ATP Recognition and sensing with a phenanthroline-containing polyammonium receptor

Article abstract of DOI:10.1039/b611031b

A new polyammonium receptor is able to selectively recognise and sense ATP among triphosphate nucleotides, thanks to ATP-induced quantitative quenching of its fluorescence emission. The Royal Society of Chemistry 2006.

Full text of DOI:10.1039/b611031b

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ATP Recognition and sensing with a phenanthroline-containing
polyammonium receptor{
Carla Bazzicalupi, Silvia Biagini, Andrea Bencini,* Enrico Faggi, Claudia Giorgi, Irene Matera and
Barbara Valtancoli
Received (in Cambridge, UK) 31st July 2006, Accepted 29th August 2006
First published as an Advance Article on the web 13th September 2006
DOI: 10.1039/b611031b
receptor range between 10.14 and 6.9 log units, higher than those
reported for 1,10-phenanthroline (log K 5 4.96).14 As already
reported for other phenanthroline-containing polyamines,15 pro-
tonation takes place on the aliphatic amine groups, while the
phenanthroline nitrogens, less basic than amine groups, are not
protonated in our experimental conditions. The hexaprotonated
receptor [H6L]6+ is the prevalent species in solution for pH ¡ 7
(Fig. 1) and displays the typical fluorescence emission band of
phenanthroline at 365 nm. Deprotonation of [H6L]6+ to give less
protonated forms leads to a complete quenching of the emission
(Fig. 1), due to the presence of deprotonated amine groups which
can quench the emission through an electron transfer process. In
particular, 1H NMR spectra recorded at different pH values show
that deprotonation of the [H6L]6+ cation to give the [H5L]5+ and
[H4L]4+ ones implies release of acidic protons from the benzylic
nitrogens N2, adjacent to phenanthroline. In fact, a marked
upfield shift (ca. 0.8 ppm) of the signal of the benzylic methylene
groups H1, adjacent to N2, accompanies deprotonation of [H6L]6+
to form the [H5L]5+ and [H4L]4+ species (see inset of Fig. 1).
Therefore, in the [H5L]5+ and [H4L]4+ cations the fluorescence
emission is quenched by the benzylic amine groups N2, through an
electron transfer process to the excited phenanthroline. Of course,
protonation of these nitrogens in the hexaprotonated receptor
[H6L]6+ leads to renewal of the fluorescence emission. In the
cations with protonation degree lower than 4, however, the N3
and N4 nitrogens may also contribute to the quenching process.
Protonation of the receptor enables L to form stable complexes
with anionic forms of triphosphate nucleotides ATP, GTP, CTP
or TTP as well as with ADP and AMP. A potentiometric study
A new polyammonium receptor is able to selectively recognise
and sense ATP among triphosphate nucleotides, thanks to
ATP-induced quantitative quenching of its fluorescence
emission.
There is a growing interest in the design of fluorogenic receptors
able to recognise and signal analytes in aqueous solutions,1 due to
their potential use in medicinal and environmental chemistry. ATP
is undoubtedly one of the preferred targets, due to its basic role as
a center for chemical energy storage and transfer in the
bioenergetics of all living organisms. Polyammonium receptors
containing fluorogenic units are often chosen as chemosensors for
anionic analytes, including ATP,2–7 since their ability to give
charge–charge interactions with anionic species may lead to the
formation of stable host–guest complexes in aqueous solutions.
Selective coordination, however, requires also the incorporation of
sites capable of interactions with the sugar moiety or the nucleic
base.8–13
Although many fluorogenic synthetic systems able to recognise
and signal ATP have been reported,2–13 in most cases they act as
selective chemosensors for ATP with respect to the less charged
ADP, AMP or inorganic phosphate anions. Selective recognition
and sensing of ATP over the other triphosphate nucleotides is a
much more difficult goal, due to the similar charge gathered on the
nucleotides; in fact, only one example of a chemosensor able to
selectively sense GTP over ATP has been recently reported.4
The new phenanthroline-containing macrocycle L (Scheme 1)
protonates in aqueous solutions affording polyammonium cations
of the type [HxL]x+ (x 5 1–6). The protonation constants of the
Scheme 1
Dipartimento di Chimica, Universita` di Firenze, Via della Lastruccia 3,
50019, Sesto Fiorentino, Firenze, Italy. E-mail: andrea.bencini@unifi.it;
Fax: +39 055 4573364; Tel: +39 055 4573371
{ Electronic supplementary information (ESI) available: Experimental
details for L synthesis, potentiometric and spectroscopic measurements
and MD simulations, protonation constants of L, formation constants of
the adducts with ATP, ADP, AMP, GTP, CTP and TTP, distribution
diagrams for each system L–nucleotide, CIS values for the receptor–
nucleotide complexes, atomic coordinates for the lowest energy con-
formers of adducts with ATP and CTP. See DOI: 10.1039/b611031b
Fig. 1 pH dependence of the fluorescence emission at 365 nm (-&-, left y
axis) and distribution diagrams of the protonated species of L (solid lines,
right y axis. Inset: pH dependence of chemical shift of the benzylic protons
H1 (-$-) superimposed on the species distribution of L (0.1 M NMe4Cl,
[L] 5 2.5 6 1025 M.
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shows that all nucleotides forms stable 1 : 1 complexes of the type
[HxLA](x24)+ (see ESI{).
The comparison between the binding constants of nucleotides to
the protonated forms of the receptor, however, is complicated by
the remarkably different acid–base characteristics of the substrates,
and by the presence of overlapping protonation equilibria
involving both receptor and anions in the same pH range. This
problem can be conveniently overcome by considering
a
competitive system containing receptor and nucleotides in
equimolar concentrations and calculating the overall percentages
of the different complexed anions over a wide pH range.16 Plots of
the percentages vs. pH produce species distribution diagrams from
which the binding ability of the receptor can be interpreted in
terms of selectivity. Fig. 2 displays the plot obtained for a
competitive system containing L, ATP, CTP, TTP and GTP in
equimolecular ratio; the formation of ATP adducts with L prevails
over a wide pH range, i.e., ATP is selectively bound with respect to
the other triphosphate nucleotides. For instance at pH 6, 80% of
ATP is complexed, while CTP, the most competitive substrate for
ATP complexation with L, is complexed at less than 15%.
A similar plot can be also obtained for a competitive system
containing ATP, ADP and AMP. In this case, ATP is selectively
complexed in the pH range 2–9 in a percentage greater than 90%.
(see ESI, Figure S7{).
Fig. 3 1H NMR spectra (aromatic region) of L (a), ATP in the presence
of L (1 : 1 molar ratio) (b), ATP (c) and 31P NMR spectra of ATP (d) and
ATP in the presence of L (1 : 1 molar ratio) (e).
formation of rather strong charge–charge contacts and weak
p-stacking interactions with the receptor.
Spectrofluorimetric measurements show that the receptor can
act as effective chemosensor for ATP in aqueous solutions. In fact,
addition of increasing amounts of ATP to a solution of L at pH 6
leads to a linear decrease of the fluorescence emission of L. As
shown in Fig. 4, the fluorescence of L is completely quenched in
the presence of 1 eq. ATP. ADP and AMP give a slight decrease of
the fluorescence emission of the receptor (max 10% in the presence
of a tenfold excess of ADP). Triphosphate nucleotides also
produce a decrease of the fluorescence emission. In this case,
however, the quenching effect is only partial even in the presence
of a large excess of CTP, GTP or TTP. As shown in the inset of
Fig. 4, the larger effect (a fluorescence emission decrease of ca.
30%) is observed in the presence of an excess of CTP. Therefore, in
the present receptor phenanthroline is not only used for ATP
selective binding, thanks to its ability to give strong p-stacking
interaction with the nucleobase, but also as signalling unit for this
substrate.
31P Spectra recorded on solutions containing the nucleotides
and the receptor in a 1 : 1 molar ratio show remarkable downfield
shifts of the 31P signals of the Pc and Pb phosphorus atoms of the
phosphate chain with respect to the nucleotides in the absence of
the receptor, indicating the formation of strong salt bridges
between the anionic triphosphate moiety and the ammonium
groups of the receptor. Complexation is also accompanied by
upfield shifts of the 1H signals of the nucleobases and the
phenanthroline of the receptor (see Fig. 3 for ATP), suggesting the
presence of p-stacking interactions between the nucleobases and
the heteroaromatic unit of the receptor.
ATP shows the most relevant complexation-induced chemical
1
shifts (CIS) in both 31P and H NMR spectra (see ESI, Table S6
and S7{). This would confirm the presence of an overall stronger
interaction of ATP with the receptor with respect to the other
nucleotides. Among the other triphosphate nucleotides, CTP, the
most competitive nucleotide toward ATP, displays the highest CIS
values for the 31P signals (3.8 and 1.2 ppm for the Pc and Pb
To clarify the binding mode of nucleotides, we carried MD
calculations on the adducts between [H6L]6+ and ATP or CTP, the
most competitive substrate for ATP coordination by L, exploring
the potential energy surface by means of simulated annealing. In
all the sampled conformers the receptor assumes a folded
conformation, with an almost perpendicular disposition between
phenanthroline and the aliphatic polyamine chain. Both ATP and
CTP display a bent conformation, with the nucleobase–sugar
1
phosphorus atoms) and the lowest one in the H signals for its
aromatic protons (20.28 and 20.20 ppm), suggesting the
Fig. 4 Fluorescence emission spectra of L in the presence of increasing
amounts of ATP at pH 6 (lexc 270 nm, NMe4Cl 0.1 M, 298.1 K,
[L] 5 2.5 6 1025). Inset: fluorescence intensity at 365 nm in the presence
of increasing amounts of ATP, TTP, GTP or CTP.
Fig. 2 Overall percentages of L complexed species with ATP, CTP, TTP
or GTP as a function of pH in a competitive system containing L, ATP,
CTP, TTP and GTP in equimolecular ratio ([L] 5 [ATP] 5 [CTP] 5
[TTP] 5 [GTP] 5 1 6 1024 M).
4088 | Chem. Commun., 2006, 4087–4089
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Therefore, while ATP gives hydrogen bonds with the benzylic
ammonium groups in both the A and B families, CTP forms
hydrogen bonds with these nitrogens only in the less populated A
family.
These results are in accord with the observed high stability of the
ATP complexes as well as with the observed complete quenching
of the fluorescent emission of phenanthroline in the ATP complex.
In the case of ATP, the nucleotide can assume a conformation
suitable to give simultaneously strong p-stacking, charge–charge
and hydrogen bonding contacts, which reinforce the overall
substrate–receptor interaction. At the same time, in the case of
ATP the hydrogen bonding P–O2 H2N interactions strongly
involve the protonated benzylic nitrogens; this can lead to partial
transfer of an acidic proton from the benzylic amines N2 to the
phosphate chain. Deprotonation of the N2 nitrogens in the
[H6L]6+ cation leads to the observed fluorescence quenching.
In conclusion, the present polyammonium receptor represents a
unique system simultaneously able not only to selectively recognise
ATP over GTP, TTP and CTP, but also to signal ATP through a
quantitative quenching of its fluorescence emission.
+
…
Fig. 5 Low energy conformers of the adducts between [H6L]6+ and ATP
in the A (a) and B (b) families and between [H6L]6+ and CTP in the A (c)
…
and B (d) families. Only hydrogen bonds with N–H O distances lower
˚
than 2 A are reported.
Notes and references
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torsion in a syn form, which allows the simultaneous interaction of
the nucleobases and of the triphosphate chain with the receptor,
the terminal c-phosphate group being encapsulated within the
macrocyclic cavity. In both ATP and CTP complexes, the different
conformers can be grouped in two different families (herein called
A and B), which differ in the interaction mode of the nucleobase
with the receptor. The low energy conformers of each family for
the ATP and CTP complexes are shown in Fig. 5. In the A family,
the most populated for the ATP complex (70%) and the less
populated one for CTP (35%), adenine and cytosine interact with
the phenanthroline unit via p-stacking. The adenosine of ATP,
however, gives a stronger p-stacking interaction than cytidine of
CTP; while adenine lies above phenanthroline (interplanar distance
˚
3.6 A) and assumes an almost parallel disposition with respect to
the phenanthroline plane (Fig. 5a), cytidine displays a smaller
‘‘overlap’’ with phenanthroline and the plane of cytidine is bent ca.
26u with respect to the phenanthroline one (Fig. 5c). The bent
conformation of the nucleotides allows the formation of hydrogen
bonding interactions of the c-phosphate groups of both ATP and
CTP with the benzylic nitrogens.
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The low energy conformer in the B family, the most populated
for the CTP complex (65%) and the less populated one for ATP
(30%), shows hydrogen bonding interactions between the N7
donor of adenine (Fig. 5b) or the N3 nitrogen of cytosine (Fig. 5d)
with an ammonium group of the aliphatic chain of [H6L]6+.
Thanks to its smaller dimensions, cytidine can assume a spatial
position closer to the macrocyclic cavity than adenine. This
determines a different disposition of the triphosphate moiety with
respect to the aliphatic chain of the receptor. In fact, the
c-phosphate of CTP is pushed away from the benzylic ammonium
groups and cannot give hydrogen bonding interactions with them,
while the c-phosphate of ATP can give a couple of strong
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Chem. Commun., 2006, 4087–4089 | 4089