binuclear copper complexes-interaction study with proteins
Post on 02-Jun-2018
224 Views
Preview:
TRANSCRIPT
-
8/11/2019 Binuclear Copper Complexes-Interaction Study With Proteins
1/13
Original article
Binuclear copper complexes: Synthesis, X-ray structure andinteraction study with nucleotide/protein by in vitro biochemicaland electrochemical analysis
M. Alagesan a, N.S.P. Bhuvanesh b, N. Dharmaraj a,*
a Inorganic and Nanomaterials Research Laboratory, Department of Chemistry, Bharathiar University, Coimbatore 641 046, Indiab Department of Chemistry, Texas A&M University, College Station, TX 77843, USA
a r t i c l e i n f o
Article history:
Received 4 July 2013
Received in revised form
17 February 2014
Accepted 14 March 2014
Available online 19 March 2014
Keywords:
Binuclear copper complex
Nucleotide and protein interaction
Cytotoxicity
a b s t r a c t
Two new, binuclear copper(II) hydrazone complexes have been synthesized and characterized by various
physico-chemical techniques including single crystal X-ray diffraction. Interaction of these complexes
with nucleotide and protein were analyzed byin vitrobiochemical and electrochemical analysis. Both the
complexes exhibited intercalative mode of binding with DNA. Further, gel electrophoresis assay
demonstrated the ability of the complexes to cleave the supercoiled pBR322 plasmid DNA to nicked
circular DNA form. Cytotoxicity of the complexes performed against a panel of cancer cell lines and a
normal cell line proved that these complexes are potentially cytotoxic against the cancerous cell lines,
particularly with IC50 as low as 0.7 mM against HeLa cell line.
2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
Consequent to the discovery and extensive use ofcis-platin as an
anticancer drug, synthesis of novel, bio-active metal complexes is
one of the pioneering topics in medicinal inorganic chemistry[1].
But, multifactorial resistance (intrinsic or acquired) and inherent
limitations such as serious side effects and general toxicity has
limited the use ofcis-platin. Therefore, considerable attempts are
being made to replace this drug with suitable alternatives by syn-
thesizing numerous transition metal complexes and tested for their
anticancer activities [2e7]. So more efcacious, less toxic, target
specic and non-covalent DNA binding anticancer drugs are to be
developed. On the other hand, copper, being a bio-essential
element and the one that accumulates in tumors due to the se-
lective permeability of cancer cell membranes as well as its com-plexes attained more signicance in nucleic acid chemistry as
compared to the heavier transition elements.
Generally, anticancer agents that are approved for clinical use
are molecules which damage DNA, block DNA synthesis indirectly
through inhibition of nucleic acid precursor biosynthesis or disrupt
hormonal stimulation of cell growth [7]. Therefore, considerable
effort has been now focused on the development of new anticancer
drugs based on transition metal complexes, particularly, bio-compatible copper(II) complexes, that bind to and cleave DNA un-
der physiological conditions [8]. Additionally, copper complexes are
also shown to up-regulate DNA-binding, a pivotal molecule in the
regulation of cell progression, cell survival and apoptosis [9].
An understanding on the binding modes to DNA would give
insights into the understanding of the biochemical mechanism of
action of the metal complexes. The chemistry of binuclear copper
complexes with ligands of biological relevance and with metal
centers at close proximity is one of the central themes of current
research [10] due to their interesting structural, electrochemical
and magnetic properties[11]and also because of their relevance to
the active sites of several metalloenzymes and greater cleaving
efciency or DNA interaction than the mononuclear complexes
[12e17]. Based on these facts, several reports are published on thesynthesis of copper(II) complexes along with their interactions
with DNA[18e20].
The continuing investigation on binuclear and polynuclear
metal complexes stems from the interest of researchers to under-
stand biological processes such as hydroxylation, oxygen transport,
electron transfer and catalytic oxidation and they give opportunity
to study the intramolecular binding, magnetic exchange in-
teractions, multi-electron redox reactions and possible activation of
small substrate molecules. Many metalloenzymes contain two
copper ions in their active site that operate cooperatively [21,22]
and consequently, complexes with two metal centers drawn a* Corresponding author.
E-mail address:dharmaraj@buc.edu.in(N. Dharmaraj).
Contents lists available atScienceDirect
European Journal of Medicinal Chemistry
j o u r n a l h o m e p a g e : h t t p : / / w w w . e l se v i e r . c o m / l o c a t e / ej m e c h
http://dx.doi.org/10.1016/j.ejmech.2014.03.043
0223-5234/
2014 Elsevier Masson SAS. All rights reserved.
European Journal of Medicinal Chemistry 78 (2014) 281e293
mailto:dharmaraj@buc.edu.inhttp://www.sciencedirect.com/science/journal/02235234http://www.elsevier.com/locate/ejmechhttp://dx.doi.org/10.1016/j.ejmech.2014.03.043http://dx.doi.org/10.1016/j.ejmech.2014.03.043http://dx.doi.org/10.1016/j.ejmech.2014.03.043http://dx.doi.org/10.1016/j.ejmech.2014.03.043http://dx.doi.org/10.1016/j.ejmech.2014.03.043http://dx.doi.org/10.1016/j.ejmech.2014.03.043http://www.elsevier.com/locate/ejmechhttp://www.sciencedirect.com/science/journal/02235234http://crossmark.crossref.org/dialog/?doi=10.1016/j.ejmech.2014.03.043&domain=pdfmailto:dharmaraj@buc.edu.in -
8/11/2019 Binuclear Copper Complexes-Interaction Study With Proteins
2/13
great deal of attention. Binuclear Cu(II) complexes have also been
reported as bio-inspired efcient catalysts in phosphoester hydro-
lysis[23].
Proteins are important biomolecules with considerable signi-
cance in biochemistry and their interactions with various mole-
cules such as drugs, dyes and metal complexes have aroused great
interest because of their importance in the explanation of the
structure and function of proteins[24,25]. It is known that serum
albumin is the main protein in blood plasma that acts as a trans-
porter and disposition of many drugs and has been frequently used
as a model protein for investigating the protein folding and ligand-
binding mechanism. The drugeprotein interaction may result in
the formation of a stable proteinedrug complex, which has
important effect on the distribution, free concentration, the meta-
bolism and the efcacy of drugs, etc. Over the past few years, much
research has been focused on the determinant factors that inu-
ence on the protein structures and functions [26e30]. In this re-
gard, bovine serum albumin (BSA) has been studied extensively,
partly because of its structural homology with human serum al-
bumin (HSA). Also, some metal ions present in blood plasma affect
the binding between drugs and serum albumins and could partic-
ipate in many biochemical processes. The phenomenon of confor-
mational alteration of serum albumin molecule caused by metalions is also well known.
Though considerable volume of research is constantly being
undertaken by several research group on the chemistry and bio-
molecular interactions of copper(II) complexes containing diversi-
ed ligand systems, corresponding studies on the binuclear
complexes appear to be limited[23,25]. Hence, the present inves-
tigation on the synthesis of two new binuclear copper(II) hydra-
zone complexes with single crystal structure determination and
interactions with DNA/protein is signicant to gain some insight
into the inuence of structural variation of the coordinated ligand
(hydrazones) on binding properties. The cytotoxicity assay per-
formed using the above binuclear copper complexes is also inter-
esting due to the fact that the former complex is very efcient
(0.7mM) to arrest the growth of cancer cells (HeLa) and a detailedprobe into the mechanism of anticancer activity would provide
some valuable information on the future prospects of these novel
binuclear copper complexes for biological evaluations.
2. Results and discussion
2.1. Synthesis and characterization
The ligands, benzoic acid (2-hydroxy-benzylidine)-hydrazone
(HL1) and 4-methyl-benzoic acid (2-hydroxy-benzylidine)-hydra-
zone (HL2) and the copper(II) complexes [CuCl(L1)]2 (1) and
[CuCl(L2)]2(2) were synthesized according to the reactions shown
inScheme 1.The structure of the binuclear complexes1and2were
characterized by various physico-chemical techniques and nallyconrmed by single crystal X-ray studies. The binuclear structure of
the complexes is stable in the solid phase and in solution medium
also which has been proved through electronic absorption spectral
and cyclic voltametric studies. The copper complexes are very
much soluble in DMF and DMSO, moderately soluble in methanol
and acetonitrile, and practically insoluble in carbon tetrachloride,
chloroform and benzene. The binucleating ligands, HL1 and HL2
coordinate to metal ions via carbonyl oxygen atoms, imine nitrogen
without undergoing enolization and hydroxyl oxygen of salicy-
laldehyde to form penta coordinated square planar binuclear cop-
per(II) complexes.
The IR spectra of the binuclear copper(II) complexes showed
bands in the region 3200e3260 cm1, indicating the presence of
NH groups. The peak at 1600e1630 cm1 due ton(C]O) and peak
forn(C]N) in the region 1460e1490 cm1 indicates the coordina-
tion of hydrazone ligand to copper metal which is in agreement
with previous report[31]. In order to obtain further structural in-
formation electronic spectra of all the complexes were recorded in
DMSOeTris buffer medium. The complexes 1 and 2 exhibit an ab-
sorption maximum in the range 550e610 nm and a shoulder band
in the range 755e785 nm, due to ded transition of the copper(II)
ion [31], indicating that the geometry around the copper ion is
square-pyramidal. The bands found in the range 245e290 nm and
320e360 nm is assigned to an intraligand transition band [32]andligand to metal charge transfer (LMCT) transition, respectively. The
ORTEP diagrams of binuclear complexes,1 and 2 are shown in Fig.1.
Crystallographic data are given in Table 1and the selected bond
lengths and bond angles are listed inTable 2. In crystal structure,
both the copper(II) ions are ve coordinated. The basal plane of
both the complexes is made of O, N and O atoms of the ligand in a
mononegative tridentate form and the fourth coordination site of
the basal plane is occupied by the oxygen atom of symmetric
[CuCl(L)] unit, and one chloride ion occupies axial position in both
the complexes. The [CuCl(L)] unit, which bridged through the ox-
ygen atom of the salicylaldehyde moiety of the ligand resulted in
centrosymmetric [CuCl(L)]2 dimer. The distortion of the coordina-
tion polyhedron from square pyramid (SP, s 0) and trigonal-
bipyramid (TBP, s 1) topologies were analyzed for both com-plexes[33], the value obtained was s 0.141 and 0.100 for1 and 2
respectively which clearly indicates that the environment around
the copper(II) ions is close to the SP topology. The non-bonded CueCu distance was found to be 3.041 A (1) and 3.043 A (2). The cop-
per(II) ion lies at about 0.301 A (1) and 0.277 A (2) above the
average basal plane towards the axial Cl atom in the complex. A
small dihedral angle of 4.82 (1) and 4.51 (2) between the mean
planes of theve member chelation ring and the six member one
ensures that the planarity of square is appreciable. Both the
hydrazone molecules in the complex are coplanar. The hydrazone
moiety possesses hydrogen bond donors and acceptors it provides
possibility of forming hydrogen bond in the crystal. The complexes
are stabilized by hydrogen bonds involving hydrogen from N2 ni-
trogen of the hydrazone ligand and coordinated chlorine atom inboth1 and 2. The bond distances and bond angles of the complex
HN
N
O
OCu
NH
N
O OCu
Cl
Cl
N
HN
OH
O
CHO
OH H2N
HN
O
+ [CuCl2(DMSO)2]
R = H or CH3
5h, reflux
EtOHDMF/MeOH
R
R
R
R
Scheme 1. Synthesis of ligands and binuclear copper(II) complexes.
M. Alagesan et al. / European Journal of Medicinal Chemistry 78 (2014) 281e293282
-
8/11/2019 Binuclear Copper Complexes-Interaction Study With Proteins
3/13
agree very well with those that are reported in related copper(II)
hydrazone complexes[34]. The distance between the bridged ox-
ygen atom and copper atoms in 1 is 1.953(3) A and 1.992(2) A and
the distance between the carbonyl oxygen and copper atom is
1.970(3)A whereas the distance between bridged oxygen atom and
copper atoms in complex 2 is 1.981(1) A and 1.964(2) A and the
distance between the carbonyl oxygen atom is 1.984(2) A.
The distance between copper and chlorine atoms is found to be
2.499(1) and 2.515(7)A in 1 and 2, respectively. Crystal lattice of the
complexes showed a two-dimensional array in which each unit of
the complex is hydrogen bonded to the other through N2 and Cl
atoms. Molecular packing suggested that the stabilization of lattice
was due to hydrogen bonds, mainly involving the N2and Cl atom of
1and2.
2.2. DNA binding properties
DNA is the primary pharmacological target of many antitumor
compounds, and hence, the interaction between DNA and metal
complexes is of paramount importance in understanding the
mechanism. Thus, the mode and propensity for binding of free li-
gands and binuclear copper(II) complexes to CT-DNA were studied
with multiple techniques such as UVevisible absorption, uores-
cence emission using ethidium bromide, cyclic voltammetry and
circular dichroism.
2.2.1. Absorption spectral studies
Interaction of metal complexes with DNA has been character-
ized through absorption titrations. The UVevisible spectra of the
investigated compounds in the absence as well as the presence ofCT-DNA (nucleotides) were obtained in DMSO:TriseHCl buffer
(5 mmol, pH 7.2) containing 50 mmol NaCl solutions. Binding of
free ligands and the corresponding binuclear copper(II) complexes
to DNA helix has been studied through the changes in intensity
absorption and shift in wavelength. Usually, a compound that
bound with DNA through intercalation exhibits hypochromism,
due to the strong stacking interaction between the planar aromatic
chromophore and the base pairs of DNA [35]. The extentof shift and
hypochromism are commonly found to correlate with the inter-
calative binding strength. But, metal complexes which bind non-
intercalatively or electrostatically with DNA may result in hyper-
chromism or hypochromism [33,36,37].
The electronic absorption spectra of the free ligands HL1 and
HL2 as well as the complexes,1 and 2 measured as a function of
increasing concentration of CT-DNA is shown in Fig. 2. Upon in-
cremental additions of DNA to the test compounds, the absorption
bands of HL1 observed at 323 and 295 nm exhibited a hypo-
chromism of about 4.1% and 3.9% without any shift in the above
band positions. Similarly, absorption bands ofHL2 found at 324 and
296 nm exhibited a hypochromism of 9% and 8.9% without any shift
in the wavelength of absorption. This fact accounts that there is an
interaction between the ligands and DNA through intercalation or
some other mode of binding. However, complex 1 exhibited a
hypochromism of about 19.7% and 19.5% with a hypsochromic shift
of 1 and 2 nm at 374 and 320 nm and the absorption bands of
complex 2 at 381 and 319 nm exhibited the same phenomenon of
hypochromism of about 26.1% and 23.4% respectively with a hyp-
sochromic shift of about 2 nm. These results suggested an intimate
association of the complexes,1 and 2 with CT-DNA, and it is also
likely that they bind to the DNA helix via intercalation [34,37]. After
the compounds intercalate to the base pairs of DNA, thep* orbital ofthe intercalated compounds could couple with p orbitals of the
base pairs, thus decreasing thep/ p* transition energies, resulted
hypochromism [1]. The complexes, 1 and 2 showed more hypo-
chromicity with red shift than the ligands (HL1 and HL2), indi-
cating that the binding strength of the copper(II) complexes is
much stronger than that of the free ligands. In order to afrm
quantitatively the afnity of the compounds bound to DNA, the
intrinsic binding constants (Kb) of the compounds with DNA was
obtained by using the following equation[38]
DNA=
a f
DNA=
b f
1=Kb
b f
Where, [DNA] is the concentration of DNA in base pairs and theapparent molar extinction coefcients a, f, and b correspond to
Aobs/[compound], the extinction coefcient of the free compound,
and the extinction coefcient of the compound when fully bound to
DNA, respectively. The plot of [DNA]/(a f) versus [DNA] gave a
slope and intercept which are equal to 1/(b f) and 1/Kb(b f),
respectively, Kbis the ratio of the slope to the intercept.
The magnitude of intrinsic binding constants (Kb) were calcu-
lated as 2.3 104 M1 (HL1) and 1.13 104 M1 (HL2) for the li-
gands and 3.46 105 M1 (1) and 1.96 105 M1 (2) for the
complexes. The observed values ofKbrevealed that the ligands and
the Cu(II) complexes bind to DNA via intercalative mode[39]and
the corresponding plot is given inFig. 3. From the results obtained,
it has been found that both complexes showed higher afnity to
binding with DNA. In order to clarify whether hypochromism and
Fig. 1. An ORTEP view of copper(II) complexes 1 and 2 with the atom numbering scheme and thermal ellipsoids drawn at 50% probability level.
M. Alagesan et al. / European Journal of Medicinal Chemistry 78 (2014) 281e293 283
-
8/11/2019 Binuclear Copper Complexes-Interaction Study With Proteins
4/13
red shift of absorption band can be used as positive criterions for
DNA binding modes, titration experiments of EBeDNA system havebeen performed.
2.2.2. EB-DNA quenching studies
Tond the ability of the compounds utilized in the above study
to displace EB from EB-DNA complex, a competitive EB binding
study was undertaken with uorescence experiments. The uo-
rescence based competitive binding serves as an indirect evidence
to understand the DNA-complex binding mode. EB, a phenan-
thridine uorescence dye, is a typical indicator of intercalation[40]
that forms soluble complex with nucleic acids and emits intense
uorescence in the presence of CT-DNA due to intercalation of the
planar phenanthridine ring between adjacent base pairs on the
double helix. The changes observed in the spectra of EB on its
binding to CT-DNA are often used for study between DNA and other
compounds, such as metal complexes. As the free ligands and the
Cu(II) complexes show no uorescence at room temperature in
solution or in the presence of CT-DNA, and their binding to DNA
cannot be directly predicted through the emission spectra. How-
ever, competitive EB binding studies could be used in order to
examine the binding of each compound with DNA. EB does not
show any appreciable emission in buffer solution due to uores-
cence quenching of the free EB by the solvent molecules. Upon
addition of the ligand or complex to a solution containing EB,
neither quenching of free EB uorescence has been observed, nor
new peak in the spectra appeared. The uorescence intensity is
highly enhanced upon addition of CT-DNA, due to its strong inter-
calation with DNA base pairs. Addition of a second molecule, which
may bind to DNA more strongly than EB, results in a decrease in the
DNA-induced EB emission due to the replacement of EB[41]. The
addition of the compounds results in a signicant decrease of the
uorescence intensity of the emission band of the DNA-EB system
(Fig. 4) indicate that the strong binding afnity of the compoundswith DNA over DNA-EB.
The observed quenching of DNA-EB uorescence for the ligand
or complex suggested that they displace EB from the DNA-EB
complex and they can interact with CT DNA probably by the
intercalative mode [42]. The quenching plots (Fig. 5) illustrated that
the quenching of EB bound to DNA by the complexes and free
ligand is in good agreement with the linear SterneVolmer equa-
tion. In the plots ofI0/Iversus [Q], Kq is given by the ratio of the
slope to the intercept. The Kq values for HL1, HL2, 1 and 2 were
found to be 2.4 103 M1, 5.6 103 M1, 5.1 104 M1 and
3.1 104 M1 respectively. Further, the binding constant (Kapp)
value obtained for the compounds using the following equation,
KEBEB Kappcompound
KEB 1.0107 M1 and [EB] 2.5 mM were 3.47 104 M1,
5.98 104 M1, 2.05 105 M1and 1.01 105 M1 forHL1,HL2, 1
and 2 respectively. The data showed that the interaction of the
Cu(II) complexes with DNA is stronger than that of the free ligand,
which is consistent with the electronic absorption spectral results.
Moreover, complex 1 showed higher DNA binding afnity
compared to complex2.
2.2.3. Cyclic voltammetry
Application of electrochemical methods to study metallo-
intercalation and binding of transitional metal complexes to DNA
is a useful complement to other investigation methods, such as
UVe
visible and uorescence spectroscopies. The cyclic voltam-mogram of binuclear copper complexes1 and2in the absence and
presence of CT-DNA is shown inFig. 6. In the CV of complex1, the
anodic (Epa) and cathodic responses (Epc) are found at 0.156,
0.168 V and 0.083, 0.100 V respectively, a characteristic of binu-
clear complex. The separations of the anodic and cathodic peak
potentials (DEp) are calculated as 73 and 68 mV, and the ratio of
cathodic to anodic peak currents ipa/ipc, are 1.9 and 1.6. The pres-
ence of DNA in the solution at the same concentration of binuclear
Cu(II) complex causes shift of 0.040 and 0.029 V in E1/2respectively,
and a change inDEof about 80 and 58 mV. The ratio of cathodic to
anodic peak currentsipa/ipc, are 1.8 and 1.5. The separations of the
anodic and cathodic peak potentials for the complex 2 (DEp) are 310
and 33 mV, and the ratio of cathodic to anodic peak currents ipa/ipc,
are 0.13 and 1.52. The formal potential (E1/2), taken as the average of
Table 1
Experimental data for crystallographic analysis.
1 2
Empirical formula C28H22Cl2Cu2N4O4 C30H26Cl2Cu2N4O4Formula weight 676.48 704.53
Wavelength (A) 1.54178 1.54178
Crystal system Triclinic Monoclinic
Space group P-1 P2(1)/c
Unit cell dimensionsA(A) 6.3713 (7) 12.4800 (11)
b(A) 8.8192 (10) 6.4162 (6)
c(A) 11.8050 (11) 17.4813 (17)
a() 86.780 (8) 90
b() 88.816 (9) 93.177 (7)
g() 79.462 (6) 90
Volume (A3) 651.07 (12) 1397.6 (2)
Z 1 2
Density (calculated)
(Mg/m3)
1.725 1.674
Absorption coefcient
(mm1)
4.279 4.013
F(000) 342 716
Theta range for
data collection
3.75e59.96 6.03e59.99
Index ranges 7 h 7, 9 k 9,
13
l
13
14 h 14, 7 k 6,
19
l
19
Independent
reections
1869 [R(int) 0.0367] 2047 [R(int) 0.0522]
Max. and min.
transmission
0.8824 and 0.7259 0.9241 and 0.6897
Data/restraints/
parameters
1869/0/181 2047/0/191
Goodness-of-t onF2 1.113 0.947
FinalR indices [I>
2sigma(I)]
R1 0.0406,
wR2 0.1064
R1 0.0256, wR2 0.0731
Rindices (all data) R1 0.0425,
wR2 0.1079
R1 0.0294, wR2 0.0746
Largest diff. peak
and hole (e.A3)
0.998 and 0.345 0.267 and 0.298
Table 2
Selected bond lengths (A) and angles () of the complexes.
1 2
Cu(1) eO(1) 1.952 (3) 1.964 (16)
Cu(1) eN(1) 1.947 (3) 1.939 (2)
Cu(1) eO(2) 1.970 (3) 1.984 (16)
Cu(1) eCl(1) 2.498 (11) 2.515 (7)
Cu(1) eO1(1) bridge 1.992 (3) 1.981 (16)
N(1)-Cu(1)-O(1) 90.82 (12) 91.09 (7)
N(1)-Cu(1)-O(2) 80.99 (12) 80.56 (7)
O(1)-Cu(1)-O(2) 164.97 (12) 165.33 (7)
N(1)-Cu(1)-O(1) bridge 156.46 (13) 159.31 (8)
O(1)-Cu(1)-O(1) bridge 79.10 (11) 79.08 (7)
O(2)-Cu(1)-O(1) bridge 103.63 (11) 104.78 (7)
N(1)-Cu(1)-Cl(1) 98.81 (10) 98.93 (6)
O(1)-Cu(1)-Cl(1) 101.19 (9) 106.34 (5)
O(2)-Cu(1)-Cl(1) 92.55 (9) 86.97 (5)
O(1) bridge-Cu(1)-Cl(1) 103.96 (9) 101.28 (5)
M. Alagesan et al. / European Journal of Medicinal Chemistry 78 (2014) 281e293284
-
8/11/2019 Binuclear Copper Complexes-Interaction Study With Proteins
5/13
EpaandEpc, is 0.15 and 0.016 V in the absence of DNA. The presence
of DNA in the solution at the same concentration of binuclearcomplex causes shift in E1/2 of 0.15 and 0.023 V and a decrease inDE
of 4 and 14 mV. The ratio of cathodic to anodic peak currents ipa/ipc,
are 0.30 and 0.047, the value ofipa/ipc also decreases with the in-
crease of the DNA concentration. The decrease in peak currents can
be explained in terms of an equilibrium mixture of free and DNA-
bound copper(II) complex to the electrode surface [43,44]. Upon
addition of DNA, the anodicand cathodic peak potentials shift more
positive values, but ipc/ipavalue decreases with the increase of the
DNA concentration. The more decrease of the peak currents
observed for 1 than that of2 upon addition of CT-DNA indicated
that the binding afnity of the former to DNA is stronger than that
of the latter.
2.2.4. Circular dichroism (CD) studies
Circular dichroic spectral analysis provides valuable information
on the binding mode of metal complexes with DNA[45]. Generally,
structural alterations of DNA caused by interaction with com-
pounds are reected as signicant changes in intrinsic CD spec-
trum. InFig. 7,the CD spectrum of free DNA showed a positive peak
at approximately 278 nm and a negative peak at 247 nm which
corresponds to B-DNA. These bands are caused by stacking in-
teractions between the bases and the helical supra structure of the
polynucleotide that provides an asymmetric environment for the
bases[41]. Simple groove binding and electrostatic interaction of
molecules show less or no perturbation on the base stacking and
helicity, while intercalation decreases or increases the intensities of
both positive and negative bands[46e
48]. The CD spectra of DNA
recorded after the addition of different concentration of binuclear
copper complexes 1 and 2 caused an increase in the intensity ofboth positive and negative bands of DNA due to an intercalative
mode of interaction between them.
2.3. Cleavage of pBR322 plasmid DNA by copper(II) complexes
To assess the DNA cleavage ability of the new Cu(II) complexes
by gel electrophoresis, supercoiled (SC) pBR322 DNA was incubated
with three different concentrations of copper complexes in 5 mM
TriseHCl/50 mM NaCl buffer (pH 7.2). Both the complexes, 1and 2
exhibited concentration-dependent nuclease activity during which
SC DNA was converted in to nicked circular (NC) DNA (Fig. 8, lanes
3e8) without any reductant. Upon increasing the concentration of
the complex solution from 2.5mM to 10mM, the extent of NC formof DNA was also increased. Moreover, at 10 mM concentration, 1
showed more cleavage efciency than2to convert SC DNA (Form I)
to NC DNA (Form II) revealing the superior performance of the
former. However, free ligands and precursor
complex [CuCl2(DMSO)2] did not exhibit any cleavage activity un-
der the same experimental conditions. Thus, the cleavage proper-
ties of the present compounds are attributed to the coordination
geometries and the proximity of the DNA-bound complexes to the
deoxyl ribose rings, as understood from the spectral and electro-
chemical properties. Similar observation was made by Yingying
Kou et al.[49],. Further, the cleavage efciency of complexes, 1and
2 remains unaffected in the presence of scavengers of hydroxyl
radicals (DMSO and mannitol), singlet oxygen (sodium azide and L-
histidine), and superoxide radical scavengers (SOD). This indicates
Fig. 2. Changes in the electronic absorption spectra of the ligands HL1 (a), HL2 (b) and complexes 1(c) and 2(d) (25 mM) with increasing concentration of CT-DNA (0e20mM).
M. Alagesan et al. / European Journal of Medicinal Chemistry 78 (2014) 281e293 285
-
8/11/2019 Binuclear Copper Complexes-Interaction Study With Proteins
6/13
that the cleavage of DNA probably follows a hydrolytic cleavage
mechanism[39,49,50]. Moreover, inhibition or promotion of DNA
cleavage was not appreciably altered under aerobic as well asanaerobic conditions and thereby conrmed that the cleavage did
not follow oxidative mechanism.
2.4. Protein binding studies
Bovine serum albumin (BSA) and human serum albumin (HSA)
are the most widely investigated proteins [51,52]. Among them,
BSA is the major soluble protein that has many physiological
functions, such as maintaining the osmotic pressure and pH of
blood and as carriers transporting a great number of endogenous
and exogenous compounds such as fatty acids, amino acids, drugs
and pharmaceuticals[53]. A useful feature of the intrinsic uores-
cence of proteins is the high sensitivity of tryptophan and its local
environment. Changes in the emission spectra of tryptophan are
common in response to protein conformational transitions, subunit
associations, substrate binding, or denaturation[54]. Therefore, the
intrinsic uorescence of proteins can provide considerable infor-
mation on their structure and dynamics and is often utilized in the
study of protein folding and association reactions. Hence, uores-
cence quenching is an important technique to study the interaction
of metal complexes with BSA because of its accuracy, sensitivity,
rapidity and convenience of usage. A solution of BSA (1 mM) was
titrated with various concentrations of the compounds (0e8mM).
The uorescence of BSA at around 345 nm was gradually quenched
upon increasing the concentration of ligands and complexes with a
little blue shift of the emission maximum wavelength as shown in
Fig. 9.
Addition of the above compounds to the solution of BSAresulted
in a signicant decrease in the uorescence intensity of BSA at
345 nm, up to 51%, 57%, 89% and 78% of the initial uorescenceintensity of BSA for HL1, HL2,1 and 2 respectively. The observed
blueshiftof 5 and 6 nmfor 1 and 2 is mainly due to the fact that the
active site in the protein is buried in a hydrophobic environment.
This result suggested a denite interaction of the compounds with
the BSA protein. Quenching may occur by different mechanism
usually classied as dynamic quenching and static quenching. Dy-
namic quenching refers to a process in which the uorophore and
Fig. 3. Plots of [DNA]/(a f) versus [DNA] for the compounds with CT-DNA.
Fig. 4. Fluorescence quenching curves of ethidium bromide bound to DNA: ligands HL1(a) and HL2(b) and the complexes 1(c) and 2 (d). [DNA] 10 mM, [EB] 10 mM, and
[compound]
0e
16mM.
M. Alagesan et al. / European Journal of Medicinal Chemistry 78 (2014) 281e293286
-
8/11/2019 Binuclear Copper Complexes-Interaction Study With Proteins
7/13
the quencher come into contact during the transient existence of
the excited state. Static quenching refers to the uorophoreequencher complex formation in the ground state. A simple method
to explore the type of quenching is UVevisible absorption spec-
troscopy. UVevisible spectra of BSA in the absence and presence of
the compounds (Fig. 10) showed that the absorption intensity of
BSA was enhanced as the compounds were added and there
observed a slight blue shift of about 2 and 3 nm for the ligand and
binuclear Cu(II) complexes, respectively. Hence, change in the
absorption spectrum ofuorophores, reveals quenching of BSA by
the compounds are static quenching processes[53]. To study the
quenching process further, uorescence quenching data were
analyzed with the SterneVolmer and Scatchard equations.
The values ofKqandKbinfor the ligands and the binuclear Cu(II)
complexes suggested that the complexes interact with BSA more
strongly than ligands. The quenching constants (Kq 9.5 106 (1),
7.2 106 M1 (2), 1.1 104 M1 (HL1), 1.7 104 M1 (HL2)) have
been calculated from the plot ofI0/Iversus [Q](Fig. 11B). Based on
the plot of log (I0 I)/Iversus log [Q](Fig. 11A), binding constant
(Kbin 1.8 106 M1 (1), 1.1 106 M1 (2), 1.2 104 M1 (HL1),
1.3 104 M1 (HL2)) have been obtained. The value of nindicates
the existence of a single binding site in BSA for the complex or
ligand. The larger values ofKqandKbinindicate a strong interaction
between the BSA and the complex over the ligands.
2.4.1. Characteristics of the synchronousuorescence spectra
Synchronous uorescence spectra provide information on the
molecular micro-environment particularly in the vicinity of the
uorophore functional groups[55]. The uorescence of BSA is due
to presence of tyrosine and tryptophan residues. Among them,
tryptophan is the most dominant uorophore, located at the sub-
strate binding sites. Most of the drugs bind to the protein in the
active binding sites. Hence, synchronous method is usually applied
to nd out the conformational changes around tryptophan and
tyrosine region. According to Miller, the difference between the
excitation and emission wavelengths (Dl lem lex) reects the
spectra of a different nature of chromophores [56]. If the Dl value is
15 nm, the synchronous uorescence of BSA is characteristic of a
tyrosine residue, whereas a larger Dl value (60 nm) is characteristic
Fig. 5. SterneVolmer plots of the uorescence titration of the ligands and the
complexes.
Fig. 6. Cyclic voltammogram of complexes in the absence and presence (inner line) of DNA (10 mM). Scan rate: 100 mV s1.
Fig. 7. Circular dichroic spectra of CT-DNA (10 mM) with the addition of different concentration of binuclear copper complexes 1 and 2 (10mM).
M. Alagesan et al. / European Journal of Medicinal Chemistry 78 (2014) 281e293 287
-
8/11/2019 Binuclear Copper Complexes-Interaction Study With Proteins
8/13
of tryptophan [57]. To investigate the structural changes that
occurred in BSA upon the addition of our compounds, synchronous
uorescence spectra of BSA were measured before and after the
addition of test compounds. The synchronous uorescence spectra
of BSA with various concentrations of test compounds were
recorded atDl
15 nm andDl
60 nm and are shown in Figs. 12and 13, respectively. In the synchronousuorescence spectra of BSA
at Dl 15, the addition of the compounds to the solution of BSA
resulted in a small decrease in the uorescence intensity of BSA at
302 nmup to30,39, 56 and 56% of the initial uorescence intensity
of BSA for the ligands and the complexes respectively, with no shift
in their emission wavelength maxima. But, in the case of the syn-
chronous uorescence spectra of BSA at Dl 60, the addition of
compounds to the solution of BSA signicantly decreased the
uorescence intensity of BSA at 342 nm, up to 16, 42, 88 and 74.1%
accompanied with a blue shift of 1 and 2 nm for the ligands and
complexes, respectively. Thus, synchronous uorescence spectral
studies suggested that the uorescence intensity of both tyrosine
and tryptophan residues were affected by increasing the concen-
tration of compounds, but the signicant decrease along with a
blue shift of the uorescence intensity of tryptophan has been
observed. These results suggested that the interaction of the ligand
and the complex with BSA affects the conformation of tryptophan
much than the tyrosine micro-region. The binding strength of the
binuclear Cu(II) complexes with BSA is signicantly higher than
that of the ligands, which can be explained by the fact that the
hydrophobicity of the complex is greater than that of the ligand. So,
the strong interaction between the compounds and BSA suggested
that these compounds can easily be stored in protein and can be
released to desired targets. Hence, we took interest to study the
cytotoxicity of the compounds.
2.5. Evaluation of in vitro anticancer activity
Cytotoxicity of the compounds were tested against a series of
cancer cell lines and a normal cell line by two different methods
such as Trypan blue dye exclusion and MTT method.
2.5.1. Trypan Blue assay
Trypan Blue, a blue acid dye with two azochromophoric groups
will not enter into the live cell, but into dead cell and makes it blue
color stain[58,59]. Thenumber of dead cells can be easily calculated
by counting stained cells through microscope. The results ofin vitro
cytotoxicity test were shown in Table 3 as their IC50 values. The
result obtained showed that both the binuclear copper(II) com-
plexes are highly toxic towards Ehrlich ascites carcinoma and Dal-
tons ascites lymphoma cell lines, whereas the ligand and
[CuCl2(DMSO)2] did not show any signi
cant activity on all the
cancer cells. From the results obtained,it is clear that coordinationof
copper atom enhanced the cytotoxic potential of free ligands[60].
2.5.2. MTT assay
The potential toxicity of the compounds towards the HeLa
(cancer cell line) and NIH 3T3 (normal cells) was further studied by
MTT assay. The complexes were dissolved in DMSO and diluted to
the requiredconcentration and same volume of DMSO was taken as
control to balance solvent activity in the cytotoxicity experiment.
The results were analyzed by means of cell inhibition expressed as
IC50 values and are given inTable 4. The IC50 values presented in
Table 4showed that the new complexes,1 and 2 are signicantly
active against HeLa, with less toxicity to normal cells. However, it is
to be noted that both the ligands and precursor complex
[CuCl2(DMSO)2] did not show any signicant activity on the above
cancer cells (IC50 above 100 mM). Hence, it is concluded that
chelation of the ligand with Cu(II) ion only responsible for the
observed cytotoxic properties of the new Cu(II) complexes. The
better cytotoxic activity of the Cu(II) complex may be attributed to
the extended planar structure induced by the p ep* conjugation
resulting from the chelation of the Cu(II) ion with ligand.
3. Conclusion
In this study, we describe the synthesis and single crystal
structure of two new, binuclear copper(II) hydrazone complexes.
Interaction of the complexes and free ligands with biomolecules
such as DNA/BSA and in vitroanticancer activity versus cancer and
normal cells were also presented. The magnitude of binding con-
stant of the test compounds with CT-DNA decreased in the orderHL2< HL1
-
8/11/2019 Binuclear Copper Complexes-Interaction Study With Proteins
9/13
used for the biological studies were of high quality and procured
commercially from the reputed suppliers.
4.2. Physical measurements
Elemental analyses (C, H and N) were performed on Vario EL III
Elemental analyzer instrument. IR spectra of the samples were
recorded as KBr pellets on a Nicolet Avatar instrument in the fre-
quency range of 400e4000 cm1. Melting points were determined
with a Lab India instrument. Absorption and emission spectra were
recorded in DMSO-buffer solution on a Jasco V-630 spectropho-
tometer and Jasco FP 6600 spectrouorometer respectively, at
room temperature. Electrochemical measurements were per-
formed in a conventional two compartment three electrode cell
with a mirror polished GCE as a working electrode, Pt wire as acounter electrode and NaCl saturated Ag/AgCl as a reference elec-
trode. The electrochemical measurements were carried out with
CHI electrochemical workstation (Model 643B, Austin, TX, USA). All
the electrochemical measurements were carried out under nitro-
gen atmosphere at room temperature. Induced Circular Dichroism
spectra were recorded on JASCO J-810 spectropolarimeter with
PMT detector in DMSO-buffer solution.
4.3. Synthesis of benzoic acid (2-hydroxy-benzylidine)-hydrazone
(HL1) and 4-methyl-benzoic acid (2-hydroxy-benzylidine)-
hydrazone(HL2) ligands
The ligands were prepared according to the literature method
with slight modi
cations[32,61]. The hydrazone ligands (HL1) and(HL2) were synthesized by mixing equimolar amounts of salicy-
laldehyde (0.122 g; 1 mM) with benzhydrazide(1 mM) or p-tol-
uichydrazide (0.150 g; 1 mM) in ethanol (50 mL) respectively. The
reaction mixturewas reuxedonawaterbathfor5handpouredinto
crushed ice. The corresponding hydrazone formed was ltered and
washed several times with distilled water and recrystallized from
ethanol with 85% yield. Thepurity of theligands was checked by TLC
and melting point has been compared with the literature[61].
4.4. Synthesis of metal complexes
4.4.1. Synthesis of [CuCl(L1)]2(1)
A warm DMF solution (20 mL) containing [CuCl2(DMSO)2]
(0.344 mM, 0.1 g) was added to a methanolic solution of
Fig. 9. The emission spectrum of BSA (1mM;lex 280 nm, lem 345 nm) in the presence of increasing amounts of the ligandHL1(a) andHL2(b) and the complexes1(c) and2(d)
(0e8 mM). The arrow shows the decreases in the emission intensity upon increasing the concentration of the compounds.
Fig. 10. Electronic absorption spectra of BSA (5 mM) with ligands and complexes
(10mM).
M. Alagesan et al. / European Journal of Medicinal Chemistry 78 (2014) 281e293 289
-
8/11/2019 Binuclear Copper Complexes-Interaction Study With Proteins
10/13
HL1(0.344 mM, 0.082 g) and reuxed for an hour. Green single
crystals suitable for X-ray studies were obtained on slow evapo-
ration of the reaction mixture over a period of 15e20 days.
Yield: 52%. Melting point:
-
8/11/2019 Binuclear Copper Complexes-Interaction Study With Proteins
11/13
bromide under a UV illuminator. The cleavage efciency was
determined based on the ability of the complex to convert the
supercoiled DNA (SC) to nicked circular form (NC).
4.8. Protein binding studies
Protein-binding studies were performed by uorescence
quenching experiments using bovine serum albumin (BSA). The
uorescence spectra were recorded with an excitation at 280 nm
andcorrespondingemission at 345 nm assignable tothat of BSA and
the ligands/complexes as quenchers with increasing concentration
[71]. The excitation and emission slit widths and scan rates were
maintained constant forall the experiments. A stocksolution of BSA
was prepared in 50 mM phosphate buffer (pH 7.2) and stored in
the dark at 4 C for further use. Concentrated stock solution of the
ligands and its copper complexes were prepared by dissolving them
in DMSO and diluted to required concentrations with phosphate
buffer (5:95). BSA solution (1 mM) was titrated by successive addi-
tions of test solutions HL1, HL2, 1 and 2 using micropipettes for all of
the experiments. Synchronous uorescence spectra was also
recorded using the same concentration of BSA and compounds as
mentioned above with two different Dl (difference between the
excitation and emission wavelengths of BSA) values such as 15 and
60 nm. Thequenching constant (Kq) canbe calculated using the plot
ofI0/Iversus [Q]. If it is assumed that thebinding of compoundswith
BSA occurs at equilibrium, the equilibrium binding constant can be
analyzed according to the Scatchard equation:
logI0I=I logKbinnlogQ
Where,Kbinis the binding constant of compound with BSA and nis
the number of binding sites. From the plot of log(I0I)/Iversus log
[Q], the number of binding sites (n) and the binding constant (Kbin)was calculated.
4.9. Evaluation of cytotoxicity
Cytotoxicity of the binuclear complexes1 and 2 as well as free
ligands HL1 and HL2 was determined by Trypan blue and MTT
assays as given below.
4.9.1. Trypan Blue assay
Trypan Blue Assay was performed against Daltons ascites
lymphoma (DAL) and Ehrlich ascites carcinoma (EAC) cells and
these cells were grown in the peritoneal cavity of mice by serial
transplantation. For the experiment, the cells were aspirated from
the peritoneal cavity of tumor bearing mice, pelleted by centrifu-gation and maintained by intraperitoneal inoculation of 10 6 cells/
mouse. The cells were washed thrice with phosphate buffer saline
and made up to a concentration of 10 million/mL. The cells
(1 million) were incubated with different concentration of drugs in
a total volume of 0.9 mL with PBS at 37 C for 3 h. After incubation,
0.1 mL of Trypan blue (1%) was added and in vitrocell viability is
measured by trypan blue exclusion test based on the ability of
trypan blue to stain dead cells. The percentage of inhibition was
calculated using the following equation.
%inhibition No: of dead cells=No: of dead cells
No of live cells 100
Fig. 12. Synchronous spectra of BSA (1 mM) in the presence of increasing amounts of the ligands HL1(a) and HL2(b) and the complexes1(c) and 2(d) (0e8 mM) at a wavelength
difference ofDl 15 nm. The arrow shows the decreases in the emission intensity upon increasing concentration of the compounds.
M. Alagesan et al. / European Journal of Medicinal Chemistry 78 (2014) 281e293 291
-
8/11/2019 Binuclear Copper Complexes-Interaction Study With Proteins
12/13
4.9.2. MTT assay
The growth inhibitory effect of the same complexes and li-
gands were also assessed versus a cancer (HeLa), and normal (NIH
3T3) by means of MTT assay[72]. For the screening experiments,
the cells were seeded into 96-well plates in 100 mL of the
respective medium containing 10% FBS, at a plating density of
10,000 cells/well. The cells were incubated at 37 C in 5% CO2and
95% air at a relative humidity of 100% for 24 h prior to the
addition of the test compounds. The compounds were dissolved
in DMSO and diluted in the respective medium containing 1% FBS.
The maximum concentration DMSO used in the experiment is
10mL. After 24 h, the medium was replaced with the respective
medium with 1% FBS containing the compounds at various con-
centrations and incubated at 37 C under conditions of 5% CO2,
95% air, and 100% relative humidity for 48 h. Triplicate cultures
were established for each treatment, and the medium not con-
taining the compounds served as the control. After 48 h, 10 mL of
MTT (5 mg/mL) in phosphate buffered saline (PBS) was added to
each well and incubated at 37 C for 4 h. The medium with MTT
was then icked off, and the formed formazan crystals were
dissolved in 100mL of DMSO. Mean absorbance for each drug dose
was expressed as a percentage of the control untreated wellabsorbance and plotted versus drug concentration. IC50 values
represent the drug concentrations that reduced the mean absor-
bance at 570 nm to 50% of those in the untreated control wells
and a graph was plotted with the percentage of cell inhibition
versus concentration. From this, the IC50 value was calculated
[73].
Fig. 13. Synchronous spectra of BSA (1 mM) in the presence of increasing amounts of the ligandsHL1(a) and HL2(b) and the complexes 1(c) and 2(d) (0e8 mM) at a wavelength
difference ofDl 60 nm. The arrow shows the decreases in the emission intensity upon increasing concentration of the compounds.
%inhibition mean OD of untreated cellscontrol=mean OD of treated cells 100
Table 3
Cytotoxicity of the complexes determined by Trypan Blue dye exclusion method.
Compound IC50 values (mM)
EAC DLA
Complex 1 4 0.50 4.1 0.57
Complex 2 5 0.86 4.1 0.61
Table 4
Cytotoxicity of the complexes determined by MTT assay.
Compound IC50values (mM)
HeLa NIH 3T3
Complex 1 0.7 0.98 22 0.54
Complex 2 18.6 0.98 54 0.98
M. Alagesan et al. / European Journal of Medicinal Chemistry 78 (2014) 281e293292
-
8/11/2019 Binuclear Copper Complexes-Interaction Study With Proteins
13/13
Supplementary material
Crystallographic data for the structure reported in this paper
have been deposited with the Cambridge Crystallographic Data
Centre (CCDC) as supplementary CCDC reference numbers of the
complexes are 852728 and 852729, respectively. Copies of the data
can be obtained free of charge from the CCDC (12 Union Road,
Cambridge CB2 1EZ, UK; Tel.: 44-1223-336408; Fax: 44-1223-
336003; e-mail: deposit@ccdc.cam.ac.uk; Web site http://www.
ccdc.cam.ac.uk/).
Acknowledgment
The corresponding author of the manuscript (N. D) acknowl-
edges the Council of Scientic and Industrial Research (CSIR),
Ministry of Human Resources Development (MHRD), Government
of India, New Delhi, for the nancial support in the form of a major
research project (CSIR sanction letter No. 01(2684)/12/EMReII
dated 03. 10. 2012). We thank prof. A. Ramu, School of Chemistry,
Madurai Kamaraj University, India, for his help to record CD spectra
and Dr. S. Abraham John, Department of Chemistry, Gandhigram
Rural Institute eDeemed University, Gandhigram, India, for elec-
trochemical measurement of samples.
References
[1] B.V. Rosenberg, L. Camp, T. Krigas, Nature 205 (1965) 698e699.[2] M. Cocchietto, G. Sava, Pharmacology & Toxicology 87 (2000) 193e197.[3] S. Zorzet, A. Sorc, C. Casarsa, M. Cocchietto, G. Sava, Metal-Based Drugs 8
(2001) 1e7.[4] R. Gagliardi, G. Sava, S. Pacor, G. Mestroni, E. Alessio, Clinical & Experimental
Metastatis 12 (1994) 93e100.[5] M. Magnarin, A. Bergamo, M.E. Carotenuto, S. Zorzet, V. Sava, Anticancer
Research 20 (2000) 2939e2944.[6] J.M.R. Lakhai, D.V.D. Bongard, D. Pluim, J.H. Beijnen, J.H. Schellens, Clinical
Cancer Research 10 (2004) 3717e3727.[7] W.O. Foye, Cancer Chemotherapeutic Agents, American Chemical Society,
Washington, DC, 1995.[8] K.M. Deck, T.A. Tseng, J.N. Burstyn, Inorganic Chemistry 41 (2002) 669e677.
[9] G.W. Verhaegh, M.J. Richard, P. Hainaut, Molecular and Cellular Biology 17(1997) 5699e5706.
[10] D. Senthil Raja, N.S.P. Bhuvanesh, K. Natarajan, European Journal of MedicinalChemistry 46 (2011) 4584e4594.
[11] E.I.Solomon, M.J.Baldwin,M.D. Lowery, Chemical Reviews 92 (1992)521e542.[12] T. Hirohama, Y. Kuranuki, E. Ebina, T. Sugizaki, H. Arii, M. Chikira, P. Tamil
Selvi, M. Palaniandavar, Journal of Inorganic Biochemistry 99 (2005) 1205e1219.
[13] Z. Liu, A. Habtemariam, A.M. Pizarro, S.A. Fletcher, A. Kisova, O. Vrana,L. Salassa, P.C.A. Bruijnincx, G.J. Clarkson, V. Brabec, P.J. Sadler, Journal ofMedicinal Chemistry 54 (2011) 3011e3026.
[14] Z. Chen, X. Wang, Y. Li, Z. Guo, Inorganic Chemistry Communications 11(2008) 1392e1396.
[15] J. Sun, S.Y. Deng, L. Zhang, J. He, L. Jiang, Z.W. Mao, L.N. Ji, Journal of Coordi-nation Chemistry 62 (2009) 3284e3295.
[16] K. Dhara, J. Ratha, M. Manassero, X. Wang, S. Gao, P. Banerjee, Journal ofInorganic Biochemistry 101 (2007) 95e103.
[17] V. Rajendiran, R. Karthik, M. Palaniandavar, H.S. Evans, V.S. Periasamy,M.A. Akbarsha, B.S. Srinag, H. Krishnamurthy, Inorganic Chemistry 46 (2007)
8208e8221.[18] D. Senthil Raja, N.S.P. Bhuvanesh, K. Natarajan, Inorganic Chemistry 50 (2011)
12852e12866.[19] D. Senthil Raja, G. Paramaguru, N.S.P. Bhuvanesh, J.H. Reibenspies,
R. Renganathan, K. Natarajan, Dalton Trans 40 (2011) 4548e4559.[20] O. Schicke, B. Faure, M. Giorgi, A.J. Simaan, M. Rglier, Inorganica Chimica Acta
391 (2012) 189e194.[21] P. Akilan, M. Thirumavalavan, M. Kandaswamy, Polyhedron 22 (2003) 1407e
1413.[22] M. Jiang, Y.T. Li, Z.Y. Wub, Z.Q. Liu, C.W. Yan, Journal of Inorganic Biochemistry
103 (2009) 833e844.[23] C. Belle, C. Beguin, I.G. Luneau, S. Hamman, C. Philouze, J.L. Pierre, F. Thomas,
S. . Torelli, E.S. Aman, M. Bonin, Inorganic Chemistry 41 (2002) 479e491.[24] C. Gerdemann, C. Eichen, B. Krebs, Accounts of Chemical Research 35 (2002)
183e191.[25] W. Sun, Y.Y. Han, K. Jiao, Journal of the Serbian Chemical Society 71 (2006)
385e396.[26] I. Petitpas, T. Grune, A.A. Bhattacharya, S. Curry, Journal of Molecular Biology
314 (2001) 955e
960.
[27] M. Dockal, D.C. Carter, F. Ruker, Journal of Biological Chemistry 275 (2000)3042e3050.
[28] B. Ahmad, B. Ankita, R.H. Khan, Archives of Biochemistry and Biophysics 437(2005) 159e167.
[29] X.M. He, D.C. Carter, Nature 358 (1992) 209e215.[30] T. Peters, All about Albumin: Biochemistry, Genetics and Medical Application,
Academic Press, Inc, New York, 1996.[31] N. Sengottuvelan, D. Saravanakumar, M. Kandaswamy, Polyhedron 26 (2007)
3825e3832.[32] E.W. Ainscough, A.M. Brodie, A.J. Dobbs, J.D. Ranford, J.M. Waters, Inorganica
Chimica Acta 267 (1998) 27e38.[33] D. SenthilRaja, E. Ramachandran, N.S.P. Bhuvanesh, K. Natarajan, European
Journal of Medicinal Chemistry 64 (2013) 148e159.[34] Z.C. Liu, B.D. Wang, Z.Y. Yang, Y. Li, D. Qin, D. Li, T.R. Li, European Journal of
Medicinal Chemistry 44 (2009) 4477e4484.[35] J.K. Barton, A.T. Danishefsky, J.M. Goldberg, Journal of the American Chemical
Society 106 (1984) 2172e2176.[36] F.Q. Liu, Q.X. Wang, K. Jiao, F.F. Jian, G.Y. Liu, R.X. Li, Inorganica Chimica Acta
359 (2006) 1524e1530.[37] D. Lawrence, V.G. Vaidyanathan, B.U. Nair, Journal of Inorganic Biochemistry
100 (2006) 1244e1251.[38] M. Alagesan, N.S.P. Bhuvanesh, N. Dharmaraj, Dalton Transactions 42 (2013)
7210e7223.[39] J. Olmsted, D.R. Kearns, Biochemistry 16 (1977) 3647e3654.[40] D. Senthil Raja, N.S.P. Bhuvanesh, K. Natarajan, Journal of Biological Inorganic
Chemistry 17 (2012) 223e237.[41] P. Krishnamoorthy, P. Sathyadevi, A.H. Cowley, R.R. Butorac, N. . Dharmaraj,
European Journal of Medicinal Chemistry 46 (2011) 3376e3387.[42] J. Liu, T. Zhang, T. Lu, L. Qu, H. Zhou, Q. Zhang, L. Ji, Journal of Inorganic
Biochemistry 91 (2002) 269e276.[43] J. Liu, T.B. Lu, H. Li, Q.L. Zhang, L.N. Ji, Transition Metal Chemistry 27 (2002)
686e690.[44] G. Xu, J. Fan, K. Jiao, Electroanalysis 20 (2008) 1209e1214.[45] S. Neidle, Nucleic Acid Structure and Recognition, Oxford University Press,
New York, 2002, pp. 89e138.[46] N. Shahabadi, S. Kashanian, F. Darabi, European Journal of Medicinal Chem-
istry 45 (2010) 4239e4245.[47] V. Rajendiran, M. Palaniandavar, P. Swaminathan, L. Uma, Inorganic Chemistry
46 (2007) 10446e10448.[48] P. Uma Maheswari, M. Palaniandavar, Journal of Inorganic Biochemistry 98
(2004) 219e230.[49] Y. Kou, J. Tian, D. Li, W. Gu, X. Liu, S. Yan, D. Liao, P. Cheng, Dalton Transactions
(2009) 2374e2382.[50] T. Kobayashi, S. Tobita, M. Kobayashi, T. Imajyo, M. Chikira, M. Yashiro, Y. Fujii,
Journal of Inorganic Biochemistry 101 (2007) 348e361.[51] F. Tan, M. Guo, Q.S. Yu, Spectrochimica Acta Part A 61 (2005) 3006e3012.[52] E.K. Efthimiadou, A. Karaliota, G. Psomas, Journal of Inorganic Biochemistry
104 (2010) 455e
466.[53] A. Sulkowska, Journal of Molecular Structure 616 (2002) 227e232.[54] G.Z. Chen, X.Z. Huang, J.G. Xu, Z.Z. Zheng, Z.B. Wang, Methods of Fluorescence
Analysis, second ed, Science Press, Beijing, 1990.[55] J.N. Miller, Proceedings of the Analytical Division of the Chemical Society 16
(1979) 203e208.[56] J.H. Tang, F. Luan, X.G. Chen, Bioorganic & Medicinal Chemistry 14 (2006)
3210e3217.[57] H.J. Phillips, E.J. Terryberry, Cell Research 13 (1957) 341e347.[58] T. Kosta, T. Maryama,M. Otagiri, Pharmaceutical Research 14 (1997) 1607e1612.[59] K.J. Obserhausen, J.F. Richardson, R.M. Buchanan, J.K. McCusker,
D.N. Hendrickson, J.M. Latour, Inorganic Chemistry 30 (1991) 1357e1365.[60] A. .Kumar, A. Mitra, A.K. . Ajay, M.K. . Bhat, C.P. . Rao, Journal of Chemical
Sciences 124 (2012) 1217e1228.[61] P. Sathyadevi, P. Krishnamoorthy, R.R. Butorac, A.H. Cowley, N.S.P. Bhuvanesh,
N. Dharmaraj, Dalton Transactions 40 (2011) 9690e9702.[62] FRAMBO v. 4.1.05 Program for Data Collection on Area DetectorsBRUKER-
Nonius Inc., 5465 East Cheryl Parkway, Madison, WI 53711e5373 USA.[63] G.M. Sheldrick, Cell_Now (Version 2008/1): Program for Obtaining Unit Cell
Constants From Single Crystal Data: University of Gttingen, Germany.[64] APEX2 Program for Data Collection and Integration on Area Detectors
BRUKER AXS Inc., 5465 East Cheryl Parkway, Madison, WI 53711-5373 USA.[65] G.M. Sheldrick, SADABS (Version 2008/1): Program for Absorption Correction
for Data from Area Detector Frames, University of Gttingen, 2008.[66] G.M. Sheldrick, Acta Crystallographica A64 (2008) 112e122.[67] P. Krishnamoorthy, P. Sathyadevi, R.R. Butorac, A.H. Cowley, N.S.P. Bhuvanesh,
N. Dharmaraj, Dalton Transactions 41 (2012) 4423e4436.[68] J. Marmur, Journal of Molecular Biology 3 (1961) 208e212.[69] M.E. Reichmann, S.A. Rice, C.A. Thomas, P.J. Doty, Journal of the American
Chemical Society 76 (1954) 3047e3053.[70] Y. Hua, Y. Liu, J. Wang, X. Xiao, S. Qu, Journal of Pharmaceutical and
Biomedical Analysis 36 (2004) 915e919.[71] N. Shahabadi, M. Maghsudi, Journal of Molecular Structure 929 (2009) 193e
199.[72] D.T. Vistica, P. Skehan, D.A. Scudiero, A. Monks, A. Pittman, M.R. Boyd, Cancer
Research 51 (1991) 2515e2520.[73] B.J. Hathaway, D.E. Billing, Coordination Chemistry Reviews 5 (1970) 143e
207.
M. Alagesan et al. / European Journal of Medicinal Chemistry 78 (2014) 281e293 293
mailto:deposit@ccdc.cam.ac.ukhttp://www.ccdc.cam.ac.uk/http://www.ccdc.cam.ac.uk/http://refhub.elsevier.com/S0223-5234(14)00256-6/sref1http://refhub.elsevier.com/S0223-5234(14)00256-6/sref1http://refhub.elsevier.com/S0223-5234(14)00256-6/sref2http://refhub.elsevier.com/S0223-5234(14)00256-6/sref2http://refhub.elsevier.com/S0223-5234(14)00256-6/sref3http://refhub.elsevier.com/S0223-5234(14)00256-6/sref3http://refhub.elsevier.com/S0223-5234(14)00256-6/sref3http://refhub.elsevier.com/S0223-5234(14)00256-6/sref3http://refhub.elsevier.com/S0223-5234(14)00256-6/sref4http://refhub.elsevier.com/S0223-5234(14)00256-6/sref4http://refhub.elsevier.com/S0223-5234(14)00256-6/sref4http://refhub.elsevier.com/S0223-5234(14)00256-6/sref5http://refhub.elsevier.com/S0223-5234(14)00256-6/sref5http://refhub.elsevier.com/S0223-5234(14)00256-6/sref5http://refhub.elsevier.com/S0223-5234(14)00256-6/sref5http://refhub.elsevier.com/S0223-5234(14)00256-6/sref6http://refhub.elsevier.com/S0223-5234(14)00256-6/sref6http://refhub.elsevier.com/S0223-5234(14)00256-6/sref6http://refhub.elsevier.com/S0223-5234(14)00256-6/sref7http://refhub.elsevier.com/S0223-5234(14)00256-6/sref7http://refhub.elsevier.com/S0223-5234(14)00256-6/sref8http://refhub.elsevier.com/S0223-5234(14)00256-6/sref8http://refhub.elsevier.com/S0223-5234(14)00256-6/sref8http://refhub.elsevier.com/S0223-5234(14)00256-6/sref9http://refhub.elsevier.com/S0223-5234(14)00256-6/sref9http://refhub.elsevier.com/S0223-5234(14)00256-6/sref9http://refhub.elsevier.com/S0223-5234(14)00256-6/sref10http://refhub.elsevier.com/S0223-5234(14)00256-6/sref10http://refhub.elsevier.com/S0223-5234(14)00256-6/sref10http://refhub.elsevier.com/S0223-5234(14)00256-6/sref11http://refhub.elsevier.com/S0223-5234(14)00256-6/sref11http://refhub.elsevier.com/S0223-5234(14)00256-6/sref11http://refhub.elsevier.com/S0223-5234(14)00256-6/sref12http://refhub.elsevier.com/S0223-5234(14)00256-6/sref12http://refhub.elsevier.com/S0223-5234(14)00256-6/sref12http://refhub.elsevier.com/S0223-5234(14)00256-6/sref13http://refhub.elsevier.com/S0223-5234(14)00256-6/sref13http://refhub.elsevier.com/S0223-5234(14)00256-6/sref13http://refhub.elsevier.com/S0223-5234(14)00256-6/sref13http://refhub.elsevier.com/S0223-5234(14)00256-6/sref14http://refhub.elsevier.com/S0223-5234(14)00256-6/sref14http://refhub.elsevier.com/S0223-5234(14)00256-6/sref14http://refhub.elsevier.com/S0223-5234(14)00256-6/sref15http://refhub.elsevier.com/S0223-5234(14)00256-6/sref15http://refhub.elsevier.com/S0223-5234(14)00256-6/sref15http://refhub.elsevier.com/S0223-5234(14)00256-6/sref15http://refhub.elsevier.com/S0223-5234(14)00256-6/sref16http://refhub.elsevier.com/S0223-5234(14)00256-6/sref16http://refhub.elsevier.com/S0223-5234(14)00256-6/sref16http://refhub.elsevier.com/S0223-5234(14)00256-6/sref16http://refhub.elsevier.com/S0223-5234(14)00256-6/sref17http://refhub.elsevier.com/S0223-5234(14)00256-6/sref17http://refhub.elsevier.com/S0223-5234(14)00256-6/sref17http://refhub.elsevier.com/S0223-5234(14)00256-6/sref17http://refhub.elsevier.com/S0223-5234(14)00256-6/sref17http://refhub.elsevier.com/S0223-5234(14)00256-6/sref18http://refhub.elsevier.com/S0223-5234(14)00256-6/sref18http://refhub.elsevier.com/S0223-5234(14)00256-6/sref18http://refhub.elsevier.com/S0223-5234(14)00256-6/sref19http://refhub.elsevier.com/S0223-5234(14)00256-6/sref19http://refhub.elsevier.com/S0223-5234(14)00256-6/sref19http://refhub.elsevier.com/S0223-5234(14)00256-6/sref20http://refhub.elsevier.com/S0223-5234(14)00256-6/sref20http://refhub.elsevier.com/S0223-5234(14)00256-6/sref20http://refhub.elsevier.com/S0223-5234(14)00256-6/sref21http://refhub.elsevier.com/S0223-5234(14)00256-6/sref21http://refhub.elsevier.com/S0223-5234(14)00256-6/sref22http://refhub.elsevier.com/S0223-5234(14)00256-6/sref22http://refhub.elsevier.com/S0223-5234(14)00256-6/sref22http://refhub.elsevier.com/S0223-5234(14)00256-6/sref23http://refhub.elsevier.com/S0223-5234(14)00256-6/sref23http://refhub.elsevier.com/S0223-5234(14)00256-6/sref23http://refhub.elsevier.com/S0223-5234(14)00256-6/sref24http://refhub.elsevier.com/S0223-5234(14)00256-6/sref24http://refhub.elsevier.com/S0223-5234(14)00256-6/sref24http://refhub.elsevier.com/S0223-5234(14)00256-6/sref25http://refhub.elsevier.com/S0223-5234(14)00256-6/sref25http://refhub.elsevier.com/S0223-5234(14)00256-6/sref25http://refhub.elsevier.com/S0223-5234(14)00256-6/sref26http://refhub.elsevier.com/S0223-5234(14)00256-6/sref26http://refhub.elsevier.com/S0223-5234(14)00256-6/sref26http://refhub.elsevier.com/S0223-5234(14)00256-6/sref27http://refhub.elsevier.com/S0223-5234(14)00256-6/sref27http://refhub.elsevier.com/S0223-5234(14)00256-6/sref27http://refhub.elsevier.com/S0223-5234(14)00256-6/sref27http://refhub.elsevier.com/S0223-5234(14)00256-6/sref28http://refhub.elsevier.com/S0223-5234(14)00256-6/sref28http://refhub.elsevier.com/S0223-5234(14)00256-6/sref28http://refhub.elsevier.com/S0223-5234(14)00256-6/sref29http://refhub.elsevier.com/S0223-5234(14)00256-6/sref29http://refhub.elsevier.com/S0223-5234(14)00256-6/sref30http://refhub.elsevier.com/S0223-5234(14)00256-6/sref30http://refhub.elsevier.com/S0223-5234(14)00256-6/sref31http://refhub.elsevier.com/S0223-5234(14)00256-6/sref31http://refhub.elsevier.com/S0223-5234(14)00256-6/sref31http://refhub.elsevier.com/S0223-5234(14)00256-6/sref31http://refhub.elsevier.com/S0223-5234(14)00256-6/sref32http://refhub.elsevier.com/S0223-5234(14)00256-6/sref32http://refhub.elsevier.com/S0223-5234(14)00256-6/sref32http://refhub.elsevier.com/S0223-5234(14)00256-6/sref33http://refhub.elsevier.com/S0223-5234(14)00256-6/sref33http://refhub.elsevier.com/S0223-5234(14)00256-6/sref33http://refhub.elsevier.com/S0223-5234(14)00256-6/sref34http://refhub.elsevier.com/S0223-5234(14)00256-6/sref34http://refhub.elsevier.com/S0223-5234(14)00256-6/sref34http://refhub.elsevier.com/S0223-5234(14)00256-6/sref35http://refhub.elsevier.com/S0223-5234(14)00256-6/sref35http://refhub.elsevier.com/S0223-5234(14)00256-6/sref35http://refhub.elsevier.com/S0223-5234(14)00256-6/sref36http://refhub.elsevier.com/S0223-5234(14)00256-6/sref36http://refhub.elsevier.com/S0223-5234(14)00256-6/sref36http://refhub.elsevier.com/S0223-5234(14)00256-6/sref37http://refhub.elsevier.com/S0223-5234(14)00256-6/sref37http://refhub.elsevier.com/S0223-5234(14)00256-6/sref37http://refhub.elsevier.com/S0223-5234(14)00256-6/sref38http://refhub.elsevier.com/S0223-5234(14)00256-6/sref38http://refhub.elsevier.com/S0223-5234(14)00256-6/sref38http://refhub.elsevier.com/S0223-5234(14)00256-6/sref38http://refhub.elsevier.com/S0223-5234(14)00256-6/sref39http://refhub.elsevier.com/S0223-5234(14)00256-6/sref39http://refhub.elsevier.com/S0223-5234(14)00256-6/sref40http://refhub.elsevier.com/S0223-5234(14)00256-6/sref40http://refhub.elsevier.com/S0223-5234(14)00256-6/sref40http://refhub.elsevier.com/S0223-5234(14)00256-6/sref41http://refhub.elsevier.com/S0223-5234(14)00256-6/sref41http://refhub.elsevier.com/S0223-5234(14)00256-6/sref41http://refhub.elsevier.com/S0223-5234(14)00256-6/sref42http://refhub.elsevier.com/S0223-5234(14)00256-6/sref42http://refhub.elsevier.com/S0223-5234(14)00256-6/sref42http://refhub.elsevier.com/S0223-5234(14)00256-6/sref43http://refhub.elsevier.com/S0223-5234(14)00256-6/sref43http://refhub.elsevier.com/S0223-5234(14)00256-6/sref43http://refhub.elsevier.com/S0223-5234(14)00256-6/sref44http://refhub.elsevier.com/S0223-5234(14)00256-6/sref44http://refhub.elsevier.com/S0223-5234(14)00256-6/sref45http://refhub.elsevier.com/S0223-5234(14)00256-6/sref45http://refhub.elsevier.com/S0223-5234(14)00256-6/sref45http://refhub.elsevier.com/S0223-5234(14)00256-6/sref46http://refhub.elsevier.com/S0223-5234(14)00256-6/sref46http://refhub.elsevier.com/S0223-5234(14)00256-6/sref46http://refhub.elsevier.com/S0223-5234(14)00256-6/sref47http://refhub.elsevier.com/S0223-5234(14)00256-6/sref47http://refhub.elsevier.com/S0223-5234(14)00256-6/sref47http://refhub.elsevier.com/S0223-5234(14)00256-6/sref48http://refhub.elsevier.com/S0223-5234(14)00256-6/sref48http://refhub.elsevier.com/S0223-5234(14)00256-6/sref48http://refhub.elsevier.com/S0223-5234(14)00256-6/sref49http://refhub.elsevier.com/S0223-5234(14)00256-6/sref49http://refhub.elsevier.com/S0223-5234(14)00256-6/sref49http://refhub.elsevier.com/S0223-5234(14)00256-6/sref50http://refhub.elsevier.com/S0223-5234(14)00256-6/sref50http://refhub.elsevier.com/S0223-5234(14)00256-6/sref50http://refhub.elsevier.com/S0223-5234(14)00256-6/sref51http://refhub.elsevier.com/S0223-5234(14)00256-6/sref51http://refhub.elsevier.com/S0223-5234(14)00256-6/sref52http://refhub.elsevier.com/S0223-5234(14)00256-6/sref52http://refhub.elsevier.com/S0223-5234(14)00256-6/sref52http://refhub.elsevier.com/S0223-5234(14)00256-6/sref53http://refhub.elsevier.com/S0223-5234(14)00256-6/sref53http://refhub.elsevier.com/S0223-5234(14)00256-6/sref54http://refhub.elsevier.com/S0223-5234(14)00256-6/sref54http://refhub.elsevier.com/S0223-5234(14)00256-6/sref55http://refhub.elsevier.com/S0223-5234(14)00256-6/sref55http://refhub.elsevier.com/S0223-5234(14)00256-6/sref55http://refhub.elsevier.com/S0223-5234(14)00256-6/sref56http://refhub.elsevier.com/S0223-5234(14)00256-6/sref56http://refhub.elsevier.com/S0223-5234(14)00256-6/sref56http://refhub.elsevier.com/S0223-5234(14)00256-6/sref56http://refhub.elsevier.com/S0223-5234(14)00256-6/sref57http://refhub.elsevier.com/S0223-5234(14)00256-6/sref57http://refhub.elsevier.com/S0223-5234(14)00256-6/sref58http://refhub.elsevier.com/S0223-5234(14)00256-6/sref58http://refhub.elsevier.com/S0223-5234(14)00256-6/sref58http://refhub.elsevier.com/S0223-5234(14)00256-6/sref59http://refhub.elsevier.com/S0223-5234(14)00256-6/sref59http://refhub.elsevier.com/S0223-5234(14)00256-6/sref59http://refhub.elsevier.com/S0223-5234(14)00256-6/sref60http://refhub.elsevier.com/S0223-5234(14)00256-6/sref60http://refhub.elsevier.com/S0223-5234(14)00256-6/sref60http://refhub.elsevier.com/S0223-5234(14)00256-6/sref61http://refhub.elsevier.com/S0223-5234(14)00256-6/sref61http://refhub.elsevier.com/S0223-5234(14)00256-6/sref61http://refhub.elsevier.com/S0223-5234(14)00256-6/sref63http://refhub.elsevier.com/S0223-5234(14)00256-6/sref63http://refhub.elsevier.com/S0223-5234(14)00256-6/sref64http://refhub.elsevier.com/S0223-5234(14)00256-6/sref64http://refhub.elsevier.com/S0223-5234(14)00256-6/sref64http://refhub.elsevier.com/S0223-5234(14)00256-6/sref65http://refhub.elsevier.com/S0223-5234(14)00256-6/sref65http://refhub.elsevier.com/S0223-5234(14)00256-6/sref65http://refhub.elsevier.com/S0223-5234(14)00256-6/sref66http://refhub.elsevier.com/S0223-5234(14)00256-6/sref66http://refhub.elsevier.com/S0223-5234(14)00256-6/sref67http://refhub.elsevier.com/S0223-5234(14)00256-6/sref67http://refhub.elsevier.com/S0223-5234(14)00256-6/sref67http://refhub.elsevier.com/S0223-5234(14)00256-6/sref67http://refhub.elsevier.com/S0223-5234(14)00256-6/sref68http://refhub.elsevier.com/S0223-5234(14)00256-6/sref68http://refhub.elsevier.com/S0223-5234(14)00256-6/sref68http://refhub.elsevier.com/S0223-5234(14)00256-6/sref69http://refhub.elsevier.com/S0223-5234(14)00256-6/sref69http://refhub.elsevier.com/S0223-5234(14)00256-6/sref69http://refhub.elsevier.com/S0223-5234(14)00256-6/sref70http://refhub.elsevier.com/S0223-5234(14)00256-6/sref70http://refhub.elsevier.com/S0223-5234(14)00256-6/sref70http://refhub.elsevier.com/S0223-5234(14)00256-6/sref71http://refhub.elsevier.com/S0223-5234(14)00256-6/sref71http://refhub.elsevier.com/S0223-5234(14)00256-6/sref71http://refhub.elsevier.com/S0223-5234(14)00256-6/sref71http://refhub.elsevier.com/S0223-5234(14)00256-6/sref71http://refhub.elsevier.com/S0223-5234(14)00256-6/sref70http://refhub.elsevier.com/S0223-5234(14)00256-6/sref70http://refhub.elsevier.com/S0223-5234(14)00256-6/sref70http://refhub.elsevier.com/S0223-5234(14)00256-6/sref69http://refhub.elsevier.com/S0223-5234(14)00256-6/sref69http://refhub.elsevier.com/S0223-5234(14)00256-6/sref68http://refhub.elsevier.com/S0223-5234(14)00256-6/sref68http://refhub.elsevier.com/S0223-5234(14)00256-6/sref68http://refhub.elsevier.com/S0223-5234(14)00256-6/sref67http://refhub.elsevier.com/S0223-5234(14)00256-6/sref67http://refhub.elsevier.com/S0223-5234(14)00256-6/sref67http://refhub.elsevier.com/S0223-5234(14)00256-6/sref66http://refhub.elsevier.com/S0223-5234(14)00256-6/sref66http://refhub.elsevier.com/S0223-5234(14)00256-6/sref65http://refhub.elsevier.com/S0223-5234(14)00256-6/sref65http://refhub.elsevier.com/S0223-5234(14)00256-6/sref65http://refhub.elsevier.com/S0223-5234(14)00256-6/sref64http://refhub.elsevier.com/S0223-5234(14)00256-6/sref64http://refhub.elsevier.com/S0223-5234(14)00256-6/sref63http://refhub.elsevier.com/S0223-5234(14)00256-6/sref63http://refhub.elsevier.com/S0223-5234(14)00256-6/sref61http://refhub.elsevier.com/S0223-5234(14)00256-6/sref61http://refhub.elsevier.com/S0223-5234(14)00256-6/sref61http://refhub.elsevier.com/S0223-5234(14)00256-6/sref60http://refhub.elsevier.com/S0223-5234(14)00256-6/sref60http://refhub.elsevier.com/S0223-5234(14)00256-6/sref60http://refhub.elsevier.com/S0223-5234(14)00256-6/sref59http://refhub.elsevier.com/S0223-5234(14)00256-6/sref59http://refhub.elsevier.com/S0223-5234(14)00256-6/sref59http://refhub.elsevier.com/S0223-5234(14)00256-6/sref58http://refhub.elsevier.com/S0223-5234(14)00256-6/sref58http://refhub.elsevier.com/S0223-5234(14)00256-6/sref57http://refhub.elsevier.com/S0223-5234(14)00256-6/sref57http://refhub.elsevier.com/S0223-5234(14)00256-6/sref56http://refhub.elsevier.com/S0223-5234(14)00256-6/sref56http://refhub.elsevier.com/S0223-5234(14)00256-6/sref56http://refhub.elsevier.com/S0223-5234(14)00256-6/sref55http://refhub.elsevier.com/S0223-5234(14)00256-6/sref55http://refhub.elsevier.com/S0223-5234(14)00256-6/sref55http://refhub.elsevier.com/S0223-5234(14)00256-6/sref54http://refhub.elsevier.com/S0223-5234(14)00256-6/sref54http://refhub.elsevier.com/S0223-5234(14)00256-6/sref53http://refhub.elsevier.com/S0223-5234(14)00256-6/sref53http://refhub.elsevier.com/S0223-5234(14)00256-6/sref52http://refhub.elsevier.com/S0223-5234(14)00256-6/sref52http://refhub.elsevier.com/S0223-5234(14)00256-6/sref52http://refhub.elsevier.com/S0223-5234(14)00256-6/sref51http://refhub.elsevier.com/S0223-5234(14)00256-6/sref51http://refhub.elsevier.com/S0223-5234(14)00256-6/sref50http://refhub.elsevier.com/S0223-5234(14)00256-6/sref50http://refhub.elsevier.com/S0223-5234(14)00256-6/sref50http://refhub.elsevier.com/S0223-5234(14)00256-6/sref49http://refhub.elsevier.com/S0223-5234(14)00256-6/sref49http://refhub.elsevier.com/S0223-5234(14)00256-6/sref49http://refhub.elsevier.com/S0223-5234(14)00256-6/sref48http://refhub.elsevier.com/S0223-5234(14)00256-6/sref48http://refhub.elsevier.com/S0223-5234(14)00256-6/sref48http://refhub.elsevier.com/S0223-5234(14)00256-6/sref47http://refhub.elsevier.com/S0223-5234(14)00256-6/sref47http://refhub.elsevier.com/S0223-5234(14)00256-6/sref47http://refhub.elsevier.com/S0223-5234(14)00256-6/sref46http://refhub.elsevier.com/S0223-5234(14)00256-6/sref46http://refhub.elsevier.com/S0223-5234(14)00256-6/sref46http://refhub.elsevier.com/S0223-5234(14)00256-6/sref45http://refhub.elsevier.com/S0223-5234(14)00256-6/sref45http://refhub.elsevier.com/S0223-5234(14)00256-6/sref45http://refhub.elsevier.com/S0223-5234(14)00256-6/sref44http://refhub.elsevier.com/S0223-5234(14)00256-6/sref44http://refhub.elsevier.com/S0223-5234(14)00256-6/sref43http://refhub.elsevier.com/S0223-5234(14)00256-6/sref43http://refhub.elsevier.com/S0223-5234(14)00256-6/sref43http://refhub.elsevier.com/S0223-5234(14)00256-6/sref42http://refhub.elsevier.com/S0223-5234(14)00256-6/sref42http://refhub.elsevier.com/S0223-5234(14)00256-6/sref42http://refhub.elsevier.com/S0223-5234(14)00256-6/sref41http://refhub.elsevier.com/S0223-5234(14)00256-6/sref41http://refhub.elsevier.com/S0223-5234(14)00256-6/sref41http://refhub.elsevier.com/S0223-5234(14)00256-6/sref40http://refhub.elsevier.com/S0223-5234(14)00256-6/sref40http://refhub.elsevier.com/S0223-5234(14)00256-6/sref40http://refhub.elsevier.com/S0223-5234(14)00256-6/sref39http://refhub.elsevier.com/S0223-5234(14)00256-6/sref39http://refhub.elsevier.com/S0223-5234(14)00256-6/sref38http://refhub.elsevier.com/S0223-5234(14)00256-6/sref38http://refhub.elsevier.com/S0223-5234(14)00256-6/sref38http://refhub.elsevier.com/S0223-5234(14)00256-6/sref37http://refhub.elsevier.com/S0223-5234(14)00256-6/sref37http://refhub.elsevier.com/S0223-5234(14)00256-6/sref37http://refhub.elsevier.com/S0223-5234(14)00256-6/sref36http://refhub.elsevier.com/S0223-5234(14)00256-6/sref36http://refhub.elsevier.com/S0223-5234(14)00256-6/sref36http://refhub.elsevier.com/S0223-5234(14)00256-6/sref35http://refhub.elsevier.com/S0223-5234(14)00256-6/sref35http://refhub.elsevier.com/S0223-5234(14)00256-6/sref35http://refhub.elsevier.com/S0223-5234(14)00256-6/sref34http://refhub.elsevier.com/S0223-5234(14)00256-6/sref34http://refhub.elsevier.com/S0223-5234(14)00256-6/sref34http://refhub.elsevier.com/S0223-5234(14)00256-6/sref33http://refhub.elsevier.com/S0223-5234(14)00256-6/sref33http://refhub.elsevier.com/S0223-5234(14)00256-6/sref33http://refhub.elsevier.com/S0223-5234(14)00256-6/sref32http://refhub.elsevier.com/S0223-5234(14)00256-6/sref32http://refhub.elsevier.com/S0223-5234(14)00256-6/sref32http://refhub.elsevier.com/S0223-5234(14)00256-6/sref31http://refhub.elsevier.com/S0223-5234(14)00256-6/sref31http://refhub.elsevier.com/S0223-5234(14)00256-6/sref31http://refhub.elsevier.com/S0223-5234(14)00256-6/sref30http://refhub.elsevier.com/S0223-5234(14)00256-6/sref30http://refhub.elsevier.com/S0223-5234(14)00256-6/sref29http://refhub.elsevier.com/S0223-5234(14)00256-6/sref29http://refhub.elsevier.com/S0223-5234(14)00256-6/sref28http://refhub.elsevier.com/S0223-5234(14)00256-6/sref28http://refhub.elsevier.com/S0223-5234(14)00256-6/sref28http://refhub.elsevier.com/S0223-5234(14)00256-6/sref27http://refhub.elsevier.com/S0223-5234(14)00256-6/sref27http://refhub.elsevier.com/S0223-5234(14)00256-6/sref27http://refhub.elsevier.com/S0223-5234(14)00256-6/sref26http://refhub.elsevier.com/S0223-5234(14)00256-6/sref26http://refhub.elsevier.com/S0223-5234(14)00256-6/sref26http://refhub.elsevier.com/S0223-5234(14)00256-6/sref25http://refhub.elsevier.com/S0223-5234(14)00256-6/sref25http://refhub.elsevier.com/S0223-5234(14)00256-6/sref25http://refhub.elsevier.com/S0223-5234(14)00256-6/sref24http://refhub.elsevier.com/S0223-5234(14)00256-6/sref24http://refhub.elsevier.com/S0223-5234(14)00256-6/sref24http://refhub.elsevier.com/S0223-5234(14)00256-6/sref23http://refhub.elsevier.com/S0223-5234(14)00256-6/sref23http://refhub.elsevier.com/S0223-5234(14)00256-6/sref23http://refhub.elsevier.com/S0223-5234(14)00256-6/sref22http://refhub.elsevier.com/S0223-5234(14)00256-6/sref22http://refhub.elsevier.com/S0223-5234(14)00256-6/sref22http://refhub.elsevier.com/S0223-5234(14)00256-6/sref21http://refhub.elsevier.com/S0223-5234(14)00256-6/sref21http://refhub.elsevier.com/S0223-5234(14)00256-6/sref20http://refhub.elsevier.com/S0223-5234(14)00256-6/sref20http://refhub.elsevier.com/S0223-5234(14)00256-6/sref20http://refhub.elsevier.com/S0223-5234(14)00256-6/sref19http://refhub.elsevier.com/S0223-5234(14)00256-6/sref19http://refhub.elsevier.com/S0223-5234(14)00256-6/sref19http://refhub.elsevier.com/S0223-5234(14)00256-6/sref18http://refhub.elsevier.com/S0223-5234(14)00256-6/sref18http://refhub.elsevier.com/S0223-5234(14)00256-6/sref18http://refhub.elsevier.com/S0223-5234(14)00256-6/sref17http://refhub.elsevier.com/S0223-5234(14)00256-6/sref17http://refhub.elsevier.com/S0223-5234(14)00256-6/sref17http://refhub.elsevier.com/S0223-5234(14)00256-6/sref17http://refhub.elsevier.com/S0223-5234(14)00256-6/sref16http://refhub.elsevier.com/S0223-5234(14)00256-6/sref16http://refhub.elsevier.com/S0223-5234(14)00256-6/sref16http://refhub.elsevier.com/S0223-5234(14)00256-6/sref15http://refhub.elsevier.com/S0223-5234(14)00256-6/sref15http://refhub.elsevier.com/S0223-5234(14)00256-6/sref15http://refhub.elsevier.com/S0223-5234(14)00256-6/sref14http://refhub.elsevier.com/S0223-5234(14)00256-6/sref14http://refhub.elsevier.com/S0223-5234(14)00256-6/sref14http://refhub.elsevier.com/S0223-5234(14)00256-6/sref13http://refhub.elsevier.com/S0223-5234(14)00256-6/sref13http://refhub.elsevier.com/S0223-5234(14)00256-6/sref13http://refhub.elsevier.com/S0223-5234(14)00256-6/sref13http://refhub.elsevier.com/S0223-5234(14)00256-6/sref12http://refhub.elsevier.com/S0223-5234(14)00256-6/sref12http://refhub.elsevier.com/S0223-5234(14)00256-6/sref12http://refhub.elsevier.com/S0223-5234(14)00256-6/sref11http://refhub.elsevier.com/S0223-5234(14)00256-6/sref11http://refhub.elsevier.com/S0223-5234(14)00256-6/sref10http://refhub.elsevier.com/S0223-5234(14)00256-6/sref10http://refhub.elsevier.com/S0223-5234(14)00256-6/sref10http://refhub.elsevier.com/S0223-5234(14)00256-6/sref9http://refhub.elsevier.com/S0223-5234(14)00256-6/sref9http://refhub.elsevier.com/S0223-5234(14)00256-6/sref9http://refhub.elsevier.com/S0223-5234(14)00256-6/sref8http://refhub.elsevier.com/S0223-5234(14)00256-6/sref8http://refhub.elsevier.com/S0223-5234(14)00256-6/sref7http://refhub.elsevier.com/S0223-5234(14)00256-6/sref7http://refhub.elsevier.com/S0223-5234(14)00256-6/sref6http://refhub.elsevier.com/S0223-5234(14)00256-6/sref6http://refhub.elsevier.com/S0223-5234(14)00256-6/sref6http://refhub.elsevier.com/S0223-5234(14)00256-6/sref5http://refhub.elsevier.com/S0223-5234(14)00256-6/sref5http://refhub.elsevier.com/S0223-5234(14)00256-6/sref5http://refhub.elsevier.com/S0223-5234(14)00256-6/sref4http://refhub.elsevier.com/S0223-5234(14)00256-6/sref4http://refhub.elsevier.com/S0223-5234(14)00256-6/sref4http://refhub.elsevier.com/S0223-5234(14)00256-6/sref3http://refhub.elsevier.com/S0223-5234(14)00256-6/sref3http://refhub.elsevier.com/S0223-5234(14)00256-6/sref3http://refhub.elsevier.com/S0223-5234(14)00256-6/sref2http://refhub.elsevier.com/S0223-5234(14)00256-6/sref2http://refhub.elsevier.com/S0223-5234(14)00256-6/sref1http://refhub.elsevier.com/S0223-5234(14)00256-6/sref1http://www.ccdc.cam.ac.uk/http://www.ccdc.cam.ac.uk/mailto:deposit@ccdc.cam.ac.uk
top related