Bright mono-aqua europium complexes based on triazacyclononane that bind anions reversibly and permeate cells efficiently

Article abstract of DOI:10.1002/chem.201301273

A series of five europium(III) complexes has been prepared from heptadentate N₅O₂ ligands that possess a brightness of more than 10 mM^-1 cm^-1 in water, following excitation over the range λ=330-355 nm. Binding of several oxy anions has been assessed by emission spectral titrimetric analysis, with the binding of simple carboxylates, lactate and citrate involving a common ligation mode following displacement of the coordinated water. Selectivity for bicarbonate allows the rapid determination of this anion in human serum, with K_d=37 mM (295 K). The complexes are internalised quickly into mammalian cells and exhibit a mitochondrial localisation at early time points, migrating after a few hours to reveal a predominant lysosomal distribution. Herein, we report the synthesis and complexation behaviour of strongly emissive europium (III) complexes that bind oxy-anions in aqueous media with an affinity and selectivity profile that is distinctively different from previously studied systems. Let there be light! A new class of luminescent anion-responsive europium complexes, which enter mammalian cells and accumulate in the lysosomes, has been synthesized. Each Eu^III complex is based on a triazacyclononane core with two alkynyl-aryl sensitizers and one coordinated water molecule (see figure). Copyright

Full text of DOI:10.1002/chem.201301273

FULL PAPER
DOI: 10.1002/chem.201301273
Bright Mono-aqua Europium Complexes Based on Triazacyclononane That
Bind Anions Reversibly and Permeate Cells Efficiently
Stephen J. Butler, Brian K. McMahon, Robert Pal, David Parker,* and
James W. Walton[a]
Abstract: A series of five europium-
(III) complexes has been prepared
from heptadentate N5O2 ligands that
possess brightness of more than
displacement of the coordinated water.
Selectivity for bicarbonate allows the
rapid determination of this anion in
exhibit a mitochondrial localisation at
early time points, migrating after a few
hours to reveal a predominant lyso-
ACHTUNGTRENNUNG
a
human
serum,
with
Kd =37 mm
ACHTUNGTRENNsUNG omal distribution. Herein, we report
10 mmÀ1 cmÀ1 in water, following excita-
tion over the range l=330–355 nm.
Binding of several oxy anions has been
assessed by emission spectral titrimet-
ric analysis, with the binding of simple
carboxylates, lactate and citrate involv-
ing a common ligation mode following
(295 K). The complexes are internal-
ised quickly into mammalian cells and
the synthesis and complexation behav-
iour of strongly emissive europium
(III) complexes that bind oxy-anions in
aqueous media with an affinity and se-
lectivity profile that is distinctively dif-
ferent from previously studied systems.
Keywords: cell imaging · europium ·
luminescence · microscopy · serum
bicarbonate
Introduction
a nonadentate ligand based on triazacyclononane effectively
shields the metal from quenching, and in which three pyri-
Luminescent lanthanide
A
dyl-arylkynyl chromophores are incorporated.[8] Herein,
ACHTUNGTRENNUNG
studied over the past decade and can be used as active com-
ponents in electroluminescent devices,[1] emissive tags for
fluorescence resonance energy transfer (FRET) assays[2] or
responsive optical probes to signal changes in the composi-
tion of the ionic environment.[3] In the latter case, modula-
tion of the emission spectral form, lifetime or relative inten-
sity has been used to allow rapid, practicable assays of bicar-
bonate,[4] citrate and lactate,[5] urate[6] or certain acute-phase
proteins[7] in diverse bio-fluids, such as urine, serum or semi-
nal fluid. Typically, coordinated water or a weakly bound
ligand donor is replaced by the added anion or protein,
leading to a change in the metal coordination environment
that is reported by changes in emission behaviour. Com-
plexes used for this purpose usually do not have overall
quantum yields that exceed 10% in the medium used. Fur-
thermore, they have been based on systems incorporating
sensitizers with an extinction coefficient of less than
10000mÀ1 cmÀ1. For excitation beyond l=340 nm, the
“brightness” B (B(l)=fe(l)) of the responsive probes stud-
ied to date has not exceeded 1000mÀ1 cmÀ1. Recently, we
these systems have been modified, creating a series of com-
plexes of heptadentate ligands with a brightness of over
10000mÀ1 cmÀ1, [Eu·Ln]+ (n=1–5). These coordinatively un-
saturated complexes bind a water molecule that can be dis-
placed by added mono- or bidentate ligand donors, such as
oxy anions or by amino-acid residues in peptides and pro-
teins.[3a,9,10]
Results and Discussion
Complex synthesis and photochemical characterisation: The
synthesis of ligands L1–L5 was undertaken either by direct
alkylation of 1,4,7-triazacyclonane with two equivalents of
the appropriate mesylate 1a–d, or by reaction with the
mono-BOC protected amine 2 (Scheme 1 and the Support-
ing Information). In the former approach, the corresponding
tris-alkylated ligands were also obtained and are reported
elsewhere.[8] The latter sequence was higher yielding, with
subsequent stepwise deprotection of the carbamate and
have reported a series of C3-symmetric europium
G
ester groups followed by reaction with EuCl
the desired complexes, [Eu·Ln
(H2O)]Cl (n=1–4). In the
case of the bis
(amide) complex, [Eu·L5(H2O)]+, the prepa-
ration involved reaction of [Eu·L4(H2O)]+ with glucamine
3ACHTUNGERTN(NUNG H2O)6 giving
plexes with a brightness exceeding 25000mÀ1 cmÀ1, in which
ACHTUNGTRENNUNG
G
ACHTUNGTRENNUNG
AHCTUNGTRENNUNG
[a] Dr. S. J. Butler, Dr. B. K. McMahon, Dr. R. Pal, Prof. D. Parker,
Dr. J. W. Walton
in the presence of the coupling agent pyBOP. The com-
plexes were purified by reverse-phase HPLC, revealing the
presence of two major diastereoisomers in ratio 7:1, as was
determined by integration of the two separate species (see
the Experimental Section). The tri-substituted series have
been shown to form the RRR and SSS enantiomers,[8,11,12] so
Department of Chemistry
Durham University, South Road, Durham, DH1 3LE (UK)
E-mail: david.parker@durham.ac.uk
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201301273.
Chem. Eur. J. 2013, 19, 9511 – 9517
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Scheme 1.
that it is assumed that the RR/SS pair will predominate
here. The isomers (RR/SS or RS at P) were not separated
deliberately in the subsequent studies discussed herein. The
centre of another molecule, displacing the coordinated
water. The q value and emission spectral form did not vary
over the concentration range 1 to 100 mm. The emission
1
2
complexes of L and L that incorporate P Ph groups were
spectrum for [Eu·L4]À was the same as that recorded for
À
sparingly soluble in water, whereas complexes of L3–L5 with
[Eu·L1À3
]
in the presence of 10 mm acetate, consistent with
+
À
the P Me substituents and more hydrophilic p-aryl-substitu-
this hypothesis.
ents dissolve readily in water. Accordingly, photophysical
data characterising their excited properties were obtained in
50% aqueous methanol in the presence of 0.1m sodium
chloride to allow a complete comparison (Table 1).
Table 1. Selected photophysical data[a] for europium complexes of L1–L5
(295 K, H2O/MeOH 50:50, pH 7.4).
Each complex possesses a broad absorption band typically
at l=332 nm (e=40 mmÀ1 cmÀ1 in H2O), and the europium
emission spectrum was similar for every complex, except
[Eu·L4]À (Figure 1). Overall emission quantum yields were
in the range 15–23%. These are the highest values recorded
Complex
k
k
q
fEu fEu
(10 mm
labs
e
(H2O) (D2O)
[nm]
[mmÀ1 cmÀ1
]
À
HCO3
)
G
2.00
1.54
2.04
1.30
2.17
0.9 23 29
1.2 19 29
0.8 19 36
0[b] 15 32
0.7 17 29
332
307
332
328
330
44.0
40.1
38.6
41.4
40.1
AHCTUNGTRENNUNG
for any europiumACHTUNGTRENNUNG(III) complex of a heptadentate ligand in
AHCTUNGTRENNUNG
aqueous media. The emission lifetimes were measured in
H2O and D2O and were typically 50% larger in the latter
solvent. This data allows the hydration state of each com-
plex to be assessed,[13] because exchangeable proximate OH
and OD oscillators have very different quenching efficien-
cies. In each case, a hydration state (q) of one was found,
except for [Eu·L4]À, for which q was zero. In this case, it is
plausible that the complex is self-associating in solution,
with the remote carboxylate group binding to the EuIII
AHCTUNGTRENNUNG
AHCTUNGTRENNUNG
[a] Radiative rate constants k (Æ10%) are reported as the mean of three
independent sets of measurements; emission quantum yields (Æ20%)
were determined by using an integrating sphere, and hydration states q
were assessed by using literature methods.[13] Similar values were record-
ed in water only. [b] This complex is self-associated, by ligation of the pe-
ripheral carboxylate to Eu; the spectral form of the complex resembled
that observed for the others in the presence of excess added benzoate or
acetate.
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Bright Mono-aqua Europium Complexes
FULL PAPER
consistent with a hydration state of zero. For [Eu·L3], values
of k were 2.2ACTHUNGTRENNUNG
added bicarbonate, citrate, lactate, phosphate, benzoate and
HSA, compared to 1.95 (Æ0.1)ꢁ103 sÀ1 in D2O.[13] Thus,
anion addition leads to displacement of the metal-coordinat-
ed water molecule, accompanied by binding of an anionic
oxygen donor.
The observed europium emission spectral form for each
complex, following addition of lactate, citrate, benzoate (or
acetate), 2-picolinate and human serum albumin, was identi-
cal, which is consistent with a common metal-coordination
environment (Figure 2). The similar spectrum observed with
Figure 1. Absorption and total emission spectrum for [Eu·L3
solution (0.1m NaCl, H2O, pH 7.4).
(H2O)]+ in
ACHTUNGTRENNUNG
The overall emission quantum yields for every complex
increased to between 29 and 36% in the presence of 10 mm
sodium bicarbonate. These quantum yields are remarkably
high and represent
a brightness in the range 10 to
14 mmÀ1 cmÀ1 (lexc =332–340 nm). These are particularly
À
high values, given that the N H oscillator of the coordinated
secondary ring nitrogen is normally as efficient as a bound
water molecule in deactivating the europium 5D0 excited
state.[13,14]
Anion-binding behaviour in solution: To gain an apprecia-
tion of the anion-binding capabilities of these new systems,
emission spectra for each europium complex were initially
recorded in the presence of a 1000-fold excess of a series of
anions and a 500-fold excess of a representative protein,
human-serum albumin (HSA; Table 2). For every added
anion, increases in intensity and changes in spectral form
were observed, which is consistent with a change in the
metal-coordination environment. Emission lifetimes were
measured in H2O and D2O in the presence of each anion
and the measured radiative rate constants k were similar,
Figure 2. Comparison of the limiting emission spectra for [Eu·L4]À (0.1m
NaCl) in the presence of : A) 5 mm NaF; B) 5 mm NaHCO3; C) 5 mm cit-
rate; D) 5 mm benzoate; E) 0.4 mm HSA; F) 5 mm HPO42À. Conditions:
pH 7.4, H2O, 5 mm complex, pH 7.4, lexc =332 nm). The same behaviour
was observed for the europium
nol or in water.
Table 2. Affinity constants, logKa, for anion complexation[a] (295 K, 50%
MeOH/0.1m NaCl). Values in parenthesis refer to measurement in water
only (0.1m NaCl solution).
(III) complexes of L2À4 in aqueous metha-
ACHTUNGTRENNUNG
Anion
[EuL1]+
E
G
[EuL4]À[c]
ACHTUNGTRENNUNG
À
HCO3
3.47
3.51
3.54
3.73
3.78
4.10
3.20
3.30
3.35
3.28
4.14
n.d.
3.72
4.20
3.90
3.70
n.d.
2.70
3.91
n.d.
3.52
3.37
2.71
2.80
3.33
3.20
3.92
n.d.
2.98
3.71
2.44
2.94
ACHTUNGTRENNUNG
added protein suggests that side-chain carboxylates of Glu
or Asp residues are most likely to bind. In this case, the
overall emission intensity decreased when protein was
added, suggesting that concomitant quenching of the chro-
mophore excited state occurs by electron transfer from the
more electron rich Tyr or Trp residues.
lactate
citrate
ACHTUNGTRENNUNG
AHCTUNGTRENNUNG
benzoate
2À
HPO4
ACHTUNGTRENNUNG
O-P-Tyr
O-P-Ser
O-P-Thr
ACHTUNGTRENNUNG
AHCTUNGTRENNUNG
AHCTUNGTRENNUNG
The spectral form associated with phosphate binding was
distinguished by the appearance of a sharp, intense band in
the DJ=2 manifold at l=613 nm. Spectra with added O-P-
Tyr, O-P-Ser and O-P-Thr were also almost identical (see
the Supporting Information) to that observed with added
HPO4 under the same conditions. Added bicarbonate gave
rise to the most pronounced change in emission spectral
form, characterised by an increase in intensity of the DJ=2
[a] Typical errors in measurement were Æ0.10 with the statistical error
associated with the fit of the data <0.05. [b] The affinity constant for
[Eu·L4]À with each anion is determined neglecting self-association; the
value found for bicarbonate in water with 0.4 mm HSA present was
logKa =2.86. [c] The affinity constant for [Eu·L4]À with HSA in water
(0.1m NaCl) was logKa =4.36 (Æ0.10). [d] The binding constant of
2À
ACHTUNGTRENNUNG
[Eu·L3(H2O)]+ with fluoride gave logKa =3.72 in 50% aqueous methanol
(0.1m NaCl), this value reduced to logKa =2.4 in water (0.1m NaCl).
n.d.=not determined.
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D. Parker et al.
band at l=622 nm, and a 30% rise in the ratio of the inten-
sities of the DJ=2/DJ=1 manifolds.
yet is not as pronounced as with the tris-HOPO diaqua-
lanthanideACTHNUTRGNEUG(N III) complexes and their analogues. In these sys-
Titrations were carried out for complexes [Eu·L1À4] with
the given anion by adding aqueous solutions of the anion in
1 mL aliquots to a solution of the complex in 50% H2O.
Changes in the ratio of selected emission spectral bands
were measured as a function of added anion concentration.
tems, displacement of the two trans-related bound waters by
added anions or protein does not apparently occur to a sig-
nificant extent[18], reflecting the different coordination ge-
ometry and the significant steric demand in these systems. If
the carboxylate and carbonate ligands chelate, a preferred
tricapped trigonal prismatic coordination geometry will be
adopted,[8,11] contrasting with the mono-capped square-anti-
prismatic coordination found in X-ray analyses of the
cyclen-based anion ternary complexes.[10]
ACTHNUTRGNEUNG
A representative titration, shown for [Eu·L3(H2O)]+ with
O-phospho-Tyr, revealed the appearance of “isoemissive”
points at l=589, 594, 685, 639 and 705 nm, accompanied by
a marked change in the form of the hypersensitive DJ=2
and DJ=4 emission bands at l=610–630 and 684-710 nm,
respectively (Figure 3). Such behaviour has previously been
The binding affinity of the series of four PV oxy anions to
each EuIII complex was also similar, with the O-P-Tyr ad-
ducts being most strongly bound, consistent with the lower
hydration energy of this more hydrophobic anion, compared
to O-phospho-serine-threonine. The complex [Eu·L4]À con-
tains a single anionic charge at pH 7.4, because the carboxy-
late groups are fully ionised. The anion affinity values were
not the lowest for this complex, suggesting that electrostatic
repulsion on anion encounter does not contribute signifi-
cantly to the overall free energy of binding. In aqueous solu-
tion, the logKa values were even lower, with citrate and bi-
carbonate the most strongly bound (Table 2 and Scheme 2).
Figure 3. Variation of the europium
ACHTUNGTRNE(NUNG III) emission spectral profile for
[Eu·L3]+ (5 mm) as
a
function of added O-phospho-tyrosine (lexc
=
332 nm; 50% MeOH/0.1m NaCl in H2O). The inset shows the fit (line)
to the data, for logKa =3.37 (+0.10).
attributed to the greater polarizability of the carbonate
oxygen donor, compared to
a “harder” carboxylate
oxygen[15], and has been reported for a large number of EuIII
complexes of heptadentate ligands based on 1,4,7,10-tetra-
ACHTUNGTRENNUNG
azacyclododecane.[4,16] An extended set of spectral titrations
is given in the Supporting Information.
Binding constants were estimated in each case for dupli-
cate runs, based on non-linear, least-squares fitting to a 1:1
binding model. Titration studies with complex [Eu·L5]+ re-
sulted in partial precipitation of the anion adduct, prevent-
ing accurate determination of binding affinities. Analysis of
the data for complexes [Eu·L1À4] revealed that the binding
affinities were similar for the lactate, benzoate and bicar-
bonate ternary adducts, typically falling in the range
logKa =3 to 4 (Table 2). Such a pattern accords with a simi-
lar coordination environment about the metal centre and
contrasts with the behaviour of the EuIII cyclen analogues, in
which the a-hydroxy-acids bound as five-ring chelates, in-
volving the hydroxyl and the carboxylate oxygen atoms.
Indeed, the affinity for simple carboxylates here is about
two orders of magnitude greater than with these ana-
logues.[16b] Presumably, the steric demand at the metal
centre is greater for these triazacyclononane-based systems,
Scheme 2.
Due to the large steric demand at the metal centre in
these systems, strong binding of small spherical anions was
expected. Therefore, binding to fluoride was examined in
one example and was found to be quite high in 50% aque-
ous methanol (logKa =3.7 with [Eu·L3]+), reducing to
logKa =2.4 in 0.1m aqueous NaCl. This is a similar overall
affinity to that measured for certain tripositive tetra-amide
europium mono-aqua complexes in Tris/HClO4 buffer (i.e.,
lacking chloride)[19] and indeed is higher than most “fluo-
ride-selective” synthetic receptors reported to date.[20]
In many biological fluids, for example, serum and cerebro-
spinal fluid, bicarbonate is present at a concentration that is
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Bright Mono-aqua Europium Complexes
FULL PAPER
10–20 times greater than that of any other oxy anion.[3a] In
aqueous solution, simulating an extracellular fluid contain-
ing HSA (0.4 mm), sodium lactate (2.3 mm), hydrogenphos-
phate (0.9 mm), citrate (0.13 mm) and sodium bicarbonate
of L3À5 were examined in Chinese hamster ovarian cells
(CHO) and mouse skin fibroblasts (NIH-3T3). First, the cy-
totoxicity of each complex was examined over 24 h by using
an (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide (MTT) assay that monitors perturbation of mitochon-
drial redox function.[23] In each case, the IC50 value was
>150 mm (24 h). No evidence was found for any perturba-
tion of cell function or change in morphology over this time
period.
The intracellular uptake of each complex into a popula-
tion of 107 mouse skin fibroblasts (NIH-3T3) was assessed
by measuring the europium concentration by ICP MS, fol-
lowing a 24 h incubation with a 10 mm solution of complex in
the growth medium.[24,25] The values found were 11 mm for
[Eu·L4]À, 70 mm for [Eu·L3]+ and 252 mm for the bis-amide
[Eu·L5]+. In each case, a cell volume of 4ꢁ103 mm3 was as-
sumed. Stained cells were also observed by laser-scanning
confocal microscopy and the brightness of the microscopic
images echoed the cell-uptake order. Because these com-
plexes are very bright, the laser power was reduced to ap-
proximately 8 mW with a scan speed of 400 Hz. Under these
conditions, images were acquired over 3.5 seconds for a typi-
cal field of view with 1024ꢁ1024 pixels (132ꢁ132ꢁ780 nm
depth) exposed to four consecutive scans. Therefore, each
pixel was subject to laser excitation for 10 ms, and cells
within the field-of-view were examined for up to 15 min at a
time.
(30 mm) and by using [Eu·Ln
ACTHNUTRGEN(UNG H2O)] (5 mm; n=1, 3 and 4),
the observed emission spectrum was the same as that ob-
served in the presence of excess bicarbonate only. This se-
lectivity for bicarbonate allowed an estimate to be made of
the concentration of bicarbonate in human-serum solution.
To remove bicarbonate, the pH of the serum sample was
lowered to five, and argon was bubbled through the solution
to displace dissolved carbon dioxide. The pH was re-adjust-
ed to 7.4 under argon, and incremental additions of sodium
bicarbonate were made up to a limiting value of 50 mm.
Emission spectra were recorded, (lexc =365 nm), and the
change in the intensity ratio of the europium DJ=2/DJ=1
manifolds plotted as a function of added bicarbonate con-
centration to generate a calibration curve. A near linear re-
sponse was observed over the range 15 to 45 mm with Kd =
37 mm ACHTUNGTRENNUNG(Figure 4). These characteristics allowed the concen-
The intracellular localisation profile was assessed by using
the organelle selective dyes, Mitotracker Green and Lyso-
tracker Green, and 4’,6-diamidino-2-phenylindole (DAPI)
was used to reveal the nuclear region. The complexes exam-
ined exhibited a predominant staining of the lysosomes after
an incubation of 1 h or more (Figure 5); at earlier time
points with [Eu·L5]+ and [Eu·L3]+, there was evidence for
Figure 4. Emission spectral data showing the changes in the EuIII emis-
sion spectrum for [Eu·L3(H2O)]+, following incremental addition of
AHCTUNGTRENNUNG
sodium bicarbonate to degassed human serum (pH 7.40 (+0.05), 5 mm
complex, 298 K, lexc =365 nm, 1 ms excitation pulse, 2 ms integration
time, 0.25 nm resolution by using a linear CCD (550 to 800 nm). The
inset is a plot of the intensity ratio (l=605–630/585-600 nm) versus
[HCO3À] that shows a fit (line) to the observed data with Kd =37 mm.
tration of bicarbonate in unknown samples to be determined
quickly and easily; the typical clinical range is 18 to 30 mm.
Indeed, in chronic kidney patients or in people with meta-
bolic acidosis, a value of 22 mm is generally considered to
mark the threshold of acidosis.[21,22] Recently, we reported a
different method for assessing bicarbonate in human serum
by measuring a ratio of Eu/Tb intensities.[4a] In that system,
the brightness of the EuIII complex used was about
600mÀ1 cmÀ1, compared to 10000mÀ1 cmÀ1 herein. Conse-
quently, much less complex is required for analysis in the
current system, the signal/noise ratio is much higher for the
same acquisition time, and only the EuIII complex is needed.
Figure 5. Confocal microscopy images revealing the distribution of
[Eu·L3]+ in CHO cells following a 3 h incubation with the complex
(10 mm, red), LysoTracker Green (LTG; 10 mm, green) and DAPI
(10 mm, blue).
ACTHNUTRGNEUNG
Cell-staining behaviour of complexes [Eu·L3À5(H2O)]+: The
complexes of L1 and L2 showed poor solubility in cell-
growth media, so that only the more soluble EuIII complexes
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D. Parker et al.
co-staining of the mitochondria. The emission spectrum of
the complex within the cells was recorded by using a home-
built, time-gated spectral-imaging microscope. The observed
spectra showed at least two DJ=0 components and were vir-
tually the same as those recorded in the cell-growth medium
at pH 7.6 and 6.0. The spectra most closely resembled a mix-
ture of the aqua complex and the serum albumin adduct.
Cell-growth medium contains a mixture of proteins (ca.
10 mgsmLÀ1) including serum albumin at a concentration of
about 5 mgmLÀ1 or 75 mm. Given that the association con-
stant measured for HSA with [Eu·L4]À was logKa =4.36, this
seems a reasonable assumption (Table 2 and Figure 6).
ring nitrogen or the p-aryl sites, the development of further
organelle-specific or responsive emissive probes may be ex-
pected, functioning at low concentration because of their
high inherent optical brightness.
Experimental Section
Details of instrumentation, ligand synthesis, methods of purification,
characterisation data and selected spectral titrations with added anions
and proteins are given in the Supporting Information.
Typical preparation of europiumACHTUNGTRNEUNG
(III) complex of L3: Synthesis of tert-bu-
toxycarbonyl-protected di-ethyl methylphosphinate ester of L3: Tert-butyl-
1,4,7-triazacyclononane-1-carboxylate (34 mg, 0.11 mmol) and ethyl(6-
(ethyl-methanesulfonate)-4-[2-(4-methoxyphenyl)ethynyl]pyridin-2-yl)-
AHCTUNGTREG(NNUN methyl)phosphinate, (96 mg, 0.23 mmol) were dissolved in CH3CN
(5 mL), and K2CO3 (53 mg, 0.39 mmol) was added. The mixture was stir-
red under argon at 658C and monitored by TLC (silica; CH2Cl2:5%
CH3OH, Rf (product)=0.48). After 12 h, the starting material had been
consumed and the reaction was cooled, the solution decanted from
excess potassium salts. The solvent was removed under reduced pressure,
and the crude material was purified by column chromatography (silica,
CH2Cl2/CH3OH 0–5% in 2% increments) to give a yellow oil (55 mg,
56%). 1H NMR (600 MHz, CDCl3) dH =7.96 (2H, m, H3), 7.65 (2H, bs,
3
3
H5), 7.44 (4H, d, JHÀH 8.5 Hz, H13), 6.87 (4H, d, JHÀH 8.5 Hz, H14), 4.11–
4.05 (2H, m, H7), 3.93, (2H, s, H1), 3.92 (2H, s, H1), 3.86–3.82 (2H, m,
H7), 3.81 (6H, s, H16), 3.35 (4H, br m, ring Hs), 3.08 (4H, br m, ring Hs),
2.68 (4H, br m, ring Hs), 1.75 (3H, d, JHÀP 15 Hz, H9), 1.74 (3H, d, JHÀP
2
2
Figure 6. Analytical reverse-phase HPLC trace of [Eu·L3
ACTHNUGTRNEUNG(H2O)]Cl. Major
isomer: tR =7.2 min; minor isomer: tR =8.5 min; [gradient: 10 to 100%
MeOH in water (0.05% formic acid) over 10 min].
15 Hz, H9), 1.46 (9H, s, H19), 1.23 (3H, t, JHÀH 7 Hz, H8), 1.22 ppm (3H,
3
t, JHÀH 7 Hz, H8); 13C NMR (151 Hz, CDCl3): d=161.6, 161.4 (d, JCÀP
3
3
20 Hz, C6), 160.5, 160.4 (s, C17), 155.6 (s, C15), 153.8, 153.6 (d, JCÀP
1
165 Hz, C2), 133.6, 133.5 (s, C15), 132.5 (br m, C4), 127.6 (br m, C3), 126.4,
126.1 (d, JCÀP 3 Hz, C5), 114.2, 114.1 (s, C14), 113.9, 113.8 (s, C12), 95.6,
4
95.3 (s, C11), 85.6, 85.4 (br m, C10),79.4 (s, C18) 62.8, 62.5 (s, C1), 60.9, 60.8
Conclusion
(d, JCÀP 3 Hz, C7), 55.3 (s, C16), 55.0–49.7 (br m, ring Cs), 28.6 (s, C19),
2
16.4 (d, JCÀP 6 Hz, C8), 13.3, 13.2 ppm (d, JCÀP 109 Hz, C9); 31P NMR
(243 MHz, CDCl3): dH =40.0, 39.9 ppm; HRMS (ESI): m/z calcd for
C47H60O8N5P2: 884.3917 [M+H]+; found 884.3937.
3
1
The europiumACHTUNGTRENNUNG(III) complexes of the five ligands examined
combine a high emission quantum yield in aqueous media
with two strongly absorbing arylkynyl–pyridyl chromo-
phores, generating “bright” complexes. Each complex pos-
sesses one coordinated water molecule in solution, except
for [Eu·L4]À, which shows evidence for self-association. The
water molecule can be displaced by a range of added anions
leading to emission intensity increases and a change in the
spectral fingerprint. In contrast to previously studied sys-
tems, simple carboxylates bind in a similar manner to a-hy-
droxy acids; proteins also seem to bind preferentially
through carboxylate ligation from Asp or Glu residues
(Scheme 2).
In solutions containing more than 10 mm bicarbonate, this
anion is bound selectively allowing its quantification in a
complex medium, such as human serum. The intensity ratio
of the emission bands centred at l=620 and 590 nm signals
the change in bicarbonate concentration for undiluted
serum samples, and it may be determined rapidly by using
time-gated emission spectroscopy, with excitation wave-
lengths in the range l=330–365 nm.
Synthesis of diethyl methylphosphinate ester of L3: The tert-butoxycarbon-
yl-protected diethyl methylphosphinate ester of L3 (65 mg, 0.074 mmol)
was dissolved in anhydrous CH2Cl2 (1 mL), and trifluoroacetic acid
(0.5 mL) was added. The solution was stirred under argon at 238C for
45 min. TLC analysis (silica; CH2Cl2:5% CH3OH), Rf (product)=0.12, Rf
(reactant)=0.52) was used to confirm that the protecting group removal
had gone to completion. The solvent was removed by using a high-
vacuum line (without heating), and the residue was re-dissolved in
CH2Cl2 (1 mL), which was again removed by using the same method.
This process was repeated five times to ensure complete removal of
excess trifluoroacetic acid. The crude material was purified by prepara-
tive HPLC to give the title product as a white solid (20 mg, 35%).
3
1H NMR (700 MHz, CDCl3): d=7.95 (2H, d, JHÀP 5.8 Hz, H3), 7.46 (4H,
3
3
d, JHÀH 8.9 Hz, H13), 7.41 (2H, bs, H5), 6.88 (4H, d, JHÀH 8.9 Hz, H14),
4.15–4.10 (2H, m, H7), 3.99 (4H, s, H1), 3.92–3.87 (2H, m, H7), 3.81 (6H,
s, H16), 3.19 (4H, br s, ring Hs), 3.07 (4H, br s, ring Hs), 2.69 (4H, br s,
ring Hs), 1.76 (6H, d, JHÀP 15.0 Hz, H9), 1.27 ppm (6H, t, JHÀH 7 Hz,
2
3
H8); 13C NMR (176 MHz, CDCl3): d=160.7 (s, C15), 159.3 (d, JCÀP 20 Hz,
3
C6), 154.4 (d, JCÀP 157 Hz, C2), 133.7 (s, C13), 133.1 (d, JCÀP 11 Hz, C4),
1
3
127.7 (d, JCÀP 20 Hz, C3), 126.3 (d, JCÀP 3 Hz, C5), 114.2 (s, C14), 113.5 (s,
2
4
C12), 96.4 (s, C11), 85.0 (s, C10), 61.1 (d, JCÀP 6.3 Hz, C7), 60.1 (s, C1), 55.3
2
(s, C16), 51.1–43.9 (br m, ring Cs), 16.5 (d, JCÀP 6 Hz, C8), 13.6 ppm (d,
3
1JCÀP 103 Hz, C9); 31P NMR (283 MHz, CDCl3): d=39.3; HRMS (ESI):
m/z calcd for C42H52O6N5P2 [M+H]+; found: 784.3387.
The more hydrophilic complexes studied herein, with
p-methoxy, p-oxyacetate or p-gluconamide substituents
readily enter mammalian cells, preferentially staining the
mitochondria at early time points, before migrating to the
lysosomes. Given the ease of attaching substituents to the
ACTHNUTRGNEUNG
Synthesis of [Eu·L3(H2O)]Cl: The diethyl methylphosphinate ester of L3
(15 mg, 20.6 mmol) was dissolved in CD3OD (3 mL), and NaOH (0.1m in
D2O, 1 mL) was added. The solution was stirred at 608C and monitored
by 1H NMR (loss of ethyl peaks) and 31P NMR spectroscopies (reactant
39.3 ppm, product 25.3 ppm) and stopped after 16 h. The pH of the solu-
9516
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ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2013, 19, 9511 – 9517

Bright Mono-aqua Europium Complexes
FULL PAPER
tion was adjusted to 7 by addition of HCl (1m). EuCl3 (H2O)6 (7.5 mg,
23 mmol) in a H2O/CH3OH solution (1:1, 0.5 mL) was added, and the
soluACHTUNGTRENNUNGtion was stirred at 508C for 24 h The solvent was removed under re-
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duced pressure and the crude material was purified by preparative re-
verse-phase HPLC [Waters XBridge RP-C18 column (5 mm, 10ꢁ100 mm)
at a flow rate of 4.4 mLminÀ1; gradient: 10–100% methanol in water
(0.05% formic acid) over 10 min] to give [Eu·L3
ACTHNUTRGNEUNG(H2O)]Cl (3.5 mg, 19%)
as a mixture of two isomers (major isomer tR =7.2 min; minor isomer
tR =8.5 min); HRMS (ESI): m/z calcd for C38H41O6N5P2151Eu: 876.1731
[M
(
151Eu)]+;
found:
876.1708;
eMeOH
tH O =0.34 ms,
tD O =0.49 ms;
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2
2
em
FMeOH=H O 1:1 =19Æ10%;
tR =7.192min.
(l=332 nm)=38645mÀ1 cmÀ1
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Received: April 4, 2013
Published online: June 6, 2013
Chem. Eur. J. 2013, 19, 9511 – 9517
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
9517