Synthesis, characterization, thermal and DNA-binding properties of new zinc complexes with 2-hydroxyphenones

Article abstract of DOI:10.1016/j.jinorgbio.2014.01.019

The neutral mononuclear zinc complexes with 2-hydroxyphenones (ketoH) having the formula [Zn(keto)₂(H₂O)₂] and [Zn(keto)₂(enR)], where enR stands for a N,N'-donor heterocyclic ligand such as 2,2′-bipyridine (bipy), 1,10-phenanthroline (phen) or 2,2′-dipyridylamine (dpamH), have been synthesized and characterized by IR, UV and ¹H NMR spectroscopies. The 2-hydroxyphenones are chelated to the metal ion through the phenolate and carbonyl oxygen atoms. The crystal structures of [bis(2-hydroxy-4-methoxy-benzophenone)(2,2′-bipyridine) zinc(II)] dimethanol solvate and [bis(2-hydroxy-benzophenone)(2,2′- bipyridine)zinc(II)] dimethanol solvate have been determined by X-ray crystallography. The thermal stability of the zinc complexes has been investigated by simultaneous TG/DTG-DTA technique. The ability of the complexes to bind to calf-thymus DNA (CT DNA) has been studied by UV-absorption and fluorescence emission spectroscopy as well as viscosity measurements. UV studies of the interaction of the complexes with DNA have shown that they can bind to CT DNA and the corresponding binding constants to DNA have been calculated and evaluated. The complexes most probably bind to CT DNA via intercalation as concluded by studying the viscosity of a DNA solution in the presence of the complexes. Competitive studies with ethidium bromide (EB) have shown that the reported complexes can displace the DNA-bound EB, suggesting strong competition with EB for the intercalation site.

Full text of DOI:10.1016/j.jinorgbio.2014.01.019

Journal of Inorganic Biochemistry 134 (2014) 66–75
Contents lists available at ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
Synthesis, characterization, thermal and DNA-binding properties of new
zinc complexes with 2-hydroxyphenones
Emina Mrkalić a,b, Ariadni Zianna a, George Psomas a, Maria Gdaniec c, Agnieszka Czapik c,
Evdoxia Coutouli-Argyropoulou d, Maria Lalia-Kantouri a,
⁎
a
Department of General and Inorganic Chemistry, Faculty of Chemistry, Aristotle University of Thessaloniki, GR-54124 Thessaloniki, Greece
b
Institute of Chemistry, Faculty of Sciences, University of Kragujevac, 34000 Kragujevac, Serbia
Faculty of Chemistry, Adam Mickiewicz University, 61614 Poznan, Poland
c
d
Department of Organic Chemistry and Biochemistry, Faculty of Chemistry, Aristotle University of Thessaloniki, GR-54124 Thessaloniki, Greece
a r t i c l e i n f o
a b s t r a c t
Article history:
The neutral mononuclear zinc complexes with 2-hydroxyphenones (ketoH) having the formula [Zn(keto)2(H2O)2]
and [Zn(keto)2(enR)], where enR stands for a N,N'-donor heterocyclic ligand such as 2,2′-bipyridine (bipy), 1,10-
phenanthroline (phen) or 2,2′-dipyridylamine (dpamH), have been synthesized and characterized by IR, UV and
1H NMR spectroscopies. The 2-hydroxyphenones are chelated to the metal ion through the phenolate and carbonyl
oxygen atoms. The crystal structures of [bis(2-hydroxy-4-methoxy-benzophenone)(2,2′-bipyridine)zinc(II)]
dimethanol solvate and [bis(2-hydroxy-benzophenone)(2,2′-bipyridine)zinc(II)] dimethanol solvate have been
determined by X-ray crystallography. The thermal stability of the zinc complexes has been investigated by simul-
taneous TG/DTG–DTA technique. The ability of the complexes to bind to calf-thymus DNA (CT DNA) has been
studied by UV-absorption and fluorescence emission spectroscopy as well as viscosity measurements. UV studies
of the interaction of the complexes with DNA have shown that they can bind to CT DNA and the corresponding
binding constants to DNA have been calculated and evaluated. The complexes most probably bind to CT DNA
via intercalation as concluded by studying the viscosity of a DNA solution in the presence of the complexes.
Competitive studies with ethidium bromide (EB) have shown that the reported complexes can displace the
DNA-bound EB, suggesting strong competition with EB for the intercalation site.
Received 11 November 2013
Received in revised form 16 December 2013
Accepted 24 January 2014
Available online 3 February 2014
Keywords:
Zinc complexes
2-Hydroxyphenones
Crystal structure
Thermal behavior
Interaction with calf-thymus DNA
Competitive studies with ethidium bromide
© 2014 Elsevier Inc. All rights reserved.
1. Introduction
the expanding interest for their use. These compounds have been effec-
tive in vitro and in vivo for the treatment of anaphylaxis, androgenesis,
Zinc is an essential trace element for the growth and development in
all forms of life and has presented beneficial, therapeutic and preventive
effects on infectious diseases [1,2], being the second most abundant
trace metal in the human body [3]. Zinc is not only an essential cofactor
with catalytic, structural or regulatory role in a plethora of enzymes but
also its complexes are often used as drugs [4–6]. In the literature, di-
verse zinc complexes showing a noteworthy biological activity have
been structurally characterized, including zinc complexes with drugs
used for the treatment of Alzheimer disease [7] and others showing bac-
tericidal [8], anticonvulsant [9], antidiabetic [5], anti-inflammatory [10],
antimicrobial [11], antioxidant [12] and cytotoxic [13–15] activity.
Benzophenone and its derivatives have been widely used in many
commercial products, e.g. sunscreens, cosmetics and plastic surface
coatings, because of their ability to absorb and dissipate ultraviolet
light A (400–315 nm) [16–18]. Besides their important commercial in-
terest, a large number of studies have been carried out in regard to the
pharmacological effects of benzophenone and its derivatives explaining
inflammation, malaria, tuberculosis and virus [18–26]; they are also
considered as inhibitors of HIV, farnesyltransferase and reverse tran-
scriptase [27–29]. On the other side, benzophenones may be absorbed
through human skin resulting in a possible bioaccumulation in the
human body [30]. They have also been found to be endocrine disrupting
chemicals (EDCs) with some hydroxylated benzophenones, such as the
2- and 4-hydroxylated derivatives, showing both estrogenic and
antiandrogenic activities [18]. Additionally, hepatotoxicity [31] and
some incidents of photoallergic reactions in patients with suspected
clinical photosensitivity [32] have been reported. Keeping in mind
these controversial effects, it is of important significance the prepara-
tion of new benzophenone compounds (including their complexes)
which may show enhanced biological activity in comparison to the
existing compounds.
We have initiated the synthesis and characterization of transition
metal complexes with carbonyl compounds derived from salicylaldehyde
and benzophenone [33–37]. Within this context, the neutral mononucle-
ar zinc complexes with two 2-hydroxybenzophenones (abbreviated
as ketoH), 2-hydroxybenzophenone (=bpoH, 2-OH-benzophenone)
and 2-hydroxy-4-methoxybenzophenone (=opoH, 2-OH-4-MeO-
⁎
Corresponding author. Tel./fax: +30 2310 997844.
E-mail address: lalia@chem.auth.gr (M. Lalia-Kantouri).
0162-0134/$ – see front matter © 2014 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.jinorgbio.2014.01.019

E. Mrkalić et al. / Journal of Inorganic Biochemistry 134 (2014) 66–75
67
benzophenone, commercially known as benzophenone 3; see
Scheme 1(A)) were prepared and characterized. Complexes 1 and 5 of
the formula [Zn(keto)2(H2O)2] were synthesized in the absence of the
N,N′-donor heterocyclic ligand (enR), whereas complexes 2–4 and 6–8
of the formula [Zn(keto)2(enR)] were prepared in the presence of 2,2′-
bipyridine (bipy) (2 and 6), 1,10-phenanthroline (phen) (3 and 7) or
2,2′-dipyridylamine (dpamH) (4 and 8). The crystal structures of the
solvated complexes 2 and 6 have been determined by X-ray crystallog-
raphy. The thermal stability and decomposition mode for five of the
compounds were studied in nitrogen atmosphere by using the simulta-
neous technique (TG/DTG–DTA).
The interaction of transition metal complexes with DNA has been in
the center of scientific interest for many years, mainly due to their ver-
satile applications in cancer research and nucleic acid chemistry
[38–42]. Nevertheless, as revealed by a thorough search of the literature,
the interaction of DNA with 2-hydroxy-benzophenones and their metal
complexes has not been studied up to now. Therefore, in the general
context of the research of potential metallodrugs as well as biological
importance of zinc, interaction of DNA with the easily available ketoH li-
gands, opoH and bpoH, and their Zn complexes 1–8, was deemed wor-
thy of extensive studies. As continuation of our earlier research [33], the
ability of opoH, bpoH and complexes 1–8 to bind to calf-thymus (CT)
DNA has been investigated by (i) UV spectroscopic titration where the
binding constants Kb of the compounds to CT DNA have been calculated,
(ii) measurements of the viscosity of DNA solution in the presence of in-
creasing amounts of the compounds (ketoHs and their complexes) and
(iii) competitive binding titration with the classic intercalator ethidium
bromide (EB) performed by fluorescence emission spectroscopy in
order to assess the ability of the compounds to displace EB from the
EB–DNA complex as an indirect proof of a potential intercalative bind-
ing mode.
CT DNA to buffer (containing 150 mM NaCl and 15 mM trisodium citrate
at pH 7.0) followed by exhaustive stirring at 4 °C for 3 days, and kept at
4 °C for no longer than a week. The stock solution of CT DNA gave a ratio
of UV absorbances at 260 and 280 nm (A260/A280) of 1.87, indicating that
the DNA was sufficiently free of protein contamination. The DNA con-
centration per base pair was determined after 1:20 dilution by the UV
absorbance at 260 nm using ε = 6600 M−1 cm−1 [43].
2.2. Instrumentation — physical measurements
Infrared (IR) spectra (400–4000 cm−1) were recorded on a Nicolet
FT-IR 6700 spectrometer with samples prepared as KBr pellets. UV–vis-
ible (UV–vis) spectra were recorded as Nujol mulls and in DMSO solu-
tions at concentration in the range 10−5–10−3 M on a Hitachi U-2001
dual beam spectrophotometer. 1H NMR spectra were recorded at
300 MHz on a Bruker AVANCEIII 300 spectrometer using DMSO-d6 as
solvent. C, H and N elemental analyses were performed on a Perkin-
Elmer 240B elemental microanalyzer. Molecular conductivity measure-
ments were carried out with a Crison Basic 30 conductometer. Fluores-
cence spectra were recorded in solution on a Hitachi F-7000
fluorescence spectrophotometer. Viscosity experiments were carried
out using an ALPHA L Fungilab rotational viscometer equipped with
an 18 mL LCP spindle and the measurements were performed at
100 rpm. The simultaneous TG/DTG–DTA curves were recorded on a
SETARAM thermal analyzer, model SETΑRAM SETSYS-1200. The sam-
ples of approximately 10 mg were heated in platinum crucibles, in a ni-
trogen atmosphere at a flow rate of 50 ml min−1, within the
temperature range 30–1000 °C, at a heating rate of 10 °C min−1.
2.3. Synthesis of the complexes
2.3.1. Synthesis of [Zn(keto)2(H2O)2] (1 and 5)
Complexes 1 and 5 were synthesized according to our published pro-
cedure [44], by the addition of a methanolic solution (15 mL) of ketoH
(1 mmol), deprotonated with CH3ONa (1 mmol, 54 mg), to a methano-
lic solution (10 mL) of Zn(NO3)2·6H2O (0.5 mmol, 148.75 mg) at room
temperature. The reaction mixture was stirred for 2 h and then turned
into yellowish. The solution was filtered and left for slow evaporation.
After a few days a pale yellow microcrystalline product was collected
with filtration, washed with cold water and air-dried.
2. Experimental
2.1. Materials
The ketoH ligands (opoH and bpoH), the amines enR (bipy, phen
and dpamH), NaCl, CH3ONa, Zn(NO3)2.6H2O, trisodium citrate, CT
DNA and EB were obtained as reagent grade from Sigma-Aldrich Co
and used as received. Solvents for preparation and physical measure-
ments of “extra pure” grade were obtained from Merck and used with-
out further purification. DNA stock solution was prepared by dilution of
[Zn(opo)2(H2O)2], 1: Yellow microcrystalline solid, yield 63%,
174 mg, conductivity in DMSO solution 3.1 μS/cm, analyzed as
[Zn(opo)2(H2O)2], (ZnC28H26O8), (MW = 555.5): C 60.48, H 4.68,
Found: C 60.04, H 4.54. IR spectrum (KBr): selected peaks in cm−1
:
4'
A
B
5'
6'
3'
2'
3439 (medium, (m)) v(O–H) of coordinated water, 2816(m)
v(−OCH3), 1612 (strong, (s)) and 1587(s) v(C = O), 1363(s) v(C–O
→ Zn), 603(m) v(Zn–O); UV–vis: λ/nm (ε/M−1 cm−1) as Nujol mull:
288.5, 321, 350; in DMSO: 288 (2400), 319 (1650), 347(sh (shoulder))
(1200). 1H NMR spectrum in DMSO-d6 (δ/ppm), 50 °C: 7.70–7.37 (12H,
m, H6, H2′, H3′, H4′, H5′and H6′-opo), 6.60–6.45 (4H, br(broad), m, H3and
H5-opo), and 3.84 (6H, s, CH3O).
6
5
5'
3'
6'
1'
7
4
6
4'
C
1
5
4
O
N
N
3
[Zn(bpo)2(H2O)2], 5: Yellow microcrystalline solid, yield 65%,
161 mg, conductivity in DMSO solution 2.4 μS/cm, analyzed as
[Zn(bpo)2(H2O)2], (ZnC26H22O6), (MW = 495.5): C 62.96, H 4.44,
2
X
OH
3
Found: C 62.55, H 4.24. IR spectrum (KBr): selected peaks in cm−1
:
3437 (medium, (m)) v(O–H) of coordinated water, 2817(m)
v(−OCH3), 1627 (strong, (s)) and 1601(s) v(C = O), 1335(s)
v(C–O → Zn), 530(m) v(Zn–O); UV–vis: λ/nm (ε/M−1 cm−1) as
Nujol mull: 265, 323, 395; in DMSO: 265 (6400), 320 (2300), 403
(sh) (350). 1H NMR spectrum in DMSO-d6 (δ/ppm): 1H NMR spectrum
in DMSO-d6 (δ/ppm): 7.85–7.30 (14H, m, H4, H6, H2′, H3′, H4′, H5′and
H6′-bpo), and 7.05–6.87 (4H, br m, H3and H5-bpo).
6
5
C
D
5
5'
N
7
4
4'
6'
4
6
8
3
7
3'
3
N
H
N
N
N
9
2
2.3.2. Synthesis of [Zn(keto)2(enR)] (2–4 and 6–8)
A methanolic solution (10 mL) of bipy (1 mmol, 156 mg), phen
(1 mmol, 180 mg) or dpamH (1 mmol, 171 mg) was added slowly to
Scheme 1. (A) The 2-hydroxyphenone ligands (ketoH) (X = H for bpoH and X = CH3O- for
opoH) and (B)–(D) the α-diimines (enR): bipy (B), phen(C), dpamH(D) and H-atom
labeling.

68
E. Mrkalić et al. / Journal of Inorganic Biochemistry 134 (2014) 66–75
a methanolic solution (10 mL) of [Zn(keto)2(H2O)2] (1 or 5) (1 mmol)
under stirring at room temperature. The reaction mixture was stirred
for 2 h, reduced in volume and left for slow evaporation. The pale yellow
microcrystalline product was filtered off and dried under vacuum.
[Zn(opo)2(bipy)]∙2CH3OH, 2∙2CH3OH: Yellow crystals, yield 65%, 240
mg, conductivity in DMSO solution 2.1 μS/cm, suitable for X-ray
crystallography, analyzed as [Zn(opo)2(bipy)]∙2CH3OH (ZnC40H38N2O8)
(MW = 739.5): C 64.91, H 5.13, N 3.78; Found: C 64.73, H 5.07, N 3.75.
IR spectrum (KBr): selected peaks in cm−1: 3430(m) v(O–H) of crystal-
lized methanol, 1612(s) and 1587(s) v(C = O), 1568(s) v(C = N),
780(m) and 724(s) δ(C–H)pyridyl, 593(m) and 507(m) v(Zn–O), 415(m)
v(Zn–N); UV–vis: λ/nm (ε/M−1 cm−1) as Nujol mull: 275, 328, 400; in
DMSO: 275 (7300), 325 (2700), 393 (1500). 1H1H NMR spectrum in
DMSO-d6 (δ/ppm), 50 °C: 9.09 (2H, br, H2- and H9-phen), 8.50 (2H, br,
H4- and H7-phen), 8.03 (2H, s, H5- and H6-phen), 7.81 (2H, br, H3- and
H8-phen), 7.71–7.28 (14H, m, H4, H6, H2′, H3′, H4′, H5′and H6′-bpo), and
7.02–6.75 (4H, br m, H3and H5-bpo).
[Zn(bpo)2(dpamH)], 8: Yellow microcrystalline solid, yield 63%,
204 mg, conductivity in DMSO solution 1.0 μS/cm, analyzed as
[Zn(bpo)2(dpamH)], (ZnC36H27N3O4) (MW = 630.5): C 68.52, H 4.28,
N 6.66; Found: C 68.19, H 4.27, N 6.61. IR spectrum (KBr): selected
peaks in cm−1: 3303(m), 3192(m) and 3075(m) v(N–H)dpamH, 1643(s)
δ(N–H)dpamH, 1603(s) v(C = O), 1573(s) v(C = N), 1344(s) v(C–O
→ Zn), 874(m), 827(m) and 760(s), 700(s) δ(C–H)pyridyl, 593(m) and
517(m) v(Zn–O), 498(w) v(Zn–N); UV–vis: λ/nm (ε/M−1 cm−1) as
nujol mull: 268, 317, 420; in DMSO: 268 (4970), 316 (3700). 359(sh)
(800), 370(sh) (300). 1H NMR spectrum in DMSO-d6 (δ/ppm):
9.58 (1H, br s, H7-dpamH), 8.22 (2H, d, J = 4.2 Hz, H3- and H3′-
dpamH), 7.82–7.33 (18H, m, H4, H6, H2′, H3′, H4′, H5′, H6′-bpo and H5,
H5′,H6′-dpamH), and 7.06–6.83 (6H, m, H3, H5-bpo and H4, H4′-dpamH).
1542(s), 1364(s) v(C–O → Zn), 840(m), 750(m) and 700(s) δ(C–H)pyridyl
,
)
586(m) v(Zn–O), 472(weak, (w)) v(Zn–N); UV–vis: λ/nm (ε/M−1 cm−1
as Nujol mull: 290, 320, 363, 420; in DMSO: 288 (6100), 320 (5500),
365(sh) (850), 416 (530). 1H NMR spectrum in DMSO-d6 (δ/ppm):
7.67–7.30 (12H, m, H6, H2′, H3′, H4′, H5′and H6′-opo), 6.11(1H, s, H3–
opo), 5.95 (1H, d, J = 7.6 Hz, H5-opo), 3.70 (6H, s, CH3O), and 8.71
(2H, d, J = 3.8 Hz, H3- and H3′-bipy), 8.51 (2H, d, J = 7.8 Hz, H6- and
H6′-bipy), 8.08 (2H, t, J = 7.8 Hz, H5- and H5′-bipy), and 7.04 (2H, br
d, J = 7.8 Hz, H4- and H4′-bipy).
[Zn(opo)2(phen)]∙1CH3OH, 3∙1CH3OH: Pale yellow microcrystalline
solid, yield 66%, 241 mg, conductivity in DMSO solution 1.8 μS/cm, ana-
lyzed as [Zn(opo)2(phen)]∙CH3OH (ZnC41H34N2O7) (MW = 731.5): C
67.25, H 4.64, N 3.82; Found: C 67.02, H 4.57, N 3.70. IR spectrum
(KBr): selected peaks in cm−1: 3427(m) v(O–H) of crystallized metha-
nol,1612(s) v(C = O), 1567(s) v(C = N), 1360(m) v(C–O → Zn),
840(m), 751(m) and 726(s) δ(C–H)pyridyl, 605(m) v(Zn–O), 415(m)
v(Zn–N); UV–vis: λ/nm (ε/M−1 cm−1) as Nujol mull: 290, 325, 385;
in DMSO: 284 (5500), 320 (2100), 385(sh) (420). 1H NMR spectrum
in DMSO-d6 (δ/ppm), 50 °C: 9.06 (2H, br, H2- and H9-phen), 8.62 (2H,
d, J = 6.3 Hz, H4- and H7-phen), 8.09 (2H, s, H5- and H6-phen), 7.89
(2H, br, H3- and H8-phen), 7.63–7.22 (12H, m, H6, H2′, H3′, H4′, H5′and
H6′-opo), 6.49–6.22 (4H, br m, H3 and H5-opo), and 3.39 (6H, s, CH3O).
[Zn(opo)2(dpamH)], 4: Yellow microcrystalline solid, yield
68%,221 mg, conductivity in DMSO solution 0.8 μS/cm, analyzed as
[Zn(opo)2(dpamH)], (ZnC38H31N3O6) (MW = 690.5): C 66.04, H 4.49,
N 6.08; Found: C 65.73, H 4.47, N 6.01. IR spectrum (KBr): selected
2.4. X-ray crystal structure determination
Slow crystallization from the reaction mixture in methanol yielded
yellow crystals of compounds [Zn(opo)2(bipy)].2CH3OH (2 · 2CH3OH)
and [Zn(bpo)2(bipy)]. 2CH3OH (6 · 2CH3OH). The diffraction data for
single crystals were collected at 100 K with an Oxford Diffraction
Xcalibur E diffractometer using Mo Kα radiation for compound 2
· 2CH3OH, and at 130 K with an Oxford Diffraction SuperNova diffrac-
tometer using Cu Kα radiation for compound 6 · 2CH3OH. The intensity
data were collected and processed using CrysAlisPro software [45]. The
structures were solved by direct methods with the program SIR-2004
[46] and refined by full-matrix least-squares method on F2 with
SHELXL-97 [47]. The carbon-bound hydrogen atoms were refined as
riding on their carriers and their displacement parameters were set
equal to 1.5Ueq(C) for the methyl groups and 1.2Ueq(C) for the remain-
ing H atoms. The O–H group hydrogen atoms were located in electron
density difference maps and freely refined. Crystallographic data, data
collection and refinement details are given in Table 1. Molecular
graphics were generated with ORTEP-3 for Windows [48] and Mercury
3.0 software [49].
peaks in cm−1: 3317(m), 3192(m) and 3072(m) v(N–H)dpamH
,
1643(s) δ(N–H)dpamH, 1597 m v(C = O), 1580(s) v(C = N), 1359(s)
v(C–O → Zn), 842(m) and 768(s) δ(C–H)pyridyl, 599(m) and 536(m)
v(Zn–O), 498(w) v(Zn–N); UV–vis: λ/nm (ε/M−1 cm−1) as Nujol
mull: 289, 320, 355, 395; in DMSO: 287 (3200), 318 (3300), 353(sh)
(350), 370(sh) (750). 1H NMR spectrum in DMSO-d6 (δ/ppm), 50 °C:
9.20 (1H, br s, H7-dpamH), 8.22 (2H, br, H3- and H3′-dpamH), 7.88–
7.47 (16H, m, H6, H2′, H3′, H4′, H5′, H6′-opo and H5, H5,H6, H6′-dpamH),
6.86 (2H, br m, H4- and H4′-dpamH). 6.62–6.45 (4H, br m, H3and H5-
opo), and 3.85 (6H, s, CH3O).
2.5. DNA-binding studies
[Zn(bpo)2(bipy)]∙2CH3OH, 6∙2CH3OH: Yellow crystals, yield 60%,
203 mg, conductivity in DMSO solution 1.4 μS/cm, suitable for X-ray crys-
tallography, analyzed as [Zn(bpo)2(bipy)]∙2CH3OH, (ZnC38H34N2O6)
(MW = 679.5): C 67.11, H 5.00, N 4.12; Found: C 66.73, H 4.98, N 4.10.
IR spectrum (KBr): selected peaks in cm−1: 3430(m) v(O–H) of crystal-
lized methanol, 1613(s) v(C = O), 1570(s) v(C = N), 1354(m) v(C–O
→ Zn), 840(m), 762(s) and 704(s) δ(C–H)pyridyl; 587(m) and 505(m)
v(Zn–O), 422(weak, (w)) v(Zn–N); UV–vis: λ/nm (ε/M−1 cm−1) as
Nujol mull: 268, 323, 390; in DMSO: 271 (6300), 320 (3700),
380 (1200). 1H NMR spectrum in DMSO-d6 (δ/ppm), 50 °C: 8.69 (2H,
br, H3- and H3′-bipy), 8.38 (2H, d, J = 7.4 Hz, H6- and H6′-bipy),
7.93 (2H, t, J = 7.4 Hz, H5- and H5′-bipy), 7.68–7.36 (16H, m, H4, H6,
H2′, H3′, H4′, H5′, H6′-bpo and H4, H4′-bipy), and 7.06–6.89 (4H, br m,
H3and H5-bpo).
[Zn(bpo)2(phen)], 7: Yellow microcrystalline solid, yield 66%,
211 mg, conductivity in DMSO solution 2.2 μS/cm, analyzed as
[Zn(bpo)2(phen)], (ZnC38H26N2O4) (MW = 639.5): C 71.30, H 4.06, N
4.38; Found: C 71.07, H 4.05, N 4.35. IR spectrum (KBr): selected
peaks in cm−1: IR spectrum (KBr): selected peaks in cm−1: 1615(s)
v(C = O), 1573(s) v(C = N), 1352(m) v(C–O → Zn), 836(m),
In order to study the interaction of DNA with the compounds, they
were initially dissolved in DMSO (1 mM). Mixing of such solutions
with the aqueous buffer solutions used in the studies never exceeded
5% DMSO (v/v) in the final solution, which was needed due to low aque-
ous solubility of some of the complexes and ketoH. All studies were per-
formed at room temperature.
2.5.1. DNA-binding studied by UV-absorption spectroscopy
The interaction of the substituted 2-hydroxyphenones (opoH and
bpoH) and complexes 1–8 with CT DNA has been studied by UV spec-
troscopy in order to investigate the possible binding modes to CT DNA
and to calculate the binding constants to CT DNA (Kb). The UV spectra
of CT DNA in the presence of each compound have been recorded for
a constant CT DNA concentration in diverse mixing ratios (r = [com-
pound]/[CT DNA]). The binding constants of the compounds with CT
DNA, Kb, have been determined using the UV spectra of the complexes
recorded for a constant concentration in the absence or presence of CT
DNA for diverse r values. Control experiments with 5% DMSO were per-
formed and no changes in the spectra of CT DNA were observed.

E. Mrkalić et al. / Journal of Inorganic Biochemistry 134 (2014) 66–75
69
Table 1
reaction of a methanolic solution of [Zn(keto)2(H2O)2] with equivalent
amount of an enR led to the preparation of the mixed-ligand zinc com-
plexes, [Zn(keto)2(enR)] (enR = bipy, phen and dpamH), (complexes
2–4 and 6–8), according to reaction (2).
Crystallographic data, data collection and refinement details for compounds 2 · 2CH3OH
and 6 · 2CH3OH.
2 · 2CH3OH
6 · 2CH3OH
Empirical formula
CCDC no.
Formula weight
Crystal system
Temperature
Radiation
[Zn(opo)2(bipy)]·2CH3OH [Zn(bpo)2(bipy)]·2CH3OH
CCDC 970394
740.09
CCDC 970395
680.04
½ZnðketoÞ2ðH2OÞ2ꢀ þ enR→½ZnðketoÞ2ðenRÞꢀ þ 2H2O:
ð2Þ
monoclinic
100 K
monoclinic
130 K
The complexes [Zn(keto)2(enR)] are soluble in DMF, DMSO, partially
soluble in most organic solvents, but insoluble in H2O and Et2O. Evi-
dence of the coordination mode of the ligands in the new zinc com-
pounds is also arisen from the interpretation of the IR, UV–vis and 1H
NMR data of the 2-hydroxy-benzophenone ligands and the complexes.
In these complexes, the 2-hydroxy-benzophenones behave as bidentate
monoanionic ligands, coordinated through the carbonyl and the pheno-
late oxygen atoms, while the enR as bidentate neutral ligand completing
the octahedral geometry around zinc. The structure for the compounds
2 and 6 was verified by single-crystal X-ray diffraction analysis.
Mo Kα
Cu Kα
Space group
Unit cell dimensions
P21/n
P21/n
a = 9.4803(1) Å
b = 33.9218(4) Å
c = 11.1930(1) Å
α = 90°
a = 9.5980(1)Å
b = 33.6995(4) Å
c = 10.0969(1) Å
α = 90°
β = 90.668(1)°
γ = 90°
β = 93.945(1)°
γ = 90°
Volume
Z, Z′
3599.30(7) Å3
4, 1
3258.08(6) Å3
4, 1
Absorption coefficient
Independent reflections/
Rint
Parameters/restraints
GOF
0.738 mm−1
6837/0.0399
1.461 mm−1
6702/0.0199
3.2. Spectroscopy (IR, UV–vis and 1H NMR)
471/0
1.227
434/0
1.039
Final R indices: R2/wR2 (all 0.0449/0.0826
data)
0.0348/0.0858
IR spectroscopy has been used in order to confirm the deprotonation
and binding mode of 2-hydroxybenzophenones. In the spectra of the
free ketoH the intense bands of the stretching and bending vibrational
modes of the phenolic OH around 3200 cm−1 and 1410 cm−1, respec-
tively, disappear from the spectra of all complexes indicating the ligand
deprotonation [50,51]. Also, the bands originating from the C\O
stretching vibrations at 1245–1285 cm−1 exhibit positive shifts towards
1350–1380 cm−1 in the complexes, denoting coordination through the
carbonyl oxygen of the ligand. The band at ~1660 cm−1 attributable to
the carbonyl bond v(R − C = O) of the free ligand, upon coordination is
shifted to lower frequency ~1605 cm−1, thus denoting its bidentate
R1/wR1 (for I N 2σ(I))
0.0408/0.0812
0.0314/0.0831
0.40/−0.45
Largest difference peak/
0.30/−0.39
hole (e Å−3
)
2.5.2. DNA-binding studied by viscosity measurements
The viscosity of a DNA solution (0.1 mM) has been measured at
room temperature in the presence of increasing amounts of the com-
pounds (substituted 2-hydroxyphenones or complexes 1–8) up to r
value of 0.35. The obtained data are presented as (η/η0)1/3 versus r,
where η is the viscosity of DNA in the presence of compound, and η0
is the viscosity of DNA alone in buffer solution.
mono-anionic character [52,53]. Also, the intense bands at ~1580 cm−1
,
attributable to the stretching vibration of v(C_N (aromatic bond)) are
present in the mixed-ligand complexes, denoting the coordination
through the nitrogen atoms of the α-diimine ligand. The medium-to-
low intensity bands at 530–560 cm−1 and 415–470 cm−1 are attributed
to the coordination bonds (Zn–O and Zn–N, respectively) according to
the literature [51].
The ultraviolet–visible (UV–vis) spectra of the complexes have been
recorded as Nujol mull and in DMSO solution and are similar, suggesting
that the complexes retain their structure in solution. In addition, in
order to explore the stability of complexes in buffer solution, the UV–
vis spectra in the series of pH (pH range 6–8, since the biological exper-
iments are performed at pH = 7) with the use of diverse buffer solu-
tions (150 mM NaCl and 15 mM trisodium citrate at pH values
regulated by HCl solution) have also been recorded. Significant changes
(shift of the λmax or new peaks) have not been observed in the spectra,
indicating that complexes 1–8 keep their integrity in the pH range 6–8
[54,55].
2.5.3. Competitive studies with EB by fluorescence emission spectroscopy
The competitive studies of each compound with EB have been inves-
tigated by fluorescence emission spectroscopy in order to examine
whether it is able to displace EB from its CT DNA–EB complex. The CT
DNA–EB complex was prepared by adding 20 μM EB and 26 μM CT
DNA in buffer (150 mM NaCl and 15 mM trisodium citrate at pH 7.0).
The intercalating effect of the substituted 2-hydroxyphenones and com-
plexes 1–8 with the DNA–EB complex was studied by adding a certain
amount of a solution of the compound step by step into the solution
of the DNA–EB complex. The influence of the addition of each com-
pound to the DNA–EB complex solution has been obtained by recording
the variation of fluorescence emission spectra with excitation wave-
length λex = 540 nm.
3. Results and discussion
1H NMR spectroscopy has also been used in order to confirm the de-
protonation of the 2-hydroxybenzophenones and the stability of the
complexes in solution. The deprotonation of the phenolic hydrogen
can be easily seen from the absence of the −OH signal, which is obvious
to the 1H NMR spectra of the free ligand, appearing as single peak at δ =
12 ppm for opoH (Fig. S1) and at 10.54 ppm for bpoH. The 1H NMR spec-
tra are consistent with the obtained structures of 1–8. It should be men-
tioned that most spectra were taken at 50 °C because of the dynamic
line broadening of the peaks due to exchange processes. All the expect-
ed sets of signals related to the presence of the ligands in the corre-
sponding compounds are present and they are slightly shifted as
expected upon binding to zinc ion. The 1H NMR spectra of the com-
plexes give signals attributable to the protons of the benzene ring at
δ = 7.85–6.45 ppm. The absence of additional set of signals related to
dissociated ligands suggests that all complexes remain intact in solution
[56,57] (Fig. 1). Similar results have been referred for the Schiff base
3.1. Synthesis-general considerations of the complexes
The reaction of Zn(NO3)2·6H2O with two 2-hydroxyphenones
(ketoH) in methanol (deprotonated by sodium methoxide) afforded
solid microcrystalline compounds in good yield, according to reaction
(1).
ΖnðNO3Þ2:6Η2Ο þ 2ketoH þ 2CH3OΝa→½ZnðketoÞ2ðH2OÞ2ꢀ
þ 2CH3OH þ 2NaNO3 þ 4Η2Ο:
ð1Þ
The obtained zinc(II) complexes are neutral (conductivities in DMSO
solutions were found in the range 0.8–3.1 μS cm−1), and possess 1:2
metal-to-ligand composition, as it is indicated from elemental analyses.
The complexes are formulated as [Zn(keto)2(H2O)2] (ketoH = opoH
and bpoH, complexes 1 and 5, respectively), and are soluble in MeOH,
CH3CN, CH3COCH3, CH2Cl2, DMF, DMSO but not in H2O and Et2O. The

70
E. Mrkalić et al. / Journal of Inorganic Biochemistry 134 (2014) 66–75
vis spectral pattern in Nujol, in DMSO solution and in the presence of
the buffer solution and their 1H NMR spectra confirm no dissociation,
suggests that the compounds are stable and keep their integrity in solu-
tion [55,59].
3.3. Description of the structures
The complexes of the formula [Zn(keto)2(enR)] can exist as three
geometric isomers I–III (Scheme 2) that can have similar but not identi-
cal properties. All three isomeric molecules are chiral, with isomers I
and II exhibiting C2 symmetry and isomer III being asymmetric. In
case of complexes 2 and 6, X-ray crystallography has been applied to de-
termine the isomeric form of the isolated compound.
The molecular structures of [Zn(opo)2(bipy)]·2CH3OH (2 · 2CH3OH)
and [Zn(bpo)2(bipy)]·2CH3OH (6 · 2CH3OH) with the atom numbering
scheme are shown in Fig. 2 and selected bond distances and angles are
given in Table 2. The complexes crystallize as racemic compounds and
the asymmetric part of the unit cell contains one neutral complex mole-
cule and two methanol molecules linked to the phenolate oxygen atoms
via O–H⋯O hydrogen bonds. The zinc(II) cation is in a distorted octahe-
dral environment formed by two nitrogen atoms from the bipy ligand
and four oxygen atoms from the two keto ligands. The Zn–Ophenolate
bond lengths (2.015, 2.020 Å in 2 and 2.026, 2.049 Å in 6) are significant-
ly shorter than the corresponding Zn–Ocarbonyl bonds (2.148, 2.164 Å
in 2 and 2.106, 2.130 Å in 6). In both crystals, the molecules have
an approximate C2 symmetry. The arrangement of the deprotonated 2-
hydroxybenzophenone ligands around the metal center is such that
their phenolate oxygen atoms coordinate trans to the bipy N atoms
and thus both isolated compounds represent the isomer II. The six-
membered chelate rings formed by the keto ligands are strongly nonpla-
nar which allows for intramolecular π–π stacking interactions of their
phenolate fragments. The two stacked aromatic rings are inclined by
12.9 and 11.9º and their centroids are 3.934 and 3.974 Å apart in 2 and
6, respectively. As intramolecular stacking interactions can be solely
formed in isomer II, they are the most probable reason for the stability
and preferred formation of isomer II of 2 and 6.
The crystal packing of 2 · 2CH3OH is to a large extent determined by
π–π stacking interactions between the bipy ligands that organize the mol-
ecules into infinite stacking extending along the a axis, with the distances
between the centroids of the stacked pyridine rings ranging from 3.755 to
3.826 Å (Fig. 3). This stacking is additionally strengthened by C–H⋯O in-
teractions (H⋯O 2.32 Å) between one of the bipy H atoms and the pheno-
late oxygen. In 6 · 2CH3OH, the stacking interactions are not so
pronounced and occur only between a pair of pyridine rings, with their
centroids at a distance of 3.613 Å. Instead, a short contact of 2.63 Å is
formed between one C–H group of the methanol molecule and the cen-
troid of the pyridine rings not involved in stacking interactions (Fig. 3).
Fig. 1. 1H NMR spectra of (A) [Zn(opo)2(H2O)2] 1 and (B) of [Zn(opo)2 (dpamH)] 4 in d6-
DMSO.
ligand 2-hydroxy-3-methoxybenzaldehyde semicarbazone and its
complexes [58].
3.4. Thermal studies
The fact that the complexes are non-electrolytes in DMSO solution
(ΛM ≤ 3.0 μS/cm, in 1 mM DMSO solution), they have the same UV–
The thermal behavior for five compounds (1, 5, 3, 7 and 8) was studied
in nitrogen atmosphere and in the temperature range ambient to 980 °C
Scheme 2. Geometric isomers (I–III) of the molecules [Zn(keto)2(enR)].

E. Mrkalić et al. / Journal of Inorganic Biochemistry 134 (2014) 66–75
71
by using the simultaneous TG/DTG–DTA technique. The temperature
ranges, the determined percentage mass losses, and the thermal effects
accompanying the decomposition are given in Table S1. Representative
thermal curves are depicted for the complexes [Zn(bpo)2(H2O)2] 5 and
[Zn(bpo)2(dpamH)] 8 in Fig. 4. For complexes 1 and 5 (absence of enR
ligand), the decomposition takes place without melting, as it is evidence
from the DTA curves (Fig. 4(A) for complex 5), while the first mass loss
coincides with the elimination of the two coordinated water molecules.
Upon further heating, the elimination of the 2-hydroxybenzophenone
ligand takes place, as a whole molecule or in fragments (Table S1), lead-
ing to a carbonaceous ZnO. The complex nature of thermal decomposi-
tion for analogous transition metal complexes has also recently been
referred [34,44,60], while the kind of carbonaceous residue (ZnO and
un-pyrolized compounds as organic part) has been observed in analo-
gous zinc complexes with the Schiff base 5-bromosalicylaldehyde
isonicotinoylhydrazone [61].
The thermal decomposition of the complexes in the presence of enR
ligands (3, 7 and 8) is even more complicated. The decomposition hap-
pens after the melting of the complexes, evidence arising from the sharp
endothermic DTA peaks (Fig. 4(B) for 8). The melting points of the stud-
ied compounds (~210–230 °C) were also determined by automated
melting point capillary tube system in static air, confirming the melting
points found on the DTA curves. Upon further heating, the decomposi-
tion continues with the elimination of the ketone fragments with sud-
den mass loss in two stages, while in the third stage the gradually
mass loss of the enR ligand takes place in the temperature range 400–
980 °C, leading to the metal oxide, ZnO.
3.5.1. DNA-binding study with UV spectroscopy
The interaction of the compounds with CT DNA is usually monitored
by UV spectroscopy since the changes observed in the UV spectra upon ti-
tration may provide evidence of the existing interaction mode; a
hypochromism due to π → π stacking interactions may appear in the
case of the intercalative binding mode, while red-shift (bathochromism)
may be observed when the DNA duplex is stabilized [63].
The UV spectra have been recorded for a constant CT DNA concen-
tration at different [complex]:[DNA] mixing ratios (r) (up to 0.3). The
UV spectra of CT DNA in the presence of opoH and its complexes 1–4
at diverse r values have not exhibited any significant change of the in-
tensity of the DNA band at λmax = 258 nm while a slight red-shift
(b3 nm) of the λmax up to 261 nm has been observed. For bpoH and
its complexes 5–8, a hyperchromism of the DNA accompanied by a 3-
nm bathochromism band has been observed (UV spectra of a CT DNA
solution in the presence of complex 6 are shown representatively in
(Fig. S2)). The recorded behavior may be considered as an evidence of
the formation of a new adduct of the compound with double-helical
CT DNA which results in the stabilization of the CT DNA duplex [64].
In the UV region of the spectra of the free ketoHs or complexes 1–8,
the intense absorption bands observed in the spectra of the complexes
can be attributed to the intraligand transition of the coordinated ligands
[33,52,65,66]. The changes observed in the intraligand transitions bands
of complexes 1–8 upon addition of CT DNA solution in diverse r values
(up to 1/r = 2) may reveal the existence of interaction between each
complex and CT DNA and may give first indication of the possible
mode of binding. UV spectra of a DMSO solution of complexes 2 and 6
in the presence of a CT DNA solution (up to 1/r = 2) are shown repre-
sentatively in Fig. 5.
3.5. Interaction of the compounds with calf-thymus DNA
In the UV spectrum of opoH and its complexes 1–4, two intraligand
bands appear in the UV region; band I at ~288 nm and band II at
~320 nm (Fig. 5(A)). Upon addition of increasing amounts of CT DNA
these two bands exhibit a slight hypochromism up to 3% while no signif-
icant shift of the λmax of these bands occurs (Table 3). For the bpo com-
pounds, an intraligand band located in the region 315–325 nm has been
observed as in Fig. 5(B). The addition of CT DNA to bpoH or complexes
5–8 results in a slight hypochromism (b3%) or hyperchromism (up to
10%) with no shift of the position of λmax (Table 3).
The results derived from the UV titration experiments suggest that
all compounds can bind to CT DNA [67], although no safe conclusions
concerning the interaction mode of the compounds to CT DNA can be
derived. As it is known, the exact mode of interaction with DNA cannot
be concluded only by UV spectroscopic studies and more techniques
should be combined in order to come to a safe conclusion. The magni-
tude of the binding strength a compound to CT DNA may be estimated
Covalent and/or non-covalent interactions are the most usual bind-
ing modes of transition metal complexes to DNA. Non-covalent interac-
tions to DNA include the intercalation of the complex within DNA helix
via the existence of π → π stacking interaction between the complex
and DNA nucleobases, minor- or major-groove binding via the forma-
tion of van der Waals forces or hydrogen-bonding or hydrophobic
bonding along minor or major groove of DNA helix and electrostatic in-
teractions upon the appearance of Coulomb forces between cationic
metal complexes and the negatively-charged phosphate groups of
DNA. The covalent binding exists when one at least labile ligand of the
complex is replaced by a nitrogen base of DNA such as guanine N7
[62]. The DNA-interaction studies of the two substituted phenones
(opoH and bpoH) and their complexes 1–8 are of special significance
since a thorough research of the literature has not revealed any relevant
studies concerning the ketoHs used herein or any of their complexes.
Fig. 2. The molecular structures of (A) 2 · 2CH3OH and (B) 6 · 2CH3OH.

72
E. Mrkalić et al. / Journal of Inorganic Biochemistry 134 (2014) 66–75
Table 2
Selected bond distances (Å) and angles (°) for complexes 2 · 2CH3OH and 6 · 2CH3OH.
Bond distance
2 · 2CH3OH
6 · 2CH3OH
Zn(1)–O(11)
Zn(1)–O(21)
Zn(1)–O(12)
Zn(1)–O(22)
Zn(1)–N(13)
Zn(1)–N(83)
2.106(1)
2.026(1)
2.130(1)
2.049(1)
2.118(2)
2.152(2)
2.164(1)
2.020(1)
2.148(1)
2.015(1)
2.144(1)
2.142(1)
Bond angle
2 · 2CH3OH
6 · 2CH3OH
O(11)–Zn(1)–O(21)
O(11)–Zn(1)–O(12)
O(11)–Zn(1)–O(22)
O(11)–Zn(1)–N(13)
O(11)–Zn(1)–N(83)
O(21)–Zn(1)–O(12)
O(21)–Zn(1)–N(13)
O(21)–Zn(1)–O(22)
O(12)–Zn(1)–O(22)
O(12)–Zn(1)–N(13)
O(12)–Zn(1)–N(83)
O(22)–Zn(1)–N(13)
O(22)–Zn(1)–N(83)
N(13)–Zn(1)–N(83)
O(21)–Zn(1)–N(83)
83.46(6)
173.75(6)
93.18(6)
96.15(6)
90.74(6)
91.68(5)
171.03(6)
94.49(6)
83.25(5)
89.26(6)
93.54(6)
94.49(6)
170.91(6)
76.93(6)
94.10(6)
83.78(4)
175.05(4)
92.20(4)
91.84(4)
86.36(4)
93.97(4)
168.62(5)
97.23(4)
83.70(4)
91.17(4)
98.17(4)
93.43(5)
169.64(5)
76.38(5)
92.82(5)
through the calculation of the binding constant Kb, which can be obtain-
ed by monitoring the changes in the absorbance at the corresponding
λ
max with increasing concentrations of CT DNA [64]. Kb is given by the
½DNAꢀ
ratio of slope to the y intercept in plots
and S4), according to Wolfe–Shimer Eq. (3) [68]:
versus [DNA] (Figs. S3
ðεA‐εf Þ
½DNAꢀ ½DNAꢀ
1
¼
þ
ð3Þ
ðεA‐εf Þ ðεb‐εf Þ Kbðεb‐εf Þ
where [DNA] is the concentration of DNA in base pairs, εA = Aobsd/[com-
pound], εf = the extinction coefficient for the free compound and εb =
the extinction coefficient for the compound in the fully bound form. The
values of Kb for the two ketoHs and their complexes 1–8, as calculated
by Eq. (3) and the plots in Figs. S3 and S4, are given in Table 3.
The Kb values of the compounds are moderate to high suggesting
their strong binding to CT DNA [65,66], with free opoH bearing the
highest Kb value (=5.20( 0.19) × 106 M−1) of all compounds and com-
plex 3 having the highest Kb value (=4.89( 0.12) × 106 M−1) among
Fig. 4. Thermoanalytical curves (TG, DTG, DTA) for the complexes: (A) [Zn(bpo)2(H2O)2],
5 and (B) [Zn(bpo)2(dpamH)], 8, with heating rate 10 °C min−1 in N2 atm.
the complexes. Free opoH exhibits higher Kb value than its complexes
1–4, while the bpo complexes 5–7 have higher Kb values than free
bpoH. The Kb values of most compounds are similar or higher than that
of the classical intercalator EB (Kb = 1.23( 0.07) × 105 M−1) [69], a
fact that may be a first indication of the ability of the compounds to dis-
place EB from the EB–DNA compound, in the case of intercalation.
3.5.2. DNA-viscosity measurement in the presence of the compounds
The measurement of the DNA viscosity upon addition of a com-
pound provides significant aid to clarify the interaction mode of a com-
pound with DNA, since the viscosity of DNA is sensitive to DNA length
changes. The relation between relative solution viscosity (η/η0) and
DNA length (L/L0) is given by the Equation L/L0 = (η/η0)1/3, where L0
denotes the apparent molecular length in the absence of the compound
[65,66].
The interaction of a compound to DNA grooves via a partial or non-
classic intercalation (i.e. electrostatic interaction or external groove-
binding) results in a bend or kink in the DNA helix and subsequently a
slight shortening of its effective length may be provoked; in such a
case, the DNA-viscosity may show a slight decrease or may remain un-
changed. In the case of intercalative binding, the insertion of the com-
pound in between the DNA base pairs results in an increase of the
separation distance of base pairs being at the intercalation sites aiming
Fig. 3. The stacking formed by π–π interactions between bipy ligands in 2 · 2CH3OH (left)
and π–π and C–H⋯π interactions in 6 · 2CH3OH.

E. Mrkalić et al. / Journal of Inorganic Biochemistry 134 (2014) 66–75
73
0.90
0.85
0.80
0.75
A 1.0
A 1.10
1.08
0.8
0.6
0.4
0.2
0.0
opoH
1
3
1.06
1.04
1.02
1.00
0.98
2
4
280
285
290
λ (nm)
295
0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35
r = [Compound]/[DNA]
280
300
320
340
λ (nm)
360
380
400
B
B 1.10
1.4
1.08
1.06
1.04
1.02
1.00
0.98
1.2
1.0
0.8
0.6
0.4
0.2
0.0
bpoH
5
7
6
8
300
320
340
λ (nm)
360
380
0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35
r = [Compound]/[DNA]
Fig. 5. UV spectra of a DMSO solution of complex (A) 2 (2.5 × 10−5 M) and (B)
6 (2 × 10−4 M) in the presence of CT DNA at increasing amounts (up to 1/r = 2).
The arrows show the changes upon increasing amounts of CT DNA.
Fig. 6. Relative viscosity (η/ηo)1/3 of CT DNA (0.1 mM) in buffer solution (150 mM NaCl
and 15 mM trisodium citrate at pH 7.0) in the presence of (A) opoH and complexes 1–4
and (B) bpoH and complexes 5–8, at increasing amounts (r = 0–0.35).
existence of intercalation; this increase is less pronounced than that ob-
served in the recently reported Zn complexes with substituted
salicylaldehydes as ligands [33].
In conclusion, the behavior of the DNA viscosity observed upon addi-
tion of the compounds is in accordance with the indications derived
from UV spectroscopic studies, and albeit not so clear, it may be consid-
ered an evidence of the existence of an intercalative binding mode to
DNA.
to host the bound compound; therefore, the increase of the length of the
DNA helix if the reason of the DNA viscosity increase, the magnitude of
which is usually in accordance to the strength of the interaction
[33,59,65,66,70].
Viscosity measurements were carried out on CT DNA solutions
(0.1 mM) upon addition of increasing amounts of the compounds (up
to the value of r = 0.35) at room temperature (Fig. 6). The relative vis-
cosity of DNA solution upon addition of all compounds seems to be sta-
ble up to r = 0.15–0.20 suggesting a partial or non-classic intercalation,
while for r N 0.20 the DNA viscosity exhibits an increase suggesting the
3.5.3. Competitive studies with ethidium bromide
Ethidium bromide (EB = 3,8-diamino-5-ethyl-6-phenyl-phenan-
thridinium bromide) is a typical indicator of intercalation since it
emits intense fluorescence in the presence of DNA due to the strong in-
tercalation of the planar EB phenanthridine ring between adjacent base
pairs on the double DNA helix [71]. Upon the addition of a compound
which can bind via intercalation to DNA equally or more strongly than
EB to a solution containing the EB–DNA complex, a quenching of the
DNA-induced EB fluorescence emission may appear.
Table 3
Spectral features of the UV spectra of ketoH and complexes 1–8 upon addition of DNA
(band studied in λ(nm), percentage of hyperchromism or hypochromism ΔA(%), and
hypsochromism or bathochromism Δλ(nm)) and the DNA binding constants (Kb) of
ketoH and complexes 1–8.
λ(nm) (ΔA(%) a, Δλ(nm)b)
Kb (M−1
)
The two substituted 2-hydroxybenzophenones (opoH and bpoH)
and their complexes 1–8 do not show any significant fluorescence at
room temperature in solution or in the presence of CT DNA, when excit-
ed at 540 nm. The addition of these compounds to a solution containing
EB does not provoke quenching of free EB fluorescence and no new
peaks appear in the spectra. Therefore, the changes observed in the fluo-
rescence emission spectra of a solution containing the EB–DNA complex
upon addition of the compounds can be used to study the ability of the
compounds to displace EB from the EB–DNA complex [72].
Compound
opoH
288(−3, 0), 319(−2.5, 0)
288(−2.5, 0), 319(−0, 0)
288(−1, 0), 320(−0, 0)
284(−2, +1), 320(+1.5, 0)
287(−1.5, 0), 318(−2.5, 0)
319(−2.5, +1)
320(+10, 0)
320(+4, 0)
325(+5, 0)
316(−3, 0)
5.20( 0.19) × 106
3.73( 0.20) × 105
6.45( 0.21) × 105
4.89( 0.12) × 106
1.96( 0.41) × 105
9.58( 0.40) × 104
3.42( 0.30) × 105
6.89( 0.12) × 105
7.68( 0.16) × 105
6.06( 0.22) × 104
[Zn(opo)2(H2O)2], 1
[Zn(opo)2(bipy)], 2
[Zn(opo)2(phen)], 3
[Zn(opo)2(dpamH)], 4
bpoH
[Zn(bpo)2(H2O)2], 5
[Zn(bpo)2(bipy)], 6
[Zn(bpo)2(phen)], 7
[Zn(bpo)2(dpamH)], 8
The emission spectra of EB bound to CT DNA in the absence and
presence of each compound have been recorded for [EB] = 20 μM,
[DNA] = 26 μM for increasing amounts of each compound up to the
a
“+” denotes hyperchromism and“−”denotes hypochromism.
“+” denotes red-shift and“−”denotes blue-shift.
b

74
E. Mrkalić et al. / Journal of Inorganic Biochemistry 134 (2014) 66–75
Table 4
value of r = 2.5, in some cases. The addition of the compounds at di-
verse r values results in a rather significant decrease of the intensity of
the emission band (Fig. 7 and Table 4) of the EB–DNA system at
592 nm indicating the competition of the compounds with EB in binding
to DNA. The observed quenching of EB–DNA fluorescence suggests that
the compounds have moderate to significant ability to displace EB from
Quenching of EB–DNA fluorescence (ΔI/Io, %) and Stern–Volmer constants (KSV) for the
ketoH and their complexes 1–8.
Compound
EB–DNA fluorescence
quenching (ΔI/Io, %)
Ksv (M−1
)
opoH
83.0
81.5
77.2
78.8
76.9
76.0
85.0
84.5
82.5
81.5
1.01( 0.05) × 105
3.19( 0.17) × 105
4.02( 0.14) × 105
2.82( 0.13) × 105
4.90( 0.15) × 105
1.80( 0.08) × 104
3.83( 0.14) × 105
7.63( 0.26) × 104
8.02( 0.22) × 104
3.95( 0.12) × 105
[Zn(opo)2(H2O)2], 1
[Zn(opo)2(bipy)], 2
[Zn(opo)2(phen)], 3
[Zn(opo)2(dpamH)], 4
bpoH
[Zn(bpo)2(H2O)2], 5
[Zn(bpo)2(bipy)], 6
[Zn(bpo)2(phen)], 7
[Zn(bpo)2(dpamH)], 8
A
10000
8000
6000
4000
2000
0
the EB–DNA complex, thus probably interacting with CT DNA by the
intercalative mode.
The Stern–Volmer constant, KSV (in M−1), may be used to evaluate
the quenching ability of each compound [33,73] according to Eq. (4):
Io
I
¼ 1 þ KSV½Qꢀ
ð4Þ
560
600
640
680
720
λ (nm)
where Io and I are the emission intensities in the absence and the pres-
ence of the quencher, respectively, [Q] is the concentration of the
quencher (the substituted 2-hydroxybenzophenones and their com-
B
Io
100
80
60
40
20
0
plexes 1–8). KSV is obtained by the slope of the diagram vs [Q] in
I
Stern–Volmer plots of DNA–EB (Figs. S5 and S6). The experimental
data indicate that the quenching of EB bound to DNA provoked by the
compounds is in good agreement (R ≥ 0.99) with the linear Stern–
Volmer equation (Eq. (4)). The Stern–Volmer constants, KSV, of the com-
pounds are moderate to high (Table 4) showing that they can displace
EB and bind relatively tightly to DNA [33,73,74]. Additionally, the opo
compounds present higher Ksv than the bpo compounds, a feature pres-
ent in the calculated Kb values from UV spectroscopic studies.
opoH
1
3
2
4
4. Conclusions
The synthesis and characterization of mononuclear zinc complexes
with 2-hydroxybenzophenones (ketoH), (2-hydroxybenzophenone =
bpoH and 4-methoxy-2-hydroxybenzophenone = opoH) in the absence
[Zn(keto)2(H2O)2], or presence of a N,N′-donor heterocyclic ligand (enR)
such as bipy, phen or dpamH, [Zn(keto)2(enR)], have been synthesized
and characterized by IR, UV and 1H NMR spectroscopies. The 2-
hydroxybenzophenones are chelated to the metal ion through the pheno-
late and one carbonyl oxygen atoms. The crystal structures of [Zn(opo)2(-
bipy)]∙2MeOH 2∙2MeOH and [Zn(bpo)2(bipy)]∙2MeOH 6∙2MeOH have
been determined by X-ray crystallography revealing a six-coordinated
Zn(II) in a distorted octahedral environment. The thermal stability of
the zinc complexes has been investigated by simultaneous TG/DTG–DTA
technique. The stable in air complexes at room temperature are
decomposed at higher temperatures leading to the pure metal oxide
ZnO or to a mixture of ZnO plus un-pyrolized carbon.
0.0
0.1
0.2
0.3
0.4
0.5
r = [Compound]/[DNA]
C
100
80
60
40
20
0
bpoH
5
7
6
8
UV spectroscopic studies have revealed the ability of the substituted
2-hydroxybenzophenones and their complexes to bind to CT DNA with
complex 3 having the highest Kb value (=4.89( 0.12) × 106 M−1
)
0.0
0.5
1.0
1.5
2.0
2.5
among the complexes and free opoH exhibiting the highest Kb value
(=5.20( 0.19) × 106 M−1) among the compounds examined, which
are higher than the Kb value of EB. DNA viscosity measurements have
revealed an intercalative binding mode of most compounds to CT
DNA. Competitive binding studies with EB have shown a moderate to
significant ability of the compounds to displace the typical intercalator
EB from the EB–CT DNA complex, suggesting their competition with
EB for the intercalation site of DNA. In general, the opo compounds
may be considered as better DNA-binders and EB-displacing com-
pounds that the bpo compounds, since they present higher Kb and Ksv
r = [Compound]/[DNA]
Fig. 7. (A) Fluorescence emission spectra (λex = 540 nm) of a buffer solution (150 mM
NaCl and 15 mM trisodium citrate at pH 7.0) containing EB–DNA in the presence of in-
creasing amounts (up to r = 1.4) of complex 6. (B) and (C) Plot of EB–DNA relative fluo-
rescence emission intensity (%I/Io) at λem = 592 nm vs r (r = [compound]/[DNA]) in
buffer solution (150 mM NaCl and 15 mM trisodium citrate at pH 7.0) in the presence of
(B) opoH and complexes 1–4 (quenching up to 17% of the initial EB–DNA fluorescence
for opoH, 18.5% for 1, 23% for 2, 21% for 3 and 23% for 4) and (C) bpoH and complexes
5–8 (quenching up to 24% of the initial EB–DNA fluorescence for bpoH, 15% for 5, 15.5%
for 6, 17.5% for 7 and 18.5% for 8).

E. Mrkalić et al. / Journal of Inorganic Biochemistry 134 (2014) 66–75
75
[24] R.G. Ferris, R.G. Hazen, G.B. Roberts, M.H. Clair, J.H. Chan, K.R. Romines, G.A.
Freeman, J.H. Tidwell, L.T. Schaller, J.R. Cowan, S.A. Short, K.L. Weaver, D.W.
Selleseth, K.R. Moniri, L.R. Boone, Antimicrob. Agents Chemother. 203 (2005)
4046–4051.
[25] J.P. Liou, C.W. Chang, J.S. Song, Y.N. Yang, C.F. Yeh, H.Y. Tseng, Y.K. Lo, Y.L. Chang, C.M.
Chang, H.P. Hsieh, J. Med. Chem. 45 (2002) 2556–2662.
values than the bpo corresponding compounds. The existing results of
the reported substituted 2-hydroxybenzophenones and their com-
plexes as DNA intercalators are the first ever reported and are promising
revealing a new possibility for their use as potential metallodrugs.
[26] Z. Wang, H.J. Lee, L. Wang, Pharm. Res.-Dord. 26 (2009) 1140–1148.
[27] R.W. Fuller, J.W. Blunt, J.L. Boswell, J. Nat. Prod. 62 (1999) 130–132.
[28] M.I. Esteva, K. Kettler, C. Maidana, J. Med. Chem. 48 (2005) 7186–7191.
[29] J. Ren, P. Chamberlain, A. Stamp, J. Med. Chem. 51 (2008) 5000–5008.
[30] U. Hagedorn-Leweke, B. Lippold, Pharmacol. Res. 12 (1995) 1354–1360.
[31] M.C. Rhodes, J.R. Bucher, J.C. Peckham, G.E. Kissling, M.R. Hejtmancik, R.S. Chhabra,
Food Chem. Toxicol. 45 (2007) 843–851.
[32] S. Schauder, H. Ippen, Contact Dermatitis 37 (1997) 221–232.
[33] A. Zianna, G. Psomas, A. Hatzidimitriou, E. Coutouli-Argyropoulou, M. Lalia-Kantouri,
J. Inorg. Biochem. 127 (2013) 116–126.
Abbreviations
Bipy
bpoH
CT
2,2′-bipyridine
2-hydroxy-benzophenone
calf–thymus
DMF
N,N-dimethylformamide
dpamH 2,2′-dipyridylamine
EB
ethidium bromide, 3,8-diamino-5-ethyl-6-phenyl-phenan-
[34] M. Lalia-Kantouri, C.D. Papadopoulos, A.G. Hatzidimitriou, M.P. Sigalas, M. Quiros, S.
Skoulika, Polyhedron 52 (2013) 1306–1316.
[35] C.D. Papadopoulos, A.G. Hatzidimitriou, M. Quiros, M.P. Sigalas, M. Lalia-Kantouri,
Polyhedron 30 (2011) 486–496.
[36] M. Lalia-Kantouri, M. Gdaniec, T. Choli-Papadopoulou, A. Badounas, C.D.
Papadopoulos, A. Czapik, G.D. Geromichalos, D. Sahpazidou, F. Tsitouroudi, J. Inorg.
Biochem. 117 (2012) 25–34.
[37] M. Lalia-Kantouri, T. Dimitriadis, C.D. Papadopoulos, M. Gdaniec, A. Czapik, A.G.
Hatzidimitriou, Z. Anorg. Allg. Chem. 635 (2009) 2185–2190.
[38] B. Armitage, Chem. Rev. 98 (1998) 1171–1200.
thridinium bromide
N,N′-donor heterocyclic ligand
substituted 2-hydroxybenzophenones
medium
2-hydroxy-4-methoxy-benzophenone
1,10-phenanthroline
strong
enR
ketoH
m
opoH
phen
s
[39] M.J. Clarke, Coord. Chem. Rev. 232 (2002) 69–93.
sh
w
shoulder
weak
[40] B.M. Zeglis, V.C. Pierre, J.K. Barton, Chem. Commun. (2007) 4565–4579.
[41] Y.K. Yan, M. Melchart, A. Habtemariam, P.J. Sadler, Chem. Commun. (2005)
4764–4776.
[42] E. Kimura, Chem. Rev. 104 (2004) 769–787.
Appendix A. Supplementary data
[43] F. Dimiza, A.N. Papadopoulos, V. Tangoulis, V. Psycharis, C.P. Raptopoulou, D.P.
Kessissoglou, G. Psomas, Dalton Trans. 39 (2010) 4517–4528.
[44] A. Zianna, S. Vecchio, M. Gdaniec, A. Czapik, A. Hatzidimitriou, M. Lalia-Kantouri, J.
Therm. Anal. Calorim. 112 (2013) 455–464.
[45] Agilent Technologies, Program CrysAlis PRO, Ver.1.171.35, Yarnton, Oxfordshire, En-
gland, 2011.
[46] M.C. Burla, R. Caliandro, M. Camalli, B. Carrozzini, G.L. Cascarano, L. De Caro, C.
Giacovazzo, G. Polidori, R. Spagna, J. Appl. Crystallogr. 38 (2005) 381–388.
[47] G.M. Sheldrick, Acta Crystallogr. A64 (2008) 112–122.
[48] L.J. Farrugia, J. Appl. Crystallogr. 30 (1997) 565.
[49] C.F. Macrae, P.R. Edgington, P. McCabe, E. Pidcock, G.P. Shields, R. Taylor, M. Towler,
J. van de Streek, J. Appl. Crystallogr. 39 (2006) 453–457.
[50] R.M. Silverstein, G.C. Bassler, G. Morvill, Spectrometric Identification of Organic
Compounds, 6th ed. Wiley, New York, 1998.
The crystal structures of compounds 2 and 6 have been submitted to
the CCDC and have been allocated the deposition numbers CCDC
970394 and 970395, respectively. These data can be obtained free of
charge via www.ccdc.cam.ac.uk/conts/retrieving.html (or from the
Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge
CB21EZ, UK; fax: (+44) 1223-336-033; or deposit@ccdc.cam.ac.uk).
Supplementary data associated with this article can be found, on the on-
line version, at http://dx.doi.org/10.1016/j.jinorgbio.2014.01.019.
References
[51] K. Nakamoto, Infrared and Raman Spectra of Inorganic and Coordination Com-
pounds, Part B: Applications in Coordination, Organometallic, and Bioinorganic
Chemistry, 6th ed. Wiley, New Jersey, 2009.
[52] A. Tarushi, C.P. Raptopoulou, V. Psycharis, A. Terzis, G. Psomas, D.P. Kessissoglou,
Bioorg. Med. Chem. 18 (2010) 2678–2685.
[53] E.K. Efthimiadou, M. Katsarou, Y. Sanakis, C.P. Raptopoulou, A. Karaliota, N. Katsaros,
G. Psomas, J. Inorg. Biochem. 100 (2006) 1378–1388.
[54] F. Dimiza, A.N. Papadopoulos, V. Tangoulis, V. Psycharis, C.P. Raptopoulou, D.P.
Kessissoglou, G. Psomas, J. Inorg. Biochem. 107 (2012) 54–64.
[55] A. Tarushi, F. Kastanias, V. Psycharis, C.P. Raptopoulou, G. Psomas, D.P. Kessissoglou,
Inorg. Chem. 51 (2012) 7460–7462.
[1] H. Tapiero, K.D. Tew, Biomed. Pharmacother. 57 (2003) 399–411.
[2] N. Farrell, Coord. Chem. Rev. 232 (2002) 1–4.
[3] B.L. Vallee, D.S. Auld, Biochemistry 32 (1993) 6493–6500.
[4] G. Parkin, Chem. Commun. (2000) 1971–1985.
[5] H. Sakurai, Y. Kojima, Y. Yoshikawa, K. Kawabe, H. Yasui, Coord. Chem. Rev. 226
(2002) 187–198.
[6] C.F. Mills, Zinc in Human Biology, Springer, New York, 1989.
[7] M. Di Vaira, C. Bazzicalupi, P. Orioli, L. Messori, B. Bruni, P. Zatta, Inorg. Chem. 43
(2004) 3795–3797.
[8] Z.Q. Li, F.J. Wu, Y. Gong, C.W. Hu, Y.H. Zhang, M.Y. Gan, Chin. J. Chem. 25 (2007)
1809–1814.
[9] J. d'Angelo, G. Morgant, N.E. Ghermani, D. Desmaele, B. Fraisse, F. Bonhomme, E.
Dichi, M. Sghaier, Y. Li, Y. Journaux, J.R.J. Sorenson, Polyhedron 27 (2008) 537–546.
[10] Q. Zhou, T.W. Hambley, B.J. Kennedy, P.A. Lay, P. Turner, B. Warwick, J.R. Biffin, H.L.
Regtop, Inorg. Chem. 39 (2000) 3742–3748.
[11] N.C. Kasuga, K. Sekino, M. Ishikawa, A. Honda, M. Yokoyama, S. Nakano, N. Shimada,
C. Koumo, K. Nomiya, J. Inorg. Biochem. 96 (2003) 298–310.
[12] A. Tarushi, Z. Karaflou, J. Kljun, I. Turel, G. Psomas, A.N. Papadopoulos, D.P.
Kessissoglou, J. Inorg. Biochem. 128 (2013) 85–96.
[13] M. Belicchi Ferrari, F. Bisceglie, G. Pelosi, P. Tarasconi, R. Albertini, S. Pinelli, J. Inorg.
Biochem. 87 (2001) 137–147.
[56] R. Mitra, M.W. Peters, M.J. Scott, Dalton Trans. (2007) 3924–3935.
[57] D.K. Garner, S.B. Fitch, L.H. McAlexander, L.M. Bezold, A.M. Arif, L.M. Berreau, J. Am.
Chem. Soc. 124 (2002) 9970–9971.
[58] P.S. Binil, M.R. Anoop, S. Suma, M.R. Sudarsanakumar, J. Therm. Anal. Calorim. 112
(2013) 913–919 (and references therein).
[59] A. Tarushi, K. Lafazanis, J. Kljun, I. Turel, A.A. Pantazaki, G. Psomas, D.P. Kessissoglou,
J. Inorg. Biochem. 121 (2013) 53–65.
[60] A. Dziewulska-Kulaczkowska, J. Therm. Anal. 101 (2010) 19–26.
[61] L.M. Dianu, A. Kriza, A.M. Musuc, J. Therm. Anal. Calorim. 112 (2013) 585–593.
[62] Q. Zhang, J. Liu, H. Chao, G. Xue, L. Ji, J. Inorg. Biochem. 83 (2001) 49–55.
[63] E.C. Long, J.K. Barton, Acc. Chem. Res. 23 (1990) 271–273.
[64] A.M. Pyle, J.P. Rehmann, R. Meshoyrer, C.V. Kumar, N.J. Turro, J.K. Barton, J. Am.
Chem. Soc. 111 (1989) 3053–3063.
[65] E.S. Koumousi, M. Zampakou, C.P. Raptopoulou, V. Psycharis, C.M. Beavers, S.J. Teat,
G. Psomas, T.C. Stamatatos, Inorg. Chem. 51 (2012) 7699–7710.
[66] C. Tolia, A.N. Papadopoulos, C.P. Raptopoulou, V. Psycharis, C. Garino, L. Salassa, G.
Psomas, J. Inorg. Biochem. 123 (2013) 53–65.
[67] G. Pratviel, J. Bernadou, B. Meunier, Adv. Inorg. Chem. 45 (1998) 251–262.
[68] A. Wolfe, G. Shimer, T. Meehan, Biochemistry 26 (1987) 6392–6396.
[69] A. Dimitrakopoulou, C. Dendrinou-Samara, A.A. Pantazaki, M. Alexiou, E.
Nordlander, D.P. Kessissoglou, J. Inorg. Biochem. 102 (2008) 618–628.
[70] J.L. Garcia-Gimenez, M. Gonzalez-Alvarez, M. Liu-Gonzalez, B. Macias, J. Borras, G.
Alzuet, J. Inorg. Biochem. 103 (2009) 923–934.
[14] Z. Travnicek, V. Krystof, M. Sipl, J. Inorg. Biochem. 100 (2006) 214–225.
[15] J.S. Casas, E.E. Castellano, M.D. Couce, J. Ellena, A. Sanchez, J. Sordo, C. Taboada, J.
Inorg. Biochem. 100 (2006) 124–132.
[16] K. Klein, Cosmet. Toiletries 107 (1992) 45–64.
[17] T. Hayashi, Y. Okamoto, K. Ueda, N. Kojima, Toxicol. Lett. 167 (2006) 1–7.
[18] T. Suzuki, S. Kitamura, R. Khota, K. Sugihara, N. Fujimoto, S. Ohta, Toxicol. Appl.
Pharmacol. 203 (2005) 9–17.
[19] J. Chen, C. Ting, T. Hwang, L. Chen, J. Nat. Prod. 72 (2) (2009) 253–258.
[20] T.D. Venu, S. Shashikanth, S.A. Khanum, S. Naveen, A. Firdouse, M.A. Sridhar, J.S.
Prasad, Bioorg. Med. Chem. 15 (2007) 3505–3514.
[21] K. Kohring, J. Wiesner, M. Altenkamper, J. Sakowski, K. Silber, A. Hillebrecht, P.
Haebel, H.M. Dahse, R. Ortmann, H.J. Priv.-Doz, G. Klebe, M. Schlitzer,
ChemMedChem 3 (2008) 1217–1231.
[71] W.D. Wilson, L. Ratmeyer, M. Zhao, L. Strekowski, D. Boykin, Biochemistry 32 (1993)
4098–4104.
[22] J.S. Neves, L.P. Coelho, R.S. Cordeiro, M.P. Veloso, P.M. Silva, M.H. Dos Santos, M.A.
Martins, Planta Med. 73 (2007) 644–649.
[72] S. Dhar, M. Nethaji, A.R. Chakravarty, J. Inorg. Biochem. 99 (2005) 805–812.
[73] G. Psomas, J. Inorg. Biochem. 102 (2008) 1798–1811.
[23] J.J. Chen, C.W. Ting, I.S. Chen, C.F. Peng, W.T. Huang, Y.C. Su, S.C. Lin, Planta Med. 74
(2008) 1042–1051.
[74] F. Dimiza, F. Perdih, V. Tangoulis, I. Turel, D.P. Kessissoglou, G. Psomas, J. Inorg.
Biochem. 105 (2011) 476–489.