A highly selective fluorescent probe for pyrophosphate in aqueous solution

Article abstract of DOI:10.1039/b808422j

A new 4-amino-1,8-naphthalimide-based fluorescent chemosensor bearing a guanidiniocarbonyl pyrrole moiety has been synthesized. The sensor displays a selective fluorescent enhancement with pyrophosphate over ATP, ADP, AMP and other inorganic anions in aqueous solution.

Full text of DOI:10.1039/b808422j

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COMMUNICATION
www.rsc.org/obc | Organic & Biomolecular Chemistry
A highly selective fluorescent probe for pyrophosphate in aqueous solution†
Yimin Sun, Cheng Zhong, Rui Gong and Enqin Fu*
Received 19th May 2008, Accepted 4th July 2008
First published as an Advance Article on the web 21st July 2008
DOI: 10.1039/b808422j
A new 4-amino-1,8-naphthalimide-based fluorescent chemo-
sensor bearing a guanidiniocarbonyl pyrrole moiety has
been synthesized. The sensor displays a selective fluorescent
enhancement with pyrophosphate over ATP, ADP, AMP and
other inorganic anions in aqueous solution.
fluorophore is ascribed to its high fluorescence quantum yield and
long wavelength emission.15 On the other hand, Gunnlaugsson
and Pfeffer have shown that the 4-amino proton in the 4-amino-
1,8-naphthalimide structure, which is quite acidic, can be used
to enhance both the strength and selectivity for the binding of
phosphate anions.16 Therefore, we designed this naphthalimide–
hydrazino–guanidiniocarbonyl pyrrole structure (a fluorophore–
spacer–receptor structure17) to satisfy the geometrical require-
ments of PPi and enable effective fluorescent sensing. It is
expected that the electrostatic interactions and multiple hydrogen
bonds of guanidiniocarbonyl pyrrole, combined with the increased
acidity of the 4-amino proton, would favor the receptor–anion
interaction and geometrical complementarity, thus producing
effective fluorescent sensing of the recognition event. Investigation
into the fluorescent sensing property of the receptor 1 has revealed
that receptor 1 is a highly selective fluorescent probe for PPi over
other anions, including its analogues ATP, AMP, ADP and Pi in
aqueous solution.
Receptor 1 was synthesized according to Scheme 1 (see ESI†).
N-Butyl-4-bromo-1,8-naphthalimde 2 reacted with hydrazine
monohydrate to give 3 as a yellow solid in 90% yield. 5-
(Methoxycarbonyl)-1H-pyrrole-2-carboxylic acid 4 was converted
into the acyl chloride 5 by reaction with oxalyl chloride in CH2Cl2
in the presence of a catalytic amount of DMF. Without further
purification, the crude acyl chloride was then treated with 3 in
CH2Cl2 to give 6 in 55% yield. The guanidinylation of 6 was
achieved by heating the ester and an excess of guanidine in DMF
under nitrogen. Upon acidification with hydrochloric acid, the
crude product (hydrochloric salt) precipitated in 40% yield. After
reaction with picric acid, host 1 was obtained as the picrate salt.
Like other amino-containing ICT systems, the fluorescence of
1 is pH-sensitive (Fig. 1). When 6.97 < pH < 11.08, the hydrogen
bond interaction between the 4-amino proton and hydroxide
enhances the intramolecular charge transfer (ICT) of receptor
1, which leads to the fluorescence intensity increase significantly.
When pH > 11.08, the deprontonation of the 4-amino-1,8-
naphthalimide quenches the fluorescence of receptor 1, which is
the same as that described by Gunnlaugsson and Pfeffer.15,16 The
mechanism is supported by the UV-vis spectrum of receptor 1 at
different pH values (Fig. S5, S6†).
The recognition and sensing of anions have received considerable
attention for their important roles in biological, industrial and
environmental processes.1 However, there are only a few artificial
receptors that allow the recognition of anions in aqueous solution.2
The reason is that the hydrogen bond and ion pairing interaction
between host and guest molecules for molecular recognition
would be weakened significantly by the competitive influence
of protic solvents. Therefore, the receptors reported so far for
the effective recognition of target molecules in water all need a
combination of multiple nocovalent interactions, such as multiple
hydrogen bonds,3 ion pairing interaction4 or metal coordination,5
to overcome the competitive influence of water. Among them,
the guanidiniocarbonyl pyrroles introduced by Schmuck can
strongly bind carboxylate or phosphate anions even in aqueous
solvents through a combination of ion pairing and multiple
hydrogen bonds.6 Excellent work has been done in studying the
complexation between guanidiniocarbonyl pyrroles and amino
acid carboxylates by NMR spectroscopy,7 but there are few reports
about fluorescent sensors based on guanidiniocarbonyl pyrroles.8
Compared with other chemosensors, fluorescent sensors appear
to be particularly attractive due to the simplicity and sensitivity of
fluorescence, as well as providing on-line and real-time analysis,9,10
so we are interested in developing luminescent chemosensors for
the detection of anions based on the guanidiniocarbonyl pyrrole
moiety.
Among the various anionic analytes, pyrophosphate (PPi) is a
biologically important target because it is the product of ATP
hydrolysis under cellular conditions.11 PPi also plays an important
role in energy transduction in organisms and could control
metabolic processes by participating in enzymatic reactions.12
The recognition effect of guanidiniocarbonyl pyrrole receptors
with sugar phosphates in aqueous solution13 led us to expand
our approach to the detection of PPi by guanidiniocarbonyl
pyrroles. In this communication, we report the synthesis of a new
long-wavelength intramolecular charge-transfer (ICT) fluorescent
receptor 1, in which the guanidiniocarbonyl pyrrole cation is
covalently attached to a N-butyl-4-hydrazino-1,8-naphthalimide
moiety. The employment of 4-amino-1,8-naphthlimide14 as a
Knowing that changes in solvent property can have a dramatic
effect on molecular recognition, fluorescence titrations were
performed in H2O–DMSO (in the range 60–99% water (10 mM
HEPES buffer, pH = 7.4)) in an effort to gauge the effect of solvent
composition on spectroscopy and association. The spectroscopic
properties and binding constants are shown in Table 1. As DMSO
is an extremely good H-bond acceptor, its electron donor capacity
is even higher than that of water,18 so the fluorescence maxima
of 1 undergoes a red shift with increasing DMSO content. Such
behavior is also indicative of the ICT character of receptor 1 upon
Hubei Key Laboratory on Organic and Polymeric Optoelectronic Materials,
Department of Chemistry, Wuhan University, Wuhan, 430072, P. R. China.
E-mail: Fueq@whu.edu.cn; Tel: +86 27-87219044
† Electronic supplementary information (ESI) available: Experimental
details; NMR spectra of compounds 1 and 6. See DOI: 10.1039/b808422j
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Scheme 1 Synthesis of guanidiniocarbonyl pyrrole receptor 1.
Fig. 2 (a) Fluorescence titration of receptor 1 (10 lm) with PPi (2000 eq.)
in 90% water–DMSO (pH = 7.4, 10 mM HEPES buffer). (b) Fluorescence
emission changes of receptor 1 (10 lm) upon addition of sodium salts
Fig. 1 pH dependence of the fluorescence emission peak of receptor 1.
of PPi, ATP, ADP, AMP, Pi, F−, Cl−, SO42−, HCO3−, AcO− and NO3
−
(0–2000 eq.) in 90% water–DMSO (pH = 7.4, 10 mM HEPES buffer).
Table 1 Spectrum characters and association constants for receptor 1
with PPi (sodium salt) in water–DMSO
chelation-enhanced fluorescence (CHEF) effect with PPi (0–
2000 eq.). The emission maximum of 1 showed a bathochromic
shift from 510 to 514 nm. The job-plot analysis indicated that
receptor 1 formed a 1 : 1 complex with PPi (Fig. S7†); the associa-
tion constant was 78 M−1 as obtained by a nonlinear least-squares
a
b
H2O/(H2O + DMSO)
kex/nm
kem/nm
K
ass/M−1
F/F0
99%
90%
80%
70%
60%
456
460
466
472
480
505
510
518
522
525
24
4.55
2.48
1.55
1.17
1.06
78
216
ndc
ndc
fitting method. In contrast, no significant fluorescence changes
were observed when ATP, ADP, AMP, Pi, F−, Cl−, SO42−, HCO3
,
−
a Association constants Kass were calculated by nonlinear least-squares
fitting with a 1 : 1 association model; error limits in Kass were estimated to
be 10%. b Fluorescence intensities are corrected. c Not determined; the
changes in the spectra were too small to calculate the association constants
precisely.
AcO− or NO3 were added (Fig. 2(b)). These results suggested
that receptor 1 has a higher selectivity for PPi over other anions.
In order to obtain a better idea about the nature of the
interactions between the guanidiniocarbonyl pyrrole receptor 1
and anionic species considered and the high selectivity of receptor
1 for PPi, the pKa value of the guanidiniocarbonyl pyrrole receptor
1 was determined by using the Henderson–Hasselbach equation;20
molecular modeling studies (PM3 of MOPAC 2007 softwware)
were also used to obtain the possible interaction models of receptor
1 with either PPi or ATP.
−
excitation. Meanwhile, DMSO effectively displaces PPi from the
4-amino moiety, which is why the more DMSO that is present,
the less the fluorescence intensity of receptor 1 increases. The
association constants for PPi are relatively small, which may due
to the ‘salt effect’ caused by the substrate itself and the buffer.19
The recognition properties of 1 (10 lm) were studied in 90%
water–DMSO (v/v, 10 mM HEPES buffer, pH = 7.4) with
various anions as substrates. Fluorescence titration experiments
were recorded on excitation at 460 nm and emission at 510 nm,
respectively. As shown in Fig. 2(a), receptor 1 displayed a
The calculated pKa value for receptor 1 is 8.18, which is
a little higher than that of other guanidiniocarbonyl pyrrole
receptors reported by Schmuck (pKa 6.9–7.9).21 This means
that the acidity (pH 7.4) of the solution that we used for the
fluorescence titration of receptor 1 is suitable for the protonation
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of guanidiniocarbonyl pyrrole receptor 1. Under this condition,
the guanidinium cation can form an ion pair with the phosphate
anion, which is simultaneously hydrogen bonded by the pyrrole
NH and another amide NH. Meanwhile, the 4-amino moiety in
the 4-amino-1,8-naphthalimide structure is quite acid,16 favoring
the formation of a hydrogen bond between receptor 1 and PPi.
According to the molecular modeling studies (PM3 of MOPAC
2007 software), the rational preorganisation of receptor 1 provides
excellent geometric and charge complementarity to PPi (Fig. 3).
Due to the appropriate size and geometry of the pyrophosphate
anion, it is possible that the proton of the 4-amino moiety could
interact with the negative oxygen atom of PPi. The hydrogen
bond would promote the ICT of 4-amino-1,8-naphthalimide,
resulting in the fluorescence enhancement of receptor 1. Therefore,
the strength and selectivity of receptor 1 for the PPi binding
can be ascribed to the combined binding modes, namely, the
electrostatic interaction, multiple hydrogen bonds and geometric
complementarity between receptor 1 and PPi.
stable. The O–P oxygen atom of ATP cannot form a hydrogen
bond effectively with the 4-amino moiety, so the fluorescence of
receptor 1 changes by a small amount relative to that when PPi
binds. On the other hand, the total anionic charge density of the O–
P oxygen atoms involved in the complexation between ATP and
guanidiniocarbonyl pyrrole acyl hydrazine sites is smaller than
that of the O–P oxygen atoms of PPi.12 Therefore, the binding
affinity of receptor 1 to ATP is relatively weak. We have tried
to get the complexation-induced shift of receptor 1 to confirm
the interaction models, but unfortunately, the 1H NMR titrations
could not be performed because receptor 1 is not soluble enough
(millimolar concentrations would be required) in solvent systems
with the necessary higher water contents.
In conclusion, we have developed a fluorescent sensor based on
the guanidiniocarbonyl pyrrole moiety with high selectivity for
PPi in aqueous solution. The receptor shows excellent shape, size
and charge complementarity to PPi, which makes it an efficient
PPi sensor with the potential for bioanalytical applications.
Acknowledgements
We gratefully acknowledge the support from the Natural Science
Foundation of China (No. 20272045 and No. 20672085)
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