Heterocyclic thioamide/phosphine mixed-ligand silver(I) complexes: Synthesis, molecular structures, DNA-binding properties and antibacterial activity

Article abstract of DOI:10.1016/j.poly.2018.05.020

Five silver(I) mixed-ligand complexes containing a heterocyclic thioamide or thioamidate (mtdztH = 5-methyl-1,3,4-thiadiazole-2-thiol or atdztH = 5-amino-1,3,4-thiadiazole-2-thiol or mqzH = 2-mercapto-4(3H)-quinazolinone) and triphenylphosphine (PPh₃) have been synthesized and structurally characterized. Four of these compounds, namely [AgCl(PPh₃)₂(mtdztH)] (1), [AgCl(PPh₃)₂(mqzH)] (2), [Ag(PPh₃)₃(mtdzt)] (3) and [Ag(PPh₃)₃(atdzt)] (4) reveal a distorted tetrahedral coordination environment, while [Ag(PPh₃)₂(atdzt)] (5) adopts a trigonal planar arrangement. The interaction of the compounds with calf-thymus DNA was monitored via UV–Vis spectroscopy, DNA-viscosity measurements and their competition with ethidium bromide for the DNA-intercalation sites studied by fluorescence emission spectroscopy. The affinity of all the silver(I) complexes for the interaction with CT DNA is high and takes place via an intercalative mode. The new complexes show in vitro antibacterial activity against certain Gram-positive (Bacillus subtilis, Bacillus cereus, Staphylococcus aureus) bacterial strains, but are inactive against the Gram-negative Escherichia coli. Interestingly, higher activity was detected for the trigonal planar compound compared to its tetrahedrally coordinated counterparts.

Full text of DOI:10.1016/j.poly.2018.05.020

Accepted Manuscript
Heterocyclic thioamide/phosphine mixed-ligand silver(I) complexes: Synthe-
sis, molecular structures, DNA-binding properties and antibacterial activity
Despoina Varna, Eleni Kapetanaki, Alexandra Koutsari, Antonios G.
Hatzidimitriou, George Psomas, Panagiotis Angaridis, Rigini Papi, Anastasia
A. Pantazaki, Paraskevas Aslanidis
PII:
S0277-5387(18)30258-4
https://doi.org/10.1016/j.poly.2018.05.020
POLY 13167
DOI:
Reference:
Polyhedron
To appear in:
Received Date:
Accepted Date:
13 March 2018
9 May 2018
Please cite this article as: D. Varna, E. Kapetanaki, A. Koutsari, A.G. Hatzidimitriou, G. Psomas, P. Angaridis, R.
Papi, A.A. Pantazaki, P. Aslanidis, Heterocyclic thioamide/phosphine mixed-ligand silver(I) complexes: Synthesis,
molecular structures, DNA-binding properties and antibacterial activity, Polyhedron (2018), doi: https://doi.org/
10.1016/j.poly.2018.05.020
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Heterocyclic thioamide/phosphine mixed-ligand
silver(I) complexes: Synthesis, molecular
structures, DNA-binding properties and
antibacterial activity
Despoina Varna,a Eleni Kapetanaki,a Alexandra Koutsari,a Antonios G. Hatzidimitriou,a
George Psomas,a Panagiotis Angaridis,*,a Rigini Papi,b Anastasia A. Pantazakib and
Paraskevas Aslanidisa
aLaboratory of Inorganic Chemistry, Department of Chemistry, Aristotle University of
Thessaloniki, Thessaloniki 541 24, Greece
bLaboratory of Biochemistry, Department of Chemistry, Aristotle University of Thessaloniki,
Thessaloniki 541 24, Greece
Synopsis:
Silver(I) complexes with heterocyclic thioamides (or thioamidates) and phosphine ligands,
with either tetrahedral or trigonal planar coordination geometries have been synthesized and
structurally characterized. Their CT DNA binding affinity and antibacterial activity has been
examined.
[1]

Dedicated to Prof. Spyros P. Perlepes on the occasion of his 65th birthday
* Corresponding author:
Tel.: +30 2310 99 7437. E-mail address: panosangaridis@chem.auth.gr (P. Angaridis).
[2]

ABSTRACT
Five silver(I) mixed-ligand complexes containing a heterocyclic thioamide (mtdztH = 5-
methyl-1,3,4-thiadiazole-2-thiol or atdztH = 5-amino-1,3,4-thiadiazole-2-thiol or mqztH = 2-
mercapto-4(3H)-quinazolinone) and triphenylphosphine (PPh3) have been synthesized and
structurally characterized. Four of these compounds, namely [AgCl(PPh3)2(mtdztH)] (1),
[AgCl(PPh3)2(mqztH)] (2), [Ag(PPh3)3(mtdzt)] (3) and [Ag(PPh3)3(atdzt)] (4) reveal a
distorted tetrahedral coordination environment, while [Ag(PPh3)2(atdzt)] (5) adopts a trigonal
planar arrangement. The interaction of the compounds with calf-thymus DNA was monitored
via UV-vis spectroscopy, DNA-viscosity measurements and their competition with ethidium
bromide for the DNA-intercalation sites studied by fluorescence emission spectroscopy. The
affinity of all the silver(I) complexes for the interaction with CT DNA is high and takes place
via an intercalative mode. The new complexes show in vitro antibacterial activity against
certain Gram-positive (Bacillus subtilis, Bacillus cereus, Staphylococcus aureus) bacterial
strains, but are inactive against the Gram-negative Escherichia coli. Interestingly, higher
activity was detected for the trigonal planar compound compared to its tetrahedrally
coordinated counterparts.
keywords: Silver(I); heterocyclic thioamides; Crystal structures; Interaction with DNA;
Antimicrobial activity
[3]

1. INTRODUCTION
During the last few decades, coordination compounds bearing heterocyclic thioamide
ligands have attracted much research interest [1]. The ability of these molecules to coordinate
metal centers in a monodentate or a bridging mode, resulted in the synthesis of a large variety
of compounds, among them a significant number of copper(I) and silver(I) complexes
bearing bulky triarylphosphines as auxilliary ligands. The vast majority of these complexes
reveal a tetra-coordinate mononeric or dimeric nature, although there are some examples of
three-coordinate species, particularly in the presence of bulky triarylphosphines [2]. The
interest for much of the research into the coordination chemistry of heterocyclic thioamides
stems, however, from their close relevance to biological system [3]. In fact, they are regarded
as a very promising starting point for biological studies and transition metal complexes of
several imidazole-, thiazole-, oxazole-, and uracil-based derivatives have been the subject of
investigations towards pharmacological activities [4].
Silver salts are known for long time for their effectiveness as antiseptic, antibacterial and
anti- inflammatory agents and were widely used until the appearance of antibiotics.
Nowadays, with the increasing resistance of bacteria to antibiotics, research has turned
significantly towards developing of silver coordination compounds with improved
pharmacological characteristics [5]. At the same time, much attention has also been paid to
the antitumor and anti-proliferative characteristics of silver(I) complexes a particular intense
activity on the antitumor and anti-proliferative activity of silver(I) complexes is also in
progress [6], whereby the status of the respective investigations has being reviewed recently
[7].
For quite a long time, research in our laboratory largely focused on heterocyclic
thioamide/phosphine mixed-ligand copper(I) and silver(I) complexes, with the aim to
understand the factors affecting the stoichiometric and structural preferences of these
compounds. Considering silver(I) complexes bearing neutral heterocyclic thioamides and
[4]

triphenylphosphine (PPh3), the majority of the examples isolated and structurally
characterized in our laboratory so far consist both of monomeric species exhibiting a
tetrahedral or trigonal environment around the metal centre [8] and doubly S-bridged
symmetric dimmers [9]. Meanwhile, many of these compounds have been proven to show
interesting antimicrobial and biological activity; thus, we decided to look for possible
relationships between structure and bioactivity. For example, in a recent report we presented
two series of copper(I) halide complexes of N-methylbenzothiazole-2-thione (abbreviated as
mbtt), namely dinuclear [CuX(mbtt)2]2 and mononuclear [CuX(PPh3)2(mbtt)], of which only
the later showed significant antibacterial activity against several Gram-positive and Gram-
negative microorganisms [10]. Further, a remarkably high antibacterial activity was found for
the mononuclear [CuI(xantphos)(mbtt)] (xantphos=4,5-bis(diphenylphosphino)-9,9-dimethyl-
xanthene) [11]. As for the analogous silver(I) complexes, it has been found that they are more
active than the corresponding copper(I) compounds [12].
Based on this background, we decided to gain more insight into the relationship between
the molecular structures of heterocyclic thioamide/phosphine mixed-ligand silver(I)
complexes and their bioactivity. In particular, herein we report the synthesis of five new
neutral silver(I) complexes bearing specific combinations of heterocyclic thioamides (or
thioamidates), shown in Scheme 1, and a varying number of PPh3 ligands in their
coordination sphere, in the presence (or absence) of a coordinated halide ion, and to study the
effect the nature of particular thioamide ligands and the different coordination environments
of the complexes, on their interaction with calf-thymus (CT) DNA as well their antimicrobial
properties.
[5]

S
S
S
HN
S
HN
NH
HN
S
N
N
O
NH2
atdztH
mtdztH
mqztH
Scheme 1. Heterocyclic thioamides used in this work
2. EXPERIMENTAL
2.1 General procedures and chemicals
All manipulations were carried out under atmospheric conditions, unless otherwise
mentioned. Solvents were purified according to established methods and allowed to stand
over molecular sieves for 24 h. Silver(I) starting materials, i.e. silver chloride (AgCl), silver
tetrafluoroborate (AgBF4), silver nitrate (AgNO3), the thioamides 5-methyl-1,3,4-thiadiazole-
2(3H)-thione (mtdztH), 5-amino-1,3,4-thiadiazole-2(3H)-thione (atdztH) and 2-mercapto-
4(3H)-quinazolinone (mqzH), triphenylphosphine (PPh3), CT DNA, ethidium bromide (EB),
NaCl and trisodium citrate CT DNA, ethidium bromide (EB), NaCl and trisodium citrate
were obtained from commercial sources and used without any further purification.
DNA stock solution was prepared by dilution of CT DNA to buffer (containing 15 mM
trisodium citrate and 150 mM NaCl at pH 7.0) followed by vigorous stirring for three days,
and kept at 4°C for no longer than a week. This solution of CT DNA gave a ratio of UV
absorbance at 260 and 280 nm (A260/A280) of ~1.90, indicating that the DNA was sufficiently
free of protein contamination [13]. The DNA concentration was determined by the UV
absorbance at 260 nm after 1:20 dilution using ε = 6600 M-1cm-1 [14].
[6]

2.2 Syntheses
2.2.1 [AgCl(PPh3)2(κ-S-mtdztH)], 1
To a suspension of AgCl (0.072 g, 0.5 mmol) in 25 mL of CH3CN, PPh3 (0.262 g, 1.0
mmol) was added in small portions. The resulting mixture was stirred at 50οC for 24 h in dark
and then an amount of 0.066 g (0.5 mmol) of mtdztH was added. The reaction mixture was
further stirred at 60οC for 2 h in dark and then it was allowed to cool at room temperature.
After filtering the reaction mixture, the filtrate was set aside in dark to evaporate slowly at
room temperature and, over a period of 24 h, large crystals of 1 were grown which were
collected. Yield: 0.152 g (38%). Anal. Calcd for C39H34AgClN2P2S2: C, 58.54; H, 4.28; N,
3.50. Found: C, 58.24; H, 4.52; N, 3.71. FTIR (KBr, cm-1): 3044w, 2955w, 1552m, 14777vs,
1432vs, 12774vs, 1198m, 1097vs, 1049vs, 1024m, 998m, 748vs, 694vs, 504vs, 489vs. UV-
Vis (CH3CN), /nm (log ): 256 (3.66), 310 (4.03).
2.2.2 [AgCl(PPh3)2(κ-S-mqztH)], 2
A portion of 0.072 g (0.5 mmol) of AgCl was suspended in 20 mL of CH3CN and then
PPh3 (0.262 g, 1.0 mmol) was added. The resulting mixture was stirred at 50οC for 18 h in
dark and then a solution of mqztH (0.089 g, 0.5 mmol) in 20 mL of MeOH was added
dropwise. The reaction mixture was stirred at 60οC for 3 h in dark and, after allowing it to
cool at room temperature, it was filtered. The filtrate was set aside in dark to evaporate
slowly at room temperature and, over a period of 5 days, crystals of 2 were grown which
were collected. Yield: 0.178 g (42%). Anal. Calcd for C44H36AgClN2OP2S: C, 62.46; H, 4.29;
N, 3.31. Found: C, 62.21; H, 4.46; N, 3.48. FTIR (KBr, cm-1): 3050m, 2968w, 1680vs,
1615vs, 1552vs, 1479vs, 1420s, 1306m, 1267s, 1159vs, 1095s, 996m, 744vs, 515s, 503vs,
409s. UV/Vis (CH3CN), λ/nm (ε): 250 (4.32), 291 (4.36).
[7]

2.2.3 [Ag(κ-S-mtdzt)(PPh3)3], 3
A portion of AgBF4 (0.097 g, 0.5 mmol) was dissolved in a 10-mL mixture of
CH3CN:MeOH (1:1 v/v) and then PPh3 (0.262 g, 1.0 mmol) was added in small portions. The
resulting mixture was stirred at 50οC for 20 min in dark and then a 20-mL methanolic
solution of mtdzt in the same solvent mixture, obtained from the deprotonation of the
corresponding amount of mtdztH (0.066 g, 0.5 mmol) with 1.0 mL of 0.5 M solution of KOH
in MeOH, was added dropwise. After stirring at 65oC for 1 h, the resulting suspension was
filtered in order to remove a small amount of an off-white solid. The filtrate was set aside in
dark to evaporate slowly at room temperature and, after 5 days, crystals of 3 started growing,
which were collected. Yield: 0.300 g (57%). Anal. Calcd for C58H52AgN2OP3S2: C, 65.85; H,
4.95; N, 2.65. Found: C, 66.01; H, 4.70; N, 2.83. FTIR (KBr, cm-1): 3448br, 3052w, 1579m,
1481s, 1437vs, 1368m, 1307m, 1281w, 1179s, 1087s, 996s, 743vs, 698vs, 516vs, 500vs.
UV/Vis (CH3CN), λ/nm (ε): 250 (4.32), 291 (4.36). UV/Vis (CH3CN), λ/nm (ε): 257 (4.34).
2.2.4 [Ag(κ-S-atdzt)(PPh3)3], 4
To a 20-mL mixture of CH3CN/MeOH (1:1 v/v) portions of AgNO3 (0.085 g, 0.5 mmol)
and PPh3 (0.262 g, 1.0 mmol) were added and the resulting suspension was stirred at mild
temperature (~50οC) for 24 h in dark. Next, a 10-mL solution of amtdz, obtained by addition
of 1.0 mL of a 0.5 M methanolic solution of KOH into a solution of amtdztH (0.066 g, 0.5
mmol) in the same solvent mixture, was added. After stirring the reaction mixture at 50oC for
2 h in dark, it was filtered in order to remove a small amount of an off-white solid, and the
filtrate was set aside in dark to evaporate slowly at room temperature. After 3 days, crystals
of 4 were grown, which were collected. Yield: 0.272 g (51%). Anal. Calcd for
C58H50AgN4P3S2: C, 65.23; H, 4.72; N, 5.25. Found: C, 65.04; H, 4.85; N, 5.38. FTIR (KBr,
[8]

cm-1): 3054m, 1617m, 1585m, 1478vs, 1308m, 1091s, 1026m, 997m, 745vs, 694vs, 513vs,
501vs. UV/Vis (CH3CN), λ/nm (ε): 260 (4.65), 314 (3.09).
2.2.5 [Ag(κ-S-atdzt)(PPh3)2], 5
To a 10-mL mixture of CH3CN/MeOH (1:1 v/v), the amount of 0.072 g (0.5 mmol) of
AgCl was added. Next, a solution of PPh3 (0.262 g, 1.0 mmol) in 10 mL of the same solvent
mixture was added. The resulting mixture was stirred at 50°C for 20 min in dark and then a
10-mL solution of amtdzt in MeOH, which was obtained after deprotonation of amtdztH
(0.066 g, 0.5 mmol) by 1.0 mL of a 0.5 M solution of KOH in MeOH, was added dropwise.
After stirring at 65oC for 2 h, the resulting suspension was allowed to cool at room
temperature and then it was filtered. The filtrate was set aside in dark to evaporate slowly at
room temperature and, in a week, crystals of 5 were grown, which were collected. Yield:
0.151 g (36%). Anal. Calcd for C41H39AgN4OP2S2: C, 58.78; H, 4.69; N, 6.69. Found: C,
58.52; H, 4.88; N, 6.82. FTIR (KBr, cm-1): 3273br, 3050w, 1604s, 1503vs, 1479s, 1434vs,
1388vs, 1314s, 1182m, 1094s, 1027s, 998s, 743vs, 693vs, 517vs, 507vs, 485s. UV/Vis
(CH3CN), λ/nm (ε): 260 (4.31), 319 (3.39).
2.3 Instrumentation
Elemental analyses were obtained on a PerkinElmer 240B elemental microanalyzer. Infra-
red spectra were recorded on a Nicolet FT-IR 6700 spectrophotometer as KBr discs in the
region of 4000-400 cm-1. The UV-visible (UV-Vis) spectra were obtained on a Hitachi U-
2001 dual beam spectrophotometer as solutions (concentrations in the range of 10-5-10-3 M) in
CH2Cl2, CH3CN or DMSO. All fluorescence spectra were obtained on a Hitachi F-7000
fluorescence spectrometer. The viscosity experiments were carried out using an ALPHA L
[9]

Fungilab rotational viscometer equipped with an 18-mL LCP spindle and the measurements
were performed at 100 rpm.
2.4 X-ray crystal structure determinations
Single-crystals of all compounds, suitable for X-ray crystallographic analysis, were
mounted on thin glass fibers with the aid of an epoxy resin. X-ray diffraction data were
collected on a Bruker Apex II CCD area-detector diffractometer, equipped with a Mo Ka (λ =
0.71070 Å) sealed tube source, at 295 K, using the φ and ω scans technique. The program
Apex2 (Bruker AXS, 2006) was used in data collection, cell refinement, and data reduction
[15]. Structures were solved and refined with full-matrix least-squares using the program
Crystals [16]. Anisotropic displacement parameters were applied to all non-hydrogen atoms,
while hydrogen atoms were generated geometrically and refined using a riding model. Details
of crystal data and structure refinement parameters are shown on Table 1. Molecular plots
were obtained by using the program Mercury [17].
[10]

Table 1.
Crystal data, data collection and refinement parameters for compounds 1 - 5
1
2
3·CH3OH
4·CH3CN
C58H51AgN4P3S2
1068.96
Triclinic
P-1
5·CH3CN·CH3OH
C41H40AgN4OP2S2
838.74
Triclinic
P-1
Chemical formula C39H34AgClN2P2S2
C44H36AgClN2OP2S C58H49AgN2OP3S2
Formula weight
Crystal system
Space group
Temperature (K)
Unit cell
parameters
a (Å)
800.11
Monoclinic
P21/c
846.12
Triclinic
P-1
1054.96
Triclinic
P-1
295
295
295
295
295
14.4611(11)
10.2270(7)
25.2849(18)
90
99.697(1)
90
11.6739(4)
13.6222(5)
14.0160(5)
72.868(2)
82.202(2)
70.395(1)
2004.82(13)
2
13.4564(5)
13.4668(4)
14.5996(5)
78.788(1)
86.062(2)
86.000(1)
2584.74(15)
2
13.5636(6)
13.9812(5)
14.1258(6)
85.831(2)
77.430(2)
86.410 (2)
2604.61(19)
2
13.4227(10)
13.6402(10)
13.8463(10)
113.765(3)
110.771(3)
102.632(3)
1966.1(3)
b (Å)
c (Å)
α (°)
β (°)
γ (°)
Volume (Å3)
Z
3686.0(5)
4
2
Radiation type, λ
(Å)
Mo Kα, 0.71073
Mo Kα, 0.71073
Mo Kα, 0.71073
Mo Kα, 0.71073
Mo Kα, 0.71073
Absorption
coefficient
(mm−1)
0.85
0.74
0.61
0.60
0.74
Crystal size (mm) 0.25×0.19×0.17
0.33×0.29×0.25
Bruker Kappa
Apex2
0.37×0.25×0.23
Bruker Kappa
Apex2
0.21×0.19×0.15
Bruker Kappa
Apex2
0.28×0.27×0.19
Bruker Kappa
Apex2
Diffractometer
Bruker Kappa
Apex2
Absorption
correction
Tmin, Tmax
Numerical
Numerical
Numerical
0.85, 0.87
0.81, 0.83
0.86, 0.87
0.89, 0.91
0.82, 0.87
Number of
measured,
independent and
observed
43700, 10435, 8471 26934, 8255, 7048
85296, 11594, 8908 42919, 10798, 8307 38032, 11421, 7659
[I> 2.0σ(I)]
reflections
Rint
(sin θ/λ)max (Å−1)
R[F2>2σ(F2)],
wR(F2), S
0.023
0.697
0.036, 0.063, 1.00
0.012
0.631
0.020, 0.028, 1.00
0.037
0.647
0.041, 0.065, 1.00
0.013
0.630
0.031, 0.060, 1.00
0.107
0.711
0.064, 0.103, 1.00
No. of reflections 8418
No. of parameters 424
7048
469
8908
602
8307
611
7659
460
∆ρmax, ∆ρmin (e
0.56, −0.53
0.38, −0.23
1.26, −0.98
0.59, −0.48
1.51, −0.96
Å−3)
[11]

2.5 Study of the interaction of the compounds with calf-thymus (CT) DNA
In order to study in vitro the interaction of complexes 1-5 with CT DNA, the compounds
were initially dissolved (1 mM) in DMSO or, for complex 3, in CH3CN. Mixing of such
solutions with the aqueous buffer DNA-containing solutions used in the studies never
exceeded 5% DMSO (v/v) in the final solution, which was needed due to low aqueous
solubility of most compounds. Control experiments with DMSO or CH3CN were performed
and no changes in the spectra of CT DNA were observed.
The investigation of the possible DNA-binding mode of complexes 1-5 and the calculation
of the corresponding DNA-binding constants (Kb) were carried out by UV-vis spectroscopy.
The UV-vis spectra of CT DNA were recorded for a constant DNA concentration (1.2-
1.6×10-4 M) in the presence of each compound at diverse [complex]/[DNA] mixing ratios (=
r). The DNA-binding constant (Kb in M-1) is obtained by monitoring the changes in the
absorbance at the corresponding max for a constant concentration (2-5×10-5 M) with increasing
concentrations of CT DNA and it is given by the ratio of slope to the y intercept in plots
[DNA]/( - ) versus [DNA] (Fig. S2), according to the Wolfe-Shimer equation [18]:
A
f
[DNA]
[DNA]
1
=
+
(εA −εf ) (εb −εf ) Kb (εb −εf )
(eq. 1)
where [DNA] is the concentration of DNA in base pairs, = Aobsd/[compound], = the
A
f
extinction coefficient for the free compound and
compound in the fully bound form.
= the extinction coefficient for the
b
The viscosity of DNA ([DNA] = 0.1 mM) in buffer solution (150 mM NaCl and 15 mM
trisodium citrate at pH 7.0) was measured in the presence of increasing amounts of
temperature and the obtained data are presented as ( / )1/3 versus r, where is the viscosity of
0
DNA in the presence of the compound, and 0 is the viscosity of neat DNA in buffer solution.
Fluorescence emission spectroscopy was used in order to examine whether each complex
may compete with EB for the DNA-intercalating sites by displacing it from its DNA-EB
[12]

conjugate. The DNA-EB conjugate was prepared by adding 20 µM EB and 26 µM CT DNA
in buffer solution (150 mM NaCl and 15 mM trisodium citrate at pH 7.0). The possible
intercalating effect of the complexes was studied by a stepwise addition of a certain amount
of a compound’s solution into a EB-DNA solution. The resultant changes were monitored by
recording the variation of fluorescence emission spectra with excitation wavelength at 540
nm. Complexes 1-5 do not show any appreciable fluorescence emission bands at room
temperature in solution or in the presence of DNA or EB under the same experimental
conditions (
= 540 nm); therefore, the observed quenching may be attributed to the
excitation
displacement of EB from its EB-DNA conjugate. The Stern-Volmer constant (KSV, in M-1) is
used to evaluate the quenching efficiency for each compound according to the Stern-Volmer
equation (eq. 2) [19,20]:
Io
=1+ kqτ0[Q] =1+ KSV[Q]
I
(eq. 2)
where Io and I are the emission intensities of the EB-DNA solution in the absence and the
presence of the quencher, respectively, [Q] is the concentration of the quencher (i.e.
complexes 1-5), o = the average lifetime of the emitting system without the quencher and kq
= the quenching constant. KSV is calculated from the Stern-Volmer plots (Fig. S8) by the slope
of the diagram Io/I versus [Q]. Taking o = 23 ns as the fluorescence lifetime of the EB-DNA
system [21], the quenching constants (kq, in M-1s-1) of the compounds can be determined
according to eq. 3:
KSV = kqτo
(eq. 3)
2.6 Antimicrobial studies
The antibacterial activities of 1-5 against four bacterial species [Escherichia coli (E. coli),
Bacillus subtilis (B. subtilis), Bacillus cereus (B. cereus) and Staphilococcus aureus (S.
aureus)] were estimated by the minimum inhibitory concentration (MIC) method as
described earlier [22]. The cultivation media used for antimicrobial activity tests were: (i) the
[13]

Luria-Bertani broth containing1% (w/v) tryptone, 0.5% (w/v) NaCl and 0.5% (w/v) yeast
extract and (ii) the minimal medium salts broth containing 1.5% (w/v) glucose, 0.5% (w/v)
NH4Cl, 0.5% (w/v) K2HPO4, 0.1% (w/v) NaCl, 0.01% (w/v) MgSO4·7H2O and 0.1% (w/v)
yeast extract. The pH of the media was adjusted to 7.0.
3. RESULTS AND DISCUSSION
3.1 Synthesis and crystal structures
Reactions of silver(I) salts with heterocyclic thioamides ArNHS or thioamidates ArNS, in the
presence of triarylphosphines PR3, depending on the nature of thioamide and phosphine, as
well as the reactions conditions. e.g. stoichiometric ratios, can result in compounds with a
variety of structural motifs [1,2,8,9]. Aiming to the synthesis of mononuclear compounds of
silver(I) having a specific mixed set of sulfur- and phosphorus-donor atom ligands, we
attempted reactions of various silver(I) salts with three heterocyclic thioamides that differ in
their stereochemical and electronic properties, i.e mtdztH, amtdztH, and mqzH (Scheme 1),
either in their neutral or deprotonated forms, in the presence of PPh3, using different
stoichiometric reactant ratios.
In particular, the reactions of AgCl with the heterocyclic thioamides mtdztH and mqzH, in
the presence of two equivalents of PPh3, in a MeCN/MeOH mixture, afforded mononuclear
mixed-ligand complexes with the general formula [AgCl(PPh3)2(κ-S-ArNHS)], ArNHS =
mtdztH (1) and mqztH (2), in which the thioamide ligands act in an S-terminal bonding mode
(Scheme 2).
[14]

Scheme 2. Synthesis of compounds 1 and 2
Single-crystals of the two compounds, suitable for X-ray crystallographic analysis, were
obtained by slow evaporation of their corresponding reaction mixtures. Views of the
molecular structures of compounds 1 and 2 are shown in Fig. 1 and 2, while selected bond
length and angle parameters are summarized in Table 1. Both are mononuclear compounds
having the silver atoms in tetrahedral coordination environments which are completed by the
exocyclic sulfur atoms of a thioamide ligand acting in an S-terminal bonding mode, the
phosphorous atoms of two PPh3 ligands, and a halide atom. Bond angles and distances around
the central metal atom (Table 1) reveal distortions from the ideal tetrahedral geometry (for
example, P1−Ag1−P2 = 126.24(2)o and Cl1−Ag1−S1 = 96.19(2)o, for compound 1). The
Ag−P and Ag−Cl bond lengths do not differ significantly between the two complexes and
they fall in ranges that are normally found for analogous mononuclear silver(I) compounds.
The Ag−S bond lengths are slightly different, i.e. 2.7976(7) and 2.7098(4) Å for 1 and 2,
respectively, but they are close to the values found in analogous mononuclear silver(I)
complexes containing an S-bound neutral thioamide ligand. Furthermore, both compounds
are stabilized by intramolecular hydrogen-bonding interactions developed between the
coordinated chlorine atom and the nitrogen atom of the heterocyclic ring of the corresponding
thioamide ligand, as it revealed by the relative orientations of two relevant ligands and the
short Cl···N distances (at 3.10 and 3.08 Å, for compounds 1 and 2, respectively).
[15]

Fig. 1. ORTEP diagram of the molecular structure of compound 1 with displacement
ellipsoids drawn at the 35% probability level. All hydrogen atoms have been omitted for
clarity.
Fig. 2. ORTEP diagram of the molecular structure of compound 2 with displacement
ellipsoids drawn at the 35% probability level. All hydrogen atoms have been omitted for
clarity.
[16]

Table 2.
Selected bond lengths (Å) and angles (o) for compounds 1 and 2.
1
2
SL = mtdztH
SL = mqzH
bond distances
2.5706(6)
2.6142(4)
2.4551(4)
2.4597(4)
2.7098(4)
Ag1−Cl1
Ag1−P1
Ag1−P2
Ag1−S1
2.4618(5)
2.4875(5)
2.7976(7)
bond angles
112.20(2)
105.53(1)
108.65(1)
126.70(1)
102.34(1)
112.84(1)
98.32(1)
Cl1−Ag1−P1
Cl1−Ag1−P2
P1−Ag1−P2
Cl1−Ag1−S1
P1−Ag1−S1
P2−Ag1−S1
110.32(2)
126.24(2)
96.19(2)
103.74(2)
102.77(2)
The reactions of the silver(I) salts AgNO3 and AgBF4 with the heterocyclic thioamidates
mtdzt and amtdzt, respectively (which were obtained upon treatment of the corresponding
neutral thioamides mtdztH and amtdztH with equivalent amounts of KOH in MeOH), in the
presence of a two-fold excess of PPh3, in MeCN, resulted in the formation of a different class
of mononuclear silver(I) compounds with the general formula [Ag(κ-S-ArNS)(PPh3)3], ArNHS
= mtdzt (3) and amtdzt (4), as shown in Scheme 3. The formulas of the two compounds are
not in agreement with the corresponding reaction stoichiometries, since the final product
contains three PPh3 units coordinated to the central metal ion. However, it has not been
possible to identify any other product from the corresponding reaction mixtures. In contrast,
the analogous reaction of AgCl with the thioamidate salt of amtdztH with two equivalents of
PPh3, under identical experimental conditions, resulted in the synthesis of the expected
[17]

mononuclear mixed-ligand complex [Ag(κ-S-amtdzt)(PPh3)2] (5) (Scheme 3). In all cases, the
thioamidate ligands act in an S-terminal bonding mode.
Scheme 3. Synthesis of compounds 3, 4, and 5
As shown in Fig. 3 and 4, compounds 3 and 4 are mononuclear complexes with
tetrahedrally coordinated silver atoms, whose coordination sphere are completed by the
phosphorus atoms of three PPh3 molecules, and the exocyclic sulfur atom of a thioamidate
ligand (mdtzt and amtdzt, respectively). In contrast, compound 5 exhibits a trigonal planar
geometry around the metal center having coordinated two PPh3 molecules and an S-bound
amtdzt ligand (Fig. 5). All compounds exhibit slight distortions from their corresponding
ideal coordination geometries (see for example in Table 2: P1−Ag1−P2 = 113.84(3)o,
114.43(2)o, 120.61(4)o, for compounds 3, 4 and 5, respectively). In case of the tetrahedrally
coordinated compounds 3 and 4, the Ag−P bond lengths are found to fall in the range of 2.52-
2.64 Å, while in the trigonal planar compound 5 these are shorter at 2.4701(11) and
2.4881(11) Å. The Ag−S bond lengths are found to be similar among compounds 3, 4, and 5,
i.e. 2.5845(9), 2.5571(7), and 2.5238(12), respectively. Noticeably, these are shorter than the
[18]

corresponding Ag−S distances observed in the mononuclear silver(I) complexes 1 and 2, a
fact that can be ascribed to the anionic nature of the S-bound thioamidate ligands they
contain, compared to the neutral mtdztH and mqzH ligands of compounds 1 and 2 discussed
above.
Fig. 3. ORTEP diagram of the molecular structure of compound 3 with displacement
ellipsoids drawn at the 35% probability level. All hydrogen atoms have been omitted for
clarity.
[19]

Fig. 4. ORTEP diagram of the molecular structure of compound 4 with displacement
ellipsoids drawn at the 35% probability level. All hydrogen atoms have been omitted for
clarity.
Fig. 5. ORTEP diagram of the molecular structure of compound 5 with displacement
ellipsoids drawn at the 35% probability level. All hydrogen atoms have been omitted for
clarity.
[20]

Table 3. Selected bond lengths (Å) and angles (o) for compounds 3, 4, and 5.
5
3
4
L = atdzt, n = 0
L = mtdzt, n = 1 L = atdzt, n = 1
bond distances
2.6019(8)
2.5219(8)
2.6400(8)
2.5845(9)
2.5854(7)
2.5537(6)
2.6405(7)
2.5571(7)
2.4881(11)
2.4701(11)
Ag1−P1
Ag1−P2
Ag1−P3
Ag1−S1
2.5238(12)
120.61(4)
bond angles
113.84(3)
112.83(3)
110.08(3)
97.75(3)
114.43(2)
114.48(2)
111.74(2)
101.54(3)
119.50(2)
93.25(2)
P1−Ag1−P2
P1−Ag1−P3
P2−Ag1−P3
P1−Ag1−S1
P2−Ag1−S1
P3−Ag1−S1
116.65(4)
121.80(4)
114.67(3)
107.06(3)
[21]

From the aforementioned results, it is clear that reactions of silver(I) halide salts, such as
AgCl, with heterocyclic thioamides ArNHS, in the presence of two equivalents of PPh3, result
in the synthesis of mononuclear silver(I) complexes [AgCl(PPh3)2(κ-S-ArNHS)], as expected
from the reaction stoichiometry. In contrast, the analogous reactions of silver(I) salts with
heterocyclic thioamidates ArNS can lead to the synthesis of different compounds whose
identity depends on the type of the silver(I) starting materials used in each case. In particular,
the utilization of AgCl led to the synthesis of a mononuclear complex with trigonal
coordination geometry and two molecules of PPh3 coordinated to the Ag(I) ion, i.e. [Ag(κ-S-
ArNS)(PPh3)2]. However, the use of silver(I) salts which contain weakly- or non-coordinating
anions, such as BF - or NO -, give rise to complexes [Ag(κ-S-ArNS)(PPh3)3], which contain a
4
3
tetrahedrally coordinated metal ion having three molecules of PPh3 in its coordination sphere.
Corroborating evidence for the identity of the five new compounds in bulk form, as well as
verification of the coordination mode of both triplenylphosphine and the respective thioamide
ligands, has been obtained by Infra-red spectroscopy. The infrared spectra of compounds 1–5,
-1
recorded in the range 4000-200 cm are dominated by characteristic bands due to the
presence of the respective heterocyclic thioamide ligand. For example, the intense bands at
1550 cm-1 and 1268 cm-1 in the spectrum of free mtdztH, attributed to the C–N stretching
vibration of the thioamide moiety, appear in the spectrum of compound 3 shifted to 1579 cm-1
and 1307 cm-1 respectively, indicating an exclusive S-coordination of the ligand. Likewise,
the band at 1095 cm-1 assigned to the C=S stretching [23], is shifted to lower energies (1087
cm-1) due to S-coordination. Analogous conclusions are drawn from the IR spectra of the
other compounds. Analogous conclusions are drawn from the IR spectra of the other
compounds.
[22]

3.3 Interaction of the compounds with CT DNA
The mode of interaction between metal complexes and double-stranded DNA depends on
the structure of the complexes and the nature of the ligands including covalent interaction,
non-covalent interaction and cleavage of the DNA-helix [24]. Covalent interaction between
metal complexes and double-stranded DNA takes place upon the displacement of a labile
ligand of the complex by DNA-base. Non-covalent interactions are of three different types,
i.e. intercalation via • stacking interactions between the planar aromatic rings of the
complex and DNA-bases, electrostatic interactions when Coulomb forces appear between a
cationic complex and the negative phosphate groups of DNA, and groove-binding in the
presence of hydrophobic or hydrogen bonding or van der Waals forces. Herein, in order to
investigate the interaction of the compounds with CT DNA, UV-vis spectroscopy, DNA-
viscosity measurements and competitive studies with EB (by fluorescence emission
spectroscopy) have been employed.
UV-vis spectroscopy was used in order to examine the DNA-binding mode and to calculate
the DNA-binding strength. Any changes observed in the DNA UV-band or the intraligand
transition bands of the complexes may reveal the existence of any interaction and its possible
mode. The UV-vis spectra of a CT DNA solution in the presence of mtdztH and complex 5 at
increasing r values are shown representatively in Fig. 6. The band at
= 258 nm exhibits a
max
slight hypochromism accompanied by a red-shift up to 261 nm, suggesting the existence of
interaction between CT DNA and the compounds. Such interaction leads to the formation of
a new compound-DNA conjugate [25] resulting in stabilization of the CT DNA double-helix
[26]. Similar changes in the UV spectrum of a CT DNA solution are also observed in the
presence of the other compounds.
[23]

1,5
1,0
0,5
0,0
1,2
1,0
0,8
0,6
0,4
0,2
0,0
(B)
(A)
240
260
280
300
32
240
260
280
300
32
λ (nm)
λ (nm)
Fig. 6. (A) UV-vis spectra of CT DNA (0.150 mM) in buffer solution (150 mM NaCl and 15
mM trisodium citrate at pH 7.0) in the absence or presence of mtdztH. The arrows show the
changes upon increasing amounts of the compound. (B) UV-vis spectra of CT DNA (0.136
mM) in buffer solution (150 mM NaCl and 15 mM trisodium citrate at pH 7.0) in the absence
or presence of complex 5. The arrows show the changes upon increasing amounts of the
complex.
In the UV-vis spectra of the thioamides (Fig. 7(A) and Fig. S1(A) and (B)), the UV-band
located in the range 293-320 nm presents in the presence of increasing amounts of CT DNA a
hypochromism up to 29%. In the UV spectra of complexes 1-5, the intraligand band located
in the range 253-283 nm presents a moderate hyperchromism (Fig. 7(B) and (C), and Fig.
S1(C)-(E)), while the second intraligand band observed for complexes 1, 4 and 5 in the range
310-320 nm exhibits a significant hypochromism followed by its disappearance. The
significant percentage of hypochromism observed for complexes 1, 4 and 5 may be attributed
to ꢀ stackiinngg iinntteerraaccttiioonnss bbeettwweeeenn tthhee ccoommpplleexxeess aanndd DDNA-bases [27] and might be
consistent of an intercalative binding mode leading to a stabilization of the DNA helix [26].
Nevertheless, safe conclusions cannot be merely derived for all compounds from UV-vis
spectroscopic titrations studies and DNA-viscosity measurements were performed in order to
clarify the DNA-binding mode of the compounds.
[24]

1,4
1,2
1,0
0,8
0,6
0,4
0,2
0,0
0,8
0,6
0,4
0,2
0,0
(B)
(A)
260
280
300
320
340
36
280
300
320
340
360
λ (nm)
λ (nm)
1,2
1,0
0,8
0,6
0,4
0,2
0,0
(C)
240
260
280
300
320
340
λ (nm)
Fig. 7. UV-vis spectra of solutions of (A) mtdztH (5×10-5 M) in DMSO, (B) complex 1
(5×10-5 M) in DMSO and (C) complex 3 (2.5×10-5 M) in CH3CN, in the presence of
increasing amounts of CT DNA (r' = [DNA]/[compound] = 0-0.8). The arrows show the
changes upon increasing amounts of CT DNA.
The DNA-binding constants (Kb) of the compounds (Table 4) were calculated by the
Wolfe-Shimer equation [27] (eq. 1) and the corresponding plots [DNA]/( - ) versus [DNA]
A
f
(Fig. S2). The Kb constants of the compounds (Table 4) are significantly high and suggest
strong binding of the complexes to CT DNA with complex 4 (=6.35( 0.05)×107 M-1) showing
the highest affinity for CT DNA among complexes 1-5. In average, the Kb constants are
5
-1
×
similar or higher than that of the classical intercalator EB (= 1.23 10 M ) as calculated by
Dimitrakopoulou et al [28].
[25]

Table 4. UV-vis spectral features of the interaction of complexes 1-5 with CT DNA: UV-
,
A/A
,
band ( in nm) (percentage of hyper-/hypo-chromism (
0, in %), blue-/red-shift of
(
max
-1
in nm
)) and DNA-binding constants (Kb, in M ).
Compound
λ(nm) (∆A/Ao% (%)a, ∆λ(nm) b)
313 (-12, 0)
Kb (Μ-1)
mtdztH
7.34( 0.02)×107
1.24( 0.19)×106
1.01( 0.24)×106
3.63( 0.11)×106
1.27( 0.01)×107
1.62( 0.10)×106
6.35( 0.05)×107
1.78( 0.10)×104
mqztH
293 (-29, +5)
atdztH
320 (-22, -10)
266 (+11, 5), 310 (-48, elm c)
[AgCl(PPh3)2(mtdztH)] (1)
[AgCl(PPh3)2(mqztH)] (2)
[Αg(PPh3)3(mtdzt)] (3)
[Αg(PPh3)3(atdzt)] (4)
[Ag(PPh3)2(atdzt)] (5)
283 (+3.6, 0)
253 (+9, 0)
267 (+4.5, +3), 320 (-60, elm)
272 (+15, 0), 315 (-20, elm)
a “+” denotes hyperchromism, “-” denotes hypochromism
b “+” denotes red-shift, “-” denotes blue-shift
c “elm” = eliminated
In order to further investigate the interaction of the compounds with CT DNA, the changes
of the DNA-viscosity induced by the presence of the complexes in increasing amounts were
also monitored. It is known that the relative DNA-viscosity ( / ) is related to the relative
0
DNA-length (L/L0) via the equation L/L0 = ( / )1/3 [28]. Therefore, any changes of the relative
0
DNA-viscosity occurring in the presence of a DNA-binder may reveal the changes of relative
DNA-length and subsequently the possible mode of DNA-interaction. In the case of
intercalation, the DNA-viscosity presents a significant increase, while in the case of non-
classical intercalation (including groove-binding or electrostatic interaction), the DNA-
viscosity may decrease slightly or remain unchanged [29].
The changes of the viscosity of a CT DNA solution (0.1 mM) were in the presence of
increasing amounts of complexes 1-5 (up to the value of r = 0.35). For all complexes, the
DNA-viscosity has shown a considerable increase upon their addition (Fig. 8). This increase
[26]

may be attributed to intercalation of the complexes in-between DNA-bases, since in the case
of intercalation, the separation distance of the DNA bases will increase in order to
accommodate the intercalating compounds and subsequently the DNA-viscosity is increased
[29]. The existing conclusion of intercalation may clarify and enforce the preliminary
conclusions derived from the UV-vis spectroscopic studies.
1,7
(1)
1,6
1,5
1,4
1,3
1,2
1,1
1,0
(2)
(3)
(4)
(5)
0,00 0,05 0,10 0,15 0,20 0,25 0,30 0,3
r = [complex]/[DNA]
Fig. 8. Relative viscosity ( / )1/3 of CT DNA (0.1 mM) in buffer solution (150 mM NaCl and
o
15 mM trisodium citrate at pH 7.0) in the presence of complexes 1-5 at increasing amounts
versus r = [complex]/[DNA].
Ethidium bromide is a DNA-intercalator. The intercalation takes place via the planar EB-
phenanthridine ring which inserts in-between the DNA-base pairs resulting in the appearance
of an intense fluorescence emission band at 592 nm upon excitation of the EB-DNA solution
at 540 nm. When a compound able to intercalate equally or more strongly than EB is added
into the EB-DNA solution, a remarkable quenching of the EB-DNA fluorescence emission
band may appear [19,30]. Therefore, the emission spectra of pre-treated EB-CT DNA ([EB] =
20 µM, [DNA] = 26 µM) in the absence and presence of each complex were recorded in the
presence of increasing amounts of the compounds up to the value of r = 0.30 (representatively
shown for complex 3 in Fig. 9(A)).
[27]

(A)
(B)
8000
6000
4000
2000
0
100
80
60
40
20
0
1
2
3
4
5
580 600 620 640 660 680 700 720 74
0,00
0,05
0,10
0,15
0,20
0,25
0,3
λ (nm)
r = [complex]/[DNA]
Fig. 9. (A) Fluorescence emission spectra ( = 540 nm) for EB-DNA ([EB] = 20 µM,
exc
[DNA] = 26 µM) in bufffer soolluuttiioonn iinn tthhee aabbsseennccee aanndd pprreesseennccee oof increasingg aammoouunnttss ooff
complex 3 (up to the value of r = 0.28). The arrow shows the changes of intensity upon
increasing amounts of 3. (B) Plot of EB-DNA relative fluorescence intensity (I/Io%) at
=
em
592 nm versus r (r = [complex]/[DNA]) in buffer solution (150 mM NaCl and 15 mM
trisodium citrate at pH 7.0) in the presence of complexes 1-5 (quenching up to 37.6% of the
initial EB-DNA fluorescence for 1, 39.3% for 2, 25.8% for 3, 37.5% for 4 and 34.9% for 5).
The addition of the complexes resulted in a significant quenching (Fig. 9(B)) of the
emission band of the EB-DNA system at 592 nm up to 74.2% of the initial EB-DNA
fluorescence (Table 5). Since the complexes do not exhibit any fluorescence emission band at
room temperature in solution or in the presence of CT DNA under the same experimental
conditions (i.e. excitation = 540 nm), the observed quenching of the EB-DNA emission band may
be attributed to the competition of the complexes with EB for the DNA-intercalation sites.
[28]

Table 5. Fluorescence features of the EB-displacement studies of the ligands and complexes
1-5: percentage of EB-DNA fluorescence emission quenching ( I/Io, in %), Stern-Volmer
(KSV, in M-1) and quenching constants (kq, in M-1s-1).
Compound
∆Ι/Ι0 %
62.4
60.1
74.2
62.5
65.1
54.9
63.2
62.5
Κsv (M-1)
kq (M-1 s-1)
[AgCl(PPh3)2(mtdtH)] (1)
[AgCl(PPh3)2(mqztH)] (2)
[Αg(PPh3)3(mtdzt)] (3)
[Ag(PPh3)3(atdzt)] (4)
[Ag(PPh3)2(atdzt)] (5)
mtdztH
1.14( 0.03)×105
1.72( 0.06)×105
5.32( 0.15)×105
1.40( 0.03)×105
3.71( 0.09)×105
1.16( 0.02)×105
3.26( 0.10)×105
1.41( 0.07)×105
4.96( 0.13)×1012
7.46( 0.24)×1012
2.31( 0.07)×1013
6.10( 0.15)×1012
1.61( 0.04)×1013
5.03( 0.10)×1012
1.42( 0.05)×1013
6.13( 0.03)×1012
mqztH
atdztH
The linear Stern-Volmer equation (eq. 2) and the corresponding Stern-Volmer plots (Fig.
S2, R = 0.99) may confirm that the observed quenching of the EB-DNA fluorescence may be
attributed to the competition of the complexes with EB. Subsequently, it may be proposed
that the complexes may displace EB from its conjugate with DNA. Therefore, the existence
of intercalation of the complexes in-between CT DNA may be indirectly confirmed [20]. The
values of the Ksv constants of the complexes (Table 5) are high enough and confirm their
ability to bind to DNA with complex 3 bearing the highest Ksv value (=5.32( 0.15)×105 M-1)
among the complexes. The EB-DNA quenching constants (kq) for the compounds were
calculated according to eq. 3, since the EB-DNA system fluorescence lifetime of has the
-1
value o = 23 ns [21]. The kq constants (Table 5) are much higher than the value of 1010 M-1s
and may suggest that the quenching of the EB-DNA fluorescence induced by the complexes
takes place via a static mechanism [19] leading to the formation of a new conjugate, i.e.
complex-DNA.
[29]