Synthesis, luminescence properties of Eu(III) and Tb(III) complexes with a novel aromatic carboxylic acid and their interactions with bovine serum albumin

Article abstract of DOI:10.1016/j.saa.2012.08.060

A novel aromatic carboxylic acid ligand (L) was synthesized and its corresponding Eu(III) and Tb(III) complexes, Na₃EuLCl ₃·2H₂O (EuL) and Na₃TbLCl ₃·3H₂O (TbL), were successfully prepared. L and its corresponding complexes were characterized by means of MS, elemental analysis, IR, ¹H NMR and TG. The luminescence spectra of Eu(III) and Tb(III) complexes were investigated and the results showed that L was an efficient sensitizer for Eu(III) and Tb(III) luminescence. The interactions of L, EuL and TbL with bovine serum albumin (BSA) have been investigated through fluorescence spectroscopy under physiological conditions. The Stern-Volmer analysis indicated that the fluorescence quenching of BSA by L, EuL and TbL was resulted from static mechanism, and the binding constants (K_a) were 2.22 × 10⁴, 1.33 × 10⁵ and 4.27 × 10⁵ at 300 K, respectively. The binding sites (n) and the corresponding thermodynamic parameters ΔH, ΔS, and ΔG were calculated at different temperatures. According to the theoretical and experimental results, van der Waals interactions and hydrogen bonds were found to play major roles in the binding reaction. Furthermore, UV-Vis absorption spectroscopy and synchronous fluorescence spectra indicated that the conformation of BSA was changed. The results obtained in the work can help understand the action mode between L and its corresponding Eu(III) and Tb(III) complexes with BSA, and they are also expected to provide important information of designs of new inspired drugs based on Eu and Tb.

Full text of DOI:10.1016/j.saa.2012.08.060

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 98 (2012) 170–177
Contents lists available at SciVerse ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Synthesis, luminescence properties of Eu(III) and Tb(III) complexes
with a novel aromatic carboxylic acid and their interactions with
bovine serum albumin
Liqun Shen a, Zhengfa Yang b, Ruiren Tang b,
⇑
a College of Chemistry and Chemical Engineering, Guangxi University for Nationalities, Guangxi Key Laboratory of Chemistry and Engineering of Forest Products,
Nanning 530006, PR China
b School of Chemistry and Chemical Engineering, Central South University, Changsha 410083, PR China
g r a p h i c a l a b s t r a c t
h i g h l i g h t s
"
A novel ligand and its Eu(III) and
Tb(III) complexes were synthesized.
The luminescence properties of
Eu(III) and Tb(III) complexes were
studied.
The binding characteristics of L, EuL
and TbL with BSA were investigated.
UV–Vis and synchronous
CH3COCl / CH3OH
r.t. 24h
KOH / CH3OH
r.t. 24h
SOCl2
COOH
HOOC
N
COOH
H3COOC
N
1
COOCH3
H3COOC
N
2
"
HN(CH2COOCH3
)
2 / CH2Cl2
O
30% KOH / CH3OH;
0oC 24h
H3COOC
N
C
N
H3COOC
N
COCl
r.t. 1h
"
"
3
H3COOC
COOCH3
O
C
N
HOOC
N
4
fluorescence indicated the changed
conformation of BSA.
"
The binding affinity with BSA was
determined to be in the order of
TbL > EuL > L.
HOOC
COOH
L
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel aromatic carboxylic acid ligand (L) was synthesized and its corresponding Eu(III) and Tb(III) com-
plexes, Na3EuLCl3ꢀ2H2O (EuL) and Na3TbLCl3ꢀ3H2O (TbL), were successfully prepared. L and its corre-
sponding complexes were characterized by means of MS, elemental analysis, IR, 1H NMR and TG. The
luminescence spectra of Eu(III) and Tb(III) complexes were investigated and the results showed that L
was an efficient sensitizer for Eu(III) and Tb(III) luminescence. The interactions of L, EuL and TbL with
bovine serum albumin (BSA) have been investigated through fluorescence spectroscopy under physiolog-
ical conditions. The Stern–Volmer analysis indicated that the fluorescence quenching of BSA by L, EuL and
TbL was resulted from static mechanism, and the binding constants (Ka) were 2.22 ꢁ 104, 1.33 ꢁ 105 and
4.27 ꢁ 105 at 300 K, respectively. The binding sites (n) and the corresponding thermodynamic parameters
Received 3 February 2012
Received in revised form 11 August 2012
Accepted 20 August 2012
Available online 27 August 2012
Keywords:
Synthesis
Dimethyl pyridine-2,6-dicarboxylate
Coordination
Lanthanide complex
Luminescence
D
H, DS, and DG were calculated at different temperatures. According to the theoretical and experimental
results, van der Waals interactions and hydrogen bonds were found to play major roles in the binding
reaction. Furthermore, UV–Vis absorption spectroscopy and synchronous fluorescence spectra indicated
that the conformation of BSA was changed. The results obtained in the work can help understand the
action mode between L and its corresponding Eu(III) and Tb(III) complexes with BSA, and they are also
expected to provide important information of designs of new inspired drugs based on Eu and Tb.
Ó 2012 Elsevier B.V. All rights reserved.
Bovine serum albumin
Introduction
their fascinating architectures and potential application as func-
tional materials [1]. In particular, Ln(III) complexes are more pop-
In recent years, the rational design and synthesis of lanthanide
organic coordination complexes have received special attention for
ular due to their well-defined luminescence properties, including
hypersensitivity to the coordination environment, narrow band-
width and millisecond luminescence decay times. For example,
the Ln(III) complexes have been widely used in diagnostic tools,
luminescence sensors, laser systems, and optical amplification for
⇑
Corresponding author. Tel.: +86 731 88836961; fax: +86 731 88879616.
E-mail address: trr@mail.csu.edu.cn (R. Tang).
1386-1425/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.saa.2012.08.060

L. Shen et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 98 (2012) 170–177
171
telecommunications [1–4]. Nevertheless, the Ln(III) complexes
usually give weak luminescence due to the weak absorption coef-
ficient of the parity-forbidden f–f transitions which limits their
practical application considerably. This can be overcome by the
use of high absorbent chelating ligands, which served as efficient
sensitizers. Much work has been done, but the effects on the lumi-
nescence properties of rare-earth complexes still need to be further
studied.
Dimethyl pyridine-2,6-dicarboxylate 1 was prepared by literature
[16]. Other chemicals were of A.R. grade and used without further
purification. The water used in this study was doubly deionized.
Melting points were determined on a XR-4 apparatus (ther-
mometer uncorrected). Elemental analysis was carried out by a
PerkinElmer 2400 elemental analyzer. Infrared spectra (4000–
400 cmꢂ1) were recorded with KBr discs as sample by a Nicolet
NEXUS 670 FT-IR spectrophotometer. 1H NMR was measured with
a Bruker-400 MHz nuclear magnetic resonance spectrometer with
CDCl3, DMSO and D2O as solvents and TMS as internal reference.
The EI-mass spectra were recorded on a Finnigan MAT 90 instru-
ment. UV–Vis spectra were carried out by TU-1800 spectropho-
tometer made by LabTech in Beijing, China. Excitation and
Emission spectra were recorded on a Hitich F-4500 luminescence
spectrophotometer (The width of emission detector slit and the
voltage of photomultiplier tube were 2.5 nm and 700 V respec-
tively for Eu(III) complex; while 5 nm and 400 V for Tb(III) complex
correspondingly in solid state). Thermal gravimetric (TG) were per-
formed in the nitrogen atmosphere using a Netzsch TG 209 ther-
mogravimetric analyzer at a heating rate of 10 °C minꢂ1 from 20
to 700 °C.
Pyridine-2,6-dicarboxylic acid (H2DPC) possesses biological
activity in the body of the animal and plant. Its derivatives and
complexes were studied extensively and were used in all sorts of
fields [5,6]. In the past few years, many studies on the molecular
structure of metal complexes and their biological activities aroused
much interest in the field of inorganic chemistry [7,8]. It was found
that the rare earth metals possessed a special affinity to tumor
cells. For instance, 70% of 169Yb was found to accumulate in tumors
10 min after intravenous injection [9]. Also, the luminescent Eu
and Tb complexes were capable of selectively binding to ‘drug site
II’ of serum albumin and played a significant part in drug pharma-
cokinetics and pharmacodynamics [10]. Serum albumins are the
major soluble protein constituents of the circulatory system and
have many physiological functions. The most important property
of this group of proteins is that they serve as transporters for a
variety of compounds. Therefore, the study of the interactions be-
tween the rare earth metal complexes and serum albumins is
important for the exploration of their biological effects and drug
screening. BSA serves well as protein model for the study due to
its structural homology with human serum albumin (HSA)
[11,12]. Fluorescence spectroscopy is one of the powerful tools to
explore the interaction between the small molecule and bio-mac-
romolecule [13]. Thus, it is useful to study the interactions of the
small molecules and the small molecule complexes with, taking
the quenching of the intrinsic tryptophan fluorescence of BSA as
a tool. The study of the interaction mechanism between drugs or
the small molecules with serum albumins shows great significance
in pharmacokinetics, and it has proved to be one of the most
important research subjects in life science, chemistry and clinical
medicine.
Mostly, many studies focused on the introducing of the coordi-
nation function groups to the H2DPC, resulted in the sensitization
of the rare earth ions [14,15]. Nevertheless, the studies on the
asymmetric ligands containing pyridine-2,6-dicarboxylic acid unit,
are rarely reported. In this paper, a novel asymmetrical chelating
ligands containing pyridine-2,6-dicarboxylic acid unit and their
complexes with Eu(III) and Tb(III) were successfully prepared.
The luminescence spectra of Eu and Tb complexes have been stud-
ied and they were proved to be efficient sensitizers. Moreover, the
interaction mechanism between L, EuL, TbL and BSA were investi-
gated, which was of great importance to understand the general
rules of their interactions and showed the reference value to the
design of new drugs as well. The synthetic route of L was expressed
in Scheme 1.
Preparation of L
Synthesis of 6-(methoxycarbonyl) pyridine-2-carboxylic acid (2)
A solution of 1 (5.85 g, 30.0 mmol) in methanol (150 mL) was
cooled to 0 °C. After KOH pellets (1.76 g, 31.0 mmol) were added,
the reaction mixture was stirred at 0 °C for 2 h and then at room
temperature for 24 h. The solvent was removed under reduced
pressure, and the residue was suspended in H2O (100 mL) and ex-
tracted with ethyl acetate (3 ꢁ 30 mL). The aqueous layers were
acidified to pH 3 with 1 M diluted HCl solution and extracted with
chloroform (5 ꢁ 30 mL). The collected organic layers were dried
over anhydrous Na2SO4. The chloroform was removed in vacuo to
provide the desired product 2 (2.80 g, 52%) as a white solid: m.p.
144–146 °C. 1H NMR (DMSO-d6): dH 8.12–8.24 (m, Py-3,4,5,3H)
3.93 (s, –CH3,3H); 13C NMR(DMSO-d6): d = 52.2, 127.4, 127.7,
139.0, 147.4, 148.9, 164.9, 165.8 ppm; IR (KBr), v/cmꢂ1: 3073,
2964, 2852, 1725, 1581, 1325.
Synthesis of methyl 6-(chlorocarbonyl) pyridine-2-carboxylate (3)
2 (0.72 g, 4 mmol) was treated with thionyl chloride (10 mL)
and one drop of N,N-dimethylformamide, then the mixture was re-
fluxed at 70 °C for 4 h. The excess thionyl chloride was removed by
evaporation under reduced pressure. Residual thionyl chloride was
further removed by co-evaporation with 30 mL of anhydrous ben-
zene to afford the corresponding acid chloride 3. This crude prod-
uct was used in the following step without further purification.
Synthesis of methyl 6-[N,N-bis(methoxycarbonyl methyl)
carbamoyl]pyridine-2-carboxylate (4)
Dimethyl iminodiacetate (0.56 g, 3.5 mmol) and triethylamine
(0.35 g, 3.5 mmol) were dissolved in 15 mL of dichloromethane,
stirring at 0 °C, and then 3 (0.7 g, 3.5 mmol) was added in three
portions with continuous stirring at 0 °C. The reaction mixture
was stirred at room temperature for 1 h. After the solvent was re-
moved under reduced pressure, the residue was dissolved in ethyl
acetate and water (15:15, v:v). The aqueous phase was extracted
with ethyl acetate (2 ꢁ 5 mL). Then, the organic phase was com-
bined and dried by anhydrous Na2SO4. After the solvent was re-
moved under reduced pressure, the crude was further purified by
column chromatography with petroleum ether and ethyl acetate
(1:1) to give 4 (0.9 g, 81.8%) as a white solid: m.p. 80–82 °C. Anal.
Calcd for C14H16N2O7 (%): C, 51.85; H, 4.97; N, 8.64; Found: C,
51.72; H, 4.91; N, 8.62; 1H NMR (CDCl3): d 8.16 (q, J = 7.2 Hz
Py-3,5, 2H), 7.98 (t, J = 8.0 Hz, Py-4, 1H), 4.52 (s, N–CH2, 2H), 4.41
Experimental
Materials and methods
The stock solution of BSA (purity P99%, purchased from Sino-
Biotechnology Company, Shanghai, China) was prepared to be the
concentration of 1.0 ꢁ 10ꢂ5 mol Lꢂ1 by dissolving it in 0.1 M HCl–
Tirs buffer solution (pH 7.4, 50 mM NaCl), and kept it in the dark
at 0–4 °C. After L and its Eu(III) and Tb(III) complexes were dis-
solved in water, and diluted to the desired concentration, the
working solutions of L, Eu(III) and Tb(III) complexes were obtained.
The buffer Tris was purchased from Acros (Geel, Belgium).

172
L. Shen et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 98 (2012) 170–177
CH3COCl / CH3OH
r.t. 24h
KOH / CH3OH
r.t. 24h
SOCl2
COOH
HOOC
N
COOH
H3COOC
N
COOCH3
H3COOC
N
1
2
HN(CH2COOCH3)2 / CH2Cl2
O
30% KOH / CH3OH;
0oC 24h
H3COOC
N
C
N
H3COOC
N
COCl
r.t. 1h
3
H3COOC
COOCH3
O
C
N
HOOC
N
4
HOOC
COOH
L
Scheme 1. The synthetic route of L.
(s, N–CH2, 2H), 3.97 (s, Py-2-OCH3, 3H), 3.83 (s, –CH3, 3H), 3.79 (s, –
CH3, 3H); IR (KBr), v/cmꢂ1: 3093, 3013, 2954, 1748, 1719, 1635,
1214, 1189; EI-MS: m/z: 324.3[M]+.
temperatures of the samples were kept by recycled water through-
out the experiment.
UV–Vis absorption spectroscopy
Absorption spectra (UV) measurements were recorded at 300 K
in the region of 210–320 nm. The 2.5 mL of BSA stock solution was
titrated with successive additions of the working solution. The
HCl–Tris buffer solution was used as a reference solution.
Synthesis of 6-[N,N-bis(ethyloic)carbamoyl]pyridine-2-carboxylic acid
(L)
4 (0.81 g, 2.5 mmol) was dissolved in 50 mL anhydrous metha-
nol with stirring in an ice bath. 2.5 g KOH solution (30%) were
added, and the reaction mixture was stirred at 0 °C for 24 h. Then
the solvent was evaporated to give a yellow oily residue, which
was purified by a 732 cation exchange resin column to provide L
(0.68 g, 96.5%) as a white solid. m.p. 198–200 °C. Anal. Calcd for
Results and discussion
Properties of the complexes
C
11H10N2O7 (%): C, 46.82; H, 3.57; N, 9.93; Found: C, 46.91; H,
3.55; N, 9.89; 1H NMR (H2O): d 8.11-8.23 (m, Py-3,5, 2H), 7.89 (d,
J = 8.0 Hz, Py-4, 1H), 4.38 (s, N–CH2, 2H),4.28 (s, N–CH2, 2H); IR
(KBr), v/cmꢂ1: 3078, 2663, 1716, 1616, 1425, 1389, 1351; EI-MS:
m/z: 282.2[M]+.
Elemental analyses
The results of elemental analyses of C, H, and N are in accor-
dance with the theoretical values calculated, indicating that the
composition of the complexes are Na3EuLCl3ꢀ2H2O and Na3TbLCl3-
ꢀ3H2O. We suspected that the chemical structure of complexes
(Scheme 2) as given below.
Preparation of the complexes
FT-IR spectra
A mixture of L (0.8 mmol) and a solution of LnCl3ꢀ6H2O
(0.8 mmol) (Ln = Tb, Eu) in H2O (6 mL) was stirred for 10 min at
room temperature, following which the aqueous NaOH (0.01 M)
was added dropwise to pH 6.0. Then the mixture was stirred at
60 °C for 12 h. The resulting precipitate was collected by filtration,
washed three times with methanol, dried and purified by recrystal-
lization from a methanol–water mixture. Elemental analysis (%):
Calcd for Na3EuLCl3ꢀ2H2O (642.58): C, 20.54; H, 1.71; N, 4.36;
Found: C, 20.47; H, 1.74; N, 4.31; IR (KBr), v/cmꢂ1:3430, 3270,
3054, 1590, 1502, 1431, 1404, 1353, 1330, 619, 420; Elemental
analysis (%): Calcd for Na3TbLCl3ꢀ3H2O (667.52): C, 19.77; H,
The characteristic infrared absorption bands of the free L and its
Eu(III) and Tb(III) complexes which are useful for suggesting the
mode of coordination of ligands are given in Table 1. The FT-IR
spectra of the two complexes are similar, indicating that they are
structurally alike (Fig. 1). The FT-IR spectrum of the free L shows
bands at 3078 cmꢂ1 attributed to free
at 1717 and 1571 cmꢂ1 are assigned to
group and (C@N) of L respectively. In the complexes, the band
for
m
(OH) of –COOH. The bands
(C@O) of the carboxyl
m
m
m
(C@N) of the pyridine ring shows a shift to low frequency from
1571 to 1498–1502 cmꢂ1 as a result of the coordination through
1.95; N, 4.19; Found: C, 19.82; H, 1.91; N, 4.22; IR (KBr), v/cmꢂ1
:
3407, 1587, 1497, 1401, 1351, 1304, 1256, 621, 421.
Interactions with BSA
Fluorescence spectrum
N
O
O
A 2.5 mL solution containing 1.0 ꢁ 10ꢂ5 mol Lꢂ1 BSA was ti-
trated by successive additions of 1.0 ꢁ 10ꢂ3 mol Lꢂ1 stock solution
of L, EuL, TbL and the concentration of it varied from 0 to
1.0 ꢁ 10ꢂ4 mol Lꢂ1. Titrations were done manually by using mi-
cro-injector. An excitation wavelength (Ex) of 280 nm was chosen
in the experiment. The excitation and emission slit widths (5 nm
each) and scan rate (240 nm minꢂ1) were constantly maintained
for all experiments. The fluorescence spectra were recorded at
300, 310 and 320 K in the range of 300–440 nm, respectively. The
N
Ln3+
O-
O
O
O
O-
Scheme 2. The chemical structure of the complexes.

L. Shen et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 98 (2012) 170–177
173
Table 1
Characteristic IR bands (cmꢂ1) of the ligand and its complexes.
Sample
m
(OH)
m(C@O) of carboxyl
m
as(COOꢂ)
m
s(COOꢂ)
mpy(C@N)
L
3078
3430
3407
1717
1571
1502
1498
Na3EuLCl3ꢀ2H2O
1590
1587
1404
1401
Na3TbLCl3ꢀ3H2O
v
as, asymmetric; vs, symmetric.
two main successive mass loss stages in the TG curves. For Eu(III)
complexes, the first loss in the range of 30–170 °C accounts for
the release of the solvated water content (Weight loss: 5.75%,
Calcd: 5.60%), the second step occurs in the range of 250–290 °C
corresponding to the loss of L (Weight loss: 44.62%, Calcd:
43.45%). While for Tb(III) complexes, the first stage of the decom-
position appears within 30–180 °C owing to the loss of the solvated
water (Weight loss: 7.97%, Calcd: 7.79%). And the second loss stage
ranging from 310 to 390 °C is attributed to the loss of L (Weight
loss: 42.03%, Calcd: 41.83%), which was confirmed by comparing
the observed/estimated and the calculated mass of the complexes.
The detailed data are listed in Table 2. The above results prove that
the water in the complexes was the solvated water and did not
participate in coordination, because the coordinated water of the
analogous complexes is usually released over 200 °C.
(c)
(b)
(a)
Fluorescence properties of complexes
The emission and excitation spectra measured in the solid state
at room temperature are displayed in Figs. 2 and 3, respectively.
The luminescence data for the complexes are listed in Table 3.
The excitation spectra of the complexes exhibited a broad band be-
tween 250 and 400 nm which was attributable to the p–
p⁄ transi-
tion of the coordinated ligands. The emission spectra of Eu(III)
complexes, obtained with the excitation wavelength of 281 nm,
are composed of the typical emission lines for Eu(III) owing to
the transitions from the first excited state (5D0) to the ground mul-
tiplet 7FJ (J = 0–4). The maximum peak intensities at 580, 591, 612,
648, and 694 nm were observed for the J = 0, 1, 2, 3, and 4 transi-
tions, respectively. The transitions of 5D0 ! 7F0 (forbidden in
inversion center) and 5D0 ! 7F3 (magnetic and electric dipole tran-
sitions) are very weak while those of 5D0 ! 7F1 (magnetic dipole
4000
3500
3000
2500
2000
1500
1000
500
Wavenumbers (cm-1)
Fig. 1. IR spectrum of L (a), Eu(III) complex (b) and Tb(III) complex(c).
metal-nitrogen bond [17]. The band m
(C@O) at 1717 cmꢂ1 for free L
disappeared and new bands appeared at 1580–1590 cmꢂ1 and
1401–1404 cmꢂ1 which are assigned to the two characteristic
7
transition) and 5D0 ! F2 are strong. The fact that the hypersensi-
bands of asymmetric (ca. 1600 cmꢂ1
) and symmetric (ca.
7
tive 5D0 ! F2 transition is the most intensive indicates a highly
1400 cm–1) stretching vibration modes of the carboxylate group.
This indicates that the carboxylic acid group was converted into
a carboxylate anion as a result of the formation of the stable
Ln(III)-cored complexes [15]. The absorption bands assigned to
polarizable chemical environment around the europium site [18].
The emission spectra of the Tb(III) complexes, obtained with the
excitation wavelength of 301 nm, are composed of the typical
emission lines for Tb(III), which are assigned to the transitions be-
tween the first excited state (5D4) and the ground multiplet 7FJ
(J = 6–3) (Fig. 3). The emission mechanism of the Tb(III) complex
is substantially the same as that of the Eu(III) complex. The emis-
sion spectrum of the Tb(III) complex consisted of four main bands
at approximate 488 nm (5D4 ? 7F6), 544 nm (5D4 ? 7F5), 581 nm
(5D4 ? 7F4) and 618 nm (5D4 ? 7F3), and we can see that the emis-
sion band at 544 nm is obviously stronger than the other emission
bands. In addition, the intensity ratio of the two main bands at
488 nm and 544 nm is about 2.1, which indicates that the Tb(III)
ion is at the center of an asymmetric coordination field [19]. Still,
the
m(Ln–O) and m(Ln–N) stretching frequencies of the complex
are observed at 420 cmꢂ1 and 620 cmꢂ1 respectively, which also
confirmed that the oxygen and nitrogen atoms participated in
the coordination [15]. A broad band at 3431–3203 cmꢂ1 assigned
to m(OH) indicates the presence of the lattice water which was con-
firmed in thermal gravimetric analyses (TGA).
TGA
The TGA results of the two complexes Na3EuLCl3ꢀ2H2O and Na3-
TbLCl3ꢀ3H2O are given in Supplementary data (Fig. S1). There are
Table 2
TG data of Na3EuLCl3ꢀ2H2O and Na3TbLCl3ꢀ3H2O.
Complexes
Stage
Temperature range (°C)
Mass loss (%) found (calc.)
Probable lost molecules
Na3EuLCl3ꢀ2H2O
I
II
30–170
250–290
5.75 (5.60)
44.62 (43.45)
2H2O
L
Na3TbLCl3ꢀ3H2O
I
II
30–180
310–390
7.97 (7.79)
42.03 (41.83)
3H2O
L

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L. Shen et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 98 (2012) 170–177
1800
EX
EM
1800
1500
1200
900
600
300
0
a
i
1500
1200
900
600
300
200
300
400
500
600
700
300 320 340 360 380 400 420 440
Wavelength(nm)
Wavelength (nm)
Fig. 2. (Left) Excitation (5D0–7F2 transition) and (right) emission (kex = 281 nm)
spectra of Na3EuLCl3ꢀ2H2O in the solid state at room temperature.
Fig. 4. The quenching effect of
L on fluorescence intensity of BSA at 300 K;
kex = 280 nm; cBSA = 1.0 ꢁ 10ꢂ5 mol Lꢂ1; c(L)/(10ꢂ5 mol Lꢂ1).(a–i): 0, 0.8, 1.6, 2.4, 3.2,
4, 6, 8 and 10, respectively.
7000
EX
EM
the corresponding calculated results are given in Table 4. The fluo-
rescence intensity of BSA consistently decreased with the addition
of L, EuL and TbL. At the same time, a slight blue shift at the max-
imum emission wavelength of BSA was observed, which indicated
that there existed strong interaction between L, EuL and TbL and
BSA, and the microenvironment around the chromophore of BSA
was also changed [20]. The quenching can be mathematically ex-
pressed by the Stern–Volmer Eq. (1), which allows for calculating
of quenching constants [21]:
6000
5000
4000
3000
2000
1000
F0
¼ 1 þ Ksv½Qꢃ ¼ 1 þ
s
0kq½Qꢃ
ð1Þ
0
F
250 300 350 400 450 500 550 600 650
where, kq, Ksv,
s0 and [Q] are the quenching rate constant of BSA
Wavelength(nm)
(Mꢂ1 sꢂ1), the dynamic Stern–Volmer quenching constant (Mꢂ1),
the average lifetime of BSA and the concentration of quencher (M),
respectively. F0 and F are the fluorescence intensities in the absence
and presence of quencher, respectively. In fact, the fluorescence life-
Fig. 3. (Left) Excitation (5D4
spectra of Na3TbLCl3ꢀ3H2O in the solid state at room temperature.
!
7F5 transition) and (right) emission (kex = 301 nm)
time of biomolecular
s
0 is about 10ꢂ8 s [22]; the biomolecular
Table 3
quenching constant kq at pH 7.40 can be obtained by the linear
Stern–Volmer plots of F0/F against [Q] from Eq. (1) at different tem-
peratures. Representative Stern-Volmer plots for L binding to BSA
are shown in Fig. 5, and those for EuL and TbL binding to BSA are
in Fig. S3. In general, the maximum collision quenching constant
(kq) of various kinds of biomolecules is 2.0 ꢁ 1010 L molꢂ1 Sꢂ1
[22]. Obviously, the rate constants (Table 4) of BSA quenched by L,
EuL and TbL are greater than 2.0 ꢁ 1010 L molꢂ1 Sꢂ1, which sug-
gests that the quenching is initiated by static quenching resulting
from the complex formation rather than by dynamic quenching,
which is confirmed in the following UV–Vis spectra. While the re-
sults also show that the values of the Stern–Volmer quenching con-
stants Ksv and kq increased with the increased temperature. It
indicates that the probable quenching mechanism of BSA-(L, EuL
and TbL) interactions was initiated by the dynamic collision. In other
words, the fluorescence quenching of BSA results from the static
quenching, while that dynamic collision could be not negligible [23].
Luminescence data for the complexes.
Complexes
kex (nm)
kem (nm)
LI
Assignment
5D0
5D0
!
!
7F1
7F2
Na3EuLCl3ꢀ2H2O
281
591
612
1246
1773
5D4
5D4
5D4
5D4
!
!
!
!
7F6
7F5
7F4
7F3
Na3TbLCl3ꢀ3H2O
301
488
544
581
618
2387
6718
863
246
each emission band is very narrow and the widths of half bands are
about several nanometers, indicating that the complexes have high
color purity [14].
BSA binding studies
Binding constant and binding sites
Fluorescence quenching of BSA by L, EuL and TbL
The relationship between the fluorescence intensity and the
concentration of the quenchers can be described by the following
Eq. (2) for static quenching:
The fluorescence quenching mechanisms are usually classified
into dynamic quenching and static quenching. For the dynamic
quenching mechanism, as the system temperature rises, the effec-
tive collision times between molecules, the energy transfer effi-
ciency, and the fluorescence quenching constants of substances
will all increase. For the static quenching mechanism, increased
temperature reduces the stability of the complex formed, resulting
in a reduced quenching constant.
1
Ka
1
½Qꢃ
log½F=ðF0 ꢂ FÞꢃ ¼ log
þ n log
ð2Þ
where Ka is the binding constant, and n is the number of binding
sites per BSA [15]. Fig. 6 shows the double-logarithm algorithm
The effect of L on the fluorescence emission spectra of BSA at
300 K is shown in Fig. 4, EuL and TbL are shown in Fig. S2, and
curve of L-BSA system and Table
calculated results. Similar results were obtained for EuL and TbL
5 gives the corresponding

L. Shen et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 98 (2012) 170–177
175
Table 4
1.8
Stern–Volmer quenching constants Ksv and kq for binding of BSA with L, EuL and TbL
at different temperatures.
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
a
L or
T
Ksv
kq
R2
Complex
(K)
( ꢁ 104 L molꢂ1
)
( ꢁ 1012 L molꢂ1 Sꢂ1
)
L
300
310
320
0.43
0.48
0.50
0.43
0.48
0.50
0.99662
0.99662
0.99653
EuL
TbL
300
310
320
1.16
1.23
1.26
1.16
1.23
1.26
0.98994
0.99109
0.99553
300
310
320
1.17
1.27
1.35
1.17
1.27
1.35
0.98861
0.99446
0.99882
4.0
4.2
4.4
4.6
4.8
5.0
5.2
a
R2 is the correlation coefficient of Stern–Volmer equation.
log1/[Q]
Fig. 6. Double-log plot of L quenching effect on BSA fluorescence at three different
temperatures (j300 K; d310 K; N320 K).
1.5
1.4
1.3
1.2
1.1
1.0
D
G ¼
D
H ꢂ T
D
S
ð4Þ
Fig. S5, by plotting the data in Table 5, shows the values of
D
D
H
and S obtained for the binding site from the slopes and ordinates
at the origin of the fitted lines. The negative values of G, seen in
Table 5, support the assertion that the binding process is spontane-
ous. When H < 0 or S > 0, the main acting force is elec-
H ꢄ 0,
trostatic force; when H < 0, S < 0, the main acting force is van
der Waals or hydrogen bond and when H > 0, S > 0, the main
force is hydrophobic [25]. The H and S values of the interactions
between L, EuL and TbL with BSA indicate that van der Waals inter-
actions and hydrogen bonds play major roles in the binding
reaction.
D
D
D
D
D
D
D
D
D
D
0
2
4
6
8
10
5
10 c(L)/(mol/L)
Fig. 5. Stern–Volmer plots of fluorescence quenching of BSA by L at three different
temperatures (j300 K; d310 K; N320 K).
BSA conformation investigation
The synchronous fluorescence spectra can provide much valu-
able information about the microenvironment in the vicinity of
the chromophore molecules [26]. In the synchronous fluorescence
of BSA, the shift in the position of the maximum emission wave-
length corresponds to the changes of the polarity around the fluo-
(Fig. S4). These results suggest that there is a strong binding force
between L, EuL and TbL with BSA. According to Table 5, the corre-
sponding binding sites n at the temperatures of 300 K, 310 K and
320 K were roughly equal to 1.0, indicating that the interaction of
the complex with BSA has only a single binding site. The binding
constants of the interaction between L, EuL and TbL with BSA in-
crease in the following order: TbL > EuL > L at different tempera-
tures, which means that TbL has the strongest ability to bind with
BSA, while L is the weakest. The two factors may result in the bind-
ing potency of L, EuL and TbL and BSA: (1) Ln(III) ionic radius; (2)
the increasing polarity of the complex than that of the ligand
strengthening its binding affinity [23,24].
rophore of amino acid residues. When
Dk values are stabilized at
15 or 60 nm, the synchronous fluorescence of BSA is characteristic
of tyrosine residues (Tyr) and tryptophan residues (Trp), respec-
tively [27]. The effect of L on BSA synchronous fluorescence spec-
troscopy is shown in Fig. 7. As seen from it, the fluorescence of
Tyr was weak and the position of maximum emission wavelength
had no change when
Trp was strong and the maximum emission wavelength moder-
ately shifted toward short wave when k was 60 nm. This reflects
Dk was 15 nm. However, the fluorescence of
D
that the microenvironment of the Trp was significantly affected by
L binding. The slight blue shift of Trp fluorescence indicates that
the polarity around the tryptophan residue decreases and the
hydrophobicity increases [28].
Thermodynamic parameters and binding mode
The interaction forces between proteins and ligands may in-
clude hydrophobic, hydrogen bonds, van der Waals, electrostatic
interactions, etc. In order to elucidate the interaction between L,
EuL and TbL with BSA, the thermodynamic parameters were calcu-
lated from the van’t Hoff plots. If the enthalpy change (
not vary significantly in the temperature range studied, both the
UV–Vis absorption measurement is a very simple method and
applicable to exploring the structural change and to know the com-
plex formation [29]. To explore the structural change of BSA by the
addition of L, the UV–Vis spectra of BSA with various amounts of L
and its Eu(III) and Tb(III) complexes were measured. Fig. 8 shows
the UV–Vis absorption spectra of BSA from 210 to 315 nm in the
presence of different L concentrations. As shown in Fig. 8, L has al-
most non-UV absorption under the present experimental condi-
tions, while BSA has a strong absorbance with a peak at 280 nm.
DH) does
enthalpy change (
D
H) and entropy change (
D
S) can be evaluated
from the van’t Hoff equation [24]:
D
RT
H
DS
ln Ka ¼ ꢂ
þ
R
ð3Þ
where Ka is analogous to the associative binding constants at the
corresponding temperature and R is the gas constant. The calcula-
tion was carried out at three different temperatures, which are
The peak at 280 nm corresponds to the absorption of the
p
p⁄
ꢂ
transition of amino acid residues (Trp, Tyr) in protein. Upon the
addition of L to the solution of BSA, it is found that the absorption
of BSA increased regularly as the peak shows a blue shift at 280 nm
(from 278.0 nm to 271.3 nm). The hyperchromicity and the shift
300, 310, and 320 K.
DH is calculated from the slope of the van’t
Hoff relationship. The free energy change (
from the following relationship:
DG) is then estimated

176
L. Shen et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 98 (2012) 170–177
Table 5
The binding constant (Ka), binding sites (n) and relative thermodynamic parameters for the interactions of L, EuL and TbL with BSA at 300 K, 310 K and 320 K, respectively.
a
b
L or Complex
T (K)
Ka (Lꢀmolꢂ1
)
n
R2
D
H (kJ molꢂ1
)
D
S (J molꢂ1 Kꢂ1
)
D
G (kJ molꢂ1
)
R2
L
300
310
320
2.22 ꢁ 104
6.91 ꢁ 103
2.82 ꢁ 103
1.17
1.00
0.93
0.98154
0.99573
0.99653
ꢂ84.69
ꢂ83.01
ꢂ93.01
ꢂ198.95
ꢂ178.09
ꢂ202.14
ꢂ25.01
ꢂ23.02
ꢂ21.02
ꢂ29.58
ꢂ27.80
ꢂ26.02
ꢂ32.37
ꢂ30.35
ꢂ28.33
0.9987
EuL
TbL
300
310
320
1.33 ꢁ 105
4.92 ꢁ 104
1.64 ꢁ 104
1.27
1.15
1.03
0.98697
0.99749
0.99719
0.99396
0.99095
300
310
320
4.27 ꢁ 105
1.47 ꢁ 105
4.07 ꢁ 104
1.39
1.27
1.13
0.99972
0.99877
0.99827
a
R2 is the correlation coefficient of double-logarithm equation.
b
R2 is the correlation coefficient of the value of
DH, DS.
160
1200
1000
800
600
400
200
0
(a)
(b)
140
a
h
a
h
120
100
80
60
40
20
270
280
290
300
310
260
270
280
290
300
310
Wavelength(nm)
Wavelength(nm)
Fig. 7. Effect of L on the synchronous fluorescence spectra of BSA. (a)
0.3, 0.4, 0.6, 0.8, 1.0, respectively.
D
k = 15 nm, (b)
D
k = 60 nm; T = 298 K; c(BSA) = 1.0 ꢁ 10ꢂ5 mol Lꢂ1; c(L)/(10ꢂ5 mol Lꢂ1), a–h: 0, 0.1, 0.2,
complexes showed that Eu(III) and Tb(III) could be sensitized effi-
ciently by the ligand. The interactions of L, EuL and TbL with BSA
have been investigated through fluorescence spectroscopy under
physiological conditions. The probable quenching mechanism of
the fluorescence of BSA by L and its Eu(III) and Tb(III) complexes
was static quenching. Van der Waals interactions and hydrogen
bonds played major roles in the binding reaction. In addition, the
binding affinity of them to BSA was determined to be in the order
of TbL > EuL > L, indicating that the addition of the rare earth ion to
the L center is helpful to the binding of BSA. The synchronous fluo-
rescence spectra and UV–Vis absorption spectroscopy indicated
that the interaction of L and its Eu(III) and Tb(III) complexes with
BSA caused conformational change of BSA. These results are ex-
pected to give important insight into interactions of the rare earth
complexes and BSA, which show great reference value for a model
of application for drug design.
1.2
1.0
0.8
G
0.6
A
0.4
0.2
0.0
H
210
225
240
255 270
285
300
315
Wavelength(nm)
Fig. 8. UV–Vis absorption spectra of BSA in the presence of L at 298 K. Curve H:
c(BSA) = 0; c(L) = 1.0 ꢁ 10ꢂ5 mol Lꢂ1
,
Curve A ? G: c(BSA) = 1.0 ꢁ 10ꢂ5 mol Lꢂ1
,
c(L)/(10ꢂ5 mol Lꢂ1), (A–G): 0, 1, 2, 3, 5, 7 and 10, respectively.
Acknowledgement
Financial support from the National Natural Science Foundation
of China (No. 21071152) is Gratefully acknowledged.
effect suggest that the conformation of BSA was changed with the
addition of L [30].
Its Eu(III) and Tb(III) complexes presented a similar synchro-
nous fluorescence and UV–Vis absorption spectra characteristics.
The results indicate that the interactions of L and its Eu(III) and
Tb(III) complexes cause conformational change of BSA.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.saa.2012.08.060.
Conclusions
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