Inorganic Chemistry
ARTICLE
General Procedure for Deprotection of (L1 and L2). TFA
(20 mL) was added into I1 or I2 in DCM (25 mL). The solution was
stirred overnight at ambient temperature. The solvent was removed
under vacuum conditions, and thesolid waswashedwithDCM (6 times).
L1. The procedure for synthesizing L1 was followed, and 0.37 g of I1
was used. A brown solid was yielded (0.33 g, 90%). 1H NMR (400 MHz,
D2O): δ (ppm) 7.85 (2H, s), 7.14 (4H, s), 4.41ꢀ3.72 (16H, br), 3.61
(6H, s), 3.46ꢀ2.48 (32H, br). 13C NMR (400 MHz, D2O): δ (ppm)
174.8 (2 ꢁ C), 169.4 (4 ꢁ C), 164.1 (2 ꢁ C), 151.0 (2 ꢁ C), 121.8 (2 ꢁ
C), 118.9 (2 ꢁ C), 116.0 (2 ꢁ CH), 113.3 (2 ꢁ CH), 109.9 (2 ꢁ CH),
72.2 (6 ꢁ C), 61.4 (2 ꢁ CH3), 56.9 (2 ꢁ CH2), 54.5 (2 ꢁ CH2), 54.1
(2 ꢁ CH2), 52.8 (4 ꢁ CH2), 50.0 (4 ꢁ CH2), 49.5 (4 ꢁ CH2). ESI-MS
m/z: 509.3 (M þ H)2þ, 1017.5 (M þ H)þ.
interaction. The 1:1 donorꢀacceptor interaction was analyzed by
BenesiꢀHildebrand equations for spectrofluorometric titration.
ꢀ
ꢁꢀ
ꢁ
Io
a
1
¼
þ 1
I ꢀ Io
b ꢀ a KB½substrateꢂ
The 1:2 donorꢀacceptor interaction was analyzed by Benesiꢀ
Hildebrand equations for spectrofluorometric titration.
!
ꢀ
ꢁ
2
Io
a
1
¼
2 þ 1
I ꢀ Io
b ꢀ a
KB½substrateꢂ
Io and I are the luminescence intensity of the fluorogenic reagent in
the absence and presence of the substrate, respectively, a and b are
constants, and [substrate] is the concentration of the target analyte. The
binding constants KB were estimated from the ratio between the
y intercept and the slope, which was obtained from the line of best fit by
using BenesiꢀHildebrand equations depending on the 1:1 or 1:2 hostꢀ
guest interactions.34
SDS-Polyacrylamide Gel Electrophoresis Analysis. Amounts
of HSA protein of decreasing concentrations (500 ng to 3.9 ng) were
loaded into the 12% SDS-polyacrylamide gel with the protein marker
(LMW-SDS Marker Kit, GE) and separated under a constant voltage of
200 V for 45 min. The gel was then fixed and stained with dye for 30 min
and analyzed with a Typhoon Trio Imager (GE) with excitation and
emission at 457 nm and ∼620 nm, respectively.35
L2. The procedure for synthesizing L2 was followed, and 0.35 g of I2
was used. A brown solid was yielded (0.31 g, 90%). 1H NMR (400 MHz,
MeOD): δ (ppm) 7.50ꢀ7.48 (4H, d), 7.12ꢀ7.10 (4H, d), 3.93ꢀ3.88
(18H, br), 3.51ꢀ3.29 (32H, br). 13C NMR (400 MHz, MeOD): δ
(ppm) 174.7 (2 ꢁ C), 169.5 (4 ꢁ C), 162.7 (2 ꢁ C), 138.9 (2 ꢁ C),
137.3 (2 ꢁ C), 130.2 (4 ꢁ CH), 121.1 (4 ꢁ CH), 72.9 (6 ꢁ CH2), 61.9
(2 ꢁ CH2), 54.8 (8 ꢁ CH2), 52.1 (8 ꢁ CH2), 41.5 (CH2). ESI-MS m/z:
486.5 (M þ H)2þ, 971.7(M þ H)þ.
General Procedure for the Synthesis of the Ln Complexes.
L1 or L2 (0.2 g) and Ln(CO3)3 (0.1 g) were stirred in H2O (40 mL) at
65 ꢀC for 24 h. Any excess Ln(CO3)3 was filtered off, and the solvent was
removed under vacuum conditions.
EuL1 (0.23 g, 90%). ESI-MS m/z: 1329 (M þ H)þ. Elemental
Analysis: C46H62N10O16Eu2 Calcd: C, 42.62; H, 4.75; N, 10.65. Found:
C, 42.82; H, 4.60; N, 11.02.
The Cellular Uptake of Europium Complexes. To measure
the intracellular concentration of the complex, 1 ꢁ 105 cells were plated
in each well and incubated with the complex with different concentra-
tions (0.01, 0.025, 0.05, 0.1, and 0.2 mM). After coincubation, the
complex-containing cell culture medium was removed, and exposed cells
were further washed with 1 ꢁ PBS 3 times to remove any complex
adhering to the outer cell membrane. Then, the cells were harvested
from the well plates using trypsin-EDTA and dispersed into 1.5 mL of
the culture medium. The exposed cells were collected with a centrifuge,
and the cell pellet was digested in 500 μL of concentrated HNO3 at
70 ꢀC for 4 h. The intracellular concentration of Eu was determined
using an Agilent 7500 series of inductively coupled plasma mass
spectroscopy (ICP-MS). All ICP experiments were performed in
triplicate, and values obtained were averaged. The concentration of Eu
per cell was calculated by determining the concentration of Eu in the cell
lysate by ICP-MS and divided by the number of cells, which was counted
using a hematocytometer.
GdL1 (0.23 g, 90%). ESI-MS m/z: 1326 (M þ H)þ. Elemental
Analysis: C46H62N10O16Gd2 Calcd: C, 41.68; H, 4.71; N, 10.57. Found:
C, 41.65; H, 4.73; N, 10.52.
TbL1 (0.23 g, 90%). ESI-MS m/z: 1329 (M þ H)þ. Elemental
Analysis: C46H62N10O16Tb2 Calcd: C, 41.58; H, 4.70; N, 10.54. Found:
C, 41.60; H, 4.71; N, 12.02.
EuL2 (0.22 g, 85%). ESI-MS m/z: 1283 (M þ H)þ. Elemental
Analysis: C45H60N10O14Eu2 Calcd: C, 42.13; H, 4.71; N, 10.92. Found:
C, 42.56; H, 4.82; N, 11.12.
GdL2 (0.22 g, 85%). ESI-MS m/z: 1280 (M þ H)þ. Elemental
Analysis: C45H60N10O14Gd2 Calcd: C, 42.24; H, 4.73; N, 10.95. Found:
C, 42.22; H, 4.69; N, 10.91.
TbL2 (0.22 g, 85%). ESI-MS m/z: 1283 (M þ H)þ. Elemental
Analysis: C45H60N10O14Tb2 Calcd: C, 42.59; H, 4.77; N, 11.04. Found:
C, 42.72; H, 4.78; N, 11.21.
Tissue Cultures and in Vitro Microscopy Imaging. Human
cervical carcinoma (HeLa) cells were maintained in an RMPI 1640
medium supplemented with 10% fetal bovine serum (FBS) and 1%
penicillin and streptomycin in 5% CO2. Cells were passaged every 3ꢀ5
days. To study the in vitro behavior of the lanthanide complexes,
experiments were carried out with a commercially available UV confocal
microscope [Leica SP5 (upright configuration)] equipped with a xenon
lamp. For the in vitro imaging, the cells were imaged in the tissue culture
chamber (5% CO2, 37 ꢀC). The excitation beam was produced by the
xenon lamp with a power of ∼6 to 10 mW and focused on coverslip-
adherent cells using a 40ꢁ oil immersion or 60ꢁ water immersion
objective. Long pass filter (LB500) was used. An MTT viability assay was
performed, as reported in the previous literature. Briefly, 3000 HeLa
cells were seeded in 96-well plates 24 h prior to exposure to the
europium complex or DMSO as a control. After various exposure time
points, 20 μL of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-
zolium bromide] solution (5 mg/mL) was added to the culture medium
in each well and incubated for 5 h at 37 ꢀC. The medium was removed,
200 μL of DMSO solubilizing reagent was added, and incubation was
carried out for another hour to dissolve the formazan crystals. The
absorbance was measured at 570 nm on a Labsystem Multiskan
microplate reader (Merck Eurolab, Switzerland). MTT assays were
Spectroscopic and Photophysical Studies. UVꢀvisible ab-
sorption spectra in the range of 200 to 1100 nm were recorded with a HP
UV-8453 spectrophotometer. Single-photon luminescence and lifetime
spectra were recorded using an Edinburgh Instrument FLS920 Com-
bined Fluorescence Lifetime and Steady-State Spectrophotometer
equipped with a single photon counting photomultiplier in a Peltier
Cooled Housing (185ꢀ850 nm). The spectra were corrected for
detector response and stray background light phosphorescence. The
solution state quantum yields of the lanthanide complexes were mea-
sured with a Demountable 142 mm (inner)-diameter barium sulphide
coated integrating sphere supplied with two access ports.
For the spectrofluorometric titrations, all of the solvents used were of
analytical grade, and the water used was purified by double distillation.
Measurements were taken after equilibrium was attained, and the 5D0f
7F2 emission (europium) was monitored. Luminescent responses in
terms of Io/(I ꢀ Io) were plotted as a function of analyte concentration.
For the determination of binding strengths of the various analyte
adducts, a series of analyte solutions at a known concentration were
mixed with the anion solutions at various concentrations. The titration
curve was then fitted either with the 1:1 or 1:2 BenesiꢀHildebrand
equations (below) to check whether it was a 1:1 or 1:2 donorꢀacceptor
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dx.doi.org/10.1021/ic2004672 |Inorg. Chem. 2011, 50, 5517–5525