Synthesis and Evaluation of Europium Complexes that Switch on Luminescence in Lysosomes of Living Cells

Article abstract of DOI:10.1002/chem.202003992

A set of four luminescent Eu^III complexes bearing an extended aryl-alkynylpyridine chromophore has been studied, showing very different pH-dependent behaviour in their absorption and emission spectral response. For two complexes with pK_a

Full text of DOI:10.1002/chem.202003992

Accepted Article
Accepted Article
Title: Synthesis and Evaluation of Europium Complexes that Switch On
Luminescence in Lysosomes of Living Cells
Authors: David Parker, Jack D. Fradgley, Jurriaan M. Zwier, Matthieu
Starck, Robert Pal, and Laurent Lamarque
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To be cited as: Chem. Eur. J. 10.1002/chem.202003992
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01/2020

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Synthesis and Evaluation of Europium Complexes that Switch On
Luminescence in Lysosomes of Living Cells
Matthieu Starck,*[a] Jack D. Fradgley,[a] Robert Pal,[a] Jurriaan M. Zwier,[b] Laurent Lamarque,[b]
and David Parker*[a]
[a]
Dr. Matthieu Starck, Mr. Jack D. Fradgley, Dr. Robert Pal and Pr. David Parker
Department of Chemistry
Durham University
South Road, Durham, DH1 3LE, UK
E-mail: david.parker@dur.ac.uk
starck.matthieu@gmail.com
[b]
Dr. Laurent Lamarque, Dr. Jurriaan Zwier
Research and Development Cisbio Bioassays
BP 84175, 30200 Codolet, France
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.xxxxxxxxx
Abstract: A set of four luminescent Eu(III) complexes bearing an
extended aryl-alkynylpyridine chromophore has been studied,
showing very different pH-dependent behaviour in their absorption
and emission spectral response. For two complexes with pKa values
of 6.45 and 6.20 in protein containing solution, the emission lifetime
increases very significantly following protonation. By varying the gate
time during signal acquisition, the ‘switch-on’ intensity ratio could be
optimised and enhancement factors of between 250 to 1330 were
measured between pH 8 and 4. The best behaved probe showed no
significant emission dependence on the concentration of
endogenous cations, reductants and serum albumin. It was
examined in live cell imaging studies to monitor time-dependent
lysosomal acidification, for which the increase in observed image
brightness due to acidification was a factor of 50 in NIH-3T3 cells.
A common feature of the ageing process of endosomes and
phagosomes is that their pH tends to fall with time over the cell
cycle period, and endosomes may finally evolve into lysosomes.
Thus, whilst cytosolic pH is around 7.2, endosomal pH ranges
from 6.5 to 5.5 and the lowest pH is to be found in mature
lysosomes and is typically around 4.5. The processes of
internalisation and endosomal uptake can therefore be followed
in time if the species being internalized (receptor or substrate) is
for instance labelled with a luminescent pH sensitive dye whose
emission intensity or lifetime varies with pH. Ideally, the
observed change should be as large as possible;[4] in the limit,
this is akin to the idea of a ‘light switch’, although this term has
sometimes been misused in the literature, and occasionally
rather modest emission intensity variations have been unduly
celebrated as ‘switch-on’ sensors. Ratiometric systems of
course offer greater scope. For this work, we set ourselves a
target of a 100% change in lifetime over the pH range 8 to 4,
and a change in the long-lived emission intensity of two orders
of magnitude. Such desirable properties require careful
consideration of the mechanism of the process leading to
emission quenching at higher pH. In addition, the selection of
the pH range determines the pKa of the emissive species
undergoing protonation. Our initial work focused on examining
systems with a pKa around 5.5 to 6.
Introduction
We introduce a series of four Eu(III) complexes whose
luminescence emission is switched on by over two orders of
magnitude during acidification, following excitation in the range
330 to 370 nm. Such complexes have utility in monitoring
acidification in living cells.
Receptor mediated endocytosis is a ubiquitous feature of
uptake into cells, following surface receptor membrane
binding.[1] It leads to the internalization of part of the cell surface
receptors together with the bound molecular species. Thus, in a
typical clathrin-mediated cell uptake process[2] with a G-protein
coupled receptor (GPCR), binding of an agonist at the receptor
site may be followed by endosomal formation. The endosome
may either be degraded, or its contents recycled, releasing the
receptor to return to the plasma membrane. Much larger
particles and species may be internalized by phagocytosis, after
the cell surface membrane is ruffled, leading to the budding-off
of a phagosome within the cell. For species of intermediate
molecular weight (MW) and for certain other low MW molecules
and a large family of lanthanide coordination complexes,
macropinocytosis may occur following invagination of the
plasma membrane after surface receptor binding.[3] The
internalised macropinosome is considered to be more leaky than
endosomes or vesicles, and discharges its contents more readily.
Chart 1. Structures of common fluorescent pH sensitive dyes.
The majority of work reported has involved an adaptation of the
molecular structure of common fluorophores, such as rhodamine,
BODIPY and various cyanine dyes, in order to tune the pKa
value and permit conjugation, (Chart 1). Several of these
examples are now commercially available. Thus, the N,N’-
trifluoroethyl rhodamine derivative (RH-PEF) has been used to
monitor antibody internalisation,[5] the core structure has a pKa
1
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around 5.1 and the intensity increase is reported to be a factor of
58 between pH 7.4 and 5.0. Related studies have used
fluorescent cyanine dye labels and an assay was developed to
track the internalisation of the HER-2 antibody,[6] while Grover[7]
has examined GPCR labelling with cyanine dyes, giving modest
intensity enhancements of a factor of 5, between pH 5 and 8.
Arguably the most promising approach has used a modified
BODIPY core, and Nagano[8] reported switching factors of up to
300 using these systems. Here, the pKa is easily tuned by
variation of the aniline substituents R1 and R2 (Chart 1): the pKa
increases from 3.8 to 6.0 in the sequence H<Me<Et, as lone pair
conjugation is increasingly disfavoured and the conjugate acid is
less strongly stabilized by solvation. Such systems were used to
examine breast cancer tissue samples and permitted the
internalisation of the antibody HER-2 receptor complex to be
followed by microscopy in mice, by labelling the
chromophore excited state, enhancing the intensity of Eu
emission by analogy with a plethora of literature examples.[4a, 13]
Such a system was not expected to show any change in the
form of either emission or excitation spectra. With the amine
group in conjugation with one of the aromatic rings, it was
reasoned that the absorption spectrum would vary with pH and
that significant Eu lifetime and intensity change was possible, as
acidification would perturb the energy of the ligand centred and
internal charge transfer transitions that exist in related examples
of these ‘EuroTracker’ complexes.[14] The nature of the
substituents in the amine moiety was selected bearing in mind
the pKa values of simple anilines in aqueous media.
Thus, the series of Eu complexes [EuL1-4] was designed and
prepared (Chart 2) and their photophysical behaviour examined,
together with preliminary studies of their localisation and time-
dependent behaviour in living cells, in the expectation that the
best examples could be taken forward as ‘core’ complexes, from
which analogues would be designed to allow conjugation, for
future internalisation studies.
immunotherapeutic agent Herceptin (Trastuzumab) with
BODIPY dye.
a
These fluorescent systems offer neither significant lifetime
modulation, nor a ratiometric response and have the inherent
issues associated with autofluorescence. Others have therefore
sought to use luminescent lanthanide labels, and benefit from
the advantages (e.g., large Stokes’ shifts, long lifetimes),
associated with time-resolved spectroscopy or microscopy
methods. Yuan,[9] for example, has reported monitoring the
change in the red/green Eu to Tb emission intensity ratio in a
modified acyclic chelate based on a terpyridine ligand, leading to
a system with a ratiometric ratio variation of a factor of 7.
Papkovsky[10] has described a rather weakly emissive Eu(III)
DTPA complex based on a carbostyril sensitizer, in which the
lifetime and emission intensity at 614 nm changed by a factor of
7 between pH 7.5 and 6.5 (pKa 6.5, exc 370 nm), allowing
extracellular acidification to be monitored for a series of high
[10b
throughput assays. In another approach by Schäferling
a
Eu(III)chelate was coupled to carboxynaphthofluorescein to
create a ratiometric pH-sensor, but this compound is unlikely to
work in a cellular environment.
Fewer time resolved studies have examined pH change
inside cells directly, where either confocal microscopy directly or
spectral imaging can be employed, observing lifetime or
emission intensity modulation. Using kinetically stable
macrocyclic ligand systems based on triazacyclononane,
McMahon[11] devised Eu(III) complexes that permitted monitoring
of the pH of the endoplasmic reticulum in live cells, based on
emission lifetime variation; a 75% change in the europium
lifetime was observed over the pH range 6.5 to 7.5. Earlier,
Smith[12] had described ratiometric methods that allow the real-
time monitoring of lysosomal pH, using confocal microscopy,
based on analysis of the Eu/Tb emission intensity ratio, that
varied from 1.3 at pH 4.6 to 2.9 at pH 6.5 (exc 355 nm).
None of these lanthanide systems permits the conjugation of
the emissive probe to a targeting vector or protein. Such an
approach is straightforward to devise and simply requires a
suitable linkage site to be introduced into the ligand structure.
Accordingly, we set out to compare the pH response behaviour
of a set of four luminescent Eu(III) complexes, in which the site
of protonation was either in partial conjugation with the
sensitising chromophore, [EuL3,4], or where a methylene spacer
insulates the amine site, [EuL1,2]. In the latter case, it was
reasoned that acidification would lead to suppression of photo-
induced electron transfer, from the nitrogen lone pair to the
Chart 2. The europium(III) complexes discussed in this work.
Results and Discussion
Synthesis of complexes. The synthesis of the target
complexes [EuL1] and [EuL3] was undertaken as shown in
Schemes 1 and 2. The structurally related complexes, [EuL2]
and [EuL4], were made using similar methods and details are
given in the SI.
Mono-alkylation of N-methylaniline with the substituted
benzylic bromide, 1, gave the tertiary amine 2, which was
coupled with trimethylsilylacetylene under palladium catalysis to
afford the alkyne 3, in 98% yield. Following silyl deprotection
with fluoride in THF, the terminal alkyne 4 was coupled with the
4-iodo-pyridine derivative 5,[15] in a copper(I) assisted reaction to
2
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give the conjugated alkyne 6. Mesylation in THF afforded the
sulfonate ester, 7, which was used directly to alkylate
triazacyclononane in acetonitrile to yield the C3 symmetric ligand
precursor, 8. Following base hydrolysis, [EuL1] was prepared by
reaction of the ligand with EuCl3 at ambient pH, and the neutral
complex was purified by reverse phase HPLC, (Scheme 1).
In the case of [EuL3], the electron rich chromophore was
introduced in the final alkylation step to avoid undue exposure to
strong acid conditions during its preparation. The intermediate
N,N-diethyl substituted terminal alkyne, 15, was made from 3-
amino-4-methoxy iodobenzene (Scheme 2) by a similar series of
standard alkylation, coupling and deprotection steps, prior to a
Sonagashira coupling reaction with the 4-bromopyridine
derivative, 9, that has been reported earlier.[16] The resultant di-
substituted alkyne 16 was mesylated using methanesulfonic
anhydride in THF in the presence of Hunig’s base, to avoid the
presence of chloride anion which can lead to unwanted
displacement of the mesylate group, under certain conditions. In
parallel, the diester, 20, was prepared from the mesylate 18[17]
and mono-BOC-triazacyclononane 19,[18] and the Boc group
removed by treatment with TFA. The resultant secondary amine
21, was alkylated with the mesylate of the conjugated
chromophore 17, and the triethyl ester 22, was hydrolysed in
methanolic aqueous base followed by complexation with
europium at ambient pH (Scheme 2). The desired charge neutral
complex, [EuL3] was purified by reverse phase HPLC.
Scheme 1. Synthesis of [EuL1].
Scheme 2. Synthesis of [EuL3].
3
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pH Dependence of Europium Luminescence. The pH
dependence of the absorption, emission and excitation spectra
for each complex was examined, together with measurements of
the europium emission lifetime (Table 1). Considering first the
complex [EuL3], as it contains only one site of protonation at
nitrogen. Protonation shifts the position of the main *
transition from 340 nm to 320 nm, characterised by the
occurrence of two isosbestic points at 331 and 278 nm, (Figure
1). The Eu emission intensity increased markedly as the pH was
lowered from 10 to 4 (Figure 2) and was accompanied by an
increase in lifetime from 0.45 ms to 1.16 ms. Overall emission
quantum yields measured at pH 4 and pH 8 were 17% and 0.5%
respectively. By fitting the variation of the Eu emission intensity
or its lifetime with pH, a pKa value was estimated by non-linear
least squares regression analysis. A value of 6.75 (±0.05) was
found (Table 2, 295 K, 0.1 M NaCl). Identical behaviour was
found for both acidimetric and alkalimetric titrations consistent
with full reversibility.
Similar overall behaviour was found in the pH dependence of
[EuL4], for which more complex absorption spectral changes
occurred (SI). In this case, the emission quantum yield
increased from 0.1 to 17% from pH 8 to 4, and the lifetime
increased from 0.24 to 0.84 ms (Figures 3 and 4). The form of
the Eu emission spectrum for [EuL3] and [EuL4] was more or
less unchanged, with only minor changes observed in the
hypersensitive J = 2 and 4 transitions, e.g. a switching in the
relative intensity of the two main bands in the region between
610 to 625 nm.
Figure 3. Absorption, excitation and metal-based emission spectra for [EuL4],
(exc 331 nm, 295 K, ccomplex = 15 µM, 0.1 M NaCl), showing the highest energy
transitions from the 5D0 excited state.
Figure 1. Variation of the absorption spectrum of [EuL3] with pH revealing the
isosbestic points at 278 and 331 nm (295 K, ccomplex = 15 µM, 0.1 M NaCl).
Figure 4. Variation of the europium emission spectrum and lifetime with pH for
[EuL4], (exc 331 nm, 295 K, ccomplex = 15 µM, 0.1 M NaCl) showing the fit (red)
to the data points. Similar plots were obtained for excitation at 337 and 355 nm,
or using emission intensity variations.
The variation of lifetime with pH allowed an apparent pKa to
be measured for [EuL4] (Table 2). In this case, three successive
protonation steps occur, but the data was fitted to a single
protonation event. The sites of protonation are separated by at
least 15 Å, and so can be considered to act independently. The
apparent pKa was 6.21 (± 0.05) in this case, and it did not
change when 0.1% bovine serum albumin solution was present,
suggesting little interaction with this common serum protein.
However, in a cell lysate background, the pKa fell to 5.75.
Figure 2. Variation of the europium emission spectrum and lifetime with pH for
[EuL3], (exc 331 nm, 295 K, ccomplex = 15 µM, 0.1 M NaCl) showing the fit (line)
to the experimental data. Similar plots were obtained for excitation at 337 or
355 nm.
4
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The Eu complexes of L1 and L2 are sparingly soluble in
water, and slowly came out of solution on standing at higher pH,
with evidence for pH induced aggregation (SI Figure S3 and S5).
However, each complex dissolved readily in methanol, in which
they were strongly emissive, with overall photoluminescence
quantum yields of 24 and 48% respectively for the protonated
species. In water, quantum yields were lower (4 and 12% at pH
7; 12 and 19% at pH 4), as found for related EuroTrackerTM
probes that also possess a strong internal charge transfer
transition.[14a, 14b, 19] The europium emission lifetime at pH 7 was
0.70 ms for each complex, and reduced by only 10 and 14%
respectively, between pH 8 and 4. At the same time, the Eu
emission intensity showed an increase in intensity: for [EuL1],
the intensity increase was 330% from pH 8 to 4, and for [EuL2]
the corresponding change was 55%. Such behaviour accords
with predominant suppression of photoinduced electron transfer
of an intermediate ligand based excited state, following complex
protonation. Given the inferior solubility and pH dependent
switching properties of [EuL1,2], further studies focused on the
properties of the complexes of L3 and L4, and [EuL4] was
selected for particular attention by virtue of its more favourable
pKa value, with a view to its use in cellulo.
emission intensities were measured in each case. By changing
the delay time from 0.06 ms to 1 ms, the ‘switch-on’ factor
increased from 250 to 650, with an apparent pKa of 5.7 (± 0.1)
under these different sets of experimental conditions. By
choosing different time windows, a slightly greater increase in
this factor was found, with the maximum ratio found for the time
window 1.5 to 2.5 ms, notwithstanding the reduction in overall
signal intensity that occurs using an acquisition period that is
rather longer than the emission lifetime. Such behaviour augurs
well for such conditions to be employed in either a time-gated
assay, or in future time-resolved microscopy studies.
Table 3. Ratios of emission intensities (‘switch-on’ factors) for the stated single
gate delay times (upper) or differing time gate periods (lower), showing the
effect on the apparent pKa values (295 K, c = 15 µM, 0.1 M NaCl; buffers,
NH4OAc (0.1 M, pH 4), NH4HCO3, (0.1 M, pH 8).
60 µs
460 µs
1000 µs
pH 4 / pH 8
250
5.8
623
5.7
650
5.6
App. pKa
60 – 460 µs
1000 – 2000 µs
1500 – 2500 µs
Table 1. Summary of Photophysical Properties for Eu(III) Complexes (295 K,
ccomplexes = 15 µM in 0.1 M NaCl).
pH 4 / pH 8
111
5.8
560
5.7
1334
5.6
App. pKa
Complex

exc (nm)
ε (M-1.cm-1)
 (ms)
 (%)
q
B (M-1.cm-1)
8100[a]
0.70[a]
0.65[b]
0.65[a]
0.60[b]
0.53[a]
1.16[b]
0.25[a]
0.84[b]
4.0[a]
324[a]
[EuL1]
[EuL2]
[EuL3]
[EuL4]
324
340
331
331
0
0
0
0
26000[b]
21000[a]
34200[b]
12.0[b]
12.0[a]
18.7[b]
0.5[a]
3120[b]
2520[a]
6498b]
60[a]
12000[c]
17.0[b]
0.1[a]
17.0[b]
2040[b]
46[a]
10200[b]
46000[a]
60000[b]
[a] Values at pH = 8. [b] Values at pH = 4. [c] Values at the isosbestic point. [d]
experimental errors on lifetime are ± 5% and for quantum yields, ±15%.
Table 2. Summary of pKa values (± 0.05) determined for [EuL1-4] (ccomplexes = 15
µM) in the stated media.
Complex
Conditions
pKa
Figure 5. Emission intensity of [EuL4] (em 613 nm) as a function of pH with
different time periods of acquisition (blue = 60 – 460 µs, red = 1000 – 2000 µs,
green = 1500 – 2500 µs). Data are normalised to a 60 – 460 µs time window
at pH 4. Measurements were taken in aqueous solutions of NH4OAc (pH 4 and
5), MES (pH 5.5, 6 and 6.5), HEPES (pH 7) and NH4HCO3 (pH 8) buffers
(ccomplex = 15 µM, 0.1 M buffer in 0.1 M NaCl).
[EuL1]
[EuL2]
[EuL3]
0.1 M NaCl
0.1 M NaCl
n.d. (see SI)
6.48
0.1 M NaCl
0.1 M NaCl, 0.1 mM BSA
6.75
6.45
[EuL4]
0.1 M NaCl
0.1 NaCl, 0.1 mM BSA
NIH-3T3 Cell Lysate
6.21
6.20
5.75
Cell Imaging Studies and Control Experiments. Cell
uptake and co-localisation studies were undertaken for [EuL4] in
living mouse skin fibroblasts (NIH-3T3) and human breast
cancer cells (MCF-7), following methods previously reported.[3b,
14c, 19] The extent of complex uptake was monitored by assessing
image brightness as a function of time. In parallel, complex
concentrations at fixed time points were measured by analysing
the Eu concentration in these cell samples, using ICP-mass
spectrometry, (Fig S11). Co-staining experiments using
Lysotracker Green, (Figure 6), verified that the complex was
localised in the lysosomes, with higher correlation coefficients as
The large increase of the Eu emission lifetime that occurs on
protonation with [EuL4] allows the apparent switching ratio
between the ‘on’ and ‘off’ states to be varied, by changing the
time delay in signal acquisition. Two cases were investigated in
which either a fixed time delay was introduced, or a different
time window for spectral acquisition was selected, (Table 3 and
Figure 5). The emission intensities were measured in buffered
solution at pH 4 and pH 8, and the ratios of the overall europium
probe brightness increased, within
a
given cell cycle.
5
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Figure 6. Live cell images (NIH-3T3) with [EuL4] after 16 h. (Left) [EuL4] (ccomplex = 30 µM, exc = 355 nm, em = 600-720 nm); (Centre) Lysotracker Green (exc
488 nm, em = 500-530 nm); (Right) Overlay shows co-localisation (P = 0.95). Note the observation of normal cell division at 16 h.
=
Figure 7. Brightness profile of [EuL4] as a function of time for incubation concentrations of 30 µM (black) and 3 µM (red) in living NIH-3T3 cells; images shown are
for 30 µM. Scale bar is 20 µm. The cells were monitored for 72 h, when the cells were proliferating normally.
6
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The microscopy study revealed a time dependent increase in
brightness that maximised at around 16 to 18 hours. Such
behaviour coincides with the time period of mitosis for NIH-3T3
cells; during unaltered proliferation, they divide every 16 hours.
In a separate study, cell toxicity was assessed using live/dead
assay image cytometry (SI), and no evidence (± 5%) for
perturbation of natural cell homeostatic function and proliferation
was found over a period of 24 hours, for concentrations of up to
100 M.
A series of control experiments in vitro was also undertaken,
in order to verify that the changes in image brightness in the cell
imaging work were not due to any excited state quenching by
common endogenous species whose concentration could
hypothetically change in the same time period in the given
organelles. Accordingly, the Eu lifetime and observed emission
intensity were measured for [EuL4] in the presence of increasing
concentrations of different species that are likely to be present
inside the cell compartment. No significant changes in lifetime or
intensity were observed following addition of up to 5 equivalents
(corresponding to a limiting 150 micromolar concentration) of Mg,
Ca and Zn ions. Similarly, no changes were found, within
experimental error, for addition of the endogenous reductants
ascorbate, urate and glutathione that can potentially quench the
probe excited state by electron transfer. Finally, no major
After cell division, the daughter cells appeared less bright, in
line with the increase in cell volume. Each daughter cell
possesses about 50% of the internalised complex of the parent
cell, as they begin their independent life cycle. The
concentration of complex found inside the cell then increases
again, as the daughter cells mature and embark on homeostatic
uptake of nutrients and [EuL4] present in the cell growth medium. variations (± 10%) were found for added bovine serum albumin
However, this process has a time lag, resulting in the observed
time vs brightness profile, (Figure 7). Similar overall behaviour
was found in a separate study where the concentration of the Eu
complex was 10 times lower.
that is present in the cell incubation medium. The insensitivity to
change compared to the direct dependence on local pH is
highlighted below, (Figure 9).
In
complex, -[EuL5], that shows no pH dependent behaviour and
predominant lysosomal staining pattern, very different
a control experiment with a structurally analogous
a
behaviour was observed.[20] In this case (Figure 8), under the
same incubation conditions in NIH-3T3 cells, the mean image
brightness reached a plateau after 4 h, and remained constant
thereafter, with a minor inflection around 16-18 h. Such a kinetic
intensity profile is characteristic of the EuroTracker series of Eu
complexes, studied in depth for several living cells.[14, 19]
Figure 9. Total change in emission lifetime (blue) and emission intensity (red)
following addition of 5 equivalents of analyte to [EuL4] (ccomplex = 30 µM, 0.1 M
NaCl, 0.1 M NH4HCO3 buffer, pH 5) relative to the pH change observed from 8
to 4 (normalised to unity). Lifetime and intensity data are given in Figure S8.
The time dependent increase in brightness in the cell
experiments with [EuL4] in theory can be attributed both to
changes in the extent of uptake, (likely proceeding via
macropinocytosis as found in related studies),[3b, 20] and the pH
drop in the ageing endosomes as they transform into lysosomes.
Control experiments measuring the intracellular concentration of
the Eu-complex directly using ICP-MS were therefore
undertaken at 2, 4, 8, 12, 16 and 24 hours (Figure S11). These
studies showed a significantly less steep increase, with the
intracellular Eu concentration doubling between 2 and 16 hours,
while the observed image brightness increased by a factor of 50.
Such behaviour is consistent with the idea that the
brightness enhancement is not simply due to the slow increase
in the total amount of complex internalized, but directly relates to
the effect of diminishing pH in the ageing endosomes/lysosomes,
with the associated large increase of Eu emission intensity as
the percentage of the more emissive protonated complex
increases. This hypothesis is also consistent with the observed
time dependent increase in Pearson’s co-localisation coefficient
Figure 8. Brightness profile of -[EuL5] as a function of time in live NIH-3T3
cells; images shown are for a 30 µM incubation. Scale bar is 20 µm. The cells
were monitored for 48 h, and were proliferating normally.
7
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(P), in the parallel microscopy study using LysoTracker Green,
(Figure 7).
The complex [EuL4] was examined in two different cell lines
and was found to localise mainly in the cellular lysosomes,
without compromising cellular viability or proliferation. Using
laser scanning confocal microscopy, exciting the complex at 355
nm, an increase in probe brightness was observed in the living
cells as a function of time, over the period 2 h to 16 h. The
observed brightness was 25 times greater than the change
associated with the independently measured increase in probe
uptake. In control experiments with a related complex, [EuL5],
that exhibits no dependence of emission on pH, very different
behaviour was observed and no significant changes in image
brightness were observed beyond the dependence on the extent
of uptake. Taken together, such behaviour is consistent with the
monitoring of acidification of the cell’s lysosomes as they age
using this pH sensitive probe. Given that the cells were healthy,
cell division occurred after 16-18 h and after a dip in probe
brightness, the daughter cells continued to take up the complex
and their lysosomes were subsequently observed to reach
maturation.
After a separate 16 hours incubation in NIH-3T3 cells with
[EuL4] (30 M), nigericin (200 nM) was added. This is an agent
that has been shown to increase lysosomal pH quickly, from ca.
4.5 to 6.5. After 5 minutes, confocal microscopy studies showed
that the Eu complex appeared to be considerably less bright
overall, consistent with the increased size of the treated cell’s
lysosomes and the reduced probe brightness at the higher pH
value, (Figure S9). In an additional control experiment, the
internalised complex concentration of both treated and untreated
cells, for a given time incubation time point, was found to be
identical within experimental error, using ICP-MS. Parallel
results were observed, in terms of localisation behaviour and
time-dependent brightness change, using the MCF-7 breast
cancer cell line. An MTT assay assessment was also
undertaken, with no evidence found for cellular toxicity with
[EuL4] monitoring mitochondrial redox status for 24 h over the
concentration range 10 to 100 M (Figure S10).
It should be noted in a word of caution, that the limitations of
the experimental approach where additions of agents that
greatly perturb cellular permeability have been noted.[3b, 21] The
use of the fluorescent LysoSensor probe for example, has been
advocated for lysosomal pH assessment, but it is only useful for
very short perturbational periods, as it induces changes itself on
the local pH and is only applicable to imaging studies carried out
over a few minutes,[21a] or in dead (fixed) cells. Studies with fixed
cells have very obvious limitations with respect to the monitoring
of living processes. The observations reported here, of the
continuous monitoring of cellular acidification over a period of up
to 24 h in live cells, have no obvious precedent for a pH
sensitive phosphorescent probe that has no apparent effect on
cellular homeostasis.
Thus, the complex [EuL4] is well suited as the basis of probe
design for new systems that can be linked to targeting vectors
and antibodies to monitor receptor internalization. Such new
derivatives can be used to tag proteins selectively, to monitor
the time-dependence of uptake into more acidic organelles and
their recycling, in order to enhance the performance of current
state-of-the-art, time-resolved internalisation assays.[22] Such
studies have been undertaken and the promising behaviour of
these pH sensitive probes will be reported in due course.
Experimental Section
Details of general experimental methods, instrumentation, cell
microscopy and the photophysical techniques used are given in the
supplementary information. Methyl 6-(hydroxymethyl)-4-iodopicolinate
5,[15] 4-bromo-2-hydroxymethyl-6-methylphosphinate pyridine 9,[16] (6-
(ethoxy(methyl)phosphoryl)pyridin-2-yl)methyl methanesulfonate) 18,[17]
and tert-butyl 1,4,7-triazacyclonane-1-carboxylate 19,[18] were prepared
using reported procedures. Details of the syntheses of [EuL2] and [EuL4]
are given in the SI, together with spectral data for ligand intermediates,
additional spectral information for each complex, and LC-MS data for the
Eu complexes.
Summary and Conclusion
Four new luminescent Eu(III) complexes have been
prepared and evaluated in a series of comparative studies,
assessing their absorption and emission spectral behaviour as a
function of pH. The Eu complexes of L1 and L2, where the site of
protonation is separated from the main chromophore by a
methylene group, showed no significant variation in absorption
spectral form and exhibited the smallest changes in emission
intensity and lifetime. In contrast, the protonation of complexes
of L3 and L4, where the amine group is conjugated into the
phenyl ring, caused a hypsochromic shift in the main absorption
band and an increase in the Eu lifetime of 120 and 240%
respectively. By varying the delay time or the time period for
acquisition of Eu emission spectral intensity data, intensity ratios
of between 250 and 1300 were measured for [EuL4], comparing
pH 4 and pH 8 samples. The Eu emission lifetime and intensity
was more or less invariant to the concentration of Ca, Mg and
Zn ions, to the natural electron rich reductants ascorbate, urate
and glutathione and to the protein BSA, each of which may
potentially perturb the behaviour of a probe luminophore in the
living cell. Such large pH induced ‘switching on’ factors are well
suited to assays where acidification needs to be monitored, as
occurs during the maturation of endosomes or in the ageing of
lysosomes, following uptake of a labelled protein or antibody.
4-Bromo-2-(bromomethyl)-1-methoxybenzene, 1. To a solution of (5-
bromo-2-methoxy-benzyl alcohol (623 mg, 2.87 mmol) dissolved in
anhydrous DMF (15 mL) under argon was added PBr5 (1.85 g, 4.32
mmol). The solution was stirred at 50 °C for 18 h under argon. Solvent
was removed under reduced pressure to yield an orange oil. The residue
was dissolved in CH2Cl2 (20 ml) and water (20 ml) was added. The
aqueous layer was extracted with CH2Cl2 (3 × 10 mL), and the combined
organic layers were dried over Na2SO4, filtered and concentrated to
dryness to yield
a light yellow oil that was purified by column
chromatography (SiO2, CH2Cl2 100%) to yield a white solid (740 mg,
92%); Rf = 0.76 (SiO2, 100% CH2Cl2); m.p. 114-116 °C; 1H NMR (298 K,
400 MHz, CDCl3) H 7.47 (1H, d, J = 2.5 Hz), 7.40 (1H, dd, J = 9.0 Hz, J
= 2.5 Hz), 6.78 (1H, d, J = 9.0 Hz), 4.48 (2H, s), 3.88 (3H, s); 13C NMR
(298 K, 100 MHz, CDCl3) C 156.6, 133.6, 132.8, 128.3, 112.8, 112.7,
56.0, 27.7; (GCMS) tr. = 4.46 min, m/z 279.950 [M+], 199.000 [M-Br]+.,
elemental analysis calculated for C8H8Br2O (%): C = 34.32, H = 2.88;
found: C = 34.09, H = 2.88.
N-(5-Bromo-2-methoxybenzyl)-N-methylaniline, 2. Compound 1 (78
mg, 0.28 mmol) was dissolved in dry CH3CN (2 mL). N-Methyl aniline (31
µL, 0.28 mmol) and K2CO3 (78 mg, 0.56 mmol) were added under argon.
The reaction was stirred at 50 °C for 24 h under argon and progress was
8
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10.1002/chem.202003992
Chemistry - A European Journal
FULL PAPER
monitored by LC/MS. The excess inorganic salts were removed by
filtration and the filtrate was evaporated to dryness. The residue was
dissolved in a CH2Cl2 / water solution (20 mL, 1:1) and the aqueous layer
was extracted with CH2Cl2 (3 × 10 mL). The combined organic layers
were dried over Na2SO4, filtered and concentrated to dryness affording a
colourless oil (80 mg, 94%). NMR analysis and LC/MS shown that
compound 2 was sufficiently pure to be used in the next step without
52.0, 38.7; (HRMS+) m/z 417.1807 [M+H]+ (C25H25N2O4 requires
417.1814).
Methyl-4-((4-methoxy-3-((methyl(phenyl)amino)methyl)phenyl)
ethynyl)-6-(((methylsulfonyl)oxy) methyl)picolinate, 7. The alcohol 6
(30 mg, 72 µmol) was dissolved in anhydrous THF (1 mL) under argon.
Mesyl chloride (8 µL, 108 µmol) and NEt3 (34 µL, 252 µmol) were added
under argon and the reaction was stirred at ambient temperature for 2 h.
After this time, solvent was removed under reduced pressure and the
residue was dissolved in CH2Cl2/water (1:1, 20 mL). The aqueous layer
was extracted with CH2Cl2 (3 × 10 mL). The combined organic layers
were dried over Na2SO4, filtered and concentrated to dryness to yield a
pale pink oil (35.7 mg, quant) that was sufficiently pure to be used for the
next step without further purification; 1H NMR (298 K, 400 MHz, CDCl3)
1
further purification; Rf = 0.90 (SiO2, 100% CH2Cl2); H NMR (298 K, 400
MHz, CDCl3) H 7.42 (1H, d, J = 2.5 Hz), 7.36 – 7.28 (3H, m), 6.90 – 6.76
(4H, m), 4.57 (2H, s), 3.92 (3H, s), 3.13 (3H, s); 13C NMR (298 K, 100
MHz, CDCl3) C 156.2, 149.6, 130.4, 129.8, 129.3, 129.2, 116.6, 113.1,
112.1, 111.8, 55.5, 52.0, 38.7; (HRMS+) m/z 306.0490 [M+H]+
(C15H17NOBr requires 306.0494).
N-(2-Methoxy-5-((trimethylsilyl)ethynyl)benzyl)-N-methylaniline,
3.
H 8.15 (1H, d, J = 2.0 Hz), 7.70 (1H, d, J = 2.0 Hz), 7.51 (1H, dd, J = 8.0
Compound 2 (105 mg, 0.34 mmol) was dissolved in dry THF (3 mL)
followed by addition of Pd(PPh3)2Cl2 (35 mg, 0.05 mmol), TMS-acetylene
(250 µL, 1.7 mmol) and pyrrolidine (168 µL, 2 mmol) under argon. The
reaction was stirred at 50 °C for 48 h under argon. After LC/MS analysis
indicated reaction completion, solvent was removed under reduced
pressure and the residual dark brown oil was dissolved in CH2Cl2/water
(40 mL, 1:1). The aqueous layer was extracted with CH2Cl2 (3 × 10 mL)
and the combined organic layers were dried over Na2SO4, filtered and
concentrated to dryness to give a crude dark brown oil that was purified
by column chromatography (SiO2, hexane to EtOAc 2% in hexane) to
afford a yellow oil (108 mg, 98%); Rf = 0.32 (SiO2, 2% EtOAc in Hexane);
1H NMR (298 K, 400 MHz, CDCl3) H 7.40 (1H, dd, J = 8.0 Hz, J = 2.0
Hz), 7.28 – 7.21 (3H, m), 6.83 (1H, d, J = 8.0 Hz), 6.78 – 6.68 (3H, m),
4.46 (2H, s), 3.88 (3H, s), 3.05 (3H, s), 0.23 (9H, s); 13C NMR (298 K,
100 MHz, CDCl3) C 157.4, 149.8, 132.3, 130.6, 129.1, 127.0, 116.3,
115.1, 112.1, 109.8, 105.5, 92.3, 55.4, 52.2, 38.5, 0.1; (HRMS+) m/z
324.1779 [M+H]+ (C20H26NOSi requires 324.1784).
Hz, J = 2 Hz), 7.35 (1H, d, J = 2.0 Hz), 7.26 (2H, td, J = 7.0, Hz, J2 = 2
Hz), 6.93 (1H, d, J = 8.0 Hz), 6.79 – 6.69 (3H, m), 5.41 (2H, s), 4.51 (2H,
s), 4.02 (3H, s), 3.94 (3H, s), 3.17 (3H, s), 3.11 (3H, s). UPLC (ES+ MS,
CH3CN/H2O, 0.1% formic acid) tR = 3.40 min, m/z 496.517 (69%, [M+H]+),
989.170 (100%, [2M]+).
Trimethyl-6,6',6''-((1,4,7-triazacyclononane-1,4,7-triyl)tris(methylene)
)tris(4-((4-methoxy-3-((methyl(phenyl)amino)methyl)phenyl)ethynyl)
picolinate), 8. 1,4,7-Triazacyclononane trihydrochloride (5.7 mg, 24
µmol) and the mesylate 7 (35.7 mg, 72 µmol) were dissolved in
anhydrous CH3CN (2 mL) and K2CO3 (20.0 mg, 144 µmol) was added.
The mixture was stirred at 60 °C for 24 h under argon. The excess
potassium salts were removed by filtration and the filtrate was
concentrated to dryness to yield an orange oil (27 mg, 85%). This
compound was used directly in the next step without further purification;
1H NMR (298 K, 400 MHz, CDCl3) H 8.01 (3H, d, J = 2.0 Hz), 7.82 (3H,
br. s), 7.45 (3H, dd, J1 = 8.0 Hz, J = 2 Hz), 7.31 (1H, d, J = 2.0 Hz), 7.21
(6H, td, J = 7.0, Hz, J = 2 Hz), 6.89 (3H, d, J = 8.0 Hz), 6.71 – 6.68 (9H,
m), 4.48 (6H, s), 3.96 (6H, s), 3.92 (18H, s), 3.05 (9H, s), 2.92 (12H, br.
s); 13C NMR (298 K, 100 MHz, CDCl3) C 165.5, 158.1, 149.6, 147.3,
132.3, 132.0, 130.6, 129.1, 128.6, 128.5, 127.9, 127.5, 116.4, 113.8,
112.0, 110.1, 95.7, 85.3, 64.1, 55.5, 52.9, 52.1, 38.6, 31.9; UPLC
(CH3CN/H2O, 0.1% formic acid) tR = 3.59 min; (HRMS+) m/z 1324.622
[M+H]+ (C81H82N9O9 requires 1324.624).
N-(5-Ethynyl-2-methoxybenzyl)-N-methylaniline, 4. Compound 3 (335
mg, 1.04 mmol) was dissolved in dry THF (4.5 mL) before NEt3.3HF (4.5
mL, 24 mmol) was added. The solution was stirred at 30 °C for 24 h
under argon, when solvent was removed under reduced pressure and
the residual oil was dissolved in CH2Cl2/water (40 mL, 1:1). The aqueous
layer was extracted with CH2Cl2 (3 × 20 mL) and the combined organic
layers were dried over Na2SO4, filtered and concentrated to dryness to
yield a light orange oil. The residual oil was purified by column
chromatography (SiO2, hexane to 2% EtOAc in hexane) to yield a light
yellow oil (255 mg, 98%); Rf = 0.25 (SiO2, 2% EtOAc in hexane); 1H NMR
(298 K, 400 MHz, CDCl3) H 7.42 (1H, dd, J = 8.0 Hz, J = 2.0 Hz), 7.28 –
7.21 (3H, m), 6.85 (1H, d, J = 8.0 Hz), 6.78 – 6.68 (3H, m), 4.49 (2H, s),
3.90 (3H, s), 3.07 (3H, s), 2.96 (1H, s); 13C NMR (298 K, 100 MHz,
CDCl3) C 157.6, 149.6, 132.3, 130.9, 129.2, 127.2, 116.4, 114.0, 112.1,
109.9, 84.0, 75.7, 55.4, 52.0, 38.6; (HRMS+) m/z 252.1383 [M+H]+
(C17H18NO requires 252.1388).
[EuL1]. Compound 8 (27 mg, 20.4 µmol) was dissolved in a solution of
CH3OH (1 mL), and a solution of aqueous sodium hydroxide (1 mL, 0.1
M) was added to reach pH = 12. The solution was heated at 60 °C
overnight, before the pH was adjusted to 7 by addition of a 1 M HCl
solution. EuCl3.6H2O (8.5 mg, 23 µmol) was added and the solution was
stirred at 60 °C for 12 h. After this time, the solution was removed under
reduced pressure to yield the crude Eu(III) complex as a yellow solid
(bright red under long wave U.V. light). The complex was purified by
reverse phase HPLC (CH3CN/25 mM ammonium bicarbonate buffer) to
give
a yellow powder (4 mg, 14%) after freeze drying; UPLC
Methyl-6-(hydroxymethyl)-4-((4-methoxy-3-((methyl(phenyl)amino)
(CH3CN/H2O, 0.1% formic acid) tR = 3.65 min; (HRMS+) m/z 1430.471
[M+H]+ (C78H73N9O9151Eu requires 1430.473);H2O = 0.70 ms (pH 7); D2O
= 0.79 ms; MeOH = 0.85 ms; q = 0; max = 340nm; 340 nm = 34200 M-1.cm-
1 (pH 7); H2O = 4.0% (pH 7), MeOH = 24%.
methyl)phenyl)ethynyl)picolinate, 6.
Methyl 6-(hydroxymethyl)-4-
iodopicolinate 5 (64 mg, 0.22 mmol) was dissolved in dry THF (1 mL) and
NEt3 (150 µL, 1.1 mmol) was added. The solution was degassed 3 times
(freeze thaw cycles). Compound 4 (60 mg, 0.24 mmol) and Pd(PPh3)2Cl2
(21 mg, 0.03 mmol) were added and the solution was degassed once
more. CuI (11.5 mg, 0.06 mmol) was added and the reaction was stirred
at 60 °C for 24 h under argon. After this time, solvent was removed under
reduced pressure and the residual oil was purified by column
chromatography (SiO2, CH2Cl2 to 2% MeOH in CH2Cl2) to yield a red oil
(58 mg, 64%); Rf = 0.30 (SiO2, 2% MeOH in CH2Cl2); 1H NMR (298 K,
400 MHz, CDCl3) H 8.07 (1H, s), 7.60 (1H, s), 7.49 (1H, dd, J = 8.0 Hz, J
= 2.0 Hz), 7.33 (1H, d, J = 2 Hz), 7.28 – 7.23 (2H, m), 6.92 (1H, d, J = 8.0
Hz), 6.78 – 6.70 (3H, m), 4.86 (2H, s), 4.51 (2H, s), 4.00 (3H, s), 3.93 (3H,
s), 3.65 (1H, br. s), 3.10 (3H, s); 13C NMR (298 K, 100 MHz, CDCl3) C
165.3, 160.6, 158.3, 149.6, 146.9, 133.9, 132.3, 130.6, 129.2, 128.7,
128.5, 127.6, 116.4, 113.6, 112.0, 110.2, 96.3, 85.1, 64.5, 55.5, 53.0,
N,N-Diethyl-5-iodo-2-methoxyaniline, 13. 2-Iodo-2-methoxyaniline
(2.83 g, 8.03 mmol) was dissolved in anhydrous CH3CN (15 mL) and
iodoethane (7 mL, 87 mmol) and K2CO3 (6.3 g, 45.6 mmol) were added
under argon. The reaction mixture was heated at 60 °C for 48 h. After
total conversion of the starting material, the excess of K2CO3 was
removed by filtration and the filtrate was concentrated to dryness. The
residual oil was dissolved in CH2Cl2/water (40 mL, 1:1), and the aqueous
layer was extracted with CH2Cl2 (3 × 20 mL). The combined organic
layers were dried over Na2SO4, filtered and concentrated to dryness to
give a light yellow oil that was purified by column chromatography (SiO2,
100% CH2Cl2 to 2% MeOH in CH2Cl2) to afford a colourless oil (2.16 g,
62%); Rf = 0.18 (SiO2, 100% CH2Cl2). 1H NMR (298 K, 400 MHz, CDCl3)
9
This article is protected by copyright. All rights reserved.

10.1002/chem.202003992
Chemistry - A European Journal
FULL PAPER
H 7.26 (1H, dd, J = 8.0 Hz, J = 2.0 Hz), 7.17 (1H, d, J = 2.0 Hz), 6.60
(1H, d, J = 8.0 Hz), 3.83 (3H, s), 3.14 (4H, q, J = 7.0 Hz), 1.04 (6H, t, J =
7.0 Hz); 13C NMR (298 K, 100 MHz, CDCl3) C 153.6, 141.1, 131.1, 130.1,
113.5, 83.1, 55.5, 46.0, 12.0; (HRMS+) m/z 306.0353 [M+H]+ (C11H17NOI
requires 306.0355).
anhydrous THF (1.5 mL) under argon. The solution was subsequently
stirred at room temperature for 3 h. After this time, solvent was removed
under reduced pressure to yield a red oil which was dissolved in CH2Cl2
(10 mL). The organic layer was washed with water (3 × 10 mL), dried
over Na2SO4 and evaporated to dryness to afford the mesylate 17 (20 mg,
77%). The compound was used directly in the next step without further
purification; 1H NMR (298 K, 400 MHz, CDCl3) H 8.07 (1H, d, J = 6.0 Hz),
7.58 – 7.54 (3H, m), 6.89 (1H, d, J = 7.0 Hz), 4.86 (2H, s), 4.17 – 4.11
(1H, m), 4.00 – 3.85 (3H, m), 3.24 (4H, br. s), 3.19 (3H, s), 1.81 (3H, d, J
= 15.0 Hz), 1.30 (3H, t, J = 7.0 Hz), 1.09 (6H, t, J = 7.0 Hz), UPLC
(CH3CN/H2O, 0.1% FA) tR = 1.39 min.
N,N-Diethyl-2-methoxy-5-((trimethylsilyl)ethynyl)aniline,
14.
Compound 13 (1.58 g, 5.18 mmol) was dissolved in dry THF (10 mL)
before addition of Pd(PPh3)2Cl2 (364 mg, 0.5 mmol), TMS-acetylene
(3.80 mL, 25.9 mmol) and pyrrolidine (2.20 mL, 25.9 mmol). The reaction
was stirred at 50 °C for 24 h under argon. After LC/MS analysis indicated
reaction completion, solvent was removed and the residual dark brown
oil was dissolved in CH2Cl2/water (40 mL, 1:1). The aqueous layer was
extracted with CH2Cl2 (3 × 10 mL). The combined organic layers were
dried over Na2SO4, filtered and concentrated to dryness to give a crude
dark brown oil. This was purified by column chromatography (SiO2,
CH2Cl2 100%) to give a dark-brown oil (1.41 g, quant.); Rf = 0.65 (SiO2,
2% MeOH in CH2Cl2); 1H-NMR (700 MHz, CDCl3) H 7.12 (1H, d, J = 8.0
Hz), 7.03 (1H, s), 6.75 (1H, d, J = 8.0 Hz), 3.85 (3H, s), 3.14 (4H, q, J =
7.0 Hz), 1.01 (6H, t, J = 7.0 Hz), 0.24 (9H, s); 13C-NMR (176 MHz,
CDCl3) C 154.5, 139.1, 127.2, 125.3, 115.1, 111.2, 105.9, 91.9, 55.6,
46.1, 12.1, 0.3.; (HRMS+) m/z 276.1770 [M+H]+ (C16H26NOSi requires
276.1784).
Tert-butyl 4,7-bis((6-(ethoxy(methyl)phosphoryl)pyridin-2-yl)methyl)-
1,4,7-triazacyclonane-1-carboxylate, 20. 1-t-Butoxycarbonyl- 1,4,7-
triazacyclononane 19 (60 mg, 0.26 mmol) and freshly prepared
compound 18 (200 mg, 0.65 mmol) were dissolved in dry CH3CN (5 mL)
and K2CO3 (152 mg, 1.12 mmol) was added. The reaction mixture was
stirred at 60 °C for 12 h under argon at which point the mixture was
allowed to cool to room temperature. The excess potassium salts were
removed by filtration and the filtrate was concentrated under reduced
pressure. The residue was subsequently dissolved in CH2Cl2/water (40
mL, 1:1). The aqueous layer was extracted with CH2Cl2 (3 × 20 mL) and
the combined organic layers were dried over Na2SO4, filtered and
evaporated to dryness to yield an orange oil. The residual oil was purified
by reverse phase HPLC (CH3CN/water with 0.1% formic acid) to yield
compound 20 (88 mg, 54%) as an orange oil; 1H NMR (298 K, 700 MHz,
CDCl3) H 7.91 – 7.86 (2H, dd, J = 8.0 Hz, J = 6.0 Hz), 7.77 – 7.70 (2H,
m), 7.61 (1H, d, J = 8.0 Hz), 7.54 (1H, d, J = 8.0 Hz), 4.09 – 4.02 (2H, m),
3.92 (4H, s), 3.85 – 3.79 (2H, m), 3.43 – 2.54 (12H, m), 1.73 (3H, d, J =
15.0 Hz), 1.72 (3H, d, J = 15.0 Hz) , 1.43 (9H, s), 1.22 (2 × 3H, 2 × t, J =
7.0 Hz); 13C NMR (298 K, 100 MHz, CDCl3) C 165.0, 159.4 (d, J = 20.0
Hz), 158.9 (d, J = 20.0 Hz), 155.4, 153.8 (d, J = 150.0 Hz), 136.7 (d, J =
9.0 Hz) 126.3 (d, J = 3.0 Hz), 126.1 (d, J = 3.0 Hz), 126.0, 80.0, 62.4,
61.0 (d, J = 4.0 Hz), 60.9 (d, J = 4.0 Hz), 55.1, 54.3, 53.9, 53.4, 49.6,
48.9, 28.5, 16.4 (d, J = 6.0 Hz), 13.4 (d, J = 100.0 Hz); 31P{1H} NMR (298
N,N-Diethyl-5-ethynyl-2-methoxyaniline, 15. Compound 14 (1.41 g,
5.13 mmol) was dissolved in dry THF (10 mL), then NEt3.3HF (5.0 mL,
26.7 mmol) was added. The reaction was stirred at 30 °C for 24 h under
argon. After this time, solvent was removed and the residual oil was
dissolved in CH2Cl2/water (40 mL, 1:1). The aqueous layer was extracted
with CH2Cl2 (3 × 20 mL) and the combined organic layers were dried
over Na2SO4, filtered and concentrated to dryness to yield a light orange
oil that was purified by column chromatography (SiO2, hexane to 2%
EtOAc in hexane) to afford a light-brown oil (0.73 g, 75%); Rf = 0.65
(SiO2, 2% MeOH in CH2Cl2); 1H-NMR (700 MHz, CDCl3) H 7.14 (1H, dd,
J = 8.0 Hz, J = 2.0 Hz), 7.06 (1H, d, J = 2.0 Hz), 6.78 (1H, d, J = 8.0 Hz),
3.86 (3H, s), 3.15 (4H, q, J = 7.0 Hz), 2.98 (1H, s), 1.02 (6H, t, J = 7.0
Hz); 13C-NMR (176 MHz, CDCl3) C154.6, 139.2, 127.1, 125.3, 113.9,
111.3, 84.4, 75.3, 55.7, 46.1, 12.1; (HRMS+) m/z 204.1401 [M+H]+
(C13H18NO requires 204.1388).
K, 162 MHz, CDCl3) P 40.18, 40.01; UPLC (CH3CN/H2O, 0.1% FA) tR
=
1.47 min; (HRMS+) m/z 624.3088 [M+H]+ (C29H48N5O6P2 requires
624.3080).
Diethyl-(((1,4,7-triazacyclononane-1,4-diyl)bis(methylene))bis
(pyridine-6,2-diyl))bis(methylphosphinate), 21. The mono-carbamate
20 (88 mg, 140 µmol) was dissolved in dry CH2Cl2 (3 mL) before the
addition of trifluoroacetic acid (0.6 mL). The solution was stirred at room
temperature for 30 min after which the solvent was removed under
reduced pressure. The residue was treated with CH2Cl2 (2 mL) and the
solvent removed under reduced pressure again; this process was
repeated several times. The crude mixture was purified by reverse phase
HPLC (CH3CN/water with 0.1% formic acid) to afford a dark brown oil
(73.1 mg, quant.); 1H NMR (298 K, 400 MHz, CDCl3) H 7.86 – 7.78 (4H,
m), 7.44 (2H, d, J = 8.0 Hz), 4.19 (4H, s), 4.16 – 4.08 (2H, m), 3.94 –
3.88 (2H, m), 3.40 – 3.04 (12H, m), 1.74 (6H, d, J = 15.0 Hz), 1.28 (6H, t,
J = 7.0 Hz); 13C NMR (298 K, 100 MHz, CDCl3) C 158.0 (d, J = 20 Hz),
153.8 (d, J = 150 Hz), 137.3 (d, J = 9.0 Hz) 126.0 (d, J = 20.0 Hz), 125.8
(d, J = 2.0 Hz), 61.6 (d, J = 6.0 Hz), 60.0, 51.5, 48.9, 44.5, 16.4 (d, J =
6.0 Hz), 13.7 (d, J = 100 Hz); 31P{1H} NMR (298 K, 162 MHz, CDCl3) P
40.12; UPLC (CH3CN/H2O, 0.1% FA) tR = 1.09 min; (HRMS+) m/z
524.2557 [M+H]+ (C24H40N5O4P2 requires 524.2556).
Ethyl-(4-((3-(diethylamino)-4-methoxyphenyl)ethynyl)-6-(hydroxy
methyl)pyridin-2-yl)(methyl)phosphinate, 16. To
a
solution of
compound 15 (55 mg, 0.27 mmol) and compound 9 (73 mg, 0.25 mmol)
in anhydrous THF (2 mL) under argon was added pyrrolidine (250 µL)
and Pd(dppf)Cl2.DCM (20 mg, 0.03 mmol). The reaction mixture was
subsequently heated at 50 °C for 24 h. After this time, solvent was
removed under reduced pressure and the residue was dissolved in
CH2Cl2/water (20 mL, 1:1). The aqueous layer was extracted with CH2Cl2
(3 × 10 mL) and the combined organic layers were dried over Na2SO4,
filtered and concentrated to dryness to yield a crude brown residue that
was purified by reverse phase HPLC (CH3CN/water) to afford a pale
yellow oil (42 mg, 41%). 1H-NMR (600 MHz, CDCl3) H 8.03 (1H, d, J =
6.0 Hz), 7.49 (1H, s), 7.18 (1H, d, J = 8.0), 7.09 (1H, s), 6.82 (1H, d, J =
8.0 Hz), 4.80 (2H, s), 4.14 – 4.06 (1H, m), 3.87 (3H, s), 3.92 – 3.82 (1H,
m), 3.16 (4H, q, J = 7.0 Hz), 1.77 (3H, d, J = 15.0 Hz), 1.26 (3H, t, J = 7.0
Hz), 1.03 (6H, t, J = 7.0 Hz); 13C-NMR (151 MHz, CDCl3) C 160.8 (d, J =
20.0 Hz), 155.2, 153.2 (d, J = 160.0 Hz), 139.5, 133.3 (d, J = 11.0 Hz),
128.3 (d, J = 22.0 Hz), 127.2, 125.0, 124.1 (d, J = 3.0 Hz), 113.5, 111.5,
97.0, 84.9, 64.2, 61.3 (d, J = 6.0 Hz), 55.7, 46.0, 16.5 (d, J = 6.0 Hz),
13.6 (d, J = 105.0 Hz), 12.0; 31P{1H}-NMR (243 MHz, CDCl3) P 39.44;
UPLC (CH3CN/H2O, 0.1% FA) tR = 1.22 min; (HRMS+) m/z 417.1954
[M+H]+ (C22H30N2O4P requires 417.1943).
Diethyl-(((7-((4-((3-(diethylamino)-4-methoxyphenyl)ethynyl)-6-
(ethoxy(methyl)phosphoryl)pyridin-2-yl)methyl)-1,4,7-triazacyclo
nonane-1,4-diyl)bis(methylene))bis(pyridine-6,2-diyl))bis(methyl
phosphinate), 22. The disubstituted triazacyclononane 20 (8.5 mg, 16.2
µmol), mesylate 17 (16.0 mg, 32.4 µmol) and K2CO3 (5 mg, 35.4 µmol)
were combined in anhydrous CH3CN (1.0 mL) and stirred at 60 °C for 12
h under argon. After this time, the solvent was removed under reduced
pressure and the residue was dissolved in CH2Cl2/water (40 mL). The
aqueous layer was extracted with CH2Cl2 (3 × 20 mL). The combined
(4-((3-(Diethylamino)-4-methoxyphenyl)ethynyl)-6-(ethoxy(methyl)
phosphoryl) pyridin-2-yl)methyl methanesulfonate, 17. The alcohol
16 (22 mg, 0.05 mmol), methanesulfonic anhydride (22 mg, 0.11 mmol)
and triethylamine (25 µL, 0.18 mmol) were combined under argon in
10
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10.1002/chem.202003992
Chemistry - A European Journal
FULL PAPER
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4842.
organic layers were dried over Na2SO4, filtered and concentrated to
dryness. The crude product was purified by reverse phase HPLC
(CH3CN/25 mM ammonium bicarbonate buffer) to afford a yellow oil (7.4
mg, 50%); 1H NMR (298 K, 400 MHz, CDCl3) H 8.04 (1H, dd, J = 6.0 Hz,
J = 2.0 Hz), 7.93 (2H, app. t, J = 6.0 Hz), 7.83 – 7.78 (2H, m), 7.69 – 7.65
(3H, m), 7.21 (1H, br. d, J = 8.0 Hz), 7.12 (1H, d, J = 2.0 Hz), 6.87 (1H, d,
J = 8.0 Hz), 4.18 – 3.76 (15H, m), 3.20 (4H, q, J = 7.0 Hz) 2.93 – 2.89
(12H, m), 1.79 (3H, d, J = 15.0 Hz), 1.78 (6H, d, J = 15.0 Hz), 1.29 (6H, t,
J = 7.0 Hz), 1.27 (3H, t, J = 7.0 Hz), 1.07 (6H, t, J = 7.0 Hz);13C-NMR
(101 MHz, CDCl3) C 161.5 (2 × d, J = 12.0 Hz), 155.0, 153.6 (2 × d, J =
160.0 Hz), 139.4, 136.4 (d, J = 9.0 Hz), 133.3 (d, J = 16.0 Hz), 127.8 (d,
J = 20.0 Hz), 127.0, 126.4 (d, J = 4.0 Hz), 125.9, 125.6 (d, J = 20.0 Hz),
124.8, 113.4, 111.3, 96.4, 85.1, 64.4 (2 × s), 60.9 (2 × d, J = 6.0 Hz),
55.6, 45.9, 16.5 (2 × d, J = 6.0 Hz), 13.4 (2 × d, J = 105.0 Hz), 11.9;
31P{1H} NMR (298 K, 162 MHz, CDCl3) P 40.24 (2P), 39.99 (1P); UPLC
(CH3CN/H2O, 0.1% FA) tR = 1.35 min; (HRMS+) m/z 922.4320 [M+H]+
(C46H67N7O7P3 requires 922.4315);
Y. Urano, D. Asanuma, Y. Hama, Y. Koyama, T. Barrett, M.
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Andraud, M. Zwier Jurriaan, L. Lamarque, D. Parker, O. Maury,
Chem. Eur. J. 2014, 20, 8636-8646; b) S. J. Butler, M. Delbianco,
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[EuL3]. The ligand 22 (2 mg, 2 µmol) was dissolved in a mixture of
CH3OH/H2O (1:1, 2 mL total) and the pH was adjusted to 12 using
aqueous NaOH solution (0.1 M). The solution was stirred at 60 °C for 14
h. After cooling and adjustment of the pH to 7 using dilute hydrochloric
acid (0.1 M), EuCl3.6H2O (3 mg, 8 µmol) was added and the reaction
mixture was stirred to 60 °C for 15 h. The reaction mixture was purified
by reverse phase HPLC (CH3CN/water) to yield a white powder after
freeze drying (2 mg, 93%); (HRMS+) m/z 986.2327 [M+H]+
(C40H52N7O7151EuP3 requires 986.2340); UPLC (CH3CN/H2O, 0.1% TFA)
tR = 1.54 min; H2O = (ms) = 0.50 (pH 9), 0.53 (pH 8), 0.74 (pH 7), 1.06
(pH 6), 1.15 (pH 5), 1.16 (pH 4); MeOH = 0.89 ms; q = 0; exc = 331 nm;
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
331 nm = 12000 M-1.cm-1;H2O = 0.01% (pH 9), 6.3 % (pH 4).
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Acknowledgements
We thank EPSRC (EP/ L01212X/1) for grant and studentship
support, and Cisbio Bioassays (JDF) for partial support.
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Conflict of interest
The authors declare no conflict of interest
Keywords: Europium; pH; cell; lysosome; luminescence.
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11
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10.1002/chem.202003992
Chemistry - A European Journal
FULL PAPER
Entry for the Table of Contents
1.2
Off
1
On
0.8
0.6
R = H, pH sensor
0.4
3
5
7
9
pH
Insert text for Table of Contents here.
Watching Lysosomes Age with
a ‘Switch-On’ Optical
Sensor: four europium complexes with extended aryl-alkylnyl
pyridine antennae show very different pH-dependent behaviour.
A ‘switch-on’ of europium luminescence occurs at low pH
allowing confocal microscopy to reveal increases in image
brightness of ageing lysosomes in live cell imaging studies.
12
This article is protected by copyright. All rights reserved.