Lanthanide complexes of 1-phenyl-3-methyl-5-hydroxypyrazole-4-carbaldehyde- (4′-hydroxybenzoyl) hydrazone: Crystal structure and interaction studies with biomacromolecules

Article abstract of DOI:10.1080/15533174.2012.740753

1-phenyl-3-methyl-5-hydroxypyrazole-4-carbaldehyde-(4′-hyd roxybenzoyl) hydrazone and Ln(III) complexes have been synthesized and characterized. The interaction between these compounds and biomacromolecules was investigated by fluorescence and UV/vis abso

Full text of DOI:10.1080/15533174.2012.740753

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Lanthanide Complexes of 1-phenyl-3-methyl-5-
hydroxypyrazole-4-carbaldehyde-(4′-hydroxybenzoyl)
Hydrazone: Crystal Structure and Interaction Studies
With Biomacromolecules
Ming-Fang Wang a , Zheng-Yin Yang a , Zeng-Chen Liu a , Yong Li a & Hong-Ge Li a
a State Key Laboratory of Applied Organic Chemistry, College of Chemistry and Chemical
Engineering, Lanzhou University, Lanzhou, P. R. China
Accepted author version posted online: 10 Dec 2012.
To cite this article: Ming-Fang Wang , Zheng-Yin Yang , Zeng-Chen Liu , Yong Li & Hong-Ge Li (2013) Lanthanide Complexes
of 1-phenyl-3-methyl-5-hydroxypyrazole-4-carbaldehyde-(4′-hydroxybenzoyl) Hydrazone: Crystal Structure and Interaction
Studies With Biomacromolecules, Synthesis and Reactivity in Inorganic, Metal-Organic, and Nano-Metal Chemistry, 43:4,
483-494, DOI: 10.1080/15533174.2012.740753
To link to this article: http://dx.doi.org/10.1080/15533174.2012.740753
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Synthesis and Reactivity in Inorganic, Metal-Organic, and Nano-Metal Chemistry, 43:483–494, 2013
Copyright ꢀ Taylor & Francis Group, LLC
C
ISSN: 1553-3174 print / 1553-3182 online
DOI: 10.1080/15533174.2012.740753
Lanthanide Complexes of 1-phenyl-3-methyl-5-
hydroxypyrazole-4-carbaldehyde-(4ꢀ-hydroxybenzoyl)
Hydrazone: Crystal Structure and Interaction Studies With
Biomacromolecules
Ming-Fang Wang, Zheng-Yin Yang, Zeng-Chen Liu, Yong Li, and Hong-Ge Li
State Key Laboratory of Applied Organic Chemistry, College of Chemistry and Chemical Engineering,
Lanzhou University, Lanzhou, P. R. China
for many endogenous and exogenous compounds.[5,6] Bovine
1-phenyl-3-methyl-5-hydroxypyrazole-4-carbaldehyde-(4ꢀ-hyd
roxybenzoyl) hydrazone and Ln(III) complexes have been synthe-
sized and characterized. The interaction between these compounds
and biomacromolecules was investigated by fluorescence and
UV/vis absorption spectroscopy. The results suggested that the
compounds caused fluorescence quenching of BSA through a
static quenching procedure. Hydrophobic interaction force also
played a major role in stabilizing the compounds. Moreover,
interactions between calf thymus DNA and compound H3L and
its Ln(III) complexes were studied by spectroscopy and viscosity
measurements. These studies showed that these compounds bind
to DNA via an intercalation mode. Furthermore, antioxidant
activities of compounds were determined by hydroxyl radical
scavenging methods in detail.
serum albumin (BSA) has been studied extensively due to its
medical importance, stability, low cost, unusual ligand-binding
properties, and special structural homology with human serum
albumin (HSA).[7] BSA has two tryptophans, Trp-134 and Trp-
212, embedded in the first subdomain IA and subdomain IIA,
respectively. The primary binding site for most drugs on albu-
min is suggested to be close to the tryptophan residues 212 of
the BSA, locating in subdomain IIA.[8] Deoxyribonucleic acid
(DNA), as another kind of biomacromolecule, is an important
genetic substance in organism. Moreover, DNA is the intracellu-
lar target for a wide range of anticancer and antibiotic drugs. So
the binding studies of small molecules with DNA could provide
useful information for finding sensitive DNA molecular probes
and have important significance in developing potential drugs
for clinical applications.[9–12]
Supplemental materials are available for this article. Go to the
publisher’s online edition of Synthesis and Reactivity in Inorganic,
Metal-Organic, and Nano-Metal Chemistry to view the supplemen-
tal file.
The previously mentioned studies inspire considerable inter-
ests in the field of the biochemical behavior of the interaction be-
tween small molecules and BSA/DNA. Moreover, fluorescence
spectroscopy is essentially a probe technique sensing changes in
the local environment of fluorophore. Any possibilities of struc-
tural rearrangements may lead to a fluorescence signal. Then by
analyzing the fluorescence parameters, much information about
the structural changes can be obtained.[13,14] Fluorescence spec-
troscopy is an appropriate method to determine the interaction
between the small molecules and biomacromolecules, so does
the UV/vis spectroscopy.
It is well known that reactive oxygen species (ROS) are
generated by normal cellular metabolism and exogenous agents.
Under pathological conditions, ROS are overproduced and result
in oxidative stress in living organism. ROS are generated when
endogenous antioxidant defenses are inadequate.[15] Among all
the ROS, hydroxyl radical (HO•) is a highly reactive product.
It is involved in the pathogenesis of various diseases through
direct effects on DNA and by acting as a tumor promoter.[16]
Therefore elimination of this radical is one of the major aims of
antioxidant administration.
Keywords antioxidant activity, bovine serum albumin, calf thymus
DNA, crystal Schiff base, spectroscopy, structure
INTRODUCTION
In the past years, the interaction of small molecules with ma-
jor biopolymers such as proteins and nucleic acid has caused
much interest in the field of bioinorganic chemistry.[1–3] Among
various biomacromolecules, serum albumins (SA) are the most
abundant soluble proteins in the circulatory system of a wide
variety of organisms.[4] Serum albumins possess many indis-
pensable physiological functions, such as regulation of colloid
osmotic pressure. And the most important property is that serum
albumins served as a depot protein and a transport protein
Received 29 March 2012; accepted 13 October 2012.
This work is supported by the National Natural Science Foundation
of China (20975046) and (81171337).
Address correspondence to Zheng-Yin Yang, State Key Laboratory
of Applied Organic Chemistry, College of Chemistry and Chemical En-
gineering, Lanzhou University, Lanzhou 730000, P. R. China. E-mail:
yangzy@lzu.edu.cn
Schiff bases are a vital class of ligands in coordination
chemistry and are found to possess extraordinary bioactivities.
483

484
M.-F. WANG ET AL.
of 6600 M–1 cm–1 at 260 nm.[22] The double-distilled water was
used throughout the experiments.
In addition, the bioactivities of lanthanide complexes such as
antimicrobial, antitumor, antivirus, and antioxidant activity have
been explored in recent decades.[17,18] Based on the previous
considerations, lanthanide complexes with Schiff bases such
as 1-Phenyl-3-methyl-4-benzoyl-5-pyrazolone (PMBP) and
1-Phenyl-3-methyl-4-formyl-5-pyrazolone (PMFP) have been a
major concern in our group.[19,20] In this article, three lanthanide
complexes with a new Schiff base, 1-phenyl-3-methyl-5-
hydroxypyrazole-4-carbaldehyde-(4ꢁ-hydroxy-benzoyl)hydra-
zone, are synthesized. Their BSA and DNA interaction
modes are investigated by fluorescence and UV/vis absorption
spectra. Antioxidant properties of the ligand and its lanthanide
complexes are also studied.
Physical Measurement
Carbon, hydrogen, and nitrogen were performed using an
Elemental Vario EL analyzer (Germany). The metal contents of
the complexes were determined by titration with EDTA (xylenol
orange tetrasodium salt used as an indicator and hexamethyli-
dynetetraimine as buffer). The melting point of the ligand was
obtained on a Beijing XT4–100X microscopic melting point
apparatus (China). The IR spectra were recorded in KBr discs
on a Thermo Mattson FTIR spectrophotometer (USA) in the
1
4,000–400 cm–1 region. H NMR spectra were recorded on a
VarianVR300MHz spectrometer (USA) inDMSO-d6 (dimethyl
sulfoxide) with TMS (tetramethylsilane) as an internal standard.
Conductivity measurements were determined in DMF (N,N-
dimethyl formamide) solution with a DDS-11C conductome-
ter (Shanghai Analytical Instrument Factory, China) at 25.0◦C.
The UV/Vis spectra were taken on a Perkin–Elmer Lambda 35
UV/vis spectrophotometer (USA). Fluorescence emission spec-
tra were obtained with a RF-5301PC spectrofluorophotome-
ter (Shimadzu, Japan) equipped with a xenon lamp source and
1.0 cm quartz cells. An electronic thermo regulating water bath
(NTT-2100, EYELA, Japan) was used to control the tempera-
ture of the samples. Viscosity experiments were conducted on
an Ubbelodhe viscometer (China), immersed in a thermostated
water-bath maintained at 25 0.1◦C. The antioxidant activi-
ties were performed in DMF with a 721-E spectrophotometer
(Shanghai Analytical Instrument Factory, China).
EXPERIMENTAL
Materials
All chemicals used were of analytical grade. Bovine serum
albumin (BSA, Fraction V, essentially fatty acid free, approx-
imately 99%) was purchased from the Kehao Biotechnology
Company, China. BSA stock solution (3 × 10–6 M, based on
its molecular weight of 68,000) was prepared in the Tris–HCl
buffer solution and kept in the dark at 4◦C. Tris–HCl buffer
solution for BSA consists of Tris (0.2 M) and HCl (0.1 M),
and the pH was adjusted to 7.4 by adding NaOH. NaCl (1.0 M)
solution was used to maintain the ion strength at 0.1 M. Calf thy-
mus (DNA) was purchased from Sigma Chemical Co. (USA).
Solution of DNA in 5 mM Tris and 50 mM NaCl (pH = 7.2)
gave ratios of UV absorbance of about 1.8–1.9:1 at 260 and
280 nm, indicating that the DNA was sufficiently free of pro-
tein.[21] The DNA concentration per nucleotide was determined
Preparation of the Ligand (H3L)
As shown in Figure 1, 1-phenyl-3-methyl-4-formyl-2-
spectrophotometrically by employing an extinction coefficient pyrazolin-5-one (PMFP) was prepared according to the
FIG. 1. The synthetic routes for ligand.

INTERACTION STUDIES WITH BIOMACROMOLECULES
485
literature.[23,24] Synthesis of the ligand (H3L) was in accordance
with the following method: the ethanol solution (10 mL) con-
tained 4-Hydroxybenzhydrazide (1.67 g, 11 mmol) was added
dropwise to the PMFP (2.02 g, 10 mmol) dissolved in ethanol
(20 mL) with stirring at 60◦C for 3 h, then a large amount
of yellow precipitate appeared. The yellow solid was filtered
off and recrystallized from ethanol, then dried in a vacuum.
Yield: 90% m.p. 281–282◦C. IR νmax (cm–1): ν(C(13) O):1656,
ν (C N): 1594, ν(C(5) O): 1533, ν(N–H): 3069 cm–1. 1H NMR
(300 MHz, DMSO-d6, ppm): 10.31 (s, 1H, H16), 8.06 (s, 1H,
H7), 7.95 (dd, 2H, H14, H18, J = 5.4Hz, 16.8Hz), 7.82 (dd, 2H,
H15, H17, J = 5.4Hz, 16.8Hz), 7.78 (d, 1H, H10, J = 8.7Hz), 7.41
(m, 2H, H9, H11), 7.26 (s, 1H, H6), 6.90 (dd, 2H, H8, H12, J =
13.2Hz, 8.7Hz), 2.18 (s, 3H, CH3-pyrazole). The label numbers
of H-atoms were shown in Figure 1.
Absorption correction was employed using semiempirical meth-
ods from equivalents.
Experimental Procedure
Binding mode between all compounds and BSA
Fluorometric titration experiments: 3.0 mL solution contain-
ing appropriate concentration of BSA was titrated manually by
successiveadditionsofstocksolutionofallcompounds(togivea
final concentration of 1.67 × 10–6 to 16.70 × 10–6 M) with trace
syringes. Fluorescence experiments were performed at three
different temperatures (292, 301, and 310 K). The fluorescence
spectra from 290 to 500 nm of the above solution were collected
with the excitation wavelength at 280 nm. The fluorescence is
attributed to a tryptophan residue.[26] Fluorescence quenching
was calculated using the well-known Scatchard’s equation[27,28]
:
Preparation of the Rare Earth Complexes
r/Df = nK − rK [1]
The ligand H3L (0.35 mmol, 0.117 g) and the Eu(III) nitrate
(0.2 mmol, 0.089 g) were added together in methanol (20 mL).
The solution was refluxed on a water bath for 12 h with stirring
and there was a yellow precipitate in the solution. The precipitate
was separated from the solution by suction filtration, purified
by washing several times with water, and dried in a vacuum.
The Tb(III) and Dy(III) complexes were prepared by the same
method. Anal. Calcd. for Eu(III) complex C37H34N9O10Eu: C,
48.48; H, 3.74; N, 13.75; Eu, 16.58. Found: C, 48.52; H, 3.66;
Where r (r = ꢁF / F0) represents the number of moles of bound
small molecules per mole of protein, Df represents the molar
concentration of free drug, and n and K are the number of bind-
ing sites and binding constant, respectively. UV/vis absorption
spectra were recorded by the same titration experiments at a
constant room temperature.
DNA binding procedures
N, 13.79; Eu, 16.70. IR νmax (cm–1): ν(13)C O: 1612, νC
:
N
Electronic absorption titration experiments are performed
with fixed concentration of drugs (1.0 × 10–6 M), with grad-
ually increasing the concentration of DNA. When measuring
the absorption spectra, an equal amount of DNA is added to
both the sample and the reference solutions to eliminate the ab-
sorbance of DNA itself. To compare quantitatively the affinity
of these compounds binding to DNA, the intrinsic binding con-
stant (Kb) is estimated using the following equation through a
1571,ν(5)C O: 1456, νNO : 1522, 1358, 1174, 818, νM-O: 621,
3
νM-N: 424 cm–1. Umax (nm): 351 nm. ꢀm (S cm2 mol–1): 36.6.
Anal. Calcd. for Tb(III) complex C36H30N9O9Tb: C, 48.50; H,
3.39; N, 14.14; Tb, 17.82. Found: C, 48.52; H, 3.36; N, 14.28;
Tb, 17.84. IR νmax (cm–1): ν(13)C O: 1613, νC N: 1568,ν(5)C
:
O
1455, νNO : 1520, 1357, 1174, 819, νM-O: 621, νM-N: 427 cm–1.
3
Umax (nm): 351 nm. ꢀm (S cm2 mol–1): 40.9. Anal. Calcd for
Dy(III) complex C36H32N9O10Dy: C, 47.35; H, 3.53; N, 13.80;
Dy, 17.79. Found: C, 47.52; H, 3.41; N, 13.85; Dy, 17.80. IR
plot of [DNA] / (εa−εf) vs. [DNA][29]
:
νmax (cm–1): ν(13)C O: 1613, νC N: 1569,ν(5)C O: 1457, νNO
:
3
1521, 1357, 1145, 818, νM-O: 621, νM-N: 419 cm–1. Umax (nm):
353 nm. ꢀm (S cm2 mol–1): 33.7.
[DNA]/(εa − εf) = [DNA]/(εb − εf) + 1/[Kb(εb−εf)] [2]
Where [DNA] is the concentration of DNA in base pairs,
εa, εf, and εb are the apparent extinction coefficient (Aobsd
/
X-Ray Crystal Structure Determination
[compound]), the extinction coefficient for free compound, and
the extinction coefficient for the compound in the fully bound
form, respectively. In plots of [DNA] / (εa−εf) versus [DNA],
Kb is given by the ratio of the slope to the intercept.
Further support for the ligand and complexes binding to DNA
via intercalation is given through the emission quenching exper-
iment. A solution (2.0 mL) of 5 μM DNA and 0.4 μM EB (at
saturating binding levels) was titrated by 2.5–35 μM the lan-
thanide complexes and ligand. The fluorescence spectra from
540 to 700 nm of the solution were collected with the exci-
tation wavelength at 525 nm. Quenching data were analyzed
according to the Stern−Volmer equation, which could be used
A yellow crystal of Eu(III) complex was obtained by slow
vapor diffusion of methanol/ether at room temperature. X-ray
diffraction data for the crystal were performed with graphite-
monochromated Mo-K radiation (0.71073 Å) on a Bruker APEX
area-detector diffractometer (Germany) and collected by the
ω/2θ scan technique at 298(2) K. The crystal structure was
solved by direct methods. The positions of non-hydrogen atoms
were determined from successive Fourier syntheses. The hydro-
gen atoms were placed in their geometrically calculated posi-
tions. The positions and anisotropic thermal parameters of all
non-hydrogen atoms were refined on F2 by full-matrix least-
squares techniques with the SHELX-97 program package.[25]

486
M.-F. WANG ET AL.
to determine the fluorescent quenching mechanism:
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 nitrate
F0/F = 1 + Kq[Q]
[3]
groups. ν(hydrazonic)C
and ν(carbonyl)C
vibrations of the
O
O
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.[30]
free ligand appear at 1651 and 1537 cm–1, respectively;
whereas for the complexes these peaks shift to about 1613,
1456 cm–1, ꢁν(ligand-complexes) (hydrazonic)C is equal to 38 cm–1,
O
ꢁν(ligand-complexes) (carbonyl)C is equal to 81 cm–1, respectively.
O
The big gap of ꢁν(ligand-complexes) (carbonyl)C
suggests that the
O
Viscosity experiments were conducted on an Ubbelodhe vis-
cometer, immersed in a thermostated water bath maintained to
oxygen atoms of pyrazole ring may take part in coordination
by the enol form. The band at 1594 cm–1 for the free ligand is
25.0
0.1◦C. Titrations were performed for the ligand and
assigned to νC stretch, which shifts to about 1569 cm–1 for
N
complexes (0.5–4 μM), and each compound was introduced
into a DNA solution (5 μM) present in the viscometer. Data
were analyzed 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.[31,32] Viscosities were calculated from the observed flow
time of solutions containing DNA corrected for the flow time of
buffer alone (t0), η = t – t0.
the complexes, and ꢁν(ligand-complexes) is equal to 25 cm–1. In the
complexes, the band at 621 cm–1 or so are assigned to νM–O
,
demonstrating that the oxygen atoms of carbonyl have formed
a coordinative bond with the rare earth ions. Weak bands at
424 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.[34]
The absorption bands of the coordinated nitrates are observed
at about 1521 (νas) and 818 (νs) cm–1. The ν3 (Eꢁ) free nitrates
appear at 1384 cm–1 in the spectra of the rare earth complexes.
In addition, the separation of the two highest frequency bands
1521 cm–1 and 1358 cm–1 resulting in |ν4 – ν1| is approximately
163 cm–1, and accordingly the coordinated nitrate group in the
complex is a bidentate ligand. In order to make sure the precise
structures of the complexes, the crystal structure of the Eu(III)
complex is analyzed as follows.
Antioxidant activity determination
In antioxidant activity experiments hydroxyl radical (HO•) in
aqueous media was generated through the Fenton reaction. So-
lution of the test compounds was prepared with DMF. The reac-
tion mixture contained 2.0 mL 0.15 M phosphate buffer (pH =
7.4), 1.0 mL 114 μM safranin, 1 mL 945 μM EDTA–Fe(II),
1 mL 3% H2O2, and 30 μL the test compound solution (the
final concentration: Ci (i = 1–6) = 1.0, 2.0, 3.0, 4.0, 5.0, 6.0 μM).
The sample without the test 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 scavenging ratio is defined as
The Eu(III) complex crystallized in the triclinic lattice with
a space group P-1. Each unit cell contained two molecules (Z =
2). The X-ray diffraction data for [Eu(H2L)2(NO3)(CH3OH)]
are given in Table 1. Selected bond lengths and angles are
summarized in Table 2. The crystal structure in Figure 2
shows that the composition of the Eu(III) complex is
[Eu(H2L)2(NO3)(CH3OH)]. Eu ion is nine coordinated with two
ligands, one methanol molecule, and one nitrate. Each ligand
act as a tridentate ligand, binding to Eu atom through the nitro-
gen atom from –C N– group of Schiff base, oxygen atoms from
1-phenyl-3-methyl-5-hydroxypyrazole-4-carbaldehydeunit and
O C–NH– of the 4-Hydroxybenzhydrazide side chain. The dis-
tances for C38–O51, C43–O4, and C29–O8, C201–O3 are 1.284
Å, 1.266 Å, 1.270 Å, and 1.263 Å, respectively, which are be-
tween the C–O (1.41–1.44 Å) and C O (1.19–1.23 Å) dis-
tances,[35] suggesting that the oxygen atoms of pyrazole ring
take part in coordination by the enol form. Besides the dis-
tances for C38–C31 and C43–C55 are 1.424 Å and 1.393 Å,
respectively, which are between the C–C (1.47–1.53 Å) and
C C (1.32–1.38 Å) distances, demonstrating the previous con-
clusion. So the ligand (H3L) displays in the enol form (I), in
the action it losses one proton and acts as a tridentate chelat-
ing agent H2L–1 coordinated to the ion in the solid state. The
physical analytical results indicate that the crystal structure has
a 1:2 metal to ligand stoichiometry, the reason for this might
be attributed to the fact that the bonding effect of nitrate anion
Suppression ratio (%) = [(Ai − A0)/(Ac − A0)] × 100% [4]
Where Ai is absorbance in the presence of the test compound;
A0 is absorbance of the blank in the absence of the test com-
pound; Ac is absorbance in the absence of the test compound,
EDTA–Fe(II), and H2O2.
The antioxidant activity was expressed as the 50% inhibitory
concentration (IC50). IC50 values were calculated from regres-
sion lines where x was the test compound concentration in μM
and y was percent inhibition of the test compounds.
RESULTS AND DISCUSSION
Characterization of Ligand and Complexes
The complexes are air-stable for extended periods and sol-
uble in DMSO, DMF, methanol, and ethanol; slightly soluble
in acetonitrile; and insoluble in water, acetone, and ethyl ether.
The molar conductivities of the complexes are around 33.7–40.9
S cm2 mol–1 in DMF solution, showing that the complexes are
nonelectrolytes.[33]

INTERACTION STUDIES WITH BIOMACROMOLECULES
487
TABLE 1
Crystal data and experimental data of Eu(III) complex
TABLE 2
Selected bond lengths (Å) and angles (◦) for Eu(III) complex
Sum formula
C76H72Eu2N18O21
1877.46
2(C37H33EuN9O10)C2H6O
Yellow
Bond name Bond length Bond name
Bond angle
Formula weight
Moiety formula
Crystal color
Crystal size (mm)
Temperature (K)
Wavelength (Å)
Radiation
Crystal system
Space group
Z
Eu1–O51
Eu1–O4
2.284(7)
2.318(8)
2.409(7)
2.425(8)
2.433(7)
2.531(8)
2.576(8)
2.595(9)
2.610(8)
2.630(10)
2.304(7)
2.319(8)
2.415(7)
2.442(7)
2.443(8)
2.550(8)
2.584(8)
2.618(8)
1.424(13)
1.284(12)
1.257(12)
1.266(13)
1.393(15)
1.247(12)
1.480(15)
1.270(11)
1.269(12)
1.263(13)
1.383(15)
1.236(12)
O51–Eu1–O4
O51–Eu1–O117
O4–Eu1–O117
O51–Eu1–O1B
O4–Eu1–O1B
O117–Eu1–O1B
O51–Eu1–O11
O4–Eu1–O11
O117–Eu1–O11
O1B–Eu1–O11
O51–Eu1–O5
O4–Eu1–O5
95.9(3)
76.9(2)
134.9(2)
145.9(2)
82.2(3)
80.6(3)
135.1(2)
75.4(3)
139.2(3)
77.7(3)
77.1(3)
146.8(2)
75.8(2)
121.5(3)
86.7(3)
120.9(3)
142.2(3)
68.3(3)
72.2(3)
72.3(3)
49.5(3)
71.3(3)
72.1(3)
63.3(3)
75.8(3)
140.2(3)
132.6(3)
127.5(3)
Eu1–O117
Eu1–O1B
Eu1–O11
Eu1–O5
0.35 × 0.32 × 0.21
296(2)
0.71073
Mo Kα
Triclinic
P—1
Eu1–O2
Eu1–N10
Eu1–N9
Eu2–N11
Eu2–O8
Eu2–O3
2
a (Å)
b (Å)
14.9536(13)
21.111(2)
22.440(2)
c (Å)
Eu2–O6
O117–Eu1–O5
O1B–Eu1–O5
O11–Eu1–O5
O51–Eu1–O2
O4–Eu1–O2
O117–Eu1–O2
O1B–Eu1–O2
O11–Eu1–O2
O5–Eu1–O2
O51–Eu1–N10
O4–Eu1–N10
O117–Eu1–N10
O1B–Eu1–N10
O11–Eu1–N10
O5–Eu1–N10
N10–Eu1–N9
α (◦)
89.800(2)
Eu2–O10
Eu2–O41
Eu2–O7
β (◦)
89.7420(10)
69.5120(10)
6635.8(10)
0.940
0.523
1896.0
2.08–27.49
–19 ≤ h ≤ 19
–23 ≤ k ≤ 27
–29 ≤ l ≤ 28
30459
γ (◦)
Volume (Å3)
D (Calc) (g/cm3)
μ (mm–1)
Eu2–O23
Eu2–N14
C38–C31
C38–O51
C26–O11
C43–O4
C43–C55
C49–O117
C29–C41
C29–O8
C32–O10
C201–O3
C201–C52
C37–O6
F (000)
θmin/max (◦)
Index ranges
Reflections collected
Independent reflections
Refinement method
Data/restraints/parameters
Goodness-of-fit on F2
Final R indices [I>2σ (I)]
R indices (all data)
12949 (Rint = 0.0635)
Full matrix least-squares on F2
3900/51/1036
1.093
R1 = 0.0864, ωR2 = 0.2702
R1 = 0.1999, ωR2 = 0.3292
FIG. 2. The molecular structure of Eu(III) complex (color figure available online).

488
M.-F. WANG ET AL.
FIG. 3. Fluorescence spectra of BSA in the present of the ligand (a), Eu(III) (b), Tb(III) (c) and Dy(III) (d) complexes at 293K, pH = 7.40, c(BSA) = 3 × 10–6
M, c(compounds): 0, 1.67, 3.33, 5, 6.67, 8.33, 10, 11.67, 13.33, 15, 16.7 × 10–6 M.
with the metal ions are stronger than the ligand. Another reason Hoff equation:
may be that the coordination modes are different and the final
complexes tend to form the most stable compound.
−ꢁH
ꢁS
R
InK =
+
[5]
RT
Where K is the binding constant at absolute temperature T and R
is gas constant. The free energy change (ꢁG) is then estimated
from the following relationship:
Binding Mode Between All Compounds and BSA
The effect of the ligand and its Ln(III) complex on BSA
fluorescence intensity is shown in Figure 3. When BSA is titrated
with different amounts of complexes, a remarkable intrinsic
fluorescence decrease of BSA is observed. Figure 4 displays the
Scatchard plots of the quenching of BSA fluorescence in the
presence of the ligand and Ln(III) complex.
ꢁG = ꢁH − T ꢁS
[6]
On the basis of the binding constants obtained at the three pre-
As reported, there are essentially four types of noncovalent vious temperatures, the thermodynamic parameters are deter-
interactions that could play a role in small molecules binding mined and summarized in Table 3. The binding process is al-
to proteins. These are hydrogen bonds, van der Waals forces, ways spontaneous, as evidenced by the negative sign of ꢁG
electrostatic, and hydrophobic interaction.[36] In order to elu- values.[38]
cidate the interaction between each compound and BSA, the
According to the theory of Ross et al.,[39] for typical hy-
thermodynamic parameters are calculated from the Van ’t Hoff drophobic interactions, both enthalpy change (ꢁH) and entropy
plots.[37] The values of ꢁH and ꢁS are obtained from the slope change (ꢁS) are positive, while the negative values of ꢁH and
and intercept of linear Van ’t Hoff plot, according to the Van ’t ꢁS are associated with hydrogen binding and van der Waals

INTERACTION STUDIES WITH BIOMACROMOLECULES
489
TABLE 3
Binding parameters and thermodynamic parameters of the ligand–BSA and the complexes—BSA
T (K)
K (105M–1)
n
ꢁG◦ (kJ·mol–1)
ꢁS◦ (J·mol–1·K–1)
ꢁH◦ (kJ·mol–1)
–3.845
292
301
310
292
301
310
292
301
310
292
301
310
0.8393
0.8023
0.7654
2.017
1.739
1.165
2.065
1.757
1.502
1.848
1.633
1.441
1.328
1.331
1.322
1.173
1.222
1.234
1.191
1.221
1.285
1.223
1.254
1.275
–23.673
–24.403
–25.133
–20.571
–21.205
–21.839
–19.044
–19.630
–20.217
–16.400
–16.905
–17.410
Ligand
81.061
Eu(III) complex
Tb(III) complex
Dy(III) complex
70.419
56.119
65.182
–9.062
–13.335
–10.407
FIG. 4. The Scatchard plot for the binding of the ligand (a), Eu(III) (b), Tb(III) (c), and Dy(III) (d) complexes (pH = 7.40). The insert shows Van ’t Hoff plot for
the interaction of BSA and the compounds (color figure available online).

490
M.-F. WANG ET AL.
FIG. 5. UV/vis absorbance spectra of BSA in the presence of the ligand (a) and Eu(III) complex (b) at pH = 7.40, a–h, c(BSA) = 3.0 × 10–6 M; c(compounds):
0, 1.67, 3.33, 5, 6.67, 8.33, 10, 11.67, 13.33, 15, 16.7 × 10–6 M. The insert shows the UV/vis absorbance spectra ranging from 250 to 370 nm and arrow shows
the absorbance changes with a blue shift upon increasing compounds concentration.
FIG. 6. Electronic absorption spectra of the ligand (a), Eu(III) (b), Tb(III) (c), and Dy(III) (d) complexes in the absence (top spectrum) and presence of increasing
amounts of DNA (2.5, 5.0, 7.5, 10.0, 12.5, 15.0, 17.5, 20.0 and 22.5 × 10–6 M; subsequent spectra). The arrow shows the absorbance changes upon increasing
DNA concentration.

INTERACTION STUDIES WITH BIOMACROMOLECULES
491
FIG. 7. The emission spectra of DNA-EB system λex = 525 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, and 35.0 μM
ligand (a), Eu(III) (b), Tb(III) (c), and Dy(III) (d) complexes. Arrow shows the emission intensity changes upon increasing ligand and complexes. (c) Plot of F0/F
versus [Q] for the titration of all compounds with DNA-EB.

492
M.-F. WANG ET AL.
interactions; finally, the very low positive or negative ꢁH and
positive ꢁS values are characterized by electrostatic interac-
tions.[40] As shown in Table 3, ꢁH and ꢁS have a small neg-
ative value and a large positive value, respectively. The main
source of ꢁG value is derived from a large contribution of ꢁS
term with little contribution from the ꢁH factor, namely the
binding process is entropy driven. It is more likely hydrophobic
interaction is involved in the binding process.
For reconfirming the binding properties with BSA by the
addition of the complexes, the UV/vis absorbance spectroscopy
is employed. Figure 5 displays that the absorption intensity of
BSA increases regularly with increasing concentration of the
ligand and Eu(III) complex. The absorption band of 210 nm is
characteristic of α-helix structure of BSA and around 280 nm
is mainly due to the presence of tryptophan residues. Further,
the blue shifts in absorption maximum around 280 nm indicate
the changes in polarity around tryptophan residue and changes
in the peptide strand of BSA molecule,[41,42] indicating effect
occurred between the two substances.
FIG. 8. Effects of increasing amounts of the ligand and its lanthanide com-
plexes on the relative viscosity of CT DNA at 25.0 0.1◦C.
quenching constants Kq are used to evaluate the quenching ef-
ficiency discussed by Stern-Volmer’s equation. The Kq values
of the ligand, Eu, Tb, and Dy complexes are 2.646 × 104 M–1,
3.445 × 104 M–1, 3.725 × 104 M–1, and 3.726 × 104 M–1,
respectively. The data show that the interaction of the Ln(III)
complexes with DNA is stronger than that of the free lig-
and, which is consistent with the electronic absorption spectral
results.
The effects of the ligand and its Ln(III) complexes on the
viscosity of DNA are shown in Figure 8. The relative viscosi-
ties of DNA increase steadily with increasing concentrations of
the compounds. The increased degree of viscosity depends on
the binding affinity of the compounds to DNA. Viscosity mea-
surements clearly show that all the compounds can intercalate
between adjacent DNA base pairs, causing an extension in the
helix and thus increase the viscosity of DNA, and the complexes
can intercalate more strongly and deeply than the free ligand.
DNA Binding Properties
Electronic absorption spectroscopy is one of the most univer-
sal techniques in DNA binding studies of compounds. Electronic
absorption spectra of the ligand and its lanthanide complexes in
the absence and presence of DNA (at a constant concentration
of the compounds) are obtained (Figure 6). From the figure, we
could obtain the relation that with increasing DNA concentra-
tions the absorption bands showed hypochromism. In general,
the reason these spectral characteristics are the strong stacking
interaction between an aromatic chromophore and the base pairs
of DNA, suggesting that the compounds may bind to DNA by an
intercalative mode.[43] After intercalating the base pairs of DNA,
the π∗ orbital of the intercalated ligand could couple with π or-
bit of base pairs, thus, decreasing the π–π∗ transition energy,
and further resulting in the bathochromism. On the other hand,
the coupling π∗ orbital was partially filled by electrons, decreas-
ing the transition probabilities, and concomitantly resulting in
the hypochromism.[44] Kb values of the ligand, Eu, Tb, and Dy
complexes are 7.72 × 104 M–1, 1.20 × 105 M–1, 1.22 × 105 M–1,
and 1.04×105 M–1, respectively. These results show that the lig-
and and its Ln(III) complexes can bind to DNA, especially the
Tb(III) complex binds more strongly than the free ligand.
EB (a typical indicator of intercalation) quenching assay is
performed to further confirm the binding mode and compare
their binding affinities. The emission spectra of EB bound to
DNA in the absence and in the presence of the ligand and
Eu(III) complex are given in Figure 7. The emission band at
589 nm of the DNA-EB system decreases in fluorescence inten-
sity upon addition of every compound at diverse r ( = [Com-
pound]/[DNA]) values indicating that the competition of the
compounds with EB in binding to DNA. The observed quench-
ing of DNA-EB fluorescence intensity for the compounds sug-
gests that they displace EB from the DNA-EB system and they
can interact with DNA probably via the intercalative mode. The
FIG. 9. Scavenging effects of the ligand and its lanthanide complexes on HO•.

INTERACTION STUDIES WITH BIOMACROMOLECULES
493
TABLE 4
The influence of investigated compounds for HO•
Average inhibition (%) for HO· (μM)
Compound
1.0
2.0
3.0
4.0
5.0
6.0
Equation
IC50 (μM)
R2
H2L
9.70
16.22
14.19
15.54
23.03
30.41
32.43
35.14
27.27
37.16
43.24
47.30
35.76
47.30
53.38
58.78
46.06
54.05
61.49
62.16
52.73
60.14
66.22
68.24
y = 53.25x + 7.06
y = 55.86x + 14.27
y = 67.63x + 12.95
y = 68.43x + 15.27
6.40
4.36
3.53
3.22
0.973
0.990
0.998
0.998
Dy(III) Complex
Eu(III) Complex
Tb(III) Complex
IC50 values were calculated from regression lines where x was log of the tested compound concentration and y was percent inhibition of the
tested compounds. When the percent inhibition of the tested compounds was 50%, the tested compound concentration was IC50. R2 = correlation
coefficient.
The results obtained from the viscosity validate those obtained indicate that the ligand and the complexes bond to DNA by an
from the spectroscopic studies.
intercalation mode. Additionally, both the ligand and the com-
plexes have some antioxidant properties of scavenging hydroxyl
radicals. The complexes show stronger scavenging effects than
the ligand. The binding study of inorganic compounds with
proteins and nucleic acid is of great importance in pharmacy,
pharmacology and biochemistry. Information obtained from this
work may be helpful to the development of biomacromolecule
probes and new therapeutic reagents for some diseases. Further
investigations on the pharmacodynamics and physiology should
be lucubrated.
Antioxidant Activity Determination
Because the synthesized ligand and its rare earth com-
plexes exhibit good binding affinity with biomacromolecule it is
considered worthwhile to investigate their other biological ac-
tivities, such as antioxidant activity. In this paper, the antioxidant
activity of the ligand and its rare earth complexes is studied by
comparing their scavenging effects on hydroxyl radical (HO•).
The data of the suppression ratio for HO• are listed in
Table 4. IC50 values for the ligand and complexes are 6.40 and
3.22–4.36 μM, respectively (Figure 9). The inhibitory effect
of the compounds is marked and the average suppression ratio
for HO• increases with the increasing compound concentration.
The average suppression ratio of the lanthanide complexes is
higher than the ligand and this is due to the chelation of or-
ganic molecules to lanthanide ions, which can exert differential
and selective effects on scavenging radicals of the biological
system. It is reported that IC50 value of ascorbic acid (Vc, a
standard agent for nonenzymatic reaction) for HO• is 1.537 mg
mL–1 (8.727 mmol),[45] notably, the investigated ligand and lan-
thanide complexes have much stronger scavenging abilities for
HO• radical than the standard antioxidant (vitamin C).
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