Cationic Europium Complexes for Visualizing Fluctuations in Mitochondrial ATP Levels in Living Cells

Article abstract of DOI:10.1002/chem.201801008

The ability to study cellular metabolism and enzymatic processes involving adenosine triphosphate (ATP) is impeded by the lack of imaging probes capable of signalling the concentration and distribution of intracellular ATP rapidly, with high sensitivity. We report here the first example of a luminescent lanthanide complex capable of visualizing changes in the concentration of ATP in the mitochondria of living cells. Four cationic europium(III) complexes [Eu.1–4]⁺ have been synthesized and their binding capabilities towards nucleoside polyphosphate anions examined in aqueous solution at physiological pH. Complexes [Eu.1]⁺ and [Eu.3]⁺ bearing hydrogen bond donor groups in the pendant arms showed excellent discrimination between ATP, ADP and monophosphate species. Complex [Eu.3]⁺ showed relatively strong binding to ATP (logK_a=5.8), providing a rapid, long-lived luminescent signal that enabled its detection in a highly competitive aqueous medium containing biologically relevant concentrations of Mg²⁺, ADP, GTP, UTP and human serum albumin. This Eu^III complex responds linearly to ATP within the physiological concentration range (1–5 mm), and was used to continuously monitor the apyrase-catalyzed hydrolysis of ATP to ADP in vitro. We demonstrate that [Eu.3]⁺ can permeate mammalian (NIH-3T3) cells efficiently and localize to the mitochondria selectively, permitting real-time visualization of elevated mitochondrial ATP levels following treatment with a broad spectrum kinase inhibitor, staurosporine, as well as depleted ATP levels upon treatment with potassium cyanide under glucose starvation conditions.

Full text of DOI:10.1002/chem.201801008

A Journal of
Accepted Article
Title: Cationic Europium Complexes for Visualizing Fluctuations in
Mitochondrial ATP Levels in Living Cells
Authors: Romain Mailhot, Thomas Traviss-Pollard, Robert Pal, and
Stephen Jonathan Butler
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Cationic Europium Complexes for Visualizing Fluctuations in
Mitochondrial ATP Levels in Living Cells
Romain Mailhot[a], Thomas Traviss-Pollard[a], Robert Pal[b] and Stephen J. Butler*[a]
Abstract: The ability to study cellular metabolism and enzymatic
processes involving adenosine triphosphate (ATP) is impeded by the
lack of imaging probes capable of signalling the concentration and
distribution of intracellular ATP rapidly, with high sensitivity. We
report here the first example of a luminescent lanthanide complex
capable of visualizing changes in the concentration of ATP in the
mitochondria of living cells. Four cationic europium(III) complexes
[Eu.1–4]+ have been synthesized and their binding capabilities
towards nucleoside polyphosphate anions examined in aqueous
solution at physiological pH. Complexes [Eu.1]+ and [Eu.3]+ bearing
hydrogen bond donor groups in the pendant arms showed excellent
discrimination between ATP, ADP and monophosphate species.
the mitochondria by oxidative phosphorylation. Numerous
enzymes utilize ATP as a substrate, including ATPases, kinases,
and RNA polymerases; thus ATP plays a key role in signalling
and the regulation of enzyme function. 1, 2, 6 The release of ATP
to the extracellular space has been identified in both damaged
and apoptotic cells.2 Extracellular ATP also serves as
a
signalling molecule by binding to purinergic receptors,3 thereby
mediating an immunological or nervous response. The
concentration of ATP varies considerably, from nanomolar
extracellular concentrations to millimolar concentrations in the
cytosol and certain organelles (e.g. mitochondria).1, 2 To gain a
better understanding of the wide range of dynamic processes
involving ATP, non-invasive imaging probes are required that
can signal changes in ATP levels by modulation of
luminescence, thereby providing spatial and temporal
information rapidly, with high sensitivity.5a, 6a
Complex [Eu.3]+ showed relatively strong binding to ATP (log Ka
5.8), providing a rapid, long-lived luminescent signal that enabled its
detection in highly competitive aqueous medium containing
=
a
biologically relevant concentrations of Mg2+, ADP, GTP, UTP and
human serum albumin. This Eu(III) complex responds linearly to
ATP within the physiological concentration range (1–5 mM), and was
used to continuously monitor the apyrase-catalyzed hydrolysis of
ATP to ADP in vitro. We demonstrate that [Eu.3]+ can permeate
mammalian (NIH-3T3) cells efficiently and localize to the
mitochondria selectively, permitting real-time visualization of
elevated mitochondrial ATP levels following treatment with a broad
spectrum kinase inhibitor, staurosporine, as well as depleted ATP
levels upon treatment with potassium cyanide under glucose
starvation conditions.
Adenosine
A
O
O
P
O
O
O
O
P
O
O
O
O
HN
HN
O
NH
NH
O
O
P
H
H
ATP
O
O
O
O
N
O
N
N
N
N
N
O
water, pH 7.4
S3+ N
S3+ N
Eu
Eu
N
N
N
R
R
N
N
N
N
H
N
H
Weakly emissive [Eu.3]+
Highly luminescent
[Eu.3]+-ATP complex
CO2
B
Introduction
O
HN
OH2
OH2
O
O
R
R
Adenosine triphosphate (ATP) is the most abundant nucleoside
polyphosphate (NPP) anion in cells1,2 and serves as the
chemical energy source for most biological processes, including
organelle transport, muscle contraction and maintenance of
neuronal membrane potential.2–4 Despite its importance, there
are surprisingly few imaging probes capable of signalling the
presence of ATP rapidly, reversibly and selectively under
physiological conditions.5, 6 We have addressed this challenge
by creating a luminescent europium(III) complex capable of
imaging dynamic changes in the concentration of ATP in the
mitochondria of living cells. The majority of ATP is generated by
N
N
N
N
N
N
N
N
Eu
Eu
N
N
N
N
R
R
O
O
O
NH
O
O
[Eu.1]+ R =
[Eu.3]+ R =
N
N
O2C
H
H
[Eu.2]+ R = H
[Eu.4]+ R = H
Figure 1. (A) Signalling mechanism and proposed binding mode between
[Eu.3]+ and ATP; (B) Structures of cationic Eu(III) complexes [Eu.1–4]+.
Current methods for monitoring the concentration of ATP in
living cells have limitations.2, 5a The enzyme firefly luciferase can
be expressed in cells to measure ATP levels indirectly, by
catalyzing a chemiluminescent reaction between ATP and
luciferin.7 However, this method consumes ATP, which may
[a]
[b]
Mr. R. Mailhot, Mr. T. Traviss-Pollard and Dr. S. J. Butler
Department of Chemistry
Loughborough University
Epinal Way, Loughborough, LE11 3TU, UK
E-mail: s.j.butler@lboro.ac.uk
Dr. R. Pal
perturb the cellular energy status, preventing accurate
9
quantification of ATP.8,
Genetically encoded fluorescent
biosensors (e.g. ATeam6a, PercevalHR6b) have been developed
to measure ATP successfully within specific cellular
compartments. However, biosensors encoded within cells can
require time consuming protein expression and maturation
procedures and are intrinsically pH sensitive;6b small changes in
Department of Chemistry
Durham University
South Road, Durham, DH1 3LE, UK
Supporting information for this article is given via a link at the end of
the document.
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intracellular pH (<0.1 units) have been shown to generate bias in
the emission response, which can complicate interpretation of
the observed signal.9
energy transfer to the nucleotide base25, 27 or displacement of a
coordinated sensitizing ligand from the Ln(III) ion
(decomplexation).28,29 Probes that utilise quenching of
luminescence are less desirable for use in cellular imaging as
other competitive quenching processes may generate the
observed decrease in emission intensity.14b
An attractive alternative strategy involves the creation of discrete
ATP-responsive synthetic receptors that exhibit intrinsic cellular
5a
uptake and localization behaviour.1,
The majority of synthetic
receptors for ATP comprise a fluorescent organic indicator
linked to a positively charged recognition group, such as
imidazolium or guanidinium units, or coordinated zinc(II) ions,
which form strong electrostatic or metal-ligand interactions with
the triphosphate fragment of ATP.10 Only a few ATP-selective
probes have been successfully applied to imaging ATP in living
cells.5a-d Each of these probes emits a short-lived fluorescence
signal, typically at wavelengths between 375 and 540 nm.
Consequently, their signal overlaps with background
autofluorescence arising from endogenous molecules, which
can complicate intensity-based measurements.11 In two cases,
the detection range of the receptor is very low (0.1–10 µM),5a, 5b
and the fluorescence response is saturated at ATP
concentrations below those expected in cells (1–5 mM).12 Chang
and co-workers developed a probe capable of detecting ATP
In this work, we report a series of luminescent cationic Eu(III)
complexes [Eu.1–4]+ that bind reversibly and with differential
affinities to NPP anions in aqueous solution at physiological pH
(Figure 1). Each Eu(III) complex is based on a C2-symmetric
octadentate ligand bearing two coordinating quinoline groups
that sensitize Eu(III) luminescence. Addition of ATP to
complexes [Eu.1]+ and [Eu.3]+ results in rapid displacement of
the coordinated water molecule, giving rise to dramatic
enhancements in Eu(III) luminescence. Complex [Eu.3]+ is very
effective at binding to ATP in water at physiological pH, by
means of
a
strong metal-ligand interaction, possibly
strengthened by hydrogen bonds to the quinoline amide arms.
[Eu.3]+ can discriminate effectively between ATP and ADP in the
presence of high concentrations of Mg2+ ions, permitting real-
time analysis of the enzymatic hydrolysis of ATP to ADP in vitro.
We demonstrate that [Eu.3]+ can signal changes in the ratio of
ATP/ADP in a simulated intracellular fluid containing biorelevant
concentrations of several NPP anions including GTP and UTP,
as well as Mg2+ ions and protein. [Eu.3]+ was shown to permeate
NIH-3T3 cells efficiently and localize selectively to the
mitochondria, providing a strong luminescent signal that can
report on dynamic changes in mitochondrial ATP levels within
living cells. This new class of Eu(III) complexes offers a number
of improvements in performance compared to existing probes for
ATP, including a long-lived luminescence signal that is sensitive
to ATP levels within the physiological range (1–5 mM), with
minimal interference from biomolecule autofluorescence or
within the 2–10 mM range, in
a
viscous medium of
glycerol/water (60:40).5c However, this probe is unable to
respond to ATP in 100% aqueous solution at physiological pH,
hence its utility in live-cell imaging is limited and dependent on
variations in viscosity within the cell.
Importantly, during in vitro testing, anion affinity and selectivity is
rarely assessed in a competitive ionic medium that resembles
intracellular fluid.5c This is critical, because it defines the target
concentration range and the nature and abundance of potentially
interfering species, including other phosphoanions (e.g. ADP,
AMP, GTP, HPO42-), cations that bind strongly to ATP (e.g. Mg2+
and Ca2+ ions), and proteins, which can interact with the probe
causing either quenching or enhancement of emission. The
influence of such a competitive ionic environment on the probe’s
anion affinity and selectivity should be studied, to optimize its
practical utility in live-cell imaging experiments.13
changes in cellular pH. In addition, [Eu.3]+ possesses
a
distinctive subcellular localization profile that allows ATP levels
to be monitored within a specific region of the cell.
In recent years, stable luminescent europium(III) and terbium(III)
complexes have emerged as attractive tools for use in cellular
imaging.14 Notably, a series of the brightest Eu(III) complexes
Results and Discussion
have been developed as efficient cellular stains, designed to
Complex Design and Synthesis. Each Eu(III) complex
contains two coordinating quinoline groups that act as efficient
sensitizers of Eu(III) emission.30 Complexes [Eu.1]+ and [Eu.2]+
were synthesised previously20a and possess two negatively
charged carboxylate groups that coordinate to the Eu(III) ion,
whereas complexes [Eu.3]+ and [Eu.4]+ possess two neutral
carbonyl amide donor groups, functionalized with terminal
carboxylate moieties to increase water solubility. The carbonyl
amide groups in [Eu.3]+ and [Eu.4]+ were expected to increase
the electropositive nature of the Eu(III) metal compared to
[Eu.1]+ and [Eu.2]+, thus strengthening the electrostatic
interaction between the Eu(III) ion and negatively charged NPP
anions in aqueous solution. Additionally, it was envisaged that
the increased local positive charge of [Eu.3]+ and [Eu.4]+,
together with the amphipathic nature of the surrounding ligands,
would facilitate cellular uptake of the Eu(III) complexes, and
16
localize preferentially in specific organelles.15,
Stable Eu(III)
and Tb(III) complexes offer significant advantages over
conventional fluorescent organic dyes, including large
a
separation between the absorption and emission bands, long
emission lifetimes17 (within the millisecond range) that enable
complete removal of background autofluorescence by using
time-resolved imaging techniques, and well-defined emission
bands that permit ratiometric analysis.18 Certain Eu(III) and
Tb(III) complexes have been shown to bind reversibly to anions
in aqueous media, including bicarbonate,19 fluoride,20 lactate21
,
phosphate22 and phosphorylated peptides23. Anion binding can
be signalled by changes in the intensity ratio of two Ln(III)
emission bands or by changes in emission lifetime of the
complex.13, 24 However, examples of Ln(III) complexes that bind
to NPP anions such as ATP are rare.25–28 Binding to ATP is
usually weak and causes quenching of luminescence due to
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potentially promote selective localization to the negatively
charged inner membrane of the mitochondria.16, 5a
groups of the two carbonyl amide arms were hydrolyzed using
0.5 M NaOH solution. Subsequent addition of one equivalent of
EuCl3 in a mixture of water/methanol at pH 7–8 gave the water
soluble Eu(III) complexes [Eu.3]+ and [Eu.4]+, after purification by
preparative reverse-phase HPLC.
Complexes [Eu.1]+ and [Eu.3]+ possess hydrogen-bond donor
(amide) groups at the 7-position of the coordinated quinoline
chromophores. We envisaged that NPP anions such as ATP
would coordinate to the Eu(III) ion via the terminal phosphate
group, enabling the adjacent phosphate group to engage in
hydrogen bonding interactions with quinoline amide N–H groups
(Figure 1B), thereby enhancing selectivity over monophosphate
species. Indeed, we have shown recently that the cooperative
use of metal-ligand interactions in combination with hydrogen
bonding can provide excellent selectivity for nucleoside
polyphosphate anions over monophosphate anions (e.g. AMP,
cAMP, HPO42-, phosphorylated amino acids), where no such
hydrogen-bonding interactions can occur.31, 32 To investigate the
function of the hydrogen bond-donor groups in the ATP
recognition process, we prepared two control complexes, [Eu.2]+
and [Eu.4]+, which lack quinoline amide groups.
Table 1 shows selected photophysical data for [Eu.1–4]+. The
UV-Vis absorption spectra of [Eu.1]+ and [Eu.3]+ are similar and
comprise of a broad band centred at 332 nm and 330 nm
respectively, tailing out to 370 nm, whereas [Eu.2]+ and [Eu.4]+
each feature two narrow bands with maxima at 303 and 318 nm
(Fig. S4-S7). The emission spectra for each complex in aqueous
buffer (10 mM HEPES, pH 7.4) was well separated from the
absorption spectra, as illustrated by [Eu.3]+ in Figure 2. The
emission spectra of [Eu.1]+, [Eu.2]+ and [Eu.4]+ showed
similarities, displaying at least three components in the ΔJ = 1
(585–605 nm) band, and two distinguishable components within
the ΔJ = 2 (605–630 nm) band, consistent with each complex
adopting a structure of low symmetry in water (Fig. S8). For
[Eu.3]+, a rather different emission spectrum was obtained,
characterised by a pronounced ΔJ = 2 band around 605–630 nm
and two discernable components within the ΔJ = 4 band around
675-705 nm in the red region of the visible spectrum (Figure 2).
Emission quantum yields were in the range 7–23% and emission
lifetimes in H2O were measured to be approximately 0.5 ms, and
at least 50% larger in D2O. The number of coordinated water
molecules, q, was determined to be one for each Eu(III)
complex.33 These data suggested that complexes [Eu.1]+ and
[Eu.3]+ were most suited for use in live-cell imaging experiments,
due to their high water solubility, long-lived luminescence and
sufficiently long excitation wavelengths of over 350 nm, thereby
matching the optics of standard fluorescence microscopes.
Table 1. Selected photophysical data for complexes [Eu.1–4]+,
measured in 10 mM HEPES, pH 7.0, 25°C.
λmax
/
Complex
ε/M-1cm-1
φem/%[a]
τ
(H2O)/ms
τ(D2O)/ms
q[b]
nm
[Eu.1]+
[Eu.2]+
[Eu.3]+
[Eu.4]+
332
318
330
318
0.00125
0.00118
0.00077
0.00210
7.0
23
0.49
0.51
0.56
0.60
1.38
1.37
1.22
1.29
1.2
1.1
0.8
0.7
6.5
18
[a] Errors in quantum yields and lifetimes are ±20%; [b] Values of
hydration state, q (±20%) were derived using literature methods.33
Anion Binding Studies at Physiological pH. The Eu(III)
emission spectra of [Eu.1–4]+ was recorded in pH 7.0 (10 mM
HEPES) in the presence of a range of anions (1 mM) (Fig. S9–
S12). Addition of 1 mM ATP to [Eu.1]+ or [Eu.3]+ resulted in an
immediate 10-fold enhancement in Eu(III) emission intensity of
the hypersensitive ΔJ = 2 band (605–630 nm), whereas adding
3.0
2.5
2.0
1.5
1.0
0.5
0.0
35
30
25
20
15
10
5
ADP induced a 14-fold increase in emission intensity (Fig. S9
2-
and S10). AMP and HPO4
induced
a
much smaller
(approximately 2-fold) increase in emission intensity while other
monophosphate anions including cyclic AMP and
phosphorylated amino acids (pSer, pThr, pTyr) induced
negligible spectral responses, as did chloride, sulphate, lactate,
acetate, glutathione, Na+, K+, Zn2+, Mg2+ and Ca2+ ions. The
guanosine phosphate anions, GTP, GDP and GMP induced
similar spectral responses to those observed for ATP, ADP and
AMP respectively. The only other anions tested that induced a
significant spectral response were citrate and bicarbonate, which
caused a maximum 4-fold and 5-fold increase in the ΔJ = 2
emission band, respectively. Control complexes [Eu.2]+ and
[Eu.4]+, which lacked quinoline amide groups, showed a much
lower level of discrimination between NPP anions: a maximum
3-fold enhancement in intensity of the ΔJ = 2 emission band was
observed in the presence of 1 mM ATP, ADP and AMP (Fig.
S11 and S12). Analysis of the relative change in total emission
0
270
330
390
450
510
570
630
690
750
Wavelength / nm
Figure 2. Absorption (blue) and emission (red) spectra of [Eu.3]+, recorded in
buffered aqueous solution (10 mM HEPES) at pH 7.0. λexc = 330 nm, 25°C.
Details of the synthesis and characterization of complexes
[Eu.3]+ and [Eu.4]+ are provided in the supporting information
(Fig. S1–S3 for synthetic schemes). Briefly, a cyclen derivative
bearing two trans-related secondary amines was reacted with an
appropriately functionalized 2-methylquinoline mesylate ester or
2-(chloromethyl)quinoline in the presence of K2CO3, to give the
protected macrocyclic ligand. The terminal ethyl ester protecting
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intensity (570–720 nm) resulted in a very similar anion selectivity
profile for each Eu(III) complex (Fig. S13–S16).
[a] Each value represents the average of at least two duplicate titration
experiments. Errors in measurements were ± 0.1 with the statistical error
associated with the fit of the data <0.05. n/d = not determined.
The emission intensity of [Eu.3]+ was found to be particularly
sensitive to ATP concentration. Figure 3 shows the increase in
emission spectra of [Eu.3]+ in the presence of increasing ATP
levels (0–180 µM), revealing a dramatic 17-fold enhancement in
intensity at 614 nm within the ΔJ = 2 band. Notably, the emission
spectral shape of [Eu.3]+ did not change significantly, suggesting
that only minor changes in conformation of [Eu.3]+ occur upon
binding to ATP. Emission lifetimes of [Eu.3]+ measured in H2O
and D2O in the absence and presence of ATP were consistent
with a hydration state, q, of 0.8 (±20%) and zero respectively
(Table S1), establishing that ATP displaces the coordinated
water molecule from the Eu(III) metal, accompanied by a
pronounced increase in luminescence.
Addition of ATP (and ADP) to complex [Eu.3]+ also caused a
considerable increase in intensity of the hypersensitive ∆J = 4
2
band (675–705 nm), whereas AMP and HPO4 caused much
smaller changes (Fig. S17 and S18). Moreover, the ATP and
ADP adducts of [Eu.3]+ could be discriminated by the variation in
spectral shape of the ∆J = 4 band; the ATP bound species is
characterized by two discernable lines centred at 688 and 697
nm respectively, whereas the ADP adduct showed at least 4
components within the ∆J = 4 region. The structurally related
complex [Eu.4]+ showed similar distinctive changes in the shape
of the ∆J = 4 band in the presence of ATP and ADP; however,
only minor increases in emission intensity took place (Fig. S17
and S18).
Figure 3. Change in emission spectra of [Eu.3]+ (5 µM) as a function of added
ATP (0–180 µM) in aqueous solution at pH 7.0; (Inset) Binding isotherm and fit
to the experimental data, for log Ka = 5.8 (± 0.1). Conditions: 10 mM HEPES
buffer, pH 7.0, λexc = 330 nm, 25°C.
Apparent binding constants were determined for complexes
[Eu.1–4]+ and a range of NPP anions by following the change in
the intensity ratio of the ΔJ = 2/ΔJ = 1 (605–630/580–600 nm)
emission bands as a function of anion concentration, followed by
a non-linear, least squares curve-fitting procedure based on a
1:1 binding model (Table 2 and Fig. S19–S49).‡ [Eu.3]+ showed
the strongest binding to ATP and ADP (log Ka = 5.8 and 5.7
respectively), approximately 10 times stronger than to AMP and
pyrophosphate (PPi), and 100 times stronger than to HPO42- and
Table 2. Apparent binding constants (log Ka) determined for [Eu.1–4]+ and
different anionic species in aqueous solution (10 mM HEPES, pH 7.0, 25°C).
[a]
Log Ka
Anion
[Eu.1]+
4.4
4.6
3.4
3.5
4.4
4.6
3.4
3.6
4.2
2.7
2.1
3.0
[Eu.2]+
3.0
3.3
3.3
2.4
n/d
n/d
n/d
n/d
n/d
n/d
n/d
n/d
[Eu.3]+
5.8
5.7
4.8
4.7
5.3
5.2
3.9
4.9
5.4
4.3
2.7
3.0
[Eu.4]+
3.8
3.8
3.5
3.4
n/d
n/d
n/d
n/d
n/d
n/d
n/d
n/d
-
HCO3 (log Ka = 2.7 and 3.0 respectively). [Eu.1]+ showed a
similar level of selectivity between ATP, ADP, AMP and PPi,
however the affinity for each anion was approximately one order
of magnitude lower, and HPO42- was bound weakly (log Ka = 2.7).
The higher affinity of [Eu.3]+ for ATP compared to [Eu.1]+ can be
attributed to replacement of the negatively charged carboxylate
donors in [Eu.1]+ with neutral carbonyl amide donors in [Eu.3]+,
increasing the electropositive nature of the Eu(III) ion, causing it
to interact more strongly with ATP.
ATP
ADP
AMP
PPi
GTP
GDP
GMP
UTP
UDP
UMP
HPO4
The selectivity of [Eu.1]+ and [Eu.3]+ for ATP and ADP over
monophosphate anions can be attributed to increased
Coulombic attraction as well as stabilizing hydrogen bonding
interactions between the coordinated phosphate fragment of
ATP (and ADP) and the amide N-H groups of the quinoline
chromophores (Figure 1). In support of this, [Eu.2]+ and [Eu.4]+,
which lack amide groups, showed significantly lower affinities
and essentially no selectivity between ATP, ADP, AMP and PPi.
Hence, binding of NPP anions to [Eu.1]+ and [Eu.3]+ is not
governed solely by favourable electrostatic interactions, but
involves additional stabilization via hydrogen bonding that leads
to enhanced selectivity towards ATP and ADP over
monophosphate species.
2-
-
HCO3
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Complexes [Eu.1]+ and [Eu.3]+ were found to bind to the
guanosine and uridine phosphate anions with a similar level of
selectivity to that determined for the adenosine phosphate
series; the general order of affinity was found to be
NTP≈NDP>NMP. The nucleoside moiety appears to contribute
towards anion binding, as both [Eu.1]+ and [Eu.3]+ bind to NMP
anions but show relatively weak affinity to HPO42- (log Ka = 2.1–
2.7 respectively), and bind to nucleoside diphosphate anions 10
times more strongly than to pyrophosphate. Due to the lack of
binding selectivity and small spectral responses exhibited for
[Eu.2]+ and [Eu.4]+ with adenosine phosphate anions, affinity
constants for other NPP anions were not determined.
Effect of Mg2+ ions in solution. The majority of intracellular
ATP exists in the form Mg-ATP2-.34 Therefore, a cellular imaging
probe for ATP should ideally be able to bind to the Mg-ATP2-
complex. Indeed, enzymes that require ATP as a substrate,
such as ATPases and kinases, utilize Mg2+ ions to provide
additional stabilization of the ATP-enzyme complex.35 The
binding interaction between Mg2+ ions and ATP in water at
physiological pH is approximately 50 times stronger than the
interaction with ADP, with log Ka (Mg-ATP) = 4.2 and log Ka (Mg-
ADP) = 3.6.36 The presence of Mg2+ ions in aqueous solution
was expected to influence the ability of complexes [Eu.1]+ and
[Eu.3]+ to discriminate between ATP and ADP. Indeed,
previously reported synthetic receptors have shown limited
discrimination between ATP and ADP at enzyme relevant
concentrations of Mg2+ ions.26, 37 We showed recently that [Eu.1]+
Data in support of 1:1 binding between [Eu.3]+ and ATP or GTP
was provided by high resolution (ESI) mass spectrometry, which
gave intense signals at m/z
= 741.6588 and 749.6564,
corresponding to the doubly charged species [Eu.3+ATP+5H]2+
and [Eu.3+GTP+5H]2+ respectively (Fig. S50 and S51).§ Mass
spectral data showing the formation of stable ternary adducts of
[Eu.1]+ and ATP or ADP was reported previously.31
can
discriminate
A
30
25
20
15
10
5
B
C
30
25
20
15
10
5
35
30
25
20
15
10
5
ATP
ADP
ATP
2-
AMP, HPO4
[Eu.3]+
0
0
0
0
0.5
1
1.5
2
2.5
3
3.5
550
575
600
625
650
675
700
550
575
600
625
650
675
700
Wavelength / nm
Wavelength / nm
[ATP] / mM
Figure 4. Selective spectral response of [Eu.3]+ for ATP in the presence of Mg2+ ions. (A) Change in emission spectra of [Eu.3]+ (5 µM) in the presence of 2 mM
2-
ATP, ADP, AMP and HPO4 in a fixed background of 5 mM MgCl2; (B) Variation in emission spectra of [Eu.3]+ upon addition of ATP (0–3 mM) in a fixed
background of 5 mM MgCl2; (C) Increase in intensity of the ΔJ = 2 band (605–630 nm) of [Eu.3]+ as a function of ATP concentration. Conditions: 10 mM HEPES
buffer, pH 7.4, λexc = 330 nm, 25°C.
constants were determined to be log Ka = 4.6 and 3.8 for ADP
effectively between ATP and ADP in a buffered aqueous
solution containing 3 mM Mg2+ ions: adding 1 mM ADP caused
an 8-fold increase in overall Eu(III) emission intensity compared
to a much smaller 2.5-fold intensity increase in the presence of 1
mM ATP.24 Excellent discrimination between ATP and ADP was
attributed to the slightly higher affinity of [Eu.1]+ for ADP (Table
1) as well as the higher competition between Mg2+ and ATP
compared to ADP. This allowed the change in the ratio of
ATP/ADP to be dynamically followed during the course of a
kinase-catalyzed phosphorylation reaction.
and AMP respectively, approximately 1 order of magnitude lower
than those determined in the absence of MgCl2.
A titration of ATP in a fixed background of 5 mM Mg2+ ions
generated an isotherm consistent with the occurrence of two
distinct emissive species (Figure 4C). Incremental addition of 0–
1.5 mM ATP gave rise to a 4-fold increase in emission intensity
of the ΔJ = 2 band, and subsequent addition of 1.5–3.0 mM ATP
induced a further 3-fold enhancement in Eu(III) emission, with no
discernable change in spectral form. This can be ascribed to the
formation of two discrete ternary adducts, each exhibiting a
similar coordination environment at the Eu(III) ion. It is
hypothesized that [Eu.3]+ binds to either ATP or the Mg-ATP2-
complex; with the binding geometry between [Eu.3]+ and Mg-
ATP2- being similar to that of ATP, and stabilization of the
negatively charged triphosphate fragment provided by the Mg2+
ion (Figure 1B). During the ATP titration, the concentration of
Mg-ATP2- increases, leading to the formation of a highly
emissive ternary complex. High resolution mass spectrometric
data supported binding of [Eu.3]+ to Mg-ATP2-, giving an intense
signal at 752.6439 corresponding to the doubly charged species
[Eu.3+ATP+Mg+3H]2+, which was in excellent agreement with
the calculated isotopic distribution (Fig. S52). A titration of ATP
Complex [Eu.3]+ showed a different discriminatory behaviour
between ATP and ADP in the presence of Mg2+ ions. Figure 4A
shows the emission spectral response of [Eu.3]+ in the presence
of 2 mM ATP, ADP, AMP and HPO42- in a fixed background of 5
mM Mg2+ ions. Adding 2 mM ATP resulted in a substantial 24-
fold enhancement in intensity of the ΔJ = 2 emission band,
whereas ADP caused
a
smaller 13-fold increase in
luminescence. Only minor changes in emission spectra took
place in the presence of AMP and HPO42-. Titrations of ADP and
AMP into an aqueous solution containing [Eu.3]+ and 5 mM Mg2+
ions resulted in standard hyperbolic curves that were fitted to a
1:1 binding model (Fig. S56 and S57). Apparent association
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emission intensity ratio at 614/594 nm versus mole fraction of ADP, in the
absence of apyrase (total nucleotide, [ATP]+[ADP], is constant at 2 mM).
Conditions: 10 mM HEPES, pH 7.0, [Eu.3]+ (5 µM), apyrase (80 or 160 mU),
ATP (2 mM), MgCl2 (5 mM). λexc= 330 nm, 25°C.
in a fixed background of NaCl (100 mM) revealed a classic
isotherm for a simple 1:1 binding system, with an apparent
binding constant of log Ka = 5.0 (Fig. S58). This confirms that
the changes in emission spectra of [Eu.3]+ in the presence of
Mg2+ ions cannot be simply a result of the additional ionic
strength of the medium, rather it is the specific interactions
between Mg2+, ATP and the Eu(III) complex that modulates the
equilibrium speciation and resulting emission response.
Given that [Eu.3]+ binds strongly to ATP, the possibility that the
probe lowers the effective concentration of ATP must be
considered, as this would influence the rate of reaction. However,
anion binding to [Eu.3]+ is fast and reversible, and due to the
highly sensitive nature of the luminescence response, the
amount of [Eu.3]+ required to monitor the ATPase assay is much
lower (5 µM) compared to the concentration of ATP (2 mM). This
ensures that the rate of ATP hydrolysis is not perturbed by the
presence of [Eu.3]+.
Figure 5C shows the linear decrease in emission intensity ratio
at 614/594 nm during the initial stages (0–9 minutes) of each
enzyme reaction. Reducing the amount of enzyme from 160 mU
to 80 mU resulted in a decrease in the initial rate of ATP
hydrolysis by a factor of two. Thus, [Eu.3]+ is able to directly and
continuously monitor ATPase activity by providing an
instantaneous and ratiometric luminescent signal, without the
need to chemically modify the enzyme or its substrate.38
Monitoring ATPase Activity in Real-Time. Having established
that [Eu.3]+ exhibits distinctive spectral responses towards ATP
and ADP in a background of 5 mM Mg2+ ions (Figure 4A), we
demonstrated the ability of the Eu(III) complex to monitor the
apyrase-catalyzed hydrolysis of ATP to ADP in real-time.
Apyrase is an enzyme that catalyzes the conversion of ATP into
ADP, releasing energy in the process. To a buffered aqueous
solution (10 mM HEPES, pH 7.0, 5 mM MgCl2) containing
[Eu.3]+ (5 µM) and ATP (2 mM) was added different amounts of
apyrase (80–160 mU, ATP/ADP selectivity ratio, 10:1). The
conversion of ATP to ADP and HPO42- caused a time-dependent
decrease in emission intensity of the ΔJ = 2 band centred at 614
nm and a smaller increase in intensity at 594 nm within the ΔJ =
1 band (Figure 5A). These changes in spectral form are
consistent with competitive displacement of the bound ATP by
ADP, as the concentration of ADP increases. Plots of the
emission intensity ratio at 614/594 nm as a function of time
revealed that the rate of ATP hydrolysis was directly proportional
to the amount of enzyme added (Figure 5B). The background
reaction in the absence of enzyme showed essentially no
change in emission intensity during the time frame of the
experiment. The ratiometric changes in emission intensity
observed during the enzyme reaction were in good agreement
with those obtained in a simulated ATPase reaction, in which the
ratio of ATP/ADP was varied systematically (Figure 5D).
Evaluating Affinity to Protein. Encouraged by the ability
[Eu.3]+ to monitor the enzymatic conversion of ATP to ADP in
real-time, we wished to evaluate the ability of [Eu.3]+ monitor
fluctuations in ATP levels in living mammalian cells. A cellular
imaging probe for ATP must be able to operate in the presence
of proteins. Emissive metal coordination complexes are known
to interact non-covalently with proteins, often causing quenching
or enhancement of emission.39 We measured the affinity of
complexes [Eu.1]+ and [Eu.3]+ for human serum albumin (HSA),
the most abundant protein in human blood plasma. Addition of
HSA (0–0.4 mM) to [Eu.1]+ at pH 7.0 resulted in a 3.5 fold
increase in overall Eu(III) emission intensity and distinctive
changes in spectral form (Figure 6A). A plot of the intensity ratio
of the ΔJ = 2 / ΔJ = 1 emission bands versus HSA concentration
allowed an estimation of the association constant, log Ka = 4.8
(Kd = 16 µM) (Fig. S59). Luminescence lifetimes for the protein
bound species in H2O and D2O were found to be 0.92 and 1.73
ms respectively, corresponding to a hydration state, q = 0. Thus,
binding of [Eu.1]+ to HSA involves displacement of the bound
water molecule, possibly by coordination of an aspartate or
glutamate residue of HSA.
A
B
50
40
30
20
10
0
10
8
[Eu.1]+
HSA (0−0.4 mM)
+
ATP
6
4
[Eu.3]+
0.4 mM HSA
+
2
0
550
575
600
625
650
675
700
550
575
600
625
650
675
700
Wavelength / nm
Wavelength / nm
Figure 5. Continuous monitoring of apyrase catalyzed hydrolysis of ATP. (A)
Change in emission spectra of [Eu.3]+ (570–630 nm) during the apyrase (80
mU) catalyzed hydrolysis of ATP to ADP; (B) Time-trace plots showing the
decrease in emission intensity ratio at 614/954 nm in the presence of different
amounts of apyrase (0, 80 or 160 mU); (C) Linear change in emission intensity
of [Eu.3]+ during the initial stages of the enzyme reaction; (D) Plot of the
Figure 6. Differential responses of [Eu.1]+ and [Eu.3]+ to human serum
albumin. (A) Change in emission spectra of [Eu.1]+ in the presence of
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increasing amounts of HSA (0–0.4 mM); (B) Increase in emission spectra of
[Eu.3]+ upon titration of ATP (0–0.5 mM) in a fixed background of 0.4 mM HSA.
Conditions: HEPES buffer (10 mM, pH 7.0), λexc = 330 nm, 25°C.
Eu(III) emission intensity was approximately linear over a
biologically relevant ATP concentration range of 0.3 to 8.0 mM,
corresponding to an increase in the ATP/ADP ratio from 3 to 80
(Figure 7, inset). These competition experiments strongly
indicated that [Eu.3]+ could be used to signal changes in the
In sharp contrast, incremental addition of HSA to [Eu.3]+ caused
a minor (10%) increase in Eu(III) emission intensity and no
change in spectral form (Fig. S60). Analysis of the luminescence
lifetimes of [Eu.3]+ in the presence of HSA in H2O and D2O
suggested displacement of the bound water molecule. A lower
apparent binding affinity was estimated between [Eu.3]+ and H
SA (log Ka = 3.2), which is approximately 2.5 orders of
magnitude lower than the binding constant determined for this
complex and ATP, under the same conditions. The minimal
interaction observed between [Eu.3]+ and HSA could be
tentatively attributed to the presence of two ancillary carboxylate
groups in the macrocyclic ligand, which could minimize the
occurrence of hydrophobic interactions between the Eu(III)
complex and the protein. Gratifyingly, addition of increasing
amounts of ATP (0–0.5 mM) to [Eu.3]+ in a fixed background of
0.4 mM HSA gave rise to a measurable 3-fold increase in
emission intensity of the ΔJ = 2 band (Figure 6B), consistent
with competitive displacement of the bound protein from [Eu.3]+,
upon binding to ATP. The apparent binding constant was
estimated to be log Ka = 4.0 (Figure S61), around two orders of
magnitude lower than that determined between [Eu.3]+ and ATP
in the absence of protein. Conversely, [Eu.1]+ was unable to
signal the presence of ATP under the same conditions.
ATP/ADP ratio in cellulo, allowing
processes to be followed in real-time.
a range of biological
20
17
15
16
ATP
12
10
12
7
0
2
4
6
8
8
[ATP] / mM
4
0
570
595
620
645
670
695
720
Wavelength / nm
Figure 7. Detection of ATP in a simulated intracellular fluid. Change in
emission spectra of [Eu.3]+ (50 µM) upon titration of ATP over the
physiological concentration range (0–8 mM); (Inset) Linear increase in Eu(III)
emission intensity at 614 nm as a function of added ATP. Conditions: HEPES
buffer (10 mM, pH 7.4), ADP (0.1 mM), GTP (0.5 mM), UTP (0.5 mM),
Na4P2O7 (0.05 mM), Na2HPO4 (0.9 mM), NaCl (10 mM), KCl (110 mM), CaCl2
(2.5 mM), MgCl2 (8 mM), Na2SO4 (0.5 mM), sodium lactate (2.3 mm), sodium
citrate (0.13 mM), glutathione (3 mM), HSA (0.4 mM). λexc = 330 nm, 25°C.
ATP Recognition in Simulated Intracellular Fluid. The above
competition experiments, involving biologically relevant amounts
of protein and Mg2+ ions, encouraged us to examine the ability of
[Eu.3]+ to detect ATP in an aqueous medium that mimics the
complex ionic environment within cells. In a healthy cell, the
most abundant NPP anion is ATP, which is estimated to be
present in concentrations between 1–5 mM (average 2.5 mM).1,
Before undertaking cellular imaging studies, the pH dependence
of the emission response of [Eu.3]+ in the presence of 2 mM
ATP was assessed. The emission intensity and spectral form
was essentially unchanged between pH 6.4 and 8.6 (Fig. S62),
suggesting that the luminescence signal should not be affected
by pH fluctuations around normal mitochondrial or cytoplasmic
pH, estimated to be near 7.3 and 8.0, respectively.34 This is
significant, as previously reported ATP-selective imaging probes
have been shown to be sensitive to changes in intracellular pH.6,
12
The majority of ATP is generated by the mitochondria by
oxidative phosphorylation. Importantly, ADP is maintained at a
significantly lower concentration (50–200 µM), the majority of
which is bound strongly to protein, such that the ATP/ADP ratio
ranges between 5 and 100.40 This ATP/ADP ratio acts as a
critical modulator of a variety of cellular events. Other NPP
anions including GTP, UTP and CTP are estimated to be
present in concentrations 5-fold lower than that of ATP. An
intracellular probe must be able to respond selectively to ATP
under these conditions.
Using the above concentrations of NPP anions, we prepared a
simulated intracellular fluid based on a modified Krebs saline
solution, which is commonly used in cell and tissue culture
experiments. The solution contains ADP (0.1 mM), GTP (0.5
mM) and UTP (0.5 mM), NaH2PO4 (0.9 mM), PPi (0.05 mM), KCl
(110 mM), NaCl (10 mM), CaCl2 (2.5 mM), MgCl2 (8 mM),
Na2SO4 (0.5 mM), sodium lactate (2.3 mM), sodium citrate (0.13
mM), glutathione (3 mM) and HSA (0.4 mM), buffered at pH 7.4
using 10 mM HEPES. Under these conditions, incremental
addition of 0–6 mM ATP to [Eu.3]+ (50 µM) resulted in a
reproducible 55% enhancement in emission intensity of the ΔJ =
2 band centred at 614 nm (Figure 7). Crucially, the increase in
7c
Additionally, certain existing methods for measuring
intracellular ATP are dependent on dissolved oxygen
concentration (e.g. the bioluminescent luciferase reaction).9 To
confirm that [Eu.3]+ is not sensitive to changes in dissolved
oxygen, emission spectra were recorded for [Eu.3]+ alone, and in
the presence of 2 mM ATP, in air-equilibrated and degassed
aqueous solution. The Eu(III) complex showed less than 5%
variation in emission intensity under these conditions (Figure
S63), with no significant change in emission intensity being
observed after bubbling of the air-equilibrated sample with
oxygen gas for 10 minutes.
Imaging Mitochondrial ATP In Living Cells
Cellular Uptake and Localization Studies. The cellular uptake
behaviour of [Eu.3]+ was examined in NIH-3T3 cells using
fluorescence and laser scanning confocal microscopy (LSCM).42
Incubation of [Eu.3]+ (50 µM) in NIH-3T3 cells for 2 hours
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resulted in uptake of the Eu(III) complex and predominant
localization to the mitochondria (λexc 355 nm, λem 605–720 nm),
verified by co-localization studies using MitoTracker Green (λexc
488 nm, λem 500–530 nm, Pearson’s correlation coefficient, P =
0.91) (Figure 8). The preferential distribution of [Eu.3]+ in the
mitochondria could be tentatively attributed to the overall
positive charge of the Eu(III) complex and the amphipathic
nature of the macrocyclic ligand.16 Imaging was possible over
extended time periods (up to 8 hours), during which time the
brightness of the observed images did not vary significantly
(±10%) and the cells appeared to be healthy and proliferating
(see supporting information for detailed analysis of probe
brightness within cells).
time period (Fig. S64). ICP-MS studies of cells incubated with
[Eu.3]+ and staurosporine for 2 hours revealed essentially no
change in the concentration of accumulated Eu(III) metal,
compared to cells grown in the absence of the kinase inhibitor
(Figure S65). Therefore, treatment with staurosporine does not
appear to perturb cellular uptake or efflux of the Eu(III) complex
from cells.
A
B
C
A
0 min
30 min
Figure 8. LSCM images showing mitochondrial localization of the Eu(III)
complex. (A) Localization of [Eu.3]+ (50 µM) in the mitochondria of NIH-3T3
cells (λexc 355 nm, λem 605–720 nm); (B) MitoTracker Green (λexc 488 nm, λem
500–530 nm); (C) RGB merged image verifying co-localization (P = 0.91).
60 min
90 min
Cytotoxicity and vitality studies were undertaken for [Eu.3]+ at 24
hours using image cytometry assays, involving DAPI and
Acridine Orange stains, which revealed an IC50 value of greater
than 200 µM. Considering that the incubation concentration of
[Eu.3]+ is four times lower than this value, it can be assumed
that the Eu(III) complex is essentially non-toxic during the time
frame of the imaging experiments. Analysis of ICP-MS data
showed that for 4 × 106 NIH-3T3 cells incubated with [Eu.3]+ (50
µM) for 2 hours, a given cell contained 69 µM (±5%) of Eu(III)
metal, consistent with the accumulation of [Eu.3]+ within the
mitochondria during the incubation period.
B
180
170
160
150
140
130
120
110
100
90
Staurosporin
(0 min refers to 2h
probe pre-incubation)
Imaging elevated mitochondrial ATP levels. We evaluated
the ability of [Eu.3]+ to visualize changes in the concentration of
ATP in NIH-3T3 cells upon treatment with staurosporine, a
broad-spectrum inhibitor of kinase activity that eventually
induces cell apoptosis. Figure 9A shows time-lapsed images of
cells stained with [Eu.3]+ (50 µM) before (0 min) and after
treatment with staurosporine (10 nM). The images revealed a
gradual increase in the observed emission intensity (λem 605–
720 nm) in the mitochondria over a 90-minute period. At 60
minutes post-staurosporine treatment, the Eu(III) emission
intensity had increased by approximately 65%, after which time
the observed signal reached a plateau (Figure 9B). No obvious
signs of cell apoptosis were evident during the time scale of the
experiment, and the Eu(III) complex remained localized to the
mitochondria. As a control, cells incubated with [Eu.3]+ without
the addition of staurosporine were examined, revealing less than
10% variation in the Eu(III) emission intensity over the same
0
15 30 45 60 75 90 105 120 135 150 165 180
time / [min]
Figure 9. Real-time monitoring of elevated levels of ATP in mitochondria. (A)
Time-lapsed LSCM images of NIH-3T3 cells stained with [Eu.3]+ (50 µM, λexc
355 nm, λem 605–720 nm) before (0 min) and after treatment with
staurosporine (10 nM),
a potent inhibitor of protein kinases; (B) Time-
dependent increase in emission intensity (605–720 nm) of cells stained with
[Eu.3]+ following treatment with staurosporine (10 nM).
These imaging experiments indicate that the concentration of
ATP in the mitochondrial region gradually increased during the
preapoptotic period (150 min) following treatment with
staurosporine. This is in agreement with previous reports of
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elevated ATP levels in the cytosol7 and mitochondria5a upon
incubation with staurosporine. The mitochondria remained intact
during this time, suggesting that mitochondrial functional
integrity is important during the early stages of apoptosis. This is
consistent with the notion that apoptosis is an energy requiring
process; the concentration of mitochondrial ATP increases to
supply chemical energy for a variety of intracellular processes,
such as enzymatic hydrolysis of macromolecules.7
concentration of ATP in the mitochondria upon incubation with
an ATP synthesis inhibiter (KCN) and a kinase activity inhibitor
(staurosporine), and could provide a versatile imaging tool for
studying cell metabolism and other ATP-requiring processes in
real-time, within a targeted organelle.
A
Monitoring depleted ATP levels in living cells. Next, the
change in mitochondrial ATP levels was monitored after
treatment of cells with potassium cyanide, an inhibitor of
oxidative phosphorylation.6a Initially, NIH-3T3 cells were
incubated with [Eu.3]+ in a glucose-free growth medium, in order
to inhibit ATP production via glycolysis. Under these conditions,
a 20% decrease in emission intensity was observed after 2
hours compared to cells grown in the presence of glucose (Fig.
S66). Subsequent addition of potassium cyanide (0.1 mM)
resulted in a significant and rapid decrease in the observed
emission intensity, with almost 85% of luminescence lost after
10 minutes (Figure 10). These results suggest that mitochondrial
ATP is depleted substantially in the presence of KCN, due to
inhibition of oxidative phosphorylation, which is consistent with
previous studies that show a reduction in intracellular ATP under
similar conditions.6a ICP-MS analysis of cells incubated with
[Eu.3]+ for 30 minutes with KCN under glucose starvation
conditions showed less than 10% variation in the concentration
of accumulated Eu(III) metal, relative to untreated cells grown in
the presence of glucose. Therefore, the possibility that
incubation with KCN promotes efflux of [Eu.3]+ from the cells can
be ruled out.
0 min
5 min
10 min
20 min
B
80
60
40
20
0
KCN
(0 min refers to 2h
probe pre-incubation
Glucose-free)
The inhibition of oxidative phosphorylation may result in
perturbation of mitochondrial bicarbonate concentration, as
bicarbonate stimulates the enzymatic production of cyclic AMP,
which in turn activates mitochondrial kinase activity, regulating
ATP production.43 Having already shown that the binding of
bicarbonate to [Eu.3]+ is approximately 100 times weaker than
that of ATP in buffered aqueous solution (Table 1), we wished to
investigate further the effect of varying intracellular bicarbonate
concentration on the emission intensity of [Eu.3]+. Following a
procedure described previously,19a NIH-3T3 cells were loaded
with [Eu.3]+ (50 µM) and the percentage of atmospheric CO2 in
the cell imaging chamber was varied between 2% and 7% CO2.
The Eu(III) emission intensity observed in the mitochondria of
NIH-3T3 cells after an incubation period of 60 minutes was
compared to those obtained when the incubation was performed
at a constant atmospheric CO2 of 5% for the same time period.
The LCMS images revealed less than 5% modulation of
emission intensity upon variation of the atmospheric CO2 (Figure
S67). These microscopy experiments indicate that [Eu.3]+ does
not respond significantly to fluctuations in the equilibrium cellular
bicarbonate concentration, which is expected considering the
lower apparent binding constant of [Eu.3]+ to bicarbonate, and
the larger emission intensity of the ATP-bound Eu(III) complex.
Taken together, these live-cell imaging experiments
demonstrate that [Eu.3]+ is capable of signalling changes in the
0
5
10
15
20
time / [min]
Figure 10. Real-time monitoring of depleted mitochondrial ATP levels. (A)
Time-lapsed LSCM images of NIH-3T3 cells stained with [Eu.3]+ (50 µM, λexc
355 nm, λem 605–720 nm) under glucose starvation conditions, before (0 min)
and after treatment with KCN (0.1 mM), an inhibitor of oxidative
phosphorylation; (B) Time dependent decrease in emission intensity of cells
stained with [Eu.3]+ following treatment with KCN (0.1 mM).
Conclusions
We have developed a discrete, cationic Eu(III) complex for
monitoring dynamic changes in the concentration of ATP within
the mitochondria of living cells. A series of luminescent Eu(III)
complexes, [Eu.1–4]+, was synthesised that bind reversibly to
nucleoside polyphosphate anions with differential affinities in
buffered aqueous solution at physiological pH. The affinity of the
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Eu(III) complexes towards NPPs and the magnitude of the
emission spectral response is tunable by making modifications
to the ligand structure. Hydrogen bond donor groups were
introduced into the quinoline units of [Eu.1]+ and [Eu.3]+ to
enhance selectivity towards ATP and ADP over monophosphate
anions. Complex [Eu.3]+, bearing two neutral amide donors,
binds most strongly to ATP (log Ka = 5.8), forming a stable
ternary complex that exhibits intense, long-lived Eu(III)
luminescence. [Eu.3]+ can discriminate effectively between ATP,
ADP and AMP in a competitive aqueous medium that simulates
the complex ionic environment present in cells. The probe
spectroscopy. RP would like to acknowledge the Royal Society
URF.
Keywords: adenosine triphosphate (ATP) • live cell imaging •
anion receptor • lanthanide • luminescence
References:
‡ Anion affinity constants for [Eu.3]+ were determined by plotting the change in
the ratio of the ΔJ = 2/ΔJ = 0 (605–630/575–580 nm) emission bands as a
function of anion concentration.
§ Major ESI mass spectral signals were also obtained for the ternary adducts
of [Eu.4]+ bound to 1 molecule of ATP or GTP (Figures S53 and S54).
provides
a linear, ratiometric emission response that is
proportional to the ratio of ATP/ADP, enabling the enzymatic
hydrolysis of ATP to ADP to be precisely monitored in real-time.
Cellular localization studies revealed that [Eu.3]+ preferentially
stains the mitochondria of mammalian cells, and is retained
within this organelle over extended time periods. We have
shown that [Eu.3]+ can detect an increase in mitochondrial ATP
concentration following treatment of cells with the kinase
inhibitor staurosporine. Additionally, [Eu.3]+ was able to visualize
a rapid decrease in mitochondrial ATP following treatment with
KCN, an inhibitor of oxidative phosphorylation. Complex [Eu.3]+
offers a several advances in performance compared to existing
ATP-responsive probes, including a luminescence signal that is:
1) sensitive to ATP within the biologically relevant concentration
range (1–5 mM); 2) minimally perturbed by changes in pH,
dissolved oxygen or the presence of protein; and 3) sufficiently
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long-lived
to
avoid
interference
from
UV-induced
autofluorescence arising from biomolecules. Such probes offer a
new versatile tool for studying metabolism and a range of
biological processes involving ATP, with subcellular resolution.
The strategy employed here will be explored further to design
Eu(III) probes capable of monitoring spatio-temporal ATP
dynamics within different cellular compartments, providing a
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This work was supported by a Wellcome Trust Seed Award
(204500/Z/16/Z) and a Royal Society of Chemistry Research
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Romain Mailhot, Thomas Traviss-
Pollard, Robert Pal and Stephen J.
Butler*
Page No. – Page No.
Cationic Europium Complexes for
Visualizing Fluctuations in
Mitochondrial ATP Levels in Living
Cells
We report a short series of cationic Eu(III) complexes for visualising dynamic
changes in the concentration of ATP within the mitochondria of living cells. Complex
[Eu.3]+ binds reversibly to ATP and responds linearly to this anion within the
physiological range (1–5 mM). The probe was used to visualize elevated ATP levels
following treatment with a broad spectrum kinase inhibitor, staurosporine, as well as
depleted ATP levels upon treatment with KCN under glucose starvation conditions.
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