Study on interaction of N, N′-(2-hydroxy-3-methoxybenzyl) diamine cerium(IV) with bovine serum albumin

Article abstract of DOI:10.1080/00958972.2013.850496

Complexes of cerium(IV) with bis(N,N′-(2-hydroxy-3-methoxybenzyl) ethane-1,2-diamine, bis(2-hydroxy-3-methoxybenzyl) propane-1,2-diamine and bis(2-hydroxy-3-methoxybenzyl) propane-1,3-diamine were synthesized by reaction of cerium(IV) nitrate and the liga

Full text of DOI:10.1080/00958972.2013.850496

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Study on interaction of N,N′-(2-
hydroxy-3-methoxybenzyl) diamine
cerium(IV) with bovine serum albumin
Hai-Tao Xiaa, Yu-Fen Liua & Wei-Xing Maa
a School of Chemical Engineering, Huaihai Institute of Technology,
Lianyungang, China
Accepted author version posted online: 03 Oct 2013.Published
online: 07 Nov 2013.
To cite this article: Hai-Tao Xia, Yu-Fen Liu & Wei-Xing Ma (2013) Study on interaction of N,N′-(2-
hydroxy-3-methoxybenzyl) diamine cerium(IV) with bovine serum albumin, Journal of Coordination
Chemistry, 66:21, 3706-3721, DOI: 10.1080/00958972.2013.850496
To link to this article: http://dx.doi.org/10.1080/00958972.2013.850496
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Journal of Coordination Chemistry, 2013
Vol. 66, No. 21, 3706–3721, http://dx.doi.org/10.1080/00958972.2013.850496
Study on interaction of N,N′-(2-hydroxy-3-methoxybenzyl)
diamine cerium(IV) with bovine serum albumin
HAI-TAO XIA*, YU-FEN LIU and WEI-XING MA
School of Chemical Engineering, Huaihai Institute of Technology, Lianyungang, China
(Received 11 June 2013; accepted 30 August 2013)
Complexes of cerium(IV) with bis(N,N′-(2-hydroxy-3-methoxybenzyl) ethane-1,2-diamine, bis
(2-hydroxy-3-methoxybenzyl) propane-1,2-diamine and bis(2-hydroxy-3-methoxybenzyl) propane-
1,3-diamine were synthesized by reaction of cerium(IV) nitrate and the ligands. The obtained coordi-
nation compounds have also been characterized by elemental analysis, IR spectroscopy, and single
crystal X-ray diffraction. The interactions of these complexes with bovine serum albumin (BSA)
were investigated using fluorescence spectroscopy under the simulated physiological conditions.
Fluorescence titration revealed that the complexes can quench the intrinsic fluorescence of BSA
through static quenching mechanism. The static quenching constants, the binding constants, and the
binding sites were also acquired according to the Stern–Volmer equation and the corresponding ther-
modynamic parameters ΔH, ΔS, and ΔG were calculated through the van’t Hoff equation. Hydrogen
bonds and van der Waals forces were the predominant intermolecular forces between these
complexes and BSA. The distances between BSA and these complexes were less than 8 nm, indicat-
ing that energy transfer between the donor and acceptor occurs with high probability. Synchronous
fluorescence studies indicate that binding of these complexes with BSA changes the polarity around
tyrosine residues rather than tryptophan residues.
Keywords: Ce(IV) complex; Crystal structure; Bovine serum albumin; Fluorescence spectrum
1. Introduction
Lanthanides have many biochemical and pharmacological actions. Lanthanide coordination
chemistry is challenging due to their unique structures and their chemical, industrial,
biochemical, and medicinal applications [1, 2]. Cerium is the most abundant and most
widely used lanthanide metal. It has an important effect on the growth and function of
blood, central nerve and immune systems, hair, skin, bone, cerebrum, liver, heart, etc.
[3, 4]. Cerium is widely used in various fields of chemical engineering, luminescence, catal-
ysis, nuclear energy, metallurgy microelectronics, therapeutic application, magnetism, etc.
[5]. Ce(III/IV) complexes have been applied as versatile oxidative reagents in organic syn-
thesis, photocatalytic oxidation, electrochemistry, and biochemistry [6]; the ligands are
mainly O, N species [7–11]. o-Vanillin is an important raw material widely used in biology,
pharmacology, catalysis, organic synthesis, chemical industry, analysis, etc. o-Vanillin dia-
mine derivatives are interesting ligands that possess conformational nonrigidity, multifunc-
tion, and potential tetradentate donation (O/N) and are analogous to the biological
*Corresponding author. Email: xht161006@hhit.edu.cn
© 2013 Taylor & Francis

Ce(IV) complex
3707
environment to some extent. Hence, o-vanillin is an optimal candidate for synthesizing Ce
(IV) complexes with important bioactivities. Some studies indicate that incorporation of
biological molecules, such as proteins and drugs, has the potential to enhance biological
activities [12]. Serum albumins are the most abundant proteins in the circulatory system,
playing an important role for transport, distribution, and deposition of a variety of endoge-
nous and exogenous substances [13–15]. Various drugs injected into the body are combined
with serum albumin; through the storage and transport of the plasma, they reach the recep-
tor site to produce pharmacological effects. Many researches focus on synthesis of lantha-
nide complexes; however, research on the interaction mechanisms between serum albumins
and lanthanides has lagged compared with research on the synthesis of lanthanide com-
plexes, restricting the development of new types of lanthanide complexes possessing higher
biological activity. Hence, it was important to investigate the interaction mechanisms
between lanthanide elements and plasma proteins. In the present study, the synthesis and
characterization of Ce(IV) complexes with o-vanillin diamine derivatives have been
described. The binding properties between the Ce(IV) complexes and bovine serum albumin
(BSA) were studied by fluorescence spectroscopy. The binding constants, the number of
binding sites, and thermodynamic parameters between BSA and the Ce(IV) complexes are
calculated at different temperatures; the interactions and binding average distance between
BSA and the Ce(IV) complexes are discussed.
2. Experimental
2.1. Materials
BSA was purchased from the Beijing Biosea Biotechnology Company; its molecular weight
is 67,000. All BSA solutions are prepared in pH 7.4 buffer solution, and BSA stock solu-
tion (1.0 × 10−4 M L−1) is kept in the dark at 4 °C; NaCl (analytical grade, 0.5 M L−1) is
used to maintain the ion strength. Buffer solution consisted of Tris (0.05 M L−1) and HCl
(0.05 M L−1), and the pH is adjusted to 7.4 by adding 0.1 M L−1 NaOH at 298 K. The
Ce(IV) complex solutions (3.0 × 10−5 M L−1) were prepared in ethanol. All other materials
were of analytical reagent grade. All solutions are prepared with doubly-distilled water.
2.2. Physical measurements
Elemental analyses were measured by a Perkin–Elmer 2400c Element analyzer. Infrared
spectra were recorded on a Nicolet 5DX FT-IR spectrophotometer using KBr disks from
4000 to 400 cm−1. Fluorescence spectra were measured with a Shimadzu RF-5301 fluoro-
photometer equipped with a 150 W Xenon lamp and 1.0 cm quartz cell. Absorption spectra
were obtained by a Shimadzu UV-2550 PC spectrophotometer.
2.3. Preparation of complexes
The preparation of ligands follows the procedure in the literature [16, 17]. Cerium(IV)
complexes were obtained according to the following general procedure: Ce(NO3)3·6H2O
(5 mM) was dissolved in 10 mL ethanol, followed by addition of the ligand L1 (bis
(N,N′-(2-hydroxy-3-methoxybenzyl) ethane-1,2-diamine, 10 mM) or L2 (bis(2-hydroxy-3-
methoxybenzyl) propane-1,2-diamine, 10 mM) or L3 (bis(2-hydroxy-3-methoxybenzyl)

3708
H.-T. Xia et al.
propane-1,3-diamine, 10 mM). The reaction mixture was stirred in air for 3–4 h and then
filtered. Crystals of [Ce(L1)2] and [Ce(L2)2] suitable for crystallographic characterization
and solids of [Ce(L3)2] were obtained by evaporation of an ethanol solution. The synthetic
pathway for the complexes may be represented by scheme 1. Results of elemental analyses:
Anal. Calcd for [Ce(L1)2] (1) [C36H44N4O8Ce] (%): C, 53.99; H, 5.54; N, 7.00. Found
(%): C, 53.86; H, 5.49; N, 6.92. IR (KBr, ν(cm−1)): 3256, 2944, 2854, 1590, 1479, 1443,
1308, 1278, 1239, 1077, 927, 840, 735.
Anal. Calcd for [Ce(L2)2] (2) [C38H48N4O8Ce] (%): C, 55.06; H, 5.84; N, 6.76. Found
(%): C, 54.91; H, 5.77; N, 6.63. IR (KBr, ν(cm−1)): 3250, 2944, 2842, 1593, 1476, 1380,
1275, 1233, 1074, 978, 843, 729.
Anal. Calcd for [Ce(L3)2] (3) [C38H48N4O8Ce] (%): C, 55.06; H, 5.84; N, 6.76. Found
(%): C, 54.95; H, 5.73; N, 6.68. IR (KBr, ν(cm−1)): 3406, 2944, 2854, 1596, 1548, 1422,
1383, 1236, 1071, 957, 840, 732.
2.4. Crystal structure determination
Dark red crystals of [Ce(L1)2] (1) crystallized in the monoclinic space group P21/c, whereas
dark red crystals of [Ce(L2)2] (2) crystallized in the orthorhombic space group Pbca
.
Diffraction data were collected with a Bruker SMART 1000 CCD diffractometer by the use
of graphite monochromated Mo Kα radiation (λ = 0.71073 Å) at 298(2) K. The crystal
structure was solved by direct methods and all nonhydrogen atoms were located with
successive difference Fourier syntheses. The structure was refined by full-matrix least
squares on F2 with anisotropic thermal parameters for all nonhydrogen atoms. The
hydrogens were added according to theoretical models. All calculations were performed
using the programs in SHELXL [18, 19].
OCH3
OCH3
OCH3
HO
OH
OH
(H2N)2R
NaBH4
N
N
R
CHO
R
NH
O
HN
OCH3
OCH3
HO
OH
O
O
O
Ce(NO3)3
air
H2O
O
O
H
N
Ce
H
N
O
O
R
HN
NH
R
H
2, CH2(CH3)C
, CH CH CH
R=CH2CH2
2
2
2
Scheme 1. The synthetic pathway for the cerium(IV) complexes.

Ce(IV) complex
3709
2.5. Fluorescence spectra
The fluorescence measurements of complex-BSA solutions are performed at different
temperatures by keeping the concentration of BSA fixed at 4 × 10−6 M L−1 while varying
the Ce(IV) complex concentration from 0 to 10 × 10−6 M L−1. The widths of both the exci-
tation slit and the emission slit are set to 10 and 5.0 nm. Fluorescence spectra were recorded
from 300 to 500 nm at an excitation wavelength of 285 nm.
2.6. Energy transfer
The absorption spectra of Ce(IV) complexes were recorded at 298 K from 300 to 500 nm.
The emission spectrum of BSA was also recorded at 298 K in the same range. Then, the
overlap of the UV absorption spectrum of Ce(IV) complexes with the fluorescence emission
spectrum of BSA wase used to calculate the energy transfer.
2.7. Synchronous fluorescence spectra
The synchronous fluorescence spectra were collected with Δλ = 15 and Δλ = 60 nm,
respectively, in the same experimental conditions.
3. Results and discussion
3.1. Crystal structure description
Details of the diffraction experiment, structure refinement, and selected bond distances and
angles for [Ce(L1)2] (1) are presented in tables 1 and 2. The crystal structure of 1 has been
reported [20]. Complex 2 is formed by complexation of Ce(IV) with two ligands. Each
Table 1. Crystal data and structure refinement for 1 and 2.
Formula
Formula weight
C36H44N4O8Ce (1)
800.87
C38H48N4O8Ce (2)
828.92
Temperature (K)
298(2)
298(2)
Wavelength (Å)
0.71073
0.71073
Crystal system
Space group
Monoclinic
P21/c
Orthorhombic
Pbca
Unit cells and dimensions (Å, °)
a
23.166(3)
11.4384(16)
b
17.803(2)
14.1272(18)
c
27.883(4)
46.433(3)
α
90
90
β
110.116(3)
90
γ
90
90
Volume (Å3), Z
10,798(2), 12
1.478
4920
0.39 × 0.35 × 0.32
1.38 – 25.01
53,789
7503.3(15), 8
1.468
3408
0.44 × 0.28 × 0.12
1.98 – 25.01
30,706
Calculated density (Mg m−3
F (0 0 0)
)
Crystal size (mm3)
θ Range for data collection (°)
Reflections collected
Independent reflection
Refinement method
18,893 [R(int) = 0.0868]
Full-matrix least-squares on F2
18,893/48/1328
0.936
6182 [R(int) = 0.1287]
Full-matrix least-squares on F2
6182/0/466
Data/restraints/parameters
Goodness-of-fit on F2
1.029
Final R indices [I > 2σ(I)]
Largest diff. peak and hole (eÅ−3
R1 = 0.0409, wR2 = 0.0680
1.404 and −1.021
R1 = 0.0994, wR2 = 0.1857
2.073 and −2.325
)

3710
H.-T. Xia et al.
Table 2. The selected bond lengths (Å), hydrogen bond lengths (Å), and bond angles (°) for 2.
Ce1–O3
Ce1–O5
Ce1–O7
Ce1–O1
2.200(10)
2.205(11)
2.220(10)
2.224(10)
Ce1–N3
Ce1–N2
Ce1–N4
Ce1–N1
2.586(14)
2.598(13)
2.604(13)
2.605(13)
O3–Ce1–O5
O3–Ce1–O7
O5–Ce1–O7
O3–Ce1–O1
O5–Ce1–O1
O7–Ce1–O1
O3–Ce1–N3
O5–Ce1–N3
O7–Ce1–N3
O1–Ce1–N3
O3–Ce1–N2
O5–Ce1–N2
O7–Ce1–N2
O1–Ce1–N2
90.4(4)
96.6(4)
149.0(4)
150.3(12)
97.0(4)
91.7(4)
74.2(4)
71.6(5)
139.3(4)
80.9(4)
72.4(4)
73.5(4)
79.9(4)
137.3(4)
N3–Ce1–N2
O3–Ce1–N4
O5–Ce1–N4
O7–Ce1–N4
O1–Ce1–N4
N3–Ce1–N4
N2–Ce1–N4
O3–Ce1–N1
O5–Ce1–N1
O7–Ce1–N1
O1–Ce1–N1
N3–Ce1–N1
N2–Ce1–N1
N4–Ce1–N1
130.7(5)
81.9(4)
138.1(5)
72.9(4)
73.3(4)
66.6(5)
139.9(4)
138.8(4)
81.9(4)
72.9(4)
70.9(4)
138.4(4)
66.6(4)
128.9(5)
D–H⋯A
D–H
0.91
0.91
0.91
0.91
0.97
H⋯A
2.29
2.65
2.26
2.47
2.69
D⋯A
∠ D–H⋯A
169
N1–H1⋯O8
N2–H2⋯O6
N3–H3⋯O4
N4–H4⋯O2
C4–H4A⋯O7i
3.188(17)
3.530(18)
3.148(18)
3.358(18)
3.332(18)
162
166
167
125
Symmetry codes: (i) −x + 1/2, y + 1/2, z.
ligand forms four coordinate bonds to the central ion (figure 1). The Ce–O and Ce–N
distances vary between 2.200(10) and 2.224(10) Å and between 2.586(14) and 2.605(13) Å,
respectively. The Ce–O distances for 2 are longer than for 1 and are similar to those
observed for [CeC40H40N4O4] (2.148(7)–2.241(9) Å) [21]. The Ce–N bond lengths are
shorter than those found for 1 and are similar to CeC40H40N4O4 [21] of (2.525(11)–2.619
(9) Å). The shorter Ce–N distances indicated that the coordination of N are strengthened
owing to the repulsive electron effect of methyl, and elongations of Ce–O bonds are a result
of shortened Ce–N bonds.
The angles formed by the same donors are similar for both ligands. The O–Ce–O angles
are the largest, 149.0(4) and 150.3(4)° for O5–Ce1–O7 and O3–Ce1–O4, respectively. The
angles formed by the adjacent oxygens of different ligands are approximately vertical, vary-
ing between 90.4(4) and 91.7(4)°, and are similar to those of 90.74(14) and 90.12(13)°
observed in 1. Due to two ligands in the sphere, the angles between the coordination bonds
formed by different ligands vary from 72.9(4)° for O7–Ce1–N1 to 139.9(4)° for N2–Ce1–N4.
The dihedral angles between phenyl ring planes are 42.1(4)° for C1–C19 and 38.2(4)°
for C20–C38. The dihedral angles between adjacent phenyl rings of different ligands are
19.0(6)° for C5–C10 and C32–C37 rings, and 19.4(5)° for C13–C18 and C24–C29 rings,
while others are 48.4(4)° for C5–C10 and C24–C29 rings, and 40.9(4)° for C13–C18 and
C32–C37 rings. The dihedral angle between CeN2O2 coordination planes formed by the
two ligands is 84.4(2)°. The dihedral angles between the CeN2O2 coordination plane and
the ring planes are 35.1(5) and 40.6(4)° for C5–C10 and C13–C18 rings for the first ligand,
and 33.7(5) and 39.3(4)° for C24–C29 and C32–C37 rings of the second ligand.
Molecules of 2 are linked into chains by C–H⋯O intermolecular hydrogen bonds (table
2). There are direction-specific interactions between adjacent chains in the 3-D network
structure.

Ce(IV) complex
3711
Figure 1. Molecular structure of 2.
3.2. Fluorescence spectroscopy
Interactions of 1, 2, and 3 with BSA have been studied from tryptophan emission quench-
ing experiments. BSA contains three fluorophores, tryptophan, tyrosine, and phenylalanine;
the intrinsic fluorescence of BSA is mainly contributed by tryptophan. BSA has two trypto-
phan residues along the chain. Trp-134 is located on the surface of the protein and Trp-212
is located within a hydrophobic binding pocket of the protein. The tryptophan residues can
bind reversibly to a large number of exogenous and endogenous compounds. The change in
the emission spectra of tryptophan is primarily due to protein conformation transitions,

3712
H.-T. Xia et al.
subunit association, substrate binding, or denaturation [22]. Figure 2 shows the fluorescence
emission spectra of BSA in the absence and presence of the complexes. BSA exhibits a
strong fluorescence emission at 346 nm due to tryptophan residues when excited at 285 nm.
When a different concentration of the complexes solution was titrated into a fixed concen-
tration of BSA, a decrease in the fluorescence intensity of BSA was observed, indicating
that there were interactions and energy transfer between the complexes and BSA.
Fluorescence quenching refers to the decrease in the fluorescence intensity of a fluoro-
phore induced by molecular interactions with quencher molecule. The mechanism of the
quenching may be either through static or dynamic quenching. Static quenching refers to
quenching through fluorophore–quencher complex formation and dynamic quenching refers
to a process where the fluorophore and the quencher come into contact during the transient
existence of the excited state. Fluorescence quenching can be analyzed by using the
Stern-Volmer equation [23]:
F0=F ¼ 1 þ Kqs0½Qꢀ ¼ 1 þ Ksv½Qꢀ
where F0 and F represent the fluorescence intensities of BSA solution at 346 nm in the
absence and presence of quencher, respectively. Kq is the quenching rate constant, Ksv is the
dynamic quenching constant, τ0 is the average lifetime of the biomolecule without quencher
and taking as fluorescence lifetime of tryptophan in BSA at around 10−8 s, and [Q] is the
concentration of quencher. Figure 3 displays the Stern-Volmer plots of the quenching of
BSA fluorescence by complexes at different temperatures. The Stern-Volmer quenching
constant Ksv can be obtained by the slope of the diagram F0/F versus [Q], and subsequently
the approximate quenching constant Kq may be calculated. Quenching mechanism can be
predicted from Stern-Volmer plots. As shown in figure 4, the plot exhibited a good linear
relationship, suggesting that a single quenching mechanism, either static or dynamic, occurs
at the experiment concentrations [24]. The upper limit of Kq expected for a diffusion-con-
trolled bimolecular process is 2.0 × 1010 M L−1 s−1 [25]; the high magnitude of all the Kq
values in the present study (1012 L M−1 s−1, table 3) indicates that fluorescence quenching
effects of complexes are not initiated by dynamic collision but by formation of the
complex.
3.3. Binding constants and the number of binding sites
For static quenching, the binding constant and the number of binding sites can be
determined by the following equation [23]:
log½ðF0 ꢁ FÞ=Fꢀ ¼ log Kb þ n log½Qꢀ
where Kb and n are the binding constant and the number of binding sites, respectively.
According to the equation, binding parameters can be obtained by a plot of the double-loga-
rithm curve log [(F0 – F)/F] versus log [Q] (figure 4). The values of Kb and n were calcu-
lated from the values of intercept and slope of the plots, respectively; the corresponding
results are summarized in table 3. The binding constants decreased with increasing tempera-
ture, indicating that stability of the complex-BSA system is reduced. They are probably
associated with hydrogen bonding and weakening of the complex-BSA stability. The values
of n at the experimental temperatures were approximately equal to 1, indicating that there is
one class of binding site on BSA for 1, 2, and 3. The values of Kb indicate that there is a

Ce(IV) complex
3713
250
200
150
100
50
(a)
0
300
350
400
450
wavelength/nm
250
200
150
100
50
(b)
0
300
350
400
450
wavelength/nm
(c)
200
100
0
300
350
400
450
wavelength/nm
Figure 2. Emission spectra of BSA in the presence and absence of 1, 2 and 3. [BSA]: 4 × 10−6 M L−1; [complex]:
0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 × 106 M L−1. λex = 285 nm; λem = 346 nm; pH = 7.4; T = 298 K.

3714
H.-T. Xia et al.
1.5
1.4
1.3
1.2
1.1
1.0
(a)
291k
298k
305k
-6
-6
-6
-6
-5
0.0
2.0x10
4.0x10
6.0x10
8.0x10
1.0x10
-1
[Q] mol L
1.5
1.4
1.3
1.2
1.1
1.0
(b)
291k
298k
305k
2.0x10-6
4.0x10-6
6.0x10-6
8.0x10-6
1.0x10-5
0.0
[Q] mol L-1
1.6
1.5
1.4
1.3
1.2
1.1
1.0
(c)
291k
298k
305k
2.0x10-6
4.0x10-6
6.0x10-6
8.0x10-6
1.0x10-5
0.0
[Q] mol L-1
Figure 3. The Stern-Volmer plots for the interaction of complexes with BSA.

Ce(IV) complex
3715
-0.2
-0.4
-0.6
-0.8
-1.0
-1.2
-1.4
(a)
291k
298k
305k
-6.0
-5.8
-5.6
-5.4
-5.2
-5.0
log[Q]
-0.2
-0.4
-0.6
-0.8
-1.0
-1.2
-1.4
(b)
291k
298k
305k
-6.0
-5.8
-5.6
-5.4
-5.2
-5.0
log[Q]
-0.2
-0.4
-0.6
-0.8
-1.0
-1.2
-1.4
(c)
291k
298k
305k
-6.0
-5.8
-5.6
-5.4
-5.2
-5.0
log[Q]
Figure 4. Plots of log(F0 − F)/F vs. log[Q].

3716
H.-T. Xia et al.
Table 3. Binding constants and thermodynamic parameters.
Kq
Ksv
Kb
ΔH
ΔG
ΔS
Complex
T (K)
(L M−1
)
(L M−1 s−1
)
(L M−1
)
n
(kJ M−1
)
(kJ M−1
)
(J M−1 K−1
)
291
298
305
3.87 × 104
3.73 × 104
2.97 × 104
3.87 × 1012
3.72 × 1012
2.97 × 1012
2.39 × 104
1.42 × 104
5.19 × 103
0.97
0.92
0.85
−24.61
−23.27
−21.93
1
−80.27
−191.18
−346.31
−148.65
291
298
305
3.44 × 104
3.02 × 104
3.55 × 104
3.44 × 1012
3.02 × 1012
3.55 × 1012
5.97 × 103
4.32 × 103
5.77 × 102
0.84
0.83
0.63
−21.73
−19.31
−16.88
2
3
−122.56
−69.72
291
298
305
4.15 × 104
4.93 × 104
5.07 × 104
4.15 × 1012
4.93 × 1012
5.07 × 1012
5.72 × 104
2.66 × 104
1.53 × 104
1.03
0.95
0.90
−26.44
−25.40
−24.36
moderate interaction between complexes and BSA. The binding propensity of 3 was higher
than 1 and 2.
Compared with studies dealing with copper complexes-BSA interaction [23, 26, 27], few
studies on cerium complexes have been reported. Comparing Kb and n values of 1, 2, and 3
with those of copper complexes, including analogous ligands, we find that the Kb and n val-
ues of the copper complexes [Cu2(oxbpa)(phen)(H2O)](pic)·2H2O (Kb, 2.09 × 106 L M−1; n,
1.23) [26] and [C20H32Cu2I3N4]n (Kb, 5.95 × 108 L M−1; n, 1.68) [23] are both higher,
implying that interactions of this class of complexes toward BSA depend on the nature of
metal ions and ligands; medicinal properties of complexes may be tuned by changing the
metal ions or ligands.
3.4. Binding mode
The interaction between any drug and biomolecule include hydrogen bonds, van der Waals
forces, electrostatic forces, and hydrophobic interaction. These forces can be identified from
studies on thermodynamics of binding of drug molecules with proteins. In order to elucidate
the interaction between the complexes and BSA, the thermodynamic parameters were calcu-
lated on the basis of the van’t Hoff equation [23]. The thermodynamic parameters for the
interaction of the complexes with BSA are shown in table 3. The negative values of ΔG
indicate that the binding process is spontaneous. According to the rules summarized by
Ross and Subramanian [28], negative values of enthalpy (ΔH) and entropy (ΔS) indicate
that the hydrogen bonds and van der Waals forces played a major role in the binding of 1,
2, and 3 with BSA.
3.5. Synchronous fluorescence
Synchronous fluorescence spectra of BSA can be used to detect the change in conformation
of BSA. When Δλ between excitation wavelength and emission wavelength is fixed at 15 or
60 nm, synchronous fluorescence can provide information on the environment of tyrosine
and tryptophan residues in BSA [29]. The synchronous fluorescence spectra of BSA in the
presence of the complexes are shown in figure 5. The maximum emission wavelength of
tyrosine residue shows an obvious red shift in the presence of 1, 2, and 3, suggesting that
the polarity around the tyrosine residues decreased. In contrast, no significant shift was
observed when Δλ = 60 nm suggesting that interaction of the complexes with BSA does
not affect the polarity and conformation of the tryptophan residue micro-region.

Ce(IV) complex
3717
(a)
= 15 nm
30
20
10
0
280
300
320
340
wavelength/nm
(b)
= 15 nm
30
20
10
0
280
300
320
340
wavelength/nm
(c)
30
20
10
0
= 15 nm
280
300
320
340
wavelength/nm
Figure 5. Synchronous fluorescence spectra of interaction between BSA and complexes. [BSA]: 4 × 106 M L−1
[complex]: 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 × 10−6 M L−1; pH = 7.4; T = 298 K.
;

3718
H.-T. Xia et al.
(a)
= 60 nm
200
100
0
320
340
wavelength/nm
360
380
250
200
150
100
50
(b)
= 60 nm
0
320
340
360
380
wavelength/nm
250
200
150
100
50
(c)
= 60 nm
0
320
340
360
380
wavelength/nm
Figure 5. (Continued).

Ce(IV) complex
3719
0.5
0.4
0.3
0.2
0.1
0.0
120
80
(a)
b
40
a
0
300
300
300
350
400
450
450
450
500
500
500
wavelength/nm
0.5
0.4
0.3
0.2
0.1
0.0
120
(b)
b
80
40
a
0
350
400
wavelength/nm
0.5
0.4
0.3
0.2
0.1
0.0
120
(c)
b
80
40
0
a
350
400
wavelength/nm
Figure 6. Spectral overlap of complexes absorption (a) with BSA fluorescence (b) [BSA] = [complexes]: 1 × 105
M L−1 (T = 291 K).

3720
H.-T. Xia et al.
Table 4. Relevant parameters of complexes-BSA systems.
Complex
J (cm3 L M−1
)
E (%)
R0 (nm)
r (nm)
1
2
3
8.58 × 1015
6.09 × 1015
8.90 × 1015
0.12
0.14
0.13
4.38
4.13
4.40
6.11
5.59
6.04
3.6. Energy transfer between the complexes and BSA
The fluorescence quenching of BSA in the presence of complexes indicated that energy
transfer between 1, 2, 3, and BSA occurred. According to Förster nonradiative energy
transfer theory, the energy transfer efficiency is described by the following equation:
6
E ¼ 1=½1 þ ðr=R0Þ ꢀ ¼ 1 ꢁ ðF=F0Þ
where E is the efficiency of energy transfer between the donor and acceptor, F and F0 are
the fluorescence intensities of BSA in the presence and absence of the complexes, r is the
distance between the donor and acceptor, and R0 is the critical distance at which transfer
efficiency equals 50%. The value of R0 can be calculated using the following equation:
R60 ¼ 8:79 ꢂ 10ꢁ25ðK2 ꢃ Nꢁ4 ꢃ U ꢃ JÞ
where K2 is the spatial orientation factor of the dipole, N is refractive index of the medium,
Φ is fluorescence quantum yield of the donor, and J is overlap integral of the fluorescence
emission spectrum of donor and absorption spectrum of the acceptor. The UV–Vis absorp-
tion spectrum of the complexes with the fluorescence emission spectrum of BSA is shown
in figure 6. J can be calculated by the following equation:
R1
FðkÞeðkÞk4dk
0
R
J ¼
1 FðkÞdk
0
where F(λ) is the fluorescence intensity of the donor in the wavelength range λ–λ + Δλ and
ε(λ) is the molar absorption coefficient of the acceptor at wavelength λ. In the present case,
K2 = 2/3, N = 1.336, Φ = 0.15 [23]. From the overlapping spectrum, J can be evaluated by
integrating the spectra for λ = 300–500 nm; the corresponding results are summarized in
table 4. Obviously, the distance between the donor BSA and the acceptor (complex) is less
than 8 nm, and 0.5 R0 < r < 1.5 R0, indicating that the binding of 1, 2, and 3 with BSA
occurs through energy transfer.
4. Conclusions
Three eight-coordinate Ce(IV) complexes with N,N′-(2-hydroxy-3-methoxybenzyl) diamine
were synthesized and the crystal structures of 1 and 2 were determined by single crystal
X-ray diffraction. The interactions of the complexes with BSA in physiological buffer
solution were studied by fluorescence spectroscopic methods. Decreasing values of binding
constants of 1, 2, and 3 with increasing temperature indicated interaction with BSA through
a static quenching procedure. The values of n revealed the presence of one binding site on
BSA. The thermodynamic parameters of the binding interaction were determined and their

Ce(IV) complex
3721
values suggested that hydrogen bonds and van der Waals forces played major roles in the
interactions of Ce(IV) complexes (1, 2, and 3) with BSA. The binding distances r between
Ce(IV) complexes and BSA indicate that energy transfer from BSA to Ce(IV) complexes
occurs. The synchronous fluorescence spectroscopy indicates that the conformation of BSA
changed in the presence of Ce(IV) complexes.
Supplementary material
Crystallographic data for the structures in this article have been deposited with the Cambridge
Crystallographic Data Center as supplementary publication CCDC Nos. 635372 and 653349 for 1 and
2. Copies of the data can be obtained, free of charge, on application to CCDC, 12 Union Road,
Cambridge CB2 1EZ, UK.
References
[1] O.-S. Jung, S.-H. Park, Y.-A. Lee, D.-C. Kim. J. Mol. Struct., 645, 281 (2003).
[2] I. Kostova, I. Manolov, G. Momekov, T. Tzanova, S. Konstantinov, M. Karaivanova. Eur. J. Med. Chem., 40,
1246 (2005).
[3] J.-M. Li, W.-T. Wei. J. Rare Earth, 28, 387 (2010).
[4] M.Y. Khotimchenko, E.A. Kolenchenko, Y.S. Khotimchenko, E.V. Khozhaenko, V.V. Kovalevb. Colloids
Surf., B, 77, 104 (2010).
[5] Z.V.P. Murthy, A. Choudhary. Desalination, 279, 428 (2011).
[6] L. Li, F. Yuan, T.-T. Li, Y. Zhou, M.-M. Zhang. Inorg. Chim. Acta, 397, 69 (2013).
[7] I. Kostova, G. Momekov. Eur. J. Med. Chem., 43, 178 (2008).
[8] M.R. Ganjali, M. Hosseini, Z. Memari, F. Faridbod, M. Hakimi, E. Motieiyan, A. Shokrollahi, A.N. Haghighi.
Inorg. Chim. Acta, 385, 140 (2012).
[9] C.M. Kizas, C. Papatriantafyllopoulou, M.J. Manos, A.J. Tasiopoulos. Polyhedron, 52, 346 (2013).
[10] Nibha, I.P.S. Kapoor, G. Singh, R. Fröhlich. J. Mol. Struct., 1034, 296 (2013).
[11] T.V. Balashova, A.P. Pushkarev, V.A. Ilichev, M.A. Lopatin, M.A. Katkova, E.V. Baranov, G.K. Fukin,
M.N. Bochkarev. Polyhedron, 50, 112 (2013).
[12] S.R.S. Ting, J.M. Whitelock, R. Tomic, C. Gunawan, W.Y. Teoh, R. Amal, M.S. Lord. Biomaterials, 344, 377
(2013).
[13] N. Shahabadi, M. Maghsudi. J. Mol. Struct., 929, 193 (2009).
[14] J.-N. Tian, J.-Q. Liu, Z.-D. Hu. Bioorg. Med. Chem., 13, 4124 (2005).
[15] T.-H. Wang, Z.-M. Zhao, B.-Z. Wei, L. Zhang, L. Ji. J. Mol. Struct., 970, 128 (2010).
[16] H.-T. Xia, Y.-F. Liu, S.-P. Yang, D.-Q. Wang. Acta Cryst., E62, o5864 (2006).
[17] H.-T. Xia, Y.-F. Liu, S.-P. Yang, D.-Q. Wang. Acta Cryst., E63, o239 (2007).
[18] G.M. Sheldrick. SHELXS-97, Program of X-ray Crystal Structure Solution, University of Göttingen, Göttin-
gen, Germany (1997).
[19] G.M. Sheldrick. SHELXL-97, Program for X-ray Crystal Strucuture Refinement, University of Göttingen,
Göttingen, Germany (1997).
[20] Y.-F. Liu, H.-T. Xia, D.-Q. Wang, S.-P. Yang. Acta Cryst., E63, m484 (2007).
[21] E. Szłyk, A. Wojtczak, L. Dobrzańska, M. Barwiołek. Polyhedron, 27, 765 (2008).
[22] Y.-Q. Wang, H.-M. Zhang, G.-C. Zhang, W.-H. Tao, Z.-H. Fei, Z.-T. Liu. J. Pharm. Biomed. Anal., 43, 1869
(2007).
[23] Y.-F. Liu, H.-T. Xia, D.-F. Rong. J. Coord. Chem., 65, 2919 (2012).
[24] F. Xue, C.-Z. Xie, Y.-W. Zhang, Z. Qiao, X. Qiao, J.-Y. Xu, S.-P. Yan. J. Inorg. Biochem., 115, 78 (2012).
[25] N. Shahabadi, M. Maghsudi. J. Mol. Struct., 929, 193 (2009).
[26] X.-L. Wang, M. Jiang, Y.-T. Li, Z.-Y. Wu, C.-W. Yan. J. Coord. Chem., 66, 1985 (2013).
[27] Y.-F. Liu, X.-Y. Xu, H.-T. Xia, D.-Q. Wang, H. Liu. Synth. React. Inorg. Met.-Org. Nano-Met. Chem., 39, 400
(2009).
[28] P.D. Ross, S. Subramanian. Biochemistry, 20, 3096 (1981).
[29] G. Vignesh, S. Arunachalam, S. Vignesh, R.A. James. Spectrochim. Acta, Part A, 96, 108 (2012).