Sensitization of Eu(III) luminescence by donor-phenylethynylfunctionalized DTPA and DO3A macrocycles

Article abstract of DOI:10.1016/j.crci.2010.01.008

The synthesis and complexation to Eu(III) of two macrocyclic ligands functionalized donor-phenylethynyl-moieties as sensitizer is presented. The two ligands based on DTPA bis-amide ([Eu(L¹)]) and DO3A ([Na][Eu(L ²)]) have been chosen because of their increased stability in water for potential applications as luminescent bioprobes for one-or twophoton excited scanning microscopy. The DTPA and DO3A ligands possess oxygen or sulfur donor atoms, respectively, connected to a triethyleneglycol (PEG) chain to ensure the water solubility. The optical properties were studied and reveal that ([Eu(L¹)]) present a lower brightness than the ([Na][Eu(L ²)]) counterpart because of its inner sphere water molecule coordination that quenches the emission. On the other hand, [Na][Eu(L ²)] exhibits excellent spectroscopic properties with high quantum yield φ = 0.284, and brightness B = 10,220M^-1cm ^-1(defined as e.φ). Unfortunately, the two-photon crosssection of this last complex was measured to be only 4 GM limiting its potential applications to linear microscopy.

Full text of DOI:10.1016/j.crci.2010.01.008

C. R. Chimie 13 (2010) 681–690
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´
Full paper / Memoire
Sensitization of Eu(III) luminescence by donor-phenylethynyl-
functionalized DTPA and DO3A macrocycles
a
a
a
b
´
Anthony D’Aleo , Mustapha Allali , Alexandre Picot , Patrice L. Baldeck ,
Loı¨c Toupet c, Chantal Andraud a, Olivier Maury a,
*
a
´
CNRS–Ecole normale supe´rieure de Lyon, UMR 5182, laboratoire de Chimie, universite´ de Lyon, 46, alle´e d’Italie, 69007 Lyon, France
b Laboratoire de spectrome´trie physique, universite´ Joseph-Fourier, 38402 Saint-Martin-d’He`res, France
c
´
ˆ
Institut de physique–IPR–UMR CNRS 6251, universite de Rennes, 1, batiment 11A, 35042 Rennes cedex, France
A R T I C L E I N F O
A B S T R A C T
Article history:
The synthesis and complexation to Eu(III) of two macrocyclic ligands functionalized
donor-phenylethynyl-moieties as sensitizer is presented. The two ligands based on DTPA
bis-amide ([Eu(L1)]) and DO3A ([Na][Eu(L2)]) have been chosen because of their increased
stability in water for potential applications as luminescent bioprobes for one- or two-
photon excited scanning microscopy. The DTPA and DO3A ligands possess oxygen or sulfur
donor atoms, respectively, connected to a triethyleneglycol (PEG) chain to ensure the
water solubility. The optical properties were studied and reveal that ([Eu(L1)]) present a
lower brightness than the ([Na][Eu(L2)]) counterpart because of its inner sphere water
molecule coordination that quenches the emission. On the other hand, [Na][Eu(L2)]
Received 14 December 2009
Accepted after revision 14 January 2010
Available online 1 March 2010
Keywords:
Luminescence
Spectroscopy
Macrocyclic ligand
Nonlinear optics
Two-photon absorption
exhibits excellent spectroscopic properties with high quantum yield
brightness B = 10,220 Mꢀ1cmꢀ1(defined as
f = 0.284, and
e.f). Unfortunately, the two-photon cross-
section of this last complex was measured to be only 4 GM limiting its potential
applications to linear microscopy.
´
ß 2010 Academie des sciences. Published by Elsevier Masson SAS. All rights reserved.
´
´
R E S U M E
Cet article de´crit la synthe`se et la complexation a` l’europium (III) de deux ligands
Mots cle´s :
1
2
Luminescence
´
macrocycliques de type DTPA bis-amide ([Eu(L )]) et DO3A ([Na][Eu(L )]) fonctionnalises
Spectroscopies
Ligands macrocycliques
`
`
´ ´
par des groupements donneur-phenylene-ethynylene. Ces macrocycles ont ete choisis en
raison de leur stabilite´ reconnue dans les milieux biologiques afin de concevoir de nouvelles
´
Optique non lineaire
´
´ ´
bio-sondes luminescentes pour la microscopie biphotonique. L’etude des proprietes
`
Absorption a deux photons
´
spectroscopiques de ces deux complexes a mis en evidence l’excellent rendement quantique
= 0,284 ainsi que la brillance B = 10 220 Mꢀ1cmꢀ1(definie comme le
´
de luminescence,
produit
d’absorption a deux photons limitera ses potentialites en microscopie non lineaire.
f
e.f
) accrue du compose´ [Na][Eu(L2)]. Cependant, sa faible section efficace
`
´
´
´
´
´
´
ß 2010 Academie des sciences. Publie par Elsevier Masson SAS. Tous droits reserves.
1. Introduction
Luminescent lanthanide complexes have attracted
much recent attention because of their use in a wide
variety of bio-imaging applications such as biofluoroim-
munoassays [1–3], as biosensors [4–9], or for two-photon
* Corresponding author.
E-mail address: olivier.maury@ens-lyon.fr (O. Maury).
1631-0748/$ – see front matter ß 2010 Acade´mie des sciences. Published by Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.crci.2010.01.008

682
A. D’Ale´o et al. / C. R. Chimie 13 (2010) 681–690
bioprobes for scanning microscopy [10–15]. In most cases,
these complexes consist of a lanthanide ion coordinated to
a ligand that shields the metal ion from water coordination
and bears a sensitizing chromophore which transfers its
excitation energy to the luminescent lanthanide ion. The
presence of the organic chromophores able to strongly
absorb UV-visible light and to transfer this energy to the
lanthanide ion overcomes the limitation of an intrinsically
small molar absorption coefficient of the forbidden f-f
therefore understand the limitation residing in the
studied systems.
We recently showed that the water soluble functiona-
lized dipicolinate ligand can possess absorption transitions
in the UV-visible (up to 400 nm) with high molar absorption
coefficient and can efficientlysensitizeEu(III)[12,20], Yb(III)
or Nd(III) [20] ions. In the mean time, these complexes
present large two-photon cross-section values. This exalta-
tion is induced by intraligand charge transfer (ILCT) state.
Unfortunately, despite a stability good enough to make in
vitro measurements, the previously presented chelators are
not stable enough for in vivo applications. In the progress of
our research, we looked for a new scaffold to improve the
stability of the complexes. In this context, we decided to
base our complexes on the DTPA [21,22] and DO3A [23,24]
macrocyclic units well known to lead to the formation of
complexes with enhanced stability in aqueous media.
Here, we report on the synthesis and complexation to
Eu(III) of two new ligands featuring donor-phenylethynyl
functionnalities that are based on DTPA bis-amide
([Eu(L1)]) and DO3A ([Na][Eu(L2)]) (Scheme 1). Those
ligands possess electron donating oxygen or sulfur donor
transitions (
e
about 1–10 L.Molꢀ1.cmꢀ1). This so-called
‘‘antenna effect’’ results in the considerable increase of the
brightness defined as the product of the luminescence
quantum yield and the absorption coefficient, B = e.F. In
addition, the luminescence quantum yield and lifetime are
highly sensitive to the inner coordination sphere and
dramatically decrease when OH vibrators are coordinated.
Furthermore, for in vivo applications, the ligands have to
bind strongly the lanthanide ion in order to form
kinetically stable complexes in a biological medium to
avoid any release of toxic-free Ln(III) ions.
The unique properties of these ions include line-like
emission, long luminescence lifetimes (in the
ms-ms
range), the sensitivity to the surrounding symmetry and
the relative insensitivity of their emission to the presence
of dioxygen (that is due to very fast energy transfer from
the transferring excited state to the f-f emitting state)
[16,17]. Despite extensive research, the optimization of
the energy transfer for the sensitization of the lanthanide
ion by exciting complexed chromophores is still not fully
understood and remains a challenging task. In this
respect, the europium ion is an interesting probe since
Beeby et al. [18] and Werts et al. [19] have shown that the
steady state luminescence spectrum and the lumines-
cence lifetime of the europium ion can be used to
evaluate the efficiency of the sensitization process and
atoms, respectively, connected to a
p-conjugated skeleton
to induce charge transfer transition [20,25] and
a
additional triethyleneglycol (PEG) chain to insure the
water solubility. The optical properties were studied and
reveal that the DTPA bis-amide derivative ([Eu(L1)]) is
much less bright than the DO3A derivative ([Na][Eu(L2)])
because of the presence of a coordinated water molecule
that quenches the emission. On the other hand,
[Na][Eu(L2)] reaches excellent photophysical properties
almost as good as our benchmark compound [12].
However, the two-photon cross-section of this complex
was measured to be only 4 GM limiting its potentialities for
two-photon applications.
Scheme 1. Structures of the investigated europium(III) complexes.

A. D’Ale´o et al. / C. R. Chimie 13 (2010) 681–690
683
2. Results and discussion
12 was purified using column chromatography on alumina
oxide. After saponification of the four ester moieties, a one
pot complexation to Eu(III) ion was carried out in a
buffered solution at pH = 6.
2.1. Synthesis
The synthesis of the DTPA derivative (Scheme 2) was
readily started with the opening of the bis-anhydride 1 by
the addition of 4-iodoaniline giving the DTPA bis-amide
derivative 2. After esterification of the three free carboxylic
acid functions, the resulting tri-ester 3 was coupled to the
acetylene derivative 4 [26] using a Pd-catalyzed Sonoga-
shira cross-coupling reaction under standard conditions
yielding 5 after column chromatography with an accept-
able yield (66%). The final ligand L1 was obtained after
deprotection of the ester moieties in basic methanol-THF
solution (1 M sodium hydroxide). Complete analyzes are
compiled in the experimental section. Mixing the ligand L1
with one equivalent of EuCl3ꢁ6H2O in aqueous solution
followed by extraction with dichloromethane leads to the
formation of analytically pure [Eu(L1)]ꢁ6H2O.
2.2. Crystal structure of the Eu-DTPA model
Due to the presence of the long PEG chains, we were not
able to prepare good quality crystals of [EuL1]. Therefore,
white crystals of the model complex [Eu(2)] featuring
iodophenyl pendant arm were obtained upon cooling
down a hot methanol solution (see experimental section
for the complex preparation). The complex crystallized as a
methanol adduct with two methanol and one diethyl ether
interstitial solvent molecules. At the molecular level (Fig. 1,
top), the wrapping of the DTPA ligand around the central
Eu(III) ion orientates the two iodophenyl in almost the
same direction (the angle (I1N1),(I2N5) is estimated to be
128). It is worth noting that the two phenyl rings are not
parallel and form an angle of 328, a distortion certainly
related to the steric hindrance induced by the coordinated
methanol molecule. The central europium ion is nona-
coordinated by three nitrogen, the carboxylato- and two
amido-oxygen atoms, the last position being occupied by
an oxygen atom from a methanol molecule. The coordina-
tion polyhedron (Fig. 1, bottom) is a distorded tricapped
trigonal prism with the Eu,O3,O9,N3 atoms lying in the
The DO3A derivative was prepared starting from the
iododipicolinic diester 6 [27] (Scheme 3). The key step of
this synthesis was the desymmetrisation of
6 into
carboxylic acid-alcohol 7 thanks to a kinetically controlled
monoreduction by sodium borohydride at room tempera-
ture in methanolic solution. After Sonogashira cross-
coupling with 8 [26], the chromophore 9 was obtained
in good yield (84%) after column chromatography. The
alcohol function was then activated with mesyl chloride
(10) and further attached to cyclen macrocycle giving the
monosubstituted species 11. The remaining free amine
functions of 11 were then alkylated by bromoacetate in
basic medium and the final functionalized DO3A derivative
˚
equatorial plane (maximum standard deviation < 0.1 A).
Such type of structure is relatively frequent for
lanthanide DTPA complexes also dimer formation can be
a
Scheme 2. Synthetic pathway for the preparation of [Eu(L1)].

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A. D’Ale´o et al. / C. R. Chimie 13 (2010) 681–690
Scheme 3. Synthetic pathway for the preparation of [Na][Eu(L2)].
observed depending on the sterical hindrance afforded by
the DTPA functionalization [28] (Table 1).
700–900 nm (i.e. when doubled: 350–450 nm). Further-
more, the observed cut-off of the UV/visible absorption for
[Eu(L1)] (at 350 nm) precludes any measurement of the
two-photon excitation spectrum. In addition, the molar
absorption coefficients of [Eu(L1)] and [Na][Eu(L2)] are
17,650 and 36,000 Mꢀ1cmꢀ1, respectively. In this case, the
molar absorption coefficient is in favor of [Na][Eu(L2)] that
will give an advantageous brightness for this complex.
2.3. UV-visible absorption spectroscopy
The UV/visible absorption data for each of the Eu(III)
complexes in water are depicted in Fig. 2 and all the data
are summarized in Table 2. The two complexes possess
their lowest absorption transition in the visible with
maxima at 300 and 338 nm for [Eu(L1)] and [Na][Eu(L2)],
respectively. This red shift observed for [Na][Eu(L2)]
as compared to [Eu(L1)] will favor the former complex
for two-photon applications since measurements
are performed using Ti-sapphire laser sources between
2.4. Luminescence of Eu(III) complexes and determination of
the sensitization parameters
The steady state emission spectra, the luminescence
quantum yields and luminescence lifetimes of the Eu(III)

A. D’Ale´o et al. / C. R. Chimie 13 (2010) 681–690
685
Table 1
Crystal data and refinement parameters.
Formula
C34H55EuI2N5O13
f.w. (g.molꢀ1
Cryst. Syst.
Space group
)
1147.59
Monoclinic
P21/n
˚
a (A)
10.4117(2)
14.7816(4)
26.6463(6)
90.0
˚
b (A)
˚
c (A)
a
b
g
(o)
(o)
(o)
99.2700(10)
90.0
3
˚
V (A )
Z
4047.35(16)
4
T (K)
120
˚
l
(MoK
D (g.cmꢀ3
(mmꢀ1
R(F)a, I > 1
a
) (A)
0.71069
1.883
)
m
)
3.143
s
(Fo)
0.037
R
w(F2)b, I > 1
s(Fo)
0.094
S
0.67
Rint
0.056
u
h
k
l
max
32.25
ꢀ15 ! 14
ꢀ22 ! 13
ꢀ37 ! 38
496
Parameters
Measured reflections
37666
Independent reflections
12950
Reflections with I > 2.0
s(I)
9882
˚
Drmin
Drmax
ꢀ2.095 e A-1
˚
2.217 e A-1
a
R(F) =
S
S
jjFoj ꢀ jFcjj/
S
jFoj.
b
Rw(F) =
[w ((Fo2 ꢀ Fc2)2/S wFo4]1/2
.
complexes were measured in water (Table 2). The relevant
radiative and non-radiative parameters were also deter-
mined following the works of Werts et al. [19] and Beeby
et al. [18], and a complete summary of these data are
reported in Table 3. As can be seen from Fig. 3, the spectra
of the two Eu(III) complexes are very similar with intense
J = 2 and J = 4 transitions. In both spectra, all transitions are
split in numerous transitions revealing the low symmetry
Fig. 1. ORTEP drawing for [EuI2C27H33N5O9.CH3OH].3CH3OH.C4H10O.
Interstitial solvent molecules were omitted for clarity (top).
Coordination polyhedron around the Eu(III) ion (bottom). Selected
˚
bond lengths (A): (amino) Eu-N2 = 2.755(3); Eu-N3 = 2.640(3); Eu-N4 =
2.673(3); (amido) Eu-O1 = 2.485(2); Eu-O8 = 2.481(2); (carboxylato) Eu-
O7 = 2.394(2); Eu-O5 = 2.344(2); Eu-O3 = 2.331(3); (methanol) Eu-O9 =
2.483(3).
Fig. 2. UV/visible absorption spectra in water of [Eu(L1)] (__) and [Na][Eu(L2)] (–).

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A. D’Ale´o et al. / C. R. Chimie 13 (2010) 681–690
Table 2
Optical data in water for the two complexes.
Complex
Absorption
Luminescence
Ha
tEu ms
Da
tEu ms
lmax nm
e
M
ꢀ1cmꢀ1
Cut-off nm
fEu
B Mꢀ1cmꢀ1
[Eu(L1)]
[Na][Eu(L2)]
300
338
17,650
36,000
350
405
0.019
0.284
0.59
1.39
1.87
n.d.
335
10,220
a
H
D
tEu and tEu represent the luminescence lifetime in H2O and D2O, respectively.
Table 3
Calculated luminescence parameters
[I(0,1)/Itot] in water.
tR, kr, Sknr, hEu, and hsens for the two investigated Eu(III) complexes using the experimental quantities fEu, tEu and
Complex
fEu
tEu/ms
tR/ms
hEu
hsens
[I(0,1)/Itot
]
kr/sꢀ1
S
knr/sꢀ1
[Eu(L1)]a
[Na][Eu(L2)]
[Eu(L1)]a,b
0.019
0.284
0.078
0.59
1.39
0.98
4.58
4.24
3.86
0.129
0.328
0.254
0.147
0.866
0.305
0.148
0.137
0.152
218
236
259
1477
483
761
a
Not taking the J = 3 into account because of the overlap with the 2
Measured in dichloromethane solution.
l of the excitation.
b
Fig. 3. Normalized luminescence spectra (on J = 2) of [Na][Eu(L2)] (bottom, lex = 335 nm) and [Eu(L1)] (top, lex = 325 nm) in water.
of those complexes [29]. In the two cases, the J = 0 seems to
be a single transition which is typical of the presence of only
one species in solution (full-width-at-half-maximum of the
J = 0 transitions are 750 and 450 cmꢀ1 for [Eu(L1)] and
[Na][Eu(L2)], respectively). Thisobservation isconfirmed by
the presence of a mono-exponential decay for both samples.
The luminescence quantum yields measured in water are
very different about 2% for [Eu(L1)] up to 28% for
[Na][Eu(L2)] with luminescence lifetimes of 0.59 and
1.39 ms, respectively. Such differences in terms of lumines-
cence quantum yields and lifetimes suggest the presence of
water molecules in the inner sphere of [Eu(L1)] while the
complex [Na][Eu(L2)] most likely presents a saturated
coordination sphere. In order to determine the number of
water molecules in the inner sphere of [Eu(L1)], the
luminescence lifetime was also measured in deuterated
water, yielding a value of 1.87 ms, which is far higher than
that in water. The q number can therefore be obtained
(Table 3) using the improved phenomenological Horrocks
equation [30], revealing a q = 1.2 (substantially one water
molecule) in agreement with the crystal structure of the
model complex [Eu(2)] containing one solvent molecule.
Similar water molecule coordination has already been
observed in related complexes [31].
To understand the limitation and advantage of such
ligands, the radiative and non-radiative parameters were
determined. As can be seen in Table 3, the symmetry of
both complexes are almost equivalent giving identical
ratio of intensities between the J = 1 and the whole
spectrum. Consequently, both complexes exhibit identi-
cal radiative lifetimes (tr) and radiative decay rate (kr)
[31]. Here, it has to be highlighted that both complexes
possess a coordination number of 9 giving the possibility
of a similar symmetry around the metal (in the case of
[Eu(L1)], the ninth site being occupied by the water
molecule). Therefore, for [Eu(L1)], the large decrease
of the luminescence lifetime induces a drop of the
europium centered efficiency (hEu = 0.129) compared to

A. D’Ale´o et al. / C. R. Chimie 13 (2010) 681–690
687
Fig. 4. a: two-photon excitation spectrum of [Na][Eu(L2)] in water solution (&, upper abscissa). Superimposed on this plot is the single-photon absorption
spectrum in wavelength doubled scale (__, lower abscissa); b: two-photon excitation spectrum of [Na][Eu(L2)] in water solution (&).
[Na][Eu(L2)] (
h
Eu = 0.328) that is followed by a large
be partially rationalized by the presence of only one
chromophore while three of such chromophores were
present in the previously cited studies.
difference in the non-radiative decay rate (483 sꢀ1 vs.
1477 sꢀ1 for [Na][Eu(L2)] and [Eu(L1)], respectively). As
can be seen in the Table 3, all parameters related to the
europium centre (tEu and hEu) are very low in the case of
3. Conclusion
[Eu(L1)]. This can be explained by the inner sphere water
molecule bound to the metal cation (also increasing the
In conclusion, this article reported the first attempts at
the design of europium complexes featuring high stability
in biological media and excellent one- or two-photon
brightness, which are the key requirements for a lumines-
cent bioprobe for biphotonic microscopy. To that end, well-
known macrocycles, namely DTPA and DO3A, were
functionalized by already described two-photon antenna
chromophores. In the case of DTPA-bis amide derivative
[Eu(L1)], the presence of one water molecule in the first
coordination sphere results in a strong quenching of the
europium luminescence. On the other hand, the functio-
nalized DO3A complex Na[Eu(L2)] presents an excellent
one-photon brightness, but the presence of only one
antenna chromophore reduces dramatically its two-
photon absorption efficiency. New macrocycles functio-
nalized by several two-photon antenna chromophores are
currently being prepared in our group towards the design
of this new generation of Ln-based bioprobes.
non-radiative decay rate (
the sensitization efficiency (
S
h
knr)). It is worth noting that
sens), that is independent of
the presence of inner sphere water molecule, is low for
[Eu(L1)] (0.147) while it is almost quantitative for
[Na][Eu(L2)] (0.866). In the dichloromethane solution,
this sensitization efficiency remains rather low
(h
sens = 0.305) proving the inefficiency of L1 ligand to
induce Eu(III) luminescence.
2.5. Two-photon absorption properties
Since [Eu(L1)] has its UV/visible absorption cut-off at
350 nm and is poorly emissive (f = 0.019), no measure-
ment of the two-photon excitation spectrum was per-
formed. On the other hand, the two-photon absorption
cross-sections (s
2PA) of [Na][Eu(L2)] have been deter-
mined in water using the two-photon excited fluorescence
technique in the 700–900 nm wavelength range with a
femtosecond Ti-Sapphire pulsed laser source, according to
the experimental protocol described by Xu and Webb
4. Experimental
(coumarin-307 as
a standard) [32]. The two-photon
excitation spectrum matches the wavelength-doubled
scale single-photon one (Fig. 4a). This similarity indicates
that the low lying excited state in energy is one- and two-
photon allowed and confirms that the europium lumines-
cence is induced by a two-photon antenna effect with a
similar photophysical sensitization process (the same
excited states are involved in the one- or two-photon
sensitization process). As can be seen in Fig. 4, the s2PA
found for [Na][Eu(L2)] in water is rather small compared to
the value reported for the europium (III) dipicolinate
derivatives [12,33] revealing a maximum of 4 GM. This can
4.1. Crystal structure analysis
The samples were studied on a Oxford Diffraction
Xcalibur Saphir 3 diffractometer with graphite monochro-
matized MoKa radiation. Cell parameters were obtained
with Denzo and Scalepack with 10 frames (psi rotation: 18
per frame). The structure was solved with SIR-97 [34],
which reveals the non hydrogen atoms of structure. After
anisotropic refinement, many hydrogen atoms may be
found with a Fourier Difference. The whole structure
was refined with SHEL-XL97 [35] by the full-matrix

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A. D’Ale´o et al. / C. R. Chimie 13 (2010) 681–690
least-square techniques (use of F magnitude; x, y, z,
a
ij for
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 an internal
standard. Low resolution mass spectrometry was carried
out on an Agilent 1100 Series LC/MSD apparatus. High-
resolution mass spectrometry measurements and elemen-
tal analyses were performed at the Service Central
d’Analyse du CNRS (Vernaison, France).
C, N and O atoms, x, y, z in riding mode for H atoms). ORTEP
views were made with PLATON 98 [36]. All calculations
were performed on a Silicon Graphics Indy computer.
Crystallographic datum for the structural analysis has been
deposited with the Cambridge Crystallographic Data
Centre, CCDC 601476.
4.2. UV/visible absorption measurements
4.6. Synthesis
UV/visible absorption measurements were recorded on
a JASCO V550 absorption spectrometer. The spectra were
corrected using a solution of water as a reference.
1. In a 250 mL round bottom flask, a suspension of DTPA
(49 g, 125 mmol) in acetic anhydride (53 mL, 560 mmol)
and dry pyridine (62 mL, 770 mmol) was heated at 65 8C,
while stirring for 24 h. After cooling to room temperature,
the precipitate formed was filtered off and then washed
with diethyl ether. After drying under vacuum, pure DTPA-
bis(anhydride) 4 was obtained (43 g, 120 mmol, 96%). 13C-
NMR ([d6]-DMSO): 50.78, 51.72, 52.71, 54.73, 165.88,
171.82 ppm.
4.3. Luminescence measurements
The luminescence spectra were measured using a
Horiba-Jobin Yvon Fluorolog-3 spectrofluorimeter (see
the preceding article for details). Phosphorescence life-
times (> 30 ms) were obtained by pulsed excitation using
an FL-1040 UP xenon lamp. Luminescence decay curves
were fitted by least-squares analysis using Origin. Fluo-
rescence quantum yields, Q, were measured in a diluted
solution with an optical density lower than 0.1 using the
2. In a 50-ml round bottom flask, iodoaniline was
solubilized in DMF (20 mL). Pyridine was added to the
solution followed by 1. The solution was stirred for 1–2 h at
room temperature. The DMF was removed by adding
toluene, and the solid obtained was cleaned twice
with dichloromethane (2 ꢂ 150 mL). Yield 85%. 1H-NMR
(200.13 MHz, [d6]-DMSO): 10.12 (d, 2H), 7.55 (d,
3J = 8.8 Hz, 4 H), 7.45 (d, 3J = 8.8 Hz, 4H), 3.43 (m, 10H),
2.95 (m, 4H), 2.90 (m, 4H).
3. In a 250-ml round bottom flask, 2 was suspended in
anhydrous MeOH (150 mL), 2 mL of concentrated H2SO4
was added and the solution was refluxed for 16 h. After a
cooling period, the acid was neutralised by adding
NaHCO3. The solution was filtered and the solvent was
evaporated. Yield 95%. 1H-NMR (200.13 MHz, CDCl3): 9.77
(bs, 2H, NH), 7.57 (d, 3J = 6.8 Hz, 4H), 7.40 (d, 3J = 6.8 Hz,
4H), 3.68 (m, 9H), 3.48 (m, 10H), 2.71 (m, 8H); 13C-NMR
(50.332 MHz, CDCl3): 177.06, 175.80, 174.23, 172.30,
141.64, 141.44, 126.05, 125.95, 90.86, 50.78, 50.36,
51.63, 51.21, 52.91, 52.49, 52.06, 55.00; m/z 838 (MH+
requires 838), 860 (MNa+ requires 860).
following equation: Qx/Qr = [Ar(
where A is the absorbance at the excitation wavelength
), n is the refractive index, and D is the integrated
l)/Ax(l
)][nx2/nr2][Dx/Dr],
(l
luminescence intensity. The ‘‘r’’ and ‘‘x’’ stand for reference
and sample. Here, references are Ru(bpy)3 2Cl complexes
in a water solution (Qr = 0.028) [37] and [Na]3[EuL1G
(Qr = 0.157) [12] for each complex.
]
3
4.4. Two-photon excited luminescence measurements
The TPA cross-section spectra were obtained by up-
conversion fluorescence using a Ti:sapphire femtosecond
laser in the range of 700–900 nm. The excitation beam
(5 mm diameter) is focalized with a lens (focal length
10 cm) at the middle of the fluorescence cell (10 mm). The
fluorescence, collected at 908 to the excitation beam, was
focused into an optical fiber (diameter 600
mm) 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 fluorescence 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 700–900 nm Coumarin-
307 two-photon absorption spectrum60 (Coumarin-307
quantum yield 0.56 in ethanol). The measurements were
done at room temperature in dichloromethane and at a
concentration of 10ꢀ4 M.
5. In a 100-mL schlenk flask, 3 (0.25 g, 0.30 mmol) was
dissolved in a mixture of THF and triethylamine (15 mL/
15 mL). The solution was thoroughly degassed by argon
bubbling for 20 min and 4 [33] (0.75 mmol, 2.5 equiv.),
copper iodide (0.2 equiv.) and (Ph3P)PdCl2 (0.l equiv.) were
added under a flux of argon. The reaction was stirred at
40 8C for 14 h. The compound was purified by column
chromatography on silica using gradient ethyl acetate/
methanol (9/1 to 7/3) as eluent. This yields a brown oil
(0.22 g, 66%). 1H-NMR (200.13 MHz, CDCl3):
d = 9.82 (bs,
2H), 7.59 (d, 3J = 8.6 Hz, 4H), 7.44 (m, 8H), 6.88 (d,
3J = 8.7 Hz, 4H), 4.12 (t, 3J = 5.6 Hz, 4H), 3.84 (t, 3J = 5.5 Hz,
4H), 3.75 (s, 9H), 3.40–3.72 (m, 26H), 3.54 (m, 2H), 3.36 (s,
6H), 2.74 (m, 8H); EI-MS: [M + H]+: 1110, [M + Na]+: 1132.
4.5. General procedure
4
and 8 were prepared following the published
procedure [33]; all other starting materials were commer-
cially available. All solvents for synthesis were analytic
grade. For the spectroscopy, spectroscopic grade solvents
were used. NMR spectra (1H and 13C) were recorded at
room temperature on a BRUKER AC 200 operating at
200.13 and 50.32 MHz for 1H and 13C, respectively. Data
L1. In
a 100-mL round bottom flask, 5 (0.236 g,
0.230 mmol) was dissolved in 30 mL of methanol and
stirred at room temperature. Subsequently, 10 mL of a 1 M
aqueous sodium hydroxide solution is added and the
reaction is stirred at room temperature for 2 h. The
solution is extended to 50 mL by adding water and the

A. D’Ale´o et al. / C. R. Chimie 13 (2010) 681–690
689
methanol is removed under vacuum. The aqueous solution
is extracted with ethyl acetate (2 ꢂ 50 mL), then acidified
to pH = 2 by adding 1 M HCl aqueous solution, and
extracted with dichloromethane (3 ꢂ 50 mL). The dichlor-
omethane solutions are regrouped and dried over sodium
sulfate and the dichloromethane was evaporated yielding
the pure 8 as a light yellow solid (0.22 g, 93%). 1H-NMR
iodide (0.112 g, 0.59 mmol, 0.2 eq) and Pd(PPh3)2Cl2
(0.207 g, 0.295 mmol, 0.1 eq). The dark brown mixture is
heated in the dark at 40 8C while stirring for 20 h. After
cooling to room temperature, the black precipitate is
filtered and triturated with Et2O (2 ꢂ 40 mL). The remain-
ing organic phase is washed with saturated aqueous
ammonium chloride solution (2 ꢂ 50 mL), and brine
(50 mL). The organic layer is dried (Na2SO4) and evaporat-
ed under vacuum. The crude residue is purified by flash
chromatography (CH2Cl2/MeOH 49/1) yielding a light
yellow oil (1.267 g, 84%). 1H NMR (200.13 MHz, CDCl3):
7.90 (d, 4J = 1.2 Hz, 1H), 7.56 (d, 4J = 1.2 Hz, 1H), 7.30 (d,
3J = 8.5 Hz, 2H), 7.17 (d, 3J = 8.5 Hz, 2H), 4.75 (d, 3J = 4.3 Hz,
2H), 4.29 (t, 3J = 4.4 Hz, 1H), 3.84 (s, 3H), 3.61–3.38 (m,
10H), 3.24 (s, 3H), 3.04 (t, 3J = 6.8 Hz, 2H); 13C-NMR
(50.332 MHz, CDCl3): 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
(MH+ requires 446), 468 (MNa+ requires 468).
10. To a stirred solution of 9 (1.10 g, 2.56 mmol, 1 eq)
and triethylamine (0.777 g, 7.68 mmol, 3 eq) in CH2Cl2
(100 mL) in a 250-mL round bottom flask, is added
methanesulfonyl chloride (0.440 g, 3.84 mmol, 1.3 eq)
drop by drop at room temperature. After 30 min, the
reaction is quenched by addition of saturated NaHCO3
solution (100 mL). The organic phases is extracted, dried
over Na2SO4, and evaporated. The crude material is
purified by chromatography on silica (CH2Cl2/MeOH 97/
3) to afford the product as a bright yellow oil (1.19 g, 92%).
1H NMR (200.13 MHz, CDCl3): 8.04 (d, 4J = 1.2 Hz, 1H), 7.62
(d, 4J = 1.2 Hz, 1H), 7.37 (d, 3J = 8.5 Hz, 2H), 7.22 (d,
3J = 8.5 Hz, 2H), 5.32 (s, 2H), 3.91 (s, 3H), 3.66–3.42 (m,
10H), 3.28 (s, 3H), 3.13–3.04 (m, 5H); 13C-NMR
(50.332 MHz, CDCl3): 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 (MH+ requires 524), 546 (MNa+ requires 546), 562
(MK+ requires 562).
11. To a stirred solution of 1,4,7,10-tetraazacyclodode-
cane (1.92 g, 11.2 mmol, 4 eq) in CHCl3 (100 mL) in a 250-
mL round bottom flask, is added a solution of 10 (1.46 g,
2.8 mmol, 1 eq) in CHCl3 (100 mL) drop by drop over 3 h at
room temperature. After 20 h of stirring, the mixture is
evaporated and the crude material is purified twice by
chromatography on aluminum oxide (CH2Cl2/MeOH 97/3)
to afford the product as a bright yellow oil (1.00 g, 60%). 1H
NMR (200.13 MHz, CDCl3): 7.93 (d, 4J = 1.1 Hz, 1H), 7.47 (d,
43J = 1.1 Hz, 1H), 7.33 (d, 3J = 8.5 Hz, 2H), 7.20 (d, 3J = 8.5 Hz,
2H), 5.31 (b, 3H), 3.90–3.84 (m, 5H), 3.64–3.40 (m, 10H),
3.26 (s, 3H), 3.06 (t, 3J = 6.8 Hz, 2H), 2.65-2.90 (m, 16H);
13C-NMR (50.332 MHz, CDCl3): 165.2, 160.0, 147.5, 139.2,
133.3, 132.2, 127.7, 127.4, 125.7, 118.5, 95.1, 86.4, 71.8,
70.51, 70.50, 70.4, 69.6, 59.5, 59.0, 53.0, 51.8, 47.2, 45.7,
45.3, 32.1; LRMS m/z: 600 (MH+ requires 600), 622 (MNa+
requires 622).
(200.13 MHz, CD3OD):
d
= 7.56 (d, 3J = 8.0 Hz, 4H), 7.33 (m,
8H), 6.82 (d, 3J = 8.1 Hz, 4H), 4.06 (m, 4H), 3.89 (m, 4H),
3.31–3.83 (m, 59H); 13C-NMR (50.332 MHz, CD3OD):
d
= 171.4, 169.1, 168.1, 1158.9, 137.5, 132.7, 131.6,
115.5, 114.6, 88.7, 87.4, 71.6, 70.4, 70.1, 69.4, 67.4, 59.7;
54.9, 52.7, 50.8; m/z (ESI) 1068 (MH+ requires 1068), 1090
(MNa+ requires 1090); elemental analysis: Calcd for
C
56H69N5O16ꢁ6H2O: C, 57.18; H, 6.94; N, 5.95; found: C,
56.85; H, 6.94; N, 5.66.
[Eu(L1)]. In a 100-mL round bottom flask, L1 (1 eq) and
sodium carbonate (5 eq) are solubilized in water (50 mL).
Europium(III) chloride hexahydrate (1 eq) is solubilized in
water (10 mL) and added to the solution. The reaction flask
is stirred for 1 h at room temperature before being
extracted with dichloromethane (4 ꢂ 100 mL). The organic
phases are combined, dried over sodium sulfate, and
evaporated under vacuum giving a slightly yellow solid
(0.22 g, 93%). m/z (ESI) 1218 (MH+-H2O requires 1218),
1239 (MNa+ requires 1239); elemental analysis: Calcd for
C56H66N5O15Euꢁ6H2O: C, 50.75; H, 5.93; N, 5.28; found: C,
50.46; H, 5.50; N, 5.15.
[Eu(2)]. In a 100-mL round bottom flask, 5 (651 mg,
0.819 mmol) was dissolved in basic water (30 mL with
K2CO3 (302 mg, 2.19 mmol)). Then, EuCl3ꢁ6H2O (305 mg,
0.832 mmol) was added and the solution was stirred for 3 h
at room temperature. The precipitate was filtered and the
white solid was cleaned with cold water and with ether.
(764 mg, 98.8%) (M = 944.3 g.molꢀ1
[M + H]+, 966.0[M + Na]+.
) M(HPLC-MS) = 945.2
7. To a stirred mixture of insoluble dimethyl 4-
iodopyridine-2,6-dicarboxylate 6 (2 g, 6.23 mmol, 1 eq)
and MeOH (40 mL) in a 250-mL round bottom flask, is
added NaBH4 (473 mg, 12.5 mmol, 2 eq) at room tempera-
ture in one portion. The colorless solution becomes red
while H2 is released. As soon as
6 is solubilized
(approximately 20 s), the mixture is quenched by addition
of saturated NaHCO3 solution (40 mL). The MeOH is
evaporated under vacuum, and the aqueous solution is
extracted with ethyl acetate (3 ꢂ 100 mL). The combined
organic extracts are dried (Na2SO4), and the solvent
evaporated under vacuum. The crude material is purified
by chromatography on silica (CH2Cl2/Acetone 9/1) to
afford the product as
a white crystalline powder.
Depending on the activity of the NaBH4 and the length
of the reaction, the yields average from 50 to 76%. 1H NMR
(200.13 MHz, CDCl3): 8.37 (s, 1H), 7.95 (s, 1H), 4.81 (d,
3J = 4.9 Hz, 2H), 3.99 (s, 3H), 3.09 (t, 3J = 4.9 Hz, 1H);
13C-NMR (50.332 MHz, CDCl3): 164.4, 161.4, 147.2, 133.2,
132.9, 106.8, 64.2, 53.2; LRMS m/z: 294 (MH+ requires
494), 316 (MNa+ requires 316).
9. To a degassed solution of 7 (0.865 g, 2.95 mmol, 1 eq)
and (4-ethynylphenyl)(2-(2-(2-methoxyethoxy)ethoxy)
ethyl)sulfane 8[33] (0.858 g, 3.25 mmol, 1.1 eq) in THF
(20 mL) and Et3N (10 mL) under argon, is added copper
12. To a stirred solution of 11 (0.852 g, 1.42 mmol, 1 eq)
and Cs2CO3 (1.48 g, 4.55 mmol, 3.2 eq) in CH3CN (100 mL)
in a 250-mL round bottom flask, is added methyl 2-
bromoacetate (0.782 g, 5.11 mmol, 3.6 eq). After stirring
for 20 h at room temperature, CH2Cl2 (100 mL) is added
and the solution is filtered. After evaporation of the

690
A. D’Ale´o et al. / C. R. Chimie 13 (2010) 681–690
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solvents, the crude material is purified by chromatography
on aluminum oxide (CH2Cl2: MeOH 39/1) to afford the
product as a bright yellow oil (1.08 g, 93%). 1H NMR
(500 MHz, CDCl3): 7.92 (s, 1H), 7.44 (s, 1H), 7.33 (d,
3J = 8.4 Hz, 2H), 7.19 (d, 3J = 8.4 Hz, 2H), 3.87 (s, 3H), 3.67 (s,
3H), 3.59 (t, 3J = 6.8 Hz, 2H), 3.52-3.46 (m, 12H), 3.44–3.36
(m, 2H), 3.24 (s, 3H), 3.06 (t, J = 6.8 Hz, 2 H), 3.2–2.2 (m,
24H); 13C-NMR (50.332 MHz, CDCl3): 173.5, 172.4, 165.0,
159.2, 147.7, 139.5, 134.0, 132.2, 129.0, 127.5, 125.3, 118.0,
96.2, 85.9, 71.7, 70.31, 70.29, 70.26, 69.4, 59.1, 58.8, 55.2,
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/dm3
molꢀ1 cmꢀ1 27400); LRMS m/z: 816 (MH+ requires 816),
838 (MNa+ requires 838); HRMS m/z: 816.3848 (MH+ cald
for C40H58N5O11S requires 816.3854).
[Na][Eu(L2)]. To a stirred solution of 12 (122 mg,
0.15 mmol, 1 eq) in H2O (10 mL) in a 50-mL round bottom
flask, is added NaOH (36 mg, 0.9 mmol, 6 eq). After stirring
for 1 h at 50 8C, the solution is cooled down to room
temperature and acidified to pH = 6 by addition of 0.1 M HCl
solution. At that stage, a solution of EuCl3ꢁ6H2O (66 mg,
0.18 mmol, 1.2 eq) in H2O (10 mL) is slowly added while the
pHofthereactioniskeptatpH = 6byadditionof0.1 MNaOH
solution. Once the pH of the reaction is stable, the reaction is
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dm3 molꢀ1 cmꢀ1 36100); LRMS m/z: 910 (MHH+ requires
910), 932 (MHNa+ requires 932), 948 (MHK+ requires 948),
954 (MNaNa+ requires 954); HRMS m/z: 954.1859 (MNaNa+
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The authors thank the ANR LnOnL NT05-3_42676 for
financial support and for grants to AD and MA. We are also
grateful to ENS Lyon for AP’s grant.
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