Design and synthesis of europium luminescent bio-probes featuring sulfobetaine moieties

Article abstract of DOI:10.1016/j.tetlet.2014.01.025

Herein we report the straightforward preparation of chromophore- functionalized TACN ligand via Cu-free cross-coupling reactions using a common halogenated platform. This versatile methodology allows the preparation of original macrocyclic ligand featurin

Full text of DOI:10.1016/j.tetlet.2014.01.025

Tetrahedron Letters 55 (2014) 1357–1361
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Design and synthesis of europium luminescent bio-probes featuring
sulfobetaine moieties
Virginie Placide a, Delphine Pitrat a, Alexei Grichine b, Alain Duperray b, Chantal Andraud a,
Olivier Maury a,
⇑
a Laboratoire de Chimie, UMR 5182, CNRS-ENS Lyon-Université Lyon 1, 46 Allée d’Italie, 69364 Lyon Cedex 07, France
b Institut Albert Bonniot, INSERM-U823-CHU Grenoble-EFS, University Joseph Fourier—Grenoble I, BP170, 38042 Grenoble, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Herein we report the straightforward preparation of chromophore-functionalized TACN ligand via
Cu-free cross-coupling reactions using a common halogenated platform. This versatile methodology
allows the preparation of original macrocyclic ligand featuring both optimized antenna for the sensitiza-
tion of europium luminescence and sulfobetaine zwitterionic groups to ensure water solubility of the
complex. In addition preliminary two-photon excited microscopy imaging experiments of fixed cells
reveal that sulfobetaine groups are able to limit undesirable non specific interactions with biological
surrounding.
Received 19 November 2013
Revised 23 December 2013
Accepted 7 January 2014
Available online 13 January 2014
Keywords:
Lanthanide
Triazacyclonane macrocycle
Sulfobetaine
Ó 2014 Elsevier Ltd. All rights reserved.
Luminescence
Bio-imaging
The peculiar photophysical properties of f-block elements make
them attractive candidates for the design of lanthanide lumines-
cence bioprobes (LLB) for biological imaging or sensing applica-
tions.1 An optimized LLB must fulfill a set of requirements
namely (i) solubility and stability in water, and (ii) optimized chro-
mophore antenna (Chrom) to ensure efficient sensitization. In this
context, triazacyclononane macrocycle has become a platform of
choice for the design of LLB for the following reasons: (i) its tris
N-alkylation by pyridine carboxylate or phosphinate leads to the
formation of a nonadentate ligand which can saturate the coordi-
nation sphere of lanthanide ion and form a stable complex in aque-
ous solutions.2 (ii) In addition, following pioneering works of
Takalo,3 functionalization of the para position of the pyridine by
chromophore antenna (Fig. 1, Chrom = aromatics, p-substituted
aryl-ethynyl) enhances the absorption in the visible, while making
it possible to tune its position. Recently, this strategy led to the
preparation of Yb-bioprobes for thick tissue biphotonic microscopy
imaging4 and Eu-bioprobes with optimized brightness for one pho-
ton imaging5 and sensing applications.6 In each case, the ligand’s
synthesis proceeds according to the same convergent procedure
(Fig 1A), involving the preparation of a Chrom-substituted picolinic
arm followed by alkylation of the TACN moieties. This last step
requires the use of excess Chrom-picolinic arm, which multi-
step synthesis requires lots of efforts, and which is hard to recover
during purification.
In this Letter we describe an alternative versatile synthesis of
various chromophore-functionalized triazacyclonane ligand
Ln(COOMe), (n = 1–3) using a divergent approach (Fig. 1B) involving
the key synthon LI3 (COOMe). The versatile character of the new
synthetic methodology is first illustrated by the preparation of
L1,2
featuring methoxy donor groups, via Suzuki–Miyaura
(COOMe)
and copper-free Sonogashira cross-coupling reaction. In a second
time, this procedure is applied for the design of the advanced
ligand L3
containing sulfobetaine end-groups. This zwitter-
(COOMe)
ionic fragment has been widely used for the hydrosolubilization
of quantum dots or magnetic nanoparticles for bio-imaging appli-
cations7 and it has been reported that the covering of the surface
nanoparticles by sulfobetaine moieties presents the additional
advantage of limiting the non specific interactions with the
lipophilic biological surrounding compared to other neutral
(poly-ethylene glycol) or charge (sulfonate, carboxylate, or ammo-
nium) hydrosolubilizing functions.7,8 This issue is crucial for any
practical biological applications (imaging, sensing, and bioconjuga-
tion. . .) but has rarely been addressed for molecular probes. In this
context, sulfobetaine groups are only present as polar fragments in
the now commercially available amphiphilic membrane chromo-
phore di-4-ANEPPS,9 and have been recently introduced as
hydrosolubilizing moieties in functional BODIPY dyes by Ziessel,
Ulrich and co-workers.10 To the best of our knowledge, this
zwitterionic function has never been used in combination with lan-
⇑
Corresponding author.
E-mail address: olivier.maury@ens-lyon.fr (O. Maury).
0040-4039/$ - see front matter Ó 2014 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetlet.2014.01.025

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V. Placide et al. / Tetrahedron Letters 55 (2014) 1357–1361
Figure 1. Retro-synthethic scheme for the preparation of target chromophore-TACN ligands.
thanide complexes. Here we described the design of [EuꢀL3(COO)], a
LLB featuring sulfobetaine moieties enabling both hydrosolubiliza-
tion and limitation of undesirable non-specific interactions with
bio-molecules or lipophilic part of cells. The photophysic properties
of the complex and preliminary two-photon bio-imaging experi-
ments11 are reported.
The ‘classical’ convergent synthesis illustrated in Figure 1,
involving the alkylation of the TACN macrocycle in the last step,
is not compatible with the presence of the dimethylamino periph-
eral moieties precursor of the sulfobetaine. Therefore an alterna-
tive synthetic pathway has been envisaged to tackle this issue,
using the LI3
ligand as a key functional platform. Moreover,
(COOMe)
this intermediate is an excellent starting point for a divergent and
versatile one step preparation of various chromophore-ligands
combinations via cross-coupling reaction with different Chrom-Y
(Y is cross-coupling compatible function, typically terminal alkyne,
boronic or stannane groups). This new synthetic pathway allows
performing a systematic study of the influence of the antenna on
the luminescence of the related Ln(III) complexes.
This LI3
scaffold was obtained via a classical synthetic
(COOMe)
strategy, involving the alkylation of triazacyclononane trihydro-
chloride 1 by the mesylated derivative 2 in dry acetonitrile in the
presence of sodium carbonate (Scheme 1). The synthesis was opti-
mized and scaled-up to ca. 1.5 g (57%) after purification by simple
precipitation in ethyl acetate.12 This procedure was further
Scheme 1. Synthesis of the iodo containing platform.
of the expected L1
ligand in 40% yield after column
(COOMe)
chromatography.13 On the other hand, Sonogashira coupling with
extended to the preparation of the related dissymmetric LI2
anisole acetylene (4) under classical conditions (PdCl2(PPh3)2,
CuI, THF–NEt3) was not successful. However, alternative conditions
with PdCl2(PPh3)2 as the catalyst in the absence of Cu(I) gave the
(COOMe)
platform starting from the mono-boc protected TACN macrocycle,
10.5b,6a
expected L2
ligand in 40% yield (Scheme 3). This result can
The LI3
platform was first engaged in model cross cou-
(COOMe)
(COOMe)
be explained by the parasitic complexation of Cu(I) in the macrocy-
clic precursor. The apparently modest 40% yield is in fact the
average of three coupling reactions in each picolinic moiety of
the ligand and the effective yield of each coupling is around 75%
which is classical for this type of ligands.14 After optimization of
the reaction conditions, we applied this synthetic procedure to
pling reactions to check its reactivity. Two kinds of cross-coupling
reactions were envisioned, namely a Suzuki–Miyaura and a Sono-
gashira coupling to introduce the chromophore onto the triiodo li-
gand (Scheme 3). The Suzuki–Miyaura coupling with the
commercially available anisole boronic acid (3) under classical
conditions (Pd(PPh3)4, Na2CO3, DMF, 90 °C) led to the formation

V. Placide et al. / Tetrahedron Letters 55 (2014) 1357–1361
1359
Scheme 2. Preparation of ligand L1,2
using the synthetic pathway B via copper free Pd-catalyzed cross coupling reactions.
(COOMe)
the introduction of different antenna containing various hydrosol-
ubilizing groups like a-D-galactose derivative or sulfobetaine ones.
In the following, we will only describe the synthesis of ligand L3 -
(
featuring zwitterionic sulfobetaine substituent and that of
COOMe)
the related [EuꢀL3(COO)] complex (Scheme 3).
The Chrom antenna 8 was first synthesized. First the terminal
dimethylamino function was introduced by alkylation of 4-iodo-
phenol by 1-dimethylamino-3-chloropropane. The resulting com-
pound 6 was involved in a Sonogahira cross-coupling under classi-
cal conditions with trimethylsilylacetylene giving 7 in 84% yield
after column chromatography (SiO2, eluent CH2Cl2/MeOH gradi-
ent). Finally, the TMS group was removed to give 8 that was di-
rectly involved in the next step without purification. The
Sonogashira coupling between LI3
and 8 (4 equiv) was car-
(COOMe)
ried out using the copper free procedure described above and led
to the formation of 9 in 65% yield after purification by three succes-
sive precipitations (AcOEt/C5H12). The final step was the quatern-
ization of the dimethylamine moieties by opening of the 1,3-
propanesultone. It is important to note that 9 presents a priori
three sites for this quaternization reaction namely, the dimethyl-
amino peripheral moieties, the tertiary amine of the TACN macro-
cycle, and the imino moieties of the picolinic ring. At room
temperature, the opening of the 1,3-propanesultone occurs only
on the less sterically hindered site, the dialkylamino one. Conse-
quently the desired L3
ligand was obtained and isolated in
(COOMe)
85% yield after washing and trituration in dichloromethane. The fi-
nal ligand and all intermediates were characterized by NMR and
high-resolution mass spectrometry.15 Complex [EuꢀL3(COO)] was
prepared after in situ hydrolysis of the ester moieties and further
addition of europium salt using reported procedure and was puri-
fied by dialysis in water.4,5 Its structure is supported by mass
spectrometry.16
This complex is perfectly soluble in water allowing to measure
its spectroscopic properties. It presents a broad absorption spec-
trum centered at 337 nm (e
= 32000 Mꢁ1 cmꢁ1) characteristic for
this type of antenna.5,17 Excitation in this transition resulted in
the expected threefold symmetric Eu(III) luminescence profile
(Fig. 2). The quantum yield (18%) and lifetime (0.8 ms) are in the
same range although slightly lower than the corresponding poly-
ethylene glycol substituted analogous (25%, 1.06 ms)5 indicating
Scheme 3. Synthesis of LI3
.
(COOMe)

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V. Placide et al. / Tetrahedron Letters 55 (2014) 1357–1361
Figure 2. Comparison of the luminescence spectrum obtained in solution using a Jobin-Yvon Fluorolog apparatus (in red) and the two-photon excited luminescence spectrum
obtained by the microscope using the spectral scanning mode. Inset from right to left: biphotonic image, phase contrast image and merged image of fixed human epithelial
bladder cancer cell line (ATCC No HTB-4) cells stained by [EuꢀL3(COO)] using a microscope LSM-DuoScan NLO (Carl Zeiss) equipped with a Ti:Sa fs tunable laser (Chameleon,
Ultra II, Coherent) (excitation 700 nm).
that modification of the peripheral solubilizing moieties does not
significantly affect the complex photophysical properties. Fixed
Furthermore the synthesis of the di-halogenated LI2
plat-
(COOMe)
form opens the way to the design of di-functionalized, or dissym-
metric tri-functionalized triazacyclononane ligands for sensing
applications6 or bio-conjugation.5 This synthetic methodology
T24 cancer cells were stained with a solution of [EuꢀL3
]
(COO)
(cꢂ10ꢁ5 mol Lꢁ1) and two-photon cell imaging experiments were
successfully performed (k2 = 700 nm, Fig. 2).18 The spectral analysis
was successfully applied to the preparation of the ligand L3
(COOMe)
of the red glowing pixels corresponds well with the [EuꢀL3
]
containing hydrosolubilizing zwitterionic sulfobetaine moities. The
related europium(III) complex exhibits good emission properties in
water and was involved in fixed cell imaging experiments using
biphotonic microscopy. The staining was reversible upon rinsing
indicating that the presence of sulfobetaine moieties limits
strongly the non specific interactions with biological surrounding
responsible for the accumulation of lipophilic organic dyes or com-
plexes in membranes or organelles. The limitation of these non
specific interactions is also a crucial issue for bioconjugation of
emissive dyes or complexes with proteins or other biomacromole-
cules and the use of sulfobetaine moieties in this context is under
study in our laboratory.20
(COO)
luminescence spectra recorded independently, clearly confirming
that the LLB is not altered in the cell. The localization of the sulf-
obetaine containing complex in the fixed cell seems to be identical
to what had been previously observed with PEG or methyl substi-
tuted complexes.4,5,19 However, the images look more diffuse, due
to the ubiquitous presence of [EuꢀL3(COO)] in the endoplasmic retic-
ulum without any specific localization. This behavior was con-
firmed by the complete disappearance of the complex, when the
cells are rinsed with the medium. These observations are in
marked contrast with PEG or methyl substituted and indicate that
[EuꢀL3(COO)] does not accumulate in the lipophilic part of the cell.
The non-specific interactions responsible for this complex accumu-
lation are less prone to occur with [EuꢀL3(COO)] and this behavior
can be ascribed to the presence of the zwitterionic sulfobetaine
moieties (Scheme 2).
Acknowledgments
Authors thank Lyon Science Transfer Agency for financial sup-
port (grant to V.P.) and C. Chapelle for assistance in the project.
In conclusion, this article describes a new versatile synthesis of
chromophore functionalized triazacyclononane macrocycles using
a common halogenated platform, LI3
by Suzuki–Miyaura or
References and notes
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