.3
t
(HC@N), 1432
t
asy
absorption at a given DNA concentration,
cient of the complex free in solution, , the extinction coefficient
of the complex when fully bound to DNA, K , the intrinsic binding
constant and [DNA], the concentration in nucleotides [23]. A plot
of [DNA]/( ) versus [DNA], gives K as the ratio of the slope
to the intercept.
Viscosity experiments were carried out in an Ostwald viscome-
ter, immersed in a thermostated water-bath maintained at a con-
stant temperature at 30.0 ± 0.1 °C. CT DNA samples of
approximately 0.5 mM were prepared by sonication in order to
minimize the complexities arising from CT DNA flexibility [24].
Flow time was measured with a digital stopwatch three times for
ef, the extinction coeffi-
ꢁ
ꢁ
(
cm
COO ); 1378
t
sy(COO ), (
t
e
b
ꢁ
1
1
;
H NMR (DMSO-d
6
b
@
CH@N), 2.4 (s, AC@CH
CH ), 2.7 (t, CH
DMSO-d
3
3
13
e
a
ꢁ
e
f
b
2
2
3
(
(
(
6
3
NACH
CANH
3
), 169.7 (C@O), 161.3 (CH@N), 15.7–35.4 (Met C), 55.3
), 175.3 (COO ) ppm. Anal. Calc. for C28
ꢁ
2
H
36
N
6
O
7
2
S Zn: C,
4
4
K
8.17%; H, 5.20%; N, 12.04%; S, 9.19%; Zn, 9.36%. Found: C,
8.15%; H, 5.19%; N, 12.00%; S, 9.16%; Zn, 9.31%. MS: m/z 700.
ꢁ3
ꢁ1
ꢁ1
2
m
ꢂ 10 (O mol cm ) 14.56; kmax in DMSO 340 and 283 nm.
2
[
CuL (Met)
disc): 3328
(HC@N), 1412
2
] (4) Yield: 79%. M.Wt: 681, m.p: 186 °C; FT-IR (KBr
t
(NH
2
), 1069
t
(ACAOACA), 1645
t
D
(C@O), 1596
= 36); 529
39CuN : C,
1.12%; H, 5.77%; N, 10.28%; S, 9.41%; Cu, 9.33%. Found: C,
1.10%; H, 5.74%; N, 10.26%; S, 9.38%; Cu, 9.31%. MS: m/z 682.
each sample and the average flow time was calculated at both
ꢁ
ꢁ
1/3
t
t
asy(COO ), 1376
tsy(COO ),
(
t
pH. Data were presented as (
the metal(II) complexes, where
the presence of complex, and
Viscosity values were calculated after correcting the flow time of
buffer alone (t ), )/t
g
/
g
g
o
)
versus the concentration of
ꢁ1
(
M@O), 459 (M@N) cm ; Anal. Calc. for C29
H
5
O
S
6 2
is the viscosity of CT DNA in
g is the viscosity of CT DNA alone.
o
5
5
ꢁ3
ꢁ1
ꢁ1
2
K
m
ꢂ 10 (O mol cm ) 9.42;
l
eff (BM) 1.89; kmax in DMSO
0
g
ꢁ (t ꢁ t
0
0
.
2
82, 374 and 817 nm.
Cyclic voltammetry was performed on a CHI620C electrochem-
ical analyzer with three electrode system of glassy carbon as the
working electrode, a platinum wire as auxiliary electrode and Ag/
AgCl as the reference electrode. The supporting electrolyte was
5 mM Tris–HCl/50 mM NaCl buffer at pH 7.4 and 4.7. Solutions
were deoxygenated by purging with nitrogen gas for 15 min prior
2
[
CoL (Met)
2
] (5) Yield: 76%. M.Wt: 676, m.p: 172 °C; FT-IR (KBr
(NH
disc): 3324
t
2
), 1067
t
(ACAOACA), 1647
t
D
(C@O), 1613
= 46); 540
39CoN : C,
1.47%; H, 5.81%; N, 10.35%; S, 9.48%; Co, 8.71%. Found: C,
1.43%; H, 5.79%; N, 10.32%; S, 9.47%; Co, 8.68%. MS: m/z 677.
ꢁ
ꢁ
t
(HC@N), 1421
t
asy(COO ), 1375
tsy(COO ),
(
t
ꢁ1
(
M@O), 456 (M@N) cm ; Anal. Calc. for C29
H
5 6 2
O S
5
5
to measurements. During measurement a stream of N
passed over the solution.
2
gas was
ꢁ3
ꢁ1
ꢁ1
2
K
m
ꢂ 10 (O mol cm ) 12.28;
leff (BM) 4.89; kmax in DMSO
8
04, 589 and 339 nm.
The extent of pBR322 DNA cleavage in the presence of an acti-
vator H as oxidizing agent was monitored by using agarose gel
electrophoresis. In reactions using super coiled pBR322 plasmid
DNA Form I (2 L,10 M) in Tris–HCl buffer (50 mM) with
50 mM NaCl (pH 7.4 and 4.7) which was treated with metal com-
plex (25 M) and activating agents (100 M) followed by dilution
with the Tris–HCl buffer to a total volume of 20 L. The samples
were incubated for 1 h at 37 °C. A loading buffer containing 25%
bromophenol blue, 0.35% xylene cyanol, 30% glycerol (3 L) was
2
[
ZnL (Met)
disc): 3316
(HC@N), 1430
M@O), 462 (M@N) cm ; H NMR (DMSO-d
multiplet), 9.5 (s, ACH@N), 3.9 (s, AOCH ), 2.4 (s, ACACH
), 2.7 (t, CH ) 2.3 (s, SACH
Met protons)] ppm; C NMR (DMSO-d ): d = 114.02–140.14 (aro-
), 14.8 (ACACH ), 40.2 (NACH ), 168.2
), 176.0
2
] (6) Yield: 81%. M.Wt: 683, m.p: 193 °C; FT-IR (KBr
2 2
O
t
(NH
2
), 1068
t(ACAOACA), 16,753
t
D
(C@O), 1618
ꢁ
ꢁ
t
t
asy(COO ); 1383
t
sy(COO ), (
t
= 47); 543
l
l
ꢁ1 1
(
6
) d: 6.8–7.6 (phenyl
), 3.5
l
l
3
3
(
(
s, NACH
3
); [3.9 (t, CH), 2.1 (dd, CH
2
2
3
)
l
1
3
6
matic C), 56.8 (AOCH
3
3
3
l
(
(
C@O), 160.4 (CH@N), 15.7–35.4 (Met C), 55.3 (CANH
2
added and electrophoresis was performed at 40 V for each hour
in Tris–Acetate–EDTA (TAE) buffer using 1% agarose gel containing
ꢁ
COO ). Anal. Calc. for C29
H
39
N
5
O
6
2
S Zn: C, 50.99%; H, 5.75%; N,
1
1
0.25%; S, 9.39%; Zn, 9.57%. Found: C, 50.97%; H, 5.73%; N,
1.0 lg/mL ethidium bromide. The gel was visualized by photo-
ꢁ3
ꢁ1
ꢁ1
0.21%; S, 9.36%; Zn, 9.52%. MS: m/z 685.
K
m
ꢂ 10 (O mol
graphing the fluorescence of intercalated ethidium bromide under
a UV illuminator. The cleavage efficiency was measured by deter-
mining the ability of the complex to convert the super coiled
(SC) DNA to nicked circular form (NC) and linear form (LC). Inhibi-
tion reactions were carried out by prior incubation of the SC
pBR322 DNA (10 mM) with DMSO (2 mL).
2
cm ) 15.23; kmax in DMSO 341 and 270 nm.
DNA binding and cleavage experiments
Concentrated stock solutions of metal complexes were prepared
by dissolving them in 2% DMF/5 mM Tris HCl/50 mM NaCl buffer
(
pH 7.4; blood and 4.7; stomach) and diluting suitably with corre-
Ex vivo antimicrobial assay
sponding buffer to the required concentrations for all the experi-
ments. For absorption spectral experiments, the DNA solutions
were pretreated with the solutions of metal complexes to ensure
no change in the concentration of the metal complexes. The
absorption spectra were recorded on a Shimadzu model UV-1601
spectrophotometer using cuvettes of 1 cm path length. Absorption
spectral titration experiments were performed by maintaining a
constant concentration of the complex and varying the nucleic acid
concentration. It was achieved by dissolving an appropriate
amount of the metal complex and DNA stock solution while main-
taining the total volume constant (1 mL). The absorbance (A) of the
most red-shifted band of each complex was recorded after succes-
sive addition of CT DNA. A fixed concentration value of complex
The synthesized ligand and its complexes were tested for their
ex vivo antimicrobial activity against two Gram-negative (Esche-
richia coli and Pseudomonas aeruginosa) and two Gram-positive
(Bacillus subtilis and Staphylococcus aureus) bacterial strains and
for ex vivo antifungal activity against Aspergillus niger, Aspergillus
flavus, Rhizoctonia bataicola and Candida albicans by disc diffusion
method using potato dextrose agar as medium. The stock solution
(10 mol L ) was prepared by dissolving the compounds in
DMSO and the solutions were serially diluted in order to find out
the Minimum Inhibitory Concentration (MIC) values. In a typical
procedure [25], a disc was made on the agar medium inoculated
with microorganisms. The disc was filled with the test solution
using a micropipette and the plate was incubated, 24 h for bacteria
ꢁ3
ꢁ1
(
10 lM) was titrated with increasing amounts of DNA over the