Di- and polynuclear silver(I) saccharinate complexes of tertiary diphosphane ligands: Synthesis, structures, in vitro DNA binding, and antibacterial and anticancer properties

Article abstract of DOI:10.1007/s00775-013-1052-y

A series of new silver(I) saccharinate (sac) complexes, [Ag ₂(sac)₂(μ-dppm)H₂O]·H₂O (1), {[Ag₂(μ-sac)₂(μ-dppe)]·3H ₂O·CH₂Cl₂} _n (2), [Ag ₂(μ-sac)₂(μ-dppp)] _n (3), and [Ag(sac)(μ-dppb)] _n (4) [dppm is 1,1-bis(diphenylphosphino) methane, dppe is 1,2-bis(diphenylphosphino)ethane, dppp is 1,3- bis(diphenylphosphino)propane, and dppb is 1,4-bis(diphenylphosphino)butane], have been synthesized and characterized by C, H, N elemental analysis, IR spectroscopy, ¹H NMR, ¹³C NMR, and ³¹P NMR spectroscopy, electrospray ionization mass spectrometry, and thermogravimetry-differential thermal analysis. Single-crystal X-ray studies show that the diphosphanes act as bridging ligands to yield a dinuclear complex (1) and one-dimensional coordination polymers (2 and 4), whereas the sac ligand adopts a μ₂-N/O bridging mode in 2, and is N-coordinated in 1 and 4. The interaction of the silver(I) complexes with fish sperm DNA was investigated using UV-vis spectroscopy, fluorescence spectroscopy, and agarose gel electrophoresis. The binding studies indicate that the silver(I) complexes can interact with fish sperm DNA through intercalation, and complexes 1 and 3 have the highest binding affinity. The gel electrophoresis assay further confirms the binding of the complexes with the pBR322 plasmid DNA. The minimum inhibitory concentrations of the complexes indicate that complex 1 exhibits very high antibacterial activity against standard bacterial strains of Escherichia coli, Salmonella typhimurium, and Staphylococcus aureus, being much higher than those of AgNO₃, silver sulfadiazine, ciprofloxacin, and gentamicin. Moreover, complexes 1-3 exhibit very high cytotoxic activity against A549 and MCF-7 cancer cell lines, compared with AgNO₃ and cisplatin. The bacterial and cell growth inhibitions of the silver(I) complexes are closely related to their DNA binding affinities.

Full text of DOI:10.1007/s00775-013-1052-y

J Biol Inorg Chem (2014) 19:29–44
DOI 10.1007/s00775-013-1052-y
ORIGINAL PAPER
Di- and polynuclear silver(I) saccharinate complexes of tertiary
diphosphane ligands: synthesis, structures, in vitro DNA binding,
and antibacterial and anticancer properties
•
Veysel T. Yilmaz Elif Gocmen Ceyda Icsel
•
•
•
Murat Cengiz Sunde Y. Susluer Orhan Buyukgungor
•
Received: 10 July 2013 / Accepted: 28 September 2013 / Published online: 17 October 2013
Ó SBIC 2013
Abstract A series of new silver(I) saccharinate (sac)
complexes, [Ag2(sac)2(l-dppm)H2O]ꢀH2O (1), {[Ag2(l-sac)2
(l-dppe)]ꢀ3H2OꢀCH2Cl2}n (2), [Ag2(l-sac)2(l-dppp)]n (3),
and [Ag(sac)(l-dppb)]n (4) [dppm is 1,1-bis(diphenyl-
phosphino)methane, dppe is 1,2-bis(diphenylphosphino)
ethane, dppp is 1,3-bis(diphenylphosphino)propane, and dppb
is 1,4-bis(diphenylphosphino)butane], have been synthesized
and characterized by C, H, N elemental analysis, IR spec-
N/O bridging mode in 2, and is N-coordinated in 1 and 4. The
interaction of the silver(I) complexes with fish sperm DNA
was investigated using UV–vis spectroscopy, fluorescence
spectroscopy, and agarose gel electrophoresis. The binding
studies indicate that the silver(I) complexes can interact with
fish sperm DNA through intercalation, and complexes 1 and 3
havethehighestbindingaffinity.Thegelelectrophoresisassay
further confirms the binding of the complexes with the
pBR322 plasmid DNA. The minimum inhibitory concentra-
tions of the complexes indicate that complex 1 exhibits very
high antibacterial activity against standard bacterial strains of
Escherichia coli, Salmonella typhimurium, and Staphylococ-
cus aureus, being much higher than those of AgNO3, silver
sulfadiazine, ciprofloxacin, and gentamicin. Moreover, com-
plexes 1–3 exhibit very high cytotoxic activity against A549
and MCF-7 cancer cell lines, compared with AgNO3 and
cisplatin. The bacterial and cell growth inhibitions of the sil-
ver(I) complexes are closely related to their DNA binding
affinities.
1
troscopy, H NMR, 13C NMR, and 31P NMR spectroscopy,
electrospray ionization mass spectrometry, and thermogravi-
metry–differential thermal analysis. Single-crystal X-ray
studies show that the diphosphanes act as bridging ligands to
yield a dinuclear complex (1) and one-dimensional coordi-
nation polymers (2 and 4), whereas the sac ligand adopts a l2-
V. T. Yilmaz (&) ꢀ E. Gocmen ꢀ C. Icsel
Department of Chemistry,
Faculty of Arts and Sciences,
Uludag University,
16059 Bursa, Turkey
e-mail: vtyilmaz@uludag.edu.tr
Keywords Silver(I) complexes ꢀ Saccharinate ꢀ
Tertiary diphosphanes ꢀ DNA binding ꢀ Antibacterial
activity
M. Cengiz
Department of Pharmacology and Toxicology,
Faculty of Veterinary Medicine,
Uludag University,
Introduction
16059 Bursa, Turkey
S. Y. Susluer
Elemental silver and silver(I) compounds have been used
as antimicrobial agents for decades [1–4], and the anti-
microbial activity depends on the release of Ag? ions.
Silver sulfadiazine (AgSD) was introduced as an anti-
bacterial and antifungal agent to prevent bacterial infec-
tions in cases of severe burns [5–11]. AgSD has a
polymeric structure, in which the silver(I) ions are coor-
dinated by the nitrogen atoms of the pyrimidine ring and
Department of Medical Biology,
Medical Faculty, Ege University,
35100 Izmir, Turkey
O. Buyukgungor
Department of Physics,
Faculty of Arts and Sciences,
Ondokuz Mayis University,
55139 Samsun, Turkey
123

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J Biol Inorg Chem (2014) 19:29–44
sulfonyl oxygen atoms of sulfadiazine molecules [12, 13].
Its effectiveness as an antimicrobial agent is due to its
tendency to dissociate in solution, providing a steady
supply of silver(I) ions over a period of time [6]. The
mechanism for the antimicrobial action of silver(I) ions is
not properly understood; however, it is generally accepted
that they interfere with cell growth in three ways: (1)
inhibition of transport functions in the cell wall, (2)
interruption of cell metabolism, and (3) interaction with
DNA [14–16]. Moreover, it was concluded that the
magnitude of antimicrobial properties of silver complexes
is related to the ease with which they participate in ligand
exchange reactions [17]. The biological activity of sil-
ver(I) complexes with oxygen-donor ligands comes from
the weaker bonding property of the Ag–O bonds. In the
biological system, the ease of ligand replacement of the
silver(I) complexes will result in further replacement with
biological ligands as protein, enzymes, and membrane-
containing thiol groups in their active sites [18–21].
The water-soluble alkali salts of saccharin (also named
1,1-dioxo-1,2-benzothiazol-3-one or o-benzosulfimide) are
widely used as a noncaloric artificial sweetener and food
additive. The imino hydrogen of saccharin is acidic and
thus in solution the molecule can be easily converted into
the corresponding nitranion, saccharinate (sac). Sac has
different coordination sites, such as one negatively charged
imino nitrogen atom, one carbonyl oxygen atom, and two
sulfonyl oxygen atoms. As a polyfunctional ligand, it is
able to form metal complexes from mononuclear to coor-
dination polymers, but in the case of bulky ligands, it
sometimes remains outside the coordination sphere as a
counterion [22]. Although a large number of metal com-
plexes of sac have been reported in the literature, their
biological activities have received less attention and only a
few metal complexes of sac with anticancer activity have
been reported. For example, K[Pt(sac)3(H2O)] and
[(PTA)Au(sac)] (PTA is 1,3,5-triaza-7-phosphaada-
mantane) exhibited strong antigrowth effects on HeLa and
A2780 cells, respectively [23, 24]. We recently reported a
number of highly cytotoxic palladium(II) and platinum(II)
complexes of sac with bis(2-pyridylmethyl)amine [25],
2,20:60,200-terpyridine [26–28], 2-(hydroxymethyl)pyridine
[29], and 2-(2-hydroxyethyl)pyridine [29]. The DNA
binding studies indicated that the metal sac complexes with
2,20:60,200-terpyridine interact with fish sperm DNA (FS-
DNA) strongly via both intercalation and coordination as
dual-function metallointercalators [30].
and antitumor properties of silver(I) compounds. The lit-
erature shows that in some cases, silver(I) complexes
exhibit significant anticancer activity compared with cis-
platin, a clinically used anticancer drug.
The synthesis and structures of silver(I) sac complexes
with phosphine ligands were not studied extensively, and
there is only one example, [Ag(sac)(PPh3)2] [33]. In addi-
tion, to the best of our knowledge, there is as yet little
information available concerning biological evaluation of
silver(I) complexes of sac, and only two studies of their
antimicrobial activities have been reported [34, 35]. It was
found that [Ag(sac)]n was very effective on a wide range of
bacteria [35]. The encouraging results with regard to the
antibacterial and anticancer activities of the metal complexes
of sac led us to design a series of new silver(I) complexes
bearing both sac and diphosphine ligands (named as phos-
phane by IUPAC), namely, [Ag2(sac)2(l-dppm)H2O]ꢀH2O
(1), {[Ag2(l-sac)2(l-dppe)]ꢀ3H2OꢀCH2Cl2}n (2), [Ag2(l-
sac)2(l-dppp)]n (3), and [Ag(sac)(l-dppb)]n (4) [dppm is
1,1-bis(diphenylphosphino)methane,
dppe
is
1,2-
bis(diphenylphosphino)ethane, dppp is 1,3-bis(diphenyl-
phosphino)propane, and dppb is1,4-bis(diphenylphos-
phino)butane] (Fig. 1)]. The interactions of these complexes
with DNA were studied using absorption and fluorescence
spectroscopy, and gel electrophoresis measurements. Fur-
thermore, in vitro antibacterial activity tests were performed
on Gram-negative and Gram-positive bacteria, and in vitro
anticancer activities were tested against two human cancer
(CH2)n
n = 1 dppm
P
P
n = 2 dppe
n = 3 dppp
n = 4 dppb
3
4
2
5
1
6
Sac
O
O
S
C
O
N
In addition to their antimicrobial properties, the anti-
cancer activities of silver(I) complexes have received great
attention in the last decade. The main advantage of the use
of silver(I) complexes in the development of new anticancer
drugs is their low toxicity toward humans [31]. Recently,
Banti and Hadjikakou [32] reviewed the antiproliferative
Fig. 1 Tertiary diphosphane and saccharinate (sac) ligands used in
the synthesis of [Ag2(sac)2(l-dppm)H2O]ꢀH2O (1), {[Ag2(l-sac)2(l-
dppe)]ꢀ3H2OꢀCH2Cl2}n (2), [Ag2(l-sac)2(l-dppp)]n (3), and
[Ag(sac)(l-dppb)]n (4), and the numbering scheme for NMR
spectroscopy
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J Biol Inorg Chem (2014) 19:29–44
31
cell lines (A549 and MCF-7) as well as a normal cell line
(WI-38).
(m, 28H, Ph-sac and Ph-dppm), 2.05 (s, 2H, CH2-dppm).
13C NMR (100 MHz, DMSO-d6, d, ppm): 168.33 (C=O-
sac), 144.19 (C6-sac), 133.72 (d, Cipso-P), 133.27 (d, C4-
sac), 133.12 (C3-sac), 132.89 (C1-sac), 132.05 (Ph-Cortho
-
Materials and methods
P), 131.21 (Ph-Cpara-P), 129.06d (Ph-Cmeta-P), 123.83
(C2-sac), 120.46 (C5-sac). 31P NMR (200 MHz, DMSO-
1
All chemicals used in the experiments were commercial
products and were used without further purification.
Elemental analyses for carbon, hydrogen, and nitrogen
were performed using a Costech elemental analyzer. UV–
vis spectra were measured with a PerkinElmer Lambda
35 spectrophotometer. IR spectra were recorded from 400
to 4,000 cm-1 with a Thermo Nicolet 6700 Fourier
transform (FT) IR spectrophotometer and are reported
with the following abbreviations: vs (very strong), s
d6, d, ppm): 8.82 [t, J(31P-Ag) = 504, 688 Hz]. FT-IR
(KBr, cm-1): 3,044 (w) m(Carom-H)], 2,913 (w), 2,844
(vw) m(Calf-H), 1,639 (vs) m(C=O), 1,332 (m) ms(CNS),
1,291 (vs), 1,258 (s) mas(SO2), 1,153 (vs) ms(SO2), 1,111
(s), 960 (s) mas(CNS), 754 (s), 691 (m) m(C–P), 679 (m),
601 (s), 510 (m). ESI? mass spectrometry (MS) (MeOH)
m/z: 491.0 [Ag(dppm)]?, 634.9 [Ag2(sac)(MeOH)Na]?,
781.9 [Ag2(sac)(dppm)]?, 1,019.0 [Ag2(dppm)2Cl]?,
1,166.0 [Ag2(sac)(dppm)2]?, 1,309.9 [Ag3(sac)3(dppm)
(MeOH)Na]?, 1,547.0 [Ag2(sac)(dppm)3]. KM (MeOH,
25 °C, 10-3 M) 58 S cm2 mol-1. KM (DMSO, 25 °C,
1
(strong), m (medium), w (weak), and vw (very weak). H
NMR and 13C NMR spectra were recorded with a Varian
Mercuryplus spectrometer at 400 and 100 MHz, respec-
tively, in dimethyl-d6 sulfoxide (DMSO-d6) using tetra-
methylsilane as an internal reference at room temperature,
whereas 31P NMR spectra were recorded with an INO-
VA-500 spectrometer at 200 MHz in DMSO-d6. Thermal
analysis curves [thermogravimetry (TG) and differential
thermal analysis (DTA)] were obtained using a Seiko
Exstar TG/DTA 6200 thermal analyzer in a flowing air
atmosphere with a heating rate of 10 °C min-1 using a
sample size of 5–10 mg and platinum crucibles. Fluo-
rescence spectra were recorded at room temperature with
a Varian Cary Eclipse spectrophotometer equipped with a
xenon pulse lamp of 75 kW. For all fluorescence mea-
surements, both slits were maintained at 5 nm. The
electrospray ionization (ESI) mass spectra were recorded
using a Bruker Daltonics micrOTOF II ESI time-of-flight
mass spectrometer. The electrical conductivity measure-
ments of MeOH and DMSO solutions of the sil-
ver(I) complexes were performed with an Inolab Cond
730 conductimeter at room temperature and are reported
as KM (S cm2 mol-1).
10-3 M) 14 S cm2 mol-1
.
Synthesis of 2
Na(sac)ꢀ2H2O (0.06 g, 0.25 mmol) in water (5 mL) was
added to a solution of AgNO3 (0.04 g, 0.25 mmol) in water
(5 mL). The solution immediately became milky. The addi-
tion of MeCN (10 mL) to the milky suspension resulted in a
clear solution. The addition of dppe (0.05 g, 0.125 mmol) in
CH2Cl2 (5 mL) to the solution resulted in a biphasic solution,
which turned into a monophasic clear solution on addition of a
mixture of DMSO and MeCN (10 mL, 2:1). The resulting
solution was allowed to stand in darkness at room tempera-
ture. A colorless polycrystalline powder precipitated within
4 days. Yield 0.096 g, 70 %. Melting point 276–285 °C
(decomp.). Anal. Calcd for C41H40Ag2Cl2N2O9P2S2 (%): C,
44.10; H, 3.61; N, 2.50. Found (%): C, 44.61; H, 3.89; N, 2.44.
1H-NMR (400 MHz, DMSO-d6, d, ppm): 7.95–7.36 (m, 28H,
Ph-sac and Ph-dppe), 5.77 (s, 2H, CH2Cl2), 2.57 (s, 4H, CH2-
dppe). 13C NMR (100 MHz, DMSO-d6, d, ppm): 167.54
(C=O-sac), 144.17 (C6-sac), 133.38 (d, Cipso-P), 133.19 (C4-
sac), 133.03 (C3-sac), 132.31 (d, Ph-Cortho-P), 132.02 (Ph-
Synthesis of 1
Cpara-P), 131.34 (C1-sac), 129.49 (d, Ph-Cmeta-P), 123.80
(C2-sac), 120.42 (C5-sac), 55.37 (C-CH2Cl2). 31P NMR
(200 MHz, DMSO-d6, d, ppm): 10.04 [d, 1J(31P-
Ag) = 579 Hz]. FT-IR (KBr, cm-1): 3,085 (w), 3,056 (w),
3,015 (vw) m(Carom-H), 2,909 (w), 2,852 (vw) m(Calf-H), 1,655
(vs) m(C=O), 1,332 (m) ms(CNS), 1,279 (vs), 1,246 (vs)
mas(SO2), 1,140 (vs) ms(SO2), 1,111 (s), 960 (vs) mas(CNS), 747
(s), 731 (s), 690 (s) m(C–P), 669 (s), 600 (s), 510 (m). ESI?-
MS (MeOH) m/z: 461.1 [Ag2(sac)(MeOH)2]?, 507.0
[Ag(dppe)]?, 706.0 [Ag3(sac)2(H2O)]?, 752.0 [Ag3(sac)2
(MeOH)2]?, 795.9 [Ag2(sac)(dppe)]?, 859.2 [Ag2(sac)(dp-
pe)(MeOH)2]?, 905.2 [Ag(dppe)2]?. KM (MeOH, 25 °C,
10-3 M) 29 S cm2 mol-1. KM (DMSO, 25 °C, 10-3 M)
A solution of Na(sac)ꢀ2H2O (0.06 g, 0.25 mmol) in
MeCN/MeOH (10 mL, 1:1) was added dropwise to a
solution of AgNO3 (0.04 g, 0.25 mmol) in MeOH (5 mL)
and the mixture was stirred for 30 min at room tempera-
ture. Then, dppm (0.05 g, 0.125 mmol) in MeCN (5 mL)
was added to the solution. The resulting clear solution was
stirred for 2 h and allowed to stand in darkness at room
temperature. Colorless crystals were formed after 3 days.
Yield 0.180 g, 72 %. Melting point 256–272 °C (de-
comp.). Anal. Calcd for C39H34Ag2N2O8P2S2 (%): C,
46.80; H, 3.43; N, 2.80. Found (%): C, 46.84; H, 3.61; N,
1
2.83. H-NMR (400 MHz, DMSO-d6, d, ppm): 7.87–7.55
9 S cm2 mol-1
.
123

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J Biol Inorg Chem (2014) 19:29–44
824.0 [Ag2(sac)(dppb)]?, 1,103.1 [Ag2Cl(dppb)2]?, 1,250.2
[Ag2(sac)(dppb)2]?, 1,394.1 [Ag3Cl(sac)(dppb)]?, 1,535.9
[Ag3(sac)2(dppb)2]?. KM (MeOH, 25 °C, 10-3 M) 46 S
Synthesis of 3
To a solution of AgNO3 (0.04 g, 0.25 mmol) in MeOH
(5 mL), Na(sac)ꢀ2H2O (0.06 g, 0.25 mmol) in MeOH/
MeCN (10 mL, 1:1) was added. After 30 min stirring at
room temperature, the addition of dppp (0.052 g,
0.125 mmol) in MeCN (5 mL) resulted in the formation of a
clear solution. The evaporation of the solution at room
temperature gave a white powder. The powder was washed
with water and dried at room temperature. Yield 0.099 g,
80 %. Melting point 230–235 °C. Anal. Calcd for
C41H34Ag2N2O6P2S2 (%): C, 43.13; H, 3.14; N, 2.82. Found
(%): C, 43.00; H, 2.50; N, 2.88. 1H-NMR (400 MHz,
DMSO-d6, d, ppm): 7.86–7.36 (m, 28H, Ph-sac and Ph-
cm2 mol-1. KM (DMSO, 25 °C, 10-3 M) 14 S cm2 mol-1
.
X-ray crystallography
The intensity data for complexes 1, 2, and 4 were collected
with an STOE IPDS 2 diffractometer with graphite-mono-
˚
chromatized Mo Ka radiation (k = 0.71073 A) at
296 (2) °C. The structures were solved by direct methods
and refined on F2 with the SHELX-97 program [36]. All non-
hydrogen atoms were found from the difference Fourier map
and were refined anisotropically, whereas the hydrogen
atoms bound to carbons were refined using a riding model,
but those of water were refined freely. Compounds 1 and 2
contain highly disordered water molecules, which were
eliminated from the refinement of these structures by means
of the SQUEEZE subroutine of PLATON [37], and the hkl
intensities were modified accordingly. In addition, the
crystal of 2 was a nonmerohedral twin (twin lattice quasi-
symmetry) crystal with two reciprocal lattices differently
oriented, giving rise to double diffraction spot sets. The data
sets of the twin parts for two different crystals were inte-
grated separately and then scaled to give the hkl set used.
But, because the partial overlapped reflections could not be
integrated separately, the reflection count and reflection
angle are less than 85 and 65 %, respectively. Crystal data
and refinement details are summarized in Table 1.
dppp), 2.62 (q, 4H, CH2-dppp), 2.05 (s, 2H, CH2-dppp). 13
C
NMR (100 MHz, DMSO-d6, d, ppm): 167.23 (C=O-sac),
143.88 (C6-sac), 133.43 (d, Cipso-P), 133.19 (Ph-Cortho-P),
132.79 (C4-sac), 132.50 (C3-sac), 132.19 (d, C1-sac),
131.21 (Ph-Cpara-P), 129.43 (d, Ph-Cmeta-P), 123.85 (C2-
sac), 120.45 (C5-sac). 31P NMR (200 MHz, DMSO-d6, d,
ppm): 5.86 (sbr). FT-IR (KBr, cm-1): 3,074 (vw), 3,055 (w),
3,010 (vw) m(Carom-H), 2,921 (vw), 2,864 (vw) m(Calf-H),
1,635 (s) m(C=O), 1,329 (m) ms(CNS), 1,283 (vs), 1,251
(s) mas(SO2), 1,148 (vs) ms(SO2), 1,119 (s), 953 (s) mas(CNS),
744 (s), 693 (s) m(C–P), 677 (vs), 597 (vs), 513 (s). ESI?–MS
(MeOH) m/z: 519.0 [Ag(dppp)]?, 809.9 [Ag2(sac)(dppp)]?,
933.2 [Ag(dppp)2]?, 1,014.9 [Ag2(sac)2(dppp)Na]?, 1,222.1
[Ag2(sac)(dppp)2]?, 1,365.9 [Ag(dppp)3(H2O)]?. KM
(MeOH, 25 °C, 10-3 M) 21 S cm2 mol-1. KM (DMSO,
25 °C, 10-3 M) 11 S cm2 mol-1
.
Crystallographic data (without structure factors) for the
structures reported in this article have been deposited with
the Cambridge Crystallographic Data Centre (CCDC) as
supplementary publication no. CCDC 949365–949367.
Copies of the data can be obtained free of charge from the
CCDC via http://www.ccdc.cam.ac.uk/data_request/cif.
Synthesis of 4
Complex 4 was prepared by the same procedure as described
for 2 with Na(sac)ꢀ2H2O (0.03 g, 0.125 mmol) and dppb
(0.054 g, 0.125 mmol). Colorless crystals were formed after
2 days. Yield 0.070 g, 78 %. Melting point 279–287 °C
(decomp.). Anal. Calcd for C35H32AgNO3P2S (%): C,
58.70; H, 4.50; N, 1.95. Found (%): C, 59.22; H, 4.79; N,
DNA binding studies
The concentrations of the complexes in various solutions
used in this work were based on their empirical formula. FS-
DNA solutions of various concentrations (0–100 lM) dis-
solved in a tris(hydroxymethyl)aminomethan (Tris)–HCl
buffer (pH 7) were added to complexes 1–4 (10 lM dis-
solved in MeOH). The amount of MeOH in these final
solutions was 4.0 %. The values of r (ratio of complex
concentration to DNA concentration) ranged from 0.1 to 1.0.
Absorption spectra were recorded after equilibrium had been
attained at 20 °C for 10 min. The intrinsic binding constant
(Kb) was determined using the following equation [38]:
1
1.99. H-NMR (400 MHz, DMSO-d6, d, ppm): 7.84–7.38
(m, 24H, Ph-sac and Ph-dppb), 2.36 (s, 4H, CH2-dppb), 1.55
(s, 4H, CH2-dppb). 13C NMR (100 MHz, DMSO-d6, d,
ppm): 167.60 (C=O-sac), 144.14 (C6-sac), 133.39 (d, Cipso
-
P), 133.15 (C4-sac), 133.03 (C3-sac), 132.99 (d, C1-sac),
132.75 (Ph-Cortho-P), 131.14 (Ph-Cpara-P), 129.44 (d, Ph-
C
meta-P), 123.75 (C2-sac), 120.36 (C5-sac). 31P NMR
(200 MHz, DMSO-d6, d, ppm): 5.86 [d, 1J(31P-
Ag) = 488 Hz]. FT-IR (KBr, cm-1): 3,065 (w), 3,007 (vw)
m(Carom-H), 2,926 (w), 2,897 (w), 2,856 (vw) m(Calf-H),
1,643 (s) m(C=O), 1,328 (m) ms(CNS), 1,281 (vs), 1,254
(m) mas(SO2), 1,151 (vs) ms(SO2), 1,115 (s), 957 (s) mas(CNS),
744 (s), 695 (s) m(C–P), 600 (m), 514 (m). ESI?–MS
(MeOH) m/z_ 535.1 [Ag(dppb)]?, 677.0 [Ag2Cl(dppb)]?,
½DNAꢁ=ðea ꢂ efÞ ¼ ½DNAꢁ=ðeb ꢂ efÞ þ 1=Kbðeb ꢂ efÞ ð1Þ
where [DNA] is the concentration of DNA in base pairs, ea,
ef, and eb correspond to Aobs/[M], the extinction coefficient
123

J Biol Inorg Chem (2014) 19:29–44
33
Table 1 Crystallographic data and structure refinement for complexes 1, 2, and 4
1
2
4
Empirical formula
Formula weight
Crystal system
Space group
C39H32Ag2N2O7P2S2
982.47
C20H16AgNO3PS
489.25
C35H32AgNO3P2S
716.49
Monoclinic
P21/c
Triclinic
P-1
Monoclinic
P21/c
˚
a (A)
12.297 (2)
15.466 (2)
21.449 (4)
90
8.6916 (15)
11.293 (4)
11.632 (2)
91.36 (2)
106.884 (15)
102.57(2)
1,061.7 (4)
2
20.3361 (4)
14.9758 (3)
27.2484 (6)
90
˚
b (A)
˚
c (A)
a (°)
b (°)
c (°)
96.204 (15)
90
126.417 (1)
90
3
˚
V (A )
4,055.6 (12)
4
6,677.9 (2)
8
Z
qcalcd (g cm-3
)
1.609
1.530
1.425
l (mm-1
F(000)
h (°)
Collected reflections
Data/restraints/parameters
Goodness of fit on F2
R1 (I [ 2r)
)
1.197
1.141
0.797
1,968
490
2,928
1.6–26.5
37,270
1.8–26.0
8,285
1.2–26.5
99,408
8,424/4/493
1.01
2,706/6/244
0.89
13,833/0/755
0.97
0.065
0.060
0.041
wR2
0.110
0.108
0.086
See Fig. 1 for the structures of the complexes
of the free metal(II) complex, and the extinction coefficient
of the complex in the fully bound form, respectively. The
ratio of the slope to the intercept in the plot of [DNA]/
(ea - ef) versus [DNA] gives the value of Kb.
KA were determined from Eq. 3 [40] and Eq. 4 [41],
respectively:
KEB½EBꢁ ¼ Kapp ½complexꢁ
ð3Þ
Ethidium bromide (EB)–FS-DNA fluorescence quench-
ing experiments were conducted in the buffer by keep-
ing the ratio of the DNA concentration to the EB
concentration at 10 (50 lM DNA, 5 lM EB), and then
the silver(I) complexes (2.5–50 lM) in MeOH were
added to this solution. The buffer used in the binding
studies was 20 mM Tris–HCl (pH 7.0), containing
20 mM NaCl. All solutions were allowed to equilibrate
thermally at 20 °C for about 30 min before measure-
ments. The fluorescence spectra of the solutions were
recorded in the range from 500 to 750 nm with an
excitation wavelength of 295 nm. The quenching ability
of the complexes was evaluated by the Stern–Volmer
constant (KSV) [39]:
in which the complex concentration is that which results in
a 50 % reduction of the fluorescence intensity of EB and
KEB = 1.0 9 107 M-1 (5.0 lM EB);
log(F0 ꢂ FÞ=F ¼ n logKA ꢂ n logf 1/[complex]
ꢂ ½EB ꢂ DNAꢁðF0 ꢂ FÞ=F0g
ð4Þ
where [EB-DNA] and [complex] are the total concentra-
tion of EB-DNA and silver(I) complexes, respectively.
The plot of log(F0 - F)/F versus log{1/([complex] -
[EB-DNA](F0 - F)/F0)} was drawn and fitted linearly,
and then the slope gives n, which is the number of binding
sites per nucleotide.
The interaction of the silver(I) complexes with super-
coiled pBR322 plasmid DNA was monitored using agarose
gel electrophoresis. The plasmid DNA (10 lM) in Tris–
HCl/EDTA buffer (pH 7.6) was treated with different
amounts of the silver(I) complexes (10–100 lM) in MeOH,
followed by dilution with the Tris–HCl buffer to a total
volume of 10 lL. Then, the samples were incubated at
37 °C for 1.5 h. A dye solution, containing 0.05 % bro-
mophenol blue, 40 % sucrose, 0.5 % sodium lauryl sulfate,
I0=I ¼ 1 þ KSV ½complexꢁ
ð2Þ
where I0 and I are the emission intensities in the absence
and presence of the complexes, respectively. KSV depends
on the ratio of the concentration of bound EB to the
concentration of DNA and is also called the quenching
constant. On the other hand, the binding constants Kapp and
123

34
J Biol Inorg Chem (2014) 19:29–44
and 0.1 M EDTA, was added to the reaction mixture prior
to electrophoresis. Then, the samples were electrophoresed
on 1 % agarose gel with 1 lg/mL EB, using 0.59 Tris–
borate–EDTA buffer (pH 8.0) for 1 h at 120 V at room
temperature. Finally, the gel was photographed under UV
light.
After the incubation periods, 10 lL of WST-1 solution was
added to each well, followed by incubation at 37 °C for
1 h. The absorbance (optical density) of each well was read
at wavelengths of 450 and 630 nm using a microplate
reader (Multiskan FC, Thermo Scientific). Each compound
was tested at every concentration in triplicate in a single
experiment, which was repeated three times. AgNO3 and
cisplatin were used as reference cytotoxic agents. Values
obtained for the vehicle-treated cells were used as the
reference for 100 % viability. IC50 values were determined
by sigmoidal dose–response functions using GraphPad
Prism.
Antibacterial activity
Bacterial strains of Escherichia coli (ATCC 25922 and
O157:H7), Salmonella typhimurium (ATCC 14028), and
Staphylococcus aureus (ATCC 25923 and ATCC 33591)
were used.
Broth microdilution testing was performed to determine
the minimum inhibitory concentrations (MICs) of cipro-
floxacin, gentamicin, and the silver(I) complexes according
to the guidelines of the Clinical Laboratory Standards
Institute [42]. The bacterial cultures were prepared in
Mueller–Hinton broth at 37 °C for 20 h. All compounds
were dissolved in DMSO. Freshly prepared stock solutions
were sterilized using 0.20-lm single-use filter units (Min-
isart, Sartorius Stedim Biotech). Dilutions ranging from
0.007 to 775 lM were prepared in Mueller–Hinton broth,
and inocula with a density equivalent to 0.5 McFarland
turbidity were added to tubes containing the dilutions of the
compounds. After incubation at 37 °C for 20 h, the MICs
were defined as the minimum concentration of the com-
pound that inhibited growth of the organism. The optical
densities of the cultures were measured at a wavelength of
595 nm (iMark, Bio-Rad). All MIC determinations were
performed in duplicate.
Results and discussion
Synthesis and characterization
The complexes were synthesized by the reaction of
Na(sac)ꢀ2H2O with AgNO3 in the presence of the equiva-
lent amount of the tertiary diphosphanes in solution at
room temperature. The analytical data confirmed the stoi-
chiometry of complexes 1–4. The structures of complexes
1, 2, and 4 were confirmed by X-ray crystallography.
Complex 1 has a dinuclear structure, whereas complexes 2
and 4 are coordination polymers. In some cases, the
molecular formula differs as a result of recrystallization.
For example, the polycrystalline powder of 2 has three
water molecules and a CH2Cl2 solvate in the empirical
formula, but it contains only a water molecule in the crystal
form, as will be discussed below. Regrettably our attempts
to grow X-ray-quality crystals of complex 3 failed; how-
ever, on the basis of elemental analysis, IR spectroscopy,
and NMR spectroscopy, complex 3 is assumed to have a
molecular structure similar to that of complex 2, but it does
not contain any solvent molecules such as water or CH2Cl2
present in 2. All complexes were obtained in high yields
(over 70 %). Complexes 1–3 are stable to light for a long
time, but complex 4 begins to turn brown after 2 weeks. On
the other hand, the solutions of the complexes in MeOH
and DMSO were kept for up to 72 h at room temperature
Cytotoxic activity
The samples for in vitro tests were obtained from 50 mM
stock solutions of the complexes in neat DMSO. The stock
solutions were then sequentially diluted with the required
amount of cell culture medium in order to obtain the
solutions studied, the concentration of which ranged from
1 nM to 100 lM. Untreated cells were used as a control
group (0.2 % DMSO vehicle).
1
WST-1 viability assay (4-[3-(4-iodophenyl)-2-(4-nitro-
phenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate sodium
salt, Roche Applied Science) was performed to examine
the cytotoxic effects of the concentrations of complexes 1–
4 that cause 50 % inhibition of cells (IC50) on A549
(human lung carcinoma), MCF-7 (human breast adeno-
carcinoma), and WI-38 (normal human fibroblasts) cell
lines. The assay is based on the cleavage of the tetrazolium
salt by viable cells into a colored formazan product that can
be measured spectrophotometrically. For this purpose,
these cell lines were incubated at a density of 1 9 104
cells per milliliter using 96-well plates for 24, 48, and 72 h.
and were checked by H and 13C NMR spectroscopy. The
observations showed that these solutions did not change
color during that time and also that there was no decom-
position product in the NMR spectra. Moreover, a com-
parison of the initial 1H and 13C NMR spectra of the
solutions of the silver(I) complexes with those after 72 h
indicated that the spectra were identical and the chemical
1
shifts (d) differ by only 0.03 ppm for the H NMR spectra
and 2.0 ppm for the 13C NMR spectra. All the complexes
are nonhygroscopic and highly soluble in MeOH, MeCN,
and DMSO. The electrical conductivity measurements
indicate that the complexes exhibit a nonelectrolytic
123

J Biol Inorg Chem (2014) 19:29–44
35
behavior, with KM ranging from 21 to 58 S cm2 mol-1 in
MeOH, and from 9 to 14 S cm2 mol-1 in DMSO [43].
The thermal stability of the silver(I) complexes was
studied by TG–DTA. Complex 1 dehydrates between 57
and 157 °C with a mass loss of 3.0 % (calculated 2.9 %),
involving the lost of both the lattice and coordination water
molecules. Then, the anhydrite complex begins to decom-
pose exothermically at around 260 °C, and the decompo-
sition ends at approximately 580 °C. The total mass loss of
76.7 % is consistent with the formation of Ag2O (the cal-
culated mass value is 77.8 %). The mass loss up to 86 °C is
due to the removal of the CH2Cl2 solvate in 2, followed by
dehydration. The complex decomposes exothermically
between 270 and 700 °C to give Ag2O (total mass loss,
found 80.1 % and calculated 79.3 %). Complexes 3 and 4
are stable up to approximately 300 °C, and their decom-
positions end at around 800 °C. The mass losses are in
accord with the structures of these complexes and suggest
the formation of Ag2O as the final decomposition product.
Complexes 1–4 were characterized by the positive-ion
ESI–MS spectra. The different cationic species present in
MeOH solution of the complexes were identified, and most
relevant data are reported in ‘‘Materials and methods.’’ The
isotopic distribution of these species is consistent with the
calculated composition. The strongest peaks for these
complexes usually correspond to dinuclear silver(I) species
such as [Ag2(sac)(dppm)3]?, [Ag2(sac)(dppp)2]?, and
[Ag2(sac)(dppb)2]? in accordance with possible aggrega-
tion in solution and formation of these species. However, in
the case of 2, the [Ag(dppe)2]? cation is the most abundant.
In addition, the ESI–MS spectra also exhibit several less
intense peaks related to the presence of mononuclear
[Ag(diphos)]? or [Ag(diphos)2]? and dinuclear
[Ag2(sac)]? cations, arising from fragmentation under the
conditions used [44–49].
The spectral characterizations were performed using
polycrystalline powders of the silver(I) complexes. In the
IR spectra of complexes 1–4, the bands between 2,852 and
3,085 cm-1 are due to the absorption of the phenyl and
CH2 groups. The C=O vibration of the sac ligand is
observed as a very sharp band at in the frequency range
from 1,635 to 1,655 cm-1. The asymmetric stretching of
the SO2 group appears to be split into two strong bands at
approximately 1,280 and 1,250 cm-1, whereas the sym-
metric stretching is observed as a sharp band at approxi-
mately 1,150 cm-1. A 10-cm-1 decrease in ms(SO2) in the
spectrum of 2 is evidence for the coordination via the
sulfonyl group in this complex. The bands at 1,425–1,450
and 1,000 cm-1 are usually assigned to the P–C vibrations
of the free forms of the phosphane ligands [50, 51].
However, in the case of mixed-ligand metal complexes, it
is very difficult to assign these bands, and therefore the
very strong absorption bands centered at around 690 cm-1
were attributed the P–C bonds in the free forms of the
ligands and their complexes [52].
1
The H, 13C, and 31P NMR spectra of complexes 1–4
were measured at room temperature in DMSO-d6 solution.
The aromatic protons of both sac and diphosphanes appear
Fig. 2 Molecular structure of 1.
All C–H hydrogen atoms are
omitted for clarity. The
probability level of thermal
ellipsoids is 40 %
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36
J Biol Inorg Chem (2014) 19:29–44
between 7.36 and 7.95 ppm as multiplets. The bridging
methylene protons in 1 and 2 are observed as a singlet at
2.05 and 2.57 ppm, respectively, whereas those in 3 and 4
are centered at 2.05–2.62 and 1.55–2.36 ppm, respectively,
as doublets. The methylene protons of the diphosphanes
generally experience deshielding on complexation. In
addition, there was a detectable signal at d 5.77 ppm cor-
responding to the protons of the CH2Cl2 solvate in 2. In the
13C NMR spectra of the complexes, the carbonyl group of
the sac ligand occurs at approximately d 167.50 ppm,
whereas the chemical shifts for the phenyl carbons are
observed in the range from 120.0 to 144.0 ppm. The 31P
NMR spectra of complexes of silver(I) with phosphorous-
1
donor ligands usually exhibit splitting owing to the J(P-
Ag) coupling. The phosphorous nuclei in the diphosphane
ligands experience deshielding by approximately
22.0–32.0 ppm at room temperature compared with those
in the free forms of the ligands. The 31P NMR spectrum of
complex 1 consists of a triplet centered at 8.82 ppm with
two coupling constants of 504 and 688 Hz. These different
Fig. 3 The coordination
environment around
silver(I) and the one-
dimensional polymeric structure
of 2. Hydrogen atoms were
omitted for clarity. The
probability level of thermal
ellipsoids is 40 %. Symmetry
code: -x ? 1, -y ? 1, -z ? 1
123

J Biol Inorg Chem (2014) 19:29–44
37
coupling constants are believed to arise from the different
chemical environments around the phosphorous nuclei in 1,
as will be shown in the X-ray structure of the complex. On
the other hand, the spectra of 2 and 4 reveal well-resolved
doublets at 10.04 and 5.86 ppm, respectively. The calcu-
lated coupling constants of 1J(31P-107,109Ag) are 579 Hz for
2 and 488 Hz for 4, being typical of silver(I) complexes
containing the corresponding diphosphane ligands [47, 48].
In particular, the 31P NMR spectrum of 3 consists of a
broad singlet at 5.86 ppm, presumably in consequence of
reasonably fast exchange equilibrium at room temperature.
bridging ligands as expected [53, 54], whereas the sac
ligand exhibits different coordination modes [22]. Complex
1 has a dinuclear structure, in which the dppm ligand
bridges the two silver(I) centers. In addition, each sil-
ver(I) ion is coordinated by a nitrogen-donor sac ligand.
These two silver(I) ions exhibit different coordination
geometry: one has a linear geometry with an angle of
175.49 (17)°, whereas the other one displays a T-shaped
geometry with the additional coordination of an aqua
ligand (Fig. 2). The Ag–N bond distances of 2.151 (5) and
˚
2.132 (5) A are typical for silver(I) complexes containing a
nitrogen-bonded sac ligand [35, 55–66], whereas both Ag–
˚
P bond distances are identical, being 2.3550 (17) A and
Description of crystal structures
similar to those found in the silver(I) complexes of dppm
[67–74], but significantly shorter than the corresponding
bonds in silver(I) complexes containing the same ligand
[44–46, 49, 72, 75–96]. These short Ag–P bond distances
in complex 1 may be due to the low coordination number
The molecular structures of complexes 1, 2 and 4 together
with their numbering schemes are given in Figs. 2, 3, and
4. Selected bond lengths and angles are given in Table 2.
The diphosphanes in these silver(I) complexes behave as
Fig. 4 View of the
coordination environment
around silver(I) and the one-
dimensional polymeric structure
of 4. Hydrogen atoms were
omitted for clarity. The
probability level of thermal
ellipsoids is 40 %. Symmetry
codes: (i) –x ? 2, y ? 1/2,
-z ? 3/2; (ii) –x ? 1, y ? 1/2,
-z ? 1/2
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38
J Biol Inorg Chem (2014) 19:29–44
˚
of silver(I) and relatively less steric hindrance of the sac
ligand compared with the co-ligands in the other sil-
ver(I) complexes of dppm. The individual molecules of 1
are connected by strong intermolecular O–HꢀꢀꢀO hydrogen
bonds between the aqua and sac ligands in the adjacent
species. Moreover, relatively weak interactions such as
Table 2 Selected bond lengths (A) and angles (°) for complexes 1, 2,
and 4
1
2
4
Ag1–N1
2.151 (5)
2.160 (8)
2.313 (2)
2.336 (2)
2.4115 (8)
2.4198 (8)
2.4212 (8)
2.4300 (8)
–
Ag2–N2
2.132 (5)
–
Ag1–P1
2.3550 (17)
2.356 (3)
˚
C–HꢀꢀꢀO [CꢀꢀꢀO bond distance 3.464 (8) to 3.485 (7) A]
Ag1–P2
2.3552 (17)
–
˚
and p(sac)ꢀꢀꢀp(sac) [CgꢀꢀꢀCg bond distance 3.811 A (x,
Ag2–P3
–
–
-1 ? y, z); Cg, ring centroid] are also present in 1.
Ag2–P4
–
–
Complex 2 is a one-dimensional coordination polymer
of {[Ag2(l-sac)2(l-dppe)]ꢀH2O}n. The sac ligands adopt a
l2-bridging mode via the imino nitrogen and the sulfonyl
oxygen atoms, forming dinuclear [Ag(sac)]2 units with a
chairlike eight-membered ring. The coordination geometry
around each silver(I) ion is T-shaped. The [Ag2(sac)2] units
are further bridged by the dppe ligands, leading to a linear
polymeric chain of 2 (Fig. 3). The Ag–N and Ag–O bond
Ag1–O1W
Ag1–O2i
2.594 (4)
–
–
2.527 (7)
–
N1–Ag1–P1
N1–Ag1–P2
N2–Ag1–P2
N1–Ag1–O1W
N1–Ag1–O2i
P1–Ag1–O1W
P1–Ag1–P2
N2–Ag2–P3
N2–Ag2–P4
P3–Ag2–P4
P1–Ag1–O2i
169.11 (13)
156.0 (2)
112.70 (7)
106.67 (7)
–
–
–
175.49 (17)
–
95.27 (16)
–
–
–
95.6 (3)
–
˚
distances are 2.160 (8) and 2.527 (7) A, respectively, and
95.40 (12)
–
–
–
–
–
–
–
–
140.17 (3)
112.39 (7)
107.54 (7)
139.59 (3)
–
are in the range of those reported for silver(I) complexes
containing N,Osulfonyl bridging sac ligands [56, 97–99]. The
˚
Ag–P bond distance in complex 2 is 2.356 (3) A (Table 2),
–
–
being similar to the bond distances found in the sil-
ver(I) complexes of dppe reported in [70, 100], but it is
obviously shorter than the bond distances for sil-
ver(I) complexes of dppe reported in [44, 47, 49, 91, 101–
–
108.37 (18)
Symmetry code: -x ? 1,-y ? 1, -z ? 1
Fig. 5 UV–vis spectra of complexes 1–4 (10 lM) in MeOH solution in the presence of increasing amounts of fish sperm DNA (0–100 lM). The
arrows show the changes in absorbance with increasing amounts of fish sperm DNA. Insets [DNA]/(ea - ef) versus [DNA]
123

J Biol Inorg Chem (2014) 19:29–44
39
DNA binding
110]. As mentioned earlier, the complex contains a highly
disordered water molecule, which was removed during the
refinement. The presence of the lattice water molecule is
important for hydrogen bonding between the chains in 2,
but the exclusion of the water molecule makes the dis-
cussion of the crystal packing meaningless. However, it
can be said that in addition to the possible hydrogen-
bonding interactions involving the water molecules, the
chains are connected by p(sac)ꢀꢀꢀp(sac) stacking interac-
Electronic absorption titration is one of the commonest
methods to investigate the interactions between metal
complexes and DNA. The intense absorption band centered
at approximately 260 nm was used to monitor the interac-
tion of the silver(I) complexes with FS-DNA. The elec-
tronic absorption spectra of complexes 1–4 in the absence
and presence of increasing amounts of FS-DNA are shown
in Fig. 5. The absorption band of DNA decreases in inten-
sity with increasing DNA concentration. This is a typical
‘‘hypochromic effect,’’ suggesting that the complexes most
likely bind to DNA through intercalation. The hypo-
chromicity of complexes 1–4 is 33.3, 23.5, 32.1, and
28.8 %, respectively, at r = 0.3. The intrinsic binding
constants (Kb) of the complexes determined from Eq. 1 are
given in Table 3 and Fig. 5. The Kb values are in the range
from 2.00 9 104 to 3.00 9 104 M-1 and suggest a signif-
icant association of these complexes with FS-DNA. Com-
plexes 1 and 3 exhibit the highest binding affinity; however,
the binding constants are much lower than that reported for
the classic intercalator EB (Kb = 1.4 9 106 M-1) [113].
To further clarify the nature of the binding interactions
between the silver(I) complexes and DNA, competitive
binding experiments were performed. The studies involved
the addition of the complexes to solutions of DNA pre-
treated with EB, and then the measurement of the emission
intensities of EB in the range from 600 to 620 nm. Fluo-
rescence quenching of the EB–DNA complex in the pre-
sence of a new molecule can be used to realize the binding
mode of the molecule. The emission spectra of EB binding
to FS-DNA in the absence and presence of complexes 1–4
are shown in Fig. 6. A reduction in the fluorescence inten-
sity is observed on the addition of the complexes to FS-
DNA pretreated with EB. From the Stern–Volmer plots in
Fig. 6, the quenching constants (KSV) of the complexes
were calculated (Table 3). The KSV values indicate that
complexes 1 and 4 exhibit greater quenching efficiency than
˚
tions of 3.65 (1) A (symmetry code 1 - x, -y, 1 - z) and
the structure of 2 is further reinforced by C–Hꢀꢀꢀp(dppe)
and C–HꢀꢀꢀO interactions, ranging from 3.00 (3) to
˚
3.28 (2) A.
As shown in Fig. 4, complex 4 has two species in its
asymmetric unit with a similar conformation. The struc-
ture of 4 consists of polymeric chains of [Ag(sac)(l-
dppb)]n, which are built up from the bridging of the sil-
ver(I) ions by the dppb ligands (Fig. 4). The trigonal
geometry around silver(I) is completed by the coordination
of the sac ligand via the nitrogen atom. Although the
synthesis of 4 is similar to that of 2, it has a different
polymeric structure in which the bridging of sac is prob-
ably avoided by the dppb ligand containing a much longer
ethylene chain than those of dppe in 2. The Ag–N bond
distances in Table 2 are similar to those of previously
reported silver(I) complexes of sac [55–66]. On the other
hand, the Ag–P bond distances are in the range from
˚
2.4115 (8) to 2.4300 (8) A, and are similar to those found
in silver(I) complexes of dppb reported in [48, 49, 110,
111], but are noticeably longer than the corresponding
bonds in silver(I) complexes containing dppb reported in
[70, 112]. In addition, a few examples of silver(I) com-
plexes of dppb with much longer Ag–P bond distances
have also been reported [47, 84]. The polymeric chains of
4 are connected with each other by a number of weak
˚
C–HꢀꢀꢀO [CꢀꢀꢀO bond distance 3.32 (2) to 3.38 (3) A], and
˚
C–Hꢀꢀꢀp [HꢀꢀꢀCg bon distance 2.67 (2) to 2.96 (2) A]
interactions in the solid state.
Table 3 Quenching constant (KSV) and binding constants (Kapp and KA) for binding of complexes 1–4 and other compounds with fish sperm
DNA
Compounds
KSV (9104 M-1
)
Kapp (9106 M-1
)
KA (9104 M-1
)
Number of binding
sites per nucleotide
Kb (9104 M-1
)
1
1.85 0.13
0.63 0.14
1.00
a
1.02 0.24
0.07 0.02
0.76 0.06
0.04 0.02
0.10 0.07
1.40 0.22
1.01
1.40
1.04
1.47
1.20
1.02
2.86 0.16
2.50 0.22
3.00 0.12
2.00 0.17
0.83 0.07
3.75 0.21
2
–
a
3
0.87
1.07
0.46
1.97
0.13
0.21
0.01
0.25
–
a
4
–
a
AgNO3
AgSD
–
0.57
AgSD silver sulfadiazine
a
These values could not be determined because the corresponding compounds did not cause a 50 % reduction in the fluorescence intensity of the
ethidium bromide–DNA solutions
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40
J Biol Inorg Chem (2014) 19:29–44
Fig. 6 Fluorescence spectra of ethidium bromide (EB)–DNA in the
presence of increasing amounts of complexes 1–4 (5.0 lM EB,
50.0 lM DNA). The arrows show the changes in intensity on
increasing amounts of the complexes. The ratio of the complex
concentration to the DNA concentration is r. Insets Stern–Volmer plot
of the fluorescence data
not be determined, because increasing their concentrations
did not cause a 50 % reduction in the fluorescence intensity
of the EB–DNA solutions. The interaction of complexes 1
and 3 results in the highest binding constants (KA), and this
observation is consistent with the findings (Kb) obtained
from the absorption studies, suggesting that complexes 1
and 3 strongly bind to FS-DNA by an intercalative mode.
However, all binding constants for AgSD were higher than
those for the silver(I) complexes.
When a molecule binds to DNA it affects the size or shape
of the DNA, and it will affect the electrophoretic mobility of
DNA. The electrophoretic patterns of pBR322 plasmid DNA
incubated with complexes 1–4 at various concentrations are
shown in Fig. 8. The double-stranded plasmid pBR322
exists in a compact supercoiled conformation (form I), which
has faster mobility compared with its closed circular (form
II) and linear (form III) forms, resulting from the cleavage of
one or two of the strands of the double helix. As shown in
Fig. 8, complexes 2 and 4 do not have any impact on DNA
mobility at the concentrations investigated, whereas com-
plexes 1 and 3 exhibit noticeable retardation of the super-
coiled DNA band with increasing complex concentration,
indicating unwinding of the plasmid DNA owing to the
binding of these complexes, since an intercalator is expected
to unwind the DNA by lengthening and stiffening it [114].
Fig. 7 Fluorescence quenching plots obtained from Eq. 4, giving the
binding constants (KA) and the number of binding sites (n) for
complexes 1–4
the other complexes. The DNA binding constants (Kapp and
KA) are listed in Table 3, and the corresponding plots
obtained from the experimental quenching data are shown
in Fig. 7. Kapp is the apparent binding constant and is based
on the concentration of the complex that causes a 50 %
reduction of the fluorescence intensity of the EB–DNA
adduct (Eq. 3), and KA is the intrinsic binding constant
obtained from the fluorescence quenching studies using
Eq. 4. The Kapp values of complexes 2–4 and AgNO3 could
123

J Biol Inorg Chem (2014) 19:29–44
41
silver(I) compounds for susceptibility testing. Three bac-
terial species were used in this study. Among the bacterial
species, E. coli (O157:H7) and methicillin-resistant S.
aureus (ATCC 33591) are considered to be two of the most
virulent microorganisms for the human population. The
effect of the solvent (DMSO) on bacterial growth was
tested and was found to be negligible. Complex 4 is totally
inactive, whereas the MICs of complexes 1–3 range from
15.9 to 116.4 lM. The highest antibacterial activity is
observed for complex 1 against all bacterial strains, except
for E. coli (ATCC 25922), compared with AgNO3 and
AgSD (Table 4). Moreover, complex 3 also exhibits better
activity against S. typhimurium and S. aureus than the
antimicrobial agents and AgNO3, but it has less activity
than AgSD and AgNO3 in the case of E. coli. On the other
hand, complex 2 exhibits higher activity against S. ty-
phimurium and S. aureus than the antimicrobials. The
relatively high bacterial growth inhibition of these com-
plexes is consistent with their DNA binding affinities.
Although silver(I) complexes with Ag–P bonds generally
exhibit weak antibacterial activity [74, 115, 116], complex
1 may be a candidate for clinical applications after further
tests.
Cytotoxic activity
We tested the in vitro cytotoxic activity of complexes 1–4
against two human cancer cell lines (A549 and MCF-7)
and a normal cell line (WI-38). The IC50 values were
determined from the dose dependence of surviving cells
after exposure to the silver(I) complexes for 24, 48, and
72 h (Fig. 9). The inhibition of cell proliferation by the
complexes after 72 h is presented in Table 5 and is com-
pared with inhibition by AgNO3 and the clinically used
anticancer agent cisplatin. The antigrowth effect of the
complexes increases with increasing incubation time
(Fig. 9). As shown in Table 5, the IC50 values of these
complexes range from 0.74 to 86.40 lM and indicate that
complexes 1 and 3 exhibit very high cytotoxic activity
Fig. 8 Electrophoretic mobility of pBR322 plasmid DNA (10 lM) in
the absence of the silver(I) complexes (lane 0) and in the presence of
the silver(I) complexes (a 1, b 2, c 3, and d 4) at 10, 20, 50,and
100 lM (lanes 1–4)
Antibacterial activity
The antibacterial activities of complexes 1–4, AgSD,
AgNO3, and some licensed antimicrobials against various
bacterial strains are given in Table 4, as estimated by the
MIC. AgNO3 and AgSD were used as reference
Table 4 Antibacterial activities (minimum inhibitory concentration, lM) of complexes 1–4 and other compounds
Escherichia coli
(ATCC 25922)
Escherichia coli
(O157:H7)
Salmonella typhimurium
(ATCC 14028)
Staphylococcus aureus
(ATCC 25923)
Staphylococcus aureus
(ATCC 33591)
1
15.9
15.9
31.9
15.9
15.9
2
116.4
32.3
116.4
32.3
58.2
58.2
58.2
3
32.3
32.3
32.3
4
C1,000
23.5
C1,000
23.5
C1,000
47.1
C1,000
94.2
C1,000
94.2
AgNO3
AgSD
22.4
22.4
44.8
22.4
22.4
Ciprofloxacin
Gentamicin
0.05
96.6
772.6
536.0
772.6
134.0
772.6
134.0
33.5
134.0
123

42
J Biol Inorg Chem (2014) 19:29–44
Fig. 9 The antigrowth effect of
complexes 1–4, AgNO3, and
cisplatin on A549, MCF-7, and
WI-38 cells treated with the
compounds for 24, 48, and 72 h.
IC50 concentration that causes
50 % inhibition of cells
Table 5 Cytotoxic activities (half-maximal inhibitory concentration,
lM) of silver(I) complexes 1–4, AgNO3, and cisplatin after 72 h
incubation
containing phosphane ligands has received less attention so
far. The results of the present study indicate that further
studies are warranted to assess their pharmacological
properties and elucidate the actual mechanism of their
biological activity.
A549
MCF-7
WI-38
1
2.58 0.83
9.11 2.32
1.51 0.57
86.4 4.75
21.51 3.63
10.92 1.81
2.32 1.00
3.18 1.01
0.74 0.24
5.30 1.61
2.62 1.44
4.58 1.25
9.54 2.40
2.61 1.16
1.05 0.53
10.78 2.22
8.14 1.88
2.62 1.04
2
3
Conclusion
4
AgNO3
Cisplatin
Four new silver(I) complexes, [Ag2(sac)2(l-dppm)H2O]ꢀ
H2O (1), {[Ag2(l-sac)2(l-dppe)]ꢀ3H2OꢀCH2Cl2}n (2),
[Ag2(l-sac)2(l-dppp)]n (3), and [Ag(sac)(l-dppb)]n (4),
have been synthesized and fully characterized. The struc-
tures of complexes 1, 2, and 4 were determined by X-ray
diffraction. All phosphane ligands act as bridging ligands.
The DNA binding affinity of the complexes was studied by
spectroscopic methods such as absorption and fluorescence
titrations. The results show that the complexes bind to
DNA by intercalation. The binding of the complexes to the
pBR322 plasmid DNA was further confirmed by gel
electrophoresis measurements. The biological activities of
the silver(I) complexes were tested against five bacterial
strains and two human cancer cell lines. Among the com-
plexes, complex 1 exhibits powerful antibacterial activity
against the licensed antimicrobials, being much more
powerful than AgNO3 and AgSD. The cytotoxicity studies
indicate that complexes 1 and 3 are very effective against
against all the cell lines, compared with AgNO3 and cis-
platin. The high inhibition activity of these complexes is in
accord with their relatively high propensity to bind DNA.
Among the silver(I) complexes, complex 4 is the least
active against the A549 cell line, whereas its antigrowth
effect on MCF-7 cells is close to that of cisplatin. The IC50
values of cancer and control cell lines were found to be
similar. Cytotoxicity in the WI-38 cell line was evaluated
as untoward side effects. Only one report on the anticancer
activity of cationic silver(I) complexes with diphosphane
ligands, namely, [Ag(R2P(CH2)nPR2)2]NO3 (R is Ph or Et
and n = 2), has appeared in the literature [117] and sig-
nificantly low IC50 values were reported for B16 melanoma
cells. The anticancer activity of silver(I) complexes
123

J Biol Inorg Chem (2014) 19:29–44
43
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values are much smaller than those of AgNO3 and cis-
platin. It may be concluded that the high antibacterial and
anticancer activities of the silver(I) complexes correlate
well with their DNA binding affinities.
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