A Multisite-Binding Switchable Fluorescent Probe for Monitoring Mitochondrial ATP Level Fluctuation in Live Cells

Article abstract of DOI:10.1002/anie.201510003

Adenosine triphosphate (ATP), commonly produced in mitochondria, is required by almost all the living organisms; thus fluorescent probes for monitoring mitochondrial ATP levels fluctuation are essential and highly desired. Herein, we report a multisite-binding switchable fluorescent probe, ATP-Red 1, which selectively and rapidly responds to intracellular concentrations of ATP. Live-cell imaging indicated that ATP-Red 1 mainly localized to mitochondria with good biocompatibility and membrane penetration. In particular, with the help of ATP-Red 1, we successfully observed not only the decreased mitochondrial ATP levels in the presence of KCN and starvation state, but also the increased mitochondrial ATP levels in the early stage of cell apoptosis. These results indicate that ATP-Red 1 is a useful tool for investigating ATP-relevant biological processes. Illuminating the ATP: A multisite-binding, switchable fluorescent probe can selectively and rapidly respond to ATP at intracellular concentrations. In live-cell imaging, the probe was successfully applied to monitor fluctuations in mitochondrial ATP levels.

Full text of DOI:10.1002/anie.201510003

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International Edition: DOI: 10.1002/anie.201510003
German Edition: DOI: 10.1002/ange.201510003
Fluorescent Probes
A Multisite-Binding Switchable Fluorescent Probe for Monitoring
Mitochondrial ATP Level Fluctuation in Live Cells
Lu Wang, Lin Yuan,* Xian Zeng, Juanjuan Peng, Yong Ni, Jun Cheng Er, Wang Xu,
Bikram Keshari Agrawalla, Dongdong Su, Beomsue Kim, and Young-Tae Chang*
Abstract: Adenosine triphosphate (ATP), commonly pro-
duced in mitochondria, is required by almost all the living
organisms; thus fluorescent probes for monitoring mitochon-
drial ATP levels fluctuation are essential and highly desired.
Herein, we report a multisite-binding switchable fluorescent
probe, ATP-Red 1, which selectively and rapidly responds to
intracellular concentrations of ATP. Live-cell imaging indi-
cated that ATP-Red 1 mainly localized to mitochondria with
good biocompatibility and membrane penetration. In partic-
ular, with the help of ATP-Red 1, we successfully observed not
only the decreased mitochondrial ATP levels in the presence of
KCN and starvation state, but also the increased mitochondrial
ATP levels in the early stage of cell apoptosis. These results
indicate that ATP-Red 1 is a useful tool for investigating ATP-
relevant biological processes.
molecular probes to track and image mitochondrial ATP and
further elucidate its contributions to physiological states.
Several off-line analytic methods have been applied to
measure ATP concentration of cell extracts.[6] Compared with
these methods, fluorescence imaging shows specific advan-
tages in the research of functional- and molecular-recognition
events in live cells.[7] Recently, several genetically encoded or
aptamer-based fluorescent ATP probes have been developed
and applied in live-cell imaging.[8] However, small-molecule
probes show complementary properties, such as ease of
operation and low cost. Thus, some small-molecule fluores-
cent ATP probes, mainly based on complexation with
imidazolium[9] or metal ions,[10] have been developed. How-
ever, owing to the challenges in overcoming issues of
selectivity and response concentration, only a few ATP
sensors have been applied for cell imaging.[9,10c,e,l] In partic-
ular, fewer probes are suitable to monitor the increased ATP
levels because of the narrow response range and low
saturation concentration.[10l] At the same time, despite the
essential roles of mitochondrial ATP, only two recent
fluorescent probes were successfully used for mitochondrial
ATP imaging.[10c,l] Thus, preparing a good sensor for monitor-
ing the fluctuation of mitochondrial ATP levels remains
a challenge.
The current design strategy for sensing ATP is mainly
based on the electrostatic interaction between negatively
charged phosphates of ATP and positively charged recogni-
tion groups, such as imidazolium and metal ions. However, in
most cases, the stability and selectivity of these probes were
not satisfying owing to the interference from nucleoside
polyphosphates (NPPs) or negatively charged biomolecules.
To tackle these problems, herein, a multisite-binding strategy
was applied. Considering the three main parts of NPPs
(vicinal diol, nitrogenous base, and phosphates; Figure 1A),
we envisioned that covalent bonding, p-p interactions, and
electronic attraction could cooperate to achieve the specific
recognition of ATP. It has been reported that phenylboronic
acid can reversibly react with diols, and such motifs have been
applied to detect carbohydrates inside cells.[11] Therefore,
a phenylboronic acid was introduced into Rhodamine B
linked at ortho-, meta-, and para-positions, respectively, to
obtain ATP-Red 1–3 (Supporting Information, Scheme S1).
It was hypothesized that these probes would be non-
fluorescent when forming a ring-closed structure; in the
presence of ATP, however, a covalent bond between boronic
acid and ribose, p-p interaction between xanthene and
adenine, and electrostatic interactions between amino and
phosphate groups function cooperatively to facilitate the ring-
opening structure of the probe, thereby generating strong
A
denosine triphosphate (ATP), an indispensable biomole-
cule, is shared by almost all independently living organisms on
Earth. As the molecular unit of currency, ATP is the primary
energy source for cellular processes.[1] ATP also functions as
a signaling molecule for regulating cell movement,[2] neuro-
transmission,[3] and ion channels.[4] In mitochondria, ATP is
recharged by the addition of phosphate to adenosine diphos-
phate (ADP) through oxidative phosphorylation (OXPHOS).
Thus, mitochondrial ATP plays a central role in regulating the
cellular energy status for metabolic activities in healthy and
diseased states.[5] Therefore, there is a compelling need for
[*] L. Wang, Prof. Dr. L. Yuan, J. C. Er, W. Xu, B. K. Agrawalla,
Prof. Dr. Y.-T. Chang
Department of Chemistry and Medicinal Chemistry Programme
National University of Singapore
Singapore 117543 (Singapore)
E-mail: chmcyt@nus.edu.sg
Prof. Dr. L. Yuan
State Key Laboratory of Chemo/Biosensing and Chemometrics
College of Chemistry and Chemical Engineering
Hunan University
Changsha 410082 (PR China)
E-mail: lyuan@hnu.edu.cn
X. Zeng
Department of Pharmacy
National University of Singapore
Singapore 117543 (Singapore)
Dr. J. Peng, Dr. Y. Ni, Dr. D. Su, Dr. B. Kim, Prof. Dr. Y.-T. Chang
Laboratory of Bioimaging Probe Development
Singapore Bioimaging Consortium
Singapore 138667 (Singapore)
Supporting information and ORCID(s) from the author(s) for this
article are available on the WWW under http://dx.doi.org/10.1002/
anie.201510003.
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Figure 1. A) Structures of ATP and ATP probes. B) Proposed mecha-
nism for sensing ATP.
Figure 2. UV absorption (A) and fluorescence (B) spectra of ATP-Red
1 (10 mm) responding to ATP at intracellular concentrations (0–
10 mm). C) Fluorescence titration curve of ATP-Red 1 upon the addi-
tion of ATP (blue), ADP (red) and AMP (black). [Analytes]=0–10 mm.
D) Time-dependence of the fluorescence intensity change before and
after treatment of ATP. [ATP]=5 mm. Time interval: 10 s. F/F0 repre-
sents the fluorescence intensity ratio (590 nm) and F0 is the initial
fluorescence intensity (590 nm) of ATP-Red 1 in the absence of nucleo-
side polyphosphates. Conditions: lex: 510 nm, [ATP-Red 1]=10 mm,
Glycerol/Krebs buffer solution (60/40, pH 7.8), 258C.
fluorescence (Figure 1B; Supporting Information, Table S1).
ATP-Red 1–3 and a control probe ATP-Red C were prepared
and characterized by 1H NMR, 13C NMR, and high resolution
mass spectrometry (Supporting Information).
Initially, the fluorescence response of ATP-Red 1–3
(10 mm) toward ATP were evaluated in buffered (Krebs,
pH 7.8) water/glycerol (40:60, v/v) solution, which mimics
mitochondrial pH and viscosity.[12] As shown in Figure S1,
ATP-Red 1 displayed the strongest fluorescence response to
ATP. This may be because the larger steric hindrance of
phenylboronic acid at ortho position is favorable for the
fluorescent ring-opening structure in the presence of ATP.
Thus, ATP-Red 1 was chosen as an ATP sensor and tested in
the subsequent experiments.
capability of ATP-Red 1 to ATP at physiological pH (5.5–7.8;
Figure S7).
The selectivity evaluation of ATP-Red 1 toward ATP
against various biomolecules was performed. The fluores-
cence intensity of ATP-Red 1 increased 5.6-fold in the
presence of 5 mm ATP (Figure 3), while no significant
increase in fluorescence was observed after addition of
other biomolecules, such as metal ions and carbohydrates
(entries 1–20). Notably, NPPs, which easily compete with
ATP detection, only caused slight fluorescence intensity
increase in this work (Figure 3, entries 21–32). Moreover, it
Absorption and fluorescence spectra of ATP-Red 1 were
recorded with titration of ATP from 0.5 to 10 mm, a range that
is comparable to intracellular ATP concentrations.[13a] An
increased absorption band at l = 570 nm was observed upon
the addition of ATP (Figure 2A), which supports the
assumption of the ring-opening process. A solution of 10 mm
ATP-Red 1 was weakly fluorescent, while addition of 10 mm
ATP led to an 18.1-fold turn-on response (Figure 2B). Similar
fluorescence enhancements were also observed not only in
buffer solution containing high concentration of carbohy-
drates (Figure S2), but also in buffer solution mimicking
intracellular protein and ion concentrations (Figure S3). ADP
and AMP (adenosine monophosphate), partially dephos-
phorylated products of ATP, are persistent sources of
interference in ATP detection. Thus, the fluorescence inten-
sity of ATP-Red 1 was tested by titrating with ADP and AMP,
which showed qualitatively similar, but much smaller changes
in the spectra (Figure 2C; Supporting Information, Fig-
ure S4). The corresponding dissociation constant (Kd) of
ATP-Red 1 with ATP, ADP, and AMP were shown in
Figure S5. Importantly, the fluorescence response of ATP-
Red 1 was also obtained in the buffer solution with different
ATP/ADP ratios (Figure S6). Except for the spectral signa-
tures, the sensing process could be achieved within 20 seconds,
thus enabling monitoring of intracellular ATP (Figure 2D).
Furthermore, pH titration experiments indicated the response
Figure 3. A) Fluorescence response of ATP-Red 1 to various biomole-
cules. Analytes (5 mm): 1) buffer, 2) Zn2+, 3) Mg2+, 4) Ca2+, 5) Na+,
6) K+, 7) GSH, 8) HOCl, 9) H2O2, 10) d-arabinose, 11) l-arabinose,
12) d-galactose, 13) d-glucose, 14) d-fructose, 15) d-ribose, 16) l-
ribose, 17) l-sorbose, 18) sucrose, 19) d-xylose, 20) heparin, 21) AMP,
22) ADP, 23) ATP, 24) CMP, 25) CDP, 26) CTP, 27) UMP, 28) UDP,
29) UTP, 30) GMP, 31) GDP, 32) GTP. F/F0 represents the fluores-
cence intensity ratio (590 nm) and F0 is the initial fluorescence
intensity (590 nm) of ATP-Red 1 in the absence of biomolecules.
Conditions: [ATP-Red 1]=10 mm, Glycerol/Krebs buffer solution (60/
40, pH 7.8). lex: 510 nm, 258C.
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was reported that intracellular ATP is two-fold higher in
mitochondrial membrane potential,[15] high viscosity, and
concentration than UTP and GTP, and more than five-fold
higher than other NPPs.[13] Under these conditions, ATP-
Red 1 showed excellent selectivity for ATP over other NPPs
(Figure S8), indicating that ATP-Red 1 is a promising fluo-
rescent probe for detecting intracellular ATP.
ATP concentration in mitochondria (Figure S15). The cell
experiment also indicated good membrane permeability of
ATP-Red 1, which would benefit live-cell imaging.
We therefore examined the applicability of ATP-Red 1 to
monitor mitochondrial ATP levels in living cells. As shown in
Figure 5Aa, OSCC cells incubated with 5 mm ATP-Red 1
To demonstrate the function of the covalent bond the in
multisite binding process, control probe ATP-Red C without
boronic acid was synthesized.[14] The fluorescence of ATP-
Red C remained weak in the presence of ATP (Figure S9).
The proposed covalent bond between boronic acid and diol of
ATP was further demonstrated by MALDI-TOF mass
spectrometry (Figure S10). We assumed that p-p stacking
between xanthene and adenine is stronger than other nitro-
genous bases, which would benefit the recognition of adeno-
sine polyphosphates. Furthermore, the positively charged
amino group of the probe forms stronger electrostatic
interactions with the negatively charged phosphate anions
of ATP than that of ADP and AMP, because ATP has more
negative charges at physiological pH. In particular, the
proposed multisite-binding was demonstrated by theoretical
calculations (Figure S11). The reversibility of the sensing
process was indicated by addition of apyrase, which catalyzes
the hydrolysis of ATP to yield AMP. As shown in Figure S12,
after ATP consumption, the fluorescence intensity decreased
significantly owing to the recovery of the non-fluorescent
ring-closed structure.
To evaluate the biocompatibility of ATP-Red 1, an MTT
assay was performed and indicated no significant effects on
cell viability at the concentration of 1–8 mm after incubation
for 3 h or 24 h (Figure S13). Intracellular distribution of ATP-
Red 1 was examined in oral squamous cell carcinoma (OSCC)
cells (Figure 4) and HeLa cells (Figure S14). The strong
fluorescence signal of ATP-Red 1 colocalized well with that
of Mito-Tracker Green in mitochondria (Pearsonꢀs Coeffi-
cient: 0.894) but not that of Lyso-Tracker Green in lysosome,
or Hoechst in nucleus. The specific mitochondrial distribution
may result from the cooperatively function of the negative
Figure 5. A) Fluorescence images of ATP-Red-1 (5 mm, 20 min)-stained
live OSCC cells in the presence/absence of KCN (0.5 mm, 0.5 h) and
starvation conditions. B) Fluorescence images of ATP-Red 1 (2.5 mm,
20 min)-stained live OSCC cells incubated with/without camptothecin
(10 mm) for 2, 4, and 6 h, respectively. Green: Annexin V (cell apoptosis
tracker); red: ATP-Red 1. Arrows indicate the cell apoptosis process.
Scale bar: 30 mm.
presented strong fluorescence in the red channel. To verify
that the fluorescence was induced by mitochondrial ATP,
glucose, the precursor for the synthesis of ATP, was removed
from the medium.[8c] As expected, a slight fluorescence
decrease was observed (Figure 5Ab; Supporting Information,
Figure S16). OSCC cells were further incubated with KCN, an
inhibitor of OXPHOS, under glucose starvation condi-
tions.[10f, 16] Subsequent ATP-Red 1 staining showed much
weaker fluorescence (Figure 5Ac; Supporting Information,
Figure S16), indicating the significantly reduced mitochon-
drial ATP levels after KCN-induced inhibition of OXPHOS.
A similar effect was also observed in live HeLa cells
(Figure S17).
To demonstrate the capability of ATP-Red 1 to monitor
increased ATP levels, camptothecin was used to induce cell
apoptosis because elevation of intracellular ATP levels is
a requisite to the apoptotic cell death process.[17] Time lapse
imaging displayed that OSCC cells incubated with campto-
thecin showed much stronger fluorescence (red), which
indicates higher ATP concentration in mitochondria during
apoptosis process (Figure 5B and S18). This conclusion was
also demonstrated by flow cytometry analysis (Figure S19).
To confirm the apoptotic process, Annexin V, a cell apoptosis
tracker, was administered. No signal from Annexin V was
observed in the untreated control group, while camptothecin-
Figure 4. Intracellular localization of ATP-Red 1 in OSCC cells. Live
OSCC cells were prestained with ATP-Red 1 (5 mm, 15 min) and further
treated with Mito-Tracker Green (0.25 mm, 15 min), Lyso-Tracker Green
(1 mm, 60 min), or Hoechst (1 mm, 15 min), respectively. Yellow:
merged signal. Green: MitoGreen and LysoGreen; blue: Hoechst; red:
ATP-Red 1. Scale bar: 10 mm.
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In conclusion, a multisite-binding, switchable fluorescent
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sense ATP in living cells. On account of the multisite-binding
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Acknowledgements
This work was supported by intramural funding from
A*STAR (Agency for Science, Technology and Research,
Singapore), Biomedical Research Council, and National
Medical Research Council grant (NMRC/CBRG/0015/2012).
Keywords: ATP · cell imaging · fluorescent probes ·
multisite-binding
How to cite: Angew. Chem. Int. Ed. 2016, 55, 1773–1776
Angew. Chem. 2016, 128, 1805–1808
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