A novel strategy for the design of 8-hydroxyquinolinate-based lanthanide bioprobes that emit in the near infrared range

Article abstract of DOI:10.1002/chem.200600964

A new polydentate tripodal ligand T2soxMe was synthesized to take advantage of the chelating effect of tridentate 8-hydroxyquinolinate sub-units. Potentiometric and spectrophotometric titrations reveal seven pK_a values of between 3.7 and 10.2. In water, the use of T2soxMe leads to thermodynamically stable and soluble Ln^III complexes at physiological pH, with conditional stability constants in the range log β₁₁= 7.8-8.6. The chelates are resistant toward hydrolysis and show interesting photophysical proper ties, particularly in the near infrared (NIR) range. The emission lifetimes of the Nd^III and Yb^III complexes recorded in D₂O and H₂O suggest the absence of water molecules in the first coordination sphere of the metal ions. Moreover, the low energy of the triplet state allows efficient energy transfer from the ligand to the metal ions: in waterat pH 7.4, the sensitization efficacy of the NIR luminescence reaches 75 and ≈ 100% for Nd^III and Yb^III, respectively, leading to overall quantum yields of 0.027 and 0.13%; Er ^III luminescence is also detected. According to the WST-1 test, the Yb^III podate at concentrations of up to 250 μM does not display sizeable cytotoxicity for Jurkat cells after 24 h of incubation. Finally, the same podate is shown to couple to human serum albumin, leading to an increase of 50% in the NIR-luminescence intensity.

Full text of DOI:10.1002/chem.200600964

DOI: 10.1002/chem.200600964
A Novel Strategy for the Design of 8-Hydroxyquinolinate-Based Lanthanide
Bioprobes That Emit in the Near Infrared Range
Steve Comby, Daniel Imbert,* Caroline Vandevyver, and Jean-Claude G. Bünzli[a]
Abstract: A new polydentate tripodal
ligand T2soxMe was synthesized to
take advantage of the chelating effect
of tridentate 8-hydroxyquinolinate sub-
units. Potentiometric and spectrophoto-
metric titrations reveal seven pKa
values of between 3.7 and 10.2. In
water, the use of T2soxMe leads to
thermodynamically stable and soluble
LnIII complexes at physiological pH,
with conditional stability constants in
the range logb11 =7.8–8.6. The chelates
are resistant toward hydrolysis and
show interesting photophysical proper-
ties, particularly in the near infrared
(NIR) range. The emission lifetimes of
the NdIII and YbIII complexes recorded
in D2O and H2O suggest the absence
of water molecules in the first coordi-
nation sphere of the metal ions. More-
over, the low energy of the triplet state
allows efficient energy transfer from
the ligand to the metal ions: in water
at pH 7.4, the sensitization efficacy of
the NIR luminescence reaches 75 and
ꢀ100% for NdIII and YbIII, respective-
ly, leading to overall quantum yields of
0.027 and 0.13%; ErIII luminescence is
also detected. According to the WST-1
test, the YbIII podate at concentrations
of up to 250 mm does not display sizea-
ble cytotoxicity for Jurkat cells after
24 h of incubation. Finally, the same
podate is shown to couple to human
serum albumin, leading to an increase
of 50% in the NIR-luminescence in-
tensity.
Keywords: bioprobes · hydroxyqui-
noline · lanthanides · ligand effects ·
luminescence
Introduction
them, the induced-fit concept,[9] sometimes combined with
the use of self-assembly processes,[6,7] has proven to be suc-
cessful in the tailoring of adequate receptors. However,
chemical requirements are not the only constraints in the
design of efficient lanthanide-based luminescent probes:
photophysical aspects must be taken into account, and these
can be summarized simply as follows. Firstly, because f–f
transitions have low oscillator strengths due to their forbid-
den nature, excitation of the luminescent centers relies on
energy transfer from their environment. In molecular com-
pounds, an important path involves the triplet state of the li-
gand(s), so that the coordinated ligand should have high in-
tersystem-crossing efficiency, in addition to a triplet-state
The large degree of attention devoted presently to molecu-
lar compounds that emit in the near infrared (NIR) region
originates from the requirements of medical diagnostics, cell
imaging, and photonic materials, such as planar optical am-
plifiers or light-emitting diodes, for use in the telecommuni-
cations industry.[1–4] The intrinsic photophysical properties of
trivalent lanthanide ions, narrow and easily recognizable
emission lines, relatively long lifetimes of their excited
states, and large Stokesꢀ shifts upon excitation through indi-
rect processes,[5] make them ideal candidates as emitting
centers for these applications. Trivalent lanthanides are hard
spherical ions without stereochemical preferences and,
therefore, their specific insertion into stable and kinetically
inert environments, a requirement for efficient luminescent
probes, necessitates carefully planned strategies.[6–8] Among
energy E
(3pp*) close to, but larger than, the emissive excit-
A
ed state of the metal ion. Energy transfer from the ligand
singlet state is also feasible, particularly for NdIII, but is usu-
ally less efficient. For YbIII, which, contrary to most other
lanthanide ions, possesses only two electronic levels, the sit-
uation is more complex and either phonon-assisted transfer
from the triplet state (or metal-to-ligand charge-transfer
states) or an internal double-electron transfer mechanism
[a] S. Comby, Dr. D. Imbert, Dr. C. Vandevyver, Prof. Dr. J.-C. G. Bünzli
Laboratory of Lanthanide Supramolecular Chemistry
École Polytechnique FØdØrale de Lausanne (EPFL)
BCH 1402, 1015 Lausanne (Switzerland)
2
account for the population of its F5/2 fluorescent state.[10,11]
Fax : (+41)21-693-9825
Secondly, the metal ion environment must be rigid enough
and devoid of high-energy oscillators to avoid de-excitation
through vibrational processes. In the case of NIR-emitting
E-mail: Daniel.Imbert@epfl.ch
Supporting information for this article is available on the WWW
under http://www.chemeurj.org/ or from the author.
936
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Chem. Eur. J. 2007, 13, 936 –944

FULL PAPER
ions, such as NdIII, ErIII, and YbIII, the presence of high-
droxyquinoline subunits was achieved by using oleum
(H2SO4, SO3), as already reported for 2-carboxy-8-hydroxy-
quinoline,[15] giving an overall yield of 27%.
ꢁ
ꢁ
ꢁ
energy oscillators (C H, N H, O H) in the ligand(s), al-
though outside the inner coordination sphere, leads to dra-
matic extinction of the luminescence. As a consequence, the
overall quantum yield QLLn, obtained by excitation in the
ligand levels, remains very small, particularly in water, usu-
ally <0.1% for NdIII, <0.001% for ErIII, and <0.1% for
YbIII. Indeed, this parameter is linked to the sensitization ef-
Solution study: thermodynamic aspects
Ligand deprotonation constants: The fully protonated
T2soxMe ligand, denoted (H10L)4+, possesses ten deprotona-
tion sites, three pyridinium groups, and one tertiary nitrogen
atom, three hydroxyl oxygen atoms, and three sulfonate
groups. The three sulfonate groups are totally deprotonated
under the conditions used; the starting species is generally
denoted (H7L)+ in this paper. The deprotonation constants
were determined separately by potentiometry and spectro-
photometry. The extracted pKai values obtained by titrations
in the 1.5–12.5 pH range are defined by Equations (1) and
(2):
ficacy of the ligand, hsens, and the intrinsic quantum yield
Ln
Q , determined upon direct excitation in the LnIII levels
Ln
and which consequently reflects the deactivation processes:
QLLn ¼ hsens Á QLLnn
The sensitization efficacy of the ligand is well established,
with hsens commonly much larger than 50%, as we have re-
cently shown with an appended terpyridine–boradiazainda-
cene dye used both as chelating and sensitizing unit.[12] This
is not yet the case for the deactivation processes, particularly
for the luminescent ions that have a small energy gap be-
tween the emitting level and the next electronic level of
lower energy, unless substantial modifications of the ligand,
such as deuteration or fluorination, are performed.[3]
However, by taking into account the fact that YbIII ap-
pears to be the lanthanide ion best adapted for biomedical
applications, we recently showed that TsoxMe, a tetrapodal
ligand featuring bidentate 7-carboxy-8-hydroxyquinolinate
coordinating units, meets both the chemical (pLn=15–16)
and photophysical (hsens ꢀ70–100% for NdIII and YbIII) re-
quirements for a NIR-emitting luminescent probe, with a
quantum yield of up to 0.37% in water for YbIII.[13] Here,
we tested a novel strategy based on the same chromophore,
but in which the chromophore was connected by its 2-posi-
tion, which renders it tridentate, and to a smaller frame-
work, namely tris(ethylamine) rather than N,N’-bis-
H8ꢁiLð2ꢁiÞþ Ð H7ꢁiLð1ꢁiÞþHþ
ð1Þ
ð2Þ
Kai ¼ ½H7ꢁiLð1ꢁiފ Á ½HþŠ=½H8ꢁiLð2ꢁiÞþ
Š
The potentiometric titration of the protonated form
(H7L)+ of the ligand with NaOH in 0.1m KCl at 258C (see
Supporting Information, Figure S1) allowed the determina-
tion of four deprotonation constants. Analysis of the poten-
tiometric titration curve over the pH range 2.5–12.26 gave
four pKai values (Table 1), defined by Equations (1) and (2).
Spectrophotometric titration carried out in the pH range
1.04–12.99 at lower concentration (Figure 1) allowed us to
1) establish the pKa values that could not be determined by
potentiometry, and 2) confirm the values already obtained
in the higher-pH range. The absorption spectra (Figure 1a)
show the presence of several isosbestic points at 253, 254,
263, 297, 329, 334, and 410 nm. The best refinement allowed
us to determine all the pKa values, except for pKa3 and pKa4,
for which only the sum could be calculated (Table 1). The
calculated spectra (Figure 1b) are in good agreement with
(propyl)ethylenediamine. In addition to the thermodynamic
and photophysical studies of the resulting podates (Ln =
Pr, Nd, Gd, Er, Yb) in water, we also tested the cytotoxicity
of the YbIII chelate, as well as
its ability to couple with human
serum albumin (HSA).
Results and Discussion
Ligand T2soxMe was obtained
according to the synthetic path-
way depicted in Scheme 1. The
starting triamine is a commer-
cial product and was methylat-
ed as previously described.[14] In
the next step, this product was
condensed with activated 2-car-
boxy-8-hydroxyquinoline
give the tripodal
to
ligand
T2oxMe. Finally, the regiospe-
Scheme 1. Synthetic pathway used to obtain ligand T2soxMe: i) a) EtCO2Cl, H2O, toluene; b) LiAlH4, THF;
cific sulfonation of the 8-hy- ii) 2-carboxy-8-hydroxyquinoline, N,N’-carbonyl diimidazole (CDI), THF; iii) H2SO4, SO3 (20%).
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937

D. Imbert et al.
Table 1. Deprotonation constants of (H7L)+ (Æs, see Equations (1) and
(2) for the definition of Kai).
case, four pKa values are between 6.85 and 10.23, the first
being attributed to the central nitrogen as it is close to the
value reported for O-Trensox. The three other dissociation
constants have an average of 9.09 and are due to the hy-
droxyl moieties. The corresponding values for O-Trensox
are between 7.44 and 8.62 with an average of 8.07, whereas
that for 5-sulfo-8-hydroxyquinoline is 8.42. This difference
can be attributed to the position of the carboxylation in
T2soxMe, position two instead of position seven in the case
of O-Trensox and, moreover, the three pKa values differ
from the statistical factor of log3=0.48 (the value for nonin-
teracting arms). In contrast to the situation for O-Trensox,
this indicates cooperativity between the arms of the tripodal
ligand, which is probably due to the presence of the methyl
groups and of intra- or intermolecular hydrogen bonds. The
corresponding distribution curves are presented in Figure 2.
[a]
[b]
[a,b]
logb10
n
pKa1
–3.7(1)
47.0(1)
pKa2
pKa3 +pKa4
pKa4
pKa5
pKa6
–4.62(7)
–11.41(6)
6.85(9)
8.33 (9)
8.70(9)
43.29(7)
38.67(6)
–34.11(9)
7.88(6)
27.26(9)
18.93(9)
10.23(8)
9.01(5)
pKa7
10.23(8)
10.05(3)
[a] Potentiometric data: [L]0 =5.98·10ꢁ4 m; m=0.1m (KCl); T=258C.
[b] Spectrophotometric data: [L]0 =6.5·10ꢁ6 m; m=0.1m (KCl); T=
25.08C.
Figure 2. Distribution curves of the ligand computed from the pKa values
at pH 2–12.
Interaction with trivalent lanthanide ions: To unravel the in-
fluence of pH on the speciation, the equilibria of the differ-
ent metal complexes present in solution were studied by
performing potentiometric and spectrophotometric titra-
tions. The potentiometric titration curve of a 1:1 solution of
YbIII and T2soxMe did not enable us to determine the
global constants defined by Equations (3) and (4), in spite
of several models tested to refine the titration data:
Lnaq þ HnL Ð LnðHnꢁiLÞ þ iHþ
Figure 1. a) UV/Vis absorption spectra of (H7L)+ as a function of pH in
water: [T2soxMe]=6.5·10ꢁ6 m; T=25.0Æ0.18C; m=0.1m (KCl). b) Recal-
culated spectra of the various species.
i
½LnðHnꢁiLފ Á ½HþŠ
ð3Þ
ð4Þ
KLnLH
¼
nꢁ1
½HnLŠ Á ½LnaqŠ
LnðHLÞ þ nH2O Ð LnLðOHÞn þ nHþ
n
½LnLðOHÞnŠ Á ½HþŠ
the experimental data. Extractions at different wavelengths
are presented in the Supporting Information, Figure S1.
The three lowest pKa values are attributed to the pyridini-
um nitrogen atoms by comparison with the pKa values of O-
Trensox (1.83, 2.55, and 3.01),[16] a tripodal ligand based on
sulfonated 7-carboxy-8-hydroxyquinoline, and with the value
of 3.92 published for 5-sulfo-8-hydroxyquinoline.[17] In our
KLnLðOHÞ
¼
n
½LnðHLފ
Despite the complexity of the system, it is clear from the
spectrophotometric data that at physiological pH only one
major species is present. The stability of this species appears
to be comparable to the one determined for the tetrapodal
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Lanthanide Bioprobe Near-Infrared Emitters
FULL PAPER
complexes with TsoxMe (e.g., pEu=14.9[13]). For instance,
the addition of two equivalents of the tetrapodal ligand
leads to partial decomplexation of [Ln–T2soxMe] only. On
the other hand, the addition of one equivalent of diethylene-
triaminopentaacetic acid (DTPA) (pEu=19.6 computed
from known stability constants[17]) results in a large dissocia-
tion of the EuIII tripodal chelate. Therefore, we estimate
that the pEu value for the T2soxMe chelate is between 15
and 19, and the corresponding values for the other lantha-
nide ions studied here should be similar, referring to the
range of values obtained for TsoxMe (15–16).
served during UV-visible titration experiments because the
concentrations were too low. The same evolution was ob-
served for the signals in the aliphatic part of the NMR spec-
tra. Other titration experiments with the paramagnetic pra-
seodymium cation gave similar results (Figure 4). In addi-
tion, one signal is present for each proton of the 8-hydroxy-
quinoline subunit, indicating that the three arms are coordi-
nated to the lanthanide ion and, thus, are equivalent on the
NMR timescale.
Next, the interaction between LnIII ions (Ln = Nd, Gd,
Dy, Yb) and the ligand was monitored by UV-visible spec-
trophotometry (Figure 3) by using dilute aqueous solutions
Figure 4. Evolution of the 1H NMR spectra of T2soxMe in buffered D2O
(Tris-DCl, pD 7.5) as a function of added PrIII
.
Photophysical properties of T2soxMe: In water, the absorp-
tion spectra of the ligand features two main bands (with ad-
ditional shoulders) located at around 38000 and 26500 cmꢁ1
and assigned to p!p* and n!p* transitions. The high-
energy band is blue-shifted by 1400 cmꢁ1 in progressing
from the acidic to the basic forms of the ligand. The low-
energy absorption appears to be more sensitive to the de-
protonation process of T2soxMe (a shift of 5000 cmꢁ1).
Under excitation at these wavelengths, an emission band ap-
pears, centered at 19000 cmꢁ1, which disappears upon en-
forcement of a time delay and is consequently assigned as
Figure 3. Evolution of the UV/Vis absorption spectra of a solution of
T2soxMe in buffered water (pH 7.4, HBS) upon addition of increasing
amounts of GdIII
.
at pH 7.4 in Hepes-buffered saline (HBS buffer). The varia-
tion in absorbance versus R=[LnIII]tot/[L]tot indicates the
presence of one species, the 1:1 complex, in the stoichiome-
try range investigated (0<R<4) for which [L]0 =6.5·10ꢁ6 m,
pH 7.4, and T=25.08C (20.08C for YbIII). The conditional
stability constants extracted from these data are logb11 =
8.2(5), 8.5(7), 8.6(6), 7.8(2) for Ln = Nd, Gd, Dy, and Yb,
respectively.
1
arising from a pp* state. At low temperature (77 K, in solu-
tions containing 10% glycerol), a second emission band ap-
pears in the range 16500–17500 cmꢁ1, corresponding to
phosphorescence from the triplet state(s). The ligand-cen-
tered luminescence spectra of the 1:1 complexes with LaIII,
EuIII, GdIII, and LuIII, both at room temperature in aqueous
solutions and at 77 K in a frozen water/glycerol mixture,
have essentially the same features, with slight shifts of the
maxima (Figure 5). The phosphorescence decays are mono-
exponential functions, with associated lifetimes of 29(1),
0.51(2), and 21.5(5) ms for the complexes of LaIII, GdIII, and
The complexation of T2soxMe with PrIII, NdIII, and LuIII
1
was also investigated by conducting H NMR experiments in
which the spectra of the ligands (10ꢁ3 m in D2O) were moni-
tored as a function of aliquots of cations added, the pD
being fixed at 7.5 by using Tris-DCl buffer (0.1m). For dia-
magnetic lutetium (Figure S2, Supporting Information), a
rapid evolution was observed during the addition of LuIII,
until 0.8 equivalents of metal had been added. Further addi-
tions did not result in an observable spectral change. Clear-
ly, only one species is formed, with a stoichiometric ratio of
1:1. For Ln:L ratios larger than 1, the peaks remained iden-
tical and a new one appeared at 9.15 ppm after addition of
between 1.5–3 equivalents of LuIII. This could indicate the
possible formation of polynuclear species that were not ob-
3
LuIII, respectively. In the case of the EuIII complex, the pp*
emission is not detected, and the characteristic emission
lines of the metal ion are observed, reflecting a complete
3pp*-to-EuIII transfer at low temperature. At room tempera-
ture, the EuIII luminescence is not observable, due to the
energy gap DE
(3pp*–5D0) being too small and allowing effi-
G
cient back-transfer onto the ligand.
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939

D. Imbert et al.
Figure 5. Luminescence spectra of T2soxMe and its 1:1 complexes at
77 K in 6·10ꢁ5 m in HBS buffer pH 7.4 containing 10% glycerol, without
(b) and with (g) a 0.05 ms time delay, and at RT (c).
Figure 6. Left: Normalized absorption and excitation spectra (g, lan
=
1540 (Er), 1063 (Nd), and 976 nm (Yb)). Right: Emission spectra in the
NIR region (lex =417 nm) of the 1:1 chelates of ErIII, NdIII, and YbIII. All
solutions were in water at pH 7.4 and RT.
Sensitization of lanthanide-centered NIR emission by
T2soxMe: All the chelates with NdIII, ErIII, and YbIII display
metal-centered NIR luminescence in aqueous solutions at
physiological pH. Moreover, the ligand luminescence almost
disappears, with only a faint emission from the singlet state
(ꢀ5–10% compared with the free ligand), which indicates
an efficient energy-transfer process from the ligand levels to
the acceptor levels of the metal ions. At room temperature
and upon broad band excitation through the ligand levels
both at 274 (36500 cmꢁ1) and 417 nm (23980 cmꢁ1), the lu-
minescence spectra display the peaks corresponding to the
expected transitions. The YbIII complex emits in the 920–
1100 nm range, with a sharp band at 977 nm assigned to the
Table 2. Metal-ion-centered lifetimes (t) for Nd
N
N
AHCTREUNG
D2O solution, absolute quantum yields (Q, relative uncertainty Æ20%),
and number of water molecules bound in the inner coordination sphere
(q).
Ln[a]
lan
[nmꢁ1
]
t[ms]
QLLn [%]
t[ms]
QLLn [%]
q[b]
H2O
H2O
D2O
D2O
Nd
Er
Yb
1063
1540
976
0.15(2)
0.24(2)
2.47(6)
0.027(5)
–2.55(2)
0.13(3)
0.91(4)
3.5·10
26.6(6)
0.075(15)
0.31
–
0.16
ꢁ3
1.5(3)
[a] [Ln–T2soxMe]=6.5·10ꢁ5
m (Ln = Er, Yb, Nd). [b] From Equa-
2
2F5/2! F7/2 transition and a broader vibronic component[18]
tions (5) and (6); see text for uncertainties.
at longer wavelength. Under the same conditions, the NdIII
complex displays NIR luminescence in the 840–1430 nm
range, the main band being centered at 1064 nm. The three
bands occur in the ranges 840–940, 1020–1135, and 1290–
longer in deuterated water. The former lifetimes are in line
with those published for polycarboxylate complexes derived
from DTPA,[19] 0.58, 1.46, and 10.4 ms for NdIII, ErIII, and
YbIII, respectively, and more recently, from the ligand
TsoxMe: 0.61(1), 2.31(2), and 14.6(1) ms for NdIII, ErIII, and
YbIII, respectively.[13]
The number of bound water molecules around the metal
ions in the Ln–T2soxMe podates with NdIII and YbIII was
determined by using Equations (5) and (6), proposed by
Faulkner and co-workers[20,21] for NdIII aminocarboxylates
(q=0–2), and by Beeby and co-workers[22] for YbIII com-
plexes (qꢂ1):
4
1370 nm, and are assigned to transitions from the F3/2 level
4
4
4
to the I9/2, I11/2, and I13/2 sublevels, respectively. Finally, the
luminescence spectrum of the ErIII complex in solution was
4
4
recorded, displaying the typical I13/2! I15/2 transition cen-
tered at 1540 nm. If water is replaced by deuterated water,
the shape of the luminescence spectra remains unchanged,
as expected, but the overall intensity increases considerably.
The close similarity in the absorption and excitation spectra
(Figure 6, left) of the three lanthanide chelates (NdIII, ErIII,
and YbIII) clearly indicates sensitization of the NIR lumines-
cence by the ligand.
q ¼ AðDkobsÞꢁC for NdIII
q ¼ AðDkobsꢁBÞ for YbIII
Furthermore, we determined the lifetimes (Table 2) of the
ð5Þ
ð6Þ
Nd
A
G
N
tion by using the Nd:YAG line at 355 nm. The luminescence
decays are all monoexponential and the corresponding life-
times in water are 0.15(1), 0.24(1), and 2.47(1) ms for NdIII,
ErIII, and YbIII, respectively. These lifetimes are appreciably
in which A=130 ns (NdIII) or 1 ms (YbIII), B=0.2 msꢁ1, and
C=0.4; Dkobs is given in nsꢁ1 and msꢁ1 for NdIII and YbIII, re-
940
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Chem. Eur. J. 2007, 13, 936 –944

Lanthanide Bioprobe Near-Infrared Emitters
FULL PAPER
spectively, kobs =1/tobs, and Dkobs =kobs
G
(D2O). By
using these Equations, we obtain q=0.31 and 0.16 for NdIII
and YbIII, respectively. These values are much smaller than
unity, indicating complexes with no coordinated water mole-
cules around the metal ion. This is in line with a complete
coordination of the three pendant arms of the podand that
acts consequently as a nonadentate host. Indeed, given the
uncertainty of q (Æ0.1–0.3), we do not think that the residu-
al values reflect equilibria involving hydrated species, but,
rather, that they arise from second-sphere effects not taken
into account by parameters B and C.
To quantify the ability of the chromophoric subunits of
T2soxMe to sensitize the NIR-emitting lanthanides, the ab-
solute quantum yields of the Ln–T2soxMe podates (Ln =
Nd, Er, Yb) were determined in aqueous solutions upon
ligand excitation. Under the experimental conditions used
(6.5·10ꢁ5 m HBS-buffered solutions, pH 7.4), the quantum
yields are 0.027 and 0.13% for NdIII and YbIII, respectively,
and these increase by 2.8- and 11.5-fold in deuterated water,
respectively. ErIII luminescence in water is too weak for a
quantum yield to be determined, however, the correspond-
ing value could be measured in D2O. These quantum yields
are large relative to published data and are of the same
order of magnitude to those reported for TsoxMe,[13] partic-
ularly for YbIII and for aqueous solutions in which the pres-
ence of proximate OH vibrations in the second coordination
sphere induces a large quenching effect for the ions that
have a small energy gap. If we use, as a first approximation,
the radiative lifetimes of 0.42 and 2 ms for NdIII and YbIII
Figure 7. Cell viability values (%) obtained by using the WST-1 test of
Jurkat cells for different concentrations of the Yb–TsoxMe complex after
24 h incubation.
first equivalent of HSA and remained constant upon further
additions. This indicates that addition of this protein to the
medium does not quench the luminescence of the complex,
but rather, that interaction between the complex and the
protein takes place, resulting in an increase in luminescence
intensity. On the other hand, the lifetime of the YbIII level
does not change significantly in response to HSA addition
(between 2.47 and 2.52 ms for 0.1–100 equivalents of HSA).
corresponding to the aquo ions,[13] we find that hsens =QLLn/
Ln
Q
ꢀ75 and 100% for the ligand sensitization efficacy in
Conclusion
Ln
the NdIII and YbIII chelates, respectively. That is, the modest
overall quantum yields are essentially due to deactivation
processes involving vibrational oscillators located outside
the first coordination sphere.
The tripodal ligand proposed here perfectly meets the chem-
ical requirements for the design of an in vitro luminescent
probe in that it wraps around the LnIII ions, yielding hydro-
lysis-resistant, stable podates at physiological pH. The
Cytotoxicity and interaction with HSA: To quantify the influ-
ence of the Yb–T2soxMe complex on the viability of the
cells, the WST-1 test was used. To determine the optimal in-
cubation time, a preliminary experiment was performed by
taking measurements after 0.5, 1, 2, 3.5, and 4.5 h (Figure S3,
Supporting Information). An increase in absorbance (450–
650 nm) is observed over time, reflecting the proliferation of
the cells (Figure S4, Supporting Information). However, for
concentrations of Yb–T2soxMe complex >125 mm, a nega-
tive influence on cell proliferation is observed and is con-
firmed by measurements after 24 h. Compared to the cells
not incubated with the complex, there is no significant
change in cell viability for concentrations up to 125 mm.
However, the viability drops to 90% for 250 mm and to 70%
for 500 mm (Figure 7), indicative of a toxic effect occurring
at only high concentrations of the Yb–T2soxMe chelate.
Finally, we measured the luminescence intensity and the
lifetimes of the Yb–T2soxMe complex in a buffered medium
containing different quantities of HSA, from 0 to 100 equiv-
alents with respect to the complex, as reported previously.[23]
The luminescence increased by 50% upon addition of the
podand acts as a nonadentate receptor, as demonstrated by
the equivalence of the three arms in the NMR spectra and
by the absence of water molecules in the inner coordination
sphere. Thus, one of the main deactivation paths of the LnIII
NIR luminescence is avoided.[3] Relative to our previous ap-
proach using TsoxMe, a tetrapodal ligand fitted with biden-
tate coordinating pendant arms,[13] the present strategy
making use of a tris(tridentate) tripodal ligand is to some
extent easier with respect to ligand synthesis and it yields
chelates with similar stability. This strategy could be extend-
ed easily to the design of similar podates featuring a carbon
anchor offering various derivatization possibilities. On the
other hand, the photophysical properties are not as good, as
exemplified by the shorter lifetimes of the tripodal deriva-
tives relative to the tetrapodal chelates. In fact, the photo-
physical parameters are comparable to those obtained for
the chelates with Tsox, the unmethylated analogue of
TsoxMe, that is, they are still better than the ones reported
for most of the complexes published to date.[3] The YbIII
podate appears to be particularly promising as its cytotoxici-
ty is low (Jurkat cells, 24 h incubation, >90% cell viability
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941

D. Imbert et al.
sulfonic acid (HEPES, 10 mm) and a solution of NaCl (150 mm, in doubly
distilled water). Stock solutions of lanthanides were prepared just before
use in freshly boiled, doubly distilled water from the corresponding per-
at concentrations <250 mm), and because it couples to HSA
with a concomitant increase in luminescence intensity. This
opens real perspectives for the development of bioprobes
based on this system. Indeed, improvement of the photo-
physical properties by taking advantage of the heavy-atom
effect of the bromine substituents[24] on the 8-hydroxyquino-
chlorate salts Ln(ClO4)3·nH2O (Ln = La, Nd, Eu, Gd, Dy, Er, Yb, and
R
Lu; n=3–6). Caution! Perchlorate salts combined with organic ligands
are potentially explosive and should be handled in small quantities and
with adequate precautions.[26,27] These salts were prepared from their
oxides (Rhône–Poulenc, 99.99%) in the usual way.[28] Concentrations of
solutions were determined by performing complexometric titrations using
a standardized Na2H2EDTA solution in urotropine-buffered medium and
with xylenol orange as indicator.[29] UV/Vis absorption spectra were mea-
sured by using a Perkin–Elmer Lambda 900 spectrometer with quartz Su-
prasil cells of 0.2- and 1-cm path length.
ꢁ
linate moiety is within reach. Furthermore, proximate C H
vibrators could be removed by substitution with fluorine.
These improvements are in progress in our laboratory and
we are also working towards the development of a cell-
imaging system based on this and other[13] YbIII luminescent
stains.
Spectrophotometric titrations: Spectrophotometric titrations were per-
formed by using a J & M diode array spectrometer (Tidas series) con-
nected to an external computer. All titrations were performed in a ther-
mostated (25.0Æ0.18C) glass-jacketed vessel at m=0.1m (KCl). In a typi-
cal experiment, a ligand solution (50 mL, 6.5·10ꢁ6 m) was titrated with
freshly prepared sodium hydroxide solutions at different concentrations
(4, 1, 0.1, and 0.01m). After each addition of base, the pH of the solution
was measured by a KCl-saturated electrode and the UV/Vis absorption
spectrum was recorded by using a 1-cm Hellma optrode immersed in the
thermostated titration vessel and connected to the Tidas spectrometer.
By using the same equipment, conditional stability constants were deter-
mined by titration of T2soxMe by LnIII at fixed pH (0.1m HBS buffer,
pH 7.4). Factor analysis[30] and mathematical treatment of the spectropho-
tometric data were performed by using the SPECFIT program.[31,32]
Experimental Section
Tetrahydrofuran (THF, >99%) from Acros and methanol (99.9%) from
Merck were purified by usual methods.[25] 1H and 13C NMR spectroscopy
was performed by using a Bruker Avance DRX 400 spectrometer at
258C, with deuterated solvents [D1]CHCl3 and [D6]DMSO (dimethyl sulf-
oxide, 99.8 atom% D) from Armar Chemicals, and deuterium oxide
(99.9 atom% D) from Aldrich as internal standards. Mid-infrared spectra
were recorded by using a Perkin–Elmer Spectrum One spectrometer
equipped with a Universal Attenuated Total Reflection sampler. ESI-MS
spectra were obtained by using a Finningan SSQ 710C spectrometer with
10ꢁ4–10ꢁ5 m solutions in acetonitrile/H2O/acetic acid (50:50:1) or MeOH,
Potentiometric titrations: T2soxMe (H7L)+ was titrated by using a 5 mL
sample ([H7L+]tot =5.98·10ꢁ4 m in H2O) in a thermostated (25.0Æ0.18C)
glass-jacketed vessel under Ar atmosphere. The ionic strength was fixed
with KCl (m=0.1m). The solution was acidified to pH~2.0 with 37%
HCl 30 min before titration. Titrations were carried out by using an auto-
matic Metrohm Titrino 736 GP potentiometer (resolution 0.1 mV, accura-
cy 0.2 mV) linked to an IBM PS/2 computer and by using constant
volume addition (0.025 mL). An automatic burette (Metrohm 6.3013.210,
10 mL, accuracy 0.03 mL) was used along with a Metrohm 6.0238.000
glass electrode. The standard base solution (NaOH 0.1m) was added
inside the solution through a capillary tip attached to the automatic bu-
a
capillary temperature of 2008C, and an acceleration potential of
4.5 keV. This instrument was calibrated by using horse myoglobin and all
analyses were conducted in the positive mode; the ion-spray voltage was
4.6 keV. The samples were introduced through a syringe pump operating
at 20 mLminꢁ1. Elemental analyses were performed by Dr. H. Eder from
the Microanalytical Laboratory of the University of Geneva.
Podand T2oxMe: 2-Carboxy-8-hydroxyquinoline (1 g, 5.3 mmol) was dis-
solved in 100 mL of freshly distilled THF. A solution of N-hydroxysucci-
nimide (0.66 g, 5.7 mmol) and 1,3-dicyclohexylcarbodiimide (1.16 g,
5.7 mmol) in THF (50 mL) was added to the stirred solution. After 48 h
of stirring at RT, a solution of compound 1 (0.25 g, 1.7 mmol) in dry THF
(50 mL) was added within 60 min. The mixture was stirred for an addi-
tional 72 h and evaporated. The resulting residue was dissolved in chloro-
form (250 mL), successively washed with saturated NH4Cl, brine, and
water (4”250 mL), dried with MgSO4, and then evaporated to dryness.
The residue was applied three times to a Sephadex LH-20 column (50%
MeOH/CH2Cl2). An orange foam 3, eluted as a single band, was obtained
(445 mg, 37%). 1H NMR (400 MHz, [D6]DMSO): d=2.87 (t, 6H; CH2),
2.91 (s, 9H; CH3), 3.75 (m, 6H; CH2), 7.26 (d, 3H; ArH), 7.44 (d, 3H;
ArH), 7.65 (t, 3H; ArH), 8.24 (d, 3H; ArH), 8.36 (d, 3H; ArH),
8.45 ppm (t, 3H; NH); IR (ATR): n˜ =3450–3230, 1616, 1453 cmꢁ1; ESI-
MS: m/z: calcd [M+H]+: 701.30; found: 702.35; calcd [M+2H]2+: 351.65;
found: 351.85; elemental analysis calcd (%) for C39H39N7O6·MeOH: C
65.47, H 5.91, N 13.36; found: C 65.12, H 5.87, N 13.28.
rette. The data (200 points, drift<1 mVminꢁ1
) were mathematically
treated by the program HYPERQUAD2000[33] by using a Marquardt al-
gorithm, and the distribution of species was calculated by using the pro-
gram HYSS2. Calibration of the pH meter and the electrode system was
performed prior to each measurement by using a standardized HCl solu-
tion at 25.08C: 10 mL of the latter was titrated with a standardized 0.1m
NaOH solution, and the electrode potential readings were converted to
pH values. The ion product of water (pKw =13.91) and electrode poten-
tial were refined by using the program Scientist by Micromath (Version
2.0).
Luminescence measurements: Low-resolution luminescence measure-
ments (spectra and lifetimes) were recorded by using a Fluorolog FL 3-
22 spectrometer from Spex-Jobin-Yvon-Horiba with double-grating emis-
sion and excitation monochromators, and a R928P photomultiplier. For
measurements in the NIR spectral range, the spectrometer was fitted
with a second measuring channel equipped with a FL-1004 single-grating
monochromator and light intensity was measured by two Jobin–Yvon
solid-state InGaAs detectors 1) DSS-IGA020L, cooled to 77 K (range
800–1600 nm) and 2) DSS-IGA020A (range 800–1700 nm) working at RT
and inserted into an LN2 housing including an elliptical mirror (908
beam path) and coupled to a Jobin–Yvon SpectrAcq2 data acquisition
system. The equipment and experimental procedures for luminescence
measurements in the visible and NIR ranges have been published previ-
ously.[13] All spectra were corrected for the instrumental functions. Phos-
phorescence lifetimes were measured in frozen glycerol/water solutions
(10:90%) in time-resolved mode. They are averages of three independent
measurements that were taken by monitoring the decay at the maxima of
the emission spectra, enforcing a 0.05–0.5 ms delay. The monoexponential
decays were analyzed by using the package Origin7.0. Quantum yields of
the complexes at pH 7.4 (HBS buffer) and at RT were determined rela-
Podand T2soxMe: T2oxMe (430 mg, 0.613 mmol) was placed in a round-
bottomed flask and was covered with a minimum of oleum (H2SO4, SO3,
30%). The mixture was stirred at RT overnight and then poured onto
ice, yielding a precipitate that was filtered and washed with cold water.
The collected solid was dried under vacuum for one week to give a
yellow powder (530 mg, 92%). 1H NMR (400 MHz, D2O, pH 13): d=
2.91 (m, 9H; CH3), 2.97 (m, 6H; CH2), 3.51 (m, 6H; CH2), 3.52 (m, 4H;
CH2), 6.43 (d, 3H; ArH), 8.02 (d, 3H; ArH), 8.23 (d, 3H; ArH),
9.04 ppm (d, 3H; ArH); IR (ATR): n˜ =3600–3150, 1637, 1602, cmꢁ1; ESI-
MS: m/z: calcd [M+H]+ 941.17; found: 942.20; elemental analysis calcd
(%) for C39H39N7O15S3·2H2O: C 47.90, H 4.43, N 10.03; found: C 47.53,
H 4.43, N 10.08.
Physicochemical measurements: Analytical-grade solvents and chemicals
(Fluka AG) were used without further purification. HBS buffer was pre-
pared by mixing a solution of 4-(2-hydroxyethyl)piperazine-N’-2-ethane-
942
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Chem. Eur. J. 2007, 13, 936 –944

Lanthanide Bioprobe Near-Infrared Emitters
FULL PAPER
tive to [Yb
C
G
Acknowledgements
This research was supported through grants from the Swiss National Sci-
ence Foundation. We thank Mr. FrØdØric Gumy for his help with the lu-
minescence measurements.
Cell culture and complex loading: The influence of the Yb–T2soxMe
complex (1 mm in HBS) on cell viability was tested by using the human
T leukaemia cell line Jurkat (ATCC TIB152). The cells were cultivated
in 75 cm2 tissue-culture flasks by using RPMI 1640 supplemented with
10% foetal calf serum, 2 mm l-glutamine, 1 mm sodium pyruvate, 1%
nonessential amino acids, and 1% HEPES (all from Gibco 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 cells were ready for use. Cell density and viabil-
ity (defined as the ratio of the number of viable cells to the total number
of cells) of the cultures were determined by using trypan-blue staining
and a Neubauer improved hemacytometer (Blau Brand, Wertheim, Ger-
many). Prior to each viability test, the cells were harvested and diluted at
a density of 7.5·105 cellsmLꢁ1. The cell suspension was seeded into 96-
well plates at 100 mL/well 2 h prior to addition of the Yb–T2soxMe com-
plex and WST-1 reagent.
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3.5, 4.5, and 24 h). The WST-1 (Cell Proliferation Reagent WST-1,
Roche, Germany) is a slightly red tetrazolium salt that is reduced only in
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ductase system).
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cells could be quantified spectrophotometrically. The test was performed
as follows: Cells were seeded in a 96-well tissue-culture microplate at a
concentration of 7.5·104 cells/well in 100 mL of a culture medium and in-
cubated for 2 h at 378C with 5% of CO2. The Yb–T2soxMe complex was
dissolved at a concentration of 1 mm in fresh HBS at RT, and 100 mL was
added to each well (final concentrations: 500, 250, 125, and 50 mm); 20 mL
of the WST-1 reagent was added to each well and the plate was shaken
for 1 min on a microtiter-plate shaker (450 rpm). The plate was further
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absorbance values as follows:
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ðA450 nmꢁA650 nmÞexp
ð7Þ
viabilityWST ½%Š ¼
ꢃ 100
ðA450 nmꢁA650 nmÞmedium
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in which A450 nmꢁA650 nm is the difference in absorption at 450 and 650 nm
for the cells that were in contact with the Yb–T2soxMe complex (“exp”)
and for the “medium” containing the Yb–T2soxMe complex at different
concentrations. The results are expressed as an average over eight nomi-
nally identical measurements.
Chem. Eur. J. 2007, 13, 936 –944
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943

D. Imbert et al.
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Received: July 6, 2006
Published online: October 31, 2006
944
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Chem. Eur. J. 2007, 13, 936 –944