Modulating the photophysical properties of azamacrocyclic europium complexes with charge-transfer antenna chromophores

Article abstract of DOI:10.1021/ic200227b

Two europium complexes with bis(bipyridine) azamacrocyclic ligands featuring pendant arms with or without p-conjugated donor groups are synthesized and fully characterized by theoretical calculations and NMR spectroscopy. Their photophysical properties, i

Full text of DOI:10.1021/ic200227b

ARTICLE
pubs.acs.org/IC
Modulating the Photophysical Properties of Azamacrocyclic
Europium Complexes with Charge-Transfer Antenna Chromophores
Adrien Bourdolle,† Mustapha Allali,‡ Jean-Christophe Mulatier,† Boris Le Guennic,† Jurriaan M. Zwier,‡
Patrice L. Baldeck,§ Jean-Claude G. B€unzli,^ Chantal Andraud,† Laurent Lamarque,*,‡ and Olivier Maury*,†
‡Cisbio Bioassays, Parc Marcel Boiteux, BP 84175, 30204 Bagnols-sur-Cꢀeze Cedex, France
†Universitꢁe de Lyon 1, CNRS UMR 5182, Ecole Normale Supꢁerieure de Lyon, 46 allꢁee d’Italie, 69007 Lyon, France
§Laboratoire de Spectromꢁetrie Physique, Universitꢁe Joseph Fourier, CNRS UMR 5588, BP 87, F-38402 Saint Martin d'Hꢀeres, France
^
ꢁ
Institute of Chemical Sciences and Engineering, Ecole Polytechnique Fꢁedꢁerale de Lausanne, BCH 1402, CH-1015 Lausanne, Switzerland
S Supporting Information
b
ABSTRACT:
Two europium complexes with bis(bipyridine) azamacrocyclic ligands featuring pendant arms with or without π-conjugated donor
groups are synthesized and fully characterized by theoretical calculations and NMR spectroscopy. Their photophysical properties,
including two-photon absorption, are investigated in water and in various organic solvents. The nonfunctionalized ligand gives
highly water-stable europium complexes featuring bright luminescence properties but poor two-photon absorption cross sections.
On the other hand, the europium complex with an extended conjugated antenna ligand presents a two-photon absorption cross
section of 45 GM at 720 nm but is poorly luminescent in water. A detailed solvent-dependent photophysical study indicates that this
luminescence quenching is not due to the direct coordination of OꢀH vibrators to the metal center but to the increase of
nonradiative processes in a protic solvent induced by an internal isomerization equilibrium.
’ INTRODUCTION
Significant σ2 values (between 100 and 300 GM) were obtained,
but the poor solubility and stability of these complexes in water
preclude any practical applications as bioprobes. These limitations
were partially overcome by the design of tris(dipicolinate) lantha-
nide complexes [Ln(DPA)3]3ꢀ (DPA = pyridinedicarboxylate),
where the DPA ligand was functionalized by π-conjugated donor
moieties decorated with hydrosolubilizing poly(ethylene glycol)
(PEG) end groups (Chart 1), leading to complexes soluble
enough in water so that the first two-photon scanning microscopy
experiment on fixed human cancer cells could be successfully
performed.2b Moreover, fine tuning of the π-conjugated antenna
allowed us to reach very important two-photon absorption cross
sections, up to 775 GM.9 However, the stability of this class of
complexes in water remains moderate, and the complexes tend to
dissociate in dilute solutions (30% of dissociation in the 10ꢀ4ꢀ10ꢀ5
The sensitization of europium or terbium luminescence by
nonlinear two-photon excitation is recently becoming a promis-
ing strategy for the design of a new generation of bioprobes for
multiphoton microscopy imaging techniques.1,2 These new
probes should combine the intrinsic advantages of two-photon
excitation,3 such as 3D resolution, low-energy excitation localized
in the biological window, reduced photodamage, and photobleach-
ing, with those of lanthanide luminescence, namely, sharp emission
bands with large apparent Stokes’ shifts, longexcited-statelifetimes,
and sensitivity to the local environment.1b,4 The proof-of-concept
of the biphotonic sensitization of lanthanide luminescence by
organic ligands was established in the early 2000s by Lakowicz
and co-workers.5 The two-photon absorption efficiency estimated
by the measure of the two-photon cross section (σ2) was then
improved using ligands featuring charge-transfer transitions like
Michler’s ketone,6 2-(diethylanilin-4-yl)-4,6-bis(3,5-dimethyl-
pyrazolyl)triazine,7 or functionalized pyridinedicarboxamide.8
Received: February 2, 2011
Published: May 09, 2011
r
2011 American Chemical Society
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Chart 1. Structures of the Europium Tris(dipicolinate) Ligand, the Target Macrocyclic Ligands (L1 and L2), and Related
Europium(III) Complexes
M concentration range), limiting their use in biologically relevant
applications.10,2b In this context, we started a research project
aimed at incorporating our two-photon antenna onto a macro-
cyclic ligand widely encountered in lanthanide coordination
chemistry to improve its stability in a biological medium. Follow-
ing this idea, we recently reported the functionalization of
diethylenetriaminepentaacetic acid (DTPA) and DOTA macro-
cycles by two-photon antenna, but the nonlinear optical properties
were rather modest.11 To combine the stability and photophysical
requirements, we now propose using the bis(bipyridine) azama-
crocyclic platform known since the pioneering work of Lehn et al.
oneuropium tris(bipyridine)cryptates,12 which yields highly stable
complexes presently used and commercialized in homogeneous
time-resolved fluorescence bioassays and related applications.13,14
Saturation of the coordination sphere is ensured by two additional
pyridinecarboxylate pendant arms known to act as efficient chelat-
ing units,15 which can be easily modified to behave as two-photon
antenna according to the aforementioned strategy.9,11 In this
article, we report the synthesis of two new macrocyclic ligands,
L1ꢀ2 (Chart 1), and their related europium(III) complexes.
Whereas the nonfunctionalized [EuL1]þ complex is highly
luminescent and stable in aqueous or buffer media, its functio-
nalized counterpart [EuL2]þ retains its emission properties only
in organic solvents. The surprising difference between the
photophysical properties in water of these two complexes is
discussed on the basis of density functional theory (DFT)
calculations, solution NMR studies, and solvent-dependent
photophysical measurements. Finally, the two-photon absorp-
tion properties of [EuL2][TFA] (TFA is trifluoroacetate) in
organic media are presented and discussed.
’ RESULTS AND DISCUSSION
Synthesis. Synthesis of the pendant chelating units 4 and 11
(Scheme 1) involves a key step in the desymmeterization of the
diester derivatives 2 and 7, respectively. These monoreductions,
adapted from literature procedures,16 were successfully achieved
using NaBH4 (1.5 equiv) in dry methanol and scaled up to several
grams. The (alkylthio)phenylethynyl fragment 9, containing a
2-[2-(2-methoxyethoxy)ethoxy]ethyl moiety (Peg) that ensures
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Scheme 1. Synthesis of the Chromophore Antenna Fragments
Scheme 2. Synthesis of the Ligands L1ꢀ2
the final solubility in water, was coupled to 8 via a classical
palladium-catalyzed Sonogashira cross-coupling reaction, lead-
ing to the final functionalized pendant chromophore 11 after
mesylation.11
azamacrocyclic precursor 13, featuring two nonconjugated bi-
pyridine subunits tethered to each other via a nitrogen-contain-
ing hydrocarbon chain,17 followed by saponification of the ester
moieties in basic media (Scheme 2). The final europium complexes
[EuL1ꢀ2][TFA] (Chart 1) were prepared in an acetonitrile
solution by mixing 1 equiv of ligands L1ꢀ2 with an excess of
Synthesis of the macrocyclic ligands L1ꢀ2 was achieved in
small amounts with good yield by double alkylation of the
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0
Figure 1. DFT-optimized structures of [YL1]þ (left) and [YL2 ]þ (right), with the C2 symmetry axis being vertical (up) or perpendicular to the figure
(bottom). Nitrogen, oxygen, and sulfur atoms are drawn in blue, red, and yellow, respectively.
europium chloride (3 equiv) in the presence of Na2CO3. All
syntheses were carried out on a small scale, and therefore the
ligands 14, 15, and L1ꢀ2 and related complexes [EuL1ꢀ2][TFA]
were purified, at each step, by high-performance liquid chromato-
graphy (HPLC; see the Experimental Section for details and the
Supporting Information, Figure S1) and further characterized by
high-resolution mass spectrometry (HRMS) analysis. In addition,
the final complexes were characterized by 1H NMR spectroscopy
and will be detailed later. It is worth noting that a counter-
ion-exchange reaction from chloride to triflate, unambiguously
identified by 19F NMR spectroscopy, occurs during purification
of the complexes because of the presence of trifluoroacetic acid in
the eluent.
fragment. The paramagnetic EuIII ion was replaced by the
diamagnetic YIII or LuIII ion for calculation facilities.18 The
optimized structures are represented in Figure 1 in the case of
yttrium (see the Supporting Information, Figure S2, for lutetium
complexes), and selected bond lengths are compiled in Table 1.
The yttrium and lutetium complexes are isostructural. The only
observed difference lies in the shortening of the metal-to-ligand
bond lengths when YIII is replaced by LuIII, featuring a smaller
ionic radius (Table 1). It is also worth noting that ligand
functionalization by a π-conjugated chromophore has a negligible
impact on the coordination polyhedron (see the comparison
between [ML1]þ and [ML2]þ in Table 1). In all complexes, the
central metal ion is bound to the 10 donor atoms of the ligands,
among which 8 nitrogen atoms are from the bis(bipyridyl)
moieties (4Nbipy), the azamacrocycle (2Ncy), and the picolinic
acid pendant arm (2Npa). The coordination sphere is completed
by two oxygen atoms from the carboxylate functions. The
DFT Geometry Optimizations. In order to interpret the
spectroscopic data, DFT geometry optimizations (see the Com-
putational Details section) were undertaken on [ML1]þ and
0
model [ML2 ]þ (M = Y, Lu) featuring a shorter thioalkyl
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bis(bipyridine) azamacrocycle can be considered as a platform
filling half of the coordination space with a rather small angle
(60ꢀ) between the two bipyridyl fragments. The two picolinic
acid pendant arms act as a scorpionate ligand lying above the
macrocyclic platform in a syn-like conformation. Such types of
conformations are generally encountered for related complexes
based on triazacyclononane15c,d or crown ether15e,f platforms.
Because of the lack of flexibility of the bis(bipyridine) azamacro-
cycle, each bipyridine ligand is slightly distorted with a twist angle
of ca. 10ꢀ. As a consequence, the complexes present a low C2
symmetry, where the symmetry axis is perpendicular to the
pseudoplane made by the four Nbipy atoms (Figure 1). The lack
of macrocyclic ligand flexibility combined with the rather unusual
10-coordinate structure15f in [LuL1]þ is also responsible for the
significantly longer metal-to-ligand bond lengths.19 As an example,
the LuꢀNbipy bond lengths are found in the 2.6ꢀ2.7 Å range and
the LuꢀNcy distances are even longer at about 2.84 Å. On
the contrary, the more flexible pendant pyridinecarboxylate arms
are more strongly coordinated with a LuꢀNpa bond length of
around 2.55 Å in [LuL2]þ in the range of those measured for
related 1,7-diaza-12-crown-415e or triazacyclononane15c plat-
forms (2.4ꢀ2.6 Å).
room temperature, the [EuL1][TFA] complex exhibits a well-
resolved spectrum, spread over 40 ppm because of the para-
magnetism of europium (Figure 2). Each proton of the azama-
crocycle and the benzylic protons of the pendant arms are
diastereotopic, a signature of the rigidification of the macrocyclic
ligand upon complexation. As a consequence, the azamacrocyclic
protons, in the closer vicinity of the metal, are strongly high-field
shifted (in the [ꢀ5, ꢀ20] ppm range; Figure 2) and exhibit 2Jgem
coupling constants in the range of 14.4ꢀ16.0 Hz. In addition,
three different pyridinyl systems can be identified but not
unambiguously assigned, which reveals that the nonequivalence
of each pyridine subunit of the bipyridine moieties is preserved in
solution. In marked contrast, the spectrum of [EuL2][TFA] in
D2O is only composed of broad signals at room temperature, a
signature of the dynamic behavior of the complex in this solvent.
On the other hand, in CDCl3, the spectrum becomes well
resolved (Figure 3) and the para-functionalization of the pico-
linic acid pendant arms allows a definitive assignment of each
signal by 2D COSY experiments. These experiments unravel two
sets of signals for the bipyridine fragment and the equivalence of
the picolinic acid moieties. It is worth noting that two NMR
signals belonging to one pyridine of the bipyridine ligand are
merged at 6.3 ppm. This observation is unambiguously con-
firmed thanks to variable-temperature measurements: because of
the temperature dependence of the paramagnetic chemical shift,
these twosignals are wellresolved by increasing the temperatureto
40 ꢀC. Therefore, the C2 symmetry of both complexes observed in
the optimized geometries is confirmed in solution by 1H NMR.
Photophysical Properties of Europium Complexes. The
absorption and emission properties of the two complexes were
investigated in water and in phosphate or HEPES buffers.
[EuL1][TFA] presents absorption bands localized in the
UV region with a modest extinction coefficient (λmax = 308 nm,
εmax = 18 000 Mꢀ1 cmꢀ1) that are classically observed for πꢀπ*
transitions of pyridinic/picolinic ligands (Figure 4). As expected,
Solution Structures of [EuL1ꢀ2][TFA] Complexes. These
structures were investigated by 1H NMR spectroscopy. In D2O at
Table 1. Selected Bond Lengths (Å) of the DFT-Optimized
0
Complexes [ML1]þ and [ML2 ]þ
0
0
distance (Å)
[YL1]þ
[YL2 ]þ
[LuL1]þ
[LuL2 ]þ
MꢀNbipy
2.658
2.700
2.614
2.834
2.279
2.703
2.659
2.589
2.833
2.282
2.663
2.615
2.578
2.844
2.216
2.670
2.621
2.551
2.843
2.219
0
MꢀNbipy
MꢀNpa
MꢀNcy
MꢀO
Figure 2. 1H NMR (500 MHz, 20 ꢀC) spectrum of [EuL1][TFA] in D2O with magnification of the aromatic region. The aliphatic protons are indicated
in red, pink, and cyan, whereas the pyridinyl ones are in blue, green, and yellow.
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Figure 3. 1H NMR (500 MHz, 20 ꢀC) spectrum of [EuL2][TFA] in CDCl3. The inset displays a portion of the variable-temperature 1H NMR spectrum
illustrating the fusion of two signals in one broad peak. The aliphatic protons are indicated in red, pink, and cyan, whereas the pyridinyl ones are in blue,
green, and yellow.
scence quantum yield efficiency and a long luminescence lifetime
(1.8 ms) in water. The same lifetimes and quantum yields were
obtained in HEPES or phosphate buffer solutions. It is also worth
noting that the introduction of potassium fluoride in the solution
mixture has no effect on the luminescence properties indicating
that the coordination sphere of the central metal ion is fully
saturated by the macrocyclic ligand. In addition, the lumine-
scence lifetime remains constant upon dilution of the complex
from 10ꢀ5 up to 10ꢀ7 M or when further diluted to 6 nM in
HEPES and phosphate buffers including 0.1% bovine serum
albumin (BSA), which is under experimental conditions appro-
priate for bioassays. Moreover, upon the addition of 20 mM
EDTA to this solution, no loss of luminescence was observed,
underlining that, under these experimental conditions, no dis-
sociation occurs. These results confirm the NMR observations
and clearly establish that the introduction of pyridinecarboxylate
pendant arms strongly stabilizes the [EuL1][TFA] complex in
water compared to analogous chelates featuring bipyridine or
phenanthroline pendant arms.22 The [EuL1][TFA] emission
spectrum is very well resolved (Figure 5, top) and shows the
Figure 4. Absorption spectra of [EuL1][TFA] (dashed line) and
[EuL2][TFA] (solid line) in water.
the introduction of donor-functionalized π-conjugated moieties
in [EuL2][TFA] results in the appearance of an additional broad
5
7
bands corresponding to the D0 f FJ (J = 0ꢀ4) transitions.
The splitting of these transitions and their relative intensities
are the signature of the ligand-field effect and are directly related
to the symmetry of the coordination polyhedron. In the present
case, the observed splitting of the J = 2 transition in several bands
is a signature of a low-symmetry complex, but resolution of the
spectrum recorded in water is not sufficient to draw any definitive
conclusion (vide infra).
intense transition (λmax = 337 nm, εmax = 53 000 Mꢀ1 cmꢀ1
)
assigned to an intraligand charge-transfer (ILCT) transition from
the thioalkyl electron-donating group to the picolinic electron-
accepting fragment,20 in addition to the 308 nm feature (εmax
≈
61 000 Mꢀ1 cmꢀ1). It is now well established that such ILCT
transitions are necessary to ensure efficient two-photon absorption
by the complex6ꢀ9 and are further able to induce an efficient
europium sensitization via an alternative “ILCT sensitization
pathway”.1b,20,21
In marked contrast, the emission of the functionalized analo-
gue [EuL2][TFA] is very weak in aqueous media, as is illustrated
by the low luminescence quantum yield (<0.02), the shorter
luminescence lifetime, and the less resolved spectrum with
Excitation in the πꢀπ* transitions of [EuL1][TFA] results in
bright-red europium luminescence with a reasonable 0.13 lumine-
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Table 3. Calculated Values of τr, kr, ∑knr, ηEu, and ηsens
for [EuL2][TFA] (ca. 10ꢀ6 M) in Various Solvents and
[EuL1][TFA] (ca. 10ꢀ6 M) in Water Using the Room
Temperature Experimentally Determined Quantities φEu,
τobs, and [I(0,1)/Itot]
b
τobs
τr
[I(0,1)/
Itot
kr/
∑knr/
102 sꢀ1 102 sꢀ1
a
solvent φEu
(ms) (ms) ηEu ηsens
]
[EuL2][TFA]
1.24 4.24 0.29 0.38
2.66 5.15 0.52 0.33
1.88 4.18 0.45 0.22
DCM
0.11
0.17
0.17
0.18
0.17
0.18
0.22
2.3
1.9
2.4
1.8
1.9
1.5
5.7
1.8
MeCN 0.17
DMSO 0.10
2.9
MeOH 0.008 0.80 5.40 0.15 0.05
EtOH 0.025 0.81 5.23 0.15 0.16
10.7
10.4
17.2
H2O
0.013 0.53 6.84 0.08 0.02
[EuL1][TFA]
H2O
0.13
1.80 7.42 0.24 0.53
0.24
1.3
4.2
a λex = 360 nm. b λex = 370 nm.
Figure 5. Normalized luminescence spectra of [EuL1][TFA] (top) and
[EuL2][TFA] (bottom) in a phosphate buffer (pH = 7; λex = 330 nm).
The small peak at 660 nm corresponds to the excitation wavelength
harmonic.
Figure 6. Emission spectrum of [EuL2][TFA] in DCM at room
temperature (λex = 330 nm). Inset: magnification of the first component
of the D0 f F1 transition and fit using Gaussian functions (green
Table 2. Photophysical Data of [EuL1ꢀ2][TFA] (ca. 10ꢀ6 M)
in Water at Room Temperature
5
7
curves; sum in red).
[EuL1][TFA]
τobs (ms)
1.80
[EuL2][TFA]
τobs (ms)
into the differences observed between [EuL1][TFA] and
[EuL2][TFA], an extensive study of the influence of the solvent
on the photophysical properties of [EuL2][TFA] was undertaken.
Structural Information Probed by Luminescence Mea-
surements. Owing to the presence of the Peg functionalization,
[EuL2][TFA] is very soluble in most organic solvents like dichloro-
methane (DCM), acetonitrile (MeCN), dimethyl sulfoxide
(DMSO), methanol (MeOH), or ethanol (EtOH) and slightly
soluble in methyltetrahydrofuran (MeTHF). In all solvents, very
well-resolved emission spectra characteristic of an EuIII-centered
emission were recorded. The respective luminescence quantum
yields and lifetimes are compiled in Table 3. Figure 6 illustrates
a representative emission spectrum recorded using a 0.3-nm
resolution, the highest afforded by our experimental setup.
Excitation in the antenna broad absorption band at 330 nm
gives an emission spectrum featuring the 5D0 f 7FJ transitions
(J = 0ꢀ4) characteristic of EuIII, with 0.1, 1, 2.7, 0.2, and 2.0
relative corrected intensity patterns, signature of a low-symmetry
solvent
φEu
φEu
H2O
0.13
a
0.013
0.017
0.53
0.65
a
D2O
a Not measured.
broader emission bands (Table 2 and Figure 5). This emission
quenching cannot be simply attributed to coordination of the
water molecules because the lifetime in D2O (0.65 ms) is close to
that in H2O (0.53 ms) (Table 2). The hydration number qcorr can
be estimated as ca. 0.04 on the basis of Horrocks’ phenomeno-
logical hydration second equation, taking into account the
correction factor describing the outer-sphere water molecule
contribution,23 qcorr =A0(Δkꢀ R) with A0 = 1.11msfor EuIII, Δk=
1/τobs(H2O) ꢀ 1/τobs(D2O), and R = 0.31 msꢀ1. This result
suggests that no water molecule is directly bonded to the metal
center, with the lifetime variation being mainly due to second or
outer-sphere water molecules. In order to gain deeper insight
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Figure 7. (Top) Emission spectra of [EuL2][TFA] in MeOH/EtOH (1/4, v/v) at room temperature (black curve) and in frozen glass at 77 K (red
curve) using λex = 330 nm. Inset: magnification on the 5D0 f 7F0 transition and fit using Gaussian functions (green curves; sum in red). (Bottom)
Magnification of the J = 4 transition and decomposition into nine Gaussian curves (green curves; sum in blue).
environment of the emitting center. In addition, in the case of
europium, the splitting of each 5D0 f 7FJ transition is correlated
to ligand- (crystal-)field effects and is therefore representative of
the local symmetry. For a C2 compound, as anticipated for the
[EuL2][TFA] complex according to DFT and NMR data, a
splitting of each transition in 2J þ 1 sublevels is predicted by
theory,24 in line with experimental data.25 In the present case,
whereas the J = 1 transition is split into three main sublevels, its
first component (Figure 6, inset) is clearly the sum of two
individual transitions and the resulting global splitting is then
larger than the prediction. In order to obtain a better resolution,
low-temperature emission spectra (77 K) were recorded in
EtOH/MeOH (1/4, v/v) frozen organic glasses.26 Lowering
the temperature results in a ca. 10 times enhancement of the
emission intensity, as illustrated by the relative areas of the room
temperature and 77 K spectra (Figure 7, top), accompanied by a
large increase of the lifetime from 0.85 ms at room temperature
up to 2.4 ms at 77 K. Indeed, the vibronic broadening of the
transition is strongly reduced and the full width at half-maximum
(fwhm) of the hypersensitive J = 2 transition decreases from
42 cmꢀ1 at room temperature to 25 cmꢀ1 at 77 K, leading to
enhanced resolution. The J = 2 transition is now clearly split into
five sublevels, and the transition to the J = 4 level exhibits nine
sublevels after decomposition into Gaussian components
(Figure 7, bottom), as is expected for a C2-symmetric complex.
Furthermore, the J = 1 transition is clearly composed of three
main components, but the first one appears as a doublet. The
explanation of this doublet can be found in a careful examination
of the J = 0 transition (inset of Figure 7, top): this band cannot be
satisfactorily fitted by a single Gaussian and is better described as
the sum of two contributions. This result suggests the presence of
two emitting species at low temperature, both presenting very
similar local environments with overall C2 symmetry, with the
only observable emission difference being the presence of a
doublet in the first component of the J = 1 transition. These two
species may originate from an internal isomerization dynamic
equilibrium between a 10-coordinated species [Eu10-L2]þ and a
nine-coordinated isomer [Eu9-L2]þ in which the longest Eu-
ꢀNcy bond is broken (Figure 8). This dynamic equilibrium could
be assisted by additional exocyclic hydrogen-bond formation
between a protic solvent (water, D2O, alcohols, etc.) and the
uncoordinated nitrogen lone pair. This dynamic equilibrium is
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Figure 8. Hypothesis for the internal isomerization dynamic equilibrium.
too fast to be observed by 1H NMR at low temperature in aprotic
CDCl3. On the other hand, in protic solvents like D2O, [EuL2]þ
presents only broad 1H NMR signals similar to those observed in
the case of coalescence.27
Solvent Dependence of the Photophysical Properties. In
order to gain deeper insight into these solvent effects, the
relevant radiative and nonradiative parameters were deduced
from experimental data (spectra, quantum yields, and lifetimes)
according to the works of Werts et al.28 and Beeby et al.29 Using
this method, the overall europium quantum yield of lumine-
scence (φEu) is defined as the product of the efficiency of
sensitization (ηsens, i.e., here the fraction of energy transferred
from the donor state to the EuIII accepting levels) and the
quantum efficiency of the metal-centered luminescence upon
direct excitation into the f levels (ηEu).
due to the direct coordination of OꢀH vibrators to the europium
emitting ion but to the dramatic increase of alternative non-
radiative deactivation processes like the above-mentioned inter-
nal isomerization equilibrium between [Eu10-L2]þ and [Eu9-L2]þ.
Other potential contributions could also come from proximate
high-energy OꢀH vibrators located in the outer coordination
sphere.23b As a consequence, lowering the temperature to 77 K
slows the dynamic equilibrium kinetics, resulting in reduced
contributions of the related nonradiative processes and, hence, in
the recovery of the luminescence intensity (Figure 7, top).
Finally, applying the same procedure to [EuL1][TFA] in water
(Table 3) clearly indicates that the nonradiative rates (∑knr) in
water are far smaller than those for the functionalized counter-
part (420 vs 1723 sꢀ1). This result indicates that the internal
isomerization equilibrium does not occur for [EuL1][TFA],
which explains the enhanced luminescence quantum yield of
this compound in water. Finally, both NMR and luminescence
experiments unambiguously demonstrate that the lateral donor
π-conjugated functionalization in [EuL2][TFA] induces more
lability in the complex, which therefore presents a more pro-
nounced binding exchange dynamic behavior, i.e., the internal
isomerization equilibrium in protic solvents. The electronic
reasons behind this difference are not well understood and are
currently under investigation in our group.
jEu ¼ ηsensηEu
In this equation, ηEu = τobs/τr, where τobs represents the
experimentalluminescencelifetime of the complex and τr, thepure
radiative lifetime, calculated from kr = 1/τr = A(0,1)[Itot/I(0,1)].
The constant A(0,1) is the spontaneous emission probability
5
7
of the D0 f F1 transition, equal to 39.4 sꢀ1 in DCM, and
Itot/I(0,1) is the ratio of the total integrated emission intensity to
5
7
the intensity of the D0 f F1 transition. Finally, ∑knr can be
deduced knowing kr and τobs, from the relationship ∑knr = 1/τobs
ꢀ1/τr. This procedure was applied to [EuL2][TFA] in various
solvents and to [EuL1][TFA] in water. All data are summarized
in Table 3.30 In all organic solvents, the symmetries of the
complexes appear to be identical, as inferred from almost
constant [I(0,1)/Itot] ratios (0.17ꢀ0.18); henceforth, almost
negligible variations in the radiative lifetime (τr) or radiative
constant rate (kr) are observed. In protic solvents (water and
alcohol), a strong decrease in the luminescence lifetime induces a
drop in ηEu, which decreases to 0.08ꢀ0.15 compared to that in
aprotic solvents of various polarity (DCM, MeCN, DMSO, etc.),
where ηEu is in the range of 0.29ꢀ0.52. As a consequence, the
nonradiative decay rates (∑knr) are far larger in protic solvents.
These observations explain the important decrease of the lumine-
scence quantum yield efficiency in protic solvents (Table 3) with,
for example, a drop from 0.17 in MeCN to less than 0.01 in
MeOH. Interestingly, this decrease is not, as is usually observed,
Two-Photon Absorption Properties. The two-photon absorp-
tion properties of [EuL2][TFA] featuring a π-conjugated antenna
were studied in a DCM solution by monitoring the two-
photon excited luminescence in the 700ꢀ900 nm spectral range.
Coumarin-307 was used as the standard, according to the
experimental protocol described by Xu and Webb,31 and excita-
tion was achieved with a femtosecond Ti:sapphire pulsed laser.
The two-photon excitation spectrum (Figure 9) matches the
wavelength-doubled single-photon one. This result indicates that
transitions to the lowest-energy excited state, namely, a charge-
transfer state (ILCT), are one- and two-photon-allowed, in
agreement with the symmetry of the complex and with the
two-photon selection rules in the case of noncentrosymmetric
derivatives. At 720 nm, [EuL2][TFA] presents a two-photon
cross section of 45 GM. It is worth noting that the maximal
absorption wavelength is located out of our laser excitation range
and that only the red tail of the two-photon absorption spectrum
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Low-resolution mass spectrometry was carried out on a Agilent
1100 series LC/MSD apparatus. The compounds (14 and 15), ligands
(L1 and L2), and related europium complexes ([Eu(L1)][TFA] and
[Eu(L2)][TFA]) were analyzed by reversed-phase HPLC (RP-HPLC)
using a Waters Alliance 2695 system coupled with Waters 996 photodiode
array detector and a Micromass ZQ Waters 2000 mass spectrometer. The
procedure developed was 0.1% formic acid in water/MeCN as the mobile
phase using a XBridge C18, 3.5 μm, 4.6 ꢁ 100 mm as the column.
Purifications have been carried out using preparative RP-HPLC Shimadzu
LC-8A and XBridge C18, OBD, 19 ꢁ 100 mm as the column with 0.1%
TFA in 95% water/MeCN isocratic flow as the mobile phase. HRMS was
performed using a Bruker MicroTOF-Q II apparatus (precision 1ꢀ5 ppm)
equipped with an electrospray source using highly diluted samples in a
MeOH/CDM/water/formic acid mixture (46.1:38.4:15.4:0.1, v/v). The
experimental sequences for the preparation of compounds 4,16 7,32 9,9
and 1317 were reproduced from literature data.
Computational Details. DFT geometry optimizations with no
symmetry constraint were carried out with the Gaussian 03 (revision D.01)
package,33 employing the hybrid functional PBE0.34 The “Stuttgart/
Dresden” basis sets and effective core potentials were used to describe
lanthanide atoms,35 whereas all other atoms were described with the
D95 basis sets.36
Figure 9. Two-photon excitation spectrum of [EuL2][TFA] in a DCM
solution (b, upper abscissa). Superimposed on this plot is the single-
photon absorption spectrum (—, lower abscissa).
was measured. These two-photon photophysical properties are
very similar to those observed for the tris(dipicolinate)europium
complex, featuring a similar π-conjugated donor antenna with a
σ2 value of 53 GM at 700 nm.9
Luminescence. Emission spectra were measured using a Horiba-
Jobin-Yvon Fluorolog-3 spectrofluorimeter, equipped with three-slit
double-grating excitation and emission monochromators with dispersions
of 2.1 nm/mm (1200 grooves/mm). The steady-state luminescence was
excited by unpolarized light from a 450 W continuous-wave xenon lamp
and detected at an angle of 90ꢀ for diluted solution measurements
(10 mm quartz cell) by a red-sensitive Hamamatsu R928 photomultiplier
tube. Spectra were reference-corrected for both the excitation source
light-intensity variation (lamp and grating) and the emission spectral
response (detector and grating). Phosphorescence lifetimes (>30 μs)
were obtained by pulsed excitation using a FL-1040 UP xenon lamp.
Luminescence decay curves were fitted by least-squares analysis using
Origin. Luminescence quantum yields Q were measured in a diluted
water solution with an absorbance lower than 0.1 using the equation
Qx/Qr = [Ar(λ)/Ax(λ)][nx2/nr2][Dx/Dr], where A is the absorbance at
the excitation wavelength (λ), n the refractive index, and D the
integrated luminescence intensity. “r” and “x” stand for the reference
and sample. Here, the reference is quininebisulfate in a 1 N aqueous
sulfuric acid solution (Qr = 0.546). Excitation of the reference and
sample compounds was performed at the same wavelength.
Lifetime measurements at low concentrations (6ꢀ10 nM) were
performed in 0.1 M phosphate (pH 7) or 0.05 M HEPES (pH 7) with
or without 0.4 M KF. All buffers contained 0.1% BSA. The solutions
were distributed in Costar 96 black well flat bottom plates, and
luminescence decay was measured on a Rubystar (BMG labtech) plate
reader at 620 nm (exc 337 nm) using the “advanced mode” option.
Two-Photon Excited Luminescence Measurements. The
TPA cross-sectional spectrum was obtained by up-conversion lumines-
cence using a Ti:sapphire femtosecond laser in the range of
700ꢀ900 nm. The excitation beam (5 mm diameter) is focalized with
a lens (focal length 10 cm) at the middle of the 10-mm cell. Emitted light
was collected at 90ꢀ and was focused into an optical fiber (600 μm
diameter) connected to an Ocean Optics S2000 spectrometer. The
incident beam intensity was adjusted to 50 mW in order to ensure an
intensity-squared dependence of the luminescence over the whole
spectral range. The detector integration time was fixed to 1 s. Calibration
of the spectra was performed by comparison with the published 700ꢀ
900 nm Coumarin-307 two-photon absorption spectrum31 (quantum
yield = 0.56 in EtOH). The measurements were done at room
temperature in DCM and at a concentration of 10ꢀ4 M.
’ CONCLUSION
A new europium-based chelate system has been designed,
aiming to combine high thermodynamic stability in water with
efficient two-photon absorption and luminescence properties,
two aspects that are essential for an optical bioprobe. To that end,
two new bis(bipyridine) azamacrocylic ligands featuring or not
π-conjugated donor picolinic pendant arms were prepared. The
nonfunctionalized ligand leads to the formation of a highly water-
stable europium complex featuring bright luminescence proper-
ties but a poor two-photon absorption cross section. On the
other hand, the europium complex with the ligand based on the
extended conjugated antenna presents a two-photon absorption
cross section of 45 GM at 720 nm but is not very luminescent in
water. A detailed photophysical study conducted in various
solvents indicates that this luminescence quenching is not due
to the direct coordination of OꢀH vibrators to the metal center
but to the increase of nonradiative processes in a protic solvent
induced, in part, by an internal isomerization equilibrium. It
appears that π-electron-donating functionalization has a negative
influence on the complex dynamics. This effect is presently not
fully understood and will have to be overcome in the design of
opti-
mized lanthanide-based bioprobes for two-photon scanning laser
microscopy imaging.
’ EXPERIMENTAL SECTION
General Procedures. NMR spectra (1H and 13C) were recorded at
room temperature on a Bruker AC 200 spectrometer operating at 200.13
and 50.32 MHz for 1H and 13C, respectively, and on a Bruker Advance
1
spectrometer operating at 500.10 and 125.75 MHz for H and 13C,
respectively. Data are listed in parts per million (ppm) and are reported
relative to tetramethylsilane (1H and 13C); residual solvent peaks of the
deuterated solvents were used as internal standards. UV/vis absorption
measurements were recorded on a Jasco V670 absorption spectrometer.
Synthesis. Compound 8. To a suspension of dimethyl 4-iodopyr-
idine-2,6-dicarboxylate (7; 2 g, 6.23 mmol) in MeOH (40 mL) cooled at
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ARTICLE
0 ꢀC was added in one portion of NaBH4 (473 mg, 12.5 mmol). The
mixture was stirred at 0 ꢀC for 30 min and left to reach room temperature
over 30 min. The mixture was quenched by the addition of a 10% HCl
solution (10 mL). The volatile components were evaporated under
vacuum, and the aqueous solution was extracted with EtOAc (3 ꢁ
100 mL). The organic phases were combined, dried over Na2SO4,
filtered, and concentrated in vacuo. The crude material was purified by
flash chromatography on silica (98:2 CH2Cl2/MeOH) to afford the
desired product as a white crystalline powder (1.15 g, 63%). 1H NMR
(200.13 MHz, CDCl3): δ 8.37 (s, 1H), 7.95 (s, 1H), 4.81 (d, J = 4.9 Hz,
2H), 3.99 (s, 3 H), 3.09 (t, J = 4.9 Hz, 1H). 13C NMR (50.33 MHz,
CDCl3): δ 164.4, 161.4, 147.2, 133.2, 132.9, 106.8, 64.2, 53.2. LRMS:
m/z 294 ([M þ H]þ), 316 ([M þ Na]þ).
temperature for 1 h. The solvent was evaporated, and the aqueous phase
was extended to 50 mL and then was extracted with EtOAc (2 ꢁ 25 mL).
The aqueous phase was acidified with HCl (10%) to pH 1ꢀ2 and was
extracted with CH2Cl2 (3 ꢁ 25 mL). The organic phases were combined,
dried over Na2SO4, filtered, and concentrated in vacuo. Purifications of
the crude material were carried out using preparative RP-HPLC, and the
purities of the compounds were unambiguously established on the basis of
the HPLC chromatogram (see the Supporting Information, Figure S1).
L1. Yield: 93%. HRMS: m/z 665.2595 ([C38H32N8O4]þ, [M þ H]þ)
(calcd m/z 665.2624).
L2. Yield: 87%. ESþ-LRMS: m/z 1220 ([C68H68N8O10S2]þ), 1221
([M þ H]þ), 1243 ([M þ Na]þ).
General Procedure for Synthesis of the Complexes. To a suspension
of free ligand (0.04 mmol) and Na2CO3 (0.084) mmol) in dry MeCN
Compound 10. To a degassed solution of 8 (0.865 g, 2.95 mmol) in
THF (20 mL) and 9 (0.858 g, 3.25 mmol) in Et3N (10 mL) was added
CuI (0.112 g, 0.59 mmol) and Pd(PPh3)2Cl2 (0.207 g, 0.29 mmol). The
dark-brown mixture was heated in the dark at 40 ꢀC for 20 h. After
cooling to room temperature, the black suspension was filtered and
triturated with Et2O (2 ꢁ 40 mL). The filtrate was washed with
saturated aqueous NH4Cl (2 ꢁ 50 mL) and brine (50 mL). The organic
layer was dried over Na2SO4, filtered, and concentrated in vacuo. The
crude material was purified by flash chromatography (49:1 CH2Cl2/MeOH),
(60 mL) was added EuCl3 6H2O (0.12 mmol). The solution was heated
3
at reflux for 30 h under an inert atmosphere. The mixture was then
cooled to room temperature and filtered off, and the filtrate was
concentrated in vacuo. Purification of the complexes was carried out
by preparative RP-HPLC, and the purities of the compounds were
unambiguously established on the basis of the HPLC chromatogram
(see the Supporting Information, Figure S1).
[Eu(L1)][TFA]. Yield: 78%. 1H NMR (500.10 MHz, D2O): δ 18.16
(s, 2H), 9.59 (t, 3J = 8 Hz, 2H), 9.33 (t, 3J = 7.7 Hz, 2H), 9.08 (m, 4H),
8.73 (d, 3J = 7.7 Hz, 2H), 7.9 (d, 3J = 7.5 Hz, 2H), 7.38 (t, 3J = 7.5 Hz,
2H), 7.2 (d, 3J = 8 Hz, 2H), 3.14 (d, 3J = 7.5 Hz, 2H), ꢀ0.57 (s, 2H),
ꢀ7.84 (d, 2J = 16 Hz, 2H), ꢀ11.09 (d, 2J = 16 Hz, 2H), ꢀ13.14 (d, 2J =
14.4 Hz, 2H), ꢀ15.84 (d, 2J = 14.4 Hz, 2H). 19F NMR (188.29 MHz,
D2O): δ ꢀ75.5. UV/vis: λmax = 308 nm (ε = 18 000 L molꢀ1 cmꢀ1).
HRMS: m/z 815.1584 ([EuC38H30N8O4]þ) (calcd m/z 815.1602).
1
yielding a light-yellow oil (1.267 g, 84%). H NMR (200.13 MHz,
CDCl3): δ 7.90 (d, J = 1.2 Hz, 1H), 7.56 (d, J = 1.2 Hz, 1H), 7.30
(d, J = 8.5 Hz, 2H), 7.17 (d, J = 8.5 Hz, 2H), 4.75 (d, J = 4.4 Hz, 2H), 4.29
(t, J = 4.4 Hz, 1H), 3.84 (s, 3H), 3.61ꢀ3.38 (m, 10H), 3.24 (s, 3H), 3.04
(t, J = 6.8 Hz, 2H). 13C NMR (50.33 MHz, CDCl3): δ 165.1, 161.5,
147.0, 139.2, 133.3, 132.2, 127.6, 125.4, 125.3, 118.5, 95.0, 86.6, 71.8,
70.51, 70.47, 70.40, 69.6, 64.6, 58.9, 52.9, 32.1. LRMS: = m/z 446
([M þ H]þ), 468 ([M þ Na]þ).
1
[Eu(L2)][TFA]. Yield: 90%. H NMR (500.10 MHz, CDCl3): δ
Compound 11. To a solution of 10 (1.10 g, 2.56 mmol) and Et3N
(0.777 g, 7.68 mmol) in CH2Cl2 (100 mL) was added dropwise
methanesulfonyl chloride (0.440 g, 3.84 mmol). After 30 min, the
reaction was quenched by the addition of a saturated NaHCO3 solution
(100 mL). The organic phase was separated, dried over Na2SO4, filtered,
and concentrated in vacuo. The crude material was purified by flash
chromatography on silica (97:3 CH2Cl2/MeOB.L.GH) to afford the
31.87 (s, 2H), 12.15 (s, 2H), 10.22 (s, 2H), 8.85 (s, 2H), 8.74 (s, 2H), 8.4
(s, 2H), 8.01 (d, 3J = 7.8 Hz, 4H), 7.65 (d, 3J = 7.8 Hz, 4H), 6.86 (s, 2H),
6.3 (s, 4H), 3.9ꢀ3.4 (m, 30H), 2.93 (s, 2H), ꢀ6.08 (s, 2H), ꢀ7.65
(s, 2H), ꢀ10.13 (s, 2H), ꢀ19.7 (s, 2H). 19F NMR (188.29 MHz,
CDCl3): δ ꢀ74.9. UV/vis: λmax = 308 (ε = 61 000 L molꢀ1 cmꢀ1
)
and 337 nm (ε = 53 000 L molꢀ1 cmꢀ1). HRMS: m/z 1373.3584
([EuC68H68N8O10S2]þ) (calcd m/z 1373.3712).
1
product as a bright-yellow oil (1.19 g, 92%). H NMR (200.13 MHz,
CDCl3): δ 8.04 (d, J = 1.2 Hz, 1H), 7.62 (d, J = 1.2 Hz, 1H), 7.37 (d, J =
8.5 Hz, 2H), 7.22 (d, J = 8.5 Hz, 2H), 5.32 (s, 2H), 3.91 (s, 3H),
3.66ꢀ3.42 (m, 10H), 3.28 (s, 3H), 3.13ꢀ3.04 (m, 5H). 13C NMR
(50.33 MHz, CDCl3): δ 164.7, 154.7, 147.9, 139.6, 134.1, 132.3, 127.6,
126.6, 126.4, 118.2, 96.1, 86.1, 71.9, 70.56, 70.55, 70.53, 70.46, 69.6, 59.0,
53.1, 38.0, 32.1. LRMS: m/z 524 ([M þ H]þ), 546 ([M þ Na]þ), 562
([M þ K]þ).
’ ASSOCIATED CONTENT
S
Supporting Information.
Semipreparative HPLC
b
analysis, optimized geometry for the lutetium complexes, Cartesian
coordinates for all optimized geometries, and a low-temperature
emission spectrum of [EuL2][TFA] in MeTHF. This material is
available free of charge via the Internet at http://pubs.acs.org.
General Procedure for Alkylation Reactions. To a solution of
macrocycle 13 (0.06 mmol) in dry MeCN (75 mL) were added a
mesylate derivative (0.12 mmol) and Na2CO3 (0.12 mmol) . The
resulting mixture was heated at reflux for 6 days and then filtered off.
The filtrate was concentrated in vacuo, and the oily residue was
partitioned between water (50 mL) and CH2Cl2 (50 mL). The aqueous
layer was extracted with CH2Cl2 (2 ꢁ 25 mL). The organic phases were
combined, dried over MgSO4, filtered, and concentrated in vacuo.
Purification of the sodium complexes was carried out using preparative
RP-HPLC, leading to the desired compounds. The purity of the
compounds was unambiguously established on the basis of the HPLC
chromatogram.
’ AUTHOR INFORMATION
Corresponding Author
*E-mail: llamarque@cisbio.com (L.L.), olivier.maury@ens-lyon.fr
(O.M.).
’ ACKNOWLEDGMENT
B.L.G. thanks the P^ole Scientifique de Modꢁelisation
Numꢁerique (PSMN) at Ecole Normale Supꢁerieure de Lyon for
the computing facilities. We thank F. Albrieux, C. Duchamp, and
N. Henriques from the Universitꢁe de Lyon for assistance and
access to the mass spectrometry facility.
Compound 14. Yield: 67%. ESþ-LRMS: m/z 692 ([C40H36N8O4]þ),
715 ([M þ Na]þ).
Compound 15.Yield: 78%. ESþ-LRMS: m/z1248 ([C70H72N8O10S2]þ),
1271 ([M þ Na]þ).
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ARTICLE
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