Eu(III), Tb(III) complexes with novel ligands containing pyridine-2,6-dicarboxylic acid unit: Synthesis, characterization, fluorescence properties and application in biological imaging

Article abstract of DOI:10.1016/j.saa.2011.09.036

Three novel ligands containing pyridine-2,6-dicarboxylic acid unit, trans-4 -(4′-methoxystyryl) pyridine-2,6-dicarboxylic acid, trans-4-(4′- (dimethylamino)styryl)pyridine-2,6-dicarboxylic acid, and trans-4-(4′- (diphenylamino)styryl)pyridine-2,6-dicarbox

Full text of DOI:10.1016/j.saa.2011.09.036

Spectrochimica Acta Part A 84 (2011) 238–242
Contents lists available at SciVerse ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Eu(III), Tb(III) complexes with novel ligands containing pyridine-2,6-dicarboxylic
acid unit: Synthesis, characterization, fluorescence properties and application in
biological imaging
Haibo Xiao∗, Minjuan Chen, Chong Mei, Hongyao Yin, Xiaoying Zhang, Xiaoxia Cao
Department of Chemistry, Shanghai Normal University, Shanghai 200234, PR China
a r t i c l e i n f o
a b s t r a c t
Three novel ligands containing pyridine-2,6-dicarboxylic acid unit, trans-4 -(4ꢀ-methoxystyryl)
pyridine-2,6-dicarboxylic acid, trans-4-(4ꢀ-(dimethylamino)styryl)pyridine-2,6-dicarboxylic acid, and
trans-4-(4ꢀ-(diphenylamino)styryl)pyridine-2,6-dicarboxylic acid were synthesized and their complexes
with Eu(III), Tb(III) ions were successfully prepared. The ligands and the corresponding metal complexes
were characterized by means of MS, elemental analysis, IR, 1H NMR and TG–DTA. The luminescence
spectra of Eu(III) and Tb(III) complexes in solid state were studied. The strong luminescence emitting
peaks at 615 nm for Eu(III) and 545 nm for Tb(III) can be observed. The applications in cell imaging of the
europium and terbium complexes were investigated.
Article history:
Received 27 June 2011
Accepted 12 September 2011
Keywords:
Pyridine-2,6-dicarboxylic acid
Tb(III) complexes
Eu(III) complexes
Biological imaging
© 2011 Elsevier B.V. All rights reserved.
1. Introduction
2. Experimental
2.1. Materials and apparatus
The unique luminescence properties of LnIII ions (sharp emis-
sion, large Stokes shift, insensitivity to oxygen) and particularly
their long excited state lifetime ranging from microseconds (Yb,
Nd) to milliseconds (Eu, Tb) triggered the development of time-
resolved spectroscopy or microscopy for applications in biological,
environmental, or clinical analysis [1,2]. These techniques con-
sist of the introduction of a time delay before the detection of
the lanthanide luminescence in order to eliminate parasitic scat-
tering and short-lived luminescence resulting in an enhanced
signal/noise ratio [1]. The absorption coefficients of the optical
transitions for lanthanide ions are, however, very low which lim-
its their practical application considerably. This drawback can be
overcome through the use of highly absorbent chelating ligands,
which serve as efficient sensitizers [3]. Pyridine-2,6-dicarboxylic
(dipicolinic) acid and its derivatives are highly useful tridentate
ligands, they can form nine-coordinate complexes with lanthanide
ions [4–6]. In this paper, we designed and synthesized a series
of novel chromophores containing dipicolinic acid unit, namely,
trans-4-(4ꢀ-methoxystyryl) pyridine- 2,6-dicarboxylic acid, trans-
4-(4ꢀ-(dimethylamino)styryl)pyridine-2,6- dicarboxylic acid, and
trans-4-(4ꢀ-(diphenylamino)styryl)pyridine-2,6-dicarboxylic acid
(Scheme 1), studied the fluorescence properties of their Eu(III),
Tb(III) complexes. In addition, the applications in cell imaging of
the europium and terbium complexes were investigated.
All reagents and solvents were commercial reagents of analyti-
cal grade and were used as received. Further purification and drying
by standard methods were employed and they were distilled prior
to use when necessary.
Trans-dimethyl-4-(4ꢀ-methoxystyryl)pyridine-2,6-
dicarboxylate,
trans-dimethyl-4-(4ꢀ-(dimethylamino)styryl)py-
ridine-2,6-dicarboxylate, and trans-dimethyl-4-(4ꢀ-(dipheny-
lamino)styryl)pyridine-2,6-dicarboxylate were prepared starting
from 2,6-dimethylpyridine according to the previous method [4,7].
Melting points were determined with an XT4A apparatus and
are uncorrected. Mass spectral studies were carried out using
VG12-250 mass spectrometer. 1H NMR and 13C NMR spectra were
obtained on a Bruker DRX 400 MHz spectrometer. Chemical shifts
were reported in ppm relative to a Me4Si standard. IR spectra
were obtained on a Nicolet Avatar 370 DTGS spectrometer. Ele-
mental analyses were performed by Atlantic Microlab. Steady-state
emission and excitation spectra were recorded on PerkinElmer
LS55 instrument. Visible absorption spectra were determined on
PerkinElmer Lambda 35 spectrophotometer.
2.2. Synthesis of the ligand (L)
2.2.1. Trans-4 -(4ꢀ-methoxystyryl) pyridine-2,6-dicarboxylic acid
(H2L1)
0.327 g
dimethyl-4-(4ꢀ-methoxystyryl)pyridine-2,6-
∗
dicarboxylate (1 mmol), 160 mg NaOH (4 mmol) and 1 mL H2O
Corresponding author. Tel.: +86 21 64838189.
E-mail address: xiaohb@shnu.edu.cn (H. Xiao).
were added to 10 mL THF and the mixture was refluxed at 80 ◦C
1386-1425/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.saa.2011.09.036

H. Xiao et al. / Spectrochimica Acta Part A 84 (2011) 238–242
239
COOH
COOCH3
1) NaOH /THF
X
X
N
N
2) H
COOH
COOCH3
X=OCH3, N(CH3)2, N(Ph)2
Scheme 1. Synthesis of the ligands.
Table 1
for 12 h. Then the solvent was completely removed under reduced
pressure. The residue was acidified to pH ≈ 2 by dropwise addition
of 1 N HCl solution, stirred at room temperature for 2 h and
gave 0.275 g orange solid in 92% yield. m.p. > 250 ◦C. EI–MS:m/z
299(M+).1H NMR (400 MHz,DMSO-d6, ppm) ı: 13.36 (s,2H), 8.36
(s, 2H), 7.72–7.76 (d, 1H, J = 16 Hz), 7.69–7.67 (d, 2H, J = 8 Hz),
7.35–7.31(d, 1H, J = 16.4 Hz), 7.01–6.99 (d, 2H, J = 8.4 Hz), 3.79 (s,
3H). 13C NMR (DMSO-d6, ıppm)ı:166.16, 160.90, 149.21, 148.93,
135.67, 129.72, 129.27,124.37, 122.99, 115.09, 56.02.
Elemental analytical data for the complexes.
Compounds
C (%) found
(calc.)
H (%) found
(calc.)
N (%) found
(calc.)
H6Eu(L1)3Cl3·16H2O
H6Eu(L2)3Cl3·6H2O
39.68(39.91)
46.73(47.00)
52.12(52.42)
42.20(42.42)
49.79(50.08)
4.89(4.95)
4.59(4.64)
4.95(5.00)
4.56(4.61)
4.72(4.77)
2.94(2.91)
6.51(6.45)
4.58(4.53)
8.82(8.73)
6.55(6.49)
H6Eu(L3)3Cl3·16H2O
H6Tb(L2)3(NO3)3·9H2O
H6Tb(L3)3 (NO3)3·16H2O
3. Results and discussion
2.2.2.
Trans-4-(4ꢀ-(dimethylamino)styryl)pyridine-2,6-dicarboxylic acid
(H2L2)
3.1. Properities of the complexes
Compound H2L2 was obtained following the same procedure
described as H2L1. Yellow solid in 93% yield: 1H NMR(400 MHz,
DMSO-d6, ppm)ı: 9.41(s,2H), 8.33(s,2H), 8.09 (d,2H, J = 4.8 Hz),
7.71–7.68 (d,1H, J = 13.2 Hz), 7.29–7.26 (d,1H, J = 13.6 Hz), 6.98
(d,2H, J = 4.4 Hz), 3.00 (s, 6H). 13C NMR (DMSO-d6, ppm)ı:166.22,
151.38, 148.49, 133.88, 132.76, 132.22, 132.13, 129.52,129.40,
128.81, 21.14.
The result of elemental analysis (Table 1) indicated that the
composition of the complexes conforms to H6Eu(L1)3Cl3·16H2O
(5),
H6Eu(L2)3Cl3·6H2O
(1),
H6Eu(L3)3Cl3·16H2O
(3),
H6Tb(L2)3(NO3)3·9H2O (2), H6Tb(L3)3(NO3)3·16H2O (4), respec-
tively. The complexes are soluble in DMSO, DMF, slightly in ethanol
and acetone, and insoluble in diethylether and tetrhydrofuran.
Because of the insolubility of complexes in suitable solvents we
were unsuccessful in growing crystals for single crystal X-ray
structural studies.
2.2.3.
Trans-4-(4ꢀ-(diphenylamino)styryl)pyridine-2,6-dicarboxylic acid
(H2L3)
3.2. IR spectra
Compound H2L3 was obtained following the same procedure
described as H2L1. Dark red solid in 92% yield. 1H NMR(300 MHz,
DMSO-d6, ppm)ı: 12.94 (s, 2H), 8.36 (s, 2H), 7.75–7.70 (d,
1H, J = 15.6 Hz), 7.63–7.61(d, 2H, J = 8.4 Hz), 7.37–7.28 (m, 5H),
7.13–7.06 (m, 6H), 6.97–6.94 (d, 2H, J = 8.4 Hz). 13C NMR(DMSO-
d6, ppm) ı191.04, 153.30, 146.07, 133.78, 132.76, 132.52, 132.03,
131.93, 130.52, 129.30, 129.18, 126.90, 125.97, 118.70.
The bands useful for suggesting the mode of coordina-
tion of ligands are listed in Table 2. The uncoordinated
ligands show the bands of middle intensity at 3059 cm−1(H2L1),
3016 cm−1(H2L2), 3055 cm−1(H2L3) attributed to free ꢀ(OH)
of COOH, and strong and sharp bands at 1725 cm−1(H2L1),
1703 cm−1(H2L2), 1728 cm−1(H2L3) assignable to ꢀ(C O). The bands
at 1598 cm−1(H2L1), 1583 cm−1(H2L2), 1586 cm−1(H2L3) were
assigned to pyridine ring stretching [ꢀ(C N)]. In the complexes,
2.3. Synthesis of the complexes
the bands for ꢀ(C
of pyridine ring shows a shift to a lower fre-
N)
quency about 89–154 cm−1 as a result of the coordination through
metal-nitrogen bond [8]. The bands for ꢀ(C of carboxylic groups
To a stirring solution of the ligand (0.160 mmol) in methanol,
the methanol solution of EuCl3·6H2O (19.5 mg, 0.053 mmol) or
Tb(NO3)3·6H2O (24.5 mg, 0.053 mmol) was added and refluxed
for 24 h under nitrogen atmosphere. The precipitate was filtered,
washed with methanol and air-dried to give the desired complex
(typically about 54% yield).
O)
are disappeared completely, thus indicates that all the carboxylic
groups take part in coordination. Two strong absorption bands
lying at 1636–1627 cm−1 and 1400–1381 cm−1 were observed in
complexes, which were attributed to the asymmetric vibration
absorption ꢀas(COO
−
and symmetric vibration absorption ꢀs(COO
−
)
)
Table 2
Characteristic IR bands (cm−1) of the ligands and their complexes.
Compounds
ꢀ (C O)
ꢀ (OH)
Py(C N)
ı( C–H)
ꢀas (COO−
)
ꢀs (COO−
)
ꢁꢀas-s
H2L1
H2L2
H2L3
Na2 L1
Na2 L2
Na2 L3
1727
1729
1728
3059
3016
3055
1598
1583
1586
1552
1599
1535
1436
1448
1446
1445
1445
966
968
963
1027
1024
1020
1023
1023
1000
1023
1001
1634
1625
1628
1629
1627
1636
1633
1633
1385
1384
1406
1384
1384
1400
1381
1400
249
241
222
245
243
236
252
233
H6Eu(L1)3Cl3·16H2O (5)
H6Eu(L2)3Cl3·6H2O (1)
H6Eu(L3)3Cl3·16H2O (3)
H6Tb(L2)3(NO3) 3·9H2O (2)
H6Tb(L3)3(NO3)3·16H2O(4)

240
H. Xiao et al. / Spectrochimica Acta Part A 84 (2011) 238–242
5D4 7F5
Table 3
The fluorescence spectra of the europium and terbium complexes.
Compounds
ꢂex (nm)
ꢂem (nm)
Assignment
5D4 7F6
277
591
615
698
5D0 7F1
5D0 7F2
5D0 7F4
H6Eu(L1)3Cl3·16H2O (5)
5D4 7F4
5D4 7F3
275
275
276
590
615
699
5D0 7F1
5D0 7F2
5D0 7F4
H6Eu(L2)3Cl3·6H2O (1)
H6Eu(L3)3Cl3·16H2O (3)
H6Tb(L2)3(NO3)3·9H2O (2)
4
2
591
615
691
5D0 7F1
5D0 7F2
5D0 7F4
489
545
584
620
5D4 7F6
5D4 7F5
5D4 7F4
5D4 7F3
400
450
500
550
600
650
700
272
488
545
584
621
5D4 7F6
5D4 7F5
5D4 7F4
5D4 7F3
wavelength / nm
H6Tb(L3)3(NO3)3·12H2O (4)
Fig. 2. Emission spectra of complexes with Tb(III) ions.
(Fig. 2). The components at 489 nm, 580 nm and 620 nm are
7
7
7
assigned to the 5D4 → F6, 5D4 → F4, 5D4 → F3 transition, respec-
of the carboxylic groups. For ligands L2 and L3, the asymmetric
stetching vibration frequencies ꢀas(COO in the complexes show
tively. And the intensity of 5D4 → F5 transition is far stronger than
7
−
)
that of 5D4 → F6, the intensity ratio value (5D4 → F5/5D4 → F6)
7
7
7
a shift to a higher frequency, meanwhile the symmetric stretching
vibration frequencies ꢀs(COO show a shift to a lower frequency.
is 2.0, indicating a low symmetry for the electrostatic field
−
)
The determined ꢁꢀas-s for the Ln(III) complexes is larger than
that of Na2L. This state clearly that the carboxlic group acting as
a single dentate chelate coordinated to the lanthanide ions in the
100
1
complexes[9,10]. The ꢀas(COO ) of the L1 ligand in coordination com-
−
90
pound was shifted to a lower frequency region as compared with
free ligand, therefore the ꢁꢀas-s was decreased and the carboxylic
group was coordinated with a Eu(III) ion as a form of a symmetri-
cal chelating bi-dentate [11]. The broad bands at about 3430 cm−1
in all complexes indicate the presence of crystal water molecules
[12,13]. The IR spectra and elemental analytical data suggest that
the coordination numbers of Eu(III) and Tb(III) ions is nine.
80
70
2
3
60
50
3.3. Fluorescence properties of complexes
4
40
5
Fluorescence properties of complexes in solid were shown in
Table 3, Figs. 1 and 2. The europium and terbium complexes exhib-
ited strong characteristic luminescence of europium and terbium
ions when excited at about 276 nm. The most intense transition of
30
0
200
400
600
800
Temperature/°C
the Tb(III) complexes is observed at 545 nm due to the 5D4 → F5
7
Fig. 3. TG analysis of complexes.
5D0 7F2
5D0 7F1
5
0
1
5D0 7F4
3
-5
5
-10
-15
-20
-25
-30
1
2
3
5
4
0
450
500
550
600
650
700
750
800
850
0
200
400
600
800
wavelength / nm
Temperature/°C
Fig. 1. Emission spectra of complexes with Eu(III) ions.
Fig. 4. DTA analysis of complexes.

H. Xiao et al. / Spectrochimica Acta Part A 84 (2011) 238–242
241
Fig. 5. Fluorescence (left) and phase contrast (right) images of sertoli cell fixed in paraformaldehyde and loaded with 4.
surrounding Tb(III) [14]. From Fig. 1, visible bright emission peaks,
which centered at 615 nm were observed in europium complexes
phase contrast image clearly indicates that the complex is mainly
localized in a perinuclear region. Bright spots are seldom observed
in the nucleus, indicating that the complex difficultly targeted small
organelles called nucleoli. When a comparison is made between
the fluorescence images, we can find that the fluorescence inten-
sity of the complexes in cells increases with the ligands in the
order L3 > L2 > L1.The promising results suggest their potential in
cell imaging techniques.
7
and assigned to 5D0 → F2. In all europium and terbium complexes,
the ligand-luminescence is completely suppressed, indicating that
the ligands are comparative good organic chelators to absorb
energy and transfer them to Ln(III) ions (Tb(III) ion or Eu(III) ion).
3.4. TG–DTA analysis
Thermal decomposition of all of the studied complexes was car-
ried out using TG and DTA techniques. Samples of about 10 mg
were placed in a crucible, and heated up to 800 ◦C at the rate of
10 ◦C/min in an air atmosphere at ambient pressure, using ␣-Al2O3
as reference material.
4. Conclusions
The preparation, characterication, fluorescence properties and
cell fluorescence imagings of the europium(III) and terbium(III)
complexes of three novel ligands containing dipicolinic acid unit
(L1, L2 and L3) are reported. Difference in the IR spectra of the
free ligands and the metal complexes indicated that coordination
of the Ln(III) ions to the ligands were in the form of a tridentate
ligand. The Eu(III) and Tb(III) complexes exhibited characteristic
luminescence of Eu(III) and Tb(III) ions. TG–DTA curves indicated
that all complexes are thermally stable and L2-based complexes
exhibit better thermal stability. The results of cell fluorescence
imaging show that the fluorescence intensity of the complexes in
cells increases with the ligands in the order L3 > L2 > L1. These com-
plexes would be potential candidate materials for application in
bioimaging.
TG and DTA curves of the complexes are presented in
Figs. 3 and 4, respectively. The mass loss observed in the first
step of decomposition of all complexes corresponds to the loss
of water of crystallization (50–150 ◦C) and coordination water
(150–200 ◦C) [15,16]. After a complete dehydration stage, the
subsequent mass loss stages were attributed to decomposition
and/or elimination of ligands. The mass loss starts again at first
smoothly and then steeply from 450 ◦C to 560 ◦C and then smoothly
to end up at 800 ◦C in the case of H6Tb(L2)3(NO3)3·9H2O or
H6Eu(L2)3Cl3·6H2O, respsctively. Concerning H6Eu(L3)3Cl3·16H2O
and H6Tb(L3)3(NO3)3·16H2O, the second stage begin first steeply
from 230 ◦C to 330 ◦C and then gradually up to 800 ◦C. The
decomposition process of H6Eu(L1)3Cl3·16H2O is similar to that
of H6Eu(L3)3Cl3·16H2O or H6Tb(L3)3(NO3)3·16H2O, albeit steeper
decomposition pathways were observed for H6Eu(L1)3Cl3·16H2O.
The TG curves of free ligands show that the residues obtained
are close to 0% at 800 ◦C. The TG results were confirmed
by the endothermic peaks observed in DTA curves. TG–DTA
curves indicated that L2-based complexes exhibit better thermal
stability.
Acknowledgement
This work was financially supported by the Natural Science
Foundation of Shanghai City of China (no. 11ZR1426200).
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