Influence of terminal substitution on structural, DNA, Protein binding, anticancer and antibacterial activities of palladium(ii) complexes containing 3-methoxy salicylaldehyde-4(N) substituted thiosemicarbazones

Article abstract of DOI:10.1039/c1dt11838b

The variable chelating behavior of 3-methoxysalicylaldehyde-4(N)- substituted thiosemicarbazones was observed in equimolar reactions with [PdCl₂(PPh₃)₂]. The new complexes were characterized by various analytical, spectros

Full text of DOI:10.1039/c1dt11838b

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PAPER
Influence of terminal substitution on structural, DNA, Protein binding,
anticancer and antibacterial activities of palladium(II) complexes containing
3-methoxy salicylaldehyde-4(N) substituted thiosemicarbazones†
P. Kalaivani,a R. Prabhakaran,*a,b E. Ramachandran,a F. Dallemer,b G. Paramaguru,c R. Renganathan,c
P. Poornima,d V. Vijaya Padmad and K. Natarajan*a
Received 28th September 2011, Accepted 1st November 2011
DOI: 10.1039/c1dt11838b
The variable chelating behavior of 3-methoxysalicylaldehyde-4(N)-substituted thiosemicarbazones was
observed in equimolar reactions with [PdCl2(PPh3)2]. The new complexes were characterized by various
analytical, spectroscopic techniques (mass, 1H-NMR, absorption, IR). All the new complexes were
structurally characterized by single crystal X-ray diffraction. Crystallographic results showed that the
ligands H2L1 and H2L4 are coordinated as binegative tridentate ONS donor ligands in the complexes 1
and 4 by forming six and five member rings. However, the ligands H2L2 and H2L3 bound to palladium
in 2 and 3 as uninegative bidentate NS donors by forming a five member chelate ring. From this study,
it was found that the substitution on terminal 4(N)-nitrogen may have an influence on the chelating
ability of thiosemicarbazone. The presence of hydrogen bonding in 2 and 3 might be responsible for
preventing the coordination of phenolic oxygen to the metal ion. The interaction of the complexes with
calf-thymus DNA (CT-DNA) has been explored by absorption and emission titration methods. Based
on the observations, an electrostatic binding mode of DNA has been proposed. The protein binding
studies were monitored by quenching of tryptophan and tyrosine residues in the presence of complexes
using Lysozyme as model protein. Antibacterial activity studies of the complexes have been screened
against pathogenic bacteria such as Enterococcus faecalis, Staphylococcus aureus, Escherichia coli,
Klebsiella pneumonia and Pseudomonas aeruginosa. MIC50 values of the complexes showed that they
exhibited significant activity against the pathogens and among them, 3 exhibited higher activity.
Further, anticancer activity of the complexes on the lung cancer cell line A549 has also been studied.
The versatility of the thiosemicarbazone ligands for binding to
the metal ion has been well documented. Besides the enormous
1. Introduction
structural diversity exhibited by the metal complexes,3 they possess
a wide range of biological properties such as antiviral,4 antifungal,5
antibacterial,6 antitumor,7 anticarcenogenic8 and insulin mimetic
properties.9 For the last decade, our group has been actively
engaged in the synthesis and characterization of N-substituted
thiosemicarbazone complexes with various transition metals.2d,2h,10
In the continuation of our efforts in understanding the coordi-
nation propensities of thiosemicarbazones, we have carried out
the structural characterization of new palladium(II) complexes
containing 3-methoxysalicylaldehyde-4(N)-substituted thiosemi-
carbazones (H2L1–4). The study on the interaction of transition
metal complexes with DNA is a vibrant area of research.11 In
this area, the ability to selectively target and cleave DNA with
high affinity and to report on the binding event by changes in
luminescence is of great current interest.12 An advantage of using
transition metal complexes in such studies is that the ligands and
metal ions in such complexes can be conveniently varied to suit
individual applications due to the ability to inhibit the biosynthesis
of DNA, possibly by binding to the nitrogen base of DNA or
Among the sulfur family, thiosemicarbazones are unique and
multifaceted ligands that possess a variety of flexible donor sets
and are capable of coordinating to the metal ion through the sulfur
and one of the hydrazinic nitrogen atoms (N1 or N2), leading to
the formation of either a five or a four member chelate ring.1,2
aDepartment of Chemistry, Bharathiar University, Coimbatore, 641 046,
India. E-mail: rpnchemist@gmail.com,k_natraj6@yahoo.com; Fax: +91-
422-2422387; Tel: +91-422-2428319
bLaboratoire Chimie Provence-CNRS UMR6264, Universite´ of Aix-
Marseille I, II and III – CNRS, Campus Scientifique de Saint-Je´roˆme,
Avenue Escadrille Normandie-Niemen, F-13397, Marseille, Cedex 20,
France
cDepartment of Chemistry, Bharathidasan University, Trichirappalli, 620
015, India
dDepartment of Biotechnology, Bharathiar University, Coimbatore, 641 046,
India
† Electronic supplementary information (ESI) available. CCDC ref-
erence numbers 815366–815369 for [Pd(Msal-tsc)(PPh3)](1), [Pd(H-
Msal-mtsc)Cl(PPh3)](2), [Pd(H-Msal-etsc)Cl(PPh3)](3) and [Pd(Msal-
ptsc)(PPh3)](4). For ESI and crystallographic data in CIF or other
electronic format see DOI: 10.1039/c1dt11838b
2486 | Dalton Trans., 2012, 41, 2486–2499
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Scheme 1 Preparation of new palladium(II) complexes.
RNA, which may hinder or block base replication.13 Cisplatin is
one of the most widely used anticancer drugs.14 In general, nuclear
DNA is the cellular target associated with the therapeutic action of
cisplatin.15 One of the major drawbacks of the platinum antitumor
drugs is its resistance, either developed or intrinsic.16 Phenoxide
compounds appended with thiosemicarbazone pharmacophore
have been known to exert cytotoxicities towards many cancer
cells. With the above facts in mind, new palladium(II) complexes
containing substituted thiosemicarbazones have been synthesized,
characterized and their cytotoxicity has been tested on lung
cancer cell line A549 and also been subjected to test their
potential activity against various pathogenic bacteria such as
Enterococcus faecalis, Staphylococcus aureus, Escherichia coli,
Klebsiella pneumonia and Pseudomonas aeruginosa. In this paper,
we are reporting the synthesis, characterization, crystallography,
DNA, protein binding ability and cytotoxic activities of new Pd(II)
thiosemicarbazone complexes.
ligands H2L1–4, has completely disappeared in the spectra of all
the new complexes and the appearance of a new band at 739–
743 cm-1 due to n(C–S) indicated the coordination of the sulfur
atom after enolisation followed by deprotonation.2h Moreover, the
characteristic absorption bands due to triphenylphosphine were
also present in the expected region.18 The electronic spectra of
the complexes have been recorded in dichloromethane and they
displayed three to five bands in the region around 234–418 nm.
The bands appearing in the region 234–386 nm have been assigned
to intra ligand transition19 and the bands around 394–418 nm have
been assigned to ligand to metal charge transfer transitions.19a,20
The 1H-NMR spectra of H2L1–4 and the corresponding complexes
recorded in DMSO showed all the expected signals (Fig. S1–S8†).
In the spectra of H2L1–4, a singlet appearing in the range 9.13–
10.0 ppm has been assigned to the N(2)HCS group.21 However,
in the spectra of all the four complexes (1–4), there was no
resonance attributable to N(2)H, indicating the coordination of
ligands in the anionic form after deprotonation at N(2). A sharp
singlet corresponding to the phenolic –OH group has appeared
at 11.34–11.76 ppm in the free ligands. In the spectra of 2 and
3 the appearance of a singlet at 11.30 ppm indicated the non-
participation of phenolic oxygen from coordination.2b But the
same singlet has completely disappeared in 1 and 4, confirming
the involvement of phenolic oxygen in the coordination. In the
spectra of H2L1–4 and in all the four complexes, a complex multiplet
appeared at 6.48–7.7 ppm due to aromatic protons of the ligands
and triphenylphosphine2b,2e and a singlet corresponding to the –
OCH3 group occurred at the 3.61–3.81 ppm range. Two singlets
observed at 8.37–8.40 ppm and 8.37–9.40 ppm have been assigned
to azomethine and terminal –NH protons for H2L2–4. Two broad
singlets appeared at 7.8 and 8.0 ppm corresponding to NH2
protons for H2L1. Though the spectra of 2 and 3 showed a singlet
at 8.3–8.5 ppm corresponding to the azomethine proton, the same
has been observed as a doublet at 8.2–8.6 ppm for 1 and 4, which
may be due to the coupling with the phosphorus atom of the
triphenylphosphine.10h In the spectra of the complexes 2, 3 and
4, a sharp singlet was observed around 7.74–8.40 ppm due to
the terminal –NH protons of the substituted thiosemicarbazone
ligands. In the spectra of H2L2, H2L3 and the complexes (2
and 3), a triplet was observed around 1.03–2.90 ppm due to
the methyl group of protons. In addition, a multiplet at 3.12–
3.58 ppm corresponding to the methylene group of protons was
observed in the spectra of H2L3 and 3.22 In order to confirm the
composition of the new complexes, ES mass spectra were recorded
in positive mode. The molecular ion peaks (M+) observed at
m/z = 591, 668, 642 and 656 supporting the molecular formulae.
In addition, few representative fragment peaks were seen in the
2. Results and discussion
The reaction between [PdCl2(PPh3)2] and
4(N)-substituted thiosemicarbazones
(H2L1–4
a
)
series of
in 1 : 1
ethanol/dichloromethane resulted in the formation of new
complexes (Scheme 1), the analytical data of which confirmed the
stoichiometry of the complexes (1–4). The complexes are soluble in
common organic solvents such as dichloromethane, chloroform,
benzene, acetonitrile, ethanol, methanol, dimethylformamide and
dimethylsulfoxide.
Spectroscopic studies
The IR spectra of the ligands H2L1–4 and the corresponding
complexes provided significant information about the metal ligand
bonding. A band in the region 3339–3458 cm-1 due to a hydrogen
bonded –OH group in the free ligands (H2L1–4) was not observed
in the IR spectra of complexes 1 and 4. This indicates that the
phenolic oxygen is deprotonated and coordinated in the complexes
1 and 4. This was further supported by the increase in the value
of phenolic C–O stretching frequency from 1272 to 1307 cm-1
and 1273 to 1313 cm-1 in the complexes 1 and 4.10a However,
the n(O–H) stretching frequency found at 3424 and 3310 cm-1
for the complexes 2 and 3 indicated the non-participation of
phenolic oxygen in coordination to the palladium ion. A strong
vibration observed at 1536–1593 cm-1 in the ligands corresponding
to n(C
was shifted to 1582–1596 cm-1 in all the complexes
N)
indicating the participation of azomethine nitrogen in bonding.17
A sharp band observed at 771–795 cm-1, ascribed to n(C in the
S)
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spectra of all the four complexes. These fragments indicated the
coordination of thiosemicarbazone ligands to the palladium metal
atom.
X-ray crystallography
¯
Complex 1 crystallized in the triclinic space group P1 whereas the
complexes 3 and 4 crystallized in the monoclinic space groups
P21/c and P21 respectively. The complex 2 crystallized in the
orthorhombic space group P212121. The crystallographic data
and bonding parameters and hydrogen bonding of the complexes
are given in Tables 1–3. The molecular structures and hydrogen
bonding diagrams are depicted in Fig. 1–7. In the complexes 1
and 4, the Pd(II) ion is coordinated to the negatively charged
tridentate ligand through the thiolate sulfur (Pd–S bond distances
˚
˚
of 2.250(2) A and 2.242(1) A, respectively), phenolic oxygen (Pd–
˚
˚
O bond distances of 2.010(6) A and 2.020(4) A, respectively)
Fig. 1 ORTEP diagram of (1).
˚
and the nitrogen atom (Pd–N bond distances of 2.014(8) A and
˚
2.019(4) A, respectively). The remaining binding site is occupied by
the triphenylphosphine unit (Pd–P(1) bond distances of 2.287(3)
˚
˚
A and 2.280(1) A, respectively) with a bite angle [S(1)–Pd(1)–
N(1)] of 83.6(2)◦ for 1 and 84.1(1)◦ for 4. The [S(1)–Pd(1)–
O(1)] bond angles found are 176.0(2)◦ for 1 and 176.5(1)◦ for
4 and the [P(1)–Pd(1)–N(1)] bond angles are 175.2(2)◦ for 1
and 172.2(1)◦ for 4, which deviate considerably from the ideal
angle of 180◦ causing significant distortion in the square planar
geometry of the complexes. It is observed from the trans angle
[P(1)–Pd(1)–N(1)] that the deviation from the ideal geometry is
more in 4 than in 1. In the complexes 2 and 3, the mono basic
bidentate ligand coordinated to Pd through the thiolate sulfur
˚
˚
(Pd–S bond distances of 2.249(2) A for 2 and 2.248(1) A for
˚
3), and the nitrogen atom (Pd–N bond distances of 2.103(4) A
for 2 and 2.115(3) A for 3, respectively). The remaining binding
˚
Fig. 2 ORTEP diagram of (1) with hydrogen bonding pseudo binuclear
structure.
sites are occupied by one chlorine atom (Pd–Cl bond distances
˚
˚
of 2.338(2) A for 2 and 2.341(1) A for 3, respectively) and
the triphenylphosphine unit (Pd–P(1) bond distances of 2.259(2)
˚
˚
A for 2 and 2.269(1) A for 3) with the bite angle of 83.5(1)
and 83.18(9), respectively, causing considerable distortion of the
PdNSClP coordination sphere. All the bond lengths fall in the
range of reported values.2h The P(1)–Pd(1)–N(1) bond angles
[171.6(1) for 2 and 176.11(9) for 3] and Cl(1)–Pd(1)–S(1) bond
angles [173.22(6) for 2 and 172.35(4) for 3] deviate considerably
from the ideal angle of 180◦. The variation in trans angles in
the complexes indicate considerable distortion from square planar
geometry around palladium. In compound 1, one of the hydrogen
atoms of each amino group is engaged in intermolecular hydrogen
bonding with the N2 nitrogen atom of a second molecule N(3A)–
H(32A) ◊ ◊ ◊ N(2B) and N(3B)–H(32B) ◊ ◊ ◊ N(2A) (Fig. 2) and the
second hydrogen is non bonded. This intermolecular hydrogen
bonding gives a binuclear-like structural appearance. In complex 2,
two crystallographically distinct molecules are linked by hydrogen
bonding. One is an intramolecular hydrogen bond, which is
Fig. 3 ORTEP diagram of (2).
intra- and one intermolecular hydrogen bond were observed like in
the case of 2. This hydrogen bonding leads to the formation of 1D
chain and each 1D chain is connected to two other chains through
terminal NH groups and O donor atoms of adjacent complexes
˚
formed by an OH group (O(1A) ◊ ◊ ◊ N(2A) 2.51(7) A) while the
second, which is intermolecular, is formed by the NH group
˚
(N(3A) ◊ ◊ ◊ O(2) 2. 98(7) A. Symmetry code: (x, y, z); - 1/2 +
˚
x, 1.5 - y, 1 - z; 1/2 + x, 1.5 - y, 1 - z; 1/2 + x, 1.5 - y, 1 -
z; 1 + x, y, z. The complex 2 formed a layer structure through
hydrogen bonding (Fig. 4). The result is the assembly of discrete
complexes into a supramolecular 1D chain. In the complex 3, one
[N(3A)–H(3A) ◊ ◊ ◊ O(2B) 3.03(0) A], symmetry code: (x, y, z); 1 -
x, -1/2 + y, 1.5 - z; 1 - x, 1/2 + y, 1.5 - z; x, -1 + y, z] (Fig. 6).
This net of hydrogen bonds allows the assembly of individual 1D
chains into a zigzag 2D supramolecular network.
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◦
˚
Table 2 Selected bond lengths (A) and angles ( ) for Pd(II) thiosemicarbazone complexes
Atoms
[Pd(Msal-tsc)(PPh3)]
[Pd(H-Msal-mtsc)Cl(PPh3)]
[Pd(H-Msal-etsc)Cl(PPh3)]
[Pd(MSal-ptsc)(PPh3)]
Pd–S(1)
Pd–P(1)
Pd–O(1)
Pd–N(1)
2.250(2)
2.287(3)
2.010(6)
2.014(8)
—
92.63(8)
176.0(2)
83.6(2)
90.8(2)
175.2(2)
92.9(3)
—
2.250(2)
2.259(2)
—
2.104(4)
2.338(2)
91.62(6)
—
83.6(1)
—
171.7(1)
—
2.248(1)
2.269(1)
—
2.115(3)
2.341(1)
93.16(4)
—
83.18(9)
—
176.11(9)
—
2.242(1)
2.280(1)
2.020(3)
2.019(4)
—
93.31(4)
176.5(1)
84.1(1)
90.1(1)
172.2(1)
92.5(1)
—
Pd–Cl(1)
S(1)–Pd (1)–P(1)
S(1)–Pd(1)–O(1)
S(1)–Pd(1)–N(1)
P(1)–Pd(1)–O(1)
P(1)–Pd(1)–N(1)
O(1)–Pd(1)–N(1)
Cl(1)–Pd(1)–N(1)
Cl(1)–Pd(1)–P(1)
Cl(1)–Pd(1)–S(1)
95.6(1)
90.01(5)
173.25(6)
94.63(9)
88.83(4)
172.35(4)
—
—
—
—
◦
˚
Table 3 Hydrogen bond lengths (A) and angles ( ) for 1–3
D–H ◊ ◊ ◊ A
d(D–H)
d(H ◊ ◊ ◊ A)
d(D ◊ ◊ ◊ A)
<(DHA)
1a
N(3A)–H(32A) ◊ ◊ ◊ N(2B)
1.14(4)
1.94(0)
1.94(0)
1.14(4)
3.08(1)
3.08(1)
175.05
175.05
N(3B)–H(32B) ◊ ◊ ◊ N(2A)
2b
D–H ◊ ◊ ◊ A
d(D–H)
0.82(0)
0.86(0)
0.82(0)
0.86(0)
d(H ◊ ◊ ◊ A)
1.70(9)
2.22(7)
1.70(9)
2.22(7)
d(D ◊ ◊ ◊ A)
2.51(7)
2.98(7)
2.51(7)
2.98(7)
<(DHA)
167.91
147.35
167.91
147.35
O(1A)–H(1A) ◊ ◊ ◊ N(2A)
N(3B)–H(3B) ◊ ◊ ◊ O(1A)
O(1B)–H(1B) ◊ ◊ ◊ N(2B)
N(3A)–H(3A) ◊ ◊ ◊ O(1C)
3c
D–H ◊ ◊ ◊ A
d(D–H)
0.82(0)
0.86(0)
d(H ◊ ◊ ◊ A)
1.75(1)
2.17(3)
d(D ◊ ◊ ◊ A)
2.55(3)
3.03(0)
<(DHA)
165.58
174.01
O(1A)–H(1A) ◊ ◊ ◊ N(2A)
N(3A)–H(3A) ◊ ◊ ◊ O(2B)
a Symmetry operation for 1: (x, y, z); (-x, -y, -z). b Symmetry operation for 2: (x, y, z); -1/2 + x, 1.5 - y, 1 - z; 1/2 + x, 1.5 - y, 1 - z; 1/2 + x, 1.5 - y, 1
- z; 1 + x, y, z. c Symmetry operation for 3: (x, y, z); 1 - x, -1/2 + y, 1.5 - z; 1 - x, 1/2 + y, 1.5 - z; x, -1 + y, z.
Fig. 4 ORTEP diagram of (2) with hydrogen bonding 1D network.
DNA binding studies
non- covalent DNA interactions include intercalative, electrostatic
and groove binding of metal complexes outside of the DNA helix.
The DNA binding properties of the new Pd(II) complexes has
been investigated by absorption and emission spectroscopy (Fig.
8a–8d). The spectra have been recorded for a constant CT-DNA
concentration (3.5 mM) with different concentrations of complexes
The interaction of transition metal complexes with DNA takes
place via both covalent and/or non-covalent interactions.23 In the
case of covalent binding, the labile ligand of the complexes is
replaced by a nitrogen base of DNA such as guanine N7, while the
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Fig. 5 ORTEP diagram of (3).
Fig. 7 ORTEP diagram of (4).
(1 mM–40 mM). The changes observed in the absorption spectra
of CT-DNA in the presence of various concentrations of these
complexes, i.e. the increase of the intensity at lmax = 262 nm, which
is accompanied with a blue shift of lmax up to 232 nm, after mixing
with each concentration of the new complexes (1–4), indicate that
all the complexes had interaction with CT-DNA.
Table 4 Binding constant for interaction of complexes with CT-DNA
System
Kb (¥ 105 M-1)
CT-DNA + 1
CT-DNA + 2
CT-DNA + 3
CT-DNA + 4
0.502
1.445
1.697
0.230
For complex 1, the intensity of the band at 262 nm (A =
0.1012) for CT-DNA increased upon the addition of increasing
concentrations of the complex (1 mM–40 mM), while a blue shift
was observed up to 242 nm (A = 1.5294) accompanied by a
hyperchromism (Fig. 8a). The binding behaviour of remaining
complexes (2–4) is also quite similar. The band intensity of CT-
DNA at lmax = 262 nm (A = 0.1012) presents a blue shift up to
232 nm (A = 1.6126) for 2, 232 nm (A = 1.8057) for 3 and 240 nm
(A = 1.9216) for 4 (Fig. 8b–8d). The resultant hyperchromic shift
suggests that all the complexes bind to CT-DNA by the external
contact, possibly due to electrostatic binding.24 The intrinsic
binding constant Kb is a useful tool to monitor the magnitude of
the binding strength of compounds with CT-DNA (Table 4). It can
be determined by monitoring the changes in the absorbance at the
corresponding lmax with increasing concentration of the complex
and is given by the ratio of slope to the Y intercept in plots of
[complex]/(ea-ef) vs. [complex] (insets in Fig. 8a–8d). From the
binding constant values, it is inferred that the complex 3 exhibited
better binding than other complexes. Based on the Kb value, we
can arrange the complexes in the following order with respect to
the electron donating ability of the ligand i.e. the substitution on
N-terminal nitrogen atom, 3 (ethyl) > 2 (methyl) > 1 (hydrogen)
> 4 (phenyl).
In the emission spectra, the binding behaviour of complexes 1–
4 is quite similar. The intensity of the band at lmax = 289 nm for
CT-DNA is decreased from 1 mM to 25 mM by a blue shift for
the complexes 1–4 (276, 257, 258 and 280 nm, respectively). The
observed blue shift is an indication of hypsochromism resulting
in electrostatic interaction between the complexes with CT-DNA.
Further increase in the concentration of the complexes (30 mM,
35 mM and 40 mM) brought red shift with hypsochromism. This
indicates, at higher concentrations of the complexes the binding
mode turned from electrostatic to intercalative.
Protein binding studies
Fluorescence spectroscopy. Qualitative analysis of the binding
of complexes to lysozyme can be detected by examining the
Fig. 6 ORTEP diagram of (3) with hydrogen bonding zigzag network.
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Fig. 8 (a) UV-vis and emission spectra of complex 1 in DMSO. (b) UV-vis and emission spectra of complex 2 in DMSO (c) UV-vis and emission spectra
of complex 3 in DMSO. (d) UV-vis and emission spectra of complex 4 in DMSO.
emission spectra. Fluorescence measurements provide informa-
tion about the binding of small molecules to a protein, such
as the binding mechanism, binding mode, binding constants
and binding sites. A variety of molecular interactions can result
in quenching, including excited state reactions, molecular rear-
rangements, energy transfer, ground-state complex formation, and
collisional quenching. The different mechanisms of quenching are
usually classified as either dynamic quenching or static quenching.
Dynamic quenching refers to a process where the fluorophore and
the quencher come into contact during the transient existence of
the excited state. Static quenching refers to fluorophore–quencher
complex formation.
The effect of the complex 4 on the photoluminescence intensity
of lysozyme is shown in Fig. 9a. On increasing the concentration of
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Fig. 9 (a) The fluorescence quenching of lysozyme (1 ¥ 10-6 M; lexi = 280 nm; lemi = 347 nm) in the absence and presence of various concentrations
of 4 (0–5 ¥ 10-5 M). (b) The fluorescence quenching of lysozyme (1 ¥ 10-6 M; lexi = 280 nm; lemi = 347 nm) in the absence and presence of various
concentrations of 1 (0–5 ¥ 10-5 M). (c) The fluorescence quenching of lysozyme (1 ¥ 10-6 M; lexi = 280 nm; lemi = 347 nm) in the absence and presence
of various concentrations of 2 (0–5 ¥ 10-5 M). (d) The fluorescence quenching of lysozyme (1 ¥ 10-6 M; lexi = 280 nm; lemi = 347 nm) in the absence and
presence of various concentrations of 3 (0–5 ¥ 10-5 M).
complex 4, a progressive decrease in the fluorescence intensity was
observed, accompanied by a blue shift. The observed blue shift
may be due to the binding of 4 with the active site in lysozyme.25
This quenching effect indicates the interaction of lysozyme with
4. The other complexes 1, 2 and 3 also gave a similar type of
fluorescence behavior (Fig. 9b–9d). The fluorescence quenching
data have been analyzed by Stern Volmer equation (eqn (1)).
A common method to distinguish between static and dynamic
quenching is by careful examination of the absorption spectra
of the lysozyme in the presence of complexes. The absorption
spectra of lysozyme, complex 4 and that of 4 + lysozyme have
been depicted in Fig. 11. The changes in the absorbance spectra
for 4 + lysozyme indicate that 4 interacts with the lysozyme.
The absorption spectra of all the other complexes also show
a similar type of change, which indicates their interaction with
lysozyme. When small molecules bind to active site of Lysozyme,
the equilibrium binding constant and the number of binding sites
can be analyzed by using Scatchard equation (eqn (2)).
I0/I = 1 + KSV [Q]
(1)
Where I0 and I are the fluorescence intensities of the fluorophore
in the absence and presence of quencher, KSV is the Stern–Volmer
quenching constant and [Q] is the quencher concentration. The
plot of I0/I vs. [Q] gave upward curvature suggesting the static
nature of the quenching,26 as shown in Fig. 10. Hence, the
fluorescence quenching results from the formation of a complex
between lysozyme and complex 4.
⎡
⎢
⎤
⎥
F −F
F
0
log
= logK + nlog[Q]
(2)
⎢
⎣
⎥
⎦
Where K is the binding constant of quencher with lysozyme, n is
the number of binding sites, F0 and F are the fluorescence intensity
in the absence and presence of the quencher. The value of K can be
determined from the slope of the plot of log[(F0 - F)/F] vs. log[Q]
as shown in Fig. 12. The calculated value of the binding constant
(K) and the number of binding sites (n) are listed in Table 5. The
data obtained indicate that the complex 3 has higher magnitude of
binding than 2. This indicates that the binding ability to lysozyme
increases by increasing the aliphatic chain in the amino group.
Since all other groups are the same in both 3 and 2, they differ
only in the alkyl substituent. The free amino group in 1 has a lower
binding value than the N-substituted complexes, which confirms
the effect of substitution on binding with lysozyme. The complex 4,
which contains a phenyl substituent, has a similar binding constant
to 1. From this, it is inferred that the increase in electron donating
ability of the substituent at the terminal nitrogen of the ligands
Fig. 10 Plot of I0/I vs. log[Q].
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Table 5 Binding constant and number of binding sites for interaction of
complexes with lysozyme
System
K (¥ 105 M-1)
n
Lysozyme + 1
Lysozyme + 2
Lysozyme + 3
Lysozyme + 4
1.98
2.06
3.07
1.94
1.1971
1.2151
1.2388
1.1929
Fig. 13 Synchronous spectra of lysozyme (1 ¥ 10-6 M) in the absence and
presence of 4 (0–5 ¥ 10-5 M) in the wavelength difference of Dl = 60 nm.
Hence, the results clearly indicate that the complexes bind to the
active site in the protein showing them as potential molecules for
biological applications.
Anticancer activity
Preliminary screening of anticancer activity for the new complexes
was performed by MTT assay. All the samples were found to be
cytotoxic to lung cancer cell line A549 (Fig. 14). The complex
3 showed higher cytotoxic effect followed by the complexes 1
and 2. It is interesting to note that the complexes 1, 2 and 3
exhibited better activity than cisplatin. The highest activity was
noted for the complex 3 and the least activity was observed with
the complex 4. The IC50 value (50% inhibition of cell growth for
24 h) for the complexes 1–4 was determined as 18, 18, 15 and 30
mM, respectively, and for cisplatin it was found as 25 mM. The
higher cytotoxic effects on lung cancer cells with lower IC50 values
of the complexes indicate their efficiency in killing the cancer
cells even at lower concentrations than the conventional standard
cisplatin. Many researchers have reported the cytotoxicity effects
of complexes with longer incubation period. But the present
data shows that the complexes with lower incubation period
with higher cytotoxic effects can replace the compounds that
need longer time periods. As the longer incubation period may
result in the development of cellular resistance for that particular
compound and also have harmful effects such as affecting
Fig. 11 The absorption spectra of (1) Lysozyme (1 ¥ 10-6 M), (2) 4 (1 ¥
10-5 M) and (3) Lysozyme - 4 [lysozyme = 1 ¥ 10-6 M and 4 = 1 ¥ 10-5 M].
Fig. 12 Plot of log[(F0 - F)/F] vs. log [Q].
increases the protein binding ability of the complexes. This may be
due to the increase in the electron density on the electron deficient
metal centers.
The binding of small molecules to lysozyme could induce a
conformational change of protein, because the intramolecular
forces involved in maintaining the secondary structure could be
altered. Spectroscopic methods are usually applied to study the
conformation of protein. The fluorescence of lysozyme is due
to tryptophan and tyrosine residues. Among them, tryptophan
lies in the active site of the protein. Synchronous fluorescence
spectra provide the information on the molecular microenviron-
ment, particularly in the vicinity of the fluorophore functional
groups.26 In synchronous fluorescence spectroscopy, according
to Miller,27 the difference between excitation wavelength and
emission wavelength (Dl = lemi - lexc) reflects the nature of the
chromophores in the spectra. With larger Dl values such as 60
nm, the synchronous fluorescence of lysozyme is characteristic of
the tryptophan residue, while smaller Dl values such as 15 nm are
characteristic of tyrosine.28 The synchronous fluorescence spectra
of lysozyme with various concentrations of 4 were recorded at
Dl = 60 nm and Dl = 15 nm. The fluorescence intensity of both
tryptophan and tyrosine showed a decrease in intensity but the
tryptophan spectrum is accompanied with blue shift (Fig. 13).
It reveals that the binding around Trp residues is strengthened.
Fig. 14 The treatment of complexes exert an antiproliferative effect on
lung cancer cells: A549 cells were treated with complexes (0.1, 1.0, 10,
25 and 50 mM) for 24 h. The control received appropriate carriers. Cell
viability was assessed by MTT cell proliferation assay. The results shown
are Mean SD (n = 9), which are three separate experiments performed in
triplicate.
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non-target sites in the body when they are used for clinical
purposes, the compounds that show activity in shorter time
periods are preferred. Our data are highly significant when
compared with the results of Beckford et al.29 They have reported
the 50% inhibitory concentration of different complexes after
exposure for 72 h in mM concentrations. Moreover, the IC50 values
of our compounds are comparable with the reported IC50 values
of standard anticancer drugs such as cisplatin and doxorubicin.
As our samples are of high antiproliferative/anticancer activity,
they may find their role in medicine. Further molecular studies are
required to exploit our complexes in the pharmaceuticals.
Antibacterial activity studies
Infections due to different pathogenic bacteria are a dreadful
threat to the human race. Virulent strains of E. coli can cause
gastroenteritis, urinary tract infections, and neonatal meningitis.
Enterococcus faecalis can cause endocarditis, as well as bladder,
prostate, and epididymal infections; nervous system infections
are less common. In infants, the S. aureus infection can cause
a severe disease staphylococcal scalded skin syndrome (SSSS),
and it can cause mastitis in cows. K. pneumoniae leads to the
dreadful disease pneumonia and P. aeruginosa is an opportunistic
pathogen in immuno-compromised individuals.31–35 Since these
bacterial species develop resistance to the existing antibacterial
agents, new compounds with more effective cytotoxic/cytostatic
effects on the pathogenic bacteria are of urgent need in the medical
field. There are hundreds of antibacterial agents but their use is
limited due to a low spectrum of activity and good activity in
high concentrations, which may also be toxic to the non targets.
Antibacterial activities for palladium complexes have not been
examined well. Here in our study, we made an attempt to explore
the toxic effects of the newly synthesized palladium complexes
on five different bacterial species. As described in the materials
and methods, different concentrations of the complexes were
used to find out their minimum inhibitory concentration on the
bacterial species like E. faecalis, S. aureus, E.coli, K.pneumoniae
and P. aeruginosa. Complex 3 was found to be more effective on
S. aureus, E. coli, K. pneumoniae and P. aeruginosa with lower
minimal inhibitory concentrations, followed by complex 2 (Fig.
16). Complex 1 was observed to be more toxic to E. faecalis when
compared to the other three complexes. E. faecalis was sensitive
to 2, 3 and 4 with the same inhibitory concentration. 4 and 1
showed similar minimum inhibitory concentrations on E.coli and
K. pneumoniae. Complex 4 was observed to be less effective on
S. aureus and P. aeruginosa. The activity for the metal chelates
can be explained on the basis of chelation theory.36 Chelation
considerably reduces the polarity of the metal ion because of
the partial sharing of its positive charge with the donor groups
Cellular uptake study
The intracellular uptake of a specific drug plays a vital role in
ameliorating treatment of several diseases. Since the IC50 values
are critical when compared to the normal cells in the human body,
the present study is focused on the concentrations that showed 50%
inhibition for the lung cancer cells. The intracellular concentration
of the complexes 1–4 and the standard doxorubicin were found out
using the method described in the methodology.30 The intracellular
concentration of the complexes after the incubation period of 24 h
was found to be 35.622, 49.938, 48.086 and 72.796%, respectively,
for the complexes 1–4 and for the standard doxorubicin it was
found as 59.16% (Fig. 15). With their IC50 values of 18, 18, 15,
30 and 5 mM, the intracellular concentration of the different
complexes used for the study was found to be approximately
half of their IC50. It is clear from the results that even a low
concentration of the complexes is cytotoxic to the lung cancer
cells when they are completely absorbed by the cells. Hence,
further studies to improve their cellular uptake may be helpful
for their use in the clinical world. A549 cells were treated with
the different complexes for 24 h and their IC50 values and the
concentrations of complexes in the cell lysates were measured
with a fluorescence spectrophotometer at their maximum ex-
citation/emission wavelengths. All the complexes entered the
cancer cell, and the uptake levels were dependent on the dose of
each complex used. This also indicated that their cytotoxicities
as determined by the MTT assay were not disproportionately
influenced by the complexes having different cellular uptake levels.
Results in their percentage uptake are shown in Fig. 15, which are
Mean SD (n = 9), in three separate experiments performed in
triplicate.
Fig. 16 Minimal inhibitory concentration of new Pd(II) complexes: the
new Pd(II) complexes are exposed at different concentrations overnight and
the minimal inhibitory concentration of each complex is given, considering
that the control sets gave null values.
Fig. 15 Intracellular concentration of complexes in A549 cells.
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and possible p-electron delocalization over the chelate ring. Such
chelation could increase the lipophilic character of the central
metal atom, which subsequently favors the permeation through the
lipid layer of cell membrane. The mode of action of the complexes
may involve the formation of the hydrogen bond through the
azomethine group (>C N) with the active centers of the cell
constituents resulting in the interference with normal cell process.37
The variation in the effectiveness of the different complexes
against different organisms depends on the impermeability of
the cells of microbes or difference in ribosome of the microbial
cells.38
Synthesis of 3-methoxysalicylaldehyde thiosemicarbazone
[H2-Msal-tsc] (H2L1)
Thiosemicarbazide (0.92 g, 10 mmol) was dissolved in 40 mL
of methanol with continuous stirring and it was gently heated
for a period of 30 min. To this, methanolic solution (10 mL)
of 3-methoxy salicylaldehyde (1.53 g, 10 mmol) was added and
the mixture was refluxed by stirring for 2 h. Upon cooling, a
white crystalline product begins to separate. This was collected by
filtration, washed well with cold methanol and dried in vacuum.
The product dissolves in common organic solvents such as
acetone, methanol, ethanol, dichloromethane, chloroform, DMF
and DMSO. Yield: 38%. Anal. calcd for C9H11N3O2S: C 47.98;
H 4.92; N 18.65; S 14.23. Found: C 47.76; H 5.00; N 18.67; S
14.20%. FT-IR (cm-1) in KBr: 3458 (nOH), 1593 (nC N), 1272 (nC–O),
771(nC S); 1H NMR (DMSO-d6, ppm): 11.36 (s, 1H, OH), 9.13 (s,
1H, NHCS), 8.38 (s, 1H, CH N), 7.84 and 8.06 (2br s, 1H each,
NH2), 3.79 (s, 3H, OCH3), 6.73–7.52 (m, 3H, aromatic).
3. Conclusion
Various N-substituted thiosemicarbazones containing 3-
methoxysalicylaldehyde were reacted with [PdCl2(PPh3)2].
N-methyl and N-ethyl substituted thiosemicarbazones yielded
palladium complexes with NS chelation by forming a stable five
member ring with N(1) hydrazinic nitrogen and thiolate sulfur
atoms. However, the unsubstituted and N-phenyl substituted
thiosemicarbazones yielded the ONS chelate with the formation
of five and six member rings by utilizing phenolic oxygen,
N(1) nitrogen and thiolate sulfur atoms. The new complexes
have been characterized by X-ray crystallography. Further, the
complexes (1–4) have been subjected to DNA and protein binding
studies. From the results, it is observed that the complexes
significantly bind to the DNA and the protein. Among the
complexes, 3 exhibited better binding affinity over the DNA and
lysozyme. From the results of cytotoxicity studies, it is found
that the complexes 1, 2 and 3 exhibited better activity than the
standard cisplatin. The complex 3 showed higher activity than
the remaining complexes. In order to know the uptake of the
complexes into the cells, cellular uptake studies were done and
it was found that the uptake levels were dependent on the dose
of each complex used. This designated that their cytotoxicities,
as determined by the MTT assay, were not disproportionately
influenced by the complexes having different cellular uptake
levels. Antibacterial screening studies were also done to know
about the effectiveness of the complexes on various pathogenic
bacteria. All the complexes exhibited significant activity and the
complex 3 showed better activity than all other complexes. From
the overall results of the biological studies, the better activity
of complex 3 may be due to the presence of more electron rich
substitution on the terminal nitrogen. In general, the order of
activity of the complexes may be assigned as 3 > 2 > 1 > 4.
Hence, it is concluded that the substitution on the N-terminal
nitrogen may have a significant effect in tuning the structural
and biological properties of the complexes formed in the given
experimental condition.
A similar method as described above was followed for the
preparation of all other thiosemicarbazone ligands.
3-methoxysalicylaldehyde-4(N)-methylthiosemicarbazone
[H2-Msal-mtsc] (H2L2)
The ligand [H2-Msal-mtsc] was prepared from 4(N)-
methylthiosemicarbazide (1.05 g, 10 mmol) and 3-
methoxysalicylaldehyde (1.53 g, 10 mmol). Yield: 58%. Anal.
Calcd for C10H13N3O2S: C,50.19; H, 5.47; N, 17.56; S, 13.40.
Found: C, 50.15; H, 5.40; N, 17.49; S, 13.31%. FT-IR (cm-1) in
1
KBr: 3338 (nOH), 1554 (nC N), 1276 (nC–O), 780 (nC S); H NMR
(DMSO-d6, ppm): 11.40 (s, 1H, OH), 9.13 (s, 1H, NHCS), 8.37
(s, 1H, NHCH3), 8.36 (s, 1H, CH N), 3.80 (s, 3H, OCH3),
6.75–7.54 (m, 3H, aromatic), 2.99 (d, 3H, CH3).
3-methoxysalicylaldehyde-4(N)-ethylthiosemicarbazone
[H2-Msal-etsc] (H2L3)
The ligand [H2-Msal-etsc] was prepared from 4(N)-
ethylthiosemicarbazide (1.19 g, 10 mmol) and 3-
methoxysalicylaldehyde (1.53 g, 10 mmol). Yield: 64%. Anal.
calcd for C11H15N3O2S: C 55.21; H 6.31; N 16.58; S 12.65. Found:
C 55.15; H 6.27; N 16.50; S 12.59%. FT-IR (cm-1) in KBr: 3310
(nOH), 1536 (nC N), 1276 (nC–O), 795 (nC S); 1H NMR (DMSO-d6,
ppm): 11.34 (s, 1H, OH), 9.14 (s, 1H, NHCS), 8.42 (s, 1H,
NHC2H5), 8.40 (s, 1H, CH N), 3.80 (s, 3H, OCH3), 6.75–7.53
(m, 3H, aromatic), 3.55–3.58 (m, 2H, CH2), 1.13 (t, 3H, CH3).
3-methoxysalicylaldehyde- 4(N)-phenylthiosemicarbazone
[H2-Msal-ptsc] (H2L4)
The ligand [H2-Msal-ptsc] was prepared from 4(N)-
phenylthiosemicarbazide (1.67 g, 10 mmol) and 3-
methoxysalicylaldehyde (1.53 g, 10 mmol). Yield: 54%. Anal.
calcd for C15H15N3O2S: C 59.81; H 5.12; N 13.95; S 10.64. Found:
C 59.65; H 4.99; N 13.78; S 10.43%. FT-IR (cm-1) in KBr: 3339
(nOH), 1589 (nC N), 1273 (nC–O), 782 (nC S); 1H NMR (DMSO-d6,
ppm): 11.76 (s, 1H, OH), 10.00 (s, 1H, NHCS), 9.20 (s, 1H,
NHPh), 8.50 (s, 1H, CH N), 3.81 (s, 3H, OCH3), 6.76–7.68 (m,
8H, aromatic).
4. Experimental
Materials
The ligands [H2L1–4] and the palladium complex [PdCl2(PPh3)2]
were synthesized according to the standard literature procedures.39
All the reagents used were analar grade, were purified and dried
according to the standard procedure.40
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Preparation of [Pd(Msal-tsc)(PPh3)] (1)
(nC–O), 743 (nC–S), 1433, 1098, 694 cm-1 (for PPh3); UV-vis (CH2Cl2),
l
max: 242 (54368), 292 (25117), 324 (29457), 348 (23960), nm (dm3
An ethanolic (25 mL) solution of [PdCl2(PPh3)2]
(0.200 g; 0.285 mmol) was slowly added to 3-
methoxysalicylaldehydethiosemicarbazone [H2-Msal-tsc] (0.064
g, 0.285 mmol) in dichloromethane (25 mL). The mixture was
allowed to stand for 4 d at room temperature. A yellowish orange
solid formed was filtered, washed with petroleum ether (60–80
◦C) and recrystallized from dichloromethane an◦d acetonitrile to
yield orange red crystals. Yield: 55%. M.p. 236 C. Anal. calcd.
for C27H24N3O2SPdP: C 54.78; H 4.09; N 7.09; S 5.41. Found:
C 54.70; H 4.01; N 7.00; S 5.38%. FT-IR (cm-1) in KBr: 1596
(nC N), 1307 (nC–O), 742 (nC–S), 1432, 1097, 695 cm-1 (for PPh3);
UV-vis (CH2Cl2), lmax: 234 (14879), 302 (9609), 342 (9900) nm
(dm3 mol-1 cm-1) (intra-ligand transition); 394 (4931), 418 (2102)
nm (dm3 mol-1 cm-1) (LMCT s→d); 1H NMR (DMSO-d6, ppm):
8.24 (d (J = 14), 1H, CH N), 3.60 (s, 3H, OCH3), 6.48–7.70 (m,
aromatic), m/z = 592 (M+).
mol-1 cm-1) (intra-ligand transition); 412 (15475) nm (dm3 mol-1
cm-1) (LMCT s→d); H NMR (DMSO-d6, ppm): 8.65 (d (J =
1
13.6), 1H, CH N), 9.40 (s, 1H, NHPh), 3.64 (s, 3H, OCH3),
6.54–7.73 (m, aromatic), m/z = 668 (M+).
Measurements
Infrared spectra were measured as KBr pellets on a Nicolet
instrument between 400–4000 cm-1. Elemental analysis of carbon,
hydrogen, nitrogen, and sulfur were determined using Vario EL
III CHNS at the Department of Chemistry, Bharathiar University,
Coimbatore, India. The electronic spectra of the complexes
have been recorded in dichloromethane using a JASCO V-630
Spectrophotometer in the 200–800 nm range. Emission spectra
were recorded by using a JASCO FP 6600 spectrofluorometer.
1H NMR spectra were recorded in DMSO at room temperature
with a Bruker 400 MHz instrument, chemical shift relative to
tetramethylsilane. Melting points were measured in a Lab India
apparatus.
The very similar method was followed to synthesize other
complexes.
Preparation of [Pd(H-Msal-mtsc)(PPh3)] (2)
X-Ray crystallography
The complex 2 was prepared by the procedure as used for 1,
with 3-methoxy salicylaldehyde 4(N)-methylthiosemicarbazone
[H2-Msal-mtsc] (0.068 g; 0.285 mmol) and [PdCl2(PPh3)2] (0.200
g; 0.285 mmol). Yield: 60%. M.p. 128 ◦C. Anal. calcd. for
C28H27N3O2SClPdP: C 52.38; H 4.23; N 6.54; S 4.99. Found: C
52.29; H 4.20; N 6.49; S 4.91%. FT-IR (cm-1) in KBr: 3424 (nOH),
1593 (nC N), 1310 (nC–O), 739 (nC–S), 1437, 1097, 695 cm-1 (for
PPh3); UV-vis (CH2Cl2), lmax: 234 (24713), 316 (10965), 342 (8735),
nm (dm3 mol-1 cm-1) (intra-ligand transition); 400 (4701) nm (dm3
mol-1 cm-1) (LMCT s→d); 1H NMR (DMSO-d6, ppm): 11.30 (s,
1H, OH), 8.35 (s, 1H, CH N), 8.39 (s, 1H, NHCH3), 3.61 (s, 3H,
OCH3), 6.48–7.70 (m, aromatic), 2.72 (d (J = 4.8), 3H, CH3), m/z
= 642 (M+).
Single crystal data collections and corrections for the new Pd(II)
complexes were done at 293 K with CCD kappa Diffractometer us-
41
˚
ing graphite mono chromated Mo-Ka (l = 0.71073 A) radiation.
The structural solutions were done by using SHELXTL-9742 and
refined by full matrix least square on F2 using SHELXL-97.43
DNA binding studies
For electronic absorption titration, a stock of 1 mM solution of the
complex was made up in a phosphate buffer (pH 7); 3000 mL of the
solution was loaded into an optical glass cuvette with a path length
of 1 cm, and 300 mL was removed with a micropipette and replaced
with 300 mL of the complex solution. This cuvette was then loaded
into the spectrometer sample block, controlled at 25 ◦C; 3000 mL
of the buffer was loaded to an identical cuvette and placed in
the reference cell. After the cuvettes had been allowed to reach
equilibrium over the course of 30 min, a spectrum was recorded
between 700 and 200 nm. Absorption titration experiments were
performed by maintaining the nucleic acid (30 mL) concentration
as constant (3.5 mM) and varying the metal complex concentration
(1 mM–40 mM). The spectrum was recorded after checking for
bubbles, which showed an increase in absorption indicating the
interaction between the DNA and the metal complex. The intrinsic
binding constant of the complex with CT-DNA was determined
by using modified Stern volmer eqn (3).44,45
Preparation of [Pd(H-Msal-etsc)(PPh3)] (3)
The complex 3 was prepared by the procedure as used for
1, with 3-methoxy salicylaldehyde 4(N)-ethylthiosemicarbazone
[H2-Msal-etsc] (0.072 g; 0.285 mmol) and [PdCl2(PPh3)2] (0.200
g; 0.285 mmol). Yield: 65%. M.p. 234 ◦C. Anal. calcd for
C29H29N3O2SClPdP: C 53.06; H 4.45; N 6.40; S 4.88. Found: C
53.00; H 4.40; N 6.36; S 4.81%. FT-IR (cm-1) in KBr: 3317 (nOH),
1582 (nC N), 1280 (nC–O), 742 (nC–S), 1450, 1092, 697 cm-1 (for
PPh3); UV-vis (CH2Cl2), lmax: 302 (14619), 338 (14113) nm (dm3
mol-1 cm-1) (intra-ligand transition); 1H NMR (DMSO-d6, ppm):
11.30 (s, 1H, OH), 8.56 (s, 1H, CH N), 7.74 (br s, 1H, NHC2H5),
3.77 (s, 3H, OCH3), 6.78–7.76 (m, aromatic), 3.12–3.15 (m, 2H,
CH2), 1.03 (t, 3H, CH3), m/z = 656 (M+).
[Complex]/[ea-ef]) = [Complex]/[eb-ef]+1/Kb[eb-ef]
(3)
The absorption coefficients ea, ef, and eb correspond to
Aobsd/[complex], the extinction coefficient for the free DNA and
the extinction coefficient for the complex in the fully bound form,
respectively. The slope and the intercept of the linear fit of the
plot of [Complex]/[ea - ef] vs. [Complex] give 1/[ea - ef] and
1/Kb[eb - ef], respectively. The intrinsic binding constant Kb can
be obtained from the ratio of the slope to the intercept (Table
4).44 Emission measurements were carried out by using a JASCO
FP- 6600 spectrofluorometer. Tris–buffer was used as a blank
to make preliminary adjustments. The excitation wavelength was
Preparation of [Pd(Msal-ptsc)(PPh3)] (4)
The complex 4 was prepared by the procedure as used for 1,
with 3-methoxy salicylaldehyde 4(N)-phenylthiosemicarbazone
[H2-Msal-ptsc] (0.086 g; 0.285 mmol) and [PdCl2(PPh3)2] (0.200
g; 0.285 mmol). Yield: 70%. M.p. 245 ◦C. Anal. calcd. for
C33H28N3O2SPdP: C 59.33; H 4.22; N 6.29; S 4.80. Found: C 59.29;
H 4.19; N 6.22; S 4.76%. FT-IR (cm-1) in KBr: 1594 (nC N), 1313
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fixed and the emission range was adjusted before measurements.
All measurements were made at 20 ◦C. For emission spectral
titrations, DNA concentration was maintained constant as 3.5
mM and the concentration of the palladium complex was varied
from 1 mM to 40 mM. The emission enhancement factors were
measured by comparing the intensities at the emission spectral
maxima under similar conditions.
Antibacterial activity studies
MICs (minimum inhibitory concentration) of the compounds
against test organisms such as Enterococcus faecalis, Staphylo-
coccus aureus, Escherichia coli, Klebsiella pneumonia and Pseu-
domonas aeruginosa were determined by using the broth micro
dilution method.48 A broth microdilution susceptibility assay was
used as recommended by NCCLS for the determination of the
MIC. All the tests were performed in Mueller–Hinton broth
(MHB) supplemented with Tween-80 detergent (final concentra-
tion of 0.5% (v/v)). Bacterial strains were cultured overnight at
37 ◦C in MHA. Test strains were suspended in MHB to give
a final density of 5 ¥ 105 cfu mL-1 and these were confirmed
by viable counts. Geometric dilutions of the compounds were
prepared in a 96-well microtiter plate, including one growth control
(MHB + Tween 80) and one sterility control (MHB + Tween 80 +
compound). The plates were incubated under normal atmospheric
conditions at 37 ◦C for 24 h.
Proteinase binding studies
Lysozyme was purchased from Hi Media, India. Lysozyme was
prepared in phosphate buffer of pH 7.6 and stored in the dark
at 4 ◦C for use. The concentration of lysozyme was deter-
mined spectrophotometrically using e280 (lysozyme) = 37 646 M-1
cm-1.46 UV-vis absorption experiments were performed on JASCO
V-630 spectrophotometer. Emission spectra were recorded on
JASCO FP- 6600 spectrofluorometer. The excitation wavelength of
lysozyme was 280 nm and the emission was monitored at 342 nm.
The excitation and emission slit widths (each 5 nm) and scan rate
(500 nm min-1) were maintained constant for all the experiments.
A 3 mL solution, containing an appropriate concentration of
lysozyme (1 ¥ 10-6 M) was titrated with successive additions
of the complex. For synchronous fluorescence spectra, also the
same concentration of Lysozyme and THPP were used and the
spectra were measured at two different Dl (difference between the
excitation and emission wavelengths of lysozyme) values such as
15 and 60 nm.
Acknowledgements
Authors P.K. and K.N. gratefully acknowledge UGC-SAP and
CSIR, India for the financial assistance and author R.P. gratefully
acknowledge CNRS, France, for CNRS Fellowship.
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