A benzothiazole-based sensor for pyrophosphate (PPi) and ATP: Mechanistic insight for anion-induced ESIPT turn-on

Article abstract of DOI:10.1039/c4tb01109k

A benzothiazole derivative 2 bearing two 2,2′-dipicolylamine (DPA) groups was examined for its zinc-binding and subsequent anion sensing properties. The study revealed the anion sensing mechanism of polyphosphate anions via sequential binding to two zinc centers, on the basis of both ¹H NMR and mass spectral evidence. The mechanistic insight would provide valuable information for the future design of new excited state intramolecular proton transfer (ESIPT) sensors. In addition, the zinc complex exhibited solvent-switchable selectivity, responding to pyrophosphate (PPi) in EtOH and adenosine 5′-triphosphate (ATP) in water. This journal is

Full text of DOI:10.1039/c4tb01109k

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Materials Chemistry B
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PAPER
A benzothiazole-based sensor for pyrophosphate
(PPi) and ATP: mechanistic insight for anion-
induced ESIPT turn-on†
Cite this: J. Mater. Chem. B, 2014, 2,
6634
a
a
ab
*
Junfeng Wang, Xiumin Liu and Yi Pang
A benzothiazole derivative 2 bearing two 2,20-dipicolylamine (DPA) groups was examined for its zinc-
binding and subsequent anion sensing properties. The study revealed the anion sensing mechanism of
1
polyphosphate anions via sequential binding to two zinc centers, on the basis of both H NMR and mass
Received 7th July 2014
Accepted 5th August 2014
spectral evidence. The mechanistic insight would provide valuable information for the future design of
new excited state intramolecular proton transfer (ESIPT) sensors. In addition, the zinc complex exhibited
solvent-switchable selectivity, responding to pyrophosphate (PPi) in EtOH and adenosine 50-triphosphate
(ATP) in water.
DOI: 10.1039/c4tb01109k
www.rsc.org/MaterialsB
The design of uorescent chemosensors for biologically rele-
vant anions remains a challenging topic.1–3 This is due to the
lack of a general design principle that can reliably translate an
anion-binding event into a large uorescent signal response.4
One of the most successful strategies for anion recognition has
been the use of coordination complexes that bear one or two
vacant coordination sites for binding anionic guests.5–8 This
strategy can be realized via a proper arrangement of two zinc
centers, which can selectively bind anions such as pyrophos-
phate (PPi)9–16 and adenosine 50-triphosphate (ATP).4,17,18
However, this binding pattern usually induced little spectral
shi in the uorescence signal, which lowers the sensitivity and
hampers their practical applications. An ideal uorescent
sensor should exhibit a great uorescence turn-on with a large
spectral shi, in order to minimize the interference from the
free dye.19
The excited state intramolecular proton transfer (ESIPT) has
been shown to be an attractive mechanism, as the ESIPT
ON–OFF could induce a large spectral shi.20 A few attempts
have been made to incorporate the ESIPT mechanism in the PPi
sensor scheme on the basis of either a mononuclear zinc
complex2ꢀ(1a-Zn),21 which responds to diphosphate ions
(H2P2O7 and ADP), or binuclear zinc complex (1b-Zn, where
R ¼ “–CH2DPA–Zn”).22 Herein, we report the ESIPT properties of
sensor 2, a thiazole analogue of 1b, where the sulfur heteroatom
perturbs the ESIPT and related chemical processes. Interest-
ingly, the sensor exhibited solvent-switchable recognition for
Scheme 1 Structure of 1, the zinc complexes and the ESIPT OFF–ON
switch induced by H2PPi.
pyrophosphate (in alcohol) and ATP (in water). The anion
recognition is accompanied by a large spectral shi (ꢁ95 nm
shi from blue to green), which is useful for naked eye detec-
tion. In addition, the heteroatom substitution makes the two
zinc binding sites in 2 distinguishable from each other, thereby
allowing close examination of the sensing mechanism step-by-
step. This result led to a better understanding about the ESIPT
turn-on (Scheme 1).
Formation of the ligand–zinc complex
Ligand 2 was synthesized as a light yellow syrup-like oil by using
a similar strategy with a modied procedure (see ESI, Scheme
S1†).21,22 Our initial attention was paid to the process of zinc
binding-induced ESIPT turning-off, by addition of different
equiv. of Zn(NO3)2 to 2. When the rst equiv. of Zn2+ was added,
the absorption peak (lmax ¼ 343 nm) was red-shied to lmax
¼
aDepartment of Chemistry, The University of Akron, Akron, Ohio 44325, USA. E-mail:
367 nm, as a consequence of deprotonation Ph–OH / Ph–Oꢀ
(Fig. 1a).21,22 The observation clearly indicated that the rst Zn2+
predominantly bound to “the site I” to disable the ESIPT, which
caused the blue uorescent shi with great uorescence turn-
on (Fig. 1b). The second equiv. of Zn2+ induced nearly no
yp5@uakron.edu
bMaurice Morton Institute of Polymer Science, The University of Akron, Akron, Ohio
44325, USA
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c4tb01109k
6634 | J. Mater. Chem. B, 2014, 2, 6634–6638
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Fig. 1 UV-vis (left) and fluorescence (right) titration of 2 (10 mM) upon
addition of different equiv. of Zn2+ in EtOH.
spectral shi in absorption, as the Zn2+ cation was bound to the
second DPA group, resulting in the formation of complex 4
(Scheme 2). The binding of the second Zn2+ further increased
the uorescence, which is consistent with the previous data (by
removing the weak PET effect from DPA).21 Fluorescence
quantum yields of 2 (f ¼ 0.01) and 4 (f ¼ 0.17) were calcu-
lated by using quinine sulfate as the reference.
Fig. 2 1H NMR titration of 2 upon addition of different equiv. of Zn2+ in
CD3OD.
1H NMR titration further conrmed the assumption. The
proton signals at ꢁ8.5 ppm were attributed to two overlapping
doublets from the characteristic pyridine protons Ha and Ha0 ,
which are associated with different zinc binding sites in 2. The
addition of the rst equiv. Zn2+ only caused a downeld shi of
Ha at the site I (from 8.53 to 8.70), and the second equiv. Zn2+
gradually induced a downeld shi of Ha0 at the site II (from 8.56
to 8.60) (Fig. 2). Similarly, the same trend was observed from the
mass experiments (Fig. S1, ESI†). Upon addition of the
rst equiv. of Zn2+, the signal of ligand 2 (TOF-MS-ES+ at peaks
[2 + H+]+ ¼ 650.3146 and [2 + Na+]+ ¼ 672.2971) almost dis-
appeared (ESI Fig. S1†) with the formation of a new peak cor-
responding to [2 + Zn2+ ꢀ H+]+ ¼ 712.2447. Two-dimensional
ESI-traveling wave ion mobility mass spectrometry (TWIM-MS)
further revealed that the mass peak at m/z ¼ 712 included two
signals with dri times at 2.89 ms and 4.96 ms, which were
assumed to be 3b and 3a, respectively (Fig. 3). It appeared that
the mono-charged complex 3a with the dri time of 4.96 ms was
Fig. 3 Two-dimensional ESI TWIM MS plot for m/z at 712. Ion mobility
separation was effected using a traveling wave height of 9 V and a
traveling wave velocity of 350 m sꢀ1. The precursor ion from m/z at 712
gives rise to signals at 2.89 and 4.96 ms; on the basis of the isotope
patterns observed, the signals at 2.89 and 4.96 ms agree well with
[(2 + Zn2+
)
2
ꢀ 2H+]2+ (3b) and [2 + Zn2+ ꢀ H+]+ (3a).
predominant. Upon addition of the second equiv. Zn2+, the
signals of complex 3 gradually disappeared, and the di-zinc
complex 4 was detected as the predominant species with three
positive charges: [2 + 2Zn2+ ꢀ H+]3+ ¼ 258.7352 (Fig. S2, ESI†).
All the data pointed to that the zinc complex with four coordi-
nation (at the site I) are easier to form than that with three
coordination (at the site II).
Binding to phosphate anions
The anion response of the resulting zinc complex 4 was examined
by addition of 5.0 equiv. of different anions in EtOH. Much to our
delight, 4 showed very good selectivity toward H2PPi, giving
the ESIPT turn-on with bright green emission (Fig. 4a and c).
Interestingly, 4 showed selectivity toward H2ATP over the other
anions in aqueous (Fig. 4b and c). The mass spectra from the
solution of “4 + H2PPi” in EtOH detected the major species
with m/z ¼ 952.1031 and 974.1014, which matched with the
isotope patterns for [5 + H+]+ ¼ 952.0404 (C39H36N7O8P2SZn2)
and [5 + Na+]+ ¼ 974.0224 (C39H36N7NaO8P2SZn2) (ESI† Fig. S10).
Scheme 2 Structure of 2 with two different zinc-binding sites (I and II),
and its zinc complex formation.
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Fig. 4 Fluorescence response of Zn complex 4 (10 mM) with different
anions (50 mM) in ethanol (a) and different anions (2 mM) in water (b),
while the dye was excited at the isosbestic point ꢁ357 nm. The fluo-
rescence image (c) shows the response of 4 to various phosphate
anions in ethanol (top) and DI water (bottom).
The observation pointed to the formation of 5, which is essen-
tial to turn on the ESIPT.
A clear differentiation of 1H NMR signals at two different
zinc binding sites allowed us to further examine the details of
pyrophosphate binding. Upon addition of H2PPi to the zinc
complex 4 (i.e. 2–Zn2), both Ha and Ha0 (at 8.6 and 8.7 ppm)
disappeared simultaneously to form a new broad peak at ꢁ8.8
ppm. The spectral shi suggests that H2PPi was binding to both
site I (from the Ha shi) and site II (from Ha0 shi) (Fig. 5a). The
observation supports the formation of 5 (Scheme 3), which was
the key for the ESIPT turn-on mechanism. The PPi adduct 6
might also be possible, since the PPi anion can bind to a mono-
nuclear zinc center.21 The control experiment was carried out by
reaction of zinc complex 3a with PPi, which would give 7. The 1H
NMR of 7 showed that the Ha at site I would have a different
pattern if PPi was bonded to only one zinc cation (Fig. 5b). The
result thus consistently pointed to the formation of 5, and ruled
out the presence of 6 (Scheme 3).
Fig. 5 1H NMR spectra of 4 (a) and 3 (b) upon addition of H2PPi in
CD3OD.
As seen in Fig. 4b and c, the zinc complex showed an inter-
esting response to H2ATP in aqueous solution (pH z 4.0 for 2
mM H2ATP). The mass spectrum of the aqueous solution
detected the formation of a major species at m/z ¼ 1281.2192
and 1303.2233, which matched with the isotope pattern of
intermediates [8 + H+]+ and [8 + Na+]+ respectively (Fig. 6; ESI
Fig. S3, S11†), also showing the binding of H2ATP to both
binding sites I and II.
The stability of complex 4 was further examined in buffered
aqueous at different pH values. The study revealed that
complex 4 was quite stable in buffered aqueous at different pH
values (ranges from pH ¼ 4.7 to 7.7) (Fig. 7). When pH ¼ 4.7,
the ESIPT turn-on could be accomplished by H2ATP (Fig. 8).
The selectivity of 4 to phosphate anions was examined. The
result showed that H2ATP exhibited a stronger tendency to
turn-on ESIPT over other phosphate anions such as H2PPi
(Fig. 9), revealing the same trend as observed in DI water
Scheme 3 Possible formation of PPi adducts from zinc complexes.
(Fig. 4b). The reversal of selectivity in different solvents, i.e.
H2PPi in EtOH and H2ATP in water (or acidic buffer), showed
that the anion binding-induced ESIPT turn-on was quite
sensitive to the solvent environment, which should be further
examined. Clearly, the current sensor worked better under the
acidic conditions, as the ESIPT turn-on requires an H+ to
generate the phenol: Ph–Oꢀ + H+ / Ph–OH.
In conclusion, benzothiazole derivative 2 bearing two DPA
groups was examined for its zinc-binding and subsequent anion
sensing properties. The binuclear zinc complex was formed via
sequential zinc chelating at two different DPA groups. Spectral
data analysis, including UV-vis, uorescence, 1H NMR and ESI-
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Fig. 6 Possible formation of H2ATP adducts from zinc complexes in
water.
Fig. 9 Fluorescence intensity of 4 (10 mM) in PBS buffer (pH ¼ 4.7),
upon addition of 2 mM of phosphate anions.
insight would provide a useful guide for the future design of
new ESIPT sensors.
Acknowledgements
This work was supported by National Institute of Health (Grant
no: 1R15EB014546-01A1). We also thank the Ohio Department
of Development and Coleman endowment from the University
of Akron for partial support.
Fig. 7 Fluorescence intensity of 4 in PBS buffer at different pH values.
The inset plots the stability of the complex at different pH values
(4.7–7.7).
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