Bright, highly water-soluble triazacyclononane europium complexes to detect ligand binding with time-resolved FRET microscopy

Article abstract of DOI:10.1002/anie.201406632

Luminescent europium complexes are used in a broad range of applications as a result of their particular emissive properties. The synthesis and application of bright, highly water-soluble, and negatively charged sulfonic- or carboxylic acid derivatives of

Full text of DOI:10.1002/anie.201406632

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Communications
DOI: 10.1002/anie.201406632
Lanthanides
Bright, Highly Water-Soluble Triazacyclononane Europium Complexes
To Detect Ligand Binding with Time-Resolved FRET Microscopy**
Martina Delbianco, Victoria Sadovnikova, Emmanuel Bourrier, Gꢀrard Mathis,
Laurent Lamarque, Jurriaan M. Zwier,* and David Parker*
Abstract: Luminescent europium complexes are used in
a broad range of applications as a result of their particular
emissive properties. The synthesis and application of bright,
highly water-soluble, and negatively charged sulfonic- or
carboxylic acid derivatives of para-substituted aryl–alkynyl
triazacyclononane complexes are described. Introduction of
the charged solubilizing moieties suppresses cellular uptake or
adsorption to living cells making them applicable for labeling
and performing assays on membrane receptors. These
europium complexes are applied to monitor fluorescent
ligand binding on cell-surface proteins with time-resolved
Fçrster resonance energy transfer (TR-FRET) assays in plate-
based format and using TR-FRET microscopy.
describe the synthesis of europium TACN complexes and
their bioconjugates with improved water solubility that are
incapable of entering or adhering to cells at concentrations
less than 1 mm. The bioconjugates were evaluated in time-
resolved Fçrster resonance energy transfer (TR-FRET)
ligand-binding assays on living cells expressing the cholecys-
tokinin-2 (CCK2) receptor both in plate-based format as well
as using TR-FRET microscopy.
As the cell membrane consists of phospholipids and is
considered to be negatively charged, we developed TACN
complexes which are highly water soluble and negatively
charged at physiological pH (Scheme 1). We reasoned that
this should decrease interactions with the cell membrane as
a result of repulsive Coulombic interactions. Moreover, the
hydrophilic nature of the sulfonate and carboxylate groups
should mask the inherent hydrophobic nature of the three
aryl–alkynyl groups. The emission spectra and photophysical
properties of the parent complexes, for example [EuL1] and
ring-substituted analogues such as [EuL2][16], do not change
significantly, indicating that the local C3 symmetry around the
Eu3+ ion is not compromised. Thus, bioconjugation of the
Luminescent lanthanide complexes are used in an impressive
number of applications, such as sensors,[1,2] bioassays,[3] or
OLEDs (organic light-emitting diodes),[4] because of their
peculiar photophysical properties. Their use in luminescence-
based cellular imaging has been particularly interesting, as
background fluorescence from cells can be completely
rejected using time gating.[1,2,5,6] A key challenge remains to
develop bright probes to address specific biological processes
in living cells or for cell tissue samples.[3,7,8] Recently, we have
developed very bright europium triazacyclononane (TACN)
complexes that stain various intracellular compartments, such
as mitochondria, following cell uptake by macropinocyto-
sis.[9–12] Such behavior is disadvantageous in the development
of assays for membrane proteins, such as G protein-coupled
receptors (GPCR).[13,14] With all established complexes,[9–12]
the nonspecific interactions[3,15] caused by the probe adsorb-
ing to the cell membrane or proteins within, cellular uptake,
or the labeling of immature proteins within the cytosol,
reached values that were not compatible with homogeneous
time-resolved fluorescence (HTRF) bioassays.[3] Herein, we
[*] M. Delbianco, Prof. D. Parker
Department of Chemistry
Durham University
South Road, DH1 3LE, Durham (UK)
E-mail: david.parker@dur.ac.uk
Dr. V. Sadovnikova, E. Bourrier, Dr. G. Mathis, Dr. L. Lamarque,
Dr. J. M. Zwier
Research and Development Cisbio Bioassays
BP 84175, 30200 Codolet (France)
E-mail: jzwier@cisbio.com
[**] We thank the ERC (FCC-266804) and the Fonds Unique Intermi-
nistꢀriel for support. V.S. was supported by the TOPBIO program
(FP7 ITN Marie Curie 264362).
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201406632.
Scheme 1. Structures of parent and carbon-substituted Eu complexes
[EuL1], [EuL2], [EuL3], and [EuL4].
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Scheme 2. Retrosynthesis of the target Eu conjugates [EuL5], [EuL6], and [EuL7].
complexes was initiated by using derivatives substituted at the
ring carbon atom of the TACN macrocycle, for example, those
based on [EuL3] (Scheme 1). This approach avoids varying
the nitrogen substituents on the macrocyclic ring, as asym-
metry in the aryl–alkynyl antennae complicates the syn-
thesis.[9]
Table 1: Photophysical properties and logP values of the [EuL4] com-
plexes (295 K, H2O).
Complex
lmax [nm]
t0 [ms]
F [%]
e [mmÀ1 cmÀ1
]
logP
[EuL4a]
[EuL4b]
[EuL4c]
330
330
330
1.03
1.04
1.01
24
28
26
58
58
58
+1.4
À2.2
À2.2
The synthesis of complexes [EuL5], [EuL6], and [EuL7]
was undertaken in a modular manner, using three key
intermediates (Scheme 2). The carbon-substituted TACN
intermediate, 1, was prepared by an adaptation of an
established method (Supporting Information),[16] wherein
copper binding to the three ring N atoms allows the remote
primary amine to react selectively with Boc2O (Boc = tert-
butoxycarbonyl). Copper is rapidly removed from the reac-
tion mixture by bubbling H2S through the solution, and the
three N atoms on the macrocycle were alkylated with pyridyl
mesylate derivative 2 (Scheme 2).[9,11] The benzylguanine
moiety was introduced following reaction of the correspond-
ing N-hydroxysuccinimide (NHS) ester 3 with the primary
amine substituent on the ring that had been unmasked by
deprotection of the BOC precursor (the deprotection was
achieved using trifluoroacetic acid at room temperature).
The water solubility of these and related complexes was
compared by assessing the partition coefficient (logP) of the
complexes in water/octanol mixtures. Three equimolar sol-
utions of complex were prepared in MeOH. The solvent was
removed under reduced pressure and the resulting solid was
dissolved and stirred for 24 hours in a mixture of water/
octanol (0.9 mL; ratios 2:1, 1:1, or 1:2) giving a total concen-
tration of approximately 2 mm. After equilibration, an emis-
sion spectrum for each layer was recorded in MeOH (50 mL of
solution in 1 mL of MeOH). For each mixture, the logP value
was calculated (see Table 1 for values; further details
provided in the Supporting Information).
to the parent compounds.[11] However, the overall lumines-
cence quantum yield in water drops slightly, suggesting that
the energy transfer from the ligand intramolecular charge-
transfer (ICT) excited state to Eu3+ is less efficient in water
than in methanol.[9]
After solving the problem of water solubility, we eval-
uated the complexes using SNAP-tag technology[17–21] on the
cholecystokinin-2 (CCK2) receptor, a G protein-coupled
receptor which has elevated expression levels when transi-
ently transfected in HEK293 cells. The labeling of the
benzylguanine derivatives [EuL5], [EuL6], and [EuL7] on
living HEK293 cells or HEK293 cells expressing the SNAP-
tagged CCK2 (SNAP-CCK2)[21] was measured by monitoring
the time-gated luminescence intensity at l = 620 nm (Fig-
ure 2b–d).
The introduction of sulfonate or carboxylate groups onto
the ligand enhances the water solubility of complexes [EuL4a],
[EuL4b], and [EuL4c] significantly, and decreases the logP
value from + 1.4 to À2.2 (Table 1). The absorption and
emission spectrum (shown in Figure 1 for complex [EuL4c])
and the excited-state lifetimes hardly change when compared
Figure 1. UV/Vis absorption and emission spectra for [EuL4c] (295 K,
water).
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constant (Ki) is calculated to be 6 nm, in agreement
with previously reported values.[21]
As the nonspecific labeling was suppressed, we could
also monitor the labeling of HEK293 cells expressing
the SNAP-tagged CCK2 with [EuL7] using time-
resolved microscopy. HEK293 cells (50k) were plated
on a Lab-Tek slide, transfected with SNAP-CCK2 plas-
mid, and incubated for 2 days, after which time they
were labeled with [EuL7] (200 nm) for 1 hour. Using
a delay of 100 ms and a gate time of 2 ms, a typical time-
resolved image recorded with a filter of l = 615 Æ 10 nm
is shown in Figure 3a. Labeling clearly occurs at the cell
surface and not within the cell.
It is only recently that lanthanide-based time-
resolved FRET microscopy has been reported,[3,9,22,23]
using primarily a Tb complex.[3,24,25] We tested whether
this method can also be applied with these europium
complexes to monitor ligand–receptor interactions on
GPCR. As a bandpass filter at l = 615 nm was used to
detect the TR signal, we could also use the same
procedure to monitor the time-resolved FRET signal at
l = 670 nm. Similar to the plate-based assay (Figure 2e),
the red-fluorescent agonist of CCK2 was added to the
cells labeled with [EuL7]. The TR-FRET image,
recorded at l = 670 Æ 20 nm, suggests the formation of
vesicles, indicating that many of the receptors previously
expressed at the cell surface are now internalized
together with the red-fluorescent agonist through
a ligand-induced internalization process. The image
data for both the europium and the TR-FRET channel
for the conditions with or without the red-CCK(26-33)
agonist (see Figure 3) reveals that the TR-FRET signal
under the same imaging conditions is well above the
intensity caused by donor bleed-through in the TR-
Figure 2. a) Representation of a TR-FRET ligand-binding assay on HEK293
cells expressing SNAP-CCK2,[3] showing the binding of red-CCK(26-33) (red
sphere) to the SNAP-tagged CCK2 membrane receptor (orange). The TR-FRET
signal is annihilated following the competitive binding of the natural CCK(26-
33) agonist (blue sphere). b),c),d) Saturation curves for labeling HEK293
cells expressing SNAP-CCK2 and non-transfected HEK293 cells at the cell
surface monitoring the time-gated luminescence intensity (l=620Æ5 nm,
60–460 ms) with complexes [EuL5] (b), [EuL6] (c), and [EuL7] (d). e) Binding of
red-CCK(26-33) to HEK293 cells expressing SNAP-CCK2, labeled with
[EuL7] (200 nm), monitored by following the ratio of the emission intensity at
wavelengths l=665 and 620 nm ꢁ10000. The nonspecific binding signal is
measured by adding the unlabeled CCK(26-33) compound (10 mm). f) TR-
FRET competition binding assay, monitoring the dose–response curve for the
displacement of red-CCK(26-33) by CCK2 antagonist PD135158. The ratio of
the emission intensity at wavelengths l=665 and 620 nm ꢁ10000 was
measured.
Significant labeling of non-transfected HEK293 cells
occurs with the less hydrophilic compound [EuL5], whereas
with [EuL6] and [EuL7] this nonspecific labeling is negligible.
These excellent results prompted us to perform a TR-FRET
ligand-binding assay using a fluorescent agonist of the
CCK2 receptor.
Addition of increasing concentrations of the red-fluores-
cent agonist of CCK2, red-CCK(26-33),[21] to [EuL7]-labeled
living SNAP-CCK2 cells and measuring the time-resolved
FRET at l = 665 nm gave rise to a saturating binding curve
(Figure 2e). Note that upon addition of 10 mm of the natural
agonist CCK(26-33) at the different concentrations of fluo-
rescent ligand, the TR-FRET signal is annihilated because of
the competition between the red-luminescent and the non-
labeled compounds. By applying a one-site binding model to
the specific binding data, a dissociation constant (Kd) of the
red agonist of 8 nm was calculated, similar to previously
reported data.[21] A TR-FRET competition binding assay
shows that binding of 10 nm of red-CCK(26-33) is completely
reversed in a dose-dependent way by adding excess quantities
of the antagonist PD135158 (Figure 2 f). The inhibition
Figure 3. a) Time-resolved luminescence microscopy image of SNAP-
CCK2-transfected HEK293 cells labeled with [EuL7] (200 nm) monitored
at l=615Æ10 nm (lex =337 nm, 30 Hz, tdelay =100 ms, tgate =2000 ms).
b) Bleed-through signal of the labeled cells in the TR-FRET channel
(l=670Æ20 nm). c) Eu channel image, and d) TR-FRET channel
image after adding red-CCK(26-33) to cells prepared and monitored as
detailed in (a), incubating for 20 minutes, and washing three times
with Tag-lite buffer. Scale bar=20 mm in all images.
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was added to plates containing labeled cells with Tag-lite labeling
FRET channel. Similarly the Eu channel intensity is
decreased as a result of the FRET process.
medium (100 mL), followed by the addition of the fluorescent red-
CCK(26-33) agonist (20 mL). Plates were incubated at room temper-
ature for 2 hours before signal detection.
In conclusion, we have developed bright, kinetically
stable, highly water-soluble Eu3+ complexes whose emissive
properties are unchanged following bioconjugation. The
carbon-atom substitution of the TACN ring with an amino-
alkyl group (derived from S-lysine) creates a useful synthon
that is easily functionalized with different antennae ligands[26]
for use in a variety of applications. By introducing sulfonate
or carboxylate groups onto the aryl–alkynyl antennae, non-
specific labeling of living HEK cells is suppressed, permitting
the detection of GPCR-CCK2 using derivative [EuL7]. Using
time-resolved detection, the TR-FRET ligand-binding assays
both on a plate reader and with TR-FRET microscopy are
possible. The low nonspecific interactions of these complexes
with cells will be a great advantage for the development of
novel HTRF assays where specific biological interactions are
studied. Furthermore, by introducing different electron-
donating moieties on the aryl ring, we are developing
bioconjugates whose electronic absorption bands are bath-
ochromically shifted,[9,27] thereby improving their brightness
for use in time-resolved confocal or two-photon microscopy.
Competitive binding assay: In competitive binding experiments,
a fixed concentration of the fluorescent red-CCK(26-33) agonist
(10 nm) was used in the presence of increasing concentrations of
antagonist PD135158. PD135158 (20 mL) was added to plates con-
taining labeled cells with Tag-lite labeling medium (100 mL), followed
by the addition of the fluorescent red-CCK(26-33) agonist (10 mL).
Plates were incubated at room temperature for 4 hours before signal
detection.
Signal detection and data analysis: Signal detection was per-
formed on PHERAstar FS plate reader (BMG LABTECH, Cham-
pigny-sur-Marne, France) at l = 620 nm and l = 665 nm (in TR mode:
delay = 60 ms; time gate = 400 ms) upon l = 337 nm laser excitation.
Recorded data were analyzed using GraphPad Prism (GraphPad
Software, Inc., San Diego, CA). Specific binding was determined by
subtracting the nonspecific signal from the total signal. Kd values of
the fluorescent ligand were obtained from the saturation curve of the
specific binding. Ki values were calculated from competition assay
experiments according to the Cheng and Prusoff equation.[28]
TR-FRET Microscopy: A Lab-Tek 8 chamber slide system was
precoated with poly-l-ornithine (200 mL) and incubated for
30 minutes at 378C. The slide was washed with PBS (200 mL) and
HEK293 cells were plated at a density of 5 ꢁ 104 cells per well (c/w)
and incubated overnight at 378C under 5% CO2. The following day,
transfection was performed with optiMEM medium (100 mL), Lip-
ofectamine 2000 (0.8 mL), and SNAP-CCK2 plasmid (1.2 mg per well).
The Lab-Tek slide was further incubated for 2 days at 378C under 5%
CO2. After removal of the medium, SNAP-CCK2 receptors were
labeled with [EuL7] (200 nm) for 1 hour at 378C and washed twice
with Tag-lite medium. The fluorescent red-CCK(26-33) ligand
(10 nm) was added for the FRET signal detection. Hoechst 33342
(2 mgmLÀ1 per well) was added and after 20 minutes incubation at
room temperature and washing twice with Tag-lite medium, the Lab-
Tek slide was observed with a Zeiss oil immersion objective (40 ꢁ
magnification, 1.3 F-Fluar) on the Zeiss Axiovert 200m TR-FRET
inverted microscope equipped with a pulsed nitrogen laser (l =
Materials and Methods
Reagents: The Tag-lite labeling medium (LABMED), the SNAP-
CCK2 plasmid for transient transfection of CCK2 receptors
(PSNAPCCK2), and the red-fluorescent red-CCK(26-33) agonist
(L0013RED) were obtained from Cisbio Bioassays. The CCK2
receptor antagonist PD135158 was purchased from Tocris. CKK(26-
33) was obtained from Almac (Craigavon, UK). The 96-well plates
were purchased from Greiner Bio-One (ref. 655086, Monroe, NC).
Cell culture: HEK293 wild-type cells were cultured in Dulbeccoꢀs
modified Eagleꢀs medium (DMEM) glutaMAX (1966-021; Invitro-
gen, Carlsbad, CA) supplemented with fetal bovine serum (10%),
nonessential amino acids (1%), penicillin/streptomycin (1%), and
HEPES (2 mm; HEPES = 4-(2-hydroxyethyl)piperazine-1-ethanesul-
fonic acid).
337 nm, 30 Hz) and
a cooled intensified CCD camera PI
Max 1024X1024 GenIII. For the luminescence imaging of [EuL7],
a l = 615 Æ 10 nm bandpass filter was used with tdelay = 100 ms, tgate
=
Transfection procedure: Transient transfection was performed in
96-well plates using 100000 cells per well according to a previously
described procedure.[21] Prior to cell seeding, the wells of the plates
were precoated with poly-l-ornithine (50 mL) for 30 minutes at 378C.
The transfection mixture (per individual well) was prepared by
adding the SNAP-tag-CCK2 plasmid (100 ng) to optiMEM medium
(49 mL), and Lipofectamine 2000 (0.8 mL; Invitrogen) and incubating
for 20 minutes at room temperature prior to the addition to the plates.
Subsequently, HEK293 cells (100 mL) at a density of 106 cellmLÀ1 was
distributed in each well of the plates. The plates were incubated
overnight at 378C under 5% CO2.
Receptor labeling: For the labeling of SNAP-CCK2-expressed
HEK293 cells with [EuL5], [EuL6], and [EuL7], a previously de-
scribed procedure[21] was used. A concentration series ranging from
0–500 nm was prepared in Tag-lite labeling medium. After incubation,
the transfection mixture was removed from 96-well plates, and cells
were treated with the prepared solutions (50 mL) and incubated for
1 hour at 378C under 5% CO2. The residual compounds were
removed by washing each well 4 times with Tag-lite labeling medium
(100 mL).
2000 ms, and at 60 gates per exposure. TR-FRET images were
collected with a l = 670 Æ 20 nm bandpass filter using the same
procedure. The recorded data were analyzed using ImageJ software.
Received: June 27, 2014
Published online: August 12, 2014
Keywords: europium · G protein-coupled receptors ·
.
imaging agents · macrocycles · time-resolved FRET
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