Structural Control of Cell Permeability with Highly Emissive Europium(III) Complexes Permits Different Microscopy Applications

Article abstract of DOI:10.1002/chem.201504103

Four anionic europium complexes are described based on triazacyclononane tris-carboxylate or phosphinate ligands. In each case, the three sensitising chromophores comprise a substituted aryl-alkynyl pyridine group, with complex brightness in water falling in the range 4 to 23 mM^-1 cm^-1. para-Substitution of the aryl ring with carboxymethyl groups gives complexes that are taken into cells, stain the lysosomes selectively and unexpectedly permit lifetime measurements of lysosomal pH. In contrast, the introduction of sulfonate groups inhibits cell uptake enabling the Eu complex to be used as an extracellular donor for FRET applications at the membrane surface. Using time-gated FRET microscopy, the cell membrane structure was highlighted, in which Cell Mask Deep Red was used as a membrane- localized FRET acceptor.

Full text of DOI:10.1002/chem.201504103

DOI: 10.1002/chem.201504103
Full Paper
&
Microscopy |Very Important Paper|
Structural Control of Cell Permeability with Highly Emissive
Europium(III) Complexes Permits Different Microscopy
Applications
Matthieu Starck, Robert Pal, and David Parker*[a]
Abstract: Four anionic europium complexes are described
based on triazacyclononane tris-carboxylate or phosphinate
ligands. In each case, the three sensitising chromophores
comprise a substituted aryl–alkynyl pyridine group, with
complex brightness in water falling in the range 4 to
23 mmÀ1 cmÀ1. para-Substitution of the aryl ring with carbox-
ymethyl groups gives complexes that are taken into cells,
stain the lysosomes selectively and unexpectedly permit life-
time measurements of lysosomal pH. In contrast, the intro-
duction of sulfonate groups inhibits cell uptake enabling the
Eu complex to be used as an extracellular donor for FRET ap-
plications at the membrane surface. Using time-gated FRET
microscopy, the cell membrane structure was highlighted, in
which Cell Mask Deep Red was used as a membrane-
localized FRET acceptor.
Introduction
responsive probes or emissive tags: the complexes should pos-
sess high kinetic stability in biological media with respect to
premature metal dissociation; they should be water soluble
and resist vibrational deactivation by bound or proximate OH
and NH oscillators; they should possess a high overall bright-
ness B (B=e·F) to allow detection at low concentration and,
finally, they should be excited at a wavelength above about
340 nm, to avoid the use of expensive quartz optics.
We have developed a set of four europium(III) complexes,
[Eu·L1À4]3À, with very high luminescence brightness, in which
remote sulfonate groups are introduced to suppress non-
specific cell uptake. The precursor carboxylate complexes, in
contrast, enter cells and localise to the lysosomes. Rather
surprisingly, these complexes can act as lysosomal pH sensors,
based on Eu lifetime modulation.
The selection of the ligand and the sensitising moiety
should be made with these criteria in mind. A wide variety of
lanthanide coordination complexes has been studied for this
purpose, including cryptates,[13] helicates,[14] polyaminocarboxy-
lates and aminophosphinates,[15–17] notably those based on a
9-N3 or 12-N4 ligand framework.[18–20]
Coordination complexes of the f block elements have found
diverse applications in the biological and materials sciences.[1–3]
Many of these applications harness the unique properties of f-
element spectroscopy. The large pseudo-Stokes’ shifts, follow-
ing indirect ligand excitation, and sharp emission bands allow
selective detection in biological media.[4] Their long excited-
state lifetimes permit time-resolved luminescence detection.
Such spectral and temporal resolution engenders high signal-
to-noise ratios in data acquisition, a crucial issue in imaging. In
this context, many complexes of europium and terbium(III)
have been developed as luminescent probes, for one or two-
photon cell imaging,[5–7] as responsive probes, for example, for
pH, metal ions, bicarbonate, citrate and urate measured in
vitro or in cellulo[8–11] and as long-lived, emissive tags for FRET
assays.[12]
With the known high stability of lanthanide complexes of
triazacyclononane-tris-pyridines in mind,[21] a family of europi-
um (III) complexes (EuroTracker dyes) with exceptional bright-
ness has been created.[22] They have been used to image cer-
tain cell organelles selectively, serve as FRET donors and tags
in bioassays[23] and have been modified to function as intracel-
lular pH probes.[23] Their exceptional brightness can be attribut-
ed to the very high extinction coefficient associated with the
ICT transition,[24] the efficient intramolecular energy transfer
process and effective shielding of the bound EuIII ion from
vibrational deactivation.[15,18d]
Numerous complexes have been designed that strive to
meet a set of stringent requirements for use as cellular stains,
Herein, we report the preparation of a set of four EuIII com-
plexes (Scheme 1), investigate their photophysical properties
and devise applications for each type of complex, both within
and outside a living cell.
[a] Dr. M. Starck, Dr. R. Pal, Prof. D. Parker
Department of Chemistry
Durham University
South Road, Durham, DH1 3LE (UK)
E-mail: david.parker@dur.ac.uk
Supporting information and ORCID(s) from the author(s) for this article are
available on the WWW under http://dx.doi.org/10.1002/chem.201504103.
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3À
Scheme 1. Structures of the europium(III) complexes studied, [Eu·L1À4
] .
Results and Discussion
prepared by a coupling reaction using HATU (Scheme 4);
similarly, the carboxylate complex analogue, [Eu·L3]3À, was
prepared and was also isolated as its ammonium salt.
Synthesis
The synthesis of the tricarboxylate complex, [Eu·L1]3À
(Scheme 2) began with commercially available aromatic and
heterocyclic precursors, and used standard methodology.[22]
The starting material, 1, was prepared by alkylation of the
corresponding p-iodophenol. Two Sonogashira palladium
cross-coupling reactions were used to prepare the chromo-
phore intermediate, 6.
The benzylic alcohol, 6, was converted to the corresponding
mesylate 7, which was used to alkylate 1,4,7-triazacyclononane
in acetonitrile, in the presence of potassium carbonate. Near
quantitative conversion afforded the ligand, 8, which was
hydrolysed in base and its europium(III) complex formed
following addition of europium trichloride hexahydrate at
neutral pH. The EuIII complex was isolated after purification by
preparative HPLC.
Photophysical properties of the europium(III) complexes
Measurements of the photophysical properties of the four EuIII
complexes were performed in dilute aqueous and methanol
solutions; representative data are reported in Table 1. Each
complex possesses a broad absorption band around 345 nm,
assigned to an intra-ligand charge-transfer transition (ICT),
from the electron-donating group on the aryl ring to the elec-
tron-poor coordinated pyridine fragment. As observed in
Table 1. Spectroscopic properties of europium complexes (295 K, water;
MeOH data in parentheses).
[a,b]
Complex
lmax [nm]
e [mmÀ1 cmÀ1
]
f
[%]
t [ms][c]
The synthesis of the analogous phosphinate complex
(Scheme 3) involved preparation of the p-bromopyridyl
phosphinate intermediate 9,[22] which was elaborated to the
conjugated alkynyl chromophore 10 by using a Sonogashira
reaction. Subsequent mesylation of the alcohol and alkylation
of triazacyclononane gave the trisubstituted macrocycle, 12.
Base hydrolysis removed all of the ethyl ester groups, after
which the EuIII ion was introduced and the complex purified by
reverse-phase HPLC. The sulfonated derivative, [Eu·L4]3À, was
[Eu·L1]3À
[Eu·L2]3À
[Eu·L3]3À
[Eu·L4]3À
348
340
348
340
65.0
67.1
65.1
67.0
6 (42)
0.50 (0.75)
0.81 (1.25)
0.65 (1.02)
1.07 (1.34)
16 (52)
31 (52)
34 (76)
[a] Quinine sulfate was used as a standard;excitation at 335 nm; errors on
quantum yield or lifetime are +/À15%. [b] Errors are +/À20%. [c] In
each case lifetimes in D2O were longer than in water and consistent with
the absence of a coordinated water molecule (see the Experimental
Section).
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Scheme 2. Synthesis of the europium tricarboxylate complex, [Eu·L1]3À. dppf=1,1’-bis(diphenylphosphino)ferrocene.
Scheme 3. Synthesis of the europium phosphinate complex, [Eu·L2]3À
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Scheme 4. Formation of the tris-sulfonate complex, [Eu·L4]3À. HATU =1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluoro-
phosphate; DIEA=diisopropylethylamine.
recent work,[18d] the carboxylate complex absorption was
slightly redshifted with respect to the phosphinate analogue,
Such behaviour reflects both the increased steric protection af-
forded by the methyl substituents at phosphorus—affecting
deactivation of the Eu excited state—and the sensitivity of the
energy of the ICT excited state to solvent polarity that influen-
ces the rate of intramolecular energy transfer.[18d,24] In every
case, no ligand-centred emission was observed, consistent
with efficient energy transfer to the lanthanide ion, and the
emission lifetimes and intensities were the same in aerated
and degassed solutions. The quantum yields in methanol are
particularly high; the highest value (76%) was for [Eu·L4]3À and
is associated with the longest luminescence lifetime of
1.34 ms. The quantum yield (34%) and lifetime (1.07 ms) in
water are rather lower than in methanol and are consistent
with the properties of related systems recently examined.[22–24]
The sulfonated complexes [Eu·L3]3À and [Eu·L4]3À showed no
change in emission intensity and lifetime over the pH range 4
to 8, and changes in lifetime and emission intensity following
addition of bovine serum albumin were less than 10% for
every complex. The complexes with peripheral oxy-acetate
groups showed a decrease in lifetime of 25% between pH 5
and 4 in 0.1m NaCl solution (ESI) consistent with simultaneous
protonation of the three carboxylate sites. The narrow range
(over one pH unit, rather than three pH units for a mono-pro-
tonation equilibrium) for the observed variation is consistent
with such behaviour, with an apparent pKa of 4.46 (Æ0.08)
(ESI). The reduction in lifetime may be tentatively ascribed to
complex aggregation at lower pH, leading to quenching of the
Eu excited state by exciplex formation.
in terms of both the maximal absorption wavelength (Dlmax
=
8 nm) and the position of the red tail of the absorption band
(Dlcut-off =15 nm, Figure 1). This bathochromic shift had earlier
been tentatively ascribed to a shorter Ln–Npy distance for the
carboxylate complex, supported by comparative DFT calcu-
lations. The slightly shorter distance is indicative of stronger
coordination to the europium ion, the Lewis acidity of which
enhances the accepting character of the pyridine moiety.
Figure 1. Absorption (dotted), excitation (left) and emission spectra (right) of
[Eu·L1]3À (295 K, H2O).
The emission spectral profiles of the four complexes were
very similar (Figure 1 and ESI), with an intense hypersensitive
DJ=2 transition around 610–620 nm, as expected for such
symmetric complexes incorporating polarisable donor pyridyl
groups. Each complex exhibits a high quantum yield efficiency
in MeOH (42 to 76%), with the highest value for the tris-phos-
phinate, [Eu·L4]3À, bearing remote sulfonate groups. In water,
the quantum yields and excited-state lifetimes are systemati-
cally lower, especially for the carboxylate bound complexes.
Cell microscopy studies
The Eu complexes, [Eu·L1]3À and [Eu·L2]3À were incubated for
2 h in the cell growth medium with mouse skin fibroblast cells
(NIH-3T3). Laser scanning confocal microscopy images revealed
that the complexes had been internalised and gave rise to
a punctate staining pattern (Figure 2). Parallel incubations with
LysoTracker GreenTM
,
confirmed the selective lysosomal
staining pattern. Repeating the same experiments with the
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values are believed to be about 6.6 (Æ0.1) following such
treatment.[25,26]
This comparative study demonstrated that the variation of
lysosomal pH with time can be monitored by observing the
change in Eu emission lifetime. The direct comparison with
data obtained under parallel conditions, using the fluorescent
LysoSensor probe, lends support to the in cellulo calibration
procedure, allowing the lifetime of the Eu probe to indicate
lysosmal pH. Related studies with lanthanide complexes have
used a similar approach to examine pH in the endoplasmic
reticulum,[27] or in the lysosomes—in the latter case, this was
done by measuring a ratio of Tb/Eu emission intensities in
complexes of a common ligand.[28]
Figure 2. Confocal microscopy images of an NIH 3T3 cell following incuba-
tion with [Eu·L1]3À (10 mm, 2 h; lexc =355 nm; observe 605–720 nm) (left);
LysoTracker Green (200 nm; 5 min; lexc =488 nm; observe 505–535 nm)
(centre); merged image showing co-localisation (P=0.88; scale bar
20 mm (right) (ESI for images with and without nigericin at pH 4.6 and 6.5).
sulfonated complexes gave no staining within the cell, even
after a 24 h incubation period.
The K+/H+ ionophore, nigericin, was added to the incu-
bation medium (2 mm) and sequential lifetime measurements
were made over the next 5 min, with excitation at 365 nm. The
starting Eu lifetime was 470 ms at the ‘resting’ lysosomal pH;
this value was assumed to be pH 4.6 (Æ0.1), following litera-
ture precedent.[25] At pH 6.6, the lifetime was 520 ms. The initial
Eu lifetime did not change for incubations of 2, 4, 8, or 24 h,
and the overall emission intensity (i.e. brightness) was also
very similar at each of these time points. Such behaviour is
consistent with fast, irreversible lysosomal localization, with
little egress of the complex from the cell, in the absence and
presence of added nigericin.
Time-resolved FRET studies
The sulfonated complexes [Eu·L3]3À and [Eu·L4]3À showed no
cellular uptake and internalisation, and could be observed in
the growth medium surrounding the cells by microscopy,
(Figure 4). The Eu complex can serve as a FRET donor to a suit-
able accepting dye over a distance of up to 8 nm.[12,23a] The
Using a LysoSensor Probe (5 mm, 5 min incubation, LSBY-
DND160, lexc 355 nm), a similar experiment was undertaken,
monitoring the time dependence of a neutralization process
created by addition of the ionophore nigericin (2 mm). A ratio
of band intensities was observed corresponding to the range
400–485 nm (’blue’) versus 495–600 nm (’yellow’), (Figure 3)
The final blue/yellow ratio observed was the same (Æ5%) as
that induced by a separate addition of chloroquine (100 mm)
after 4 min. Chloroquine (pKa 10.1 and 8.4) is a lysosomotropic
agent that inhibits acidification. Typically, intra-lysosomal pH
Figure 4. Laser scanning confocal microscopy images showing: the lack of
internalisation of [Eu·L3]3À (internal fluorescence is due to UV induced mito-
chondrial autofluorescence) (1 h, 20 mm, lexc =355 nm, lem =605–635 nm) in
NIH-3T3 cells pre-loaded with Cell Mask Deep Red (CMDR; 100 nm,
10 min) (top left); FRET-induced CMDR fluorescence defining only the
external membrane for which FRET is possible (lexc =355 nm, lem =660–
680 nm) (top right); observed CMDR fluorescence following direct excitation
(lexc =633 nm, lem =660–680 nm) showing some evidence for internalisation
in the punctate staining profile (lower left); cell transmission image (scale
bar 15 mm, voxel size 62”62”789 nm) (lower right).[32]
Figure 3. Variation of lysosomal pH with time, following treatment with
nigericin (2 mm) in NIH-3T3 cells, monitoring Eu lifetime for [Ln·L1]3À, or
blue/yellow emission intensity ratio (LSB/Y) of the LysoSensor DND160.
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membrane-staining dye, Cell Mask Deep Red (CMDR) can act
as a FRET acceptor for a Eu donor in this context (Figure 5).
This dye is used to stain cell membranes, but on occasion it is
internalised by the cell, causing a punctate staining pattern
(Figure 4, bottom left). In order to define better the membrane
staining of CMDR, a time-resolved FRET microscopy study was
undertaken. The lifetime of the CMDR acceptor increases dra-
matically under these conditions allowing the luminescence
from the longer-lived dye (82 ms, Figure 5) to be observed se-
lectively. Energy transfer between the extracellular dye and the
membrane-immobilised dye acceptor can only occur in the vi-
cinity of the membrane, so that the clarity of the FRET image
is quite superior to that obtained examining dye fluorescence
only, in both confocal images (Figure 4) and in a definitive
time-resolved image of the membrane, using [Eu·L4]3À as the
FRET donor (Figure 5, bottom).
Conclusions
The series of experiments described provide compelling
evidence of the advantages to be gained by working with
longer-lived emissive probes that have high inherent bright-
ness. When a europium complex, for example, is not able to
enter cells, then it can serve as a FRET donor at the membrane,
allowing FRET imaging to be used to address a suitable
acceptor anchored near the surface or on the membrane, to
a penetration depth of about 6 to 8 nm.
The pH-dependent modulation of the lifetime of [Eu·L1,2 3À
]
occurred over a narrow range in vitro, and was still observable
when the probe was trapped within cellular lysosomes, there-
by permitting real-time estimation of lysosomal pH in living
cells. Such behaviour is of interest to those interested in exam-
ining the activity of new chemical entities in cellular models of
disease, for example, for lysosomal-storage diseases, in which
elevated lysosomal pH occurs.[29] Over fifty of these hereditary
metabolic disorders have been described in which defective
lysosomal function is found. Often, mutations in lysosomal
hydrolase enzymes occur, leading to the degradation of vari-
ous cellular macromolecules.[30] Therefore, time-dependent
measurements of lysosomal pH using such probes, in the pres-
ence or absence of new potential drugs to treat the disease,
may be of some value in the future.
Experimental Section
Optical measurements
Figure 5. Reduced lifetime of [Eu·L4]3À (20 mm) when serving as a FRET donor
to Cell Mask Deep Red (CMDR) (top); the insert shows the emission
spectrum and corresponding lifetime of the pure Eu complex in water
(lexc =365 nm, observed at 610–630 nm); time-gated (tg =10 ms) spectrum of
[Eu·L4]3À (20 mm) and CMDR (200 nm) (centre); enhanced lifetime of CMDR
(200 nm) following FRET from the Eu donor (lexc =365 nm, observed at 660–
680 nm) (bottom). The spectra were recorded in NIH-3T3 cells (10 cells in the
field of view) after 10 min pre-incubation with CMDR (washed ”3 with PBS),
after loading the ‘non-internalising’ [Eu·L4]3À donor; the insert shows a time-
gated (tg =10 ms) fluorescence microscopy image (scale bar 10 m), recorded
using a 250 image accumulation sequence (lexc =365 nm, observed at
660–680 nm).[31]
Absorption spectroscopy: UV/Vis absorption measurements
were recorded using
a Perkin–Elmer Lambda 900 absorption
spectrophotometer, using matched quartz cells.
Luminescence: Emission spectra were measured using a Horiba–
Jobin Yvon Fluorolog-3 spectrofluorimeter. The steady-state lumi-
nescence was excited by unpolarised light from a 450 W xenon CW
lamp and detected at an angle of 908 for diluted solution measure-
ments (10 mm quartz cell) by a red-sensitive Hamamatsu R928
photomultiplier tube. Spectra were reference corrected for both
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the excitation source light intensity variation (lamp and grating)
and the emission spectral response (detector and grating). Phos-
phorescence lifetimes (>30 ms) were obtained by pulsed excitation
using a FL-1040 UP Xenon Lamp. Luminescence decay curves were
fitted by least-squares analysis using Origin. Luminescence quan-
tum yields Q were measured in diluted aqueous solution with an
absorbance lower than 0.1 using the following Equation (1):
The europium complex [Eu·L1]3À (20 mm) was incubated with NIH-
3T3 cells for up to 24 h to allow complex uptake within the lyso-
somes; this localisation profile was verified by co-incubation with
LysoTracker Green. The K+/H+ ionophore, nigericin, was added
(2 mm) and seven consecutive sets of EuIII probe lifetimes were
measured over the next 5 min, with excitation at 365 nm using the
modified epifluorescence microscopy set up. The starting lifetime
was 470 microseconds at the ‘normal’ lysosomal pH; this starting
value was assumed to be pH 4.6 (Æ0.1), following literature prece-
dent.[25] At pH 6.5, the lifetime was 520 microseconds. Replacement
of the nigericin-containing medium with fresh medium led to slow
restoration of the initially observed lifetime value (Æ10%), over
a period of 1.5 h.
2
Qx=Qr ¼ ½ArðlÞ=Axðlފ½nx =nr2Š½Dx=DrŠ
ð1Þ
in which A is the absorbance at the excitation wavelength (l), n
the refractive index and D the integrated luminescence intensity.
“r” and “x” stand for reference and sample. Here, reference is
quinine bisulfate in 1n aqueous sulfuric acid solution (f=0.55).
Excitation of reference and sample compounds was performed at
the same wavelength.
Using a LysoSensor Probe (5 mm, 5 min incubation, LSBYDND160,
Invitrogen, lexc =355 nm), the same experiment was performed to
follow the time dependence of the increase in pH, induced by ad-
dition of the ionophore nigericin (2 mm). In this case, the ratio of
band intensities observed corresponded to 400–485 nm (’blue’)
versus 495–600 nm (’yellow’). The final ratio measured correspond-
ed (Æ5%) to that induced independently by addition of chloro-
quine (100 mm) after 4 min. Chloroquine (pKa =10.1 and 8.4) is an
established lysosomotropic agent that accumulates in lysosomes
and inhibits acidification. Typically, intra-lysosomal pH values
are believed to lie in the range 6.5 to 6.7 following such a
treatment.[25,26]
Confocal microscopy
Cell microscopy imaging of [Eu·L1À4 3À
in cells was achieved using
]
a custom built epiflorescence microscope (modified Zeiss Axiovert
200m)1, using a Zeiss APOCHROMAT 63”/1.40 NA objective, com-
bined with a low voltage 365 nm pulsed UV LED focused and colli-
mated excitation source (1.2 W). For rapid spectral acquisition, the
microscope was equipped at the X1 port with a Peltier cooled 2D-
CCD detector (Ocean Optics), used in an inverse 100 Hz time-gated
sequence. The spectrum was recorded from 400–800 nm with a
resolution of 0.24 nm and the final spectrum was acquired using
an averaged 10000 scan duty cycle.
HPLC analysis
HPLC analysis and purifications were performed at 295 K using Shi-
madzu system (degassing unit DGU-20 A5R, Prominence semi-prep-
arative liquid chromatograph LC-20AP, Prominence UV/Vis detector
SPD-20 A and communications bus module CBM-20A). The solvent
system used was an ammonium bicarbonate buffer (25 mm, pH 7)/
acetonitrile (isocratic 10% acetonitrile in buffer (3 min), linear gra-
dient to 100% acetonitrile (10 min), isocratic 100% acetonitrile
(5 min)), flow: 2 mLminÀ1 for analytical mode on XBridge C18
column, 4.6”100 mm, i.d. 5 mm, and 17 mLminÀ1 for preparative
mode on XBridge C18 column, 19”100 mm, i.d. 5 mm.
Probe lifetimes were measured on the same microscope platform
using a novel cooled PMT detector (Hamamatsu H7155), inter-
changeable on the X1 port. Both the control and detection algo-
rithm were written in LabView2011. Time-gated images were
recorded using a high-resolution cooled EO-1312m CCD camera
(Thor labs). All duty cycle and gating sequences were established
and controlled by in-house LabView software.[31]
High-resolution LSCM images were recorded on a modified Leica
SP5 II microscope, equipped with a new SIM technique called
PhMoNa.[32] In order to achieve excitation with maximal probe
emission, the microscope was coupled by an optical fibre to a
Coherent CW laser (Nd:YAG, 355 nm), operating at 8 mW power. A
He/Ne or Ar ion laser was used when commercially available
organelle-specific stains (e.g. LysoTrackerGreen) were used to
corroborate cellular compartmentalization.
Electrospray mass spectra and accurate masses
Electrospray mass spectra and accurate masses were recorded on
a TQD mass spectrometer or a QTOF Premier mass spectrometer,
respectively, both equipped with an Acquity UPLC, a lock-mass
electrospray ion source and an Acquity photodiode array detector
(Waters Ltd, UK), acetonitrile was used as the carrier solvent. The
solvent system used for TQD was water (0.1% formic acid)/acetoni-
trile (0.1% formic acid) (5% acetonitrile (0.2 min), linear gradient to
95% Acetonitrile (3.8 min), isocratic 95% acetonitrile (0.5 min),
linear gradient to 5% acetonitrile (0.5 min)), flow: 0.6 mLminÀ1 on
Acquity UPLC BEH C18 column, 2.1”50 mm, i.d. 1.7 mm. The sol-
vent system used for QTOF was ammonium bicarbonate buffer
(25 mm, pH 7)/acetonitrile (2% acetonitrile in buffer (3 min), linear
gradient to 40% acetonitrile (6 min), linear gradient to 100% ace-
tonitrile (4 min), isocratic 100% acetonitrile (2 min)) or water (0.1%
formic acid)/acetonitrile (0.1% formic acid) (linear gradient to 99%
acetonitrile (5 min), isocratic 99% acetonitrile (1 min), linear gradi-
ent to 100% water (0.1 min), isocratic 100% water (0.9 min)), flow:
0.6 mLminÀ1 on Acquity UPLC BEH C18 column, 2.1”50 mm, i.d.
1.7 mm.
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%, reducing
signal to noise by a factor of 5. The pinhole was always determined
by the Airy disc size, calculated from the objective in use (HCX PL
APO 63”/1.40 NA aBlue), using the lowest excitation wavelength
(355 nm). Scanning speed was adjusted to 100 Hz in a unidirection-
al mode, to ensure both sufficient light exposure and enough time
to collect the emitted light from the lanthanide-based optical
probes (2048”2048 frame size, a pixel size of 62”62 nm and
depth of 789 nm). Spectral imaging on this Leica system is possible
with the xyl-scan function, using the smallest allowed spectral
band-pass (5 nm) and step-size (3 nm) settings. However, much im-
proved spectral imaging in cells was achieved using a custom built
and Peltier cooled CCD detector (Ocean Optics, HR2000plus)
synchronized to the X1 port.
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233.1178); elemental analysis calcd (%) for C14H16O3: C 72.39, H
6.94; found: C 72.05, H 6.97.
Synthesis
Details of general methods and NMR/MS instrumentation may be
traced in recent references.[18d,22] Yields for final ligands and for Eu
complexes were estimated by absorbance measurements of
weighed samples.
Compound 4: Compound 4 was prepared as described in the
literature.[33]
Methyl 4-bromo-6-(hydroxymethyl)picolinate (5): Compound 4
(550 mg, 2.0 mmol) was dissolved in anhydrous CH2Cl2 (5 mL) and
anhydrous CH3OH (3.5 mL) under argon atmosphere. The reaction
mixture was cooled down to 08C. NaBH4 (84 mg, 2.2 mmol) was
added under argon an atmosphere and the reaction was stirred at
08C. After complete consumption of compound 5 (TLC monitor-
ing), the reaction was quenched with hydrochloric acid (2 mL, 1m).
The volatile components were removed under reduced pressure
and the aqueous solution was extracted into CH2Cl2 (20 mL). The
organic layers were combined, dried over Na2SO4, filtered and con-
centrated to dryness. The crude yellowish solid was purified using
flash chromatography (silica, gradient elution starting from 100%
CH2Cl2 to 2% CH3OH in CH2Cl2) to yield compound 5 (343 mg,
70%) as a white solid. TLC analysis Rf: 0.29 (silica, 3% CH3OH in
Ethyl 2-(4-iodo-3,5-dimethylphenoxy)acetate (1): 4-Iodo-3,5-
dimethylphenol (2.02 g, 8.06 mmol) was dissolved in acetone
(10 mL). K2CO3 (1.46 g, 10.50 mmol) and ethyl bromoacetate
(2.70 mL, 24.20 mmol) were added and the reaction was heated to
reflux and stirred under argon for 24 h. The reaction was filtered to
remove the potassium salts and the solvent was removed under
reduced pressure. The residue was dissolved in CH2Cl2 (20 mL) and
washed with water (20 mL). The aqueous phase was extracted into
CH2Cl2 (3”10 mL), and combined organic layers were dried over
MgSO4, filtered and concentrated to dryness. The crude product
was purified by flash chromatography (silica, gradient elution start-
ing from 100% hexane to 4% EtOAc in hexane in 0.2% incre-
ments) to yield compound 1 as a white crystalline solid (2.36 g,
87%). TLC analysis: Rf: 0.25 (silica, 4% EtOAc in hexane); m.p. 79–
808C (hexane); 1H NMR (298 K, 400 MHz, CDCl3):dH =6.67 (2H, s),
4.58 (2H, s), 4.27 (2H, q, J=7.0 Hz), 2.43 (6H, s), 1.30 ppm (3H, t,
J=7.0 Hz); 13C NMR (298 K, 100 MHz, CDCl3): dC =168.9, 157.5,
143.2, 113.6, 98.5, 65.5, 61.5, 29.9, 14.3 ppm; (HRMS+):
m/z: 335.0153 [M+H]+ (C12H16O3127I requires 335.0144); elemental
analysis calculated for C12H15IO3 (%): C=43.13, H=4.52, measured:
C=43.36, H=4.52.
1
CH2Cl2); m.p. 131–1328C (CH2Cl2); H NMR (298 K, 400 MHz, CDCl3):
dH =8.21 (1H, s), 7.79 (1H, s), 4.88 (2H, s), 4.03 (3H, s), 2.50 ppm
(1H, br); 13C NMR (298 K, 100 MHz, CDCl3): dC =164.6, 161.9, 148.2,
134.7, 127.4, 127.3, 64.5, 53.3 ppm; (HRMS+): m/z: 245.9768
[M+H]+ (C8H9NO3Br requires 245.9766); elemental analysis calcd
(%) for C8H8BrNO3: C 39.05, H 3.28, N 5.69; found: C 39.13, H 3.29,
N 5.66.
Methyl 4-{[4-(2-ethoxy-2-oxoethoxy)-2,6-dimethylphenyl]ethyn-
yl}-6-(hydroxymethyl)picolinate (6): Alcohol 5 (95 mg, 0.39 mmol)
was dissolved in anhydrous THF (5 mL) and NEt3 (272 mL,
1.94 mmol) was added. The reaction was degassed three times
using freeze–pump–thaw. The alkyne 3 (100 mg, 0.43 mmol) was
added and the solution was degassed once more. Bis(triphenyl-
phosphine)dichloropalladium(II) (27 mg, 0.039 mmol) and CuI
(15 mg, 0.078 mmol) were added and the resulting brown solution
was stirred at 658C under argon for 36 h. The solvent was removed
under reduced pressure and the resulting brown oil was purified
by column chromatography (silica, gradient elution starting from
100% hexane to 10% EtOAc in hexane in 0.2% increments) to give
compound 6 (66 mg, 45%) as a yellow solid. TLC analysis Rf: 0.32
(silica, 4% MeOH in CH2Cl2); m.p. 164–1658C (hexane); 1H NMR
(298 K, 400 MHz, CDCl3): dH =8.08 (1H, s), 7.60 (1H, s), 6.66 (2H, s),
4.89 (2H, s), 4.65 (2H, s), 4.31 (2H, q, J=7.0 Hz), 4.03 (3H, s), 2.50
(6H, s), 1.33 ppm (3H, t, J=7.0 Hz); 13C NMR (298 K, 100 MHz,
CDCl3): dC =168.7, 165.4, 160.4, 158.4, 147.2, 143.2, 134.4, 125.6,
125.3, 115.1, 113.5, 93.8, 93.6, 65.3, 64.6, 61.6, 53.2, 21.5, 14.3 ppm;
(HRMS+): m/z: 398.1592 [M+H]+ (C22H24NO6 requires 398.1604);
elemental analysis calcd (%) for C22H23NO6·0.25H2O: C 65.74, H
5.89, N 3.48; found: C 65.85, H 5.90; N 3.30.
Ethyl 2-{3,5-dimethyl-4-[(trimethylsilyl)ethynyl]phenoxy}acetate
(2): Compound 1 (500 mg, 1.5 mmol) was dissolved in anhydrous
THF (5 mL) and the solution was degassed three times using con-
secutive freeze–pump–thaw cycles. Ethynyl trimethylsilane (7 mL,
1m in THF, 7 mmol) and triethylamine (1 mL, 7.5 mmol) were
added and the solution was degassed once more. Bis(triphenyl-
phosphine)dichloropalladium(II) (105 mg, 0.15 mmol) and CuI
(57 mg, 0.30 mmol) were added and the resulting brown solution
was stirred at 558C under argon for 60 h. The solvent was removed
under reduced pressure and the resulting brown oil was purified
by column chromatography (silica, gradient elution starting from
100% hexane to 10% AcOEt in hexane in 0.2% increments) to give
compound 2 as a colourless oil which solidified on standing as
a white solid (410 mg, 90%). TLC analysis Rf: 0.22 (silica, 10%
AcOEt in hexane); m.p. 62–638C (hexane); 1H NMR (298 K,
400 MHz, CDCl3): dH =6.57 (2H, s), 4.57 (2H, s), 4.26 (2H, q, J=
7.0 Hz), 2.39 (6H, s), 1.28 (3H, t, J=7.0 Hz), 0.25 ppm (9H, s);
13C NMR (298 K, 100 MHz, CDCl3): dC =168.8, 157.2, 142.5, 116.6,
113.0, 102.7, 101.3, 65.2, 61.4, 21.3, 14.2, 0.3 ppm; (HRMS+): m/z:
305.1574 [M+H]+ (C17H25O3Si requires 305.1573); elemental analysis
calcd (%) for C17H24O3Si: C 67.06, H 7.88; found: C 67.03, H 7.95.
Ethyl 2-(4-ethynyl-3,5-dimethylphenoxy)acetate (3): Compound 2
(400 mg, 1.3 mmol) was dissolved in anhydrous THF (10 mL). Tri-
ethylamine trihydrofluoride (6 mL, 32 mmol) was added and the re-
action was stirred at 408C under argon for 6 days. After this time
the solvent was distilled off and the residue was purified by flash
column chromatography (silica, starting from 100% hexane to 4%
AcOEt in hexane) to give compound 3 as a yellow solid (272 mg,
90%). TLC analysis Rf: 0.20 (silica, 4% AcOEt in hexane); m.p. 97.5–
Methyl 4-{[4-(2-ethoxy-2-oxoethoxy)-2,6-dimethylphenyl]ethyn-
yl}-6-{[(methylsulfonyl)oxy]methyl}picolinate (7): Alcohol
6
(43 mg, 0.11 mmol) was dissolved in dry THF (3 mL). NEt3 (52 mL,
0.385 mmol) and methanesulfonyl chloride (12 mL, 0.16 mmol)
were added. The reaction mixture was stirred at rt for 2 h and
monitored by TLC. The solvent was removed under reduced pres-
sure and the residue dissolved in CH2Cl2 (20 mL) and brine (10 mL).
The aqueous layer was extracted with CH2Cl2 (3”10 mL). The com-
bined organic layers were dried over MgSO4, filtered and concen-
trated to dryness to yield the mesylate 7 as a yellow solid (52 mg,
quantitative), which was directly used in the next step without fur-
1
99.58C (hexane); H NMR (298 K, 400 MHz, CDCl3): dH =6.59 (2H, s),
4.59 (2H, s), 4.27 (2H, q, J=7.0 Hz), 3.42 (1H, s), 2.41 (6H, s),
1.30 ppm (3H, t, J=7.0 Hz); 13C NMR (298 K, 100 MHz, CDCl3): dC =
168.9, 157.5, 143.0, 115.6, 113.2, 84.3, 81.1, 65.3, 61.5, 21.4,
14.3 ppm; (HRMS+): m/z: 233.1179 [M+H]+ (C14H17O3 requires
1
ther purification. H NMR (298 K, 400 MHz, CDCl3): dH =8.14 (1H, s),
Chem. Eur. J. 2016, 22, 570 – 580
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577
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

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7.69 (1H, s), 6.66 (2H, s), 5.43 (2H, s), 4.64 (2H, s), 4.31 (2H, q,
J=7.0 Hz), 4.03 (3H, s), 3.18 (3H, s), 2.50 (6H, s), 1.33 ppm (3H, t,
J=7.0 Hz); LCMS: m/z: 475.9 (100% [M+H]+).
showed complete conversion. Purification by HPLC gave the am-
monium salt of the [EuL3] complex as a white solid (1.1 mg, quanti-
3À
½MÀ3 HŠ
tative
conversion).
(HRMSÀ):
m/z:
550.1105
3
(C72H78151EuN9O21S3 requires 550.1153); HPLC (ammonium bicarbon-
ate buffer, 25 mm, gradient: 10–100% CH3CN in buffer over
Europium complex of 2,2’,2’’-{[({6,6’,6’’-[(1,4,7-triazacyclononane-
1,4,7-triyl)tris(methylene)]tris(2-carboxypyridine-6,4-diyl)}tris-
(ethyne-2,1-diyl))tris(3,5-dimethylbenzene-4,1-diyl)]tris(oxy)}tri-
acetate [EuL1]: 1,4,7-Triazacyclononane trihydrochloride (8 mg,
0.034 mmol) and mesylate 7 (52 mg, 0.110 mmol) were dissolved in
anhydrous CH3CN (2 mL), and K2CO3 (28 mg, 0.204 mmol) was
added. The mixture was stirred under argon at 608C for 12 h.
Excess potassium salts were filtered off and the filtrate was concen-
trated to dryness. The residue was dissolved in CH2Cl2 (20 mL) and
H2O (10 mL), and the aqueous layer was extracted with CH2Cl2 (3”
10 mL). Combined organic layers were dried over MgSO4, filtered
and concentrated to yield the ligand precursor 8 as a white
powder residue. The residue was dissolved in a solution of MeOH/
H2O, and NaOH (30 mg, 0.75 mmol) was added to reach pH 12. The
solution was heated at 608C overnight and hydrolysis of the ester
10 min): tr =6.6 min; t
=0.65 ms; t O =0.82 ms;
D2
H2Oðammonium bicarbonateÞ
tMeOH =1.02 ms; q=0; lmax =348 nm; e348 nm =65000mÀ1 cmÀ1
O =31%, fMeOH =52%.
;
f
H2
Compound 9: Compound 9 was prepared following established
procedures.[18d,22]
Ethyl 2-[4-({2-[ethoxy(methyl)phosphoryl]-6-(hydroxymethyl)pyr-
idin-4-yl}ethynyl)-3,5-dimethylphenoxy]acetate (10): Compound
9 (105 mg, 0.36 mmol) was dissolved in dry THF (1 mL) and com-
pound 3 (95 mg, 0.41 mmol) and NEt3 (250 mL, 1.8 mmol) were
added. The reaction was degassed three times using freeze–
pump–thaw.
[1,1-Bis(diphenylphosphino)ferrocene]dichloropalla-
dium(II) (30 mg, 0.036 mmol) and CuI (7 mg, 0.036 mmol) were
added and the resulting brown solution was stirred at 658C under
argon for 18 h. The solvent was removed under reduced pressure
and the resulting brown solid was purified by column chromatog-
raphy (silica, gradient elution starting from 100% CH2Cl2 to 5%
MeOH in CH2Cl2 in 0.2% increments) to give compound 10 (91 mg,
57%) as a pale-brown solid. TLC analysis Rf. 0.33 (silica, 5% MeOH
in CH2Cl2); m.p. 157–1588C (CH2Cl2); 1H NMR (298 K, 700 MHz,
CDCl3): dH =8.01 (1H, s), 7.44 (1H, s), 6.61 (2H, s), 4.80 (2H, s),4.59
(2H, s), 4.26 (2H, q, J=7.0 Hz), 3.97 (2H, dq, J=175.5 Hz, 7.0 Hz),
3.63 (1H, br. s), 2.44 (6H, s), 1.77 (3H, d, J=15.5 Hz), 1.28 (3H, t,
J=7.0 Hz), 1.26 ppm (3H, t, J=7.0 Hz); 13C NMR (298 K, 175 MHz,
CDCl3): dC =168.7, 160.5 (d, J=18.0 Hz), 158.4, 153.4 (d, J=
156.0 Hz), 143.3, 133.5 (d, J=10.0 Hz), 128.1 (d, J=22.0 Hz), 124.1,
115.1, 113.4, 93.9, 93.6, 65.3, 64.1, 61.6, 61.3 (d, J=5.0 Hz), 21.5,
16.6 (d, J=3.5 Hz), 14.3, 13.6 ppm (d, J=106.0 Hz); 31P{1H} NMR
(298 K, 162 MHz, CDCl3): dP =39.52 ppm; (HRMS+): m/z: 446.1734
[M+H]+ (C23H29NO6P required 446.1733); elemental analysis calcd
(%) for C23H28NO6P: C 62.02, H 6.34, N 3.14; found: C 61.99, H 6.13,
N 3.18.
1
groups monitored by LCMS and H NMR spectroscopy. After com-
pletion, MeOH was removed under reduced pressure and the pH
was adjusted to 7 by addition of a 1m HCl solution. Then
EuCl3·6H20 (9.5 mg, 26 mmol) was added and the solution was
stirred at 608C for 12 h. The solution was evaporated and the resi-
due was dissolved in MeOH, and sonicated. The residual solution
was filtered and evaporated to yield the crude EuIII complex as
a yellow solid, bright red under UV light. The complex was purified
by reverse-phase HPLC (ammonium bicarbonate buffer, 25 mm) to
give the ammonium salt of the europium complex (30 mg) as
a pale-yellow powder after freeze-drying. (HRMSÀ): m/z: 1287.302
[MÀH]À (C63H56N6O15151Eu requires 1287.300); HPLC (ammonium bi-
carbonate buffer, 25 mm, gradient: 10–100% CH3CN in buffer over
10 min): tr =6.2 min; tH
=0.50 ms; tD O =0.58 ms;
2Oðammonium bicarbonateÞ
2
tMeOH =0.75 ms; q=0; lmax =348 nm; e348 nm =65000mÀ1 cmÀ1
;
fH O =6%, fMeOH =42%.
2
Europium complex of 2,2’,2’’-({[({6,6’,6’’-[(1,4,7-triazacyclono-
nane-1,4,7-triyl)tris(methylene)]tris(2-carboxypyridine-6,4-diyl)}-
tris(ethyne-2,1-diyl))tris(3,5-dimethylbenzene-4,1-diyl)]tris(oxy)-
triacetamido}propane-1-sulfonate [EuL3]: Complex [EuL1] (1.0 mg,
0.77 mmol) and homotaurine (0.65 mg, 4.65 mmol) were dissolved
in DMSO (0.2 mL) and water (20 mL). HATU (2.45 mg, 4.7 mmol) and
DIEA (1.5 mL, 7.7 mmol) were added to the reaction and the mixture
was stirred for 24 h under argon, after which time, HPLC and LCMS
Ethyl 2-[4-({2-[ethoxy(methyl)phosphoryl]-6-{[(methylsulfonyl)-
oxy]methyl}pyridin-4-yl}ethynyl)-3,5-dimethylphenoxy]acetate
(11): Compound 10 (10 mg, 22.5 mmol) was dissolved in dry THF
(1.5 mL). Methanesulfonyl chloride (2.15 mL, 33.7 mmol) and trime-
thylamine (11 mL, 78.7 mmol) were added and the solution was
stirred under argon at room temperature for 2 h. Solvents were
Chem. Eur. J. 2016, 22, 570 – 580
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578
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Paper
evaporated and the residue was dissolved in CH2Cl2 (10 mL) and
brine (10 mL). The organic layer was extracted and the aqueous
layer washed with CH2Cl2 (3”10 mL). The combined organic layers
were dried over Na2SO4, filtered and concentrated to dryness to
yield the mesylate 11 as a yellow oil (11.7 mg, quantitative), which
was directly used in the next step without further purification.
LCMS (ES+ MS): m/z: 524.710 (100%, [M+H]+).
Acknowledgements
We thank the EPSRC (MS), Royal Society and ERC for support
(DP: FCC 266804).
Keywords: cell imaging · europium · luminescence FRET ·
microscopy
Triethyl-2,2’,2’’-[({(6,6’,6’’-[(1,4,7-triazacyclononane-1,4,7-triyl)-
tris(methylene)]tris{2-[ethoxy(methyl)phosphoryl]pyridine-6,4-
diyl})tris(ethyne-2,1-diyl)}tris(3,5-dimethylbenzene-4,1-diyl))tris-
(oxy)]triacetate (12): 1,4,7-Triazacyclononane trihydrochloride
(1.77 mg, 7.46 mmol) and mesylate 10 (11.7 mg, 22.5 mmol) were
dissolved in anhydrous CH3CN (0.5 mL), and K2CO3 (6 mg,
44.8 mmol) was added. The mixture was stirred under argon at
608C for 24 h. Excess potassium salts were filtered off and the fil-
trate was concentrated to dryness to yield a yellowish oil (10 mg,
95%) that was used directly in the next step without further purifi-
cation. 31P{1H} NMR (298 K, 162 MHz, MeOD/D2O): dP =41.61 ppm;
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707.5 (100%,
2þ).
½MÀ2 HŠ
2
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Europium complex of 2,2’,2’’-{[({6,6’,6’’-[(1,4,7-triazacyclononane-
1,4,7-triyl)tris(methylene)]tris{2-[hydroxy(methyl)phosphoryl]-
pyridine-6,4-diyl}}tris(ethyne-2,1-diyl))tris(3,5-dimethylbenzene-
4,1-diyl)]tris(oxy)}triacetate [EuL2]: Compound 12 was dissolved
in a solution of MeOD (1.5 mL), and a solution of NaOD in D2O
(0.5 mL, 0.1m) was added to reach pH 12. The solution was heated
at 608C overnight, and hydrolysis of ester groups was monitored
by 31P NMR. 31P{1H} NMR (298 K, 162 MHz, MeOD/D2O): dP =
26.5 ppm. After completion, solvents were removed under reduced
pressure and the pH was adjusted to 7 by addition of a 1m HCl so-
lution. EuCl3·6H20 (2.75 mg, 7.44 mmol) was added and the solution
was stirred at 608C for 12 h. After this time, the solution was
evaporated to yield the crude EuIII complex as a yellow solid,
bright red under UV light. The complex was purified by HPLC in
ammonium bicarbonate buffer (25 mm) to give the ammonium
salt of the europium complex (2.5 mg, 24%) as a pale-yellow
powder, after freeze-drying. 31P{1H} NMR (298 K, 162 MHz, MeOD):
dP = +39.3; (HRMSÀ): m/z: 1397.3254 [MÀH]À (C63H59D6N6O15P3151Eu
requires 1397.3309); HPLC (ammonium bicarbonate buffer, 25 mm,
gradient: 10–100% CH3CN in buffer over 10 min): tr =6.0 min;
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tH
bicarbonate) =0.81 ms; tD O =1.12 ms; tMeOH =1.25 ms; q=
2
2O(ammonium
0; lmax =340 nm; e340 nm =67100mÀ1 cmÀ1
; fH O =16%, fMeOH =
2
52%.
Europium complex of 2,2’,2’’-{[({6,6’,6’’-[(1,4,7-triazacyclononane-
1,4,7-triyl)tris(methylene)]tris{2-[hydroxy(methyl)phosphoryl]-
pyridine-6,4-diyl}}tris(ethyne-2,1-diyl))tris(3,5-dimethylbenzene-
4,1-diyl)]tris(oxy)triacetamido}propane-1-sulfonate [EuL4]: The
complex [EuL2] (0.9 mg, 0.65 mmol) and homotaurine (0.55 mg,
3.90 mmol) were dissolved in DMSO (0.2 mL) and water (20 mL).
HATU (2.03 mg, 3.90 mmol) and DIEA (1.26 mL, 6.50 mmol) were
added to the reaction and the mixture was stirred for 24 h under
argon after which time HPLC and LCMS showed complete conver-
sion. Purification by HPLC gave the ammonium salt of the [EuL4]
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½MÀ3 HŠ
(ESÀ, C72H81D6EuN9O21P3S3): m/z: 586.130 (100%,
3À), 879.392
3
(41%,
2À), 1760.163 (3%, [MÀH]À); HPLC (ammonium bicar-
½MÀ2 HŠ
2
bonate buffer, 25 mm): tr =6.3 min; tH (ammonium bicarbonate) =1.07 ms;
2O
tD O =1.24 ms; tMeOH =1.34 ms; q=0; lmax =340 nm; e352 nm
=
2
67100mÀ1 cmÀ1; fH O =34%, fMeOH =76%.
2
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Published online on December 16, 2015
Chem. Eur. J. 2016, 22, 570 – 580
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ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim