A versatile ditopic ligand system for sensitizing the luminescence of bimetallic lanthanide bio-imaging probes

Article abstract of DOI:10.1002/chem.200701357

The homoditopic ligand 6,6′-[methylenebis(1-methyl-1H-benzimidazole- 5,2-diyl)]bis(4-{2-[2-(2-methoxyethoxy)ethoxy]ethoxy}pyridine-2-carboxylic acid) (H₂L^C2) has been tailored to self-assemble with lanthanide ions (Ln^III), which results in the formation of neutral bimetallic helicates with the overall composition [Ln₂(L ^C2)₃] and also provides a versatile platform for further derivatization. The grafting of poly(oxyethylene) groups onto the pyridine units ensures water solubility, while maintaining sizeable thermodynamic stability and adequate antenna effects for the excitation of both visible- and NIR-emitting Ln^III ions. The conditional stability constants (log β₂₃) are close to 25 at physiological pH and under stoichiometric conditions. The ligand triplet state features adequate energy (0-phonon transition at ≈ 21 900 cm^-1) to sensitize the luminescence of Eu^III (Q = 21%) and Tb^III (11%) in aerated water at pH 7.4. The emission of several other VIS- and NIR-emitting ions, such as Sm^III (Q = 0.38%) or Yb^III (0.15%), for which in cellulo luminescence is evidenced for the first time, is also sensitized. The Eu^III emission spectrum arises from a main species with pseudo-D ₃ symmetry and without coordinated water. The cell viability of several cancerous cell lines (MCF-7, HeLa, Jurkat and 5D10) is unaffected if incubated with up to 500 μM [Eu₂(L_C2)₃] during 24 h. Bright Eu^III emission is seen for incubation concentrations above 10 μM and after a 15-minute loading time; similar images are obtained with Tb^III and Sm^III. The helicates probably permeate into the cytoplasm of HeLa cells by endocytosis. The described luminescent helical stains are robust chemical species which remain undissociated in the cell medium and in presence of other complexing agents, such as edta, dtpa, citrate or L-ascorbate. Their derivatization, which would open the way to the sensing of targeted in cellulo phenomena, is currently under investigation.

Full text of DOI:10.1002/chem.200701357

DOI: 10.1002/chem.200701357
A Versatile Ditopic Ligand System for Sensitizing the Luminescence of
Bimetallic Lanthanide Bio-Imaging Probes
Anne-Sophie Chauvin,* Steve Comby, Bo Song, Caroline D. B. Vandevyver,* and
Jean-Claude G. Bünzli*[a]
Abstract: The homoditopic ligand 6,6’-
[methylenebis(1-methyl-1H-benzimid-
under stoichiometric conditions. The
ligand triplet state features adequate
cell viability of several cancerous cell
lines (MCF-7, HeLa, Jurkat and 5D10)
is unaffected if incubated with up to
A
energy
(0-phonon
transition
at
H
ꢀ21900 cmꢁ1) to sensitize the lumines-
cence of EuIII (Q=21%) and TbIII
(11%) in aerated water at pH 7.4. The
emission of several other VIS- and
NIR-emitting ions, such as SmIII (Q=
0.38%) or YbIII (0.15%), for which in
cellulo luminescence is evidenced for
the first time, is also sensitized. The
EuIII emission spectrum arises from a
main species with pseudo-D3 symmetry
and without coordinated water. The
500 mm [Eu2
(LC2)3] during 24 h. Bright
A
boxylic acid) (H2LC2) has been tailored
to self-assemble with lanthanide ions
(LnIII), which results in the formation
of neutral bimetallic helicates with the
EuIII emission is seen for incubation
concentrations above 10 mm and after a
15-minute loading time; similar images
are obtained with TbIII and SmIII. The
helicates probably permeate into the
cytoplasm of HeLa cells by endocyto-
sis. The described luminescent helical
stains are robust chemical species
which remain undissociated in the cell
medium and in presence of other com-
plexing agents, such as edta, dtpa,
overall composition [Ln
2(LC2)3] and
N
also provides a versatile platform for
further derivatization. The grafting of
poly(oxyethylene) groups onto the pyr-
A
idine units ensures water solubility,
while maintaining sizeable thermody-
namic stability and adequate antenna
effects for the excitation of both visi-
ble- and NIR-emitting LnIII ions. The
conditional stability constants (logb23)
are close to 25 at physiological pH and
A
Keywords: helical
structures
·
tion, which would open the way to the
sensing of targeted in cellulo phenom-
ena, is currently under investigation.
heterometallic complexes · imaging
agents · lanthanides · luminescence
Introduction
sensitive for these purposes and the distinctive properties of
trivalent lanthanide ions,[7] especially their ability to lend
themselves to time-resolved detection in order to eliminate
all background signals and autofluorescence, are of particu-
lar interest. This behavior is well established for lumines-
cence immunoassays[8–11] and has been described in numer-
ous reviews during the last decade.[7–13] Extension to the use
of near-infrared (NIR)-emitting lanthanide bioprobes has
added another exciting dimension to this field.[12–14]
Some of the most important aspects of biochemistry, bioen-
gineering, drug design, and medical diagnosis include the
study and sensing of in cellulo phenomena,[1] the mapping of
selected or targeted analytes,[2,3] the elucidation of receptor–
ligand interactions,[4] and the imaging of organs and cells.[5,6]
Luminescence-based analytical methods are among the most
Several strategies have been developed to encapsulate tri-
valent lanthanide ions into functional molecular structures
in order to meet the stringent requirements imposed on lu-
minescent bioprobes, namely high thermodynamic stability
and kinetic inertness under physiological conditions, cou-
pling and targeting ability, as well as the tuning and/or
switching of key photophysical properties, in addition to
non-toxicity, cell permeability and resistance to photo-
bleaching.[15] The corresponding host molecules may be di-
vided into five categories (polyaminocarboxylates,[10] bipyri-
[a] Dr. A.-S. Chauvin, S. Comby, Dr. B. Song, Dr. C. D. B. Vandevyver,
Prof. Dr. J.-C. G. Bünzli
Laboratory of Lanthanide Supramolecular Chemistry
Øcole Polytechnique FØdØrale de Lausanne (EPFL)
LCSL-BCH 1401 (Switzerland)
Fax : (+41)21-693-9825
E-mail: anne-sophie.chauvin@epfl.ch
caroline.vandevyver@epfl.ch
jean-claude.bunzli@epfl.ch
Supporting Information for this article is available on the WWW
under http://www.chemeurj.org/ or from the author.
1726
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FULL PAPER
dine-based cryptands,[16] cyclen derivatives containing coor-
dinating and sensitizing groups,[17] aza-macrocycles incorpo-
rating a pyridine, bipyridine or terpyridine unit[18,19] and
b-diketonates[20]), which often have in common the simulta-
neous use of carboxylate (or amide) and amine coordinating
groups.
lanthanide ions[44–46] with the aim of developing heterometal-
lic bimodal probes, although this approach is currently limit-
ed to organic solvents. A second series of receptors are sym-
metrical and are based on the parent ligand H2LC
(Scheme 1), which yields very stable neutral homobimetallic
Initial experiments on cell imaging with lanthanide che-
lates have involved the staining of bacterial smears from E.
Coli cell walls,[21] time-resolved studies of 3T3 cultured
cells,[22] and the detection of malignant tumors in C-57 dark-
fur mice with the help of YbIII NIR emission.[23] EuIII probes
have helped to elucidate the nature of the binding sites on
the cell walls of Datura innoxia[24] and to map the receptor-
activated heat waves in Chinese hamster ovaries by a ther-
mal imaging technique.[25] TbIII-based chelates have also
proved useful, as exemplified by a lipid-conjugated chelate
that functions as a membrane-staining agent in morphologi-
cal studies of Swiss albino mouse 3T3 cultured cells,[22] or by
Bornhopꢁs high brightness macrocyclic labels for in vitro
and in vivo analysis of abnormal tissues.[18,26,27] More recent-
ly, Parker and co-workers have designed EuIII complexes
that specifically stain the nucleoli of several cell lines (CHO,
COS, NIH 3T3, HeLa, and HDF).[15,28–30]
Scheme 1. Structures of the homoditopic hexadentate ligands.
Most lanthanide ions are also paramagnetic and possess
an anisotropic susceptibility tensor that makes them useful
for the structural investigation of axial complexes[31] or pro-
teins[32] by NMR spectroscopy, while GdIII has emerged as a
universal contrast agent for 3D imaging of biological struc-
tures (MRI).[33,34] Combining luminescent and magnetic
properties in a single probe system is therefore, an attractive
way of integrating the advantages of both molecular imaging
techniques. This can be done by attaching an organic lumi-
nophore onto a contrast agent[35,36] or by developing recep-
tors that are able to bind GdIII and luminescent LnIII ions
while preserving the specific physical properties of the metal
ions.[19,37,38] Some of these systems bear a targeting vector
for cell internalization.[39] Alternatively, the attachment of
two magnetic or two luminescent lanthanide ions onto a bio-
logically relevant molecule, such as a protein, brings defini-
tive advantages, as demonstrated recently by the generation
of peptide-linked double lanthanide binding tags, which
proved to be superior to single tags with respect to lumines-
cence output[40] and X-ray scattering power.[41]
We are engaged in a strategy aimed at incorporating two
lanthanide ions into a single molecular triple-stranded heli-
cal structure by thermodynamically controlled self-assembly
processes.[42] The resulting homo- or heterobimetallic struc-
tures are amenable to functionalization for covalent biologi-
cal coupling and/or targeting purposes. The spectroscopic
properties of the LnIII ions inserted into these helicates[43]
are fully retained thanks to the protective wrapping of the
ligand strands around the metal ions. We have built a library
of hexadentate ditopic ligands with bis(benzimidazole)pyri-
dine cores, which induce nine-coordinate, tricapped trigonal-
prismatic environments around the 4f ions similar to those
found in aqua ions. Some of these molecules are unsymmet-
rical and are tailored for the recognition of a heteropair of
[Ln2(LC)3] helicates in water, with the EuIII complex having
particularly interesting photophysical properties (quantum
yield=24%, Eu
(5D0) lifetime=2.43 ms;[47] note that the ini-
A
tially published quantum yield is incorrect and has been re-
determined by an absolute method; see Experimental Sec-
tion).
The new chemistry we are now developing allows multi-
purpose derivatization of this initial ligand and the introduc-
tion of solubilizing, chromophoric or coupling groups. We
have recently reported that the EuIII helicate formed by the
benzimidazole-substituted
hexadentate
ligand
H2LC3
(Scheme 1) is internalized into HeLa cells by endocytosis
and stains their cytoplasm.[48] However, better results are ob-
tained by substituting the 4-position of the pyridine units
with poly(oxyethylene) groups (H2LC2),[49] which ensure
water solubility and help to avoid potential stacking of the
luminescent tags.[10] In addition, H2LC2 is easily amenable to
further derivatization for covalent coupling. We present
here a versatile synthetic procedure that leads to the isola-
tion of the ditopic receptor as well as thermodynamic, pho-
tophysical and stability studies of the resulting bimetallic
helicates. The non-cytotoxicity of the [Eu
2(LC2)3] helicate to-
R
wards four cancerous cell lines is established, and the useful-
ness of the bimetallic tags (Ln=Sm, Eu, Tb, Yb) for lumi-
nescence microscopy of living cells is demonstrated.
Results
Ligand design and characterization: H2LC2 was synthesized
according to Scheme 2. The key step in this reaction is the
introduction of poly(oxyethylene) substituents in the para
positions of the pyridine rings. Thus, chelidamic acid (1) was
Chem. Eur. J. 2008, 14, 1726– 1739
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1727

A.-S. Chauvin, C. D. B. Vandevyver, J.-C. G. Bünzli et al.
the removal of the product from the ferric aqueous phase
and preventing premature hydrolysis of the diester func-
tions, which leads to an inseparable water-soluble product.
Final hydrolysis of the diester functions was performed in a
separate step.
The ligand deprotonation constants were determined by
spectrophotometric titration of H2LC2 (2.25”10ꢁ5 m) with
sodium hydroxide at constant ionic strength (0.1m KCl) in
the pH range 0.85–12.77 (see Figure S1 in the Supporting In-
formation). The data were successfully fitted to the follow-
ing set of equations [Eqs. (1–5)], which gave four of the six
pKa values and the sum of the first two:
The corresponding distribution diagram is shown in Fig-
ure S2 in the Supporting Information. The assignments were
made by comparison with H2LC, for which only three depro-
tonation constants [pKa3 =5.4 (calculated from pKa4 by as-
suming a statistical difference) pKa4 =6.0, pKa5 =9.5 and
pKa6 =10.1] could be determined by means of several differ-
ent experimental techniques, including NMR spectroscopy,
owing to solubility problems below pH 6.[51] The largest pKa
values of H2LC2 correspond to deprotonation of the imidazo-
yl groups and the two middle pKa values to deprotonation
of the pyridyl units, and the more acidic values refer to the
carboxylic acid units. A more detailed analysis is beyond the
scope of this study, but we note sizeable differences between
H2LC2 and H2LC, in particular large deviations from the stat-
istical differences for the former, which points to the pres-
ence of intra- or possibly intermolecular interactions. In par-
ticular, hydrogen bonding takes place between the pyridini-
um proton and either the carboxylate or imidazolium
groups in the protonated subunits of the ditopic ligands. The
presence of the poly(oxyethylene) pendant arms on the pyri-
dine units may influence the energy barrier for the trans/cis
conformational change needed for formation of these bonds
considerably, thereby affecting the pKa values.
Scheme 2. Synthesis of H2LC2. Shown at the bottom is the numbering of
the methylene groups in the poly(oxyethylene) substituent. (i) H2SO4,
EtOH, 4 h reflux; (ii) DIAD, PPh3, thf, 12 h reflux; (iii) NaOH, EtOH,
H2O; (iv) SO2Cl2, CH2Cl2, dmf, then 5; (v) Fe, EtOH/H2O, HCl, then
EtOH, H2SO4; (vi) NaOH, EtOH.
converted into its diester 2, and a subsequent Mitsunobu re-
action led to the expected functionalization product in rea-
sonable yield (71%). Selective hydrolysis of 3 in the pres-
ence of a sub-stoichiometric amount of aqueous NaOH with
respect to the carboxylic acid function gave 4, which was
converted into the acyl chloride and then condensed with
bis(N-methyl nitroaniline) (5) in a modified Philips coupling
reaction.[50] The resulting disubstituted product 6 was re-
duced in the presence of iron to give the benzimidazole
units of 7. The difficulties experienced during this step were
Helicate formation in water: Self-assembly of the [Ln
2(LC2)3]
A
helicates in water was monitored by electrospray mass spec-
trometry for the lanthanides La, Eu, Gd, Tb and Lu by
adding stoichiometric amounts of H2LC2 (3”10ꢁ4 m) to the
corresponding perchlorate in the presence of 10% acetoni-
trile to favor ionization. In all cases, only one series of main
peaks, which corresponds to a parent species with overall
formula [Ln
2(LC2)3], was detected (Table 1).
A
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Chem. Eur. J. 2008, 14, 1726– 1739

Sensitizing the Luminescence of Bio-Imaging Probes
FULL PAPER
Table 1. Most abundant species in the ESI mass spectra of H2LC2 (3”
10ꢁ4 m) and of stoichiometric 2:3 (Ln:H2LC2) solutions (Ln=La, Eu, Gd,
Tb, Lu). Solvent: water/acetonitrile (9/1 v/v).
as indicated by the observation of two signals for the meth-
ylene bridge at d=3.75 and 3.85 ppm. Addition of the LuIII
salt results in the appearance of sharp signals concomitant
with a complete disappearance of the ligand resonances (at
R=0.66). Further addition of LuIII does not modify the
spectrum, which is in line with the formation of a single
main triple-stranded helical species in solution with appreci-
able stability (Figure S3 in the Supporting Information). The
pyridine protons of the helicate are shielded, as expected,
owing to coordination to the metal ion, and a large chemical
shift difference (0.47 ppm) is also observed for the nitrogen-
bound methyl group of the imidazole units. The terminal
methoxy groups (d=3.13 ppm) and the methylene bridge
(d=4.08 ppm) appear as one singlet each, which is consis-
tent with three equivalent ligand strands.
m/z
m/z
C
MW
A
A
[Da][a]
A
843.3566
1400.8818
1411.84061411.9049
843.3559
1400.9139
9
23
2800.50
2690.82
G
G
T
G
46
–
A
N
1422.7963
1437.5366
1419.3853
1441.3658
1420.8291
1431.7574
1436.9131
1447.8719
1422.8555
42
84
29
30
72
116–
24
46–
–
G
U
1437.4164
1419.4265
1441.4085
1420.9321
1431.9231
1436.9476
1447.9385
2826.84
2836.85
–
E
ACHTREUNG
E
ACHTREUNG
N
A
2838.85
G
G
R
U
2870.88
H
E
[a] Molecular weight of the helicate with formula C129H144Ln2N18O36.
The interaction of LnIII (Ln=La, Eu, Lu) with H2LC2 was
quantified by spectrophotometric titrations of the ligand
(10ꢁ5 m) with lanthanide perchlorate solutions (5”10ꢁ3 m) up
to a total concentration ratio R of 4 and at pH 7.4. The UV-
vis spectra display well-defined isosbestic points at 318, 262,
and 247 nm (Figure 2, top) and an evolving factor analysis
points to the presence of four absorbing species. Several
models were tested, but the data were best fitted by non-
linear least-squares techniques to the following set of equa-
With few exceptions, the accuracy of the m/z values is in
the range 20–50 ppm. In addition, the isotopic distributions
of these peaks are in good agreement with the simulated
ones, as shown in Figure 1. It is noteworthy that no species
corresponding to other stoichiometries (e.g. 1:2, 1:3, 2:2, or
2:1) are observed, which indicates that the helicates are the
major species in solution.
1
A H NMR titration of H2LC2 (10ꢁ3 m) with lutetium
A
perchlorate was conducted in [D11]tris/DCl buffer (0.1m in
D2O; pD 7.8, tris=trishydroxymethylaminomethane) up to
an [LuIII]t/[H2LC2]t ratio (R) of 2. The signals of the unbound
G
ligand are broadened, especially in the aromatic range,
which reflects a slow conformational exchange on the NMR
timescale between at least two conformations of the ligand,
Figure 2. Top: Analysis, by using UV-vis spectroscopy, of the titration of
Figure 1. Parts of the electrospray mass spectra of solutions containing a
2:3 stoichiometric mixture of LnIII and H2LC2 in water/acetonitrile (9/1,
v/v). Ln=Eu (bottom) and Tb (top). The calculated spectra are shown
above the experimental data. [H2LC2]t =3”10ꢁ4 m.
H2LC2 10ꢁ5 m with La
(ClO4)3·xH2O at pH 7.4 (Tris-HCl 0.1m) and 298 K;
C
G
Distribution diagram for EuIII ([H2LC2]t =10ꢁ4 m) drawn with the condi-
tional stability constants reported in Table 2.
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1729

A.-S. Chauvin, C. D. B. Vandevyver, J.-C. G. Bünzli et al.
tions [Eqs. (6–8)](protons and charges have been omitted
for clarity):
324 nm depending on the metal ion (see Figure S4 in the
Supporting Information). The 2:3 species has quite a large
molar absorption coefficient (loge=4.93), which is a definite
advantage for an analytical probe.
Excitation into the lowest energy band results in emission
from the ligand singlet state, which is observed in the form
of a structured band with two maxima at 384 (0-phonon
transition) and 404 nm; this band undergoes a slight red
shift (4–8 nm) upon complexation. An additional emission
band, which extends up to 650 nm, is observed under time-
resolved conditions at 77 K. This band displays an extended
vibronic fine structure with an average spacing of about
1400–1500 cmꢁ1, which corresponds to a ring breathing
mode. The maximum is located at 489 nm and the 0-phonon
transition at 434 nm (Figure 3), while the corresponding life-
The residuals were calculated to be 2.3”10ꢁ4 for La, 7.6”
10ꢁ4 for Eu and 4.6”10ꢁ4 for Lu. The recalculated spectra
are strongly correlated (Figure S4 in the Supporting Infor-
mation), which means that the conditional constants collect-
ed in Table 2 have to be considered with some care. There is
Table 2. Conditional stability constants (pH 7.4, Tris-HCl 0.1m; 298 K)
for the systems Ln/H2LC2 compared to logb23 for Ln/H2LC (data taken
from ref.[47]). Standard deviations are given in parentheses.
L
Constant
La
Eu
Lu
LC2
logb13
logb23
logb21
logb23
18.8(2)
24.9(4)
11.7(3)
30(1)
18.1(2)
25.5(4)
11.8(5)
26.1(4)
18.7(3)
26.3(4)
12.4(2)
27.3(6)
LC
little variation in these constants on going from La to Lu,
except for a slightly larger stability of the 2:3 helicate with
LuIII. This is not the case for H2LC, however, for which the
lanthanum helicate is significantly more stable. In summary,
the introduction of the poly(oxyethylene) substituents in
H2LC2 does not affect the stability of the helical structures
with the ions in the middle and at the end of the LnIII series
appreciably. The speciation diagram (Figure 2, bottom)
shows that the main species at a total ligand concentration
of 10ꢁ4 m is the helicate (95%), with the other two metal
complexes accounting for about 5% of the total species in
equilibrium. There is a negligible concentration of both free
ligand and EuIII. When the total ligand concentration reach-
es the millimolar range, the helicate represents more than
98% of the species in solution. These findings are consistent
with the ESI-MS and NMR spectroscopic data reported
above and suggest that 2:3 solutions are appropriate for bio-
analyses since the complex concentrations used for the cell-
staining experiments described below are in the range 2.5”
10ꢁ5 to 5”10ꢁ4 m (total ligand concentrations: 7.5”10ꢁ5 to
1.5”10ꢁ3 m).
Figure 3. Left: Absorption (c) and excitation (exc, g) spectra of
4.5”10ꢁ5 m H2LC2 and 1.5”10ꢁ5 m [Ln2(LC2)3]. Right: The corresponding
N
emission spectra upon excitation at 307–323 nm. All spectra were record-
3
ed at 295 K, except the pp* emission spectrum (77 K).
time is 1280Æ125 ms. The latter transition undergoes a red
shift to 457 nm in the 2:3 helicates with non-luminescent
metal ions (see Figure S5 in the Supporting Information),
while the lifetime is reduced to about 620 ms for the com-
pounds with LaIII and LuIII and to 8.7 ms for GdIII. The effi-
cacy of the intersystem crossing to the triplet state is very
low for the unbound ligand; the heavy-atom effect is small
for the LaIII and LuIII helicates (ratio I
and 0.3, respectively) but substantial for GdIII, for which the
3pp* emission is by far the largest (I(3pp*)/I
A
ACHTREUNG
A
ACHTREUNG
The ligand H2LC2 sensitizes the luminescence of several
lanthanide ions at room temperature, with emission in
either the visible or near-infrared ranges. The best sensitiza-
tion is obtained for EuIII and TbIII, for which the proportion
of ligand fluorescence in the total emission spectrum is less
than 1 and 2%, respectively. The metal-centered lumines-
cence is sizeable for SmIII (see Figure S6in the Supporting
Information), accounting for 64% of the total emission,
while only faint LnIII-centered emission is seen for PrIII
Photophysical properties: The uncomplexed hexadentate re-
ceptor displays a main absorption band centered at 307 nm
(loge=4.54) in addition to a shoulder at around 243 nm
(loge=4.58) at pH 7.4. According to CACheꢂ calculation,
these bands arise from p!p* transitions involving intramo-
lecular electron transfer from the benzimidazole units to the
pyridine and carboxylic acid groups. Complexation to LnIII
ions induces a bathochromic shift of about 15 nm to 320–
3
6
(3P0! F2, 3H6, 610 nm), DyIII (4F9/2! H13/2
, , 480,
6H15/2
575 nm) and HoIII (5F5! H8, 650 nm) and none for TmIII
5
(see Figure S7 in the Supporting Information). Bands char-
acteristic of PrIII (1D2! F4: 1.03 mm), NdIII (4F3/2! I9/2, I11/2
3
4
4
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Chem. Eur. J. 2008, 14, 1726– 1739

Sensitizing the Luminescence of Bio-Imaging Probes
FULL PAPER
4
and I13/2: 0.89, 1.06and 1.35 mm respectively), SmIII (4G5/2
!
Table 3. Lifetimes (at room temperature), hydration numbers and quan-
tum yields upon ligand excitation (323 and 355 nm) of stoichiometric 2:3
6H15/2, F5/2 and F7/2: 0.88, 0.94 and 1.04 mm), EuIII (5D0! F5
6
6
7
solutions (EuIII:H2LC2) at 295 K, pH 7.4 and with [H2LC2]t =4.5”10ꢁ5
m
and 7F6: 0.75 and 0.83 mm, respectively), HoIII (5F5! I7:
5
(10ꢁ4 m for NdIII). Data for the EuIII and TbIII helicates with H2LC and
0.99 mm) and YbIII (2F5/2! F7/2: 0.98 mm) are detected in the
7
H2LC3 are also given for comparison.
NIR range, while the I13/2! I15/2 transition of ErIII at
4
4
Ln
t
A
t
A
q
QLn [%]
L
1.54 mm is only seen in deuterated water.
Nd
Sm
0.21Æ0.02
30.4Æ0.4
2430Æ90
2430Æ10
2200Æ100
650Æ20
1.28Æ0.01
163Æ3
0.1
0.031Æ0.006
0.38Æ0.06
21Æ2
The EuIII emission spectrum can be interpreted as arising
from a major species having identical chemical environ-
ments for the two EuIII ions with pseudo-D3 symmetry, as in-
ferred from the high-resolution spectrum of a frozen solu-
[a]
Eu
4380Æ40
4660Æ20
4000Æ100
ꢁ0.1
[b]
0.2
24Æ2
[c]
0
11Æ1
[d]
Tb
940Æ20
11Æ2
tion of [Eu
2(LC2)3] in H2O/glycerol (9/1, v/v) shown in
G
[b]
[a]
[d]
[d]
[a]
50Æ5
1.2Æ0.2
5
7
[c]
Figure 4. The D0! F0 transition is faint and rather symmet-
390Æ40
1400Æ100
0.26Æ0.1
49.3Æ0.8
0.34Æ0.04
[a]
Dy
Yb
0.16Æ0.01
4.40Æ0.07
0.0
0.15Æ0.03
[a] Not determined. [b] Data for [Ln2(LC)3].[47] [c] Data for the main spe-
cies [Ln2
(LC3)3].[48] [d] Equation not applicable (see text).
A
As far as the hydration numbers are concerned, we note
that the usual phenomenological equations are not applica-
ble to TbIII in our case because one of the basic hypotheses
is not met, namely that quenching by OH vibrations is the
only main non-radiative deactivation process. Although far
less important than for the helicates with H2LC[47] and
H2LC3,[49] some back-transfer is operating, which explains the
Figure 4. High-resolution luminescence spectrum of a stoichiometric 2:3
solution (EuIII:H2LC2) in water/glycerol (9/1, v/v) at 10 K, under ligand
relatively short lifetime of the Tb
(5D4) level. This lifetime is
A
excitation (341 nm)
N
N
temperature dependent, reaching 2.58Æ0.05 ms in H2O and
2.67Æ0.04 ms in D2O at 77 K. The latter figure allows a q
value of around ꢁ0.2 to be estimated at this temperature,
which is in line with the other hydration numbers. The Tb-
rical, with a width at half height of 17 cmꢁ1. The energy of
this transition is 17234 cmꢁ1 at 295 K. Using a known equa-
tion[52] and nephelauxetic parameters (dcarb =ꢁ17.2 cmꢁ1 for
the carboxylate and dbzp =ꢁ15.3 cmꢁ1 for heterocyclic nitro-
gen atoms)[53] gives a n˜calc value of 17231 cmꢁ1, which is
A
ity at around 240 K upon cooling, which points to a probable
phase transition occurring at this temperature. However,
this discontinuity is attenuated upon heating from 10 K to
room temperature, and analysis of the data (see Figure S8 in
the Supporting Information) yields an activation energy of
1600–1800 cmꢁ1. This range corresponds to C=O and C=C
vibrations in the IR spectrum and is in line with a vibration-
assisted energy back-transfer process operating in this heli-
cate.
5
7
quite close to the experimental value. The D0! F1 transi-
tion displays a sharp component corresponding to a transi-
7
tion to the F1(A1) sub-level and a second transition termi-
nating on the split 7F1(E) level. The A1–E separation
(161 cmꢁ1) reflects the strength of the crystal field (B2 crys-
0
tal-field parameter of approximately ꢁ600 cmꢁ1), while the
splitting of the E level (31 cmꢁ1) is a measure of the distor-
tion from D3 symmetry.[54] The data for helicates with H2LC
III
(at 295 K) and H2LC3 (at 295 K)[49] are DE
164 cmꢁ1 and DE
N
Stabilityof the Eu helicate: The stability constants report-
N
ed above are consistent with those published for similar sys-
tems. However, since the spectra of the various species in
equilibrium are strongly correlated, we turned to lumines-
cence to check the relative stability of the EuIII helicate with
respect to a known polyaminocarboxylate, namely ethylene-
diamine tetraacetate (edta; logK(Eu)=17.3[58]). The emis-
sion intensity of a 2:3 (EuIII:H2LC2) solution was monitored
for 28 days after initial addition of 15 equivalents of edta
(t=0) and a further 85 equivalents after 44 h. The emission
Taking this distortion into account, the other features of the
spectrum are in line with group-theoretical predictions for
D3 symmetry
The luminescence decays measured for all the emitting
ions are single exponential functions. The corresponding
lifetimes are collected in Table 3 along with the hydration
numbers calculated from known phenomenological equa-
tions for NdIII, EuIII, TbIII and YbIII.[55–57] A hydration
number of almost zero is estimated in each case, which
means that the overall luminescence data clearly indicate
that the chemical environment of the two EuIII ions in [Eu2-
and excitation spectra of [Eu
A
ACHTREUNG
ing to lifetime measurements) and [Eu
AHCTREUNG
ferent (see Figure S9 in the Supporting Information), al-
A
though no change was observed in either the overall emis-
7
phy for the helicate with H2LC.[47]
sion intensity or the intensity ratios I
N
(5D0!
7F1)=7FJ/7F1 (J=0–4; see Table S1 in the Supporting Infor-
Chem. Eur. J. 2008, 14, 1726– 1739
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1731

A.-S. Chauvin, C. D. B. Vandevyver, J.-C. G. Bünzli et al.
mation). Similarly, H2LC2 was added in 2:3 stoichiometry to
an initial solution of [Eu(edta)
(H2O)n]ꢁ (3”10ꢁ5 m;
Figure 5). This latter complex is not luminescent upon exci-
shown in a preliminary communication[49] that incubation of
these cells with up to 500 mm of helicate at 378C during 24 h
does not affect their viability. The data collected in Table 4
A
ACHTREUNG
Table 4. Cell viability values [%] as determined from the WST-1 test
after incubation for 24 h at 378C in the presence of various concentra-
tions of [Eu
2(LC2)3].
A
Conc [mm]
Jurkat
5D10
100
MCF-7
HeLa
0
125
250
500
100
100
100
92Æ1693
101Æ4
107Æ4
Æ11
92Æ13
102Æ7
101Æ1
88Æ3
101Æ1
108Æ2
99Æ1
89Æ4
and Figure S10 in the Supporting Information confirm that
this is also the case for other cell types.
Luminescence microscopy was carried out with an opti-
mized set-up for monitoring the intake and localization of
the [Ln
2(LC2)3] (Ln=Sm, Eu, Tb) helicates. In particular, ex-
A
Figure 5. The increase in luminescence intensity (bottom) and the 7F4/7F2
citation at 330 nm combined with appropriate excitation and
emission filters (see Figures S11 and S12 in the Supporting
Information) yielded bright images even for an incubation
concentration of the EuIII helicate as low as 10 mm. In the
case of TbIII, bright images were obtained on the lumines-
cence microscope with a loading concentration of 125 mm
ratio (top) against time, after adding 4.5”10ꢁ5 m H2LC2 to a 3.0”10ꢁ5
m
solution of [Eu
N
N
tation at 323 nm and the rise in the luminescence intensity
7
7
was monitored with time at both 615 (5D0! F2) and 691 nm
and a specific bandpass filter of 35 nm (5D4! F5 transition).
(5D0! F4; cf. Figure 4); the “recovery” of the total lumines-
Images were also obtained with SmIII despite its low quan-
tum yield (0.38%), albeit at the cost of a higher helicate in-
cubation concentration (250 mm) and longer incubation time
(24 h; Figure 6). Owing to the large molar absorption coeffi-
cient for the maximum ligand absorption at 323 nm, enough
absorbance is left at 405 nm to acquire images with a confo-
cal microscope at this excitation wavelength (Figure 7,
bottom).
7
cence was 75.5% of the expected intensity after 12 h and
more than 92% after 21 days.
Some dissociation of the helicate is observed if edta is re-
placed by 100 equivalents of diethylenetrinitrilo pentaace-
tate (dtpa; logK(Eu)=22.4[58]): the luminescence intensity
drops by about 10% after 24 h and 20% after 30 days, with
a concomitant increase in the ligand singlet-state emission
from 0.5% of the total luminescence (in the absence of
dtpa) to 5%. The helicate also forms upon addition of
H2LC2 to the dtpa complex, although to a lesser extent than
with edta. Since living media contain anions and cations
which may compete for the LnIII ion (exchange reactions) or
for the ligand (transmetalation), we checked the impact of
citrate and l-ascorbate (which may quench Ln excited
states)[15] on the luminescent properties of the EuIII helicate;
no loss in luminescence was recorded four days after addi-
tion of 100 equivalents (1.5”10ꢁ3 m) of either of these
anions. On the other hand, a small decrease in intensity
(approx. 5%) was noted 12 h after adding 10 equivalents of
zinc (150 mm); this concentration is, however, well above
that found in blood plasma (5–15 mm). These experiments
clearly point to the EuIII helicate having a larger stability
than the chelate with edta and a comparable stability to the
dtpa complex, while resisting ligand exchange and transme-
talation reactions with analytes commonly found in living
cells.
The helicates stain the cytoplasm of the cells, as shown by
the luminescence being associated with distinct internal
structures, which suggests a specific organelle localization.
Confirmation of the localization of [Eu
2(LC2)3] in the cyto-
A
Figure 6. Images of HeLa cells obtained upon excitation at 330 nm and
with an exposure time of 60 s with [Tb2
(LC2)3] (top; 7 h incubation, emis-
2(LC2)3] (bottom; 24 h incubation, emission filter LP
Cell-imaging properties: We will now demonstrate the po-
AHCTREUNG
tentiality of the [Ln
A
sion BP 545) and [Sm
585).
ACHTREUNG
on the human cervical carcinoma cell line HeLa. We have
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Sensitizing the Luminescence of Bio-Imaging Probes
FULL PAPER
under non-optimum conditions seemed to indicate the pres-
ence of an active uptake mechanism,[49] however the present
experiments, which were carried out several times under op-
timized experimental conditions, provide unambiguous re-
sults. These findings were further confirmed in co-localiza-
tion experiments in which live cells were loaded simultane-
ously with the EuIII helicate and commercially available BI-
ODIPY FL labeled transferrin, which is a known marker of
recycling endosomes,[59] or LDL, which is a marker for late
endosomes and lysosomes.[60] Cell uptake of transferrin and
LDL is a well-established receptor-mediated process. HeLa
cells were incubated with the [Eu2
(LC2)3] helicate and the or-
G
ganic marker and imaged by luminescence microscopy using
appropriate filters (see Figure 8 and Figure S16in the Sup-
porting Information). The vast majority of compartments
which internalized the EuIII helicate also contained BIODI-
PY FL labeled LDL or transferrin (Figure 8, third column),
as evidenced by the appearance of bright yellow spots
(Figure 8, fourth column) after merging the images, and
nearly all the endocytosed EuIII helicate is internalized in re-
ceptor-containing vesicles. These observations strongly sug-
gest the uptake of the complex by a lysosomally directed
and/or a recycling endosomal pathway.
Figure 7. Top: Counterstaining experiment with acridine orange (AO);
lex =330 nm, exposure time of 10 s for EuIII ; lex =470 nm, exposure time
of 30 ms for AO. Bottom: Intensity of EuIII luminescence versus incuba-
tion concentration [Eu2
(LC2)3] as measured by using a confocal micro-
G
scope. Inset: a densitometry measurement across a cell showing the cyto-
plasmic localization of the helicate.
Helicate concentration in HeLa cells: The intracellular con-
centration of the EuIII complex was measured in a given cell
cohort using the DELFIAꢂ technique. Thus, cells were
loaded overnight with a 25 mm solution of the EuIII helicate
in a six-well cell-culture plate and harvested by trypsinisa-
tion after extensive washing with PBS. The number of la-
beled cells was estimated by staining with trypan blue. The
EuIII luminescence was measured after complete cell lysis
(Figure S17 in the Supporting Information). Each cell con-
plasm came from counterstaining experiments. Thus, live
cells were loaded with the [Eu
2(LC2)3] helicate (250 mm in
A
RPMI-1640, 5 h at 378C) and then incubated with
100 mgmLꢁ1 of the commercially available nucleus stain acri-
dine orange (5 min at room temp.) and examined using ap-
propriate filters (Figure 7): the red EuIII emission is clearly
seen in the cytoplasm of the cells while the green acridine
orange emission originates from the cell nucleus. Addition-
ally, a densitometric analysis across one cell performed with
a confocal microscope also clearly points to the helicate
being present in the cytoplasm.
tains, on average, 7.9”10ꢁ16 mol of [Eu2(LC2)3]. Taking into
A
account a cell volume of between 2.6”103 and 4.2”103 mm3,
as estimated from the cell diameters measured under the
optical microscope, this translates into an intracellular con-
In order to determine the
mechanism by which the cells
take up the helicates and their
subsequent localization within
the cytoplasm, the time course
and temperature-dependence of
the complex loading were de-
termined (Figures S13–S15 in
the Supporting Information).
The first bright spots in the cy-
toplasm of the cells can be ob-
served as early as 15 min after
incubation with the complex.
No EuIII luminescence was ob-
served when the complex was
loaded at 48C, thus indicating
that the helicate is likely to
enter the cells by endocytosis.
Initial experiments performed Third column: EuIII luminescence (lexc =365 nm, 30 s exposure time). Fourth column: merged images.
Figure 8. Co-localization of cells loaded with 250 mm [Eu2
(LC2)] and 15 mgmLꢁ1 BIODIPY FL LDL (0.5 h, top)
C
Chem. Eur. J. 2008, 14, 1726– 1739
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1733

A.-S. Chauvin, C. D. B. Vandevyver, J.-C. G. Bünzli et al.
centration of between 0.18 and 0.30 mm (assuming d=1),
which is about the same as that found for a cyclen-based lu-
minescent stain.[28]
We also checked that the helicates survive unchanged in
the cells by measuring their photophysical properties after
internalization. The overall shape of the emission spectra
for EuIII[49] and TbIII (Figure 9, middle), the profile of the
result of quenching by endogenous antioxidants[15] and will
be studied in more detail in the near future.
Another very encouraging result is the observation of in
cellulo YbIII emission after incubating HeLa cells 24 h at
378C with a 250 mm solution (Figure 9, bottom; cf. Figure S7
in the Supporting Information). The corresponding lifetime
is 4.3Æ0.1 ms, which is perfectly in line with the value found
for an aqueous solution (Table 3).
Discussion and Conclusions
The chemical rules guiding the cellular uptake characteris-
tics of LnIII complexes are not yet known, and prediction of
cellular localization is difficult because it depends on several
parameters, including chemical structure, bulkiness, hydro-
phobicity and the overall charge of the metal chelate. From
the limited amount of literature data available, it seems,
however, that there is a tendency for neutral, high molecular
weight metal complexes to be internalized in living cells by
endocytosis while an active mechanism is operative for low
molecular weight, negatively charged chelates. This was
demonstrated for the first time by the uptake of d-transition
metal complexes, namely iron complexes, by epithelial cells,
in which neutral complexes with high molecular weight li-
gands (e.g. dextran) were taken up by endocytosis and re-
tained in phagosomes whereas the internalization of com-
plexes with low molecular weight ligands, such as 8-hydroxy-
quinoline, was much faster.[62]
A similar trend is observed for lanthanide complexes,
with positively charged, low molecular weight, cyclen-based
complexes being taken up by the cells by an active mecha-
nism and being preferentially localized in the nucleus of
living cells.[28,29,63–65] The microscopy images in these latter
reports show the migration of the complexes through the cy-
toplasm, across the nuclear membrane and into the nucleus,
and show substructures within the nucleus. In these exam-
ples, the homing-in to the nucleus and localization in specif-
ic organelles such as mitochondria, which possess a negative
surface potential and contain DNA, could be a result of the
positive charge of these cyclen-based chelates. The class of
luminescent stains described herein enters into the cells by
endocytosis, as shown by co-localization and temperature-
dependence experiments, therefore interaction with specific
cellular organelles will only be possible if the helicates are
able to escape from the endosomes. It is known that argi-
nine-rich cell penetrating peptides (AR-CPPs) can increase
cellular uptake and the potential of endosomal escape, and
the effect of coupling AR-CPPs to functionalized helicates
on their subsequent cellular uptake and possible endosomal
escape is under evaluation.[66]
Figure 9. A comparison of the emission spectra of [Ln2
(LC2)3] in water
U
7
5D0! F0 transition, Yb (bottom)(initial concentration: 250 mm; incuba-
tion time: 24 h, 378C). lexc =330 nm/Bp=14 nm; lemi =930–1100 nm/Bp=
14 nm (spectra not corrected); integration time t=1 s.
7
Eu
G
tensities of the 5DJ! FJ’ transitions (Table S2 in the Sup-
porting Information) are indeed identical to those found for
the aqueous solutions. Some differences, however, are ob-
served for the lifetimes of the excited levels. In the case of
7
Eu
(5D0), a single exponential decay is measured with an as-
sociated lifetime of 1.6Æ0.2 ms, while TbIII solutions in cel-
The bimetallic luminescent probe system described herein
displays interesting sensitization properties. In the case of
EuIII, the intrinsic quantum yield (i.e. the quantum yield
upon direct metal excitation), which reflects the deactivation
processes operating in the helicate, can be calculated from
the observed and radiative lifetimes. The latter, which can
lulo present a biexponential decay with associated lifetimes
of the Tb
A
corresponds to the lifetime measured in solution at pH 7.4,
and 0.25Æ0.01 ms (45%), which is much shorter than that
of the aqua ion (0.457 ms).[61] These phenomena may be a
1734
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Chem. Eur. J. 2008, 14, 1726– 1739

Sensitizing the Luminescence of Bio-Imaging Probes
FULL PAPER
be estimated by a known procedure,[67] is 6.9 ms for [Eu2-
than H2LC3. This means that if the singlet state were also in-
volved in the overall ligand-to-metal transfer, as has been
demonstrated for some lanthanide complexes,[68] the transfer
may be substantially enhanced in the former system. Alto-
gether, and given the complexity of the ligand-to-metal
energy-transfer processes, we think that the unusually large
enhancement in TbIII luminescence is owed to a combination
of several factors that are a consequence of the connection
of an oxygen atom at position 4 of the pyridine units in
H2LC2, inducing small but significant changes in the ligand
wavefunctions.
A
N
for [Eu2(LC)3].[47] This, in turn, leads to intrinsic quantum
yields of 36.4, 35.7 and 36.7%, respectively. In other words,
substitution of poly(oxyethylene) pendants at either the
benzimidazole or pyridine positions does not affect the de-
activation processes despite the greater flexibility of the
ligand strands. On the other hand, the overall quantum
yields (QLnL) of the EuIII helicates are clearly affected, de-
creasing from 24% for helicates with H2LC to 21% and
11% for helicates with H2LC2, and H2LC3, respectively,
which means that the sensitization efficacy (hsens) provided
by these ligands decreases in the sequence 67%, 58% and
30%, respectively. This is a remarkable electronic tuning of
the photophysical properties of the helicates despite their
very similar ligand-centered photophysical properties (see
Figure S5 in the Supporting Information for a comparison
between H2LC2 and H2LC3). An even more dramatic effect is
found for the helicates with TbIII, an ion which is easily ame-
nable to back-transfer processes. The overall quantum yield
Given the high thermodynamic stability of the reported
helicates, which makes them amenable to in vitro experi-
ments, as well as their versatile and tunable photophysical
properties, which results in their sensitization if bound to
both visible (SmIII, EuIII, TbIII) and near-infrared (NdIII,
YbIII) emitting ions, the new class of bimetallic helical lumi-
nescent tags described here is certainly a valid multimodal
alternative to the existing lanthanide chelates. The reported
in cellulo YbIII emission is a rare example[23] of such an NIR
luminescence and opens wide perspectives for the design of
in vivo NIR imaging probes. The sensitivity of the reported
probes is high, as shown for the EuIII helicate, which stains
the cytoplasm of HeLa cells after only a few minutes of in-
cubation at reasonably low concentrations. The molar ab-
sorption coefficient at the maximum of absorption is around
increases dramatically for [Tb2
(LC2)3] (see Table 3) com-
T
pared to helicates with the other ligands, by factors of about
10 (H2LC)[47] and 33 (H2LC3).[48] The difference observed be-
tween the helicates with H2LC and H2LC2 is consistent with
the increase in the Tb
(5D4) lifetime from 0.05 to 0.65 ms and
N
means that the main factor here is the back-transfer process,
which is more efficient in the unsubstituted helicate. The in-
8.5”104 mꢁ1 cmꢁ1, which means that eQLn amounts to 323
L
crease in quantum yield from [Tb
G
G
(SmIII), 17850 (EuIII), 9350 (TbIII) and 128 mꢁ1 cmꢁ1 (YbIII),
the latter of which compares favorably to the xanthene-de-
rivatized dota-based complexes recently evaluated for cell-
imaging purposes, which have quantum yields of 7% (EuIII)
and 24% (TbIII) with molar absorption coefficients at
the other hand, cannot be explained similarly since the life-
time increases by a factor of only 1.7. It is noteworthy that
if the efficiency of the intersystem crossing, as estimated
from the relative triplet to singlet emission intensity ratios
at 77 K, does not vary substantially between either the two
unbound ligands (factor of about 1.7 in favor of H2LC2) or
between their complexes with LaIII and LuIII (no variation),
it increases by a factor of 4.5 in favor of H2LC2 in the GdIII
chelates. We also observe that the band envelope of the
ligand singlet emission at room temperature (Figure 10) ex-
tends more towards the visible region in the case of H2LC2
336nm of around 6000 mꢁ1 cmꢁ1 and e”QLn values in the
L
range 400–1500 mꢁ1 cmꢁ1.[69] The only drawback of the de-
scribed stains is the relatively low excitation wavelength
needed (even if confocal images can also be obtained upon
excitation at 405 nm), although this can potentially be over-
come by two-photon excitation.
The class of luminescent stains described herein has two
other fascinating features. One of these is chirality, which is
an inherent property of the helicates and means that appro-
priate derivatization of the ligand should result in solutions
enriched in one or the other (P or M) enantiomer. Secondly,
the poly(oxyethylene) arms can be easily functionalized and
we already have at hand several derivatives in which the ter-
minal OMe group (Scheme 1) has been replaced by OH,
CO2H and NH2 groups. Specific targeting experiments will
be conducted with these derivatives as well as time-resolved
imaging and in cellulo NIR detection.
Experimental Section
Figure 10. Comparison of ligand singlet (c, room temperature) and
triplet (g, time-resolved conditions, 77 K) emission bands for [Gd2-
Starting materials and general procedures: Chemicals and solvents were
purchased from Fluka A.G or Aldrich. Solvents were purified by passing
them through activated alumina columns (Innovative Technology Inc.
system).[70] Stock solutions of lanthanides were prepared just before use
A
N
A
Chem. Eur. J. 2008, 14, 1726– 1739
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1735

A.-S. Chauvin, C. D. B. Vandevyver, J.-C. G. Bünzli et al.
in freshly boiled, doubly distilled water from the corresponding Ln-
(ClO4)3·xH2O salts (Ln=La, Lu, Gd, Tb, Eu; x=2.5–4.5). These salts
were prepared from their oxides (Rhône-Poulenc, 99.99%) in the usual
way.[71] The concentrations of the solutions were determined by com-
plexometric titrations using a standardized Na2H2EDTA solution in a ur-
otropine buffered medium with xylenol orange as indicator.[72]
evaporated again. The viscous residue was neutralized with a saturated
solution of NaHCO3 to pH 8 and the aqueous solution was extracted
with CH2Cl2 (4”200 mL). The organic phase was dried with Na2SO4 and
evaporated to give 2 as a pale solid, which solidified upon standing at
room temperature (6.2 g, 95%). 1H NMR (400 MHz, [D6]DMSO): d=
7.53 (s, 2H), 4.33 (q, 3J=7.13 Hz, 4H), 1.32 ppm (t, 3J=7.13 Hz, 6H).
ESI-MS m/z calcd for [M+H+]: 240.06; found 240.32.
ACHTREUNG
Analytical measurements: NMR spectra were recorded at 258C with
Bruker Avance DRX 400 (1H: 400 MHz) and AV 600 (13C: 99.8 MHz)
spectrometers. The spectra of organic compounds were recorded for solu-
tions in CDCl3 (99.8%, Aldrich) or MeOD (99.8%, Aldrich) and those
of the helicates for solutions in D2O (99.9%, Aldrich) or NaOD (0.1m
starting from NaOD 25% from Aldrich (99.5%)); deuterated solvents
were used as internal standards and chemical shifts are given with respect
to TMS. The ESI mass spectra of the ligands were recorded with a Fin-
ningan SSQ 710C spectrometer using 10ꢁ5–10ꢁ4 m solutions in acetoni-
trile/H2O/acetic acid (50/50/1), with a capillary temperature of 2008C and
an acceleration potential of 4.5 keV. The instrument was calibrated
against the horse myoglobin standard and the analyses were conducted in
positive mode. ESI-QTof mass spectra of the complexes were measured
for solutions in water/acetonitrile (9/1 v/v) with a Q-Tof Ultima API
mass spectrometer (Micromass, Manchester, UK) equipped with a Z-
spray type ESI source. Phosphoric acid was used for the positive ion
mass calibration range of 100–2000 m/z. Data were acquired and process-
ed using Masslynx version 4.0. The electrospray conditions were as fol-
lows: capillary voltage: 2.3 kV; source temperature: 808C; cone voltage:
35 V; source block temperature: 1508C. The ESI mobilization and drying
gas was nitrogen. All experiments were performed in positive ion mode.
Diethyl 4-{2-[2-(2-methoxyethoxy)ethoxy]ethoxy}pyridine-2,6-dicarboxy-
late (3): Triphenylphosphane (7.65 g, 29.2 mmol) and 2-[2-(2-methoxye-
thoxy)ethoxy]ethanol (4.8 g, 29.2 mmol) were added to a solution of 2
(3.5 g, 14.6mmol) in thf (200 mL). DIAD (5.8 g, 29.2 mmol) was then
slowly added whilst stirring and the solution was refluxed overnight. The
solvent was evaporated, the crude product was added to 150 mL of 0.01m
NaOH and the solution was stirred 45 min, during which time white tri-
phenylphosphanyl oxide precipitated. After filtration, the filtrate was ex-
tracted three times with 150 mL of CH2Cl2, the combined organic phases
were dried over Na2SO4 and evaporated to give an oil, which was puri-
fied by chromatography (silica or neutral alumina, 100% ethyl acetate)
to provide the desired product as an oil (4.0 g, 71%). 1H NMR
(400 MHz, CDCl3): d=7.80 (s, 2H; Har), 4.47 (q, 3J=7.13 Hz, 4H;
OCH2-CH3), 4.30 (td, 3J=4.75, 4J=1.08 Hz, 2H; H1), 3.90 (td, 3J=4.75,
4J=1.08 Hz, 2H; H2), 3.72 (td, 3J=5.3, 4J=2.6Hz, 2H; H 3,4), 3.66–3.64
(m, 4H; H3,4 and H5,6), 3.53 (td, 3J=4.21, 4J=1.67 Hz, 2H; H5,6), 3.37 (s,
3H; OCH3), 1.38 ppm (t, 3J=7.13 Hz, 6H; OCH2CH3). 13C NMR
(600 MHz, CDCl3): d=166.74 (CAr-O), 164.30 (C=O), 150.13 (Car), 114.36
(CHar)„ 77.01 (OCH2), 71.87 (OCH2), 70.93 (OCH2), 70.61 (OCH2), 70.32
(OCH2), 70.20 (OCH2), 62.31 (OCH2-CH3), 58.95 (-CH2-), 14.15 ppm
(OCH2-CH3). ESI-MS m/z calcd for [M+H+]: 386.17; found 386.36.
The sample was introduced with a syringe pump at a rate of 20 mLminꢁ1
.
The spectra were simulated with Molecular Weight Calculator 6.42ꢂ. UV-
visible spectra were measured for solutions in 0.2-cm quartz Suprasilꢂ
cuvettes with a Perkin–Elmer Lambda 900 spectrometer. Molecular mod-
eling was performed with the CACheꢂ workpackage 7.5 (Fujitsu, 2000–
2006). Protonation constants for H2LC2 were determined with the help of
a J&M diode array spectrometer (Tidas series) connected to an external
6-(Ethoxycarbonyl)-4-{2-[2-(2-methoxyethoxy)ethoxy]ethoxy}pyridine-2-
carboxylic acid (4): Compound 3 (1.3 g, 3.37 mmol) was suspended in a
solution of NaOH (54 mg, 0.4 equiv) in water (200 mL) and the solution
was stirred for 2 h at room temperature. The evolution of the reaction
was followed by TLC (silica plate, CH2Cl2/MeOH 97/3 v/v). After com-
pletion of the reaction, the basic aqueous solution was washed three
times with 50 mL of CH2Cl2, acidified to pH 2.0 with 0.1m HCl, and ex-
tracted four times with 50 mL of CH2Cl2. The combined organic phases
were dried with Na2SO4 and evaporated to give 4 as an oil (0.65 g, 70%).
1H NMR (400 MHz, CDCl3): d=7.88 (s, 1H; Har), 7.85 (s, 1H; Har), 4.46
(q, 3J=7.14 Hz, 2H; OCH2CH3,), 4.33 (t, 3J=4.75 Hz, 2H; H1), 3.91 (t,
3J=4.75 Hz, 2H; H2), 3.74 (d, 3J=6.0 Hz and d, 3J=5.3 Hz, 2H; H3,4),
3.68–3.64 (m, 4H; H3,4 and H5,6), 3.54 (d, 3J=6.4 Hz and d, 3J=4.94 Hz,
computer. All titrations were performed in
a
thermostatted (25.0Æ
0.18C) glass-jacketed vessel at m=0.1m (KCl). Stability constants were
determined by titration of H2LC2 with LnIII (Ln=La, Eu, Lu) at fixed pH
(7.4 in 0.1m Tris-HCl buffer). Factor analysis[73] and mathematical treat-
ment of the spectrophotometric data were performed with the Specfitꢂ
software.[74,75] IR spectra were recorded with a Spectrum One Perkin–
Elmer FT-IR spectrometer equipped with an ATR accessory. Elemental
analyses were performed at the Microchemical Laboratory of the Univer-
sity of Geneva.
3
2H; H5,6), 3.37 (s, 3H; OCH3), 1.38 ppm (t, J=7.14 Hz, 3H; OCH2CH3).
13C NMR (600 MHz, CDCl3): d=167.92 (CAr-O), 163.70 (C=O), 148.34
(Car), 148.18 (Car), 116.11 (CHar), 111.98 (CHar), 71.91 (OCH2), 70.98
The luminescence spectra and lifetimes were recorded with either a
Horiba–Jobin Yvon FL 3-22 fluorimeter or a home-made high-resolution
set-up, according to previously published procedures.[48,49,76] Quantum
(OCH2), 70,64 (OCH2), 70.6(OCH ), 69.01 (OCH2), 68.70 (OCH2), 62.40
2
(OCH2CH3), 59.02 (CH2), 14.21 (OCH2CH3). ESI-MS m/z calcd for
[M+H+]: 358.14; found 358.31.
yields were measured by both a comparative method with [Ln
(dpa)3]3ꢁ as
A
standard[77] and by an absolute method using an integration sphere, as de-
scribed previously.[48] To demonstrate the consistency of the two methods,
Table 5 gathers the results obtained for the EuIII helicates, expressed in
terms of quantum yields relative to [Eu2(LC)3].
Diester 6: Compound 4 (800 mg, 3.11 mmol), freshly distilled thionyl
chloride (3.80 g, 31.1 mmol) and dmf (0.100 mL) were refluxed for
90 min in dry CH2Cl2 (120 mL) under an inert atmosphere. The pale solid
formed after evaporation and pumping for 1 h was redissolved in 100 mL
of dry CH2Cl2 and 2 mL of NEt3. A solution of 3,3’-dinitro-4,4’-bis(N-
methylamino)diphenylmethane (5; 283 mg, 1.2 mmol in 50 mL of
CH2Cl2), which was synthesized according to a known procedure,[79] was
then added dropwise. The resulting mixture was refluxed for 12 h under
an inert atmosphere and the solvents were then evaporated. The orange
residue was redissolved in CH2Cl2 (100 mL) and washed twice with
100 mL of half-saturated NH4Cl solution. The combined organic phases
were dried with Na2SO4, evaporated, and the resulting crude solid was
purified by column chromatography (silica gel; CH2Cl2/MeOH, 100/0!
98/2 v/v) to give 6 as an orange oil (535 mg, 60%). 1H NMR (400 MHz,
CDCl3): d=8.04 (s, 2H; Hpy), 7.79 (s, 2H; Hpy), 7.68 (s, 2H; Har), 7.32 (s,
2H; Har), 6.98 (s, 2H; Har), 4.34 (m, 4H; H1), 4.28 (q, 3J=5.18 Hz, 4H;
OCH2CH3,), 4.02 (s, 6H; NCH3), 3.89 (m, 4H; H2), 3.73 (m, 4H; H3,4),
3.67 (m, 4H; H3,4), 3.63 (m, 4H; H5,6), 3.54 (m, 4H; H5,6), 3.38 (s, 2H;
CH2), 3.35 (s, 6H; OCH3), 1.35 ppm (t, 3J=5.18 Hz, 6H; OCH2CH3).
13C NMR (600 MHz, CDCl3): d=166.74 (Car-O), 164.03 (C=O), 153.78
(Car) , 147.77 (Car), 145.61 (Car), 139.52 (Car), 134.49 (Car), 131.45 (Car),
Table 5. Relative quantum yields (reference: [Eu2(LC)3]) obtained with
the comparative method (left) and with the integration sphere (right).
Eu
Eu
Eu
Eu
Eu
Eu
Qfc
Qfc2
Qfc3
Qfc
Qfc2
Qfc3
Eu
Eu
Qfc
1
0.91
0.47
1.10
1
0.52
2.14
1.94
1
Qfc
1
0.83
0.47
1.20
1
0.561
2.13
1.77
Eu
Eu
Eu
Eu
Qfc2
Qfc3
Qfc2
Qfc3
Diethyl 4-Hydroxypyridine-2,6-dicarboxylate (2): The synthesis of this
compound was adapted from that described by Lamture et al.[78] Thus,
chelidamic acid (1; 5 g, 27.3 mmol) was suspended in absolute ethanol
(100 mL) and sulfuric acid (97%) was carefully added at room tempera-
ture with vigorous stirring. The yellow mixture was refluxed for 4 h and
the solvents evaporated. Water (100 mL) was then added and the solvent
1736
www.chemeurj.org
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2008, 14, 1726– 1739

Sensitizing the Luminescence of Bio-Imaging Probes
FULL PAPER
125.58 (CHar), 113.80 (CHar), 113.42 (CHar), 70.88 (OCH2), 70.59
(OCH2), 70.55 (OCH2), 69.10 (OCH2), 69.03 (OCH2), 68.12 (OCH2),
61.61 (OCH2CH3), 58.99 (OCH3), 40.55 (CH2), 38.55 (NCH3), 14.20 ppm
(OCH2CH3). ESI-MS m/z calcd for [M+H+]: 995.38; found 995.33. calcd
for [M+2H+]/2: 498.20; found 498.37. calcd for [M+Na+]: 1017.31; found
1017.33.
(ATCC HTB-22) were used in this study. Cells were cultivated in 75-cm2
culture flasks using RPMI 1640 supplemented with 5% foetal calf serum
(FCS), 2 mm l-glutamine, 1 mm sodium pyruvate, 1% non-essential
amino acids, 1% 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
monosodium salt (HEPES) (all from Gibcoꢂ Cell Culture, Invitrogen,
Basel, Switzerland). Cultures were maintained at 378C under 5% CO2
and 95% air atmosphere. The growth medium was changed every other
day until the time of use of the cells. The cell density and viability, de-
fined as the ratio of the number of viable cells over the total number of
cells, of the cultures were determined by trypan blue staining and with a
Neubauer improved hemacytometer (Blau Brand, Wertheim, Germany).
WST-1 cell-proliferation assay:[80,81] Cells were seeded in a 96-well tissue
culture microplate at a concentration of between 1 and 5”105 cells per
well in 100 mL of culture medium and incubated overnight at 378C and
5% CO2. The complex was dissolved in fresh RPMI medium at 378C, at
a concentration of 500 mm. The medium was removed from the cell cul-
tures and 100 mL per well of the complex was added (final concentra-
tions: 500, 250, 125 and 50 mm). WST-1 reagent (10 mL, Cell Proliferation
Reagent, Roche, Germany) was added to each well and the plate was
shaken for 1 min on a microtiter plate shaker (450 rpm). The plate was
further incubated at 378C and 5% CO2 and the absorbance of the forma-
zan product was measured at 450 nm with an ELISA reader (Spectra
MAX 340, Molecular Devices, Sunnyvale, CA, USA). The cell viability
[Eq. (9)] was calculated as the average of three nominally identical meas-
urements from the absorbance difference (A450ꢁA650) between 450 and
650 nm for the cells that were in contact with the complex (exp) and the
medium:
Diester 7: Freshly activated iron powder (0.87 g, 15.9 mmol) and HCl
(37%; 4.4 mL, 44 mmol) were added to
a solution of 6 (535 mg,
0.54 mmol) in ethanol/water (110/30 mL). The mixture was refluxed over-
night under an inert atmosphere then the unreacted iron was filtered off
after cooling and the solvents evaporated. The crude product was redis-
solved in ethanol, sulfuric acid was added carefully (2 mL) and the solu-
tion was refluxed overnight. It was then cooled and the solvents re-
moved. Water (100 mL) was added and the pH was adjusted to 6with a
saturated aqueous solution of NaHCO3. H2Na2EDTA was then added
(20 equiv), and subsequent addition of 30% H2O2 (1 mL) resulted in a
brown color. The pH was adjusted to 7 with a saturated aqueous solution
of NaHCO3 before extraction with two 250-mL portions of CH2Cl2. This
procedure was repeated twice and the organic phases were combined and
extracted again with a solution of H2Na2EDTA (20 equiv) in aqueous
NaHCO3. The pale brown crude product was collected after drying with
Na2SO4, filtration and evaporation to dryness, and was purified by
column chromatography (silica gel; CH2Cl2/MeOH, 100/0!97/3) to give
a pale-yellow solid (220 mg, 45%). 1H NMR (400 MHz, CDCl3): d=8.09
(d, 3J=2.27 Hz 2H; Har), 7.71 (d, 3J=2.27 Hz, 2H; Har), 7.69 (s, 2H;
Har), 7.35 (d, 3J=8.34 Hz, 2H; Har), 7.23 (d, 3J=8.34 Hz, 2H; Har), 4.47
(q, 3J=7.07 Hz, 4H; OCH2CH3), 4.35 (m, 10H; NCH3 and H1), 4.25 (s,
2H; CH2), 3.90 (t, 3J=4.55 Hz, 4H; H2), 3.73 (d, 3J=6.1 Hz and d, 3J=
4.8 Hz, 4H; H3,4), 3.68–3.64 (m, 8H; H2, H3,4 and H5,6), 3.52 (t, 3J=
5.05 Hz, 4H; H5,6), 3.35 (s, OCH3; 6H), 1.44 ppm (t, 3J=7.07 Hz, 6H;
OCH2CH3). 13C NMR (600 MHz, CDCl3): d=166.37 (Car-O), 164.75 (C=
O), 152.05 (Car), 149.14 (Car), 148.52 (Car), 142.35 (Car), 136.46, (Car)
135.90 (Car), 125.05 (CHar), 119.68 (CHar), 113.23 (CHar), 111.75 (CHar),
109.89 (CHar), 70.82 (OCH2), 70.50 (OCH2), 70.45 (OCH2), 69.09
(OCH2), 69.02 (OCH2), 68.10 (OCH2), 61.79 (OCH2CH3), 58.99 (OCH3),
42.10 (CH2), 32.83 (NCH3), 14.14 ppm (OCH2CH3). ESI-MS m/z calcd
for [M+H+]: 899.41; found (899.81). calcd for [M+2H+]/2: 450.21; found
450.32.
ðA450ꢁA650Þexp
ð9Þ
viability ½%Š ¼
ꢂ 100
ðA450ꢁA650Þmedium
Live-cell imaging: For luminescence microscopy, cells were seeded on
glass-bottomed cell culture dishes and loaded with the complex dissolved
in freshly prepared cell culture medium. The incubation time varied from
15 min to 24 h, and the concentration of the added complex was in the
range 10–500 mm. Cells were incubated at 37 or 48C for the indicated
time and washed at least six times with PBS before examination under a
luminescence microscope. The cells were examined with a Zeiss lumines-
cence microscope Axiovert S 100 (lens: Plan-Neofluar 20x/0.4 Korr Ph2
or 40x/0.60 Korr Ph2). Excitation was at 330 or 365 nm (BP 80 nm) for
the LnIII helicates, 470 nm (BP 40 nm) for acridine orange (AO),
BODIPY FL labeled LDL or Transferrin, and the emission filters used
were LP 585 nm for EuIII and SmIII, BP 545 (35 nm) for TbIII, and BP
515–565 nm for the organic dyes; exposure times varied from 30 ms (AO)
to 60 s. Confocal images were recorded with a Zeiss LSM 500 Meta mi-
croscope fitted with a Plan-Apochromat 63/1.30 oil objective; Eu lumi-
nescence was excited at 405 nm and measured through a LP 505 filter.
Synthesis of H2LC2: Compound 7 (220 mg, 0.24 mmol, 30 equiv) was dis-
solved in EtOH (0.2 mL), then NaOH (50 mL, 0.02m) was added and the
solution was heated to 608C for 2 h. The evolution of the reaction was
followed by TLC (silica plate; CH2Cl2/MeOH 97/3 v/v). After completion
of the reaction, the basic aqueous solution was washed three times with
50 mL of CH2Cl2, acidified to pH 2.0 with 0.02m HCl and extracted with
four 50-mL portions of CH2Cl2. The organic phase was dried with
Na2SO4 and evaporated. The ligand was further purified by column chro-
matography (silica gel; MeCN/NH4OH, 100/0!88/12 v/v) to give a pale-
yellow solid (164 mg, 80%). 1H NMR (600 MHz, MeOD): d=7.84 (d,
3J=2.18 Hz, 2H; Har), 7.72 (d, 3J=2.18 Hz, 2H; Har), 7.61 (d, 3J=
8.55 Hz, 2H; Har), 7.55 (d, 3J=8.55 Hz, 2H; Har), 7.33 (d, 3J=8.55 Hz,
1H; Har), 4.39 (d, 3J=4.37 Hz, 4H; H1), 4.30 (s, 2H; CH2), 4.29 (s, 6H;
NCH3), 3.95 (t, 3J=4.37 Hz, 4H; H2), 3.74 (d, 3J=6.4 Hz and d, 3J=
5.0 Hz, 4H; H3,4), 3.68 (d, 3J=6.4 Hz and d, 3J=5.0 Hz, 4H; H3,4), 3.64
Concentration of intracellular [Eu
2(LC2)3]: All incubations for the
T
DELFIAꢂ assay were carried out at room temperature with stirring and
a final volume of 200 mL. HeLa cells were cultivated in six-well cell-cul-
ture plates and loaded overnight with [Eu2
(LC2)3] (25 mm) dissolved in
N
fresh cell culture medium at 378C. The cells were washed with PBS at
least 10 times and harvested by trypsin treatment. The number of labeled
cells suspended in a known volume of PBS was counted by trypan blue
staining and with a Neubauer improved hemacytometer (Blau Brand,
Wertheim, Germany). Cells were seeded at 50, 500 and 1000 cells per
100 mL in a 96-well Delfiaꢂ plate and 100 mL of Delfiaꢂ Inducer was
added to induce complete cell lysis. The plate was shaken vigorously for
3
3
3
3
(d, J=6.4 Hz and d, J=5.0 Hz, 4H; H5,6), 3.52 (d, J=6.4 Hz and d, J=
5.0 Hz, 4H; H5,6), 3.35 ppm (s, OCH3; 6H). 13C NMR (600 MHz,
MeOD): d=171.78 (C=O), 168.52 (Car-O), 156.75 (Car), 152.25 (Car),
143.55 (Car), 138.99 (Car), 137.33 (Car), 126.92 (CHar), 120.21 (CHar),
113.56(CH ar), 112.94 (CHar), 112.09 (CHar), 73.43 (OCH2), 72.33
(OCH2), 72.06(OCH 2), 71.89 (OCH2), 70.90 (OCH2), 69.78 (OCH2),
59.55 (OCH3), 43.49 (CH2), 33.73 ppm (NCH3). ESI-MS m/z calcd for
[M+H+]: 843.35; found 843.37. calcd for [M+2H+]/2: 422.17; found
422.37. Elemental analysis (%) calcd for C43H50N6O12·NH4OH·0.5H2O: C
58.21, H 6.37, N 11.06; found: C 58.23, H 6.23, N 10.83.
5 min and was read with
a
1234 Delfiaꢂ fluorometer plate reader
(Perkin–Elmer) using a europium protocol (lex =320 nm, lem =615 nm,
400-ms delay, 1 ms integration time per cycle). To generate a calibration
curve, a known number of cells in 100 mL were spiked with 100 mL of
1 mm to 1 pm [Eu2
(LC2)3] in Delfiaꢂ Inducer and the Eu luminescence was
R
measured as above. Each point is the average of three or four nominally
equivalent experiments.
Cell lines: The human cervical adenocarcinoma cell line HeLa (ATCC
CCL-2), the mouse hybridoma cell line 5D10 (a gift from Prof. Dr. J.
Raus of the Biomedical Research Institute “Dr. L. Willems-Instituut”,
University of Hasselt, Belgium), the human T leukaemia cell line Jurkat
(ATCC TIB152) and the human breast adenocarcinoma cell line MCF-7
Chem. Eur. J. 2008, 14, 1726– 1739
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
1737

A.-S. Chauvin, C. D. B. Vandevyver, J.-C. G. Bünzli et al.
Physics and Chemistry and Rare Earths, Elsevier Science, Amster-
dam, 2003, Vol. 33, chap. 215.
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