Unique sandwich stacking of pyrene-adenine-pyrene for selective and ratiometric fluorescent sensing of ATP at physiological pH

Article abstract of DOI:10.1021/ja906855a

A pincer-like benzene-bridged sensor 1 with a pyrene excimer as a signal source and imidazolium as a phosphate anion receptor was synthesized and investigated for ATP sensing. A unique switch of excimer vs monomer pyrene fluorescence of 1 is observed in the presence of ATP due to the charcteristic sandwich π-π stacking of pyrene-adenine-pyrene. On the other hand, four other bases of nucleoside triphosphates such as GTP, CTP, UTP, and TTP can interact only from the outside with the already stabilized stacked pyrene-pyrene dimer of 1, resulting in excimer fluorescence quenching. The fluorescent intensity ratio of monomer-to-excimer for 1 upon binding with ATP (I ₃₇₅/I₄₈₇) is much larger than that upon binding with ADP and AMP. This difference is large enough to discriminate ATP from ADP and AMP. As one of the biological applications, sensor 1 is successfully applied to the ATP staining experiments. Sensor 1 is also applied to monitor the hydrolysis of ATP and ADP by apyrase. The results indicate that 1 is a useful fluorescent sensor for investigations of ATP-relevant biological processes.

Full text of DOI:10.1021/ja906855a

Published on Web 09/23/2009
Unique Sandwich Stacking of Pyrene-Adenine-Pyrene for
Selective and Ratiometric Fluorescent Sensing of ATP at
Physiological pH
Zhaochao Xu,† N. Jiten Singh,‡ Jeesun Lim,† Jie Pan,† Ha Na Kim,† Sungsu Park,†
Kwang S. Kim,*,‡ and Juyoung Yoon*,†
Department of Chemistry and Nano Science and Department of Bioinspired Science, Ewha
Womans UniVersity, Seoul, 120-750, Korea, and Center for Superfunctional Materials,
Department of Chemistry, Pohang UniVersity of Science and Technology, Pohang, 790-784,
Korea
Received August 12, 2009; E-mail: jyoon@ewha.ac.kr; kim@postech.ac.kr
Abstract: A pincer-like benzene-bridged sensor 1 with a pyrene excimer as a signal source and imidazolium
as a phosphate anion receptor was synthesized and investigated for ATP sensing. A unique switch of
excimer vs monomer pyrene fluorescence of 1 is observed in the presence of ATP due to the charcteristic
sandwich π-π stacking of pyrene-adenine-pyrene. On the other hand, four other bases of nucleoside
triphosphates such as GTP, CTP, UTP, and TTP can interact only from the outside with the already stabilized
stacked pyrene-pyrene dimer of 1, resulting in excimer fluorescence quenching. The fluorescent intensity
ratio of monomer-to-excimer for 1 upon binding with ATP (I375/I487) is much larger than that upon binding
with ADP and AMP. This difference is large enough to discriminate ATP from ADP and AMP. As one of the
biological applications, sensor 1 is successfully applied to the ATP staining experiments. Sensor 1 is also
applied to monitor the hydrolysis of ATP and ADP by apyrase. The results indicate that 1 is a useful
fluorescent sensor for investigations of ATP-relevant biological processes.
Introduction
Apart from its roles in energy metabolism and signaling, ATP
is also incorporated into nucleic acids by polymerases in the
The development of recognition and sensing systems for
anions has received considerable attention in recent years,1
because anions are ubiquitous in biological systems and play
significant roles in wide areas of biology, pharmacy, and
environmental sciences.2 Sensors based on anion-induced
changes in fluorescence are particularly attractive because of
the simplicity and high spatial and temporal resolution of
fluorescence.3 Adenosine-5′-triphosphate (ATP) is a multifunc-
tional nucleotide containing three negatively charged phosphate
groups. It plays several important roles in cell biology, mainly
as the universal energy currency in living cells4 and a signaling
molecule to coordinate responses to energy status, in part by
modulating ion channels5 and activating signaling cascades.6
processes of DNA replication and transcription.7 Visible imaging
of ATP can offer useful information about the production and
consumption of ATP in real time, which were suggested in some
cases to be spatially restricted in cells.8 Accordingly, there have
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† Ewha Womans University.
‡ Pohang University of Science and Technology.
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10.1021/ja906855a CCC: $40.75  2009 American Chemical Society

Selective and Ratiometric Fluorescent Sensing of ATP
A R T I C L E S
14
beenconsiderableeffortstodevelopfluorescent9orcolorimetric9e,10
sensors for ATP.
ratiometric fluorescent probes for metal ions such as Zn2+
,
16
17
Ag+,15 Pb2+
,
and Cu2+
.
In contrast, ratiometric fluorescent
Recently, Anslyn and co-workers9g reported the intelligent
combinatorial library-based sensors to differentiate between
structurally similar compounds of ATP and guanosine 5′-
triphosphate (GTP), with the help of principal component
analysis (PCA). Hamachi and co-workers utilized a zinc
dipicolyl-appended xanthone,9c acridine,9d or anthracene9i as
new fluorescent chemosensors for ATP. We also reported a new
water-soluble imidazolium anthracene derivative, which not only
differentiates the structurally similar compounds GTP and ATP
but also acts as a potential fluorescent chemosensor for GTP in
100% aqueous solution.11a Wang and Chang11b and Ramaiah
et al.11c showed excellent results regarding GTP-selective
fluorescent chemosensors. On the other hand, UTP and UDP
selective fluorescent chemosensor was reported recently by our
group.11d The latest case was reported by Yellen and co-workers
using the cpFP approach to create an improved cellular ATP
biosensor based on GlnK1.8
However, it is still a challenging task to discriminate a certain
nucleoside triphosphate among various nucleoside triphosphates
such as ATP, GTP, CTP, UTP, and TTP. Indeed, in most
reports,8-11 it is not easy to find an example in which all of
these five nucleoside triphosphates were examined to evaluate
the selectivity, because all of these sensors only contain the
recognition site for triphosphate groups. The distinguishability
of ATP from adenosine diphosphate (ADP) and adenosine
monophosphate (AMP) is also urgently desired for an improved
sensor, since ATP is made from ADP or AMP, and its use in
metabolism converts it back into these precursors. The ratio
between ATP and AMP is used as a way for a cell to sense
how much energy is available and to control the metabolic
pathways that produce and consume ATP. Moreover, there are
strong interactions between ATP and ADP on some proteins
such as PII proteins, and the competition between ATP and ADP
leads these proteins to sense the “energy charge” of the
bacteria.12 Then a fluorescent sensor, which can signal the exact
concentration of ATP free from hindrance of other nucleoside
triphosphates as well as ADP and AMP, is needed.
sensors for anions mainly focused on small-sized anions such
as F- and CN-,18d,19 but hardly were developed for nucleoside
18
triphosphates. Hamachi et al. reported sensors for ratiometric
detection of ATP derivatives,9d but the selectivity lacks for
various nucleosides. To the best of our knowledge, compound
1 is the first ratiometric fluorescent sensor for ATP with high
selectivity. It is worth mentioning that a relatively simple
imidazolium receptor can discriminate ATP efficiently from
other nucleoside triphosphates by ionic hydrogen bonding20
between imidazolium (C-H)+ and triphosphate group and by
π-π stacking interaction21 between pyrene and adenine base.
The unique binding mechanism is proposed on the basis of
fluorescence measurements, NMR experiments, and theoretical
calculations.
Results and Discussion
Synthesis. For the synthesis of sensor 1, 1-pyrenemethanol
(2) was treated with phosphorus tribromide in toluene to give
1-bromomethylpyrene 3 in 91% yield. 3 was then reacted with
3.5 equiv of 4 in THF to give monoimidazolium ion 5 in 93%
yield. This intermediate was heated at 90 °C in DMF with
compound 6 for 48 h to give the desired sensor 1 in 27% yield
(Scheme 1).
Fluorescence Behavior of 1 with Phosphate-Containing
Anions. Figure 1 explains the fluorescent changes of 1 upon
addition of H2PO4- (Pi), pyrophosphate (PPi), CTP, UTP, TTP,
GTP, and ATP. Compound 1 displays two distinct and well-
known fluorescent spectra of pyrene moieties in which a peak
at 375 nm can be attributed to the monomeric emission and
another peak at 487 nm comes from the excimer formation. As
in Figure 1, 1 displays fluorescent quenching of excimer peaks
upon addition of nucleoside triphosphates. On the other hand,
Pi or PPi shows no change or induces very small quenching
effects. The quenching effects of these excimer peaks are ATP
= GTP > TTP = UTP > CTP. However, most importantly, only
ATP induces a large enhancement in the monomeric fluorescent
peak of 1. This unique change allows an easy discrimination of
ATP from the structurally similar nucleoside triphosphates. From
Herein, we report a new ATP selective fluorescent sensor,
which can display a unique ratiometric fluorescent change only
with ATP among similar nucleoside triphosphates. The ratio-
metric fluorescence signals can effectively discriminate ATP
from ADP and AMP. Ratiometric fluorescent measurements
observe changes in the ratio of the intensities of the emission
at two wavelengths.13 Thus, ratiometric fluorescent sensors have
an important feature that they can use to evaluate the analyte
concentration and provide built-in correction for environmental
effects. Up to now, many investigations were conducted to make
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Scheme 1. Synthesis of Compound 1
Scheme 2. Proposed Binding Mode of 1 with ATP and GTP
the fluorescent titration experiments (Figure 2), the association
constant for ATP is calculated to be 1.03 × 104 M-1 (error <
15%).22
Investigation of Binding Pattern. It has been reported that
different nucleoside bases interact with anthracene with different
synergistic effects of π-stacking and electrostatic interactions.11c
Also, they induce different fluorescence responses because of
quenching by guanine and enhancing by adenine, where the
guanine and adenine are perpendicular to the anthracene with
the dipole moments of 6.37 and 1.45 D, respectively.11a As
explained in our previous study, given that the dipole moment
of solvent water is ∼3 D, molecules solvating the anthracene
with larger/smaller dipole moments than the solvent water tend
to enhance/reduce the vertical emission transition and thereby
reduce/enhance the fluorescent transition.11a In the present case,
depending on the mono and excimer fluorescence responses,
adenine displays different binding modes for the two pyrenes,
in contrast to the other four bases. As in Scheme 2, we postulate
that guanine, cytosine, thymine, or uracil interact from the
outside with the already stabilized stacked pyrene-pyrene
dimer, which quenches the excimer fluorescence because of the
large dipole moments. On the other hand, adenine can be in
between the two pyrene moieties and hence separated pyrene
moieties give rise to the pyrene monomer fluorescence.
1
To gain support for the proposed binding pattern, H NMR
titration experiments of 1 with ATP and GTP and 2D NOESY
of 1 with 1 equiv of ATP and 1 equiv of GTP are investigated
(Figures S8-S11 in the Supporting Information). Figure 3 shows
1
quite different H NMR signals of 1 with 1 equiv of ATP and
1 equiv of GTP. Addition of 1 equiv of ATP to the solution of
1 in DMSO causes downfield shifts of the protons of imidazo-
lium moieties, such as H3 (0.14 ppm), H4 (0.21 ppm), H5 (0.06
ppm), H9 (0.58 ppm), and H10 (0.62 ppm). This indicates the
strong ionic hydrogen bond interaction between imidazolium
and phosphate groups. More importantly, there are changes only
in chemical shifts of the signals, which indicate that ATP
interacts with two pyrene-bisimidazolium ligands equally. On
the other hand, upon addition of 1 equiv of GTP, on the basis
of NOESY analyses, the H1-H8 peaks of 1 split into two peaks
with different upfield chemical shifts except H4 (with downfield
shifts). Particularly, the group of H2, H3, H5 and H6 experiences
large upfield shifts of 0.81, 2.97, 1.17, and 0.53 ppm, respec-
tively. It is known in the literature that when the imidazolium
hydrogen forms the CH-π interaction, there are large upfield
chemical shifts for the imidazolium hydrogen atoms.23 These
data mean that GTP interacts with two pyrene-bisimidazolium
ligands unequally. The imidazolium ring closer to the pyrene
would have the CH-π interaction with the guanine ring, which
is possible when the guanine moiety of GTP interacts outside
the pyrene-pyrene dimer (Scheme 2). This hypothesis is
supported by theoretical calculations, which will be discussed
Figure 1. Fluorescent emission changes of 1 (10 µM) upon addition of
sodium salt of Pi, PPi, CTP, UTP, TTP, GTP, and ATP (20 equiv) at pH
7.4 (20 mM HEPES) (excitation at 345 nm).
(22) (a) Conners, K. A. Binding Constants; Wiley: New York, 1987. (b)
Association constants were obtained using the computer program
ENZFITTER, available from Elsevier-BIOSOFT, 68 Hills Road,
Cambridge CB2 1LA, United Kingdom.
(23) Dupont, J.; Suarez, P. A. Z.; De Souza, R. F.; Burrow, R. A.;
Kintzinger, J.-P. Chem.sEur. J. 2000, 6, 2377–2381.
Figure 2. Fluorescent titrations of 1 (10 µM) upon addition of sodium
salt of ATP at pH 7.4 (20 mM HEPES) (excitation at 345 nm).
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Selective and Ratiometric Fluorescent Sensing of ATP
A R T I C L E S
Figure 4. Calculated structures of 1 binding with 4Br-, ATP, and GTP (i:
stacked in between two pyrene rings; o: interacting outside two pyrene rings).
Important CH---O-/OH---O hydrogen bonds are denoted by orange dotted
lines. Ion pair interactions (between positively charged imidazolium moiety
and negatively charged oxygen atoms of phosphate group) are denoted by
gray dotted lines. CH-π/π-π interactions are denoted by black dotted lines.
MO)25 single-point energy calculations on the gas-phase-
optimized geometries. The most stable form of 1-4Br- has a
perfect π-π interaction between the two pyrene moieties. In
fact, the presence of Br- anions helps stabilize the π-π
interaction between the two pyrene moieties, and hence it gives
a strong excimer peak at 487 nm. For the 1-ATP complexation,
the 1-ATP-i complex where the adenine moiety is stacked in
between the two pyrene rings is found to be more stable (∼4.6
kcal/mol in water) than the structure where the adenine moiety
interacts outside the top of one of the already stacked pyrene
rings (1-ATP-o) (Figure 4). In the 1-ATP-i complex binding
pattern, the unique fluorescent property of the pyrene moiety
was restored, whereas in the 1-ATP-o complex binding pattern,
the fluorescent excimer property was still retained. Therefore,
with the increasing concentration of ATP in the solution of 1,
more 1-ATP-i complexes will be formed. In addition, in the
1-ATP-i complex all imidazolium moieties form (C-H)+---
O- ionic H-bonds and the H12 of the imidazole moiety of
adenine has no interaction with the pyrene ring as it is displaced
outside the stacked π-systems. On the other hand, the 1-GTP-o
complex is found to be 12.8 kcal/mol more stable in water than
the 1-GTP-i complex (Figures 4). Quite interestingly, the
pyrimidine moiety of guanine forms the CH-π interaction with
the H5 of imidazolium moiety. As the imidazolium ring is the
positively charged moiety, this kind of interaction was recently
denoted as π+-π interaction,26 which has much larger binding
energies than the conventional CH-π and π-π interactions.27
Meanwhile, the H11′ of the imidazole moiety in guanine forms
the CH-π interaction with the pyrene ring. GTP cannot be
1
Figure 3. Partial 400 MHz H NMR spectra for (a) 1 (2.4 mM), (b) 1 +
ATP (1 equiv), (c) ATP, (d) 1 + GTP (1 equiv), and (e) GTP in DMSO-d6.
ATP and GTP were dissolved in D2O as stock solution at 0.55 and 0.095
mol/L, respectively.
later. For 1-GTP complex, imidazolium hydrogens H9 and H10
disappear with solvation in D2O because of the lower solubility
of GTP in D2O than ATP. The NOE from H11 (with slight
upfield shift) of ATP to pyrene hydrogens (Hpy) (Figure S10 in
the Supporting Information) and the weak NOE from H11 of
GTP to Hpy (Figure S11 in the Supporting Information) reveal
that, in 1-ATP and 1-GTP complexes, adenine and guanine are
located closely to at least one pyrene moiety. Notably, there is
no cross-peak between H12 of ATP and Hpy, indicating that only
the pyrimidine moiety of adenine is close to the pyrene dimer.
Since there are no cross-peaks between the protons of ribose
and those of pyrenes as well as imidazolium moieties, the ribose
in ATP or GTP is outside the pincer-like receptor. All the above
NMR results support the symmetrical binding mode of 1 with
ATP and asymmetrical binding mode of 1 with GTP.
In addition, insights into the binding pattern of nucleotides
were investigated theoretically by comparing the binding
patterns and energetics of two structurally most similar nucle-
otides, ATP and GTP complexed with 1. We used the density
functional theory (B97-D/TZV2P level)24 that can describe the
π-interactions. Treatment of aqueous solvation effects are
considered by doing conductor-like screening model (COS-
(25) Klamt, A.; Schu¨u¨rmann, G. J. Chem. Soc., Perkin Trans. 2 1993, 5,
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Figure 6. Fluorescence responses of 1 to various anions at pH 7.4 (20
mM HEPES) (excitation at 345 nm). White bars represent the addition of
40 equiv of the appropriate anion to a 10 µM solution of 1. Black bars
represent the subsequent addition of 20 equiv of ATP to the solution. (1)
No anion, (2) Pi, (3) PPi, (4) CTP, (5) UTP, (6) TTP, (7) GTP.
Figure 5. Fluorescent emission changes of 1 (10 µM) upon addition of
sodium salt of AMP, ADP, and ATP (20 equiv) at pH 7.4 (20 mM HEPES)
(excitation at 345 nm). Inset: Ratiometric calibration curve I375/I487 as a
function of the concentration of AMP, ADP, or ATP.
stacked between the two pyrene rings because of the presence
of oxygen atom in the guanine moiety. We also expect the
similar binding pattern in the cases of CTP, TTP, and UTP
because of the presence of oxygen atoms in cytosine, thymine,
and uracil moieties, respectively. In fact, in the calculated
structure of the 1-GTP-i complex, the oxygen atom and
imidazole ring of guanine are quite displaced in comparison
with that of the 1-ATP-i complex. As the 1-GTP-i complex
binding mode would not form in the solution, the unique
fluorescent pattern due to the pyrene ring would not be observed
when GTP forms a complex with 1.
ATP Selectivity. To further test adenine’s unique role in this
sensing system, we examine the fluorescence of 1 with the
addition of ADP and AMP. In Figure 5, ADP and AMP display
similar trends in the fluorescent changes, but much fewer
changes. AMP induces almost negligible changes in both
monomeric and excimer emissions. ADP displays a very small
fluorescent quenching effect in excimer emission and a relatively
larger enhancement in monomeric emission. However, the ratio
for ATP (I375/I487) is much larger than that for ADP, which is
large enough to discriminate ATP and ADP. More importantly,
the curve can serve as the calibration curve for detection of
ATP. The observed selectivity for ATP over ADP and the
emission change caused by ATP, ADP, and AMP may be
ascribed to the difference in electrostatic interaction due to their
anionic charges and the difference in the adenine ring stacking
between the two pyrene moieties. In ADP and AMP, the way
that the phosphate groups interact with the imidazolium moieties
of 1 would not be in a favorable structural condition for the
adenine moiety to be stacked in between the two pyrene
moieties.
Then, the competitive binding experiments are conducted in
the presence of Pi, PPi, CTP, UTP, TTP, and GTP at 40 equiv,
respectively, with the subsequent addition of 20 equiv of ATP.
As shown in Figure 6, no significant variation in the intensity
ratio (I375/I478) is found except for ATP. Ratiometric calibration
curve I375/I487 as a function of ATP’s concentration works well
even in the presence of 40 equiv of GTP (Figure 7). 1 has the
ability to sense ATP with high selectivity even in the mixtures
with other nucleoside triphosphates. At the same time, these
results indicate again that the binding mode of adenine with
the pyrene dimer is different from those of the other four
nucleobases.
Figure 7. Fluorescent emission changes of mixture solution of 1 (10 µM)
with 40 equiv of GTP upon addition of sodium salt of ATP (20 equiv) at
pH 7.4 (20 mM HEPES) (excitation at 345 nm). Inset: Ratiometric
calibration curve I375/I487 as a function ATP’s concentration.
cells are grown with DMEM for 16 h and used at 80%
confluence. One group of cells is treated with 10 µM oligomycin
for 30 min and the other group is not. Then cells are exposed
to the 100 µM ATP sensor for 5 min and are washed with PBS.
Microscopic observation is assessed in the fluorescent micro-
scope (TE2000-U, Nikon). Images are recorded at the excitation
wavelength of 340-380 nm and the emission wavelength of
435-385 nm for blue fluorescence. As shown in Figure 8, probe
1 is successfully applied to the ATP staining experiments.
Oligomycin is known to decrease cellular ATP levels.28 Probe
1 clearly displays blue fluorescence in the HeLa cells; on the
other hand, the addition of oligomycin induces the fluorescence
quenching.
Finally, with the ability of 1 to recognize ATP from PPi-
containing anions, particularly ADP and AMP, chemosensor 1
can be applied to set up a ratiometric fluorescence assay for
ATP-relevant enzyme activity. For example, apyrase is a
hydrolytic enzyme that converts ATP or ADP into AMP and
inorganic phosphate (Pi).29 During the apyrase-catalyzed ATP
and ADP hydrolysis, the monomer emission at 375 nm decreases
and the excimer emission at 478 nm increases concurrently. In
Figure 9, the plots of the emission ratio I478/I375 show that the
reaction rate is accelerated with an increase in apyrase concen-
tration. Thus, this convenient real-time monitoring system for
(28) Gong, Y.; Sohn, H.; Xue, L.; Firestone, G. L.; Bjeldanes, L. F. Cancer
Res. 2006, 66, 4880–4887.
Biological Application of 1. We further investigate the
biological application of 1 in cultured cells (HeLa cells). HeLa
(29) Zimmermann, H. Trends Pharmacol. Sci. 1999, 20, 231–236.
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15532 J. AM. CHEM. SOC. VOL. 131, NO. 42, 2009

Selective and Ratiometric Fluorescent Sensing of ATP
A R T I C L E S
60 (230-400 mesh ASTM; Merck). 1H NMR and 13C NMR spectra
were recorded using Bruker 250 MHz or Varian 500 MHz.
Chemical shifts were given in parts per million and coupling
constants (J) in hertz. Fluorescence emission spectra were obtained
using RF-5301/PC spectrofluorophotometer (Shimadzu).
Synthesis of Compound 3. The suspension of 1-pyrenemethanol
2 (2 g, 8.6 mmol) in toluene (100 mL) was cooled to 0 °C followed
by addition of phosphorus tribromide (1 mL, 10.5 mmol) via
syringe. The mixture was stirred at 0 °C for 1 h and then warmed
to room temperature, during which the reaction became homoge-
neous. Saturated Na2CO3 solution (50 mL) was added slowly, and
the reaction was stirred until it cooled to room temperature. The
phases were separated, and the organic phase was washed with H2O
(50 mL × 2) and brine (50 mL × 2) and dried over Mg2SO4. The
yellow filtrate was concentrated to minium volume. The yellow
needlelike solid was collected and dried. The mother liquid was
concentrated again, and the crystallization process was repeated.
The total product was 2.3 g in 91% yield. Mp ) 136 °C. 1H NMR
(CDCl3, 250 MHz): δ 5.23 (s, 2H), 8.02 (m, 5H), 8.21 (m, 3H),
8.35 (d, J ) 9.3 Hz, 1H). 13C NMR (CDCl3, 62.5 MHz): δ 32.3,
122.8, 124.6, 124.8, 125.1, 125.6, 126.3, 127.3, 127.7, 128.0, 128.2,
129.0, 130.5, 130.7, 131.2, 131.9.
Figure 8. Images of HeLa cells incubated with probe 1 (100 µM) and
their images after addition of olygomicyn (10 µM).
Synthesis of Compound 5. A solution of 1-bromomethylpyrene
3 (0.2 g, 0.68 mmol) and bisimidazole 4 (0.35 g, 2.4 mmol) in 60
mL of THF was refluxed for 24 h under argon. After cooling to
room temperature, the precipitate was filtered and washed with
ether. Desired product compound 5 was obtained as a white solid
1
(283 mg, 93%). H NMR (DMSO-d6, 250 MHz): δ 6.27 (s, 2H),
6.43 (s, 2H), 7.0 (s, 1H), 7.49 (s, 1H), 7.95-8.49 (m, 12H), 9.48
(s, 1H). 13C NMR (DMSO-d6, 62.5 MHz): δ 50.3, 56.9, 119.4,
122.1, 122.3, 123.4, 123.4, 123.6, 124.1, 125.2, 125.8, 126.0, 126.7,
126.8, 127.2, 128.24, 128.3, 128.7, 128.9, 129.2, 130.1, 130.6,
131.6, 136.7, 137.7. HRMS (FAB) calcd for C24H19N4, [M - Br]+
363.1610; found, 363.1614.
Figure 9. Time-trace plots of (a) ATP and (b) ADP hydrolysis of apyrase
monitored by the emission ratio I478/I375. [1] ) 10 M, [ATP] ) [ADP] )
200 M, HEPES buffer (20 mM, pH 7.4), excitation at 345 nm.
Synthesis of Compound 1. To the solution of compound 5 (150
mg, 0.34 mmol) in 30 mL of DMF was added compound 6 (46
mg, 0.17 mmol) in 2 mL of DMF. The solution was then heated at
90 °C for 48 h under the protection of argon. The precipitate was
filtered and washed with CH2Cl2 to give mg product 1 (52 mg,
apyrase activity would be helpful to elucidate their actions in
biological systems.
Conclusions
1
27%). H NMR (DMSO-d6, 250 MHz): δ 5. 51 (s, 4H), 6.32 (s,
We have shown that a new water-soluble and fluorescent
imidazolium receptor 1 effectively and selectively recognizes
the biologically important ATP over other structurally similar
nucleoside triphosphates in aqueous solution of physiological
pH 7.4. Compound 1 displays a large fluorescent quenching
effect in its excimer emission, but a selective fluorescent
enhancement in its monomeric emission. The fluorescent
intensity ratio for ATP (I375/I487) is much larger than that for
ADP and AMP, which is large enough to discriminate ATP
from ADP and AMP. Different binding modes between ATP
and other nucleoside triphosphates are proposed on the basis
of fluorescence measurements, NMR experiments, and theoreti-
cal calculations. We believe that these novel findings will be
importantinformationtodesignsensorsfornucleosidetriphosphates.
4H), 6.63 (s, 4H), 7.52 (s, 4H), 7.88 (s, 2H), 8.03 (m, 4H),
8.10-8.42 (m, 20H), 9.56 (s, 2H), 9.64 (s, 2H). 13C NMR(CDCl3,
62.5 MHz): δ 50.9, 52.2, 58.7, 122.9, 123.0, 123.6, 123.9, 124.1,
124.6, 125.7, 126.3, 126.5, 127.2, 127.4, 127.7, 128.7, 128.8, 129.2,
129.3, 129.7, 130.6, 131.1, 132.1, 135.3, 138.3. HRMS (FAB) calcd
for C56H46Br3N8, [M - Br]+ 1069.1375; found, 1069.1378.
Acknowledgment. This work was supported by grants from
the NRL program (R04-2007-000-2007-0), the SRC program of
KOSEF/MOST (R11-2005-008-02003-0, R11-2008-052-01000),
BK21, GRL (KICOS), and WCU (R32-2008-000-10180-0) program.
Supporting Information Available: Fluorescence titration of
1
1 with PPi, CTP, UTP, TTP, GTP, AMP and ADP. H NMR
titration experiments of 1 with ATP and GTP in DMSO-d6. 500
MHz NOESY spectra of 1 with 1 equiv of ATP and GTP in
DMSO-d6. NMR spectra of 1, 3, and 5. This material is available
free of charge via the Internet at http://pubs.acs.org.
Experimental Section
Materials and Methods. Unless otherwise noted, materials were
obtained from commercial suppliers and were used without further
purification. Flash chromatography was carried out on silica gel
JA906855A
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