Comparative analysis of conjugated alkynyl chromophore-triazacyclononane ligands for sensitized emission of europium and terbium

Article abstract of DOI:10.1002/chem.201402415

A series of europium and terbium complexes based on a functionalized triazacyclononane carboxylate or phosphinate macrocyclic ligand is described. The influence of the anionic group, that is, carboxylate, methylphosphinate, or phenylphosphinate, on the photophysical properties was studied and rationalized on the basis of DFT calculated structures. The nature, number, and position of electron-donating or electron-withdrawing aryl substituents were varied systematically within the same phenylethynyl scaffold in order to optimize the brightness of the corresponding europium complexes and investigate their two-photon absorption properties. Finally, the europium complexes were examined in cell-imaging applications, and selected terbium complexes were studied as potential oxygen sensors.

Full text of DOI:10.1002/chem.201402415

DOI: 10.1002/chem.201402415
Full Paper
&
Lanthanide Complexes
Comparative Analysis of Conjugated Alkynyl Chromophore–
Triazacyclononane Ligands for Sensitized Emission of Europium
and Terbium
Marine Souliꢀ,[a] Frꢀdꢀric Latzko,[a] Emmanuel Bourrier,[a] Virginie Placide,[b]
Stephen J. Butler,[c] Robert Pal,[c] James W. Walton,[c] Patrice L. Baldeck,[b, e]
Boris Le Guennic,[d] Chantal Andraud,[b] Jurriaan M. Zwier,[a] Laurent Lamarque,*[a]
David Parker,*[c] and Olivier Maury*[b]
Abstract: A series of europium and terbium complexes
based on a functionalized triazacyclononane carboxylate or
phosphinate macrocyclic ligand is described. The influence
of the anionic group, that is, carboxylate, methylphosphi-
nate, or phenylphosphinate, on the photophysical properties
was studied and rationalized on the basis of DFT calculated
structures. The nature, number, and position of electron-do-
nating or electron-withdrawing aryl substituents were varied
systematically within the same phenylethynyl scaffold in
order to optimize the brightness of the corresponding euro-
pium complexes and investigate their two-photon absorp-
tion properties. Finally, the europium complexes were exam-
ined in cell-imaging applications, and selected terbium com-
plexes were studied as potential oxygen sensors.
Introduction
The f-block elements exhibit intrinsic spectroscopic advan-
tages for such applications. Their sharp emission bands and
large pseudo-Stokes shifts following indirect ligand excitation
facilitate selective detection in biological media even in a multi-
plexing experiment, and their long excited-state lifetimes
enable time-resolved detection of the luminescence signal.[6c]
These spectral and temporal resolutions result in a significant
increase in signal-to-noise ratio, which is crucial for imaging
purposes.[7]
In the last two decades, various aspects of f-element spectros-
copy have been extensively studied,[1] and lanthanide coordi-
nation complexes in particular have found important applica-
tions in the biological sciences.[2] In this context, they have
been used as luminescent probes for one- or two-photon
imaging,[3,4] as responsive probes able to detect and quantify
in vitro or in cellulo biological activity or the presence of
a given substrate (pH, metal ions, bicarbonate, lactate, urate)[5]
and as emissive bioconjugated tags for time-resolved Fçrster
resonance energy-transfer assays.[6]
All of these favorable properties have triggered the design
of numerous complexes that meet a stringent set of require-
ments: 1) the complexes must be strongly coordinated to min-
imize nonradiative losses and ensure their stability in biological
media, bearing in mind that coordinated water molecules are
effective quenchers of the lanthanide excited state, 2) they
must be sufficiently water soluble, and 3) they must have opti-
mal brightness B (B=eF, where e is the extinction coefficient
and F the quantum yield) for enhanced detection, ideally at
an excitation wavelength above 330–350 nm to allow the use
of glass microscopy objectives. The choice of the chelating
ligand and of the organic chromophore antenna modifies
these requirements[8] and has led to the design of several
classes of compounds, for example, cryptates,[9] helicates,[10]
polyaminocarboxylates or -phosphinates,[11] and macrocyclic
derivatives, including those based on the well-known cyclen
family.[12] In this context, taking advantage of the established
stability of lanthanide complexes of triazacyclononane tris-pyri-
dine carboxylates[13] and (methyl/phenyl) phosphinates[14]
([Ln.Lc], [Ln.Lmp], and [Ln.Lpp], respectively; Figure 1), we re-
cently reported ytterbium-based bioprobes for thick-tissue
imaging by near-infrared two-photon microscopy[15] and excep-
[a] M. Souliꢀ, F. Latzko, E. Bourrier, Dr. J. M. Zwier, Dr. L. Lamarque
Cisbio Bioassays, Parc Marcel Boiteux
BP 84175, 30200 Codolet (France)
E-mail: llamarque@cisbio.com
[b] Dr. V. Placide, Dr. P. L. Baldeck, Dr. C. Andraud, Dr. O. Maury
University of Lyon, ENS Lyon, CNRS-UMR 5182
46 Allꢀe d’Italie 69364 Lyon (France)
E-mail: olivier.maury@ens-lyon.fr
[c] Dr. S. J. Butler, Dr. R. Pal, Dr. J. W. Walton, Prof. D. Parker
Department of Chemistry, Durham University
South Road, Durham, DH13LE (UK)
E-mail: david.parker@dur.ac.uk
[d] Dr. B. Le Guennic
Institut des Sciences Chimiques de Rennes, UMR 6226 CNRS
Universitꢀ de Rennes 1
263 Avenue du Gꢀnꢀral Leclerc 35042 Rennes (France)
[e] Dr. P. L. Baldeck
Laboratoire de spectromꢀtrie Physique Universitꢀ Joseph Fourier
BP 87, 38402 Saint Martin d’Hꢁres (France)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/chem.201402415.
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Results and Discussion
Synthesis
The synthesis of the 1,4,7-triaza-
cyclononane (TACN) pyridine-
carboxylate series is depicted in
Scheme 1 and starts from the
commercially available free aryl
alkynes 1a,b,d (X=H) or trime-
thylsilyl-protected
derivatives
1e,g, which were prepared ac-
cording to literature procedures
(see Supporting Information).
Sonogashira palladium cross-
coupling with pyridine deriva-
tive 2a led to the chromophore
scaffold in good yield. Chromo-
phores 1e,g were obtained by
using a modified Sonogashira
procedure with addition of tet-
rabutylammonium fluoride to
the reaction mixture, which
allows in situ deprotection of
the trimethylsilyl group. Alterna-
tively, we demonstrated that
the trimethylsilyl alkyne moiety
Figure 1. Structures of the complexes.
could be incorporated on the
pyridine ring to give 2b in 90%
tionally bright europium-based bioprobes that can image cel-
lular mitochondria and act as FRET donors ([Eu.Lca], [Eu.Lmpa],
and [Eu.Lppa]; Figure 1).[16] Their exceptional brightness of
about 25000 molLꢀ1 cmꢀ1 on excitation at 340 nm is about
one order of magnitude higher than those previously reported
for europium complexes, and can be mainly explained by opti-
mization of the extinction coefficient of the complex by using
alkoxyphenylethynyl charge-transfer antennas.[17] In addition,
europium complexes of heptadentate ligands featuring identi-
cal antennas have been reported to show additional sensing
properties following displacement of the coordinated water
molecule.[18]
yield. However, due to the poor stability of the corresponding
free alkyne 2c, the desired compound was not obtained. We
circumvented this issue by using the previous modified Sono-
gashira cross-coupling reaction leading to chromophore 3c.
The starting material 1c was readily available by alkylation
of the corresponding iodophenol (see Supporting Information).
Activation of alcohols 3 with mesyl chloride and purification
on silica gel gave the corresponding mesylated compounds
4a–e,g. Alkylation of TACN in the presence of potassium car-
bonate in acetonitrile gave rise to ligands 5a–e,g, which were
subsequently hydrolyzed, and the europium complexes were
formed by addition of europium chloride hexahydrate. Each
complex was isolated after purification by preparative HPLC
and the structure confirmed by HRMS.
Details of the syntheses of Lppa and Lppa’ have been report-
ed earlier.[16] The synthesis of the ligands Lmpb, Lmpf, and Lppj
(Scheme 2) involved preparation of p-bromopyridyl phosphi-
nate intermediates 2d,e, which were elaborated to conjugated
alkynyl chromophores 6b,f,j in a Pd-catalyzed Sonogashira
coupling reaction. Subsequent mesylation of the pyridyl alco-
hol, alkylation with TACN, and basic hydrolysis of the phosphi-
nate ester groups provided the nonadentate ligands Lmpb,
Lmpf, and Lppj in good yield over three steps.
The great potential of this series of complexes prompted us
to gain further insight into the influence of the nature of the
antenna on their luminescence properties. Herein, we report
the synthesis and spectroscopic properties of a series of euro-
pium and terbium complexes featuring various antennas
based on the same phenylethynyl scaffold. The nature,
number, and position of electron-donating or electron-with-
drawing aryl substituents was varied systematically (Figure 1),
and the photophysical properties of [Eu.Lca], [Eu.Lmpa], and
[Eu.Lppa] are compared and discussed on the basis of their
DFT calculated structures. Finally, the europium complexes
were examined in cell-imaging applications, and selected terbi-
um complexes were studied as potential oxygen sensors.
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[Y.Lppa]and [Y.Lmpa] imposes
a smaller Cpy-P-O angle of about
1018 compared to the Cpy-C-O
angle of 1128 in [Y.Lca], and con-
sequently the five-membered
Y,Npy,Cpy,P,O chelate ring is more
constrained than that of the
analogous carboxylate chelate
(Y,Npy,Cpy,C,O). These observations
explain why the YꢀNpy distances
are longer and why the central
metal ion is more deeply enc-
aged in the phosphinate com-
plexes than in the carboxylate
complexes, as illustrated by the
variation of the distance d be-
tween the Y atom and the plane
formed by the three nitrogen
atoms of the TACN ring (Table 1).
In addition, in the phosphinate
complexes, the methyl or phenyl
substituents of the phosphorus
atom point along the C3 axis and
provide significant steric protec-
tion of the upper side of the
complexes. Moreover, in the X-
ray structure of [Eu.Lppa], only
one classical intermolecular p–p
interaction is found in the lattice,
and it involves the phenylphos-
phinate rings and not the alkynyl
moieties. The centroid–centroid
distance is 4.07 ꢂ, with the two
phenyl rings shifted by 1.90 ꢂ
from perfect stacking.
Scheme 1. Syntheses of the europium carboxylate series of complexes.
Structural comparison between [Eu.Lca], [Eu.Lppa], and
[Eu.Lmpa]
Photophysical properties of Eu complexes
Comparison between Lc, Lmp, and Lpp ligands
DFT geometry optimizations were performed to evaluate the
influence of the nature of the chelating arm, that is, carboxyl-
ate, methylphosphinate, or phenylphosphinate, on the coordi-
nation sphere of the complex (see Computational Details in Ex-
perimental Section). Model complexes [Y.Lca], [Y.Lmpa], and
[Y.Lppa], in which the paramagnetic EuIII ion was replaced by
YIII, were used to simplify the calculations.[19] Each complex
adopts a helical structure with overall C3 symmetry (Figure 2).
The coordination polyhedron is a slightly distorted tricapped
trigonal prism, composed of three nitrogen atoms of the TACN
ring, three nitrogen atoms of the pyridine fragment (Npy), and
three oxygen atoms from the carboxylate or phosphinate sub-
stituents. The calculated bond lengths and angles (Table 1)
follow a similar trend to that observed in the X-ray structure of
[Eu.Lppa][16a] and related unsubstituted complexes.[13,14] The tet-
rahedral geometry around the phosphorus atom in complexes
The photophysical properties of the complexes were studied
in dilute methanol solution or in water, and representative
data are reported in Table 2. The absorption and emission
spectra of [Eu.Lca], [Eu.Lmpa], and [Eu.Lppa], which feature
identical antennas but different chelating groups, are com-
pared in Figure 3. In the absorption spectra, each complex has
a broad structureless transition, assigned to an intraligand
charge-transfer (ICT) transition from the electron-donating me-
thoxyphenyl group(s) to the electron-withdrawing pyridine
fragment. Interestingly, the absorption of [Eu.Lca] is slightly
redshifted compared to the phosphinate analogues, in terms
of both the maximal absorption wavelength (Dlmax =8 nm)
and the red tail of the absorption band (Dlcutoff =20 nm). This
bathochromic shift can be explained by the shorter LnꢀNpy dis-
tance observed for the carboxylate compound: a shorter dis-
tance leads to stronger coordination of the metal center, the
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pounds incorporating polariza-
ble donor groups. A small in-
crease in the intensity of the
DJ=4 transition around 680–
700 nm was observed for
[Eu.Lmpa] and [Eu.Lppa] com-
pared to [Eu.Lca], which may be
tentatively associated with in-
creasing distortion from the
ideal C3 symmetry in the case of
the more bulky phosphinate de-
rivatives. Remarkable quantum
yields in MeOH of 42, 43, and
52% were found for [Eu.Lca],
[Eu.Lmpa], and [Eu.Lppa], re-
spectively (Table 2). In addition,
in water the quantum yield of
[Eu.Lmpa] is almost conserved
(39%), whereas it drops to 25%
for [Eu.Lca’], the water-soluble
analogue of [Eu.Lca]. Such be-
havior is in agreement with the
increased steric protection af-
forded by the methyl or phenyl
substituents at the phosphorus
Scheme 2. Syntheses of the europium phosphinate series of complexes.
atom (Figure 2).
Lewis acidity of which enhances the accepting character of the
pyridine fragment.
To gain deeper insight into the influence of the nature of
the coordinating group, the relevant radiative and nonradiative
parameters were deduced from experimental data (spectra,
quantum yields, and lifetimes). By using the approach initially
proposed by Werts, Jukes, and Verhoeven[20] and Beeby et al.[21]
the overall quantum yield of europium luminescence fEu is de-
fined as the product of the efficiency of sensitization hsens (i.e.,
The europium emission spectral profiles are very similar for
each complex (Figure 3), with a hypersensitive DJ=2 transition
around 610–620 nm, as expected for threefold-symmetric com-
Table 1. Optimized DFT distances [ꢂ] and angles [8] for the series of YIII
model complexes.
[Y.Lca]
[Y.Lmpa]
[Y.Lppa]
YꢀN
2.829/2.830/2.831
2.563/2.563/2.564
2.246/2.247/2.247
2.250
2.787/2.787/2.787
2.703/2.700/2.703
2.214/2.214/2.215
2.190
2.768/2.768/2.769
2.676/2.676/2.676
2.213/2.214/2.213
2.170
YꢀNpy
YꢀO
d
N-Y-N
Npy-Y-Npy
O-Y-O
N-Y-O[a]
63.4/63.4/63.3
119.8/119.8/119.6
94.5/94.3/94.3
120.0/119.9/120.1
64.7/64.7/64.7
119.7/119.7/119.7
91.4/91.5/91.5
122.3/122.3/122.3
65.0/65.0/65.0
119.8/119.8/119.8
89.8/89.8/89.8
123.6/123.6/123.6
[a] Intraligand angle.
Table 2. Calculated values of tr, kr, and Sknr for [Eu.Lca], [Eu.Lmpa], and
[Eu.Lppa] in methanol by using the room-temperature experimentally de-
termined quantities tobs and [I(0,1)/Itot].
Figure 2. DFT-optimized structure of [Y.Lca] (a, b), [Y.Lmpa] (c), and [Y.Lppa]
(d) with the C3 symmetry axis perpendicular (a) or parallel (b–d) to the plane
of the figure. Atom colors: C gray, N blue, O red, P orange, Y magenta. Hy-
drogen atoms have been omitted for clarity. e) X-ray structure of
[Eu.Lppa].[16a] f) Correspondence between X-ray (bold) and DFT (ghost) struc-
tures.
Complex
I(0,1)/Itot
tobs [ms]
tr [ms]
kr [sꢀ1
]
Sknr [sꢀ1
]
[Eu.Lca]
[Eu.Lmpa]
[Eu.Lppa]
0.087
0.079
0.086
0.99
1.18
1.30
2.72
2.47
2.68
368
405
473
642
443
396
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Table 3. Spectroscopic properties of the complexes measured at room
temperature in MeOH.
Complex
lmax
e
f
t
sTPA
(at 700 nm)[b]
[nm]
[mmꢀ1 cmꢀ1
]
[%][a,b]
[ms]
[Eu.Lca]
339
345
351
339
341
349
315[c]
332
331
340
360
58000
60000
60000
60000
60000
60000
48400[c]
58000
58000
62000
57000
42
55
45
41
8
32
22[c]
52
43
54
55
0.99
0.97
0.90
1.01
0.49
0.85
0.85[c]
1.30
1.18
1.14
1.05c
46
[Eu.Lcb]
[Eu.Lcc]
[Eu.Lcd]
30
36
[Eu.Lce]
[Eu.Lcg]
[Eu.Lcw][c]
[Eu.Lppa]
[Eu.Lmpa]
[Eu.Lmpb]
[Eu.Lmpf]
26
[a] Quinine sulfate as standard, excitation at 335 nm, errors in quantum
yield or lifetime are ꢂ15%. [b] Errors are ꢂ20%. [c] Data recorded in
water according to Ref. [13a].
Figure 3. Comparison of the absorption (top) and emission (bottom) spectra
of [Eu.Lca] (black), [Eu.Lmpa] (gray), and [Eu.Lppa] (light gray) in methanol at
room temperature (lex =340 nm).
the fraction of energy transferred from the donor state to the
EuIII accepting levels) and the quantum efficiency of the metal-
centered luminescence on direct excitation into the f levels hEu
[Eq. (1)]
europium(III) complexes was thoroughly studied. Table 3 com-
piles the spectroscopic data of compounds [Eu.Lca–g] alongside
data for the unsubstituted compound [Eu.Lcw] for comparison.
The absorption spectrum of [Eu.Lcw] shows a structured transi-
tion with a maximum at 315 nm (lcutoff =335 nm) characteristic
1
of a p–p* transition associated with a local excited state LE
(Figure 4).[22] As expected, substitution of the aromatic moiety
by electron-donating groups, for example, OMe and SMe in
[Eu.Lca–g], induces a profound modification of the absorption
spectra with the appearance of a broad structureless transition
assigned, by comparison with analogous ligands,[9c,17] to an ICT
transition. These ICT transitions are bathochromically shifted,
and the magnitude of the redshift (Da–g =l([Eu.Lca–g])ꢀl-
([Eu.Lcw]) is correlated to the strength of the donating groups.
As a consequence of the stronger donor character of the SMe
ꢀEu ¼ hsenshEu
ð1Þ
in which hEu =tobs/tr where tobs is the experimental lumines-
cence lifetime of the complex and tr the pure radiative lifetime
[Eq. (2)]
kr ¼ 1=tr ¼ Að0,1Þ½Itot=Ið0,1Þꢁ
ð2Þ
where the constant A(0,1) is the spontaneous emission proba-
group compared to OMe, Dg =34 nm is higher than Da,d
=
bility of the 5D0! F1 transition (32 sꢀ1 in methanol) and
7
19 nm for one methoxyl substituent in para or ortho position,
respectively. Introduction of additional methoxyl groups in the
nonconjugated meta position does not induce any significant
Itot/I(0,1) the ratio of the total integrated emission intensity to
5
7
the intensity of the D0! F1 transition. Finally, if kr and tobs are
known, the nonradiative decay rate constant Sknr can be de-
duced from Equation (3).
Sknr ¼ 1=tobsꢀ1=tr
ð3Þ
This procedure was used for [Eu.Lca], [Eu.Lmpa], and
[Eu.Lppa] in methanol, and the data are summarized in Table 2.
Clearly, the increases in quantum yield and experimental life-
time along the series [Eu.Lca]<[Eu.Lmpa]<[Eu.Lppa] are direct-
ly connected to the strong decrease of the nonradiative rate
constant from 642 to 396 sꢀ1. These results clearly indicate that
the increased steric protection afforded by the phosphinate
substituent (Me<Ph) contributes strongly to rigidification of
the structure of the complex, and thereby reduces nonradia-
tive de-excitation pathways.[14b]
Influence of alkynyl substitution
Figure 4. Normalized absorption spectra of complexes [Eu.Lca–w] in dilute
methanol solution (black dotted [Eu.Lcw]; black [Eu.Lca]; red [Eu.Lcb]; blue
[Eu.Lcc]; pink [Eu.Lcd]; olive [Eu.Lce]; green dashed [Eu.Lcg]).
The influence of the aromatic substitution on the photophysi-
cal properties (absorption and emission) of the related
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additional bathochromic shift (De =21 nm). On the contrary, in-
troduction of one or two weakly electron-donating methyl
groups in the ortho position induces additional redshifts of 30
and 36 nm for [Eu.Lcb] and [Eu.Lcc], respectively. Finally, intro-
duction of three methoxyl groups in conjugated ortho, ortho’,
and para positions in [Eu.Lmpf] results in the strongest batho-
chromic shift of the ICT transition, with a maximal absorption
wavelength of 360 nm and a lcutoff value of about 410 nm. It is
therefore possible to fine-tune the maximum absorption wave-
length and the cutoff wavelength in the spectral range of in-
terest centered around 337 nm by varying the aromatic substi-
tution. This ICT transition is very intense, with an extinction co-
efficient of about 60000 Lmolꢀ1 cmꢀ1 and thus of prime impor-
tance in terms of brightness optimization. All compounds
except [Eu.Lce] are strongly emissive and exhibit the classical
EuIII emission profile on irradiation in the ICT transition. In each
case, no residual ligand-centered emission was observed, that
is, the energy transfer to the lanthanide ion is almost quantita-
tive. The quantum yields in methanol are good to excellent,
and the highest value of 55% was obtained for [Eu.Lcb] and
[Eu.Lmpf], supported by long luminescence lifetimes around
1 ms. The photophysical properties of all complexes were not
evaluated in water due to their limited solubility, except for
[Eu.Lmpa]. The quantum yield (39%) and lifetime (1.01 ms) in
water are slightly lower than in methanol but remain signifi-
cant and indicate good aqueous stability of this complex.
Combined with the above-mentioned strong absorption, this
family of complexes exhibits very high brightness around
337 nm, which makes them very attractive candidates for bio-
imaging and fluoroimmunoassay applications.
Figure 5. Emission spectra of [Gd.Lmpf] (lexc =355 nm) measured at 77 K
(black dotted) and 295 K (black dashed), as well as [Gd.Lppa’](lexc 330 nm) at
77 K (black) and 295 K (gray). Recorded in an EPA glass .
The parent EuIII complexes did not show significant spectral
variation with solvent polarity, except for the Eu complex of
the 2,4,6-trimethoxyphenyl triphosphinate ligand Lmpf. In this
case, the emission intensity increased in less polar solvents
(Table 4 and Figure S1 in the Supporting Information). In sol-
Table 4. Variation of spectral behavior with solvent polarity for [Eu.Lmpf].
Solvent
ET(30)
lmax [nm]
t [ms]
Irel [%][a]
water
MeOH
EtOH
iPrOH
MeCN
DMF
1.00
0.76
0.65
0.55
0.46
0.40
360
360
360
360
350
350
0.72
1.06
1.15
1.18
1.17
1.17
10
55
71
78
81
75
Sensitization process
[a] Relative emission intensities were estimated under the assumption
that the extinction coefficient of [Eu.Lmpf] is the same in each solvent
and were calibrated with the quantum yield in MeOH; prior work has
shown that for water and methanol, the ICT extinction coefficients of the
lowest-energy band are very similar, within an error of ꢂ10%.
In related tris-dipicolinate complexes, europium sensitization
has been shown to occur by an efficient intramolecular
energy-transfer process involving a relaxed and fairly broad ICT
excited state and not via a localized ligand triplet state, nota-
bly for strongly electron donating moieties.[17b] In the case of
weak donor groups such as alkyl and alkoxyl, the ICT state lies
at higher energy, and consequently a contribution of the classi-
cal triplet-mediated sensitization process remains possible.[17a]-
Complexes [Gd.Lmpf] and [Gd.Lppa’] were prepared and inves-
tigated in order to study the influence of the chelating groups
on the sensitization process (Figure 5). At low temperature,
both complexes exhibit a broad structureless emission, charac-
teristic of an ICT state, at 415 and 375 nm for [Gd.Lmpf] and
[Gd.Lppa’], respectively, together with weaker structured emis-
sion from the ligand-centered triplet state (e.g., vibrational
overtones at 490 and 470 nm). At room temperature, only
a broad structureless ICT emission is observed around 460 nm.
This variable-temperature measurement clearly indicates the
presence of a triplet excited state at approximately the same
energy as the ICT state. Consequently, the two sensitization
pathways, that is, the triplet-state-mediated and direct ICT pro-
cesses, can be simultaneously involved in the sensitization of
europium luminescence.
vents with a Reichardt normalized polarity parameter ET(30) of
less than 0.8 (e.g., MeOH, EtOH, iPrOH, MeCN, DMF) the life-
time increased slightly from 1.06 in MeOH to 1.18 in iPrOH.
Based on the assumption that the extinction coefficients of the
lowest-energy ICT band do not vary by more than 10% in the
examined solvents, the overall emission quantum yield varied
from 55% in MeOH to 78% in iPrOH. However, in water the
lifetime was 0.70 ms, the quantum yield dropped to 5%, and
the primary absorption band of the ICT state broadened and
extended beyond 405 nm.
The variation of the emission intensity with T in the range
180–295 K was examined in EPA (diethyl ether/isopentane/eth-
anol 5/5/2 v/v/v), and the changes in intensity and lifetime
with oxygen partial pressure pO in the range 0.4—160 mmHg
2
was examined in water at ambient temperature. The tempera-
ture variation was characterized by an increase in overall emis-
sion intensity by a factor of two between 295 and 230 K fol-
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lowed by a drop in intensity at much lower temperatures (Fig-
ure S2 in the Supporting Information). The emission intensity
varied nonlinearly (Figure S3 in the Supporting Information)
Bioimaging application: mitochondrial staining in different
cell types
with pO below 35 mmHg (160 mmHg is atmospheric pressure),
In a preliminary communication, we reported that certain Eu
complexes in this family exhibit cell uptake, with a tendency to
highlight the mitochondrial network.[14a] Indeed, it was possible
to obtain both single- (lexc =355, 365 nm) and two-photon
(lexc =710–730 nm) microscopy images with several of the
complexes described herein.[24] The single-photon images are
representative and are described here. The high optical bright-
ness of the probes allows live-cell imaging at high resolution
(Figure 7). Thus, in the case of mouse skin fibroblasts (NIH-3T3)
2
with a 2.6-fold increase in intensity at 0.4 mmHg with respect
to ambient pressure.
Taken together, the absorption-, solvent-, temperature-, and
oxygen-dependent changes observed for [Eu.Lmpf] support
a sensitization mechanism involving a solvent-relaxed ICT excit-
5
ed state, which may transfer its energy to the Eu D1 and/or
5D0 excited states. The energy of this ICT excited state is low-
ered in the most polar solvent water, such that thermally acti-
vated (T-dependent) back energy transfer may occur, which ex-
tends the lifetime of the ICT state and thereby increases its
sensitivity to nonradiative deactivation.
Nonlinear optical properties
The presence of the extended p-conjugated antenna ligand
prompted us to determine the two-photon absorption cross
sections of selected complexes. To that end, two-photon excit-
ed fluorescence (TPEF) measurements, based on the calibration
of the two-photon excitation spectra in the range 700–900 nm,
were performed in dilute methanol solution by using coumar-
in 307 as external reference (see Experimental Section for de-
tails). The two-photon absorption spectra are shown in
Figure 6, and it clearly appears that the maximal absorption
Figure 7. Top left: NIH 3T3 cells stained with [Eu.Lmpb], clearly showing the
mitochondrial network at high resolution (recorded with an in-house phase-
modulation nanoscope: 2048ꢃ2048 pixels, voxel size 60ꢃ60ꢃ780 nm). Top
right: [Eu.Lmpf] in HepG2 cells (which tend to clump together, forming local
foci during culturing) in red, showing higher definition and resolution com-
pared to images with MitoTracker Green, and a co-localized image. Bottom:
[Eu.Lmpf] (30 mm loading concentration) in NIH 3T3 cells (left, 2 h loading;
30 min for MTG, middle), showing the colocalization (right) with MTG
images by RGB merge (P>0.9; 1024ꢃ1024 pixels, voxel size
120ꢃ120ꢃ780 nm, 100 Hz, four scans averaged).
stained with [Eu.Lmpb] or [Eu.Lmpf], their high brightness per-
mits use of a new experimental technique, which can be
termed phase-modulated nanoscopy, to further reduce lateral
resolution (dlat =125 nm at lexc =355 nm, 1.4 N.A.).[16] This is re-
vealed in the definition of the mitochondrial network down to
about 80 nm resolution.
The complex [Eu.Lmpf] absorbs even more strongly at
355 nm and was also examined in human liver adenocarcino-
ma cells (HepG2), where it revealed the mitochondrial distribu-
tion better than the common stain MitoTracker Green (MTG).
These liver cells form defined local foci instead of a well-dis-
persed monolayer. Therefore, we conducted a simple compari-
son of the tissue penetration of [Eu.Lmpf] versus MTG. This Eu
complex appears to permeate the foci more deeply, allowing
more effective visualization of the taller axial sections of these
densely packed cells. Following uptake through macropinocy-
tosis,[25]the complex must be shuttled to the mitochondria,
crossing the outer membrane and becoming localized in the
intermembrane space. It seems unlikely that these high molec-
ular weight (M>500) probes can cross the inner mitochondrial
Figure 6. Two-photon absorption measured by TPEF in methanol of [Eu.Lca]
(empty squares), [Eu.Lcd] (empty circles), [Eu.Lce] (filled triangles), and
[Eu.Lppa] (filled stars).
wavelength is located out of our laser excitation range and
that only the red tail of the two-photon absorption spectrum
can be measured. At 700 nm, all compounds show rather
modest two-photon absorption cross sections of between 25
and 50 GM; these values lie in the range of those of recently
described complexes with similar antenna groups.[4a,9c,17,23]
Such properties render this family of complexes suitable for bi-
photonic microscopy imaging applications.
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membrane. Such behavior may then explain their low mito-
chondrial toxicity, as revealed by IC50 values of greater than
100 mm (24 h) in the MTT assay. The MitoTracker stains are
based on substituted benzylic chlorides that irreversibly alky-
late Cys residues in various mitochondrial proteins (e.g., heat
shock protein 60, VDAC1, aldolase A)[26] and can also attack mi-
tochondrial DNA by alkylation at guanine N-7. These reactions
result in irreversible perturbation of normal organelle function,
so that these organic dyes can only penetrate and stain the
outer layers of the foci and subsequently form a perturbed cell
barrier. These shallow superficial stained cell layers appear to
prevent penetration and transport of the organic dye deeper
into the tissue. Also, it can be clearly seen in the sets of
images in HepG2 cells (Figure 7), that the Eu compound does
not stain the lipid droplets that appear as very bright spots in
the images obtained with the organic dye.
Table 5. Selected photophysical properties for phenylphosphinate-based
pyridyl-alkynyl chromophores 9b–e (295 K, MeOH, or as stated).
[a]
[b]
Compound Substituent X
lmax [nm] e [mmꢀ1 cmꢀ1
]
ET [cmꢀ1
]
[Gd.Lppa’]
(OCH2CH2)3OMe 320
25000
21300
21200
21,600
21000
21800
9b
9c
9d
9e
CO2CH2CH3
CON(CH2CH3)2
CN
314
310
312
298
31800
34600
33500
15000
CF3
[a] Errors are ꢂ5%. [b] Triplet energies ET were measured at 77 K in an
EPA glass.
Photophysical properties of terbium complexes
The effect of changing the nature of the para substituent on
the phenyl ring was considered in an attempt to make this
ligand suitable for terbium sensitization. This strategy requires
raising of the energy of the ligand singlet excited state and/or
the relative energy of the ICT and ligand-centered triplet
states. Earlier work[27] has shown that very little terbium emis-
sion was observed under ambient conditions for complexes of
the ligands with chromophores bearing electron-releasing sub-
stituents, for example, Lca and Lppa. Even in deoxygenated so-
lution, [Tb.Lppa] gave only very weak emission (fem(MeOH)=
0.3% at 295 K, Figure S4 in the Supporting Information). Sensi-
tization is much less likely to occur with the terbium ana-
logues, as the Tb ion requires that the broad ICT state lie well
Figure 8. Phosphorescence emission spectra of a series of phenylphosphi-
nate model chromophores 9b–e measured in an EPA glass at 295 K.
Oxygen sensing with Eu/Tb mixtures
The p-CF3-substituted chromophore has the highest triplet
energy of this series. Indeed, the low-T spectra show that the
triplet excited state is of lower energy than the ICT state in this
case. Accordingly, the corresponding ligand Lppj was prepared
and the spectral properties of the Tb and Eu complexes ana-
lyzed in the presence of varying partial pressures of oxygen.
5
above the terbium D4 accepting state at 20400 cmꢀ1. Indeed,
sensitization of terbium in the near-UV region by an antenna
normally requires that a significant energy gap between the
energy of the excited state of the sensitizing moiety and the
The Eu complex showed no change in intensity as pO was
2
varied, whereas the Tb analogue exhibited a strong depend-
ence on dissolved oxygen concentration (Figure 9). The emis-
5
accepting Tb D4 level.[8] In this situation, the rate of thermally
sion intensity of Tb was assessed over the pO range of 0.03 to
2
activated back energy transfer is minimized when the energy
gap is less than 8kT, that is, 1640 cmꢀ1 at 298 K. In an effort to
identify a system that may allow Tb sensitization with reduced
or zero oxygen sensitivity, the excited-state energies of a set of
chromophores 9b–e with electron-poor substituents were
measured (Scheme 3, Table 5, and Figure 8).
159 mmHg and showed a linear dependence, associated with
ꢀ1
a Stern–Volmer quenching constant K of 60 mmHg. The dif-
SV
fering behavior of the Eu and Tb complexes allows oxygen
concentrations to be measured by determining the ratio of the
green Tb emission at 545 nm to the red Eu emission at 620 nm
for 1/10 mixtures of the Eu/Tb complexes (Figure 10). Such be-
havior has previously been observed for related systems[27] in
which reversible energy transfer repopulates the sensitizer trip-
let excited state and renders it sensitive to collisional quench-
ing by triplet oxygen.
Conclusion
We have developed a modular and flexible synthetic route to
a family of ligands based on TACN that enables control of
ligand and complex absorbance in the range 330–360 nm by
variation of the substituents on the aryl ring or by changing
Scheme 3. Structure of phenylphosphinate model chromophores 9b–e fea-
turing electron withdrawing substituents.
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nature of the aryl substituents and the donor group. Very effi-
cient intramolecular energy transfer occurs with the EuIII com-
plexes, whereas for the TbIII analogues, back energy transfer
occurs rapidly in all cases, due to the broad density of states
or the relatively low energy of the triplet or relaxed ICT excited
5
state and their closeness to the terbium D4 emissive state. By
using a mixture of Tb and Eu complexes of a common ligand,
the ability to measure pO in solution was demonstrated.
2
Experimental Section
Computational details
Figure 9. Emission spectra of the mixture of complexes [Ln.Lppj] (Eu/Tb=1/
10, lexc =308 nm) showing the change in the DJ=ꢀ1 Tb emission-band in-
tensity (centered at 545 nm) compared to the DJ=2 Eu band (centered at
615 nm) as the atmospheric pressure (and hence the concentration of dis-
solved molecular oxygen) was varied from 2 to 760 mmHg.
DFT geometry optimizations of the YIII complexes were carried out
with the Gaussian 09 (revision A.02) package[28] by employing the
PBE0 hybrid functional.[29]The Stuttgart/Dresden basis sets and ef-
fective core potentials were used to describe the yttrium atom,[30]
whereas all other atoms were described with the SVP basis sets.[31]
Optical measurements
Absorption spectroscopy: UV/Vis absorption measurements were
recorded on a JASCO V670 or a PerkinElmer Lambda 900 absorp-
tion spectrophotometer by using matched quartz cells.
Luminescence: Emission spectra were measured on a Horiba-Jobin
Yvon Fluorolog-3 spectrofluorimeter. The steady-state lumines-
cence was excited by unpolarized light from a 450 W xenon CW
lamp and detected at an angle of 908 for dilute-solution measure-
ments (10 mm quartz cell) by a red-sensitive Hamamatsu R928
photomultiplier tube. Spectra were reference-corrected for both
the variation in intensity of the excitation source light (lamp and
grating) and the emission spectral response (detector and grating).
Phosphorescence lifetimes (>30 ms) were obtained by pulsed exci-
tation with a FL-1040 UP Xenon Lamp. Luminescence decay curves
were fitted by least-squares analysis with Origin. Luminescence
quantum yields Q were measured in dilute aqueous solutions with
an absorbance of less than 0.1 by using the equation Qx/Qr =[Ar(l)/
Ax(l)][n2x/nr2][Dx/Dr], where A is the absorbance at the excitation
wavelength l, n the refractive index, D the integrated lumines-
cence intensity, and “r” and “x” stand for reference and sample.
The reference was quinine bisulfate in 1N aqueous sulfuric acid so-
lution (Qr =0.546). Excitation of reference and sample compounds
was performed at the same wavelength.
Figure 10. Variation of the ratio of the terbium emission intensity
(lexc =308 nm, lem =530–560 nm) for [Tb.Lppj] versus Eu emission of
[Eu.Lppj] as a function of pO in aqueous solution (295 K).
2
the nature of the donor anionic group, as exemplified by the
behavior of carboxylate and phosphinate groups. The phosphi-
nate substituents were shown by DFT and X-ray studies to
present a steric shield that protects the lower face of the lan-
thanide complexes, enhancing their resistance to collisionally
activated quenching processes and leading to longer excited-
state lifetimes.
Two-photon-excited luminescence measurements: The TPA
cross-section spectrum was obtained by upconversion lumines-
cence with a Ti:sapphire femtosecond laser in the range 700–
900 nm. The excitation beam (5 mm diameter) was focused with
a lens (focal length 10 cm) at the middle of the 10 mm cell. Emit-
ted light was collected at 908 and was focused into an optical fiber
(diameter 600 mm) connected to an Ocean Optics S2000 spectrom-
eter. The incident beam intensity was adjusted to 50 mW 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 700–900 nm two-photon absorption spectrum of
Coumarin 307 (quantum yield=0.56 in ethanol).[32] The measure-
ments were done at room temperature in dichloromethane and at
a concentration of 10ꢀ4 m.
The europium complexes showed both high emission quan-
tum yields and high absorbance, which led to several examples
having brightness B on the order of 15 to 30 mmꢀ1 cmꢀ1, the
highest reported for Eu complexes in solution. This high
brightness permits applications in immunoassays and in
single-photon microscopy and spectral imaging that are other-
wise limited to longer acquisition times or higher complex
concentrations, and it enables live cell imaging at low incident
powers. Furthermore, the two-photon cross sections of the
chromophores, which lie in the range 30–50 GM, are sufficient
to allow excitation between 700 and 720 nm, as needed for
two-photon microscopy studies. These studies, in concert with
applications to near-IR emitting systems, will be discussed in
forthcoming work.
Variable-temperature (VT) and variable-pressure (VP) experi-
ments: Details of the experimental setups used in the VT and VP
experiments have been reported earlier.[27]
The mechanism of sensitization involves a relaxed ICT excit-
ed state or a ligand triplet intermediate, depending on the
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Confocal microscopy: Details of cell culture, epifluorescence mi-
croscopy, and assessment of complex toxicity, typically by using
the MTT assay of mitochondrial redox function, have been reported
elsewhere.[16]Cell images and co-localization experiments were per-
formed with a Leica SP5 II microscope. To achieve excitation with
maximal probe emission, the microscope was coupled by an opti-
cal fiber to a Coherent 355 nm CW (Nd:YAG) laser, operating be-
tween 4 and 8 mW power. A HeNe or Ar ion laser was used when
commercially available organelle-specific stains (e.g., MitoTracker
Green) were used to corroborate cellular compartmentalization.
The microscope was equipped with a triple-channel imaging de-
tector, comprising two conventional PMT systems and a HyD
hybrid avalanche photodiode detector. The latter part of the detec-
tion system, when operated in the BrightRed mode, is capable of
improving imaging sensitivity above 550 nm by 25% and reducing
signal-to-noise ratio by a factor of 5. The pinhole was always deter-
mined by the Airy disk size, calculated from the objective in use
(HCX PL APO 63ꢃ/1.40 N.A. LbdBlue) at the lowest excitation
wavelength (355 nm). Scanning speed was adjusted to 100 Hz in
a unidirectional mode to ensure both sufficient light exposure and
enough time to collect the emitted light from the lanthanide-
based optical probes (1024ꢃ1024 frame size, pixel size of 120ꢃ
120 nm, and depth of 0.772 mm).
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Synthesis
Details of general methods and of NMR and MS instrumentation
can be found in recent references.[2,4,7,8,16]. Experimental details of
syntheses of chromophores, ligands, and complexes are given in
the Supporting Information.
CCDC-857545 ([Eu.Lppa]) contain the supplementary crystallo-
graphic data for this paper. These data can be obtained free of
charge from The Cambridge Crystallographic Data Centre via
www.ccdc.cam.ac.uk/data_request/cif.
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Acknowledgements
We thank EPSRC, the Royal Society, and the ERC for support
(D.P., R.P., S.J.B.: FCC 266804); V.P. thanks the Lyon Science
Transfer Agency for financial support.
Keywords: imaging agents
ligands · photophysics · sensors
·
lanthanides
·
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Received: February 28, 2014
Published online on June 17, 2014
Chem. Eur. J. 2014, 20, 8636 – 8646
www.chemeurj.org
8646
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