Synthesis, characterization, dna binding properties and antioxidant activity of Ln(III) complexes with schiff base ligand derived from 3-Carbaldehyde chromone and aminophenazone

Article abstract of DOI:10.1007/s10895-010-0782-2

A novel Schiff base ligand, chromone-3-carbaldehyde-aminophenazone (L) and its Ln(III) (Ln = La, Yb) complexes were synthesized and characterized by physicochemical methods. The interaction between the ligand, Ln(III) complexes and calf thymus DNA in physiological buffer (pH=7.10) was investigated by using UV-vis spectroscopy, fluorescence spectra, ethidium bromide experiments and viscosity measurements, indicating that the studied compounds can all bind to DNA via an intercalation binding mode and the complexes have stronger binding affinity than the free ligand alone. Furthermore, antioxidant activity of the ligand and its complexes was determined by superoxide and hydroxyl radical scavenging methods in vitro, suggesting that Ln(III) complexes inhibit stronger antioxidant activity than the ligand alone and some standard antioxidants, such as mannitol and vitamin C. Springer Science+Business Media, LLC 2011.

Full text of DOI:10.1007/s10895-010-0782-2

J Fluoresc (2011) 21:1091–1102
DOI 10.1007/s10895-010-0782-2
ORIGINAL PAPER
Synthesis, Characterization, DNA Binding Properties
and Antioxidant Activity of Ln(III) Complexes with Schiff
Base Ligand Derived from 3-Carbaldehyde Chromone
and Aminophenazone
Yong Li & Zheng-yin Yang & Tian-rong Li &
Zeng-chen Liu & Bao-dui Wang
Received: 1 September 2010 /Accepted: 25 November 2010 /Published online: 15 December 2010
#
Springer Science+Business Media, LLC 2010
Abstract A novel Schiff base ligand, chromone-3-
carbaldehyde-aminophenazone (L) and its Ln(III) (Ln = La,
Yb) complexes were synthesized and characterized by
physicochemical methods. The interaction between the
ligand, Ln(III) complexes and calf thymus DNA in physio-
logical buffer (pH=7.10) was investigated by using UV–vis
spectroscopy, fluorescence spectra, ethidium bromide experi-
ments and viscosity measurements, indicating that the studied
compounds can all bind to DNA via an intercalation binding
mode and the complexes have stronger binding affinity than
the free ligand alone. Furthermore, antioxidant activity of the
ligand and its complexes was determined by superoxide and
hydroxyl radical scavenging methods in vitro, suggesting that
Ln(III) complexes inhibit stronger antioxidant activity than
the ligand alone and some standard antioxidants, such as
mannitol and vitamin C.
more and more active during the past several years [3]. DNA
binding metal complexes are being investigated in many
laboratories because of their utility as DNA structural probes,
DNA dependent electron transfer probes, and DNA foot
printing, sequence-specific cleaving agents; especially they
can be used as potential anticancer drug [4–6]. Generally,
metal complexes bind to DNA in a non-covalent way, such as
intercalation, groove-binding and external electrostatic bind-
ing for cations [7]. Considerable useful applications of the
metal complexes require that they can bind to DNA via an
intercalation binding mode inducing cellular degradation [8–
10]. The intercalation ability not only correlates to the
coordination geometry and ligand donor atom type [11, 12],
but also relates with the metal ion type and its valence [13].
Among all the metal complexes, lanthanide complexes are
widely investigated since the lanthanide complexes with
tetracycline, phenanthroline [14], adriamycin and pyridine
[15–17] have already been synthesized as a probe to study
nucleic acids. Additionally, the importance of reactive oxygen
species (ROS) which are involved in the pathogeneses of
various diseases, such as lifestyle-related diseases including
hypertension and photoaging due to exposure to ultraviolet
radiation has attracted considerable attention over the past
decades [18, 19]. In the human body, superoxide and
hydroxyl radicals are produced by the reduction of oxygen
and over production of free radicals are considered to be the
main contributor to oxidative stress [20]. Recently, studies
suggest that many metal complexes also exhibited interesting
antioxidant activities [21, 22]. Therefore, it is significant to
search for new metal complexes as potential antioxidants.
Flavonoids, occurring widely throughout the plant king-
dom, are one of the most representative families of plant
secondary metabolites and display a remarkable spectrum of
biological activities, such as developing the purpose of drug
.
Keywords Chromone-3-carbaldehyde-aminophenazone
.
.
Ln(III) complexes DNA binding properties Antioxidant
activity
Introduction
Currently, a great number of experiments indicate that the
binding between small molecules and DNA can greatly help
understand drug–DNA interactions and design new and
promising anticancer drugs for clinical use [1, 2], and then
the interaction of drug molecules with DNA has become
:
:
:
:
Y. Li Z.-y. Yang (*) T.-r. Li Z.-c. Liu B.-d. Wang
College of Chemistry and Chemical Engineering and State Key
Laboratory of Applied Organic Chemistry, Lanzhou University,
Lanzhou 730000, People’s Republic of China
e-mail: yangzy@lzu.edu.cn

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J Fluoresc (2011) 21:1091–1102
delivery or target identification [23, 24]. It should be
especially noted that developing flavonoids as anticancer
agents has interested medicinal chemists for many years.
Chromones usually are indentified as a kind of flavonoids
that are ubiquitous in nature especially in plants. They are
oxygen-containing heterocyclic compounds with a benzoan-
nelated γ-pyrone ring, with the parent compound being
chromone (4H-chromone-4-one, 4H-1-benzopyran-4-one).
Molecules containing the chromone structure have a wide
range of biological activities including tyrosine and protein
kinase C inhibitors, antifungal, antiallergenic, antiviral,
antitublin, antihypertensive, anticancer agents, as well being
active at benzoazepine receptors, lipoxygenase, cyclooxyge-
nase and modulating P-glycoprotein-mediated multidrug
resistance [25–27].
In order to give a deep research to chromone ramifications
and their lanthanide complexes, in this paper, we synthesized
and characterized a novel ligand, chromone-3-carbaldehyde-
aminophenazone (L, Fig. 1) and its Ln (III) complexes. We
described a comparative study of the interaction of novel Ln
(III) complexes and the ligand with calf thymus DNA (CT
DNA) using electronic absorption spectroscopy, fluorescence
spectra, ethidium bromide experiments and viscosity meas-
urements for the first time. The antioxidant activity of the
ligand and its Ln(III) complexes was also investigated.
Information obtained from our study would be helpful to
understand the mechanism of interactions of chromone
derivatives and their complexes with nucleic acid and should
be useful in the development of potential probes of DNA
structure and conformation, and new therapeutic reagents for
some certain diseases.
Chemical Co. Ethylenediaminetetraaceticacid (EDTA), saf-
ranin, 4-aminophenazone and Ln(NO3)3•6H2O [Ln(III) = La
and Yb] were produced in China. All chemicals used were of
analytical grade. EDTA-Fe(II) and KH2PO4-Na2HPO4 buf-
fers were prepared with doubly distilled water.
All the experiments involved with the interaction of the
ligand and its lanthanide complexes with CT DNAwere carried
out in doubly distilled water buffer containing 5 mM Tris [Tris
(hydroxymethyl)-aminomethane] and 50 mM NaCl and
adjusted to pH 7.10 with hydrochloric acid. Solution of CT
DNA in Tris–HCl buffer gave ratios of UVabsorbance of about
1.8–1.9:1 at 260 and 280 nm, indicating that the CT DNA was
sufficiently free of protein [28]. The CT DNA concentration
per nucleotide was determined spectrophotometrically by
employing an extinction coefficient of 6,600 M−1 cm−1 at
260 nm [29]. The ligand and its complexes were dissolved in
a solvent mixture of 1% DMF and 99% Tris–HCl buffer
(pH 7.10) at the concentration 1.0×10−5 M.
Carbon, hydrogen and nitrogen were determined using
an Elemental Vario EL analyzer. The metal contents of the
complexes were determined by titration with EDTA
(xylenol orange tetrasodium salt used as an indicator and
hexamethylidynetetraimine as buffer). The melting point of
the ligand was determined on a Beijing XT4-100X
microscopic melting point apparatus (the thermometer was
not corrected). The IR spectra were obtained in KBr discs
on a Therrno Mattson FTIR spectrophotometer in the
4,000–400 cm−1 region. 1H NMR spectra were recorded
on a Varian VR 200 MHz spectrometer in CDCl3-d with
TMS (tetramethylsilane) as internal standard. Mass spectra
were performed on an APEX II FT-ICR MS instrument
using methanol as mobile phase. All conductivity measure-
ments were performed in DMF solution with a DDS-11C
conductometer at 25.0 °C. The UV–vis spectra were
recorded on a Shimadzu UV-240 spectrophotometer. Fluo-
rescence measurements were recorded on a Hitachi RF-
4500 spectrofluorophotometer at ambient temperature. The
antioxidant activity was performed in DMF solution with a
721-E spectrophotometer.
Experimental Section
Materials and Instrumentation
Nitroblue tetrazolium (NBT), methionine (MET), vitamin B2
(VitB2), EB and CT DNA were purchased from Sigma
Fig. 1 Scheme of the synthesis
of the ligand

J Fluoresc (2011) 21:1091–1102
1093
Fig. 2 Electronic absorption spectra of the ligand (a), La(III) (b) and
Yb(III) (c) complexes (10 μM) in the absence (top spectrum) and
presence of increasing amounts of CT DNA (2.5, 5.0, 7.5, 10.0, 12.5,
15.0, 17.5, 20.0 and 22.5 μM; subsequent spectra). Arrows show the
absorbance changes upon increasing DNA concentration
R
a
0.24
0.20
0.16
0.12
0.08
0.04
0.00
Preparation of the Ligand (L) and its Ln(III) Complexes
Preparation of the Ligand (L)
Scheme of the synthesis of the ligand is shown in Fig. 1. 3-
carbaldehyde chromone was prepared according to the
literature methods [30]. To a solution of 3-carbaldehyde
chromone (0.870 g, 5 mmol) in ethanol (50 mL) at warming
was added 4-aminophenazone (1.117 g, 5.5 mmol) in ethanol
(20 mL). The reaction mixture was boiled on an oil bath for
8 h with stirring. After that, the yellow solution was
concentrated and cooled, a yellow solid was separated out
and filtered, washed with massive water and ethanol, and then
recrystallized from DMF and water to give the ligand. Yield:
75.63%. Mp: 269−271 °C. Anal Calcd for C21H17N3O3
requires C, 70.18; H, 4.77; N, 11.69. Found: C, 70.23; H, 4.82;
250
300
350
400
450
Wavelength (nm)
b
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
N, 11.66. IR νmax (cm−1): ν(carbonyl)C=O: 1656, ν(hydrazonic)C=O
:
1631, νC=N: 1591 cm−1. λmax(nm): 350, 277 nm. L (CDCl3-d
200 MHz): δ 9.85 (1 H, s, 9-H), 8.67 (1 H, s, 2-H), 8.30 (1 H,
d, 5-H), 7.72 (1 H, d, 8-H), 7.25–7.45 (7 H, m, 6, 7, 13, 13′,
14, 14′, 15-H), 3.16 (3 H, s, 11-H), 2.41 (3 H, s, 12-H).
Preparation of the Ln(III) Complexes
The ligand (144 mg, 0.40 mmol) was dissolved in the
mixture solution of ethanol and chloform (20 mL). After
5 min, La(III) nitrate (87 mg, 0.20 mmol) was added
quickly and the solution was refluxed on a water bath with
stirring, and the reaction temperature was kept at 90 °C for
10 h. A yellow precipitate, the La(III) complex, was
separated from the solution by suction filtration, purified
by washing several times with ethanol and dried for 24 h in
a vacuum. The Yb(III) complex was prepared by the same
way. Anal Calcd for La(III) complex C42H34N9O15La
requires: C, 48.33; H, 3.28; N, 12.08; La, 13.31; Found:
C, 48.36; H, 3.23; N, 12.03; La, 13.24. Yield: 82%. IR νmax
250
300
350
400
450
Wavelength (nm)
c
0.30
0.25
0.20
0.15
0.10
0.05
0.00
(cm−1): ν(carbonyl)C=O: 1634, ν(hydrazonic)C=O: 1611, νC=N
:
−1
NO
3
1,569 cm , n : 1479, 1385, 1320, 1184, 698, νM-O: 631,
ν
M-N: 433. λmax (nm):327, 271 cm−1. Λm (S cm2 mol−1):
87.5. Anal Calcd for Yb(III) complex C42H34N9O15Yb
requires: C, 46.80; H, 3.18; N, 11.70; Yb, 16.05; Found: C,
46.78; H, 3.25; N, 11.69; Yb, 16.08. Yield: 76%. IR
250
300
350
400
450
ν
(carbonyl)C=O: 1632, ν(hydrazonic)C=O: 1615, νC=N
:
:
Wavelength (nm)
−1
NO
3
1,577 cm , n : 1479, 1383, 1321, 1188, 702, νM-O
636, νM-N: 423. λmax (nm): 328, 270 cm−1. Λm
(S cm2 mol−1): 99.8.

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J Fluoresc (2011) 21:1091–1102
Fig. 3 The ultraviolet visible absorption spectra of 1×10−5 M EB (A);
(A)+2.5×10−5 M DNA (C); (C)+2.5×10−5 M ligand (a), La(III) (b)
and Yb(III) (c) complexes in Tris–HCl buffer (5 mM Tris–HCl,
50 mM NaCl, pH 7.10) solution
R
a
0.07
A
C
0.06
0.05
0.04
0.03
0.02
0.01
0.00
B
DNA Binding Studies
Electronic Absorption Titration
Electronic absorption titration experiments were performed
with fixed concentration drugs (10 μM), while gradually
increasing the concentration of CT DNA. When measuring
the absorption spectra, an equal amount of CT DNA was
added to both the complex solutions and the reference
solution to eliminate the absorbance of CT DNA itself. Each
sample solution was scanned in the range of 190–500 nm.
400
450
500
550
600
Wavelength (nm)
Fluorescence Spectra
b
A
B
0.07
0.06
0.05
0.04
0.03
0.02
0.01
0.00
To compare quantitatively the affinity of the compounds bound
to DNA, Kb values of the compounds to DNA were obtained
by the luminescence titration method. Fixed amounts of
compound were titrated with increasing amounts of DNA,
over a range of DNA concentrations from 2.5 to 35 μM.
Excitation wave lengths of 357 nm for La(III) complex,
359 nm for Yb(III) complex and 335 nm for L were used, and
fluorescence emission intensities were monitored at 455 nm,
458 nm and 452 nm, respectively. The concentration of the
bound compound was calculated using Eq [31]:
C
Cb ¼ Ct½ðF ꢀ F0Þ=ðFmax ꢀ F0Þꢁ;
where Ct is the total compound concentration, F is the
observed fluorescence emission intensity at given DNA
concentration, F0 is the intensity in the absence of DNA,
and Fmax is the fluorescence of the totally bound compound.
Binding data were cast into the form of a Scatchard plot of
r/Cf versus r, where r is the binding ratio Cb/[DNA]t and Cf is
the free compound concentration. All experiments were
conducted at room temperature in a buffer containing 5 mM
Tris–HCl (pH 7.10) and 50 mM NaCl concentrations.
400
450
500
550
600
Wavelength (nm)
c
A
0.07
0.06
0.05
0.04
0.03
0.02
0.01
0.00
B
C
Further support for the ligand and complexes binding to
DNA via intercalation is given through the emission
quenching experiment. EB is a common fluorescent probe
Fig. 4 The emission enhancement spectra of the free ligand (a), La
„
(III) (b) and Yb(III) (c) complexes (10 μM) in the absence (bottom
spectrum) and presence of increasing amounts of CT DNA (2.5, 5.0,
7.5, 10.0, 12.5 and 15.0 μM; subsequent spectra). Arrows show the
emission intensity changes upon increasing DNA concentration. Inset:
Scatchard plot of the fluorescence titration data of ligand and rare
earth complexes, KbðaÞ ¼ ð7:80 ꢂ 1:06Þ ꢃ 105; KbðbÞ ¼ ð2:92 ꢂ 0:40Þ
ꢃ106Mꢀ1; KbðcÞ ¼ ð1:91 ꢂ 0:23Þ ꢃ 106Mꢀ1
400
450
500
550
600
Wavelength (nm)

J Fluoresc (2011) 21:1091–1102
1095
a
120
6.0
4.5
3.0
1.5
Kb = (7.80 1.06)×105 M-1
100
80
60
40
20
0
Kb = (7.80 1.06)×105 M-1
0.7
0.8
0.9
1.0
r
1.1
1.2
1.3
400
400
400
500
600
700
Wavelength (nm)
b
150
125
100
75
16
14
12
10
8
Kb = (2.92 0.40)×106 M-1
Kb = (2.92 0.40)×106 M-1
6
50
4
25
2
0
0
0.7
0.8
0.9
1.0
r
1.1
1.2
1.3
500
600
700
Wavelength (nm)
c
300
250
200
150
100
50
12
10
8
Kb = (1.91 0.23)×106 M-1
Kb = (1.91 0.23)×106 M-1
6
4
2
0
0
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1.1
500
600
700
Wavelength (nm)
r

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J Fluoresc (2011) 21:1091–1102
Fig. 5 The emission spectra of DNA-EB system λex=525 nm,
„
for DNA structure and has been employed in examinations of
the mode and process of metal complexes binding to DNA
[32]. A 2.0 mL solution of 4 μM DNA and 0.32 μM EB (at
saturating binding levels) was titrated by 2.5–50 μM the
lanthanide complexes and ligand. Quenching data were
analyzed according to the Stern−Volmer equation which could
be used to determine the fluorescent quenching mechanism:
lem ¼ 550 ꢀ 650 nm, in the presence of 0, 2.5, 5, 7.5, 10, 12.5, 15,
17.5, 20.0, 22.5, 25, 27.5, 30.0, 32.5, 35.0, 37.5, 40.0, 42.5, 45.0, 47.5
and 50.0 μM free ligand (a) and La(III) (b) complex, and 0, 2.5, 5,
7.5, 10, 12.5, 15, 17.5, 20.0, 22.5, 25, 27.5, 30.0, 32.5 and 35.0 μM
Yb(III) (c) complex. Arrows show the emission intensity changes
upon increasing ligand and complexes. Inset: Stern-Volmer plot of the
fluorescence titration ꢀd1 ata of ligand and complexes, KqðaÞ ¼
ð1:75 ꢂ 0:03Þ ꢃ 104 Mꢀ1; KqðbÞ ¼ ð5:54 ꢂ 0:12Þ ꢃ 104 Mꢀ1; KqðcÞ ¼
ð2:26 ꢂ 0:05Þ ꢃ 104 M
F0=F ¼ 1 þ Kq½Qꢁ;
Where F0 and F are the fluorescence intensity in the absence
and presence of drug at [Q] concentration respectively; Kq is
the quenching constant and [Q] is the quencher concentration.
Plots of F0/F versus [Q] appear to be linear and Kq depends
on temperature [33].
In antioxidant activity experiments the hydroxyl radical
(HO•) in aqueous media was generated through the Fenton
reaction [37]. The solution of the tested compounds was
prepared with DMF. The reaction mixture contained 2.5 mL
0.15 M phosphate buffer (pH=7.4), 0.5 mL 114 μM
safranin, 1 mL 945 μM EDTA–Fe(II), 1 mL 3% H2O2 and
30 μL the tested compound solution (the final concentration:
Ci ði¼1ꢀ6Þ ¼ 1:0; 2:0; 3:0; 4:0; 5:0; 6:0 mM). 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. Absorbances (Ai, A0, Ac) at 520 nm were
measured. The suppression ratio for HO• was calculated
from the following expression:
Viscosity Measurements
Viscosity experiments were conducted on an Ubbelodhe
viscometer, immersed in a thermostated water-bath main-
tained to 25.0 0.1 °C. Titrations were performed for the
complexes (0.5–4 μM), and each compound was intro-
duced into a DNA solution (5 μM) present in the
viscometer. Data were presented as (η/η0)1/3 versus the
ratio of the concentration of the compound and DNA,
where η is the viscosity of DNA in the presence of the
compound and η0 is the viscosity of DNA alone [34, 35].
Suppression ratioð%Þ ¼ ½ðAi ꢀ A0Þ=ðAc ꢀ A0Þꢁ ꢃ 100%
Where Ai = the absorbance in the presence of the tested
compound; A0 = the absorbance in the absence of the tested
compound; Ac = the absorbance in the absence of the tested
compound, EDTA–Fe(II) and H2O2.
The antioxidant activity was expressed as the 50%
inhibitory concentration (IC50). IC50 values were calculated
from regression lines where: x was the tested compound
concentration in μM and y was percent inhibition of the
tested compounds.
Antioxidant Activity
In antioxidant activity experiments the superoxide radicals
ðO2ꢄꢀÞ were produced by the system MET/VitB2/NBT
ꢄꢀ
ꢄꢀ
[36]. The amount of O2 and suppression ratio for O2
can be calculated by measuring the absorbance at 560 nm,
because NBT can be reduced quantitatively to blue
formazan by O2ꢄꢀ. The solution of MET, VitB2 and NBT
were prepared with 0.067 M phosphate buffer (pH=7.8) at
the condition of avoiding light. The tested compounds were
dissolved in DMF. The reaction mixture contained MET
(0.01 mol L−1), NBT (4.6×10−5 mol L−1), VitB2 (3.3×
10−6 mol L−1), phosphate buffer solution (0.067 mol L−1)
and the tested compound (the final concentration:
Ciði¼1ꢀ5Þ ¼ 0:4; 1:0; 2:0; 4:0; 6:0 mM). After incubating
at 30 °C for 10 min and illuminating with a fluorescent
lamp for 3 min, the absorbance (Ai) of the samples was
measured at 560 nm. The sample without the tested
compound and avoiding lighꢄtꢀwas used as the control.
The suppression ratio for O2 was calculated from the
following expression:
Results and Discussion
Structure of the Ln(III) Complexes
The complexes were prepared by direct reaction of ligand
with the appropriate mole ratios of Ln(III) nitrate in the
mixture solution of chloroform and ethanol. The yields
were good to moderate. The desired Ln(III) complexes
were separated from the solution by suction filtration,
purified by washing several times with ethanol. The
complexes are air-stable for extended periods and soluble
in DMSO and DMF; slightly soluble in ethanol and
methanol; insoluble in benzene, water and diethyl ether.
The molar conductivities of the complexes are around
87.5–99.8 Scm2 mol−1 in DMF solution, showing that the
two complexes are 1:1 electrolytes [38].
Suppression ratio ð%Þ ¼ ½ðA0 ꢀ AiÞ=A0ꢁ ꢃ 100%
where Ai = the absorbance in the presence of the ligand or
its complexes, A0 = the absorbance in the absence of the
ligand or its complexes.

J Fluoresc (2011) 21:1091–1102
1097
a
2.0
1.8
1.6
1.4
1.2
1.0
800
700
600
500
400
300
200
100
0
Kq = (1.75 0.03)×104 M
-1
Kq = (1.75 0.03)×104 M -1
550
600
650
700
0
10
20
30
40
50
Wavelength (nm)
[Compound]×106 M
b
4.0
3.5
3.0
2.5
2.0
1.5
1.0
1000
800
600
400
200
0
Kq = (5.54 0.12)×104 M -1
Kq = (5.54 0.12)×104 M -1
10
20
30
40
50
0
550
600
650
700
Wavelength (nm)
[Compound]×106 M
c
1000
800
600
400
200
0
1.9
1.8
1.7
1.6
1.5
1.4
1.3
1.2
1.1
1.0
Kq = (2.26 0.05)×104 M -1
Kq = (2.26 0.05)×104 M -1
550
600
650
700
0
5
10
15
20
25
30
35
Wavelength (nm)
[Compound]×106 M

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J Fluoresc (2011) 21:1091–1102
1.8
approximately 195 cm−1, and accordingly the coordinated
NO3 anion in the complexes is a bidentate ligand [41].
The study of the electronic spectra in the ultraviolet and
visible (UV–vis) ranges for the rare earth complexes and
ligand was carried out in a buffer solution containing 5 mM
Tris–HCl (pH 7.10) and 50 mM NaCl concentrations. The
electronic spectra of the free ligand have a strong band at
ꢀ
1.7
1.6
1.5
1.4
1.3
1.2
1.1
L
La(III) Complex
Yb(III) Complex
η
η
λ
max=277 nm and a medium band at λmax=350 nm. The
complexes also yield two bands and the two bands are
shifted to 270 and 328 nm or so. These indicate that the rare
earth complexes are formed.
The electrospray ionization (ESI) mass spectra of La(III)
and Yb (III) complex were made. The mass spectra of La
(III) complex show peaks at 1043, 919 and 718, which can
be assigned to the fragments [La(III) complex + H]+, [La
(III) complex - 2NO3]2+ and [La(III) complex - 3NO3 - La],
respectively. And the mass spectra of Yb(III) complex show
peaks at 1077 and 718 which can be assigned to the
fragments [Yb(III) complex + H]+ and [La(III) complex -
3NO3 - La], respectively.
In conclusion, the elemental analyses, molar conductivities
and other structural analyses indicate that the novel ligand and
lanthanide ions can form mononuclear ten-coordination
[LnL2·(NO3)2]NO3 [Ln(III) = La and Yb, L is chromone-3-
carbaldehyde-aminophenazone] complexes with 1:2 metal-
to-ligand stoichiometry at the Ln(III) centers.
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
[Compound]/[DNA]
Fig. 6 Effects of increasing amounts of the ligand and its lanthanide
complexes on the relative viscosity of CT DNA at 25.0 0.1 °C
The IR spectra of the complexes are similar and IR spectra
usually provide a lot of valuable information on coordination
reactions. All the spectra are characterized by vibrational
bands mainly due to the C = O, C = N, M-O, M-N and NO3
groups. The ν(carbonyl)C=O and ν(hydrazonic)C=O vibrations of
the free ligand appear at 1,656 and 1,631 cm−1, respectively;
whereas for the complexes these peaks shift to 1,634, 1,611
and 1,632, 1,615 cm−1, Δν(ligand-complexes) is equal to 22, 20
and 24, 16 cm−1, respectively. In the complexes, the band at
631 cm−1 or so is assigned to νM–O, demonstrating that the
oxygen of carbonyl has formed a coordinative bond with the
rare earth ions [39]. The different shifts of the wavenumbers
indicate that the Ln-O (carbonyl) bond is a little stronger
than the Ln-O (hydrazonic) bond. The band at 1,591 cm−1
for the free ligand is assigned to νC=N stretch, which shifts to
about 1,569 cm−1 for the complexes, and Δν(ligand-complexes)
is equal to 22 cm−1. Weak bands at 433 cm−1 or so are
assigned to νM–N in the complexes. These shifts and new
bands further confirm that the nitrogen of the imino-group
bonds to the rare earth ions [40]. The absorption bands of the
coordinated nitrates were observed at about 1479 (νas) and
817 (νs) cm−1. The ν3 (E′) free nitrates appear at 1,385 cm−1
in the spectra of the rare earth complexes. In addition, the
separation of the two highest frequency bands |ν4–ν1| is
DNA Binding Studies
Electronic Absorption Spectroscopy
Before reacting the ligand and its Ln(III) complexes
interacting with CT DNA, their solution behavior in buffer
75
L
La(III) Complex
Yb(III) Complex
60
45
30
15
0
1
2
3
4
5
6
Concentration (μM)
Fig. 7 Molecular mode for the investigated compounds (via an
intercalative mode)
Fig. 8 Scavenging effects of the ligand and lanthanide complexes on
O2
ꢄꢀ

J Fluoresc (2011) 21:1091–1102
1099
ꢄꢀ
Table 1 The influence of investigated compounds for O2
R2
−·
Compound
Average inhibition (%) for O2 (μM)
Equation
IC50 (μM)
0.2
1.0
2.0
4.0
6.0
y ¼ 18:51x þ 24:72
y ¼ 18:51x þ 55:17
y ¼ 20:66x þ 46:74
L
14.08
43.66
34.27
21.13
53.70
44.60
29.11
59.28
50.71
35.68
64.79
58.22
41.78
72.59
66.20
23.214
0.492
1.438
0.946
0.963
0.957
La(III) Complex
Yb(III) Complex
solution at room temperature was monitored by UV–vis
spectroscopy for 24 h. Liberation of the ligand and its
complexes was not observed under these conditions. These
suggest that the ligand and its complexes are stable under
the conditions studied.
DNA, the π* orbital of the intercalated ligand can couple
with the π orbital of the DNA base pairs, thus, decreasing
the π→π* transition energy and resulting in the bath-
ochromism. On the other hand, the coupling π orbial is
partially filled by electrons, thus, decreasing the transition
probalities and concomitantly resuting in the hypochrom-
ism [42]. All these results suggest that the complexes can
interact with CT DNA quite probably by intercalating the
compounds into DNA base pairs, so we can conclude that
the free ligand and its metal complexes can interact with CT
DNA via a same mode (intercalation), and the complexes
bind to DNA more strongly than the free ligand alone.
EB (a typical indicator of intercalation) quenching assay
was performed to further test the binding modes of DNA to
these investigated compounds [43]. Figure 3 shows the
electronic absorption spectra when EB was employed. The
absorbance of Tris–HCl buffer solution is highest when
there is only EB alone. After adding CT DNA into the
buffer solution, the absorbance decreases. However, it
increases when the ligand and metal complexes were added
dropwise into the buffer solution. In addition, the maximal
absorption of EB at 479 nm decreases and shifts to 494 nm
in the presence of CT DNA, and that is characteristic of
intercalation mode. Curve (B) in Fig. 3 shows the
absorption of a mixture solution of EB, DNA and the
prepared compounds. It is also found that the absorption at
494 nm has increased comparing with curve (C). These
may result from two reasons: (1) EB bound to the free
ligand and metal complexes strongly, resulting in the
decrease of the amount of EB intercalated into DNA; (2)
there exists competitive intercalation between the free
ligand and metal complexes and EB with DNA, so
releasing some free EB from DNA–EB system. However,
the former reason could be precluded because there were no
new absorption peaks appearing. Thus, we can come to a
conclusion that the prepared compounds can displace EB
from its DNA-EB system. Quite similar results are also
observed for other Ln(III) complexes.
The binding abilities of the ligand and Ln(III) complexes
with CT DNA can be characterized by measuring their
effects on the UV–vis spectroscopy. Intercalative mode of
binding usually results in hypochromism and red shift
because of the strong stacking interaction between an
aromatic chromophore and DNA base pairs. The extent of
the spectral change is related to the strength of binding [32].
The electronic absorption spectra of the free ligand and its
complexes in the absence and presence of CT DNA are
given in Fig. 2. As shown, in the presence of DNA, the
absorption bands of the ligand, La(III) and Yb(III)
complexes at 350. 327 and 328 nm exhibited hypochrom-
ism of 43.89%, 53.95% and 45.80%, respectively. The
hypochromism observed for the lanthanide complexes are
all accompanied by a red shift of about 7 nm. The binding
of complexes to DNA led to decrease in the absorption
intensities with a small amount of red shifts in the UV–vis
absorption spectra. After intercalating the base pairs of
100
L
La(III) Complex
Yb(III) Complex
Mannitol
80
60
40
20
0
1
2
3
4
5
Fluorescence Spectra
Concentration (μM)
Figure 4 exemplifies the steady-state emission spectra of
10 μM solutions of lanthanide complexes in Tris–HCl
Fig. 9 Scavenging effects of the ligand, lanthanide complexes and
mannitol on HO•

1100
J Fluoresc (2011) 21:1091–1102
Table 2 The influence of investigated compounds for HO•
Compound
Average inhibition (%) for HO· (μM)
Equation
IC50 (μM)
R2
1.0
2.0
3.0
4.0
5.0
y ¼ 62:43x þ 24:04
y ¼ 53:97x þ 53:52
y ¼ 64:01x þ 45:70
y ¼ 57:26x ꢀ 0:73
L
26.60
55.32
46.81
2.30
41.49
69.15
63.83
12.52
50.00
75.53
71.28
24.63
57.45
84.04
84.04
32.91
74.47
94.74
93.62
43.09
2.605
0.861
0.932
0.955
0.971
0.958
La(III) Complex
Yb(III) Complex
Mannitol
1.167
10.190
buffer and the spectra show an increase in the emission
intensity with successive addition of CT DNA at room
temperature. According to the Scatchard equation, a plot of
r/Cf versus r gave Kb values of (7.80 1.06)×105, (2.92
0.40)×106, (1.91 0.23)×106 M−1 from the fluorescence
data for the free ligand, La(III) and Yb(III) complexes,
respectively. Results obtained from fluorescence spectra
suggest that all the compounds are protected from solvent
water molecules by the hydrophobic environment inside the
DNA helix and that the metal complexes can be protected
more efficiently than the free ligand alone. Since the
hydrophobic environment inside the DNA helix reduces
the accessibility of solvent water molecules to the com-
pound and the compound mobility is restricted at the
certain binding site, a decrease of the vibrational modes of
relaxation results. The binding of the compounds to DNA
leading to a significantly increase in emission intensity also
agrees with those observed for other intercalators [44]. The
fluorescence spectra show that the lanthanide complexes
can bind to DNA more strongly and deeply than the free
ligand alone, The higher binding affinities of the complexes
are probably attributed to the extension of the π system of
the intercalated ligand due to the coordination of metal
ions, which also leads to a planar area greater than that of
the free ligand and the coordinated ligand penetrating more
deeply into and stacking more strongly with the base pairs
of DNA.
The DNA binding mode of all the compounds was
further monitored by a fluorescent EB displacement assay.
EB is one of the most sensitive fluorescence probes that can
bind to DNA and the fluorescence of EB increases after
intercalating into DNA base pairs [45]. Competitive
binding of other drugs to DNA and EB system will result
in the decrease in the fluorescence intensity of the DNA-EB
system. The reduction of the fluorescence emission inten-
sity gives a criterion to investigate the DNA binding
propensity of the complexes and the stacking interaction
(intercalation) between the adjacent DNA base pairs [46].
Figure 5 shows the emission spectra of the DNA–EB
system with increasing amounts of the Ln(III) complexes
and free ligand. The addition of the compounds to the
DNA-bound EB solutions caused obvious reduction in
emission intensities and the quenching of EB bound to
DNA by the rare earth complexes is in good agreement
with the linear Stern-Volmer equation. In the plots of F0/F
versus [Q], Kq values are given by the ratio of the slope to
the intercept and Kq values for the ligand, La(III) and Yb
(III) complexes are (1.75 0.03)×104, (5.54 0.12)×104
and (2.26 0.05)×104 M−1. The results show that all the
compounds can bind to DNA and the complexes bind to
DNA more strongly than the free ligand, which also
validate the electronic absorption spectral results. Since
these changes indicate only one kind of quenching process,
it may be concluded that the complexes and free ligand can
bind to DNA via the same mode (intercalation).
Viscosity Measurements
Viscosity titration measurements were carried out to clarify
the interaction modes between the investigated compounds
and CT DNA. Viscosity measurements are very sensitive to
changes in the length of DNA, as viscosity is proportional
to L3 for rod-like DNA of length L, and then viscosity
measurements are regarded as the least ambiguous and the
most critical tests of a binding model in solution in the
absence of crystallographic structural data [47]. A classical
intercalation model demands that the DNA helix must
lengthen as base pairs are separated to accommodate the
binding complexes, leading to the increase of DNA
viscosity, as for the behaviors of the known DNA
intercalators [48]. In contrast, a partial and/or non-
classical intercalation of the complex could bend (or kink)
the DNA helix, reducing its viscosity concomitantly [47].
In addition, some compounds such as [Ru(bpy)3]2+, which
interacts with DNA by an electrostatic binding mode, have
no influence on DNA viscosity [49]. To further elucidate
the binding mode of the present complexes, viscosity
measurements were carried out by keeping [DNA]=5 μM
and varying the concentration of the compounds.
The changes in relative viscosity of CT DNA in the
presence of the ligand and complexes are shown in Fig. 6.
As seen from Fig. 6, upon increasing amounts of the ligand
and complexes, the relative viscosities of DNA increase
steadily with increasing concentrations of ligand and

J Fluoresc (2011) 21:1091–1102
1101
complexes. The increased degree of viscosity, which may
depend on the binding affinity of compounds to DNA,
following the order of La(III) complex > Yb(III) complex >
L. These results suggest that all the title complexes and
ligand intercalate between the DNA base pairs and the
binding affinities of the complexes are higher than that of
the free ligand, which is consistent with the above
experimental results.
On the basis of all the spectroscopic studies together
with the viscosity measurements, we find that the lantha-
nide complexes and free ligand can bind to CT DNA via an
intercalative binding mode (Fig. 7) and the complexes bind
to CT DNA more strongly and deeply than the free ligand
alone.
enhanced and the rare earth metal ions, La(III) and Yb
(III) also affect the scavenger ability. From the above
depictions, we can easily find that Ln(III) complexes
exhibit considerable scavenging activity due to the chela-
tion of organic molecules to the lanthanide ions which exert
different and selective effects on scavenging radicals of the
biological system. We also find that complexes are better
inhibitor than mannitol which is usually used as special
scavenger for HO•. It is reported that IC50 value of ascorbic
acid (Vc, a standard agent for non-enzymatic reaction) for
HO• is 1.537 mg mL−1 (8.727 mmol), and the scavenging
effect for O2 is only 25% at 1.75 mg mL−1 (9.94 mmol)
ꢄꢀ
[51]. Notably, the investigated complexes have much
stronger scavenging abilities for HO• and O2 radicals. It
ꢄꢀ
was believed that the information obtained form present
work would be useful to develop new potential antioxidants
and therapeutic agents for some diseases.
Antioxidant Activity
Due to the ligand and its Ln(III) complexes exhibit good
DNA binding affinity, it is considered worthwhile to
investigate their antioxidant activity. Reactive oxygen
species (ROS), such as superoxide anion and hydroxyl
radical, are usually generated by all aerobic cells during
normal oxygen metabolism, and cumulative information
obtained has proved that the oxidation induced by ROS is
involved in the pathogenesis of various diseases through
direct effects on DNA directly and by acting as a tumor
promoter [50]. Then in this paper, antioxidant activity of
the prepared ligand and Ln(III) complexes was investigated
by comparing their scavenging effects on superoxide anion
and hydroxyl radical.
Conclusion
In summary, a new ligand, chromone-3-carbaldehyde-
aminophenazone Schiff base, and its Ln(III) complexes
have been prepared and characterized. Their DNA binding
properties and antioxidant activities were studied systemat-
ically. Experimental results indicate that the ligand and its
complexes can bind to DNA via an intercalation binding
mode and the complexes have better DNA binding affinity
than the free ligand alone. Furthermore, the complexes have
more active scavenging effects on O2 and HO• than the
ꢄꢀ
Figure 8 and Table 1 depict the inhibitory effect of the
standard antioxidants, such as mannitol and vitamin C. Our
work clearly indicates that rare earth-based complexes have
many potential practical applications, such as understanding
the mechanisms of interaction between compounds and
DNA, the development of potential anticancer drugs and
new antioxidants.
compounds on O2ꢄꢀ. The average suppression ratio for
O2
ꢄꢀ
increases with the increasing amount of the com-
pound concentration in the range of tested concentration.
The average suppression ratio of the ligand (IC50
=
23.214 μM) is the least in all the compounds and La(III)
complex (IC50=0.492 μM) is the more effective inhibitor
than Yb(III) complex (IC50=1.438 μM). It is clear that the
Acknowledgements This work is supported by the National Natural
Science Foundation of China (20975046) and Gansu NSF
(0710RJZA012).
ꢄꢀ
scavenger effect on O2 can be enhanced by the formation
of metal-ligand complexes and the metal ions also affect the
ability. The metal ions may have different and selective
nature for scavenging O2ꢄꢀ. Due to the lower IC50 values,
they are potential drugs to eliminate the radicals.
References
The data of suppression ratio for HO• are shown in
Fig. 9 and Table 2. The inhibitory effect of all the
compounds is marked and the average suppression ratio
for HO• increases with the increasing compounds concen-
tration. The average suppression ratio of the ligand is the
poorest in all the compounds, La(III) complex (IC50=
0.861 μM) and Yb(III) complex (IC50=1.167 μM) are the
more effective inhibitors than the ligand (IC50=2.605 μM),
which illuminates that because of the formation of
coordination compounds, the scavenger effect can be
1. Blasius R, Moucheron C, Mesmasker AK (2004) Eur J Inorg
Chem 20:3971
2. Zhang GW, Guo JB, Pan JH, Chen XX, Wang JJ (2009) J Mol
Struct 923:114
3. Riahi S, Ganjali MR, Bagheri M (2009) Mater Sci Eng, C
29:1808
4. Erkkila KE, Odem DT, Barton JK (1999) Chem Rev 99:2777
5. Metcalfe C, Thomas JA (2003) Chem Soc Rev 32:215
6. Xiong Y, Ji LN (1999) Coord Chem Rev 185:711
7. Li Y, Yang ZY, Wang MF (2009) Eur J Med Chem 44:4585
8. Li Y, Yang ZY (2010) J Fluoresc 20:329

1102
9. Wang J, Yang ZY, Yi XY, Wang BD (2009) J Photochem
J Fluoresc (2011) 21:1091–1102
33. Eftink MR, Ghiron CA (1981) Anal Biochem 114:199
Photobiol, A Chem 201:183
34. Eriksson M, Leijon M, Hiort C, Norden B, Graeslund A (1994)
Biochem 33:5031
35. Xiong Y, He XF, Zou XH, Wu JZ, Chen XM, Ji LN, Li RH,
Zhou JY, Yu KB (1999) J Chem Soc, Dalton Trans: Inorg
Chem 1:19
10. Long EC (1993) J Inorg Organomet Polym 3:3
11. Mahadvan S, Palaniandavar M (1997) Inorg Chim Acta 254:291
12. Xu H, Zheng KC, Deng H, Lin LJ, Zhang QL, Ji LN (2003) New
J Chem 27:1255
13. Asadi M, Safaei E, Ranjbar B, Hasani L (2004) New J Chem 28:1227
14. Bisi CC, Carugo O (1989) Inorg Chim Acta 159:157
15. Ci YX, Li YZ, Chang WB (1991) Anal Chim Acta 248:589
16. Horne DA, Dervan PB (1990) J Am Chem Soc 112:2435
17. Moser HE, Dervan PB (1987) Science 238:645
18. Yan MX, Li YQ, Meng M, Ren HB, Kou Y (2006) Biochem
Biophys Res Commun 347:192
36. Winterbourn CC (1979) Biochem J 182:625
37. Winterbourn CC (1981) Biochem J 198:125
38. Geary WJ (1971) Coord Chem Rev 7:81
39. Marchetti F, Pettinari C, Pettinari R, Cingolani A, Leonesi D,
Lorenzotti A (1999) Polyhedron 18:3041
40. Narang KK, Singh VP (1993) Transit Met Chem 18:287
41. Wang BD, Yang ZY, Zhang DW, Wang Y (2006) Spectrochim
Acta Part A 63:213
19. Cai L (2006) Free Radic Biol Méd 41:851
20. Alexandrova ML, Bochev G (2005) Free Radic Biol Méd 39:297
21. Li Y, Yang ZY, Wang MF (2010) J Fluoresc 20:891
22. Li Y, Yang ZY (2009) Inorg Chim Acta 362:4823
23. Havsteen BH (2002) Pharmacol Ther 96:67
42. Bai GY, Dong B, Lü YY, Wang KZ, Jin LP, Gao LH (2004) J
Inorg Biochem 98:2011
43. Wilson WD, Ratmeyer L, Zhao M, Strekowski L, Boykin D
(1993) Biochem 32:4098
24. Huang W, Liu MZ, Li Y, Tan Y, Yang GF (2007) Bioorg Med
Chem 15:5191
44. Xu H, Zheng KC, Lin LJ, Li H, Gao Y, Ji LN (2004) J Inorg
Biochem 98:87
25. Sosnovskikh VY (2003) Russ Chem Rev 72:489
26. Li Y, Yang ZY, Wu JC (2010) Eur J Med Chem 45:5692
27. Walenzyk T, Carola C, Buchholz H, König B (2005) Tetrahedron
61:7366
28. Cai YZ, Luo Q, Sun M, Corke H (2004) Life Sci 74:2157
29. Heim KE, Tagliaferro AR, Bobilya DJ (2002) J Nutr Biochem 13:572
30. Nohara A, Umetani T, Sanno Y (1973) Tetrahedron Lett 14:1995
31. Howe GM, Wu KC, Bauer WR, Lippard SJ (1976) Biochem
15:4339
45. Le-Pecq JB, Paoletti C (1967) J Mol Biol 27:87
46. Lee M, Rhodes AL, Wyatt MD, Forrow S, Hartley JA (1993)
Biochem 32:4237
47. Satyanarayana S, Dabrowiak JC, Chaires JB (1993) Biochem
32:2573
48. Liu JG, Zhang QL, Shi XF, Ji LN (2001) Inorg Chem 40:5045
49. Liu JG, Ye BH, Li H, Zhen QX, Ji LN, Fu YH (1999) J Inorg
Biochem 76:265
50. Towyz RM (2004) Hypertension 44:248
51. Xing RG, Yu HH, Liu S, Zhang WW, Zhang QB, Li ZE, Li PC
(2005) Bioorg Med Chem 13:1387
32. Pyle AM, Rehmann JP, Meshoyrer R, Kumar CV, Turro NJ,
Barton JK (1989) J Am Chem Soc 111:3051