Synthesis, crystal structure, antioxidation and DNA-binding properties of the Ln complexes with 1-phenyl-3-methyl-5-hydroxypyrazole-4-carbaldhyde-(benzoyl)hydrazone

Article abstract of DOI:10.1016/j.jorganchem.2009.10.032

Two lanthanide complexes (Ln = La, Pr) with a PMFP Schiff-base, 1-phenyl-3-methyl-5-hydroxypyrazole-4-carbaldhyde-(benzoyl)hydrazone (H₂L) were synthesized and characterized. The crystal structure of the La complex was determined by single-crystal X-ray diffraction, the coordination polyhedron is a tricapped trigonal prism configuration with the nine-coordinate atoms composed of three nitrogens and six oxygens from three ligands. The complex crystallized in the monoclinic lattice with a space group P2/c. Electronic absorption titration spectra, fluorescence titration spectra, EtBr competitive experiment, viscosity measurement and CD spectra indicate that all the complexes can strongly bind calf thymus DNA, presumably via groove binding and intercalation mechanism. Furthermore, investigations of antioxidation properties show that all the complexes have some scavenging effects for hydroxyl radicals.

Full text of DOI:10.1016/j.jorganchem.2009.10.032

Journal of Organometallic Chemistry 695 (2010) 415–422
Contents lists available at ScienceDirect
Journal of Organometallic Chemistry
journal homepage: www.elsevier.com/locate/jorganchem
Synthesis, crystal structure, antioxidation and DNA-binding properties
of the Ln complexes with 1-phenyl-3-methyl-5-hydroxypyrazole-4-carbaldhyde-
(benzoyl)hydrazone
*
Hong-Ge Li, Zheng-Yin Yang , Bao-Dui Wang, Jin-Cai Wu
College of Chemistry and Chemical Engineering, State Key Laboratory of Applied Organic Chemistry, Lanzhou University, Lanzhou 730000, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 5 June 2009
Received in revised form 16 October 2009
Accepted 23 October 2009
Available online 27 October 2009
Two lanthanide complexes (Ln = La, Pr) with a PMFP Schiff-base, 1-phenyl-3-methyl-5-hydroxypyrazole-
4-carbaldhyde-(benzoyl)hydrazone (H2L) were synthesized and characterized. The crystal structure of
the La complex was determined by single-crystal X-ray diffraction, the coordination polyhedron is a tri-
capped trigonal prism configuration with the nine-coordinate atoms composed of three nitrogens and six
oxygens from three ligands. The complex crystallized in the monoclinic lattice with a space group P2/c.
Electronic absorption titration spectra, fluorescence titration spectra, EtBr competitive experiment, vis-
cosity measurement and CD spectra indicate that all the complexes can strongly bind calf thymus
DNA, presumably via groove binding and intercalation mechanism. Furthermore, investigations of antiox-
idation properties show that all the complexes have some scavenging effects for hydroxyl radicals.
Ó 2009 Elsevier B.V. All rights reserved.
Keywords:
1-Phenyl-3-methyl-5-hydroxypyrazole-4-
carbaldhyde-(benzoyl)hydrazone
DNA-binding
Antioxidant activity
Lanthanide complexes
1. Introduction
Schiff-bases metal chelates have represented good antitumor
activities against animal tumors [17,18].
It is well known that DNA is an important cellular receptor,
many chemicals exert their antitumor effects through binding to
DNA thereby changing the replication of DNA and inhibiting the
growth of the tumor cells, which is the basis of designing new
and more efficient antitumor drugs and their effectiveness depends
on the mode and affinity of the binding [1–3]. Small molecules pos-
sess DNA-binding abilities include metal complexes, porphyrins,
natural antibiotics, simple aromatic hydrocarbons and some het-
erocyclic cations [4–9]. Particularly, the DNA-binding metal com-
plexes have been extensively studied as DNA structural probes,
DNA-dependent electron transfer probes, DNA footprinting and se-
quence-specific cleaving agents and potential anticancer drugs
[10–12]. Basically, metal complexes interact with the double helix
DNA in either a non-covalent or a covalent way. The former way
includes three binding modes: intercalation, groove binding and
external static electronic effects. Among these interactions, groove
binding is one of the most important DNA-binding modes as it
invariably leads to cellular degradation.
Based on the above considerations, lanthanide complexes with
Schiff-bases derived from PMBP and chromone have always been
a major concern for our group [19–21]. In this paper, two lantha-
nide complexes (Ln = La, Pr) with
a Schiff-base 1-phenyl-3-
methyl-5-hydroxypyrazole-4-carbaldhyde-(benzoyl)hydrazone
were synthesized and their DNA-binding modes were investigated.
On the other hand, reactive oxygen species (ROS) are generated
by normal cellular metabolism and by exogenous agents. Even
though ROS may serve as cell signaling or bactericidal agents, ex-
cess ROS can cause damage to cellular macromolecules such as
polyun-saturated lipid, protein, and DNA, leading to a process
called oxidative stress [22,23]. Oxidative stress is implicated in
the development of many diseases such as macular degeneration,
cardiacdisease, drug-associated toxicity, inflammation, atherogen-
esis and premature aging [24–26].
Since among all reactive oxygen species the hydroxyl radical is
by far the most potent and therefore the most dangerous oxygen
metabolite, elimination of this radical is one of the major aims of
antioxidant administration. Although a variety of (OHÅ) scavengers
are known, their application is limited by: (1) the requirement of
unphysiologically high scavenger concentrations which depend
on the rate constant, (2) toxic side effects including initiation of
further radical chain reactions, or (3) the instability of the com-
pound in biological systems [27]. Many researchers have been
working hard to develop complexes in order to achieve the
As well as the magnetic and photophysical properties, the bio-
activities of lanthanides such as antimicrobial, antitumor, antivi-
rus, anticoagulant action, prevention from arteriosclerosis, etc.,
have been explored in recent decades [13–16]. In addition, some
* Corresponding author. Tel.: +86 931 8913515; fax: +86 931 8912582.
E-mail address: yangzy@lzu.edu.cn (Z.-Y. Yang).
0022-328X/$ - see front matter Ó 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.jorganchem.2009.10.032

416
H.-G. Li et al. / Journal of Organometallic Chemistry 695 (2010) 415–422
efficient scavenger. It has been recently demonstrated that some
minor groove binders for DNA are effective inhibitors of the forma-
tion of a DNA/TBP complex or topoisomerases [28,29]. The antiox-
idation properties of the lanthanide complexes were also
investigated in this paper.
ence or absence of compound were calculated from the following
relation [32,33].
g
¼ ðt ꢀ t0Þ=t0
where t is the observed flow time of the DNA containing solution,
and t0 is the flow time of buffer.
The CD spectra were recorded on an Olos RSM 1000 at increas-
ing complex/DNA ratio (r = 0.0 and 1.0). Each sample solution was
scanned in the range of 220–320 nm. A CD spectrum was gener-
ated which represented the average of two scans from which the
buffer background had been subtracted. The concentration of
DNA was 3.0 ꢁ 10ꢀ4 M.
2. Experimental
2.1. Materials
All chemicals used were of analytical grade and used without
further purification unless otherwise noted. Calf thymus DNA
(CT-DNA) and ethidium bromide (EtBr) were obtained from Sig-
ma–Aldrich Biotech. Co., Ltd. CT-DNA stock solution was prepared
by dissolving the solid material, normally at 0.3 mg mlꢀ1, in 5 mM
Tris–HCl buffer (pH 7.20) containing 50 mM NaCl. Whereafter, the
solution was kept over 48 h at 4 °C. Calf thymus DNA (CT-DNA) ob-
tained from Sigma. UV–Vis spectrometer was employed to check
The hydroxyl radicals (OHÅ) in aqueous media were generated
through the Fenton-type reaction [34]. The solution of the tested
compound was prepared with DMF. The 5 ml reaction mixtures
contained 2 ml of 100 mM phosphate buffer (pH 7.4), 1.0 ml of
0.10 mM aqueous safranin, 1 ml of 1.0 mM aqueous EDTA–Fe(II),
1 ml of 3% aqueous H2O2, and a series of quantitatively microad-
ding solutions of the tested compound. The sample without the
tested compound was used as the control. The reaction mixtures
were incubated at 37 °C for 60 min in a water-bath. The absor-
bances of the samples and a control were measured at 520 nm.
The suppression ratio for OHÅ was calculated from the following
Eq. (1) [35,36].
DNA purity 1.8–1.9:1 and concentration (e
= 6600 Mꢀ1 cmꢀ1 at
260 nm) [30]. All the stock solutions (1.0 mMl) of the investigated
compounds were prepared by dissolving the powder materials into
appropriate amounts of DMF or CH3OH solutions, respectively.
Double-distilled water was used to prepare buffers.
Asample ꢀ Ablank
2.2. Instrumentation and methods
Scavenging effect ð%Þ ¼
ꢁ 100
ð1Þ
Acontrol ꢀ Ablank
The melting points of the compounds were determined on an
XT4-100X microscopic melting point apparatus (Beijing, China).
The IR spectra were recorded on a Therrno Mattson FT-IR spec-
trometer using KBr disc in the 4000–400 cmꢀ1 region. 1H NMR
spectra were recorded on a Varian VR 300-MHz spectrometer with
tetramethylsilane (TMS) as an internal standard. ESI-MS (ESI-Trap/
Mass) spectra were recorded on a Bruker Esquire6000 Mass
spectrophotometer.
where Asample is the absorbance of the sample in the presence of the
tested compound, Ablank is the absorbance of the blank in the ab-
sence of the tested compound and Acontrol is the absorbance in the
absence of the tested compound and EDTA–Fe (II). IC50 value was
introduced to denote the molar concentration of the tested com-
pound which caused good inhibitory or scavenging effect on hydro-
xyl radicals.
The UV–Vis spectra were recorded on a Perkin–Elmer Lambda-
35 UV–Vis (Ultraviolet and Visible) spectrophotometer. Absorption
titration experiments were performed by maintaining the ligand
2.3. X-ray crystallography
A yellow crystal of [La(HL)3]ꢂ1.5(C2H5OH)ꢂ1.5(H2O) was ob-
tained by slow vapor diffusion of C2H5OH/ether at room tempera-
ture. X-ray diffraction data for the crystal were performed with
graphite-monochromated Mo K radiation (0.71073 Å) on a Bruker
and metal complexes concentration as constant at 10 lM while
increasing the concentration of nucleic acid. While measuring the
absorption spectra, equal quantity of CT-DNA was added to both
the complex and the reference solution to eliminate the absor-
bance of DNA itself.
APEX area-detector diffractometer and collected by the
x/2h scan
technique at 298(2) K. The crystal structure was solved by direct
methods. All non-hydrogen atoms were refined anisotropically
by full-matrix least-squares methods on F2. Primary non-hydrogen
atoms were solved by direct method and secondary non-hydrogen
atoms were solved by difference maps. The hydrogen atoms were
added geometrically and not refined. All calculations were per-
formed using the programs SHELXS-97 and SHELXL-97.
For fluorescence measurements, fixed amounts (10 lM) of the
complexes were titrated with increasing amounts of CT-DNA. The
excitation and emission wavelength were 332, 359 and 418,
474 nm for the ligand and two complexes, respectively. Excitation
and emission slit were set at 5 nm. Experiments were conducted at
room temperature in a buffer containing 5 mM Tris–HCl (pH 7.2)
and 50 mM NaCl concentrations.
EtBr–DNA competitive experiment was performed as reported
2.4. Preparation of ligand and its complexes
in a literature [31]. DNA (4.0
lM, nucleotides) solution was added
incrementally to 0.32 M EtBr solution, until the rise in the fluores-
l
2.4.1. Synthesis of the ligand (H2L)
cence (kex = 496 nm, kem = 596 nm) attained a saturation. Then,
small aliquots of concentrated compound solutions (1.0 mM) were
added till the drop in fluorescence intensity (kex = 525 nm,
kem = 589 nm) reached a constant value. Measurements were made
after 5 min at room temperature.
The ligand of 1-phenyl-3-methyl-5-hydroxypyrazole-4-car-
baldhyde-(benzoyl)hydrazone was prepared according to the liter-
ature [37].
Viscosity titration experiments were carried on an Ubbelohde
2.4.2. Synthesis of lanthanide complexes
viscometer in
25.00 0.01 °C. DNA concentration was kept constant (5
gradually increased the concentration of tested compound (0.5–
3.5 M). Data were presented as (
1/3 vs. the ratio of the com-
pound to DNA, where is the viscosity of DNA in the presence of
the compound corrected from the solvent effect, and 0 is the vis-
cosity of DNA alone. Relative viscosities for DNA in either the pres-
a
thermostated water-bath maintained at
An ethanol solution containing the La nitrate (0.2 mM, 0.086 g)
was added dropwise to a stirred yellow solution of ligand H2L
(0.35 mM, 0.112 g) in ethanol (40 ml) at 60 °C. After 12 h at this
temperature, 20 ml distilled water was added dropwise to the
reaction solution which was concentrated to 5 ml, immediately
there was a yellow precipitate in the solution. The yellow precipi-
tate was separated from the solution by suction filtration, purified
lM) and
l
g/g0)
g
g

H.-G. Li et al. / Journal of Organometallic Chemistry 695 (2010) 415–422
417
Table 1
by washing several times with water, and dried for 48 h under vac-
Crystallographic data and structure refinement for the La complex.
uum. The Pr complex were prepared by the same method.
Empirical formula
C57H54LaN12O9
Formula weight
Crystal colour
1190.03
Yellow
3. Results and discussion
Crystal size (mm)
T (K)
0.31 ꢁ 0.27 ꢁ 0.25
3.1. Description of the crystal structure
296(2)
Wavelength (Å)
Crystal system
Space group
0.71073
Monoclinic
P2/c
On the basis of the single-crystal X-ray analysis of the complex
[La(HL)3]ꢂ1.5(C2H5OH)ꢂ1.5(H2O), the La ion is located at the center
of a tricapped trigonal prism configuration coordination environ-
ment, which consists of three nitrogens and six oxygens from three
ligands. The complex crystallized in the monoclinic lattice with a
space group P2/c. Each unit cell contained four molecules. The
coordination sphere of ORTEP diagram is in Fig. 1, which presents
a pseudo helix structure. The X-ray diffraction data for the La com-
plex are given in Table 1 and the selected bond lengths and angles
are summarized in Table 2.
a (Å)
b (Å)
c (Å)
a (°)
13.1407(5)
11.5427(4)
0.6615(14)
90
90.675(2)
90
6167.1(4)
4
1.282
b (°)
g (°)
V (Å3)
Z
DCalc (Mg/m3)
Absorption coefficient (mmꢀ1
F(0 0 0)
)
0.797
2504
Ligand H2L acts as a tridentate ligand, binding to La through the
oxygen atom of pyrazole ring (enolized and deprotonated from
O@C–), and the C@N group, O@C group of the benzoylhydrazine
side chain. The distance for C12–O2, C30–O4 and C48–O6 are
1.255 (Å), 1.264 (Å) and 1.276 (Å), respectively, which are between
the C–O (1.410–1.440 Å) and C@O (1.19–1.23 Å) distances [38],
this result shows that oxygen atoms of pyrazole ring of all three li-
gands take part in coordination by the enolic form, and their active
hydrogen is replaced by La atom. But O@C group of the ben-
zoylhydrazine side chain does not enolized, which can be demon-
strated by bond lengths C7–O1 (1.234 Å), C25–O3 (1.238 Å) and
C37–O5 (1.234 Å). Moreover, there are C2H5OH and H2O solvent
molecule in the lattice.
h Range (°)
0.989–24.10
29 129/9679 [0.0567]
1.015
Reflections collected/unique [Rint
]
Goodness of-fit on F2
R indices [I > 2
r
(I)]
R1 = 0.0774, wR2 = 0.2272
vibrations of the free ligand is at 1656 cmꢀ1 but for the two com-
plexes these peaks shift to about 1618 cmꢀ1
1591 cmꢀ1 for the free ligand is assignable to the
.
The band at
m(C@N) stretch,
which shifts to 1567 and 1569 cmꢀ1 for La and Pr complexes. Weak
bands are observed at 417 and 430 cmꢀ1. These low frequency
bands indicate the Ln ion and nitrogen and/or oxygen ligands.
3.3. Biological activities
3.2. Infrared spectra
3.3.1. Electronic absorption spectroscopy
The main stretching frequencies of the IR spectra of the com-
plexes are tabulated in Table 3. On the basis of the similar IR spec-
tra of the two complexes, it may be assumed that they have similar
The application of electronic absorption spectroscopy in DNA-
binding studies is one of the most useful techniques [40]. To clarify
the interactions between the compounds and DNA, the electronic
absorption spectra of the ligand and its Ln complexes (Ln = La,
Pr) in the absence and in the presence of the CT-DNA (at a constant
concentration of the compounds) were obtained, which are shown
in Fig. 2, respectively. With increasing DNA concentrations, the
absorption bands at 239 nm and 342 nm of the ligand represent
hypochromism of 15.60% and 14.66%; the absorption bands at
240 nm and 358 nm of the La complex exhibit hypochromism of
23.06% and 23.66%; the absorption bands at 242 nm and 362 nm
coordination structures. The aqueous
complexes appear at 3408, 3356 cmꢀ1. This shows that there is
some crystal water in the complexes [33,39]. The (C@O)(hydrazonic)
m(OH) bands of La and Pr
m
of the Pr complex appear hypochromism of 22.33% and 21.50%.
*
The hypochromism observed for the
p?p transition bands indi-
cating strong binding of the ligand and complexes to DNA. The
absorption data were analyzed to evaluate the intrinsic binding
constant Kb, which can be determined from Eq. (2) [41].
½DNAꢃ=ðea
ꢀ
ef Þ ¼ ½DNAꢃ=ðeb
ꢀ
ef Þ þ 1=½Kbðeb
ꢀ
ef Þꢃ
ð2Þ
where [DNA] is the concentration of DNA in base pairs; ea
,
e
f and eb
is the apparent extinction coefficient (Aobsd/[compound]), the
extinction coefficient for free compound and the extinction, respec-
tively, coefficient for compound in the fully bound form. In plots of
[DNA]/(ea
ꢀ
ef) vs. [DNA], Kb is given by the ratio of the slope to the
intercept. Kb values of the ligand, La and Pr complexes were
2.29 ꢁ 104 Mꢀ1, 1.50 ꢁ 105 Mꢀ1 and 5.01 ꢁ 104 Mꢀ1, respectively,
which were consist with hypochromism degree. The Kb values ob-
tained here are lower than that reported for classical intercalator
(for ethidium bromide and [Ru(phen)DPPZ] whose binding con-
stants have been found to be in the order of 106–107 Mꢀ1) [42–
45]. It is clear that the hypochromism and Kb values are not enough
evidence, but these results can suggest an intimate association of
Fig. 1. Thermal ellipsoid diagram of [La(HL)3]ꢂ1.5(C2H5OH)ꢂ1.5(H2O). Hydrogen
atoms have been excluded for clarity.

418
H.-G. Li et al. / Journal of Organometallic Chemistry 695 (2010) 415–422
Table 2
Selected bond lengths and angles.
O(6)–La(1)–O(4)
78.0(2)
O(2)–La(1)–N(6)
143.4(2)
C(7)–O(1)–La(1)
127.1(5)
O(6)–La(1)–O(2)
O(4)–La(1)–O(2)
O(6)–La(1)–O(5)
O(4)–La(1)–O(5)
O(2)–La(1)–O(5)
O(6)–La(1)–O(3)
O(4)–La(1)–O(3)
O(2)–La(1)–O(3)
O(5)–La(1)–O(3)
O(6)–La(1)–O(1)
O(4)–La(1)–O(1)
O(2)–La(1)–O(1)
O(5)–La(1)–O(1)
O(3)–La(1)–O(1)
O(6)–La(1)–N(10)
O(4)–La(1)–N(10)
O(2)–La(1)–N(10)
O(5)–La(1)–N(10)
O(3)–La(1)–N(10)
O(1)–La(1)–N(10)
O(6)–La(1)–N(2)
O(4)–La(1)–N(2)
O(2)–La(1)–N(2)
O(5)–La(1)–N(2)
O(3)–La(1)–N(2)
O(1)–La(1)–N(2)
N(10)–La(1)–N(2)
O(6)–La(1)–N(6)
O(4)–La(1)–N(6)
80.4(2)
83.0(2)
130.87(19)
147.1(2)
86.9(2)
O(5)–La(1)–N(6)
O(3)–La(1)–N(6)
O(1)–La(1)–N(6)
N(10)–La(1)–N(6)
N(2)–La(1)–N(6)
C(10)–N(3)–N(4)
C(12)–N(4)–N(3)
C(12)–N(4)–C(13)
N(3)–N(4)–C(13)
C(25)–N(5)–N(6)
C(25)–N(5)–H(5A)
N(6)–N(5)–H(5A)
C(26)–N(6)–N(5)
C(26)–N(6)–La(1)
N(5)–N(6)–La(1)
C(28)–N(7)–N(8)
C(30)–N(8)–C(31)
C(30)–N(8)–N(7)
C(31)–N(8)–N(7)
C(37)–N(9)–N(10)
C(37)–N(9)–H(9A)
N(10)–N(9)–H(9A)
C(44)–N(10)–N(9)
C(44)–N(10)–La(1)
N(9)–N(10)–La(1)
C(46)–N(11)–N(12)
C(48)–N(12)–N(11)
C(48)–N(12)–C(49)
N(11)–N(12)–C(49)
128.6(2)
59.0(2)
76.1(2)
C(12)–O(2)–La(1)
C(25)–O(3)–La(1)
C(30)–O(4)–La(1)
C(37)–O(5)–La(1)
C(48)–O(6)–La(1)
O(1)–C(7)–N(1)
O(1)–C(7)–C(6)
N(1)–C(7)–C(6)
N(2)–C(8)–C(9)
N(3)–C(10)–C(9)
N(3)–C(10)–C(11)
O(2)–C(12)–N(4)
O(2)–C(12)–C(9)
N(4)–C(12)–C(9)
C(14)–C(13)–N(4)
C(14)–C(13)–C(18)
N(4)–C(13)–C(18)
O(3)–C(25)–N(5)
O(3)–C(25)–C(24)
N(5)–C(25)–C(24)
N(6)–C(26)–C(27)
N(9)–C(37)–C(38)
N(10)–C(44)–C(45)
N(11)–C(46)–C(45)
N(11)–C(46)–C(47)
O(6)–C(48)–N(12)
O(6)–C(48)–C(45)
O(30)–C(74)–O(30)#1
O(30)–C(74)–C(73)
135.9(5)
126.9(6)
133.9(6)
125.5(6)
135.0(5)
121.2(8)
119.9(8)
118.9(8)
123.2(8)
110.2(8)
121.2(9)
123.0(8)
130.9(8)
106.1(7)
120.8(9)
119.2(10)
120.0(9)
121.2(9)
119.5(9)
119.3(9)
122.8(8)
116.8(9)
123.4(8)
111.2(8)
121.0(9)
122.6(8)
130.8(8)
132(2)
116.7(2)
122.7(2)
108.0(7)
109.9(7)
128.5(7)
121.6(7)
118.3(8)
120.8
86.7(2)
130.1(2)
140.9(2)
74.2(2)
145.2(2)
86.7(2)
128.84(19)
75.6(2)
79.6(2)
70.60(19)
140.7(2)
69.1(2)
60.5(2)
71.8(2)
132.4(2)
139.9(2)
72.2(2)
70.00(19)
74.9(2)
133.2(2)
59.15(18)
119.7(2)
69.4(2)
120.8
115.3(7)
129.9(6)
114.0(5)
105.1(8)
131.2(8)
109.0(8)
119.2(8)
116.8(7)
121.6
121.6
114.3(7)
131.1(6)
114.3(5)
105.6(7)
111.2(7)
127.9(7)
120.6(7)
71.2(2)
87.5(14)
Table 3
ary structure of DNA [56]. Therefore, EtBr can be used to probe the
interaction of complexes with DNA. Fig. 4 shows the emission
spectra of DNA–EtBr system with increasing amounts of the com-
plexes. The emission intensity of the DNA–EtBr system
(kem = 589 nm) decreased apparently as the concentration of com-
plexes increased, and the quenching plots illustrate that the
quenching of EtBr bound to DNA by the complexes is in good
agreement with the linear Stern–Volmer equation [57].
Some main IR data of the ligand and its complexes.
Compound
m
(OH)
m
(NH)
m
(C@O)
m
(C@N)
m(M–N)
H2L
[La(HL)3]
[Pr(HL)3]
3116m
3062m
3061m
1656w
1618s
1618s
1591s
1567s
1569s
3408w
3343w
417w
430w
the compounds with CT-DNA and indicate that the binding strength
of complex is in the order of La complex > Pr complex > H2L.
F0=F ¼ 1 þ Kq½Qꢃ
ð3Þ
where F0 and F are the fluorescence intensity in the absence and in
the presence of compound at [Q] concentration, respectively; Kq is
the quenching rate constant. The shape of Eq. (3) plots can be used
to characterize the quenching as being predominantly dynamic or
static.
Insert figure in Fig. 4 show the plots of F0/F vs. [Q]. The data of Kq
are all at 104 Mꢀ1 level for ligand, La and Pr complexes, accordingly.
In view of the strong interaction of EtBr with DNA of which the
binding constant of EtBr with DNA is at 106 Mꢀ1 level [43], we con-
sider it is impossible for the complexes to scramble EtBr from DNA.
Similar fluorescence quenching effect of EtBr bound to DNA has
been observed for the addition of several groove-binding com-
pounds, including netropsin and distamycin A [58,59]. The ob-
served results make us to suspect that the complex may interact
with DNA through the groove binding mode or intercalation mode,
releasing some EtBr molecules from EtBr–DNA system [59–61].
3.3.2. Fluorescence spectroscopy
Fixed amounts (10 lM) of the complexes were titrated with
increasing amounts of CT-DNA. The fluorescence titrations spectra
of complexes in the absence and presence of CT-DNA are given in
Fig. 3. Compared to the complexes alone, the fluorescence intensity
of the complexes are quenched steadily with the increasing con-
centration of the CT-DNA. This phenomenon of the quenching of
luminescence of the complex by DNA may be attributed to the
photoelectron transfer from the guanine base of DNA to the excited
MLCT state of the complex [46–51]. These changes also proved that
there were conjugation functions between CT-DNA and com-
pounds. [52,53].
3.3.3. Competition experiment
In order to further investigate the interaction mode between
the complexes and CT-DNA, the ethidium bromide (EtBr) fluores-
cence competitive experiments were also employed. The intrinsic
fluorescence intensity of DNA is very low, and that of EtBr in
Tris–HCl buffer is also not high due to the quenching by the solvent
molecules. However, on addition of DNA, the fluorescence intensity
of EtBr will be enhanced because of its intercalation into the DNA.
However, the enhanced fluorescence can be quenched evidently
when there is a second molecule that can replace DNA-bound EtBr
(if it binds to DNA more strongly than EtBr) and/or accept the ex-
cited state electron from EtBr [54,55], or possibly break the second-
3.3.4. Viscosity studies
Viscosity titration measurements were carried out to further
clarify the interaction modes between the investigated compounds
and CT-DNA. Hydrodynamic measurements that are sensitive to
changes in the length of DNA (i.e. viscosity and sedimentation)
are regarded as the least ambiguous and the most critical tests of
binding in solution in the absence of crystallographic structural
data [11]. The classic intercalation model involves the insertion
of a planar molecule between DNA base pairs, which results in a

H.-G. Li et al. / Journal of Organometallic Chemistry 695 (2010) 415–422
419
0.4
0.3
0.2
0.1
0.0
(a)
500
400
300
200
100
0
(a)
400
450
500
550
Wavelength/nm
200
250
300
350
400
450
Wavelength/nm
200
150
100
50
(b)
0.6
0.5
0.4
0.3
0.2
0.1
(b)
0
400
450
500
550
600
650
Wavelength/nm
250
300
350
400
450
500
Wavelength/nm
(c)300
(c) 0.8
250
200
150
100
50
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
400
450
500
550
Wavelength/nm
Fig. 3. (a) The emission enhancement spectra of the ligand (10
of 0–20 M CT-DNA. Arrow shows the emission intensities changes upon increasing
DNA concentration. (b) The emission enhancement spectra of the La complex
(10 M) in the presence of 0–25 M CT-DNA. Arrow shows the emission intensities
changes upon increasing DNA concentration. (c) The emission enhancement spectra
of the Pr complex (10 M) in the presence of 0–17.5 M CT-DNA. Arrow shows the
lM) in the presence
200
250
300
350
400
450
500
l
Wavelength/nm
l
l
Fig. 2. (a) Electronic spectra of the ligand (10
[DNA] = 0–18 M. Arrow shows the absorbance changes upon increasing DNA
concentration. (b) Electronic spectra of the La complex (10 M) in the presence of
CT-DNA. [DNA] = 0–20 M. Arrow shows the absorbance changes upon increasing
DNA concentration. (c) Electronic spectra of the Pr complex (10 M) in the presence
M. Arrow shows the absorbance changes upon increas-
lM) in the presence of CT-DNA.
l
l
l
l
emission intensities changes upon increasing DNA concentration.
l
l
of CT-DNA. [DNA] = 0–24
ing DNA concentration.
l
gated compounds to DNA (bps) increasing, the relative viscosities
of DNA first increased and then decreased, may indicating that
these compounds can bind to DNA by groove mode and intercala-
tion mode [65,66]. The changed degree of viscosity which follows
the order of La complex > Pr complex > H2L, may depend on its
affinity to DNA, which is consistent with electronic absorption
spectroscopy studies. In addition, the viscosity changes of the Ln
(Ln = La, Pr) salts are far small than those of Schiff-base complexes.
The higher binding affinity of the complexes is probably relate to
its helix structure since the structure of the La complex is able to
decrease in the DNA helical twist and lengthening of the DNA, the
molecule will be in close proximity to the DNA base pairs as well
[32,62,63]. In contrast, molecule that binds exclusively in the
DNA grooves by partial and/or non-classical intercalation, under
the same conditions, typically cause less pronounced (positive or
negative) or no change in DNA solution viscosity [35,64]. The ef-
fects of the compounds on the viscosity of rod-like DNA at
25.00 0.01 °C are shown in Fig. 5. With the ratios of the investi-

420
H.-G. Li et al. / Journal of Organometallic Chemistry 695 (2010) 415–422
(a)140
1.8
(Pr complex)
(La complex)
(Ligand)
1.1
1.7
1.6
1.5
1.4
1.3
1.2
1.1
120
100
80
(La(NO ) )
(Pr(NO33)33)
1.0
2
4
6
8
10 12
M
[Q]/10-6
0.9
0.8
0.7
0.6
60
40
20
560
580
600
620
640
0.1
0.2
0.3
0.4
0.5
0.6
0.7
Wavelength/nm
[Complex]/[DNA]
Fig. 5. Effect of increasing amounts of the ligand, Ln complex and Ln (Ln = La, Pr)
salts on the relative viscosity of CT-DNA at 25.00 0.01 °C.
(b) 160
1.30
1.28
1.26
1.24
1.22
1.20
1.18
1.16
1.14
140
120
100
80
interactions with small molecules [67]. The changes in CD signals
of DNA observed on interaction with drugs may often be assigned
to the corresponding changes in DNA structure [68]. The circular
dichroic spectrum of CT-DNA (Fig. 6) exhibits a positive band at
270 nm and a negative band at 243 nm, and both bands increased
in intensity upon addition of the La complex to DNA. This phenom-
enon may be due to the intercalation of the complex through p-
stacking which stabilizes the right-handed B form DNA [47].
1.12
4
6
8
10 12 14
[Q]/10-6M
60
40
560
580
600
620
640
3.3.6. Hydroxyl radical scavenging activity
Wavelength/nm
Generation of reactive oxygen species (ROS) is a normal process
in the life of aerobic organisms. It has been estimated that free rad-
ical-induced DNA damage in humans is at biologically relevant lev-
els, with approximately 104 DNA bases being oxidatively modified
per cell per day. Oxidative damage to DNA has been suggested to
contribute to aging and various diseases including cancer and
chronic inflammation [69]. Since among all reactive oxygen spe-
cies, the hydroxyl radical (OHÅ) is by far the most potent and there-
fore the most dangerous oxygen metabolite, elimination of this
radical is one of the major aims of antioxidant administration
[70]. Consequently, in this paper, the ligand and its Ln complexes
(Ln = La, Pr) studied for their antioxidant activity by comparing
their scavenging effects on hydroxyl radical (OHÅ).
1.35
1.30
1.25
1.20
1.15
1.10
1.05
2
(c)140
120
100
80
4
6
8
10 12
[Q]/10-6 M
60
40
Fig. 7 shows the plots of hydroxyl radical scavenging effects (%)
for the ligand, Ln complexes and Ln salts (Ln = La, Pr). The values of
560
580
600
620
640
Wavelength
3
2
Fig. 4. (a) The emission spectra of calf thymus DNA–EtBr system in the presence of
0–30 M the ligand. Arrow shows the emission intensities changes upon increasing
ligand concentration. (Inset) Stern–Volmer plot of the fluorescence titration data of
ligand. (b) The emission spectra of calf thymus DNA–EtBr system in the presence of
l
0–27.5 lM La complex. Arrow shows the emission intensities changes upon
increasing ligand concentration. (Inset) Stern–Volmer plot of the fluorescence
1
titration data of La complex. (c) The emission spectra of calf thymus DNA–EtBr
system in the presence of 0–20 lM Pr complex. Arrow shows the emission
intensities changes upon increasing ligand concentration. (Inset) Stern–Volmer plot
0
of the fluorescence titration data of Pr complex.
-1
-2
-3
provide lots of grooving positions to stack more strongly with the
base pairs of the DNA helix [58].
3.3.5. Circular dichroism (CD) studies
220
240
260
280
300
320
Circular dichroic spectral technique is useful in diagnosing
changes in DNA morphology during drug–DNA interactions, as
the band due to base stacking (275 nm) and that due to right-
handed helicity (248 nm) are quite sensitive to the mode of DNA
Wavelength
Fig. 6. CD spectra of CT-DNA in the absence (Solid line) and presence (Dash Dot
line) of La complex at r = 1.0 (r = molar ratio [compound]/[CT-DNA]).

H.-G. Li et al. / Journal of Organometallic Chemistry 695 (2010) 415–422
421
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