Mixed-ligand copper(I) halide complexes bearing 4,5-bis(diphenylphosphano)- 9,9-dimethyl-xanthene and N-methylbenzothiazole-2-thione: Synthesis, structures, luminescence and antibacterial activity mediated by DNA and membrane damage

Article abstract of DOI:10.1016/j.poly.2014.02.002

The 1:1 M-ratio reaction between copper(I) bromide or iodide and 4,5-bis(diphenylphosphano)-9,9-dimethyl-xanthene (xantphos) in acetonitrile results in the formation of [CuX(xantphos)] (X = Br, I), which further reacts with N-methylbenzothiazole-2-thione (mbtt) to afford the mononuclear mixed-ligand complexes [CuX(xantphos)(mbtt)]. The molecular structures of the complexes, established by single-crystal X-ray diffraction, feature a distorted tetrahedral geometry around the metal center, with the diphosphane acting as a chelate. The new compounds are strongly emissive in the solid state at room temperature. The complexes were also screened for antibacterial activity and their ability to interact with CT-DNA in vitro and to produce reactive oxygen species (ROS).

Full text of DOI:10.1016/j.poly.2014.02.002

Polyhedron 72 (2014) 122–129
Contents lists available at ScienceDirect
Polyhedron
journal homepage: www.elsevier.com/locate/poly
Mixed-ligand copper(I) halide complexes bearing
4,5-bis(diphenylphosphano)-9,9-dimethyl-xanthene and
N-methylbenzothiazole-2-thione: Synthesis, structures, luminescence
and antibacterial activity mediated by DNA and membrane damage
c
a,
O. Evangelinou a, A.G. Hatzidimitriou a, E. Velali b, A.A. Pantazaki b, , N. Voulgarakis , P. Aslanidis
⇑
⇑
a Laboratory of Inorganic Chemistry, Department of Chemistry, Aristotle University of Thessaloniki, GR-541 24 Thessaloniki, Greece
b Laboratory of Biochemistry, Department of Chemistry, Aristotle University of Thessaloniki, GR-541 24 Thessaloniki, Greece
c Department of Logistics, Alexander Technological Educational Institute of Thessaloniki, GR-60100 Katerini, Greece
a r t i c l e i n f o
a b s t r a c t
Article history:
The 1:1 M-ratio reaction between copper(I) bromide or iodide and 4,5-bis(diphenylphosphano)-9,9-
dimethyl-xanthene (xantphos) in acetonitrile results in the formation of [CuX(xantphos)] (X = Br, I),
which further reacts with N-methylbenzothiazole-2-thione (mbtt) to afford the mononuclear mixed-
ligand complexes [CuX(xantphos)(mbtt)]. The molecular structures of the complexes, established by
single-crystal X-ray diffraction, feature a distorted tetrahedral geometry around the metal center, with
the diphosphane acting as a chelate. The new compounds are strongly emissive in the solid state at room
temperature. The complexes were also screened for antibacterial activity and their ability to interact with
CT-DNA in vitro and to produce reactive oxygen species (ROS).
Received 18 November 2013
Accepted 2 February 2014
Available online 11 February 2014
Keywords:
Copper(I)
N-Methylbenzothiazole-2-thione
Luminescence
Crystal structures
Antimicrobial activity
DNA interaction
Ó 2014 Elsevier Ltd. All rights reserved.
Reactive oxygen species (ROS)
1. Introduction
been tested in recent years towards pharmacological activities,
such as antiviral, antibacterial or antifungal [8–13].
Owing to the favourable soft acid–soft base interaction, the
chemistry of the univalent coinage metal ions is largely based upon
coordination by ligands containing P and/or S donor atoms. A vast
amount of copper(I) and silver(I) complexes with tertiary phos-
phanes as ligands has been accumulated over the years, many of
which were employed in catalytic processes [1–3], while some of
them were even found to exhibit antitumor activity [4]. As for
the S-donor ligands, substantial interest has been given to the ex-
tended family of heterocyclic thiones, which are regarded as a very
promising starting point for biological studies because of their
close structural resemblance to thioamides, and due to the fact that
many thione-ligated metal complexes are known to have relevance
to biological systems [5–7]. In this respect, several imidazole-,
thiazole-, thiadiazole-, oxazole-, uracil- and hydantoin-based thio-
amide derivatives as well as their transition metal complexes have
Based on the rich coordination chemistry produced so far for
each of these two classes of ligands, we have long been engaged
in the investigation of mixed-ligand copper(I) and silver(I) com-
plexes bearing both bulky triaryl phosphanes and heterocyclic thi-
one ligands aiming to gain insight into the interplay between the
ligand’s characteristics and the structural diversity observed [14].
In addition, having recognized that current interest in copper com-
plexes largely focuses on their potential use as antimicrobial, anti-
viral, anti-inflammatory and antitumor agents, we recently turned
our attention to the biochemical properties of these compounds,
reporting the synthesis and characterization of copper(I) halide
complexes of N-methylbenzothiazole-2-thione (mbtt, Scheme 1)
from type [CuX(mbtt)2]2 and [CuX(mbtt)(PPh3)2] and investigating
their antibacterial activity and their interaction with CT-dsDNA
[15]. Note that the vast majority of the hitherto tested copper-
based compounds are copper(II) complexes, whereas respective
studies on the copper(I) ones are relatively few and essentially lim-
ited to derivatives bearing ligands capable of stabilizing the low
oxidation state of the metal ion in aqueous media [16,17]. On the
ground of our very promising findings, and because of the fact that
⇑
Corresponding authors.
E-mail addresses: natasa@chem.auth.gr (A.A. Pantazaki), aslanidi@chem.auth.gr
(P. Aslanidis).
http://dx.doi.org/10.1016/j.poly.2014.02.002
0277-5387/Ó 2014 Elsevier Ltd. All rights reserved.

O. Evangelinou et al. / Polyhedron 72 (2014) 122–129
123
measured in open tubes with a STUART scientific instrument and
are uncorrected.
N
S
2.2. Crystal structure determination
S
O
Single crystals of [CuBr(xantphos)(mbtt)] (1) and [CuI(xant-
phos)(mbtt)] (2) suitable for crystal structure analysis were ob-
tained by slow evaporation of their mother liquids at room
temperature. Both crystals were taken from their mother liquor,
mounted in air and covered with epoxy glue. Diffraction measure-
ments were made on a Bruker Kappa APEX II diffractometer
equipped with a triumph monochromator. Data collection (/ and
PPh2
PPh2
Scheme 1. The heterocyclic thione N-methylbenzothiazole-2-thione (mbtt) and the
diphosphane 4,5-bis(diphenylphosphano)-9,9-dimethyl-xanthene (xantphos) used
as ligands.
x
-scans) as well as processing (cell refinement, data reduction
perceptibly stronger antibacterial activity was found for the deriv-
atives bearing the triphenylphosphane co-ligand, we decided to
further extend our studies considering bidentate P-donor ligands
with an already established chelating behavior towards copper(I).
According to our hitherto experience with the use of such
arylphosphanes in the synthesis of mixed-ligand copper(I) halide
complexes bearing heterocyclic thiones, 4,5-bis(diphenylphosp-
hano)-9,9-dimethyl-xanthene (xantphos, Scheme 1), despite of its
more or less configurationally inflexible heteroarene skeleton,
proved to be best designed to chelate to copper(I) and silver(I) both
in a tetrahedral or trigonal coordination environment, with the
coordination number apparently being determined by the nature
of the co-ligands present. In particular, the synthesis and charac-
terization of some tetrahedral complexes of the general formula
[CuBr(xantphos)(thione)] obtained by the treatment of a CuBr/
xantphos intermediate with some neutral heterocyclic thioamides
was reported some years ago [18]. More recently we were able to
isolate and structurally characterize [AgBr(xantphos)] as a mono-
mer with a roughly trigonal environment around the metal centre,
which has been further used as an intermediate for the synthesis of
tetrahedrally coordinated mixed-ligand complexes of type
[AgBr(xantphos)(thione)] [19].
and numerical absorption correction) were performed using the
APEX2 program package [22]. The structures were solved using
SUPERFLIP program [23] and refined by full-matrix least-squares
methods on F2 using the CRYSTALS program package [24]. In both
structures, all non-hydrogen atoms were refined anisotropically.
Hydrogen atoms were either located by difference maps or were
introduced at calculated positions as riding on bonded atoms. Fur-
ther crystallographic details are summarized in Table 1. Details on
the crystallographic studies as well as atomic displacement param-
eters are given as Supporting Information in the form of cif files.
Illustrations were generated using the CAMERON program [25].
2.3. Synthesis of compounds 1 and 2
A suspension of 0.5 mmol of copper(I) halide (49.5 mg for CuCl,
72 mg for CuBr, 95 mg for CuI) in 50 mL of dry acetonitrile was
stirred to give a clear solution which was then treated with solid
4,5-bis(diphenylphosphano)-9,9-dimethyl-xanthene (289.3 mg, 0.5
mmol) added in small portions. After stirring for two hours at
50 °C,
a solution of N-methylbenzothiazole-2-thione (90 mg,
0.5 mmol) dissolved in a small amount (ꢁ20 mL) of methanol
was added and the new reaction mixture was stirred for additional
two hours at 50 °C. Slow evaporation of the clear solution at
ambient afforded a pale yellow microcrystalline solid, which was
filtered off and dried in vacuo.
In this work we present two new luminescent copper(I) com-
plexes of type [CuX(xantphos)(mbtt)] and investigate their anti-
bacterial activity, the in vitro CT-dsDNA damage and the levels of
lipid peroxidation.
It is known that in aerobic microorganisms, the respiration pro-
cess has the inevitable downside of electron leakage from redox
enzymes to oxygen, forming reactive oxygen species (ROS) such
as hydrogen peroxide (H2O2), the superoxide radical (O2ꢀꢀ), and
the hydroxyl radical (OHꢀ). ROS accumulation results many biolog-
ical effects among them lipid peroxidation, and DNA damage
[20,21]. Thus, to investigate the antibacterial mechanism, we have
measured the generation of reactive oxygen species (ROS) in the
presence of these complexes measured as MDA equivalents and
we have examined the in vitro DNA damage in agarose gel
electrophoresis.
2.3.1. [CuBr(xantphos)(mbtt)] (1)
Pale yellow crystals (199 mg, 44%), m.p. 277 °C; Anal. Calc. for
C
47H39NOP2S2CuBr: C, 62.49; H, 4.35; N, 1.55. Found: C, 62.16; H,
4.23; N, 1.48%. IR (cmꢀ1): 3047m, 2954w, 1568m, 1479s, 1456s,
1434vs, 1403vs, 1346vs, 1315s, 1286vs, 1229vs, 1136s, 1097vs,
974s, 879s, 756vs, 741vs, 701vs, 691vs, 576vs, 510vs, 464s. UV–
Vis (kmax, loge): 239 (4.97), 275 (4.74), 322 (4.85).
2.3.2. [CuI(xantphos)(mbtt)]ꢂCH3CN (2)
Pale yellow crystals (203 mg, 41%), m.p. 234 °C; Anal. Calc. for
C49H42N2OP2S2CuI: C, 59.36; H, 4.27; N, 2.83. Found: C, 59.46; H,
4.22; N, 2.86%. IR (cmꢀ1): 3048m, 2968w, 1568m, 1480s, 1460s,
1432vs, 1404vs, 1350vs, 1312s, 1261s, 1226vs, 1140s, 1097vs,
2. Experimental
979s, 875m, 755vs, 745vs, 698vs, 574vs, 510vs, 462s. UV–Vis (kmax
,
2.1. Materials and instrumentation
log ): 242 (5.17), 277 (4.82), 320 (5.03).
e
Commercially available copper(I) bromide and iodide (Merck)
and 9,9-dimethyl-4,5-bis(diphenylphosphano)xanthene (Aldrich)
were used as received while N-methylbenzothiazole-2-thione (Al-
drich) was re-crystallized from hot ethanol prior to its use. All sol-
vents were purified by respective suitable methods and allowed to
stand over molecular sieves. Infra-red spectra in the region of
4000–200 cmꢀ1 were obtained in KBr discs with a Nicolet FT-IR
6700 spectrophotometer, while a Shimadzu 160A spectrophotom-
eter and a Hitachi F-7000 fluorescence spectrometer were used to
obtain the electronic absorption (UV–Vis) and emission/excitation
spectra respectively. Melting points of the compounds were
2.4. Materials and methods for biological tests
2.4.1. Materials
Agarose was purchased from BRL. Tryptone and yeast extract
were purchased from Oxoid (Unipath Ltd., Hampshire, UK). All
other chemicals were obtained from Sigma. Nucleic acids: Native
DNA (dsDNA) type I, highly polymerized from calf thymus gland
was purchased from Sigma (D-1501). The intercalative dye ethi-
dium bromide (EthBr), were purchased from Sigma. Experiments
were carried out in 50 mM Tris–Cl pH 7.5 buffer solutions to con-
trol the acidity of the reaction systems. All plastics and glassware

124
O. Evangelinou et al. / Polyhedron 72 (2014) 122–129
Table 1
Crystal data and structure refinements for [CuBr(xantphos)(mbtt)] (1) and [CuI(xantphos)(mbtt)]ꢂCH3CN (2).
1
2
Molecular formula
Formula weight
T (K)
C
47H39Br1Cu1N1O1P2S2
C49H42Cu1I1N2O1P2S2
991.41
203
903.36
295
k (Å)
0.71073
0.71073
Crystal system
Space group
a (Å)
b (Å)
c (Å)
triclinic
P-1
10.2668 (15)
10.6685 (9)
20.5421 (17)
90.107
monoclinic
P21/n
10.435 (3)
21.442 (6)
19.939 (5)
90
a
(°)
b (°)
100.076 (5)
110.849 (5)
2065.2 (4)
2
1.45
1.712
94.079 (17)
90
4450 (2)
4
1.48
c
(°)
V (Å3)
Z
Dcalc (Mg mꢀ3
)
Absorption coefficient (
F(000)
l
) (mmꢀ1
)
1.389
2008
924
Crystal size (mm)
Theta range for data collection
Index ranges
0.06 ꢃ 0.15 ꢃ 0.34
1.009–28.337°
ꢀ13 6 h 6 12,
ꢀ14 6 k 6 14,
ꢀ24 6 l 6 27
32800
0.07 ꢃ 0.11 ꢃ 0.19
1.396–26.703°
ꢀ13 6 h 6 13,
ꢀ25 6 k 6 26,
ꢀ24 6 l 6 24
51923
Reflections collected
Independent reflections (Rint
Completeness
)
10221 (0.0403)
99.6% (h = 27.77°)
5658/0/496
full-matrix l.s. on F2
1.013
9245 (0.0834)
99.7% (h = 25.902°)
5285/0/523
full-matrix l.s. on F2
1.000
Data /restraints/parameters
Refinement method
Goodness-of-fit (GOF) on F2
Final R indices [(I > 2
R indices (all data)
Final weighting scheme
r
(I)]
R1 = 0.0320, wR2 = 0.0568
R1 = 0.0588, wR2 = 0.0653
R1 = 0.0419, wR2 = 0.0884
R1 = 0.1087, wR2 = 0.1588
calc w = (1/4Fobs2) ꢃ 1
calc w = w0 ꢃ [1 ꢀ (
D
Fobs/6 ꢃ
D
Fest)2]
where
ꢀ1*
w0 = [P0T00(x) + P1T01(x) + . . .Pnꢀ1Tnꢀ1(x)]
0
Largest difference in peak and hole
0.33 e Åꢀ3, ꢀ0.31 Åꢀ3
0.71 e Åꢀ3, ꢀ0.48 Åꢀ3
*
2
Where Pi are the coefficients of a Chebychev series in ti(x), and x = Fcalc2/Fcalc max. P0 ꢀ Pnꢀ1 = 1.44 1.61 0.486.
used in the biological experiments were autoclaved for 30 min at
120 °C and 130 kPa. Heat-resistant solutions were similarly trea-
ted, while heat-sensitive reagents were sterilized by filter.
The DNA stock solution (1 mg/ml) was prepared at 0–4 °C by
dissolving the commercially purchased calf thymus DNA in buffer
A [50 mM Tris [(hydroxymethyl)aminomethane)–HCl buffer (pH
7.5)] as solvent. Stock solutions of the complexes were prepared
at a final concentration of 5 mM by dissolving the complexes in
5% DMSO–water.
illuminator which forms a fluorescent complex when it binds to
DNA.
2.4.3. Determination of antimicrobial activity by the well-diffusion
method
Antimicrobial activities of the newly synthesized complexes
were determined using Gram-negative bacteria (Escherichia coli
and Xanthomonas campestris) and Gram-positive bacteria (Bacillus
subtilis and Bacillus cereus) following a modified Kirby Bauer disc
diffusion method [26,27]. In brief, the bacteria were cultured in
LB Broth at 37 °C on a shaking incubator at 130 rpm. A lawn of bac-
2.4.2. DNA binding/cleavage experiments in agarose gel
electrophoresis
terial culture was prepared by spreading 100 ll culture broth, hav-
ing 106 CFU/ml of each test organism on solid nutrient agar plates.
The plates were allowed to stand for 10–15 min to allow for cul-
ture absorption. The 8 mm size wells were punched into the agar
with the head of sterile micropipette tips. Using a micropipette,
The cleavage/binding reaction of DNA with metal complexes
was monitored using agarose gel electrophoresis. The DNA cleav-
age/binding efficiency of the complexes was estimated by deter-
mining the degree of retardation/or acceleration (precedence of
the electrophoretic mobility reflected in an up-shift or down-shift
of the DNA to higher/or lower molecular weight DNA products,
respectively). Samples, which contained aliquots of 3 lg of the nu-
cleic acid (DNA), were incubated at a constant temperature of 37 °C
for 60 min (or as indicated in the legends) in the presence of each
5, 10 and 15
wells on all plates. After incubation at 37 °C for 24 h, the size of
the zone of inhibition was measured. A volume of 15 l of culture
ll of the complexes were poured into each of five
l
medium was run as a negative control whereas the same volume
from a solution (100 mg/ml) of the antibiotic ampicillin was used
as a positive control.
compound in a buffer A to a final volume of 20
ll. It was termi-
nated by the addition of 4 l loading buffer consisting of 0.25% bro-
l
mophenol blue, 0.25% xylene cyanol FF (acid blue 147) and 30%
glycerol in water. The products resulting from interactions of the
metal complexes with DNA were separated by electrophoresis on
2.4.4. Lipid peroxidation measurement
During the lipid peroxidation (a major indicator of oxidative
stress) of unsaturated fatty acid by oxygen based free radicals,
the malondialdehyde (MDA) is produced and the measured
intensity is related to the concentration of generated reactive
oxygen species (ROS). In a biological system, ROS is determined
by using the thiobarbituric acid reactive species (TBARS) method,
which is based on the quantification of a pink colored complex
between thiobarbituric acid and malondialdehyde (MDA) after acid
agarose gels (1% w/v), which contained 1 lg/ml ethidium bromide
in 40 ꢃ 10ꢀ3 M Tris acetate, pH 7.5, 2 ꢃ 10ꢀ2 M sodium acetate,
2 ꢃ 10ꢀ3 M Na2EDTA, at 5 V/cm. Agarose gel electrophoresis was
performed in a horizontal gel apparatus (Mini-SubTM DNA Cell,
BioRad) for about 2 h. The gels were visualized after staining with
the fluorescence intercalated dyeethidium bromideunder, a UV

O. Evangelinou et al. / Polyhedron 72 (2014) 122–129
125
hydrolysis reaction that can be quantitatively measured on a spec-
trophotometer at kmax = 532 nm [28–30]. Briefly, E. coli cells were
initially cultivated on LB medium for 24 h at 37 °C and then were
collected when culture reached exponential or stationary phase.
Cells were suspended in buffer A and aliquots containing 50 mg
cells were transferred in each tube. Exponential or stationary bac-
terial cells (50 mg) were directly exposed to 10 mM hydrogen per-
oxide (H2O2) or to increasing concentrations of each of the
complexes 1 and 2 for 1 h at 37 °C. Then cells were harvested by
centrifugation and washed twice with distilled water. The pellets
regions. Although exact assignment of the two high energy bands
is difficult since both the N-methylbenzothiazole-2-thione and
xantphos strongly absorb in the same regions, with reference to
the absorption spectrum of the uncoordinated xantphos, these
bands can be considered as intraligand p⁄
p transitions on the
phenyl groups of both the phosphane and the thione moiety and
as a phosphane-originating MLCT transition, respectively. The low-
er energy band can be attributed to a thione originating CT transi-
tion at the C@S bond which may posses some MLCT character
[33,34].
were re-suspended in 500
l
L of a 10% trichloroacetic acid (TCA)
The solid state FT-IR spectra recorded in the range 4000–
250 cmꢀ1 show all the expected strong phosphane bands, which
remain practically unshifted upon coordination. Moreover, they
contain the characteristic bands required by the presence of the
N-methylbenzothiazole-2-thione ligand. In particular, the intense
bands at 1411 and 1259 cmꢀ1 in the spectrum of free mbtt, attrib-
uted to the C–N stretching vibration, appear in the spectra of all
complexes slightly shifted to higher energies, indicating an exclu-
sive S-coordination of the ligand. On the other hand, the strong
band at 1095 cmꢀ1 assigned to the C@S stretching [35], remains
essentially unshifted upon coordination, being hereby less infor-
mative with regard to the ligand’s coordination mode.
solution and transferred to a tube containing 1.5 g of glass beads.
Cells were lysed by 15 cycles of agitation in a vortex for 20 s and
incubated on ice for 20 s, followed by 30 cycles of sonication at
225 W for 10 s and 10 s incubation on ice. The supernatants ob-
tained after centrifugation were mixed with 0.1 mL of 0.1 M EDTA,
0.6 mL of 0.1% w/v thiobarbituric acid in 0.05 M NaOH. The reac-
tion mixture was incubated at 100 °C for 15 min. The samples were
cooled, centrifuged (5900 g) for 5 min), and the appearance of MDA
was recorded by measuring in the supernatant the absorbance at
532 nm and compared to a standard curve containing 0–3.2
MDA.
lM
3. Results and discussion
3.3. Description of the structures
3.1. Preparative considerations
The X-ray crystal structures of [CuBr(xantphos)(mbtt)] (1) and
[CuI(xantphos)(mbtt)] (2) (details of crystal and structure refine-
ment are shown in Table 1) have been determined. Pale yellow
crystals of both 1 and 2 were grown from their respective acetoni-
trile/methanol mother liquid upon slow evaporation during several
The preference of a rigid bidentate ligand for a certain coordina-
tion mode (chelating or bridging) depends, to a significant degree,
on its ‘‘bite angle’’, and chelation is normally favoured for bite an-
gles close to those required by the geometry of the coordination
polyhedron being formed. The structural motifs usually adopted
by copper(I) halide complexes bearing chelating rigid diphos-
phanes in a tetrahedral coordination environment are either dis-
ꢀ
days. Compound 1 crystallizes in the triclinic system P1, with two
discrete molecules in the unit cell, while 2 crystallizes in the mono-
clinic space group P21/n, with four discrete formula units and four
lattice CH3CN molecules in the unit cell. Tables 2 and 3 list selected
distances and angles of these complexes, and perspective drawings
showing the atom numbering are shown in Figs. 1 and 2.
crete monomers, or dimers containing the M(l2-X)2M core
[31,32]. Consequently, the ability of such diphosphanes to span tet-
rahedral sites under formation of fairly constrained P–M–P angles
having values far away from their quite short natural bite angles,
should be connected with the extraordinary versatility of the d10
metal ion. In this respect, the potentiality of xantphos to chelate
copper(I), either in a tetrahedral or in a trigonal planar environ-
ment can be easily explained, while steric demands on the part
of the ligands may be responsible for the adoption of a distinct
coordination number in each case. Indeed, treatment of CuX with
xantphos, independent of the ratios of the phosphane applied, re-
sults in the formation of a three-coordinate chelate, whereas sub-
sequent addition of one equivalent of a heterocyclic thione to the
solution of [CuX(xantphos)] causes increase of the coordination
number, leading to the formation of four-coordinate xantphos/thi-
one mixed-ligand species. This was once again the case with the
reactions within the present study, where the favored four-coordi-
nation for copper(I) could be easily achieved even using the more
bulky ligand N-methylbenzothiazole-2-thione.
The two structures are similar having common the tetrahedral
coordination of the copper(I) center, which is surrounded by the
S donor of the thione ligand, two P atoms of the chelating xantphos
unit, as well as the corresponding halogen atom. In compound 1,
angular deviations from the ideal tetrahedral value of 109.4° are
quite small, thus the tetrahedral environment of the copper atom
can be considered as essentially regular when compared with sev-
eral reported, four coordinated, copper(I) halide complexes bearing
a heterocyclic thione and two monodentate arylphosphanes or a
chelating short bite angle diphosphane [31]. Contrary to that, the
I(1)–Cu(1)–S(1) and S(1)–Cu(1)–P(1) angles in 2, having values of
92.11(5)° and 119.44(7)°, respectively, deviate remarkably from
the ideal tetrahedral expectation. While the P(1)_Cu(1)_P(2) angles
of 111.52(3)° in 1 and 113.85(6)° in 2 are very close to the ligands
calculated ‘‘natural’’ bite angle of 111o [36], it is obvious that che-
lation requires some adjustment on the part of the diphosphane
backbone, to keep constraints within the tetrahedron at a mini-
mum. In fact, the Pꢂ ꢂ ꢂP distance of 4.045(1) Å of the xanthene unit
appears shortened to values of 3.799 and 3.832 Å in 1 and 2,
respectively, accompanied by a respective adaptation of the folding
of the of the molecule along the axis through the oxygen atom and
the xanthene carbon atom C(45) (141.60° in 1 and 139.55° in 2 ver-
sus 156.58° in the free xantphos [37]).
The microcrystalline solids are moderately soluble in acetoni-
trile, dichloromethane and chloroform and only slightly soluble
in acetone. Their solutions in acetonitrile are stable to air and
moisture and exhibit no conductivity. Room temperature magnetic
measurements confirm the expected diamagnetic nature of the
compounds.
In both structures, the two individual Cu–P distances are within
the limits of the range expected for tetrahedrally coordinated cop-
per(I). Likewise, the Cu–S and Cu–Br and Cu–I bond lengths are
comparable to those reported for other tetrahedrally coordinated
copper(I) complexes with terminal bromine or iodine and thione-
sulfur donors [18,38].
3.2. Spectroscopy
The electronic absorption spectra of the two complexes, re-
corded in acetonitrile at room temperature, show three intense
broad bands with maxima in the ꢁ240, ꢁ276 and ꢁ321 nm

126
O. Evangelinou et al. / Polyhedron 72 (2014) 122–129
Table 2
Selected bond distances (Å) and angles (°) in [CuBr(xantphos)(mbtt)] (1).
Cu(1)–Br(1)
Cu(1)–S(1)
Cu(1)–P(1)
Cu(1)–P(2)
S(1)–C(1)
2.4468(6)
2.3797(10)
2.3019(9)
2.2947(9)
1.671(3)
Br(1)–Cu(1)–S(1)
Br(1)–Cu(1)–P(1)
Br(1)–Cu(1)–P(2)
S(1)–Cu(1)–P(1)
S(1)–Cu(1)–P(2)
P(1)–Cu(1)–P(2)
114.85(3)
103.52(3)
111.66(3)
105.17(4)
109.79(4)
111.52(3)
I1
C21
P2
C15
P1
C27
C9
Cu1
O1
Table 3
Selected bond distances (Å) and angles (°) in [CuI(xantphos)(mbtt)]ꢂCH3CN (2).
C33
C40
C39
C38
C46
C34
C35
C41
C42
Cu(1)–I(1)
Cu(1)–S(1)
Cu(1)–P(1)
Cu(1)–P(2)
S(1)–C(1)
2.6570(9)
2.3543(19)
2.2897(17)
2.2825(17)
1.650(7)
I(1)–Cu(1)–S(1)
I(1)–Cu(1)–P(1)
I(1)–Cu(1)–P(2)
S(1)–Cu(1)–P(1)
S(1)–Cu(1)–P(2)
P(1)–Cu(1)–P(2)
92.11(5)
S1
108.58(5)
110.59(4)
119.44(7)
110.04(7)
113.85(6)
C37
C44
C45
C36
C1
S2
C43
C47
N1
C2
C7
C8
C3
C47
C46
C36
C4
C6
C37
C45
C35
C5
Fig. 2. Molecular plot of [CuI(xantphos)(mbtt)].
C43
C44
C38
C34
O1
C42
C41
C39
C33
C15
C40
C21
P1
S1
C9
P2
Cu1
Br1
C8
C27
C1
N1
C3
S2
C4
C2
C5
C7
Fig. 3. Room-temperature solid-state excitation (dashed line, kem = 534 nm) and
emission (solid line, kex = 410 nm) spectrum of [CuBr(xantphos)(bmtt)] (1).
C6
Fig. 1. Molecular plot of [CuBr(xantphos)(mbtt)].
3.4. Photoluminescence
3.5. Evaluation of biological activity
Room-temperature photoexitation of solid samples of each of
the two compounds under consideration at k = 410 nm produces
an intense broad emission band with peak maximum at k = 534
and 532 nm for 1 and 2, respectively (Fig. 3). This emission cannot
be considered as one of pure intra-ligand origin, because of its sig-
nificant red shift relative to the solid-state r.t. emission spectrum
of the free xantphos, which consists of a broad band centered
around 465 nm, being more compatible with a metal-to-ligand
charge-transfer (Cu?p⁄(xantphos) excited state, mixed with IL
3.5.1. DNA binding/cleavage experiments in agarose gel
electrophoresis
The effect of the two newly synthesized copper complexes on
the integrity of calf thymus ds DNA (CTDNA) was examined.
When ds CT-DNA was treated with low concentrations (0.5 and
1 mM) of both compounds 1 and 2 the integrity seemed to be unaf-
fected visibly in both cases (Fig. 4A). However, when CT-DNA was
treated with a concentration of 2 mM of compound 1 significant
degradation was caused, as it is deduced from the diminution of
the initial amount of CTDNA (Fig. 4B, lane 2). In addition, at a high-
er concentration of 3 mM the ds CT-DNA was completely degradat-
ed (Fig. 4B, lane 3) ending in the disappearance of the main DNA
band. In contrast, the integrity of ds CT-DNA remained unaffected
after treatment with compound 2 or with each of the ligands (mbtt
or xantphos) under the same conditions.
(p?
p⁄) transitions associated with the phenyl rings of xantphos.
Contrary to our previous results from related compounds [39–
41], no significant shift of the emission energy is observed upon
changing of the halide ligand, thus the participation of a halide-
to-ligand charge-tranfer (XLCT) in the emissive excited sites seems
here unlikely.

O. Evangelinou et al. / Polyhedron 72 (2014) 122–129
127
C
1
2
3
4
5
6
7
8
C
1
2
3
4
5
6
7
8
A
B
Fig. 4. Dose dependent DNA degradation action of compounds 1 and 2 and the free ligand xantphos and separation in agarose (1%) gel electrophoresis of ds CTDNA. Each
sample containing 3 g of ds CTDNA was treated with 0.5 mM and 1 mM of compounds 1, 2 and xantphos. (A) Lane C: control, Lanes 1–2: 0.5 and 1 mM of compound 1,
l
respectively. Lanes 3–4: 0.5 and 1 mM of compound 2, respectively. Lanes 5–6: 0.5 and 1 mM of xantphos, respectively. Lanes 7–8: 0.5 and 1 mM of mbtt, respectively. (B)
Lane C: control, Lane 1–2: 2 and 3 mM of compound 1, respectively. Lanes 3–4: 2 and 3 mM of compound 2, Lanes 5–6: 2 and 3 mM of xantphos, respectively. Lanes 7–8: 0.5
and 1 mM of mbtt, respectively.
Fig. 5. Zones of inhibition of copper complexes in representative agarose plates against B. subtilis, of the antibiotic ampicillin (100 mg/ml) as a positive control and of culture
medium as a negative control (blank). Plates A–C: A volume of 5, 10 and 15
well of the plates, wherein the bacterium B. subtilis was incorporated. (D) Bar graph representing the zone of inhibition for compounds 1 and 2 and their ligands compared to
the antimicrobial activity exhibited from 15 l of a solution of ampicillin (100 mg/ml) against B. subtilis, which was considered as 100%.
ll of a 3 mM solution of compounds 1 and 2 and the free ligand xantphos were spotted in each
l
3.5.2. Determination of antimicrobial activity by the well-diffusion
method
Fig. 5 shows representative images exhibiting the zone of inhi-
bition caused by each of the two copper complexes as well as the
The antimicrobial activities were determined against two
Gram-negative (E. coli and X. campestris) and two Gram-positive
bacteria (B. subtilis and B. cereus) following a modified Kirby Bauer
well-diffusion method.
free ligands at three different volumes (5, 10 and 15
solutions at concentration 3 mM. In the center of each plate a vol-
ume of 15 l of a 100 mg/ml solution of the known antibiotic
ampicillin was spotted and the zone of inhibition was recorded,
ll) from their
l

128
O. Evangelinou et al. / Polyhedron 72 (2014) 122–129
4. Conclusion
The aim of this work was the preparation, characterization and
the study of the antibacterial activity of copper(I) halide mixed-li-
gand complexes bearing N-methylbenzothiazole-2-thione bonded
to the metal via the thione-S atom and 4,5-bis(diphenylphosphan-
o)-9,9-dimethylxanthene as a chelating ligand. According to the
overall geometries of the two structures presented here, the xant-
phos ligand applied can be considered as best tailored for chelation
of copper(I) centres, as it seems not to be forced, upon coordina-
tion, to significantly modify its natural bite angle. The new com-
pounds show intense room temperature luminescence in the
solid state, most likely originating from emissive excited states of
mixed MLCT/IL character.
Remarkably high antimicrobial activity, significantly higher
than that of the commercially available antibiotic ampicillin against
some Gram–negative and Gram–positive bacteria is found only for
the copper bromide complex (compound 1) unlike its iodide coun-
terpart which is found to be quite inactive. This antibacterial activ-
ity is mediated by the generation of ROS and damage of bacterial
membrane. Likewise DNA degradation to a significant degree upon
interaction with CT-DNA is observed only for the copper bromide
complex. This different behavior is a rather strange finding and re-
quires to be further investigated.
Fig. 6. Increase in the MDA equivalents (nmol/ml) with exposition to (1) 3 mM
compound 1, (2) 3 mM of compound 2, (3) 3 mM of xantphos and (4) 3 mM of mbtt.
Acknowledgments
as a positive control. It should be noted that the concentration of
this ampicillin solution (molecular mass = 349.41) was about hun-
dred times higher than the concentration used for each of the com-
pounds under investigation. In addition, the culture media alone
was spotted and its antimicrobial activity was recorded as a nega-
tive control.
As it is obvious, among the compounds tested only compound 1
clearly inhibits the growth of both Gram-negative and -positive
bacteria and, in addition, the zone of inhibition increases with
the increase of the complexes amount. Moreover, it can be seen
that the same volume of a solution of compound 1 (2.71 mg/ml)
proves to be as effective as the about hundred times more concen-
trated solution of the antibiotic ampicillin (100 mg/ml), producing
the same zones of inhibition against the bacterium B. subtilis under
the same conditions. Similar results were obtained with all other
bacteria tested.
This research has been co-financed by the European Union
(European Social Fund – ESF) and Greek national funds through
the Operational Program ‘‘Education and Lifelong Learning’’ of
the National Strategic Reference Framework (NSRF) – Research
Funding Program: ARCHIMEDES III. Investing in knowledge society
through the European Social Fund.
Appendix A. Supplementary data
CCDC 972048 and 972049 contain the supplementary crystallo-
graphic data for compounds 1 and 2, respectively. These data can
be obtained free of charge via http://www.ccdc.cam.ac.uk/conts/
retrieving.html, or from the Cambridge Crystallographic Data Cen-
tre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223-336-
033; or e-mail: deposit@ccdc.cam.ac.uk.
3.5.3. Lipid peroxidation measurement
References
The oxidation of cell membrane lipids could be triggered by
reactive oxygen species like H2O2 or highly reactive hydroxyl rad-
ical and singlet oxygen, which were often reported to be generated
by toxic agents. The toxicity of the newly synthesized Cu com-
plexes was studied in E. coli also in the light of formation of reac-
tive oxygen species (ROS), measured as malondialdehyde (MDA)
equivalent by thiobarbituric acid-ROS (TBARS) assay. Higher inhi-
bition of growth for the chronic exposure batches were correlated
with higher ROS generation, which subsequently contributed to
cause membrane lipid peroxidation.
[1] R.J. Puddephatt, Chem. Soc. Rev. 12 (1983) 99.
[2] F.A. Cotton, K.R. Dunbar, M.G. Verbruggen, J. Am. Chem. Soc. 109 (1987) 5498.
[3] R.V. Kiress, R. Eisenberg, Inorg. Chem. 28 (1989) 3372.
[4] S.J. Bernes-Price, R.K. Johnson, C.K. Mirabelli, L.F. Faucette, F.L. McCabe, P.J.
Sadler, Inorg. Chem. 26 (1987) 3383. and references therein.
[5] R.H. Holm, P. Kennepohl, E.I. Solomon, Chem. Rev. 96 (1996) 2239.
[6] J.A. Garcia-Vazquez, J. Romero, A. Sousa, Coord. Chem. Rev. 193–195 (1999)
691. and references therein.
[7] P.D. Akrivos, Coord. Chem. Rev. 213 (2001) 181.
[8] B.A. Al-Maythalony, M. Monim-ul-Mehboob, M. Altaf, M.I.M. Wazeer, A.A. Isab,
S. Altuwaijri, A. Ahmed, V. Dhuna, G. Bhatia, K. Dhuna, S. Singh Kamboj,
Spectrochim. Acta, Part A 115 (2013) 641.
Compared to the control batch (i.e., without complexes or li-
gands in culture media), the MDA equivalents for the cells trea-
ted with compound 1 were higher than the MDA equivalents for
the cells treated with compound 2 and for the cells treated with
xantphos (Fig. 6). Neither compound 2 nor the ligand xantphos
seems to create any MDA equivalents. E. coli cells produce
approximately 50 nM malondialdeyde (MDA) after one hour of
exposition to compound 1, which can be attributed to lipid per-
oxidation. Similar results were obtained with the other bacteria.
It should be noted that for E. coli cells that were not exposed in
any agent, the MDA equivalents were not detectable (sample of
reference).
[9] S. Braun, A. Botzki, S. Salmen, C. Textor, G. Bernhardt, S. Dove, A. Buschauer,
Eur. J. Med. Chem. 46 (2011) 4419.
[10] C. Congiu, M.T. Cocco, V. Omnis, Bioorg. Med. Chem. Lett. 18 (2008) 989.
[11] M.S. Masoud, A.A. Soayed, A.F. El-Husseiny, Spectrochim. Acta, Part A 99
(2012) 365.
[12] Y. Liu, J. Wu, P.-Y. Ho, L.-C. Chen, C.-T. Chen, Y.-C. Liang, C.-K. Cheng, W.-S. Lee,
Cancer Lett. 271 (2008) 294.
[13] C.-R. Shih, J. Wu, Y. Liu, Y.-C. Liang, S.-Y. Lin, M.-T. Sheu, W.-S. Lee, Biochem.
Pharmacol. 67 (2004) 67.
[14] P. Aslanidis, P.J. Cox, P. Karagiannidis, S.K. Hadjikakou, C.D. Antoniadis, Eur. J.
Inorg. Chem. (2002) 2216. and references therein.
[15] M.A. Tsiaggali, E.G. Andreadou, A.G. Hatzidimitriou, A.A. Pantazaki, P.
Aslanidis, J. Inorg. Biochem. 121 (2013) 121.
[16] M. Porchia, F. Benetollo, F. Refosco, F. Tisato, C. Marzano, V. Gandin, J. Inorg.
Biochem. 103 (2009) 1644.

O. Evangelinou et al. / Polyhedron 72 (2014) 122–129
129
[17] C. Marzano, M. Pellei, S. Alidori, A. Brossa, G.G. Lobbia, F. Tisato, C. Santini, J.
Inorg. Biochem. 100 (2006) 299.
[29] L.T. Rael, G.W. Thomas, M.L. Craun, C.G. Curtis, R. Bar-Or, D. Bar-Or, J. Biochem.
Mol. Biol. 37 (2004) 749.
[18] A. Kaltzoglou, P.J. Cox, P. Aslanidis, Inorg. Chim. Acta 358 (2005) 3048.
[19] A. Kaltzoglou, P.J. Cox, P. Aslanidis, Polyhedron 26 (2007) 1634.
[20] G. Storz, J.A. Imlay, Curr. Opin. Microbiol. 2 (1999) 188.
[21] J.A. Imlay, Annu. Rev. Microbiol. 57 (2003) 395.
[30] G.J. Pacheco, R.S. Reis, A.C. Fernandes, S.L. da Rocha, M.D. Pereira, J. Perales,
D.M. Freire, Appl. Microbiol. Biotechnol. 95 (2012) 1519.
[31] P. Aslanidis, P.J. Cox, S. Divanidis, P. Karagiannidis, Inorg. Chim. Acta 357
(2004) 1063.
[22] Bruker Analytical X-ray Systems, Inc. Apex2, Version 2 User Manual, M86-
E01078, Madison, WI, 2006.
[23] L. Palatinus, G. Chapuis, J. Appl. Crystallogr. 40 (2007) 786.
[24] P.W. Betteridge, J.R. Carruthers, R.I. Cooper, K. Prout, D.J. Watkin, J. Appl.
Crystallogr. 36 (2003) 1487.
[32] P. Aslanidis, P.J. Cox, A. Kaltzoglou, A.C. Tsipis, Eur. J. Inorg. Chem. (2006) 334.
[33] A.A. Del Paggio, D.R. McMillin, Inorg. Chem. 22 (1983) 691.
[34] C. Kutal, Coord. Chem. Rev. 99 (1990) 213.
[35] F. Cristiani, F.A. Devillanova, G. Verani, Spectrochim. Acta 12 (1982) 1299.
}
[36] S. Hillebrand, J. Bruckmann, C. Kruger, M.W. Haenel, Tetrahedron Lett. 36
[25] D.W. Watkin, C.K. Prout, L.J. Pearce, CAMERON
Laboratory, Oxford, UK, 1996.
[26] A.W. Bauer, W.M. Kirby, J.C. Sherris, M. Turck, Am. J. Clin. Pathol. 45 (1966)
,
Chemical Crystallography
(1995) 75.
[37] M. Kranenburg, Y.E.M. van der Burg, P.C.J. Kamer, P.W.N.M. Leeuwen, J. Fraanje,
Organometallics 14 (1995) 308.
493.
[38] P.J. Cox, A. Kaltzoglou, P. Aslanidis, Inorg. Chim. Acta 359 (2006) 3183.
[39] P. Aslanidis, P.J. Cox, P. Tsaliki, Polyhedron 27 (2008) 3029.
[40] P. Aslanidis, P.J. Cox, A.C. Tsipis, Dalton Trans. 39 (2010) 10238.
[41] I. Papazoglou, P.J. Cox, A.G. Papadopoulos, M.P. Sigalas, P. Aslanidis, Dalton
Trans. 42 (2013) 2755.
[27] A. Azam, A.S. Ahmed, M. Oves, M.S. Khan, A. Memic, Int. J. Nanomed. 7 (2012)
3527.
[28] E.L. Steels, R.P. Learmonthy, K. Watson, Microbiology 140 (1994) 569.