A visible light excitable fluorescent sensor for triphosphate/pyrophosphate based on a diZn2+ complex bearing an intramolecular charge transfer fluorophore

Article abstract of DOI:10.1039/b914802g

Triphosphate or pyrophosphate can be recognised by a diZn²⁺ complex of bis(BPEA)-appended intramolecular charge transfer fluorophore 4-amino-7-aminosulfonyl-2,1,3-benzoxadiazole, displaying a 5-6 fold fluorescent enhancement at 576 nm.

Full text of DOI:10.1039/b914802g

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A visible light excitable fluorescent sensor for triphosphate/
pyrophosphate based on a diZn2+ complex bearing an intramolecular
charge transfer fluorophore†
Guangyu Su, Zhipeng Liu, Zhijun Xie, Fang Qian, Weijiang He* and Zijian Guo*
Received 21st May 2009, Accepted 17th August 2009
First published as an Advance Article on the web 24th August 2009
DOI: 10.1039/b914802g
Triphosphate or pyrophosphate can be recognised by
a diZn2+ complex of bis(BPEA)-appended intramolecular
charge transfer fluorophore 4-amino-7-aminosulfonyl-2,1,3-
benzoxadiazole, displaying a 5–6 fold fluorescent enhance-
ment at 576 nm.
SBD-2BPA, which was devised in the normal bis(BPA-Zn2+)-
appending strategy was also investigated for its phosphate sensing
behaviour (Scheme 1). Both SBD-2BPEA and SBD-2BPA were
prepared from 4-chloro-7-chlorosulfonyl-2,1,3-benzoxadiazole re-
acting with BPEA or BPA.16 The phosphate sensing behaviours
of their diZn2+ complexes have been determined by fluores-
cence spectroscopy. In addition, SBD-BPEA, the analogue of
SBD-2BPEA with methylamine replacing 4-BPEA, was also
prepared for comparison.
Phosphates play an essential role in the human body and other
living systems. Among them, pyrophosphate (PPi) and adenosine
triphosphate (ATP) are involved in energy transduction, metabolic
processes, extracellular signal mediations, DNA polymerisation
and cyclic adenosine monophosphate synthesis, etc.1,2 Selective
sensing and discrimination of different phosphate anions are
essential for clarifying their roles in these processes and has
been extensively studied.3–10 The fluorescent sensing strategy via
metal coordination-altered emission is found to be more effective
than that of hydrogen bonding-altered emission, since metal
coordination leads to a higher phosphate affinity in aqueous
solution.3,11 Besides the high phosphate affinity, Zn2+ does not
exhibit any emission quenching effects to fluorophore due to
its 3d104s0 configuration. Therefore, Zn2+ fluorescent complexes
with a vacant coordination site have been adopted as fluores-
cent probes for different phosphates. In fact, sensors employing
bis(BPA-Zn2+)-appended fluorophores such as (naphthalen-2-
yl)phenol, xanthone and anthracenes display selective fluorescent
response to phosphates due to the synergic Zn2+ coordination to
the fluorophore-spaced bis(BPA-Zn2+) motif (BPA, bis(pyridin-
2-ylmethyl)amine).12–14 However, their UV excited sensors of-
ten suffer from the UV irradiation-induced cell damage and
autofluorescence,10,15 which precludes their application in living
systems. Sensors derived from intramolecular charge transfer
(ICT) fluorophores may overcome these problems since they
normally display visible excitability and large Stokes shift. Their
ICT effect from its electron-donating group (D) to electron-
withdrawing group (A) are responsible for these properties.16–18
In this work, a visible light excited sensor, SBD-2BPEA, was
designed for phosphate sensing via Zn2+-mediated binding. In this
compound, the distal D and A of ICT fluorophore 4-amino-7-
aminosulfonyl-2,1,3-benzoxidiazole (ASBD) has been modified
as Zn2+ ionophore BPEA (N1,N1-bis(pyridin-2-ylmethyl)ethane-
1,2-diamine) and sulfonated BPEA, respectively. The analogue,
Scheme 1 Synthesis of SBD-2BPEA, SBD-BPEA and SBD-2BPA.
SBD-2BPEA exhibits stable fluorescence in aqueous solution
from pH 7–10 (3% DMSO, v/v; lex 468 nm, lem 591 nm; Fig. S1†),
while SBD-BPEA exhibits stable fluorescence from pH 2–10 (lex
451 nm, lem 591 nm). Both compounds undergo distinct emission
decrease at pH >10.0, which can be ascribed to the reduced
ICT effect caused by the deprotonation of sulfonamide. The
stable emission of SBD-BPEA at even lower pH implies there
should be no photo-induced electron transfer (PET) effect from
7-sulfonated BPEA, which may be due to the intramolecular
hydrogen bonding between sulfonamide and the tert-amine of
7-sulfonated BPEA. However, SBD-2BPEA displays an enhanced
emission at pH ≤6. Their different fluorescent pH-dependence
at lower pH suggests that only 4-BPEA in SBD-2BPEA has a
PET effect on the fluorophore, and that the enhanced emission of
SBD-2BPEA at low pH originates from the PET elimination
induced by the tert-amine protonation in 4-BPEA.
State Key Laboratory of Coordination Chemistry, School of Chemistry and
Chemical Engineering, Nanjing University, Nanjing 210093, P. R. China.
E-mail: heweij69@nju.edu.cn, zguo@nju.edu.cn; Fax: +86 25-83314502;
Tel: +86 25-83597066; +86 25-83594549
† Electronic supplementary information (ESI) available: Experimental
details; fluorescent, emission and ESI-MS spectra; the proposed binding
mode of 2Zn2+–SBD-2BPEA and PPi. See DOI: 10.1039/b914802g
The phosphate sensing behaviour of the diZn2+ complex of
SBD-2BPEA was investigated in buffer solution containing 10 mM
SBD-2BPEA and 20 mM Zn2+ (2Zn2+–SBD-2BPEA system,
7888 | Dalton Trans., 2009, 7888–7890
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10 mM HEPES, 1% DMSO, pH 7.2). The instant fluorescence
enhancement was observed upon triphosphate (TP) addition. The
emission increases almost linearly with the [TP]total and the emis-
sion enhancement is no longer observed when the [TP]total : [2Zn2+–
SBD-2BPEA] ratio is higher than 1 : 1. The emission enhancement
factor (EEF) induced by one equivalent TP is around six. The
results suggest that the TP binding ratio of the 2Zn2+–SBD-2BPEA
system is around 1 : 1. Moreover, the emission of SBD-2BPEA
is blue shifted from 591 to 576 nm in this process (Fig. 1(a)).
A similar fluorescent response was also observed for the PPi
anion with an EEF of ~5 accompanied by a blue shift of 10 nm
(Fig. S2†). However, the addition of monophosphate anions
electron withdrawing effect of 7-sulfonamide on 4-BPA, which
in turn invalidates the strategy of appending the bis(BPA-Zn2+)
moiety to the fluorophore for phosphate sensing. A similar case
has also been reported for its analogue, NBD-BPA, in which
the electron-withdrawing 7-NO2 decreases the electron donating
ability of 4-amine and disables 4-BPA from Zn2+ binding.18,19
The fluorescent Zn2+ titration of SBD-BPEA exhibits a linear
emission decrement with the increase of [Zn2+]total, and the emission
becomes steady when the [Zn2+]total–[SBD-BPEA] ratio reaches
higher than 1 : 1 (Fig. S3†). The result suggests a 1 : 1 Zn2+-binding
stoichiometry for SBD-BPEA, and the Zn2+-triggered sulfonamide
deprotonation should reduce the ICT effect and result in the
emission decrease similar to the decreased emission at pH >10.0.
The fluorescent Zn2+ titration of SBD-2BPEA also demon-
strates a linear emission decrease with an increase of [Zn2+]total
when the [Zn2+]total–[SBD-2BPEA] ratio is lower than 0.5 (Fig. 2).
Afterwards, the emission decrease rate becomes smaller at
-
-
HPO42-/H2PO4 and other common anions such as CH3CO2 ,
NO3 , Cl-, CO32-, SO42-, ClO4- and F- does not induce any distinct
-
emission change (Fig. 1(b)). Interestingly, TP or PPi do not induce
any evident emission enhancement of the 1Zn2+–SBD-2BPEA
system (HEPES buffer of 10 mM SBD-2BPEA and 10 mM Zn2+),
which suggests that the presence of two equivalent Zn2+ is essential
for the fluorescent discrimination of TP/PPi from monophosphate
anions. On the other hand, SBD-BPEA displays a silent response
to TP and PPi in both the 2Zn2+–SBD-BPEA (HEPES buffer
with 10 mM SBD-BPEA and 20 mM Zn2+) and the 1Zn2+–
SBD-BPEA system (HEPES buffer of 10 mM SBD-BPEA and
10 mM Zn2+). All these results demonstrate that the additional
4-BPEA in SBD-2BPEA is essential for the TP/PPi sensing ability
of the 2Zn2+–SBD-2BPEA system.
higher [Zn2+]total and reaches a steady state when the [Zn2+]total
–
[SBD-2BPEA] ratio is ~1.4. Only minor emission enhancement
can be observed when the [Zn2+]total–[SBD-2BPEA] ratio increases
from 1.4 to 4. Comparison between the fluorescent Zn2+ titra-
tion data of SBD-2BPEA and SBD-BPEA implies that Zn2+
is firstly bound to the 7-sulfonated BPEA in SBD-2BPEA at
lower [Zn2+]total, and that deprotonation of sulfonamide results
in the emission decrease. Since 4-BPEA displays the sole PET
effect in SBD-2BPEA, the second Zn2+ to 4-BPEA forming the
diZn2+ complex at higher [Zn2+]total should lead to the reduced
emission decrease rate or even emission enhancement. In fact, all
of these have been observed when the [Zn2+]total–[SBD-2BPEA]
ratio reaches higher than 0.5. The stability of the diZn2+ complex
of SBD-2BPEA should be low, therefore only minor emission
enhancement was observed even when four equivalents of Zn2+
was added. In addition, the ESI-MS data show only the peak
for the mono-Zn2+ complex (727.4, [M - H + Zn]+, Fig. S4†),
suggesting that the stability of the diZn2+ complex is poor in the
conditions used for MS determination.
Fig. 1 (a) The emission spectra of the 2Zn2+–SBD-2BPEA (10 mM)
5-
system in HEPES buffer when titrated by Na5P3O10 solution. The [P3O10
]
for each spectrum is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 and 13 mM,
respectively (from bottom to top). lex, 468 nm. Inset: the titration profile
according to the emission at 576 nm. F0 is the emission intensity of free
SBD-2BPEA at 576 nm. (b) The different emission enhancement factors
of the 2Zn2+–SBD-2BPEA system in HEPES buffer (10 mM, pH 7.2, 1%
DMSO) induced by different anions. The EEFs were obtained according
to the emission at 576 nm when irradiated at 468 nm.
Under similar conditions, neither the 2Zn2+–SBD-2BPA sys-
tem (10 mM SBD-2BPA and 20 mM Zn2+) nor the 1Zn2+–
SBD-2BPA system (10 mM SBD-2BPA and 10 mM Zn2+) displays
any fluorescent response to TP or PPi. There have been successful
examples for the design of phosphate sensors by modifying
fluorophores such as xanthone and anthracenes with an appended
bis(BPA-Zn2+) moiety,12–14 however, current data show that such a
strategy does not work for ASBD fluorophore modification.
The different phosphate sensing behaviour of SBD-2BPA and
SBD-2BPEA should be correlated to their different Zn2+ binding
behaviours. In fact, SBD-2BPA displays no evident emission
change upon Zn2+ titration, suggesting that SBD-2BPA has a
very poor Zn2+ binding ability as supported from its UV Zn2+
titration data. The poor Zn2+ binding ability is due to the
Fig. 2 The fluorescent spectra of 10 mM SBD-2BPEA in aqueous HEPES
buffer (10 mM, pH 7.2) containing 3% DMSO (v/v) obtained when titrated
by adding aliquots of 5 mL Zn(NO3)2 solution (1.2 mM). lex, 468 nm. The
top line is for free SBD-2BPEA, the bottom line is for the presence of 1.4
equivalent Zn2+. The inset displays the titration profile obtained according
to the emission intensity at 591 nm.
The TP-triggered emission enhancement of the 2Zn2+–
SBD-2BPEA system should be induced by the synergic TP
coordination to both Zn2+ centres with the two spaced BPEA
motifs to form the diZn2+ complex (Fig. 3), since the 1Zn2+–
SBD-2BPEA system does not show any response. The mono-Zn2+
complex of SBD-2BPEA alone displays lower emission than the
diZn2+ complex due to the deprotonation of sulfonamide and the
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estimated to be 4.1 ¥ 105 and 2.8 ¥ 105 M-1 (Fig. S7†). The
constants are comparable to those reported by Hamachi et al.,
which displays the confirmed synergic coordination of phosphates
to the unsaturated coordinated bisZn2+.14a
In conclusion, a novel visible light excited fluorescent TP/PPi
sensor was successfully constructed by appending the bis(BPEA-
Zn2+) motif to the ICT fluorophore ASBD. The 2Zn2+–
SBD-2BPEA system displays a selective PPi/TP-amplified flu-
orescence upon excitation at 468 nm. However, the analogous
2Zn2+–SBD-2BPA system based on the reported bis(BPA-Zn2+)
appending strategy fails to exhibit any fluorescent response. This
work provides a new promising approach for the development of
visible light excited sensors for phosphates by coupling two Zn2+
binding motifs with an ICT fluorophore. Moreover, the resulting
sensors can be fine tuned so that different phosphate anions can
be distinguished.
Fig.
3
The proposed sensing mechanism and binding mode of
2Zn2+–SBD-2BPEA system to triphosphate anion.
PET effect of 4-BPEA. The affinity of SBD-2BPEA to the second
Zn2+ could be largely enhanced by the synergic coordination
effect and the negative charges of TP. As a result, the PET effect
of 4-BPEA is effectively blocked and emission enhancement is
achieved.
We thank the financial support from the National Natural
Science Foundation of China (No. 20871066, 30370351, 90713001,
20721002, 20571043) and the Natural Science Foundation of
Jiangsu Province (BK2008015).
In order to verify the coordination mode of the ternary complex
of SBD-2BPEA, solid samples were prepared by adding TP
to the methanol solution of SBD-2BPEA and Zn(NO3)2. The
ESI-MS spectrum (negative mode, in DMSO–methanol) of the
sample exhibits an intensive signal at m/z 1047.4 (Fig. S5†).
The signal can be assigned to [2Zn2+ + SBD-2BPEA + P3O105-]-,
which confirms the binding ratio of 2 : 1 : 1 ([Zn2+]–[SBD-2BPEA]–
[P3O105-]) for the diZn2+ complex. The intramolecular synergic
coordination of TP to dual Zn2+ centers favours the stabilisation of
the diZn2+ complex even in the conditions for MS determination.
Further evidence for such a binding mode comes from the 31P
NMR spectral data. Two signals of free TP at -5.62 (distal P)
and -20.47 (middle P) ppm are shifted to -7.58 and -19.35 ppm,
respectively, when mixed with one equivalent SBD-2BPEA and
two equivalents Zn(NO3)2 (Fig. 4).
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Due to the flexibility of the BPEA motif, PPi is able to coordinate
to the two spaced (BPEA-Zn2+) motif in a similar synergic manner
2-
-
(Fig. S6†). However, HPO4 and H2PO4 fail to do so and
no emission change is observed, although their Zn2+ affinity is
comparable to that of TP and PPi. Their smaller size means that
one such anion cannot coordinate with the spaced bis(BPEA-
Zn2+) motif simultaneously. The minor emission enhancement
induced by the bulk C2O42- (EEF, ~1.5) also supports this proposal.
According to the fluorescence titration, the apparent binding
constants of diZn2+–SBD-2BPEA complex to TP and PPi were
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7890 | Dalton Trans., 2009, 7888–7890
This journal is
The Royal Society of Chemistry 2009
©