Synthesis, crystal and solution structures and antimicrobial screening of palladium(II) complexes with 2-(phenylselanylmethyl)oxolane and 2-(phenylselanylmethyl)oxane as ligands

Article abstract of DOI:10.1016/j.jinorgbio.2014.11.002

Two novel Pd(II) complexes with 2-(phenylselanylmethyl)oxolane and 2-(phenylselanylmethyl)oxane as ligands were synthesized. The crystal and molecular structure of the complexes has been determined by single crystal X-ray diffraction. It turned out for bo

Full text of DOI:10.1016/j.jinorgbio.2014.11.002

Journal of Inorganic Biochemistry 143 (2015) 9–19
Contents lists available at ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
Synthesis, crystal and solution structures and antimicrobial screening of
palladium(II) complexes with 2-(phenylselanylmethyl)oxolane and
2
-(phenylselanylmethyl)oxane as ligands
a,
a
a
a
b
Zorica M. Bugarčić ⁎, Vera M. Divac , Marina D. Kostić , Nenad Ž. Janković , Frank W. Heinemann ,
c
d
Niko S. Radulović , Zorica Z. Stojanović-Radić
Faculty of Science, Department of Chemistry, University of Kragujevac, Radoja Domanovića 12, Kragujevac 34 000, Serbia
Inorganic Chemistry, Department of Chemistry and Pharmacy, University of Erlangen-Nuremberg, Egerlandstrasse 1, Erlangen 91058, Germany
Faculty of Science and Mathematics, Department of Chemistry, University of Niš, Višegradska 33, Niš 18000, Serbia
Faculty of Science and Mathematics, Department of Biology and Ecology, University of Niš, Višegradska 33, Niš 18000, Serbia
a
b
c
d
a r t i c l e i n f o
a b s t r a c t
Article history:
Two novel Pd(II) complexes with 2-(phenylselanylmethyl)oxolane and 2-(phenylselanylmethyl)oxane as
ligands were synthesized. The crystal and molecular structure of the complexes has been determined by single
crystal X-ray diffraction. It turned out for both complexes that the two ligands are coordinated to Pd via Se
Received 2 September 2014
Received in revised form 12 November 2014
Accepted 13 November 2014
Available online 20 November 2014
\
atoms in a trans-fashion and the other two trans-positions are occupied by Cl ions. Detailed 1D- and 2D-NMR
analyses revealed the existence of equilibrating trans-diastereomeric species differing in the configuration at
four chiral centers (selenium and carbon) in the solution of the complexes. A computational study was also un-
dertaken to assess the relative stabilities of the mentioned stereoisomeric species. The antimicrobial properties
of the complexes were investigated against a series of human pathogenic bacterial and fungal strains. The com-
plexes were shown to possess promising broad spectrum moderate antimicrobial activity that is more pro-
nounced against fungal organisms. The noted activity could be completely attributed to the Pd(II) center,
whereas the ligands probably mediate the transportation of a Pd(II) species across cell membranes.
Keywords:
Organoselenium chemistry
Ligands
Palladium
Antimicrobial activity
©
2014 Elsevier Inc. All rights reserved.
1
. Introduction
against toxicity of some metals (Pb, Cd and Ag) [8–10]. In addition, the
biochemical mechanism of Au interaction with selenium-containing
proteins has been regarded as promising implication for the treatment
of RA (rheumatoid arthritis) [11]. Metal–selenium coordination in
proteins is an area for future investigation since some of these interac-
tions have been identified in certain classes of enzymes, for instance,
selenium–molybdenum in formate dehydrogenase from E. coli and
selenium–tungsten in formate dehydrogenase from Desulfovibrio gigas
[12,13]. All these circumstances have initiated developments for the
design of different transition metal complexes bearing selenium func-
tionality in their molecular structure [14–22]. Due to the interesting
properties, which selenium-metal binding can enable in biological sys-
tems, some complexes with organoselenium ligands were screened
for cytotoxic and/or antitumor, antimalarial activity, etc. [23–29].
In the light of the above-mentioned facts, herein we report
the synthesis, crystal structure and antimicrobial activity of two
novel Pd(II) complexes with 2-(phenylselanylmethyl)oxolane and
2-(phenylselanylmethyl)oxane as ligands. Since these novel com-
plexes contain organoselenium- and different heterocyclic oxygen ring
functionalities, it was of interest to compare the relationship between
their structure and activity on certain bacterial and fungal strains. For
this purpose, the two complexes were tested in a microdilution assay to
Recent advances in the area of organoselenium compounds have
been driven by their applications as useful reaction intermediates and
convenient models for the study of mechanisms in bioorganic chemis-
try. In addition, many organoselenium compounds have been described
as promising pharmaceutical agents in view of their unique biological
properties ranging from antioxidant, anti-inflammatory and neuropro-
tection to chemotherapeutic and chemopreventive activities [1].
The growing demand for incorporation of organoselenium com-
pounds in transition metal coordination chemistry is partly a result
of the finding that they can confer significantly different properties
on the resultant complexes [2–4]. Undoubtedly, they constitute a new
class of building blocks for several transition metal complexes which
catalyze various organic reactions efficiently in solution [5]. More
recently, metal coordination with selenium compounds has been pro-
posed as an additional antioxidant mechanism in DNA damage inhibi-
tion [6,7]. It has also been assumed that selenium binding protects
⁎
Corresponding author. Tel.: +381 34300252; fax: +381 34335040.
E-mail address: zoricab@kg.ac.rs (Z.M. Bugarčić).
http://dx.doi.org/10.1016/j.jinorgbio.2014.11.002
162-0134/© 2014 Elsevier Inc. All rights reserved.
0

10
Z.M. Bugarčić et al. / Journal of Inorganic Biochemistry 143 (2015) 9–19
determine their minimum inhibitory (MIC) and minimum microbicidal
concentrations (MMC).
side of the central plane, in the case of complex 2 they are now point
to opposite sites of the central plane.
2.3. NMR spectroscopic characterization
2
. Results and discussion
Ambient temperature 1H and 13C NMR spectra of [Pd(L1)
Cl
2
]
2
2
.1. Synthesis of bis(2-(phenylselanylmethyl)oxolane)dichloropalladium(II)
and [Pd(L2)
2 2
Cl ], alongside the selenoether ligands L1 and L2, have
(1) and bis(2-(phenylselanylmethyl)oxane)dichloropalladium(II) (2)
1
13
been measured in deuterated chloroform solution. The H and
chemical shifts and the Δ( H)coord and Δ( C)coord coordination shifts
determined in respect to free L1 or L2 in CDCl ) are collected in
C
1
13
Ligands L1 (2-(phenylselanylmethyl)oxolane) and L2 (2-
phenylselanylmethyl)oxane) were synthesized in high yields by the
(
3
(
1
Tables 3 and 4. The H spectra showed a high degree of peak overlap,
complex signal multiplicities, and, in the case of the complexes, signifi-
cant line broadening. The assignments of both proton and carbon-13
signals were made possible only through an extensive use of 2D
reaction of the corresponding alkenol (pent-4-en-1-ol for L1, or hex-
-en-1-ol for L2) and PhSeCl in the presence of equimolar amounts
5
of pyridine, according to the reported method (Scheme 1) [30]. It is
noteworthy to mention that this methodology can be extended to a
number of other unsaturated alcohol systems, providing a wide range
of different substituted cyclic ether precursors with a phenylseleno
part in the structure. Treatment of the ligands (L1 or L2) with an excess
1
1
spectra— H– H gDQCOSY (gradient double quantum filtered correla-
tion spectroscopy), NOESY (nuclear Overhauser effect/enhancement
1
13
spectroscopy), H– C gHMQC (gradient heteronuclear multiple quan-
1
13
tum coherence) and H– C gHMBC (gradient heteronuclear multiple
of PdCl
1
2
in EtOH/MeOH mixture at 40 °C afforded the desired complexes
2 2
bond coherence), shown in Fig. 3 for [Pd(L1) Cl ]) and a series of selec-
and 2 in high yields (Scheme 2).
tive homodecoupling experiments.
1
H and 13C NMR spectra of the ligands generally agreed with the
literature values [35,36]. The ambient temperature spectra of the com-
plexes indicated the presence of two different palladated ligand solution
2
2 2 2 2
.2. Crystal structures of [PdL1 Cl ] and [PdL2 Cl ]
1
The molecular structure of complexes 1 ([PdL1
2
Cl
2
]) and 2
species (I and II) with an approximate 1:1 signal integral ratio. The H
(
[PdL2 Cl ]) were confirmed by x-ray crystal structure determination
2
2
multiplet splittings in these spectra were not revealed due to line broad-
ening from an apparent rate process involved and/or the possible
existence of several stereoisomeric species differing only slightly in
chemical shifts values. To corroborate the first assumption, although
we could not perform a detailed temperature dependence study, on
raising the temperature of the NMR sample, the exchange broadening
of the two sets of peaks led to coalescence, resulting in a single set of
averaged signals. A subsequent cooling of this sample to room temper-
ature resulted in the starting NMR. To rule out a possible slow isomeri-
and are shown in Figs. 1 and 2. Selected bond distances and angles are
given in Tables 1 and 2 for complexes 1 and 2, respectively, and their
crystal packaging in Supplementary data. For both complexes, the coor-
dination geometry around the palladium center is distorted square-
planar. The Pd(II) is coordinated by two Se and two Cl atoms in both
complexes with each pair of selenium and chlorine donor atoms being
arranged in trans-position.
The molecular structure of 1 is characterized by a slight deviation of
only 0.0211(6) Å of the Pd atom from an ideal square planar arrange-
zation process, the 1H-NMR spectrum of [Pd(L1)
Cl ] in CDCl was
2 2 3
ment of the PdSe
ly planar. The observed Pd–donor distances for complex 1 are Pd–Cl
.3013(9) Å and Pd–Se 2.4313(3) Å (Table 1) and match nicely the
values observed earlier in square planar PdCl Se complexes [31–34],
while corresponding distances for 2 are marginally shorter (Pd–Cl
.2952(7) Å and Pd–Se 2.4271(4) Å) (Table 2). The angles Se1–Pd1–
Se1A and Cl1–Pd1–Cl1A amount to 175.95(2)° and 173.61(5)° and indi-
cate the slight but significant tetrahedral deviation of the PdSe Cl unit
2
Cl
2
unit, while PdSe
2
Cl
2
unit in the complex 2 is exact-
measured once daily (the solution was kept at room temperature) for
1 month. The appearance of the spectrum did not change significantly
in any respect. An intramolecular process that may be the cause of
these dynamic NMR features is the pyramidal inversion of the coordi-
nated selenium atom [37,38]. Since this process seems to be medium
slow on the NMR timescale at ambient temperature, and the starting
ligands were a racemic mixture, two sets of six configurational isomers
can exist for the possible cis- and trans-diastereomeric arrangements
of the ligands in the coordination sphere of palladium(II), namely, two
meso complexes, and four degenerate enantiomeric pairs. The stereoiso-
meric species for the trans-ligand arrangement in the complexes are
depicted in Scheme 3. One could expect the trans-complexes to be
both thermodynamically and kinetically favored by virtue of the high
2
2
2
2
2
2
from planarity for 1. The steric pressure exerted by the methylene
groups next to the Se atom leads to a stronger deviation from the
ideal 90° angles within the PdSe
2 2
Cl plane of 1 (Cl1–Pd1–Se1
8
3.51(2)° vs. Cl1–Pd1–Se1A 96.72(2)°) and (Cl1–Pd1–Se1 83.34(2)°
vs. Cl1–Pd1–Se1A 96.66(2)°) for complex 2. While the two phenyl
rings of the ligand L1 are arranged almost perpendicular to the central
2
steric crowding of adjacent methylenes (CH Se) that would arise in
2 2
PdSe Cl plane with the two phenyl rings pointing toward the same
the cis-forms as evidenced by the current X-ray analysis (deviation
4
3
2
'
3
'
PhSeCl/Pyridine eq.
CH Cl , r.t.
Se
1'
OH
5
2
2
2
O
4'
6
'
5
'
L1
4
5
3
PhSeCl/Pyridine eq.
CH Cl , r.t.
2'
Se 1'
6
OH
O
2
3'
2
2
4
'
6
'
5
'
L2
Scheme 1. Synthesis of ligands L1 and L2.

Z.M. Bugarčić et al. / Journal of Inorganic Biochemistry 143 (2015) 9–19
11
O
SePh
PdCl2
EtOH/MeOH, 40 C, 3 h
Se
Cl
Cl
o
O
Pd
Se
L1
O
1
PdCl2
O
o
EtOH/MeOH, 40 C, 3 h
Se
Cl
SePh
O
Pd
Cl
Se
L2
O
2
Scheme 2. Synthesis of Pd(II) complexes 1 and 2.
from ideal square planarity around Pd by steric pressure of the CH
group next to the Se atom and the chlorine atoms), or the kinetic trans
Chernyaev) effect (selenium based ligands are better trans-directing
2
while all cis-forms were much less stable that the trans-forms with
the lowest energy laying cis-form enantiomeric pair-1 (calculated
for all S-isomer) separated by more than 10 kJ/mol from the trans-
meso-1. Hence, the contribution of complexes of cis configuration to a
(
ligands than the chloride is).
In order to gain an insight into the relative stabilities of the
mentioned stereoisomers in solution, a computational study of all
possible stereoisomers of [Pd(L1) Cl ] (1) in a periodic box of chloro-
2 2
form molecules was performed using the 6-311 + G(d,p) basis set for
C, H, N, O, Cl and Se, and the Def2-TZVPD basis set for Pd. The most stable
stereoisomeric form was found to be the trans-meso-1RRSS (Scheme 3),
Fig. 1. Thermal ellipsoid representation of the molecular structure of complex 1 (50%
Fig. 2. Thermal ellipsoid representation of the molecular structure of complex 2 (50%
probability ellipsoids).
probability ellipsoids).

12
Z.M. Bugarčić et al. / Journal of Inorganic Biochemistry 143 (2015) 9–19
Table 1
Selected bond distances [Å], angles and torsion angles [°] for [PdL1
good agreement with the almost equal intensities of the signals of the
like and unlike palladated ligands. Hence, it appears that the relative
stability of the like configurational combination has been overestimated
by the theoretical study.
2 2
Cl ].
Bond distances
Pd1–Cl1
Pd1–Cl1A
Pd1–Se1A
Pd1–Se1
Se1–C6
Se1A–C1B
Se1–C1
O1–C5
Bond angles
2.3013(9)
2.3013(9)
2.4313(3)
2.4314(3)
1.925(3)
1.96(2)
Cl1A–Pd1–Se1A
Cl1–Pd1–Se1
C6A–Se1A–C1B
C6–Se1–C1
C6–Se1–Pd1
C1B–Se1A–Pd1
C1–Se1–Pd1
C2–C1–Se1
96.72(2)
96.72(2)
103.5(4)
91.5(4)
102.72(10)
105.6(6)
104.9(6)
110.4(13)
113.3(12)
A corroboration of the existing selenium inversion, which would in-
terconvert the forms, was found in the NOESY spectra (Fig. 3 B) from the
correspondence (which hydrogen becomes which upon inversion at Se)
between the signals (columns 1 I and 1 II, as well as 2 I and 2 II, in
Table 3) belonging to the two stereochemically non-equivalent bound
ligands. Both complexes 1 and 2 displayed residually broadened signals
in the NMR spectra and yielded chemical/conformational exchange
peaks in the NOESY spectrum differing in sign from the “proximity”
cross-peaks (Fig. 3 B). We could conclude that the equilibrium between
all stereoisomeric species drawn in Scheme 3 (the rate of interconver-
sion) should be in the domain of medium slow (up to about a minute)
1.97(2)
1.328(15)
1.458(13)
1.52(2)
O1–C2
C1–C2
C2B–C1B–Se1A
O1B–C2B
O1B–C5B
C1B–C2B
1.344(13)
1.484(13)
1.52(2)
Torsion angles
C6–Se1–C1–C2
Pd1–Se1–C6–C11
Pd1–Se1–C1–C2
Cl1–Pd1–Se1–C6
85.4(9)
143.6(3)
174.2(7)
92.7(2)
exchanges in CDCl
3
at 25 °C.
Symmetry transformations used to generate equivalent atoms:
1 −x þ 1; −y þ 1; z:
The palladation of the ligands resulted in a mean 1H and 13C
1
13
deshielding (mean Δ( H)coord: +0.06 − +0.12 ppm, mean Δ( C)
coord: +0.56 − +1.23 ppm) as deduced from the unassigned NMR
data (Tables 3 and 4). The observed downfield shift, in some cases for car-
#
2
bon atoms up to +6–7 ppm (CH Se), could be explained by the expected
hypothetic equilibrium between the stereoisomers in chloroform solu-
tion was a negligible one (i.e. less than 1% of the equilibrium mixture
when other isomers are taken into account). The remaining diastereo-
meric trans-complexes were less stable that the trans-meso-1 by ca.
electron-withdrawing effect of palladium(II), i.e., the overall elec-
tron density should be more toward the metal halide which in effect
would make the coordinating atoms electron-deficient compared to
the free ligands. Although on the whole Pd(II) complexation of the
two ligands produced an overall deshielding of carbons, one should
note that in both complexes signals of an analogous pair of carbons
0
3
.2, 1.3, 1.5, 3.8 and 5.4 kJ/mol, respectively, for 1SSSS, 4RRSR, 2RRRS,
RSSR and meso-2RSRS. From these data, one can conclude that the
unlike combinations of configuration at Se and C centers within a single
ligand make the complex less stable.
(
C-2 and C-1′) were significantly shifted upfield by −1.8 − −3.4
and −2.2 − −3.3 ppm, respectively.
Thus, one would expect to observe at least six (trans) solution spe-
cies, of possibly unequal populations, but the ambient temperature
The coordination of the selenoether ligands to the Pd(II) center
1
could be verified by the comparison of JC-H of the free ligands and com-
1
H-NMR spectra display only two sets of signals belonging to two dis-
2
plexes as well (Table 4). The coordination to PdCl moiety caused an
tinct palladated ligands. The chemical shifts of one ligand bound in the
complex could be anticipated to generally be unaffected by the stereo-
chemistry of the other ligand in the complex. As these are invariantly
trans one to each other and separated by two rather long Pd-Se bonds,
this is easy to understand and would have to have another corollary—
one should expect the solution to behave as if having only two different
palladated ligand species with either like (R,R− or S,S−) or unlike
overall increase in the value of coupling of all directly bonded C and H
atoms by in average 2 Hz. A maximal positive increment of 5.9 Hz was
noted for H-2′/H-6′ in the tetrahydrofurane complex 1. This is an ex-
pected outcome of the presence of an electron-pulling substituent in
such a system. Due to either signal overlap or broadening of signals,
77
1
the low-intensity satellite signals arising for coupling of Se and H
13
13
1
or C were observable only in the C{ H} NMR spectra of the uncoor-
dinated ligands as shown in Table 4.
(
R,S− and S,R−) configurations at the Se and C-2 centers. According
to their calculated relative stabilities in solution, the proportions of
all six possible NMR observable trans-species in the solution could be
estimated to be grossly: 32.6% of SSSS and RRRR pair, 20.8% of RRSR
and SSRS pair, 19.3% RRRS and SSSR pair, 17.7% of RRSS meso-1, 7.6% of
RSSR and SRRS pair and 2.0% of RSRS meso-2. Thus, the ratio of the unlike
to like combinations of configurations would be 1:2.37, which is not in
2
.4. Biological activity
Screening of compounds 1 and 2 for their in vitro antimicrobial
activity revealed that these compounds possess inhibitory action
against all tested strains in the range from 0.15 to 5.00 mg/ml. The
results are presented in Table 5 where MIC and MBC are expressed
as the averages of five repetitions. Compound 1 exhibited inhibitory
activity in a concentration range 0.31–2.50 mg/ml, but a microbicidal ef-
fect was exhibited only at higher concentrations (2.50–5.00 mg/ml).
Compound 2 inhibited the growth of microorganisms from the
tested panel at concentrations from 0.15 to 5.00 mg/ml; however,
the microbicidal action was achieved only at the highest tested concen-
tration—5.00 mg/ml. Among the tested bacterial strains, Gram-positive
bacteria were shown to be slightly more sensitive to the action of both
compounds with both inhibitory and bactericidal action observed
in the tested concentration range. Staphylococcus aureus was the most
susceptible one, whereas B. cereus was the most resistant one.
Table 2
2 2
Selected bond distances [Å], angles and torsion angles [°] for [PdL2 Cl ].
Bond distances
Pd1–Cl1A
Pd1–Cl1
Pd1–Se1
Pd1–Se1A
Se1–C7
Se1–C6
O1–C5
O1–C1
Bond angles
2.2952(7)
2.2953(6)
2.4271(4)
2.4271(4)
1.921(2)
1.960(2)
1.434(3)
1.442(3)
Cl1A–Pd1–Cl1
Cl1A–Pd1–Se1
Cl1–Pd1–Se1
Se1–Pd1–Se1A
C7–Se1–C6
C7–Se1–Pd1
C6–Se1–Pd1
C5–C6–Se1
180.0
96.66(2)
83.34(2)
180.0
95.30(9)
100.68(6)
105.91(7)
112.0(2)
120.7(2)
C12–C7–Se1
With Gram-negative bacteria, compound 1 showed higher inhibito-
ry activity in comparison to compound 2, but the tested concentration
range was not high enough to achieve bactericidal action in the case
of P. vulgaris. Compound 2 exhibited both inhibitory and bactericidal
activity against all tested Gram-negative strains, but at higher concen-
trations (2.50 and 5.00 mg/ml). The highest sensitivity to the action
of both compounds was noted for Salmonella enteritidis. On the other
hand, fungal growth was inhibited at much lower concentrations of
Torsion angles:
Cl1–Pd1–Se1–C7
Cl1–Pd1–Se1–C6
Pd1–Se1–C7–C12
Pd1–Se1–C6–C5
−78.34(6)
−177.06(7)
−41.6(2)
−85.5(2)
Symmetry transformations used to generate equivalent atoms:
1 −x þ 1; −y þ 1; −z þ 1:
#

Z.M. Bugarčić et al. / Journal of Inorganic Biochemistry 143 (2015) 9–19
13
Table 3
1
H chemical and coordination shifts (in parentheses), alongside multiplicities and coupling constants (JH-H), for the free ligands (L1 and L2) and the corresponding [PdL
and 2 in CDCl (400 MHz) at ambient temperature.
2 2
Cl ] complexes 1
3
Compound
H-2c
L1
1 Ia
1 IIb
L2
2 Ia
2 IIb
d
d
4.08, pseudoquintet;
J = 6.7 Hz
1.61, ddt;
4.03, brs
(−0.05)
1.75–1.52, m, overlapping peaks
4.20, brs
(+0.12)
3.47, dddd;
J = 12.8, 6.9, 5.6, 2.2 Hz (ax)
1.31, tdd;
~3.23 ; m,
(−0.24)
~1.37 , m;
~3.73 ; m,
(+0.26)
~1.27 , m (ax)
d
d
H-3a
J = 12.1, 8.4, 7.2 Hz
2.05, dddd;
J = 12.1, 8.3, 6.5, 5.2 Hz (−0.05)
H-4a and H-4b 1.98–1.81, m; 1.99–1.79, m;
overlapping peaks, 2H
(+0.02)
2.00, brs
J = 12.8, 10.8, 3.5 Hz (ax)
1.80–1.73, m (eq)
overlapping peaks (ax), (+0.06)
~1.59 , m (eq)
(−0.04)
d
d
H-3b
2.08, brs
(+0.03)
~1.72 , m (eq)
(−0.18)
(−0.04)
d
d
~1.47 , m;
~1.38 , m, 1H (ax) (−0.09);
d
overlapping peaks (0)
overlapping peaks (ax);
~1.51 , m, 1H (ax) (+0.04);
d
1
.86–1.80, m (eq)
~1.81 , m, 2H (eq) (+0.02)
d
d
H-5a
3.75, ddd;
3.72, brquartet;
~1.48 , m;
~1.50 , m;
J = 8.3, 7.3, 6.1 Hz
J = 7.8 Hz
overlapping peaks
overlapping peaks, 4H (−0.02)
(−0.03)
d
H-5b
H-6
3.90, ddd;
J = 8.3, 6.9, 6.3 Hz
/
3.85, brs
(−0.05)
/
~1.55 , m;
overlapping peaks
4.00, ddd;
3.96, brd;
J = 11.7, 4.2, 2.1 Hz (eq);
J = 11.0 Hz, 2H (eq) (−0.04);
3.42, td; J = 11.7, 2.6 Hz (ax) ~3.43, m;
overlapping peaks (ax) (+0.01)
~3.38 , m
d
d
CHaHbSe
CHaHbSe
H-2′, H-6′
H-3′, H-5′
H-4′
2.98, dd;
J = 12.1, 6.8 Hz
3.11, dd,
J = 12.1, 5.8 Hz
7.54–7.49, m
3.52, brs
(+0.54)
3.26, brs
(+0.15)
2.92, brs
(−0.06)
3.78, brs
(+0.67)
2.92, dd;
J = 12.1, 5.6 Hz
3.06, dd;
J = 12.1, 6.9 Hz
7.52–7.48, m
~2.72 , m (−0.20)
(+0.46)
d
d
~3.26 , m; overlapping peaks (+0.20) ~3.79 , m (+0.73)
7.90, brd, J = 7.3 Hz
+0.39)
7.95–7.85, m, overlapping peaks
(+0.40)
7.38–7.30, m
(
7.27–7.20, m;
overlapping peaks, 3H
7.35, brt, J = 7.3 Hz
7.27–7.20, m;
overlapping peaks, 3H
7.41, brt, J = 7.3 Hz (+0.14)
7.46–7.38, m (+0.14)
a,b
Sets of signals corresponding to two diastereomeric palladated ligands (I, II) differing in the configurations at Se and C-2 chiral centers.
Proton numeration follows the IUPAC numbering scheme of the ligands (see Scheme 1).
Centers of these signals were determined from gHMQC spectra.
c
d
both compounds, but only compound 1 exhibited a fungicidal effect
against one yeast species (Candida albicans). The same fungal strain
was the one more susceptible to the action of both tested compounds.
The values of MIC and MBC observed in this study are in good agree-
ment with those reported previously for a similar set of microorganisms
that were treated with Pd(II) complexes with sulfur donor ligands [39].
Table 4
1
3C chemical and coordination shifts (in parentheses), alongside multiplicities and coupling constants (JC-H and JSe-C), for the free ligands (L1 and L2) and the corresponding palladium
2 2 3
complexes [PdL Cl ] complexes 1 and 2 in CDCl (101 MHz) at ambient temperature.
Compound
C-2c
L1
1 Ia
1 IIb
L2
2 Ia
2 IIb
78.3, brd;
75.0, brd;
76.1, brd;
77.0, brd;
73.6, brd;
75.2, brd;
1
1
1
1
1
1
J
C-H = 147.9 Hz;
JC-H = 149.7 Hz
J
C-H = 149.7 Hz
J
C-H = 140.9 Hz;
J
C-H = 139.8 Hz
JC-H = 141.8 Hz
2JSe-C = 6.8 Hz
(−3.3)
(−2.2)
2JSe-C = 6.6 Hz
(−3.4)
(−1.8)
C-3
C-4
C-5
C-6
31.5, brt;
31.8, brt;
31.8, brt;
31.8, brt;
1
1
1
1
J
C-H = 131.6 Hz
J
C-H = 132.0 Hz
+0.3)
25.9, brt;
J
C-H = 126.7 Hz
JC-H = 126.5 Hz
(
(0)
25.9, brt;
23.3, brt;
23.2, brt;
1
1
1
1
J
C-H = 131.3 Hz
J
C-H = 133.1 Hz
0)
68.3, brt;
J
C-H = 125.1 Hz
JC-H = 128.3 Hz
(
(−0.1)
68.3, brt;
25.8, brt;
25.6, brt;
1
1
1
1
J
C-H = 146.0 Hz
JC-H = 146.5 Hz
J
C-H = 125.4 Hz
JC-H = 124.9 Hz
(
/
0)
(−0.2)
/
68.7, ddt;
C-H = 145.7, 137.9, 7.7 Hz
33.7, t;
68.8, brt;
1
J
JC-H = 143.1 Hz (+0.1)
CH
2
Se
33.0, tdd;
C-H = 142.0, 5.9, 3.3 Hz;
40.0, brt;
39.4, brt;
40.1, brt;
1
1
1
1
J
JC-H = 142.9 Hz
J
C-H = 142.9 Hz
J
C-H = 141.2 Hz;
JC-H = 146.5 Hz
1
1JSe-C = 65.7 Hz
J
Se-C = 65.3 Hz
(+7.0)
128.1 , brs
(−2.2)
133.4 , brd;
(+6.4)
127.4 , brs
(−2.9)
133.0 , brd;
(+6.4)
128.5 , brs
d
d
e
e
C-1′
130.3, brs;
130.8, brs;
127.5 , brs
1
1
J
Se-C = 103.8 Hz
132.5, brdt;
C-H = 160.0, 7.3 Hz;
J
Se-C = 104.0 Hz
(−2.3)
(−3.3)
f
f
g
g
C-2', C-6'
132.3, brdt;
C-H = 160.3, 6.4 Hz;
133.2 , brd;
133.8 , brd;
1
1
J
J
C-H = 165.9 Hz
J
C-H = 165.9 Hz
J
J
C-H = 164.5 Hz
JC-H = 164.0 Hz
2
2
J
Se-C = 10.8 Hz
129.0, brdd;
C-H = 160.5, 8.3 Hz;
(+0.9)
129.7, brdd;
(+0.5)
J
Se-C = 10.8 Hz
129.0, brdd;
C-H = 160.3, 8.1 Hz;
(+1.0)
129.6, brdd;
JC-H = 162.2, 7.6 Hz
(+0.6)
(+1.5)
C-3', C-5'
C-4'
J
J
C-H = 162.1, 7.7 Hz
J
3
3
J
Se-C = 2.9 Hz
126.8, brdt;
C-H = 161.0, 7.5 Hz
(+0.7)
129.9, brdt;
J
Se-C = 2.7 Hz
126.7, brdt;
C-H = 161.0, 7.3 Hz
130.2, brd;
1
J
J
(
C-H = 160.6, 6.6 Hz
+3.1)
J
JC-H = 163.4 Hz
(+3.5)
a,b
Sets of signals corresponding to two diastereomeric palladated ligands (I, II) differing in the configurations at Se and C-2 chiral centers.
Values could be interchanged.
Carbon numeration follows the IUPAC numbering scheme of the ligands (see Scheme 1).
d,e,f,g
c

14
Z.M. Bugarčić et al. / Journal of Inorganic Biochemistry 143 (2015) 9–19
This is the very first report on the antimicrobial activity of selenium
containing Pd complexes in general, and in this study, we observed
that these compounds have a moderate broad spectrum antibacterial
activity with an equally effective action against both Gram-positive
and Gram-negative bacterial strains. In addition to this, the growth of
the mold and yeast organisms tested here was significantly more affect-
ed by the presence of these two complexes in their nutritive media in
comparison to the bacteria assayed.
the inoculated media, the amount of the complexes/ligands was deter-
mined by qNMR in the medium free from microbial cells and in the cells
themselves (C. albicans was chosen for this purpose due to its noted
sensitivity to the tested complexes).
The results of the experiments involving individual pure ligands
2
(L1 and L2) and PdCl (MIC values are given in Table 5) were rather
unexpected. The ligands demonstrated no activity what so ever in the
maximal tested concentration (20 mg/ml was the highest concentration
attained due to the immiscibility of the ligands and the nutritive medi-
In order to gain some insight into the mechanism of action of these
complexes, we undertook several additional experiments: (1) to test
2
um). On the other hand, PdCl showed activity which was invariantly 2-
the possible contribution of the sole ligands or PdCl
2
to the observed
to 8-fold higher when compared to the activity of complexes 1 and 2
antimicrobial effect, these were assayed on their own under identical
experimental settings; (2) to establish the fate of the complexes in
against the same bacterial strains. The only exceptions of this pattern
were the fungal organisms for which PdCl was less effective than
2
2 2
Se, as well as C-2-5 and CH
Se) of the 2D NMR spectra (A— H– H gDQCOSY, B—NOESY, C— H– C gHMQC and D— H–13C
1
1
1
13
1
Fig. 3. Expansions (signals corresponding to H-2-5 and CH
gHMBC) of [Pd(L1) Cl ] (1) in CDCl at ambient temperature. For designations of proton and carbon resonances confer with Tables 3 and 4, and Scheme 1.
2
2
3

Z.M. Bugarčić et al. / Journal of Inorganic Biochemistry 143 (2015) 9–19
15
Fig. 3 (continued).
complexes 1 and 2. We can conclude that at the heart of the observed
activities lies the Pd(II) ion and that its activity is modified by coordinat-
ing ligands.
liberated ligand (excess of chloride ions expelled ligands L1 and L2 as vis-
2
−
ible by change of pale yellow color ([PdCl
4
]
) of the suspensions upon
addition of NaCl) was exhaustively extracted with chloroform, and after-
wards quantified by qNMR.
The uptake of the complexes by the cells of C. albicans was determined
by a qNMR methodology assuming that the ligands on their own do not
cross the cell membrane in the given time frame (determined in an ear-
lier independent experiment). After an overnight incubation of a liquid
culture C. albicans with complexes 1 and 2 at half minimal inhibitory con-
centrations, the medium was first centrifuged to remove most of the cells
and then additionally filter-sterilized. The collected cells were washed to
get rid of the possibly adhering medium and subjected a complete lysis of
the cells by ultrasonification with a saturated solution of NaCl. The medi-
um was treated in the same manner as the cell debris suspension. The
Of the total amount of the complex introduced into the broth, the
majority of the detected complex remained in the nutritive medium—
74.0% and 20.2%, respectively, for complexes 1 and 2, although in dras-
tically differing amounts. On the other side, Candida cells absorbed a
similar proportion of the applied complexes, 11.6% and 7.6%, respective-
ly, for 1 and 2. The unaccounted portion of the complexes must have
undergone some chemical alteration during the incubation period,
which is more pronounced in the case of tetrahydropyran ligand L2.
About a tenth of the amount of the applied complexes was transported

16
Z.M. Bugarčić et al. / Journal of Inorganic Biochemistry 143 (2015) 9–19
Scheme 3. Possible stereoisomers of complexes 1 and 2 arising from differing configurations at Se and C-2 chiral centers.
across the cell membrane of C. albicans, while a significant portion either
remains unchanged in the nutritive medium or is biotransformed by the
microbial cells.
mechanism of action. To fully understand these differences between
bacterial and fungal cells, additional work is necessary and underway.
However, testing of the ligands and the inorganic salt precursor do
not help to understand which isomer of the mentioned ones is the ac-
tive one. Since the isomers equilibrate readily at room temperature, all
isomers are present in the nutritive medium and could be envisaged
as potential antimicrobials. Also, one can speculate that the presence
of the ligands intervenes with the transportation of Pd(II) species across
the cell membrane. Depending on the exact modus operandi, in this spe-
cific case, this can mean that the facilitated transport of complexes 1 and
3. Conclusion
Two novel Pd(II) complexes with 2-(phenylselanylmethyl)oxolane
and 2-(phenylselanylmethyl)oxane as ligands were synthesized, and
their crystal and molecular structure has been determined by single
crystal X-ray diffraction. A detailed NMR analysis of the solution chem-
istry of the two complexes was performed revealing an array of equili-
brating trans-diastereomeric species differing in the configuration at
four chiral centers (two at Se and two at C). We have further demon-
strated that these two complexes in vitro exhibit antimicrobial activity.
These compounds have a moderate broad spectrum antibacterial activ-
ity with an equally effective action against both Gram-positive and
2
could enable Pd(II) species to enter Candida cells and produce the ob-
served increase of activity when going from PdCl to PdL Cl . However,
this does not seem to be true for the bacteria tested since all strains were
2
2
2
2 2 2
more susceptible to PdCl and less to PdL Cl and suggests a different
Table 5
Antimicrobial activity of complexes 1 and 2, the corresponding ligands L1 and L2 and PdCl
2
(mmol L−1).
Bacterial/fungal strain⁎
ATCC
Complex 1
MIC
Complex 2
PdCl
MIC
L1
L2
Positive controls⁎⁎
2
MBC
MIC
MBC
MIC
MIC
G+
Staphylococcus aureus
Bacillus subtilis
Bacillus cereus
Proteus vulgaris
Escherichia coli
Salmonella enteritidis
Candida albicans
Aspergillus niger
25923
6633
9139
8427
8739
13076
10231
16404
1.89
1.89
3.79
3.79
1.89
1.89
0.47
0.47
3.79
7.58
7.58
N7.58
7.58
7.58
3.63
3.63
7.27
7.27
7.27
3.63
0.22
0.45
7.27
7.27
7.27
7.27
7.27
7.27
N7.27
N7.27
1.75
1.75
1.75
1.75
1.75
1.75
1.75
3.50
N83.00
N83.00
N83.00
N83.00
N83.00
N83.00
N83.00
N83.00
N78.00
N78.00
N78.00
N78.00
N78.00
N78.00
N78.00
N78.00
0.20
0.88
0.88
0.43
3.51
0.43
0.84
6.75
a
a
a
G−
a
a
a
b
Fungi
7.58
N7.58
b
⁎
The plates were incubated for 24 h at 37 °C (bacteria) and 48 h at 30 °C (fungi).
Antibiotics in μmol L−
Tetracycline.
1
.
⁎
⁎
a
b
Nystatin.

Z.M. Bugarčić et al. / Journal of Inorganic Biochemistry 143 (2015) 9–19
17
Gram-negative bacterial strains. On the other hand, fungal growth was
Table 6
2 2
Crystal data collection and structure refinement details for [PdL1 Cl ].
inhibited at much lower concentrations of both compounds. Also, we
have pinpointed that the activity of the complexes could be completely
attributed to the Pd(II) center. Although, this is the very first report on
the antimicrobial activity of selenium containing Pd complexes, it repre-
sents a good foundation for future work.
Empirical formula
C22H28Cl PdSe
2
2
Formula weight
Temperature
659.66 g/mol
100(2) K
Crystal system, space group
Unit cell dimensions
Tetragonal, P-42(1)c
ꢀ
ꢀ
ꢀ
a ¼ 14:4361ð1Þå α ¼ 90
b ¼ 14:4361ð1Þå β ¼ 90
4
. Experimental
c ¼ 11:3945ð1Þå γ ¼ 90
3
Volume
2374.63(3) Å
4
.1. Materials and methods
3
Z, Calculated density
Absorption coefficient
F(000)
4, 1.845 Mg/m
4.088 mm−
1
1296
All reagents and solvents employed were commercially available
and used as received without further purification. L1 and L2 were pre-
pared according to the literature [30].
Crystal size
Theta range for data collection
Limiting indices
0.18 × 0.10 × 0.07 mm
2.00 to 29.54°
−18 b = h b = 19,
−19 b = k b = 19,
−15 b = l b = 15
40672 / 3233 [R(int) = 0.0691]
100.0%
Semi-empirical from equivalents
0.746 and 0.643
3233/0/188
Gas-liquid chromatography (GLC) analyses were performed with
1
a Deni instrument, model 2000 with capillary apolar columns.
H
and 1 C NMR spectra were run in CDCl
3
Reflections collected/unique
Completeness to theta = 27.00
Absorption correction
Max. and min. transmission
Data/restraints/parameters
3
on a Varian Gemini 200 MHz
NMR spectrometer or as detailed in the Section 4.4. IR spectra were
obtained with Perkin-Elmer Model 137B and Nicolet 7000 FT spectro-
photometers. Elemental analyses were performed by Dornis u. Colbe
Mikroanalytisches Laboratorium, Mülheim and der Ruhr, Germany.
Thin-layer chromatography (TLC) was carried out on 0.25 mm E.
Merck precoated silica gel plates (60F-254) using UV light for visualiza-
tion. For column chromatography, E. Merck silica gel (60, particle size
2
Goodness-of-fit on F
1.011
Final R indices [I N 2σ(I)]
R indices (all data)
R
R
1
= 0.0289, wR
= 0.0490, wR
2
2
= 0.0521
= 0.0577
1
−3
Largest diff. peak and hole
0.381and −0.623 e.A
0
.063–0.200 mm) was used.
parameter). The complex molecule is situated on a crystallographic
twofold rotation axis (Wyckoff position 4c) and therefore possesses C
2
4
.2. Complexation of L1 and L2 with Pd(II)
molecular symmetry. The five-membered rings of the ligands are disor-
dered. Two alternative orientations of this ring were refined having ap-
proximately the same occupancy (49.6(1)% for O1–C5 and 50.4(1)% for
O1A–C5A). Crystal data and details regarding data collection and struc-
The title complexes were obtained by the general procedure given in
the short below. Ligand (0.3 mmol, 0.072 g for L1 or 0.3 mmol, 0.077 g
for L2) was dissolved in the mixture of 8 ml of EtOH and 2 ml of
MeOH and subsequently PdCl
2
(0.33 mmol, 0.058 g) was added into
2 2
ture refinement for [PdL1 Cl ] are summarized in Table 6. CCDC-977932
solution. The resulting mixture was stirred at 40 °C for 3 h. The color
changed from dark brown to orange-red. The precipitate was filtered
off and the solution concentrated by evaporation. The obtained solid
was collected, air-dried and recrystallized from ethanol. Complex 1: yel-
for [PdL1 Cl ] contains the supplementary crystallographic data. These
data can be obtained free of charge from the Cambridge Crystallograph-
ic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.
2
2
low crystals. Yield (0.088 g, 89%). Anal. calcd. (%) for C22
659.70 g/mol): C, 40.05; H, 4.28. Found: C, 40.15: H, 4.36.
Complex 2: orange crystals. Yield (0.086 g, 83%). Anal. calcd. (%)
for C24 32Cl PdSe (687.76 g/mol): C, 41.91; H, 4.69. Found: C, 41.94:
H, 4.72.
H
28Cl
2
O
2
PdSe
2
4.3.2. Crystal data for complex [PdL2
The complex molecule is situated on a crystallographic inversion cen-
ter and exhibits C molecular symmetry. Crystal data and details regarding
data collection and structure refinement for [PdL2 Cl ] are summarized
] contains the supplementary
2 2
Cl ]
(
i
H
2
O
2
2
2
2
2 2
in Table 7. CCDC-977933 for [PdL2 Cl
4
2 2 2 2
.3. X-ray structure determination of [PdL1 Cl ] and [PdL2 Cl ]
Table 7
Crystal data collection and structure refinement details for [PdL2
2 2
Cl ].
Suitable single crystals of [PdL1
from an ethanol/methanol mixture 4:1. The complex [PdL1
lizes in the tetragonal space group P-42(1)c, whereas the complex
PdL2 Cl ] is triclinic, P-1. Intensity data were collected on a Bruker-
SMART APEX2 diffractometer for complex 1 ([PdL1 Cl ]) and Bruker
Kappa APEX 2 IμS Duo for complex 2 ([PdL2 Cl ]) at 100 K using
Mo Ka radiation (λ = 0.71073 A, graphite monochromator for complex
and QUAZAR focussing Montel optics for complex 2). Data were
2
Cl
2
] and [PdL2
2
Cl
2
] were grown
Empirical formula
C
24
H
32 Cl
2
O
2
Pd Se
2
2
Cl ] crystal-
2
Formula weight
Temperature
Crystal system, space group
Unit cell dimensions
687.72 g/mol
100(2) K
Triclinic, P-1
[
2
2
a ¼ 8:592ð2Þå α ¼ 101:065ð4Þꢀ
2
2
ꢀ
ꢀ
b ¼ 9:299ð2Þå β ¼ 107:815ð4Þ
2
2
c ¼ 9:409ð2Þå γ ¼ 110:197ð4Þ
3
Volume
633.3(2) Å
1
3
Z, Calculated density
Absorption coefficient
F(000)
Crystal size
Theta range for data collection
Limiting indices
1, 1.803 Mg/m
3.836 mm
340
corrected for Lorentz and a polarization effects, a semi-empirical absorp-
tion correction was performed on the basis of multiple scans (SADABS,
Bruker AXS, 2009). The structure was solved by direct methods and
−1
0.12 × 0.08 × 0.05 mm
2.41° to 29.64°
−11 b = h b = 11,
−
−
12863/3550 [R(int) = 0.0250]
99.5%
Semi-empirical from equivalents
0.746 and 0.661
3550/0/142
2
refined by full-matrix least-squares procedures on F SHELXTL NT 6.12
(Bruker AXS, 2002). All non-hydrogen atoms were refined anisotropical-
12 b = k b = 11,
9 b = l b = 13
ly. Treatment of hydrogen atoms: All hydrogen atoms were placed in po-
sitions of optimized geometry, their isotropic displacement parameters
were tied to those of their corresponding carrier atoms by a factor of
Reflections collected/unique
Completeness to theta = 29.64
Absorption correction
Max. and min. transmission
Data/restraints/parameters
1
.2 or 1.5. For structure solution, refinement and graphical representa-
tions the SHELXTL NT 6.12 program package has been used [40].
2
Goodness-of-fit on F
1.042
4
.3.1. Crystal data for complex [PdL1
The crystal under study turned out to be an inversion twin with a
ratio of the two twin components of 42.5(9): 57.5(9)% (see Flack
2
Cl
2
]
Final R indices [I N 2σ(I)]
R indices (all data)
Largest difference peak and hole
R
R
1
= 0.0231, wR
= 0.0295, wR
2
2
= 0.0499
= 0.0518
1
−
3
0.828 and −0.402e.A

18
Z.M. Bugarčić et al. / Journal of Inorganic Biochemistry 143 (2015) 9–19
crystallographic data. These data can be obtained free of charge from the
Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_
request/cif.
(ATCC 8427), Escherichia coli (ATCC 8739), yeast C. albicans (ATCC
10231) and mold Aspergillus niger (ATCC 16404).
Bacterial strains were maintained on nutrient agar (NA), whereas
the fungal organisms were maintained on Sabouraud dextrose agar
(
SDA) at an appropriate optimal temperature (37 °C and 30 °C, respec-
4
.4. NMR measurements
tively) in the culture collection of the Microbiology laboratory, Depart-
ment of Biology and Ecology, Faculty of Science and Mathematics,
University of Niš.
3
All NMR spectra were recorded at 25 °C in CDCl with TMS
(
tetramethylsilane) as an internal standard. Chemical shifts are report-
1
ed in ppm (δ) and referenced to TMS (δ
and/or to CDCl (δ = 77.16 ppm) in heteronuclear 2D spectra. Scalar
H
= 0 ppm) in H NMR spectra
1
3
3 C
4
.6.2. Determination of MIC and MBC/MFC (minimal fungicidal)
couplings are reported in Hertz. Ten milligrams of a compound was dis-
solved in 1 ml of the deuterated solvent, and 0.7 ml of the solution was
transferred into a 5 mm Wilmad, 528-TR-7 NMR tube.
concentrations
Antimicrobial activity determination was performed by a micro-
dilution method as described previously [41]. Briefly, overnight cultures
of microorganisms were used for the preparation of suspensions. The
1
13
The H and C NMR spectra were recorded on a Bruker Avance III
1
13
5
4
00 MHz NMR spectrometer ( H at 400 MHz, C at 101 MHz), equipped
final size of the bacterial inoculum was 5 × 10 CFU (colony-forming
13
1
1
−
1
with a 5-mm dual C/ H probe head. The H spectra were recorded with
6 scans, 1 s relaxation delay, 4 s acquisition time, 0.125 Hz digital FID
free induction decay) resolution, 51 280 FID size, with 6410 Hz spectral
width, and an overall data point resolution of 0.0003 ppm. The C spectra
were recorded with Waltz 161H broadband decoupling, 12 000 scans,
unit) ml . Stock solutions of compounds 1 and 2 were prepared in
dimethylsulfoxide (DMSO, 100%) and then serially diluted (the diluting
factor 2). Dilution series were prepared in 96-well microtitre plates
in the concentration range from 0.002 to 20.00 mg/ml. The highest
concentration of the solvent (DMSO) in any well was 10% (v/v), which
was previously confirmed as concentration that does not affect the
growth of the tested cells. After attaining the right dilutions, the inocu-
lums were added to all wells. The plates were incubated for 24 h at 37 °C
1
(
13
0
6
.5 s relaxation delay, 1 s acquisition time, 0.5 Hz digital FID resolution,
5 536 FID size, 31 850 Hz spectral width, and an overall data point
resolution of 0.005 ppm.
Standard pulse sequences were used for 2D spectra. 1H– H
1
(
bacteria) and 48 h at 30 °C (fungi). Bacterial growth was determined by
gDQCOSY and NOESY spectra were recorded at spectral widths of
adding 20 μL of 0.5% triphenyltetrazolium chloride (TTC) aqueous solu-
tion [46]. One inoculated well was included to allow control of the broth
suitability for organism growth. One non-inoculated well, free of anti-
microbial agents, was also included to ensure medium sterility. Positive
controls were tetracycline and nystatin, whereas the vehicle (DMSO)
was used as the negative control. MIC was defined as the lowest concen-
tration of the compounds inhibiting visible growth (red colour pellet on
the bottom of wells after the addition of TTC), while the MBC/MFC
was defined as the lowest compound concentration killing 99.9% of
bacterial/fungal cells. To determine MBC/MFC, the broth was taken
from each well without visible growth and inoculated in Mueller Hinton
Agar (MHA) for 24 h at 37 °C and in SDA for 48 h at 30 °C in the case of
the tested yeast. Experiments were done in quintuplicate.
5
kHz in both F2 and F1 domains; 1 K × 512 data points were acquired
with 32 scans per increment and the relaxation delays of 2.0 s.
The mixing time in NOESY experiments was 1 s. Data processing
was performed on a 1 K × 1 K data matrix. Inverse-detected 2D
heteronuclear correlated spectra were measured over 512 complex
points in F2 and 256 increments in F1, collecting 128 (gHMQC) or 256
1
13
(
H– C gHMBC) scans per increment with a relaxation delay of 1.0 s.
The spectral widths were 5 and 27 kHz in F2 and F1 dimensions, respec-
tively. The gHMQC experiments were optimized for C–H couplings
1
13
of 165 Hz; the H– C gHMBC experiments were optimized for long-
range C–H couplings of 10 Hz. Fourier transforms were performed on
a 512 × 512 data matrix. π/2-shifted sine-squared window functions
were used along F1 and F2 axes for all 2D spectra.
4
.6.3. Quantitative nuclear magnetic resonance (qNMR)
Quantitative NMR experiments were performed according to a pro-
4
.5. Computational methods
cedure described in Radulović et al. [47]. After an overnight incubation
of a liquid culture C. albicans with complexes 1 and 2 at half minimal in-
hibitory concentrations, the medium was first centrifuged to remove
most of the cells, and then additionally filter-sterilized. The collected
cells were washed to get rid of the possibly adhering medium and sub-
jected a complete lysis of the cells by ultrasonification with a saturated
solution of NaCl. The supernatant medium was treated in the same
manner as the cell debris suspension. Excess of chloride ions were
All calculations were performed with the Gaussian 09 program
package [41] using the M06 functional. This hybrid meta functional
was developed by Zhao and Truhlar as “a functional with good accuracy
across-the-board’ for transition metals, main group thermochemistry,
‘
medium-range correlation energy, and barrier heights” [42]. They
recommended this method “for application in organometallic and
inorganometallic chemistry and for noncovalent interactions” [43].
The 6-311 + G(d,p) basis set was applied for C, H, N, O, Cl and Se, where-
as the Def2-TZVPD basis set [44] was used for Pd. These triple split
valence basis sets add the polarization functions to all atoms and diffuse
functions to the heavy atoms. The structures of all investigated species
in chloroform were optimized and frequency calculations performed.
The influence of the solvent (dielectric constant = 4.7113) was taken
into account by applying the CPCM solvation model (Polarizable
Conductor Calculation Model) [45]. The obtained stationary points
were verified to be equilibrium geometries (no imaginary frequencies).
used to expel ligands L1 and L2 (visible by the change to a pale yellow
2−
color of [PdCl
4
]
) from the complexes and the liberated ligands were
SO
exhaustively extracted with chloroform, dried over anhydrous Na
2
4
and evaporated to dryness. The samples were weighted, dissolved in
deuterated chloroform and a known amount of anthracene was added
as an internal standard (no changes to the appearance of the spectra
1
were noted after the addition of the standard compound). H NMR spec-
13
tra with C decoupling and a large data set (10 points per Hz digital res-
olution) were recorded. Signal-to-noise ratio of 1000:1 or higher was
obtained for all recordings. Parameters were as follows: number of
points in the time domain = 32 k, spectral width = 10 ppm, O1 =
4
4
.6. Screening of antimicrobial activity
6
.0 ppm, p1 = 45° 1H transmitter pulse, acquisition time = 5 s and
.6.1. Microorganisms and culture conditions
Antimicrobial activity assays were performed against eight American
Type Culture Collection (ATCC) strains: Gram-positive Staphylococcus
aureus (ATCC 25923), Bacillus subtilis (ATCC 6633), Bacillus cereus (ATCC
139), Gram-negative S. enteritidis (ATCC 13076), Proteus vulgaris
number of scans = 1024. After zero-filling and phase and baseline cor-
rections, the integration of signals (8.43 ppm for anthracene; 2.98, 3.75
or 3.90 ppm for L1 and 2.92, 3.42 or 4.00 ppm for L2) was performed.
The ratio of the signal integrals was used to calculate the amount of
the two ligands in the medium and inside Candida cell. The results are
9

Z.M. Bugarčić et al. / Journal of Inorganic Biochemistry 143 (2015) 9–19
19
[
[
[
[
15] S. Dey, V.K. Jain, S. Chaudhury, A. Knoedler, F. Lissner, W. Kaim, J. Chem. Soc. Dalton
Trans. (2001) 723–728.
16] S. Dey, V.K. Jain, A. Knödler, A. Klein, W. Kaim, S. Zalis, Inorg. Chem. 41 (2002)
expressed as percentages of the initial load (amount of the complexes
added to the broths in the beginning of the experiments).
2864–2870.
17] S. Dey, L.B. Kumbhare, V.K. Jain, T. Schurr, W. Kaim, A. Klein, F. Belaj, Eur. J. Inorg.
Chem. (2004) 4510–4520.
18] W. Levason, S.D. Orchard, G. Reid, Coord. Chem. Rev. 225 (2002) 159–199.
Abbreviations
RA
rheumatoid arthritis
MIC
MMC
MFC
MHA
SDA
minimum inhibitory concentration
minimum microbicidal concentration
minimum fungicidal concentration
Mueller–Hinton agar
[19] P. Bippus, A. Molter, D. Müller, F. Mohr, J. Organomet. Chem. 695 (2010) 1657–1662.
[20] R.S. Chauhan, R.K. Sharma, G. Kedarnath, D.B. Cordes, A.M.Z. Slawin, V.K. Jain, J.
Organomet. Chem. 717 (2012) 180–186.
[
[
21] A. Bredenkamp, X. Zeng, F. Mohr, Polyhedron 33 (2012) 107–113.
22] A. Molter, F. Mohr, Coord. Chem. Rev. 254 (2010) 19–45.
Sabouraud dextrose agar
[23] T.R. Todorović, A. Bacchi, D.M. Sladić, N.M. Todorović, T.T. Božić, D.D. Radanović, N.R.
Filipović, G. Pelizzi, K.K. Anđelković, Inorg. Chem. Acta 362 (2009) 3813–3820.
gDQCOSY gradient double quantum filtered correlation spectroscopy
gHMQC gradient heteronuclear multiple quantum coherence
gHMBC gradient heteronuclear multiple bond coherence
[
[
24] G. Zhao, H. Lin, S. Zhu, H. Sun, Y. Chen, J. Inorg. Biochem. 70 (1998) 219–226.
25] N. Gligorijević, T. Todorović, S. Radulović, D. Sladić, N. Filipović, D. Gođevac, D.
Jeremić, K. Anđelković, Eur. J. Med. Chem. 44 (2009) 1623–1629.
26] P. Scovill, D.L. Klayman, C.F. Franchino, J. Med. Chem. 25 (1982) 1261–1264.
27] D.L. Klayman, J.P. Scovill, US4665173, 1987.
28] M. Carland, B.F. Abrahams, T. Rede, J. Stephenson, V. Murray, W.A. Denny, W.D.
McFadyen, Inorg. Chem. Acta 359 (2006) 3252–3256.
29] A. Molter, C.W. Lehmann, G. Deepa, P. Chiba, F. Mohr, Dalton Trans. 40 (2011)
[
[
[
qNMR
FID
quantitative nuclear magnetic resonance
free induction decay
CPCM
ATCC
TTC
polarizable conductor calculation model
American-type culture collection
triphenyltetrazolium chloride
[
9810–9820.
[
30] Z.M. Bugarcic, B.M. Mojsilovic, V.M. Divac, 272 (2007) 288–292.
NOESY nuclear Overhauser effect/enhancement spectroscopy
[31] N.D. Ghavale, A. Wadawale, S. Dey, V.K. Jain, Indian J. Chem. Sect. A 48 (2009)
510–1514.
1
[
[
32] R.K. Chadha, J.M. Chehayber, J.E. Drake, Inorg. Chem. 25 (1986) 611–615.
33] L. Vigo, M. Risto, E.M. Jahr, T. Bajorek, R. Oilunkaniemi, R.S. Laitinen, M. Lahtinen, M.
Ahlgren, Cryst. Growth Des. 6 (2006) 2376–2383.
Acknowledgement
[
[
[
[
[
[
34] R. Oilunkaniemi, J. Komulainen, R.S. Laitinen, M. Ahlgren, J. Pursiainen, J. Organomet.
Chem. 571 (1998) 129–138.
35] S. Konstantinovic, Z. Bugarcic, S. Milosavljevic, G. Schroth, M.Lj. Mihailovic, Justus
Liebigs Ann Chem 3 (1992) 261–268.
36] S. Konstantinovic, Z. Bugarcic, R. Vukicevic, W. Wisniewski, Z. Ratkovic, Z. Markovic,
M.Lj. Mihailovic, J. Serb. Chem. Soc. 62 (1997) 307–317.
37] E.W. Abel, N.J. Long, K.G. Orrell, A.G. Osborne, V. Šik, P.A. Bates, M.B. Hursthouse,
J. Organomet. Chem. 367 (1989) 275–289.
38] E.W. Abel, N.J. Long, K.G. Orrell, A.G. Osborne, V. Šik, P.A. Bates, M.B. Hursthouse,
J. Organomet. Chem. 383 (1990) 253–269.
This work was funded by the Ministry of Education and Science of
the Republic of Serbia (grant nos. 172011 and 172061).
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.jinorgbio.2014.11.002.
39] A. Garoufis, S.K. Hadjikakou, N. Hadjiliadis, Coord. Chem. Rev. 253 (2009)
1384–1397.
[
[
40] SHELXTL NT 6.12, Bruker AXS, Inc., Madison, WI, U.S.A., 2002
References
41] M.J. Frisch, G.W. Trucks, H.B. Schlegel, G.E. Scuseria, M.A. Robb, J.R. Cheeseman, G.
Scalmani, V. Barone, B. Mennucci, G.A. Petersson, H. Nakatsuji, M. Caricato, X. Li,
H.P. Hratchian, A.F. Izmaylov, J. Bloino, G. Zheng, J.L. Sonnenberg, M. Hada, M.
Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O.
Kitao, H. Nakai, T. Vreven, J.A. Montgomery Jr., J.E. Peralta, F. Ogliaro, M. Bearpark,
J.J. Heyd, E. Brothers, K.N. Kudin, V.N. Staroverov, R. Kobayashi, J. Normand, K.
Raghavachari, A. Rendell, J.C. Burant, S.S. Iyengar, J. Tomasi, M. Cossi, N. Rega, J.M.
Millam, M. Klene, J.E. Knox, J.B. Cross, V. Bakken, C. Adamo, J. Jaramillo, R.
Gomperts, R.E. Stratmann, O. Yazyev, A.J. Austin, R. Cammi, C. Pomelli, J. Ochterski,
R.L. Martin, K. Morokuma, V.G. Zakrzewski, G.A. Voth, P. Salvador, J.J. Dannenberg,
S. Dapprich, A.D. Daniels, O. Farkas, J.B. Foresman, J.V. Ortiz, J. Cioslowski, D.J. Fox,
GAUSSIAN 09 Wallingford, CT, 2009.
[1] G. Mugesh, W. du Mont, H. Sies, Chem. Rev. 101 (2001) 2125.
[2] E.G. Hope, W. Levason, Coord. Chem. Rev. 122 (1993) 109–170.
[3] M.K. Davies, M.C. Durrant, W. Levason, G. Reid, R.L. Richards, J. Chem. Soc. Dalton
Trans. (1999) 1077–1084.
[
[
4] J. Arnold, in: K.D. Karlin (Ed.), Progress in inorganic chemistry, The Chemistry of
Metal Complexes with Selenolate and Tellurolate Ligands, vol. 43, John Wiley &
Sons, Inc., 1995, p. 353.
5] A. Kumar, G.K. Rao, F. Saleem, A.K. Singh, J. Chem. Soc. Dalton Trans. 41 (2012)
11949–11977.
[6] E.E. Battin, N.R. Perron, J.L. Brumaghim, Inorg. Chem. 45 (2006) 499–501.
[7] E.E. Battin, J.L. Brumaghim, Cell Biochem. Biophys. 55 (2009) 1–23.
[8] P.D. Whanger, J. Trace Elem. Electrolytes Health Dis. 6 (1992) 209–221.
[9] H. Gurer, N. Ercal, Free Radical Biol. Med. 29 (2000) 927–945.
[42] Y. Zhao, D.G. Truhlar, Acc. Chem. Res. 41 (2008) 157–167.
[43] Y. Zhao, D.G. Truhlar, Theor. Chem. Acc. 120 (2008) 215–241.
[44] D. Rappoport, F. Furche, J. Chem. Phys. 133 (2010) 134105.
[
[
10] P.C. Hsu, Y.L. Guo, Toxicology 180 (2002) 33–44.
11] K.P. Bhabak, B.J. Bhuyan, G. Mugesh, J. Chem. Soc. Dalton Trans. 40 (2011)
[
45] J. Tomasi, B. Mennucci, R. Cammi, Chem. Rev. 105 (2005) 2999–3093.
2
099–2111.
12] V.N. Gladyshev, S.V. Khangulov, M.J. Axley, T.C. Stadtman, Proc. Natl. Acad. Sci. U. S. A.
1 (1994) 7708–7711.
13] H. Raaijmakers, S. Macieira, J.M. Dias, S. Teixeira, S. Bursakov, R. Huber, J.J.G. Moura, I.
Moura, M.J. Romão, Structure 10 (2002) 1261–1272.
[46] D. Ilić, I. Damljanović, D. Stevanović, M. Vukićević, P. Blagojević, N. Radulović, R.
Vukićević, Chem. Biodivers. 9 (2012) 2236–2253.
[47] N.S. Radulović, D.B. Zlatković, T. Ilić-Tomić, L. Senerovic, J. Nikodinovic-Runic, J.
Ethnopharmacol. 153 (2014) 125–132.
[
[
[
9
14] A. Panda, S.C. Menon, H.B. Singh, C.P. Morley, R. Bachman, T.M. Cocker, R.J. Butcher,
Eur. J. Inorg. Chem. 6 (2005) 1114–1126.