Water-soluble triarylboron compound for ATP imaging in vivo using analyte-induced finite aggregation

Article abstract of DOI:10.1002/anie.201403918

Adenosine 5′-triphosphate (ATP) is a multifunctional molecule that participates in many important biological processes. Currently, fluorescence indicators for ATP with high performance are in demand. Reported herein is a novel water-soluble triarylboron compound which displays an apparent ATP-dependent fluorescence enhancement when dispersed in water. It can selectively recognize ATP from other bioactive substances in vitro and in vivo. The ATP-induced finite aggregation endows the indicator with appreciable photostability and superior tolerance to environmental electrolytes. This indicator has been successfully applied to the ATP imaging in NIH/3T3 fibroblast cells. The difference in the ATP levels within the membrane and cytosol is clearly visible. Tracking device: The triarylboron compound in the scheme displays an apparent ATP-dependent fluorescence enhancement. The analyte, ATP, induces finite aggregation and endows the indicator with appreciable photostability and superior tolerance to environmental electrolytes, thus leading to its successful application to the monitoring of ATP levels in vitro and in vivo. ATP=adenosine 5′-triphosphate.

Full text of DOI:10.1002/anie.201403918

Angewandte
Chemie
DOI: 10.1002/anie.201403918
Fluorescence Probes
Water-Soluble Triarylboron Compound for ATP Imaging In Vivo Using
Analyte-Induced Finite Aggregation**
Xiaoyan Li, Xudong Guo, Lixia Cao, Zhiqing Xun, Shuangqing Wang, Shayu Li,* Yi Li,* and
Guoqiang Yang*
Abstract: Adenosine 5’-triphosphate (ATP) is a multifunc-
tional molecule that participates in many important biological
processes. Currently, fluorescence indicators for ATP with high
performance are in demand. Reported herein is a novel water-
soluble triarylboron compound which displays an apparent
ATP-dependent fluorescence enhancement when dispersed in
water. It can selectively recognize ATP from other bioactive
substances in vitro and in vivo. The ATP-induced finite
aggregation endows the indicator with appreciable photo-
stability and superior tolerance to environmental electrolytes.
This indicator has been successfully applied to the ATP
imaging in NIH/3T3 fibroblast cells. The difference in the ATP
levels within the membrane and cytosol is clearly visible.
and NMR spectroscopy, of cell extracts.[4] In recent years,
chemiluminescent recognition by luciferase and other
enzymes have been developed.[5] However, the chemilumi-
nescence method has a few limitations. First, suitable pH
level, sufficient substrate, luciferin, and oxygen are indispen-
sable in the recognition procedure. Second, the consumption
of the analyte ATP and longer detection time make real-time
observation difficult in practice.
Fluorescence indicators with high sensitivity, good selec-
tivity, short response time, and the advantage of direct
observation, are a powerful tool for the identification of
substances in biological systems.[6] Despite ATPꢀs significance
in essential cellular processes, there are fewer studies on the
development of effective ATP indicators in comparison to
those on bioactive ions (e.g., Zn2+, Ca2+), possibly because of
the difficulty of the mission. To date, biomolecule-based ATP
indicators, such as luminophore-decorated ATP-binding pro-
teins, DNA templates, and aptamers, have been successively
implemented in eukaryotic cells,[7] but the expensive and
difficult purification process significantly limits their practical
applications. Passable ATP detections have also been ach-
ieved through low-cost chemical indicators which employ
hydrogen bonding, electrostatic interactions, or metal–oxygen
coordination,[8] though the interference from water, electro-
lytes, or partially dephosphorylated production of ATP are
inevitable in practice.
In addition to the limitations, photostability under con-
tinual irradiation is a common challenge for conventional
fluorescence indicators.[9] A reliable indicator must be photo-
stable during the detection period, but apparent photo-
bleaching is frequently observed for organic fluorophores at
low concentrations. This situation is even more serious when
the illumination source is a laser beam. Unfortunately, the
low-concentration dye and the laser source are the most
common requirements for fluorescence live-cell imaging. In
contrast to other photophysical properties, such as absorption
energy, emission wavelength, and fluorescence efficiency,
photostability is an intrinsic property which is hard to improve
even through structural modifications. Although fluorophores
show a substantially slower bleaching rate in a solid matrix or
other environments protected by a rigid shell,[10] the inter-
actions of an indicator with an analyte are also hindered.
As a result of reducing the influences from the surround-
ing environment, nanoaggregates formed by organic fluoro-
phores generally possess higher photostability compared to
that of monodisperse molecules with the same molecular
structure.[11] And a few nanoaggregates have been applied in
long-term cell tracking.[12] However, the fluorescence quench-
ing induced by aggregation is unavoidable for most fluoro-
A
denosine 5’-triphosphate (ATP), one of the best known
biological compounds, is a multifunctional molecule found in
all living organisms. As the major energy molecule of cells,
ATP serves in a number of endothermic processes, including
biosynthesis, active transport, cell division, muscle contrac-
tion, etc.[1] ATP is also critical in signal transduction processes.
It is used as an intracellular precursor in kinase activity for
proteins or lipids to provide a phosphate group, as well as in
adenylate cyclase to produce the second messenger molecule
cyclic adenosine monophosphate (cAMP).[2] Extracellular
ATP serves as an activity-dependent signal for purinoceptors
in nerve-mediated responses.[3] Visualization of ATP levels
in vitro and in vivo can offer valuable information for precise
understanding of these biological processes.
Classic measurements of ATP are based on offline
analysis methods, such as HPLC, capillary electrophoresis,
[*] X. Y. Li, X. D. Guo, L. X. Cao, Dr. S. Q. Wang, Dr. S. Y. Li,
Prof. Dr. G. Q. Yang
Beijing National Laboratory for Molecular Sciences
Key Laboratory of Photochemistry, Institute of Chemistry
Chinese Academy of Sciences, Beijing 100190 (China)
E-mail: shayuli@iccas.ac.cn
gqyang@iccas.ac.cn
Z. Q. Xun, Prof. Dr. Y. Li
Key Laboratory of Photochemical Conversion and Optoelectronic
Materials, Technical Institute of Physics and Chemistry
Chinese Academy of Sciences, Beijing 100190 (China)
E-mail: yili@mail.ipc.ac.cn
[**] We are grateful for funding from the National Basic Research
Program (2013CB834505, 2013CB834703, 2011CBA00905, and
2009CB930802) and the National Natural Science Foundation of
China (grant nos 21233011, 91123033, 21273252, 21205122,
21261160488 and 21072196).
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201403918.
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phores because of the well-known aggregation-caused
quenching (ACQ), which is a short-range interchromophoric
energy migration and transfer.[13] To tackle the problems
fluorescence indicators, we propose herein, the novel
approach of analyte-induced finite aggregation. In this
approach, the indicator molecules are separated by non-
chromophoric analytes, so aggregation indeed happens but
the ACQ effect is avoided. Although this work is focused on
a novel ATP indicator, the approach could be extended to
other indicators with lipophilic luminogens.
Scheme S1 in the Supporting Information illustrates the
synthetic route to DPTB-IMI-EG. The DPTB-CH2Br moiety
was first synthesized and subsequently reacted with the water-
soluble IMI-EG moiety in DMF. The relatively long route is
a compromise to separate the 6- and 8-substituted pyrene
derivatives. Although DPTB is a large and lipophilic moiety,
DPTB-IMI-EG is completely soluble in very strongly polar
solvents such as DMSO, ethanol, and water, but is almost
insoluble in acetone, acetonitrile, and ethyl acetate. This
insolubility may be caused by the four positive charges of the
molecule, and was utilized for the final purification process.
DPTB-IMI-EG shows appreciable thermal stability with
a decomposition temperature of over 2208C.
A triarylboron luminogen dipyren-1-yl (2,4,6-triisopro-
pylphenyl)borane (DPTB) was synthesized and functional-
ized with the di(1H-imidazol-1-yl)methane dication (IMI)
and 1-ethoxy-2-(2-methoxyethoxy)ethane (EG) moieties
(DPTB-IMI-EG). DPTB is a typical charge-transfer (CT)
compound known for its intense luminescence.[14] DPTB-IMI-
EG also shows lower fluorescence efficiency in polar media,
just like other CT compounds. The IMI is the triphosphate-
targeting functional group which can discriminate between
tri-, di-, and monophosphate groups upon binding.[8b] And the
nonionic EG moiety improves the water solubility of the
whole molecule. Moreover, the electropositive IMI and the
lipophilic DPTB facilitate the entrance of the indicator into
cell. When DPTB-IMI-EG meets with ATP in an aqueous
environment, the electrostatic interaction will result in the
formation of charge-neutralization complexes which imme-
diately aggregate because of hydrophobic interactions
(Scheme 1). The aggregate is significantly lower in polarity
than physiological buffer because of the extrusion of water
and electrolytes. So the fluorescence of DPTB can be
increased by the so-called positive solvatokinetic effect.
Moreover, nonchromophoric ATP molecules are abundant
in the aggregates, thus preventing the approach of the
luminogens and avoiding the ACQ. The results provided in
this work indicate that DPTB-IMI-EG possesses high selec-
tivity for ATP, appreciable photostability, and superior
tolerance to environmental electrolytes. And this indicator
is also used to detect ATP in living systems.
Figures 1a,b shows the absorption and fluorescence
spectra of DPTB-IMI-EG with different amounts of ATP in
vitro. The absorption band around l = 420 nm decreases and
shows a bathochromic shift, and an isosbestic point is
observed at l = 430 nm. A typical tail absorbance becomes
Figure 1. a) UV absorption and b) fluorescence spectral changes of
DPTB-IMI-EG (10 mm) upon addition of the sodium salt of ATP (1–
5000 mm). c) Photograph of DPTB-IMI-EG in the presence and absence
of ATP (1 mm). d) Fluorescence titration curve of DPTB-IMI-EG upon
the addition of AMP (blue), ADP (red), and ATP (black). I/I0 means
the fluorescence intensity (l=510 nm) changes and I0 is the initial
fluorescence intensity (l=510 nm) of DPTB-IMI-EG in the absence of
nucleotides. Conditions: lex =440 nm, [DPTB-IMI-EG]=10 mm, pH 7.4
(10 mm HEPES), 378C.
more obvious in the long-wavelength region, thus implying
the formation of aggregates, which correlates well with
dynamic light scattering (DLS) and scan electron microscopy
(SEM) measurements (see Figure S2). The presence of ATP is
more apparent through luminescence (Figure 1c). Upon the
addition of ATP, the maximum emission wavelength shows
a slight hypsochromic shift and the fluorescence efficiency
gradually increases (Table 1). It is crucial that the absorbance
of the DPTB-IMI-EG/ATP composite is slightly longer than
that of individual molecules, so that the fluorescence differ-
ence in the absence and presence of ATP can be amplified to
over 12-fold when the excitation wavelength is l = 440 nm.
This amplification increases the detection contrast and
improves the recognition sensitivity. The apparent dissocia-
Scheme 1. The ATP indicator system.
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Table 1: Quantum yield F of DPTB-IMI-EG at different ATP levels.
c [mm]
F
0
2
5
10
20
50
0.057
100
0.293
0.072
200
0.311
0.097
500
0.334
0.146
1000
0.359
0.182
2000
0.383
0.231
5000
0.378
c [mm]
F
tion constant (Kd) of the composite is determined as 51 mm by
fitting plots of fluorescence intensity against ATP concen-
trations using the Hill equation. Thus, DPTB-IMI-EG can
reliably detect ATP at levels higher than 0.1 mm.
As the partially dephosphorylated products of ATP,
adenosine 5’-diphosphate (ADP) and adenosine 5’-mono-
phosphate (AMP), are always present to some degree in any
living cell, we further examined the specific characteristics of
DPTB-IMI-EG selectivity for ATP over ADP and AMP
in vitro. ADP results in qualitatively similar, but smaller
changes in the spectra (Figure 1d). For instance, the influence
of 200 mm ADP on the fluorescence intensity is still smaller
than that from ATP at 10 mm. The apparent dissociation
constant (Kd) of DPTB-IMI-EG/ADP is determined as
460 mm by fitting the rising part of the ADP plot in Figure 1d,
and means that compared to ATP, DPTB-IMI-EG exhibits
about a ninefold lower affinity for ADP. The fluorescence
enhancements for an ATP solution and several ATP/ADP
mixtures were also compared (see Figure S3), and taking into
account the fact that ATP is five- to tenfold higher in
concentration than ADP in living organisms,[15] we can
conclude that ADP has no apparent influence on the test
results when using the indicator DPTB-IMI-EG. The spectral
changes with the addition of AMP are small enough to be
ignored. The above results indicate that DPTB-IMI-EG as
a fluorescence indicator has sufficient specificity for ATP
under physiological conditions.
As ATP has several negatively charged groups under
physiological conditions, it may chelate metals and other
electrolytes with very high affinity.[16] The intracellular
environment is complex, with large amounts of anions,
cations, and many other charged biomolecules.[17] The inter-
ference from electrolytes is an ongoing design challenge for
ATP indicators and little progress has been made in this
respect. In our work, we recorded the fluorescence spectra of
DPTB-IMI-EG with and without ATP in the presence of
essential ions, amino acids, and proteins under physiological
conditions. Three typical amino acids, Gly, Glu, and Lys, each
with a different isoelectric point and two intracellular
proteins, RNase A and chymotrypsinogen, were chosen as
representative biomolecules and bio-macromolecules. As can
be seen from Figure 2a, only very slight fluorescence changes
are observed when the indicator or indicator–ATP is exposed
to these charged substances. This excellent tolerance for
environmental electrolytes is in line with predictions. The
neutral DPTB-IMI-EG/ATP consists of a large lipophilic
section and a hydrophilic ether chain, and is similar to the
structure of nonionic surfactants known for their negligible
response to changes in ionic strength.[18] Additionally, the
rather small Kd value of DPTB-IMI-EG/ATP is another
crucial factor in the promising anti-interference performance.
Figure 2. a) Fluorescence responses of DPTB-IMI-EG to various ions in
the absence (white bars) and presence (black bars) of ATP (1 mm) at
pH 7.4. 1) No ion, 2) ClÀ (5 mm), 3) IÀ (1 mm), 4) NO3 (1 mm),
À
2À
5) SO4 (1 mm), 6) HCO3À (1 mm), 7) PPi (1 mm), 8) HPO42À (1 mm),
9) AcOÀ (1 mm), 10) Mg2+ (0.1 mm), 11) Fe2+ (0.1 mm), 12) Ca2+
(0.1 mm), 13) Zn2+ (0.1 mm), 14) Gly (1 mm), 15) Glu (1 mm), 16) Lys
(1 mm), 17) RNase A (100 mglÀ1), 18) chymotrypsinogen
(100 mglÀ1). b) Fluorescence intensity loss (%) of DPTB-IMI-EG
(10 mm) in the presence (black) and absence (red) of ATP (1 mm) at
pH 7.4 with increasing doses of UV (l=365 nm) exposure. c) Signal
loss (%) of fluorescence emission of DPTB-IMI-EG (black) and MDRF
(red) in vivo with an increasing number of scans. Excitation wave-
length: l=405 nm (for DPTB-IMI-EG) and l=635 nm (for MDRF);
emission wavelength: l=460–550 nm (for DPTB-IMI-EG) and
l=650–730 nm (for MDRF); irradiation time: 10 s per scan.
Therefore we compared the photostability of DPTB-IMI-
EG with and without ATP in vitro with the same l = 365 nm
excitation conditions. Although the fluorescence intensity of
both systems decreases with increasing exposure, the exper-
imental results clearly demonstrate that ATP considerably
enhances the photostability of DPTB-IMI-EG (Figure 2b).
ATP presents an intensive absorption band at l = 260 nm and
no absorbance above l = 340 nm, so the enhancement of
photostability can only be derived from the aggregation of the
luminophore. As revealed in Figure S2, DPTB-IMI-EG/ATP
forms aggregates which show a monomodal size distribution
with a hydrodynamic diameter of 297 nm in DLS measure-
ments, and correlated well with SEM results. The aggregates
can prevent interactions between the internal DPTB-IMI-EG
and various external molecules, and significantly decreases
the chance of photochemical reactions between the excited
DPTB and active species such as water and oxygen. In
contrast, the aggregation partially restricts the motion of
DPTB, thus reducing the possibility of photoisomerization to
some extent.[19] Thus, the aggregated luminophore possesses
better photostability than the isolated molecule in solution.
For an indicator to be practically used in living organisms,
its biocompatibility is the first and foremost criterion to be
evaluated. The cytotoxicity of DPTB-IMI-EG on NIH/3T3
fibroblasts was examined by a standard 3-(4,5-dimethyl-2-
thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay. The
indicator DPTB-IMI-EG has no obvious effects on the cell
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viability, even at a high concentration of 4.0 mm (see Fig-
ure S4). Cell-membrane permeability is another important
factor for evaluating the in vivo indicator because an indica-
tor with poor permeability can only be used through harmful
microinjection, electroporation, or scrape loading to load
inside cells.[20] Fibroblast cells incubated with DPTB-IMI-EG
for 30 minutes present strong fluorescence in the green
channel, thus confirming the good permeability of the
indicator (Figure 3A1). DPTB-IMI-EG was then assessed
for its photostability in vivo. A mitochondria dye, Mito-
Tracker Deep Red FM (MDRF), was used as a reference
the cytoplasm. The uneven distribution of ATP is in line with
expectations when we consider that the main source of ATP is
generated by mitochondrial oxidative phosphorylation in
eukaryotic cells.[21] Moreover, there has been considerable
debate as to whether the ATP level in the cell membrane is
the same as that in cytosol.[22] Our results show that the cell
membrane has the lower ATP concentrations.
The ATP level is regulated by cell metabolism and
presents a dynamic equilibrium with oscillation character-
istics on the order of minutes.[23] To test the response of
DPTB-IMI-EG to the change of the ATP levels, apyrase was
used to treat the cells prior to the co-
staining procedure. Apyrase is an
enzyme that catalyzes the hydrolysis
of ATP to yield AMP and inorganic
phosphate. When the cells were
treated with 1 UmLÀ1 apyrase at
378C for 60 minutes, the fluores-
cence signals from DPTB-IMI-EG
almost disappear, except for those
close to the membrane (Fig-
ure 3A2), thus suggesting a signifi-
cant decrease in the ATP level in the
cytoplasm. Meanwhile, MDRF still
exhibits its specificity and sensitivity
to the mitochondria (Figure 3B2),
thus further confirming no associa-
tion between the indicator and the
Figure 3. Confocal fluorescence images of live NIH/3T3 cells incubated with (A2–D2) or without
(A1–D1) apyrase (1 UmLÀ1) in DMEM for 60 min at 378C and then incubated with DPTB-IMI-EG
and MDRF. A) Green channel for DPTB-IMI-EG (1 mm). B) Red channel for MDRF (100 nm).
C) Panels A and B merged. D) Optical image.
organelle. The low hydrolytic effect
on membrane ATP levels may seem
paradoxical by taking into account
the fact that apyrase is exogenous.
One possible explanation is that the
material because of the lack of commercially available ATP
imaging agent. The cells were co-stained with 1 mm DPTB-
IMI-EG and 100 nm MDRF for 30 minutes, and excitation
powers of two lasers (l = 405 nm and l = 635 nm) were
unified as 100 mW. The fluorescence loss of DPTB-IMI-EG
is less than 5% after 20 scans with total irradiation time of
200 seconds, thus demonstrating its greater photostability
compared to that of MDRF (see Figure 2c and Figure S5).
The visualization of ATP distribution is essential for
understanding its roles in vivo. DPTB-IMI-EG was inves-
tigated for its staining ability in living NIH/3T3 cells. The
detailed fluorescence image presents apparent fluorescence
signals only in the cytoplasm and cell membrane, but not in
the nucleus (Figure 3A1). The co-staining of DPTB-IMI-EG
with MDRF illustrates that fluorescence regions of these two
dyes seem to be highly overlapped in the cytoplasm (Fig-
ure 3C1). The fluorescence signals of the MDRF and signals
of the DPTB-IMI-EG in the cytoplasm are overlapped with
Rr = 0.86. However, the two pieces of evidence demonstrate
that the indicator is only close but not specific to the
mitochondria in the cytoplasm. Firstly, the reticulum struc-
tures of the mitochondria are clearer in the image of MDRF-
stained cell and secondly, the fluorescence intensity distribu-
tions of the two dyes are different in the pseudocolor images
(see Figure S6). These images show that the ATP near the
mitochondria is at a higher level than in other spaces within
enzyme associated with the microsomal fraction can show
a significantly higher specific activity than the soluble one.[24]
It is reasonable because apyrase can only be activated by
calcium or magnesium, which is mainly distributed in micro-
some and endoplasmic reticulum.
Since the fluorescence lifetime is insensitive to several
experimental parameters, such as excitation intensity, expo-
sure duration, and photobleaching, the fluorescence lifetime
imaging microscopy (FLIM) image may provide extra infor-
mation compared to that from the intensity-based image.
DPTB-IMI-EG exhibits dual fluorescence lifetimes originat-
ing from two different excited states of DPTB.[14] The average
lifetime of the indicator shows a strong positive correlation
with ATP levels (see Table S1). So the ATP spatial distribu-
tion was also examined with the FLIM technique. The ATP
level obtained from FLIM is almost consistent with that from
the fluorescence intensity method, though there are few
differences in several bright granular regions (Figure 4). The
intense fluorescence with short lifetime indicates that few
organelles (probably the vacuole) can accumulate DPTB-
IMI-EG through adsorption. Another piece of information
provided by FLIM is the variation of the ATP levels during
cell division. It can be seen from the intensity image
(Figure 4a) that the ATP distribution is broader in mitotic
cells than in normal cells. The FLIM image demonstrates high
intracellular ATP levels at the phase of mitosis, and is in
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a hydrophilic, lipophilic, and amphiphilic three-block struc-
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indicator for monitoring ATP levels in vitro and in vivo. It
shows high specificity for ATP rather than ADP or AMP, and
other bioactive species such as essential ions, amino acids, and
several proteins show no apparent interference. The absorb-
ance and luminescence are located in the visible region. The
ATP-induced finite aggregation significantly enhances its
photostability in the recognition process. The above advan-
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bility, all suggest DPTB-IMI-EG to be a good imaging agent
for ATP distribution and alteration tracking. The determi-
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been successfully performed with the indicator by using
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Received: April 2, 2014
Published online: June 6, 2014
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Keywords: aggregation · cell recognition · fluorescence ·
.
imaging agents · molecular recognition
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