Highly selective colorimetric sensing pyrophosphate in water by a NBD-phenoxo-bridged dinuclear Zn(ii) complex

Article abstract of DOI:10.1039/c2ob25617g

A novel NBD-phenoxo-bridged dinuclear Zn(ii) complex is found to be an effective colorimetric sensor for pyrophosphate (PPi) in pure aqueous solution over a wide pH range. This sensor shows high binding affinity (K_a ≈ 3 × 10⁸ M^-1) and high selectivity for PPi, and can be also used to assay the activity of pyrophosphatase in real time.

Full text of DOI:10.1039/c2ob25617g

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PAPER
Highly selective colorimetric sensing pyrophosphate in water by a
NBD-phenoxo-bridged dinuclear Zn(II) complex†
Shengjun Yang,a Guoqiang Feng*a and Nicholas H. Williamsb
Received 26th March 2012, Accepted 11th June 2012
DOI: 10.1039/c2ob25617g
A novel NBD-phenoxo-bridged dinuclear Zn(II) complex is found to be an effective colorimetric sensor
for pyrophosphate (PPi) in pure aqueous solution over a wide pH range. This sensor shows high binding
affinity (Ka ≈ 3 × 108 M−1) and high selectivity for PPi, and can be also used to assay the activity of
pyrophosphatase in real time.
phosphate (Pi), ADP, AMP, and particularly the equally charged
Introduction
ATP tetraanion, as well as the ability to convert PPi recognition
into a fluorescent or colorimetric signal. Hence, during the past
decade, only a few examples of effective fluorescent and colori-
metric sensors for PPi in 100% aqueous solutions have been
reported.9–12 Pioneered by the work in Czarnik’s group,10 most
effort has been devoted to the development of fluorescence chemo-
sensors for PPi in aqueous solution.11 Among them, the utiliz-
ation of metal complexes, particularly zinc complexes with a
bis-(2-pyridylmethyl)amine (DPA) unit as a binding site for PPi,
has been found to be the most successful strategy.11b–i Indicator-
displacement assays (IDA) have also been used to detect PPi
when the metal complex lacks an available fluorophore or
chromophore.13 Although fluorescent sensors for PPi have been
developed most successfully to date,11,12 effective colorimetric
sensors for PPi in pure water are rather rare.14–18 The latter has
an outstanding advantage – it can be easily observed and deter-
mined by naked eye so that the measurements by analytical
instruments may be minimized or even eliminated.
Much attention has been given in recent years to the develop-
ment of receptors and sensors for anions due to their important
roles in various chemical and biological processes.1 Among
them, pyrophosphate (P2O74−, PPi) is one of the most popular
targets for recognition studies because it plays very important
roles in several biological processes.2 PPi is a product of
hydrolysis of ATP and other nucleotide triphosphates under cel-
lular conditions, and is vital for energy transduction and the
control of metabolic processes in organisms via its participation
in various enzymatic reactions.3 The detection of the PPi concen-
tration level has been examined as a real-time DNA sequencing
method4 and recently has become an important issue in cancer
research.5 The level of PPi is also related to several diseases, for
example, a lack of PPi can lead to calcium deposits in arteries –
called Mönckeberg’s arteriosclerosis (MA)6 and too much PPi
can cause calcium pyrophosphate deposition disease (CPDD).7
PPi is also the most common cause of cultural eutrophication as
an environment pollutant.8 Therefore, the specific recognition,
detection and sensing of PPi in aqueous solution is of wide-
spread significance.9
Here we report a new NBD-phenoxo-bridged dinuclear Zn(II)
complex 1·2Zn (Fig. 1), which is an effective colorimetric
sensor for PPi, not only with high sensitivity in pure aqueous
solution of a wide pH range, but also with high selectivity for
PPi over many other anions including ATP and Pi.
Due to the strong hydration of anions in water and the struc-
tural similarity of the phosphate anion family, the creation of
effective PPi sensors in aqueous solution is challenging. It
requires both high affinity for PPi in water and, importantly, high
selectivity for PPi over other related analogues such as inorganic
aKey Laboratory of Pesticide and Chemical Biology of Ministry of
Education, College of Chemistry, Central China Normal University, 152
Luoyu Road, Wuhan 430079, P.R. China.
E-mail: gf256@mail.ccnu.edu.cn
bCentre for Chemical Biology, Department of Chemistry, University of
Sheffield, Sheffield S3 7HF, UK. E-mail: N.H.Williams@sheffield.ac.uk
†Electronic supplementary information (ESI) available: Job’s plot for
PPi and ATP with 1·2Zn, sensing selectivity of sensor 1·2Zn for PPi
over Pi, ADP and ATP, sensing of PPi at different pH values, studies of
2·Zn, some of the NMR spectra. See DOI: 10.1039/c2ob25617g
Fig. 1 Sensor 1·2Zn and colorimetric sensing of PPi.
5606 | Org. Biomol. Chem., 2012, 10, 5606–5612
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that shows a high selective coloration for PPi under physiologi-
cal conditions.
Results and discussion
Sensor 1·2Zn was prepared from 4-acetamidophenol in four
steps (Scheme 1). The chromophore 7-nitrobenz-2-oxa-1,3-
diazole (NBD) in sensor 1·2Zn was selected because it can be
easily introduced and it absorbs at long wavelengths
(∼500 nm),19 to offer a sensor which does not have much back-
ground absorption to compete with. 1·2Zn can be easily formed
by mixing ligand 1 with 2 equiv of zinc nitrate in methanol and
it dissolves readily in pure water forming a red solution. As a
control, mononuclear complex 2·Zn was also prepared in a
similar manner (Scheme 1).
We first tested whether the red color of the 1·2Zn solution can
be affected by addition of anions (sodium salts). When one
equiv. of PPi was added to sensor 1·2Zn (35 μM) in an aqueous
solution of HEPES buffer (50 mM, pH 7.4, HEPES = 2-[4-(2-
hydroxyethyl)-1-piperazinyl]-ethanesulfonic acid), the color of
the sensor solution changes immediately from red to purple
(Fig. 2). In contrast, no color changes upon addition of other
inorganic anions such as either monovalent anions H2PO4−, F−,
To shed further light on this sensor, the effect of anions on the
absorption spectrum of sensor 1·2Zn were then examined in an
aqueous HEPES buffer (50 mM, pH 7.4) at 25 °C. Under these
conditions, sensor 1·2Zn (35 μM) showed a maximum absorp-
tion band centred at 501 nm with an extinction coefficient of
22 700 M−1 cm−1. As shown in Fig. 3a, upon addition of PPi in
increasing amounts, the peak at 501 nm decreases and a new
peak appears at 526 nm. The 25 nm red shift is consistent with
the color change observed from red to purple. The absorbance at
600 nm increases linearly until it reaches a plateau with the
addition of more than one equiv. of PPi (inset in Fig. 3a), which
indicates a tight 1 : 1 binding between 1·2Zn and PPi at these
concentrations. This 1 : 1 binding stoichiometry was also
confirmed by Job’s plot (Fig. S1, ESI†). Unlike the large 25 nm
Cl−, Br−, CH3CO2−, NO3−, HCO3−, ClO4 N3 or di/trivalent
anions HPO42− (Pi), SO42−, S2O72−, C2O42−, citrate, PO43− even
up to an excess of 1000 equivalents. Other organic phosphate
anions such as AMP, ADP, ATP, phenyl phosphate (PhPi),
4-nitrophenyl phosphate (NPhPi) were also tested. Only the
addition of excess ATP induces a slight color change. As a
control, mononuclear 2·Zn does not show any observable color
changes even upon the addition of a large excess of PPi and
other anions (Fig. S4, ESI†). These results indicate that only the
dinuclear complex 1·2Zn can be used as a colorimetric sensor
−
−
Scheme 1 Synthesis scheme for 1·2Zn and 2·Zn. Reagents and con-
ditions: (a) dipicolylamine (DPA), paraformaldehyde in C2H5OH, 80 °C.
(b) aq. HCl, 100 °C. (c) 4-Chloro-7-nitro-2,1,3-benzoxadiazole
(NBD-Cl), NaHCO3, MeOH, rt. (d) Zn(NO3)2, H2O, MeOH.
Fig. 3 (a) UV-vis spectra changes of sensor 1·2Zn (35 μM) upon
addition 0–3 equiv of PPi (sodium salt). Insert: the absorbance changes
at 600 nm upon addition of PPi. (b) UV-vis spectra of sensor 1·2Zn
(35 μM) in the presence of various anions (35 μM). All the spectra were
measured in pure aqueous solution of 50 mM HEPES buffer (pH 7.4) at
25 °C. Under these conditions, sensor 1·2Zn is saturated with PPi (one
equiv.) and ATP (one equiv.), and can be saturated with ADP (more than
Fig. 2 Color changes of sensor 1·2Zn in 50 mM aqueous HEPES
buffer solution (pH 7.4), [1·2Zn] = 35 μM, [PPi] = 35 μM, [other
anions] = 175 μM. Anions from left to right: none, AMP, ADP, ATP,
2−
PO43−, HPO4 (Pi), H2PO4−, PhPi, NPhPi, PPi, F−, Cl−, Br−, I−,
NO3−, SO42−, HCO3−, CH3CO2−, citrate, N3−, ClO4−, S2O72−, C2O4
.
five equiv.) with a 4 nm red shift of its λmax
.
2−
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red shift observed with PPi, only a relatively small red shift
(10 nm) is observed with ATP upon complexation, which is why
ATP only induces a slight color change. The spectral changes
and the degrees of red shift (0–4 nm) caused by other anions are
almost negligible, so no color changes can be observed in these
systems (Fig. 2 and 3b). The different degrees of red shift
induced by PPi and other anions can be explained by the degree
of electron donation of the phenolate oxygen atom into the
phenyl ring. Only when the most charged PPi tightly binds to
1·2Zn the bond between the phenolate oxygen atom and the two
Zn2+ ions sufficiently weakened to leave a significantly more-
negatively charged phenolate oxygen atom. This donates more
electron density into the phenyl ring, and as a result, induces a
large red shift. The red shift induced by PPi was also observed
in Hong’s elegant naphthalene-DPA based fluorescent11b and
azaphenol-DPA based colorimetric14 sensors, but both of them
absorb at much shorter wavelengths. The red shift of the former
sensor occurs outside the visible region and the latter requires
higher concentration (60 μM) for naked eye detection of PPi.20
The different response relative to the similarly charged ATP can
be rationalized in terms of charge density. In PPi, both phos-
phoryl units carry two negative charges and can bind to both
Zn2+ ions, whereas in ATP the same charge is distributed across
three phosphoryl units. Hence, the same binding mode, invol-
ving four oxygen atoms with lower charge density, has less
effect on the Zn2+-ligand binding. It seems likely that an alter-
nate binding mode involving all the phosphoryl groups is steri-
cally precluded, but even in this case the α and β phosphoryl
groups will be weaker donors than the γ and PPi phosphoryl
oxygens.
Fig. 4 (a) 31P NMR spectra of PPi (4 mM) and ATP (4 mM) and with
one equiv. of sensor 1·2Zn in H2O. (b) Proposed binding modes for
1·2Zn with PPi and ATP.
To further understand this, as well as the mode of complexa-
tion, 31P NMR spectroscopy studies were undertaken. As shown
in Fig. 4a, both P atoms in PPi are magnetically equivalent and
show a single signal at −6.5 ppm. However, on binding with
1·2Zn, significant upfield shifts (Δδ = 5.30 ppm) were observed
for the 31P signal. The presence of a single 31P signal suggests
that both P atoms in the metal binded PPi are still magnetically
equivalent and therefore the two sets of oxygen anions on each P
atom of PPi are equally bound to the binuclear zinc complex. In
the case of ATP binding with 1·2Zn, upfield shifts for the three
phosphorous signals in ATP were also found, but their shift
degrees are quite different. The signals for β and γ phosphorous
are upfield shifted by 1.48 and 0.77 ppm, respectively. In con-
trast, much smaller upfield shift for α phosphorous signal (Δδ =
0.15 ppm) was observed, which indicates the α phosphorous
centre is not interacting (or has very week interactions) with the
zinc centres of 1·2Zn. Therefore, the binding force of ATP with
1·2Zn mainly come from the interactions of the oxygen anions
on β and γ phosphoryl groups of ATP with the metal centres of
1·2Zn. Based on our 31P NMR studies and the crystal structure
of the PPi complex reported by Hong et al.,14 the proposed
binding modes for 1·2Zn with PPi and ATP are shown in
Fig. 4b.
Table 1 Apparent association constant (Ka) of sensor 1·2Zn for anions
2−
−
PPi, ATP, ADP, AMP, HPO4 and H2PO4 in an aqueous HEPES
buffer (50 mM, pH 7.4) at 25 °Ca
Anion
Ka (M−1
)
Anion
AMP
HPO4
H2PO4
Ka (M−1
)
PPi
ATP
ADP
(2.9 0.3) × 108
(3.7 0.4) × 106
(4.6 0.4) × 105
(7.2 0.7) × 104
(2.3 0.2) × 104
(2.1 0.2) × 104
2−
−
a All anions were added as sodium salts. Ka for PPi and ATP were
determined by competitive binding in the presence of 1000 fold excess
of HPO42−. For other anions, Ka was calculated by fitting the curve of
absorbance changes against phosphate concentration via UV-vis
titration.21
of sensor system would be low relative to the fluorescent chemo-
sensors. In this case, achieving high-affinity binding is particu-
larly important for improving detection limits. As shown in
Table 1, 1·2Zn indeed showed the strongest binding for PPi over
other phosphates. The apparent association constant (Ka) value
for the complexation of PPi with 1·2Zn was determined to be
(2.9 0.3) × 108 M−1 in an aqueous HEPES buffer (50 mM,
pH 7.4) at 25 °C, which is about 80-fold, 630-fold, 4000-fold
and over 10 000-fold higher than for ATP, ADP, AMP and
In biological systems, PPi is released in the presence of ATP
and other phosphates, so good sensors for PPi in water must
show high selectivity for PPi over other highly anionic phos-
2−
HPO4 binding with 1·2Zn, respectively. This nearly nano-
molar affinity and the big differences in the affinity values
between phosphate anions not only mean that 1·2Zn can detect
PPi at sub-micromolar concentrations in water, but also can
detect PPi in the presence of a large excess of other phosphates,
2−
phates such as ATP and HPO4 (Pi) as well as high affinity.
Since this colorimetric sensor is based on the chromophore of
nonfluorescent NBD-phenol, the detection limitation of this kind
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even with the same charge. Indeed, an obvious color change
from red to purple was also observed when PPi was titrated into
2−
the sensor 1·2Zn solution in the presence of 1000 equiv. HPO4
,
100 equiv. ADP or 50 equiv. ATP (Fig. S2, ESI†), thus making
sensor 1·2Zn a good candidate for bioanalytical applications. The
same arguments presented above concerning the degree of red
shift also apply for explaining why stronger binding is obtained
for 1·2Zn with PPi compared to ATP.11b To the best of our know-
ledge, our system is the first colorimetric sensor that not only has
nanomolar affinity for PPi in pure water solutions, but also can
effectively discriminate PPi from both Pi and ATP.22
We also tested the effect of pH on PPi sensing to evaluate the
functional pH ranges of the sensor. Over the pH range 5.5–10.0,
addition of PPi causes obvious color changes with 18–25 nm red
shift of the maximum absorption band of 1·2Zn, and these
changes are still evident in the presence of a wide range of com-
peting anions (Fig. S3, ESI†). This means 1·2Zn works over a
wide pH range, and even if the external pH is disturbed, 1·2Zn
still can detect PPi in the presence of various anions.
To demonstrate the potential of sensor 1·2Zn in a practical
application, a real-time assay was constructed to monitor the
activity of inorganic pyrophosphatase (PPase). Inorganic PPase
is essential in many important biosynthetic reactions and cata-
lyses the hydrolysis of PPi into phosphate.23 Based on our
results, we thought sensor 1·2Zn may provide an easy way to
monitor the activity of inorganic PPase, even simply by obser-
vation of color change. Thus, the activity of inorganic PPase was
tested in a real-time assay using an aqueous HEPES buffer con-
taining 50 μM 1·2Zn and 50 μM PPi. The reactions were moni-
tored at 600 nm as a function of time immediately after addition
of the given units of inorganic PPase. As shown in Fig. 5a,
decays at 600 nm were observed over time after the addition of
PPase, and the reaction accelerates with an increase in concen-
tration of PPase. More importantly, as shown in Fig. 5b, this
assay was accompanied by an obvious color change of the reac-
tion mixture from purple to red, which means 1·2Zn can be used
to detect whether a given inorganic PPase sample is active or not
without using any analytical instruments. Fig. 5c shows scanning
kinetics of the assay in the presence of 0.2 units of inorganic
PPase. Clearly, the UV-vis spectra changes of the PPase cata-
lysed reaction in the presence of 1·2Zn are just opposite to that
of the titration experiments showed above in Fig. 3a. Therefore,
we proved that 1·2Zn can be used as a simple and convenient
chemosensor for real-time probing the activity of inorganic
PPase.
Fig. 5 (a) Kinetics of real-time monitoring PPi (50 μM) hydrolysis cat-
alyzed by inorganic PPase in the presence of 50 μM 1·2Zn. Reactions
are monitored at 600 nm. Concentrations of inorganic PPase: (1) 0 units;
(2) 0.1 units; (3) 0.2 units; (4) 0.4 units. (b) Color changes of the reac-
tion catalysed by PPase. (c) Scanning kinetics of real-time monitoring
PPi (50 μM) hydrolysis catalyzed by 0.2 units inorganic PPase. The
UV-vis spectra were recorded every minute. All the reactions were
recorded in an aqueous HEPES buffer (50 mM, pH 7.5) at 25 °C.
Experimental
TLC analysis was performed using precoated plates. Column
chromatography was performed using silica gel (200–300 mesh)
using eluents in the indicated v : v ratio. IR spectra were
recorded on a Perkin-Elmer Spectrum BX FT-IR spectropho-
tometer as KBr pellets and were reported in cm−1. NMR spectra
were measured on Varian Mercury 400 and 600 instruments,
General
Starting materials were purchased from commercial suppliers
and were used without further purification. All solvents were
purified by the most used methods before use. All solutions and
buffers were prepared with using distilled water that had been
passed through a Millipore-Q ultrapurification system. Buffers
used for different pHs were 2-(N-morpholino)ethanesulfonic
acid (MES), N-(2-hydroxyethyl)piperazine-N′-(2-ethane-sulfonic
acid) (HEPES), 2-(N-cyclohexylamino)ethanesulfonic acid
(CHES) and 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS).
1
operating at 400 or 600 MHz for H NMR and 100 or 150 MHz
for 13C NMR. Coupling constants (J values) are reported in
hertz. Electrospray mass spectra (ESI-MS) were acquired on
Agilent 1100 Series LC/MS ion trap mass spectrometers and
6530 Accurate-Mass QTOF spectrometer coupled to an Agilent
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HPLC 1200 series (Agilent Technologies). UV-vis spectra were
recorded on an Analytik Jena Specord 210 spectrophotometer.
Kinetics of real-time assay of pyrophosphatase were recorded on
an Agilent Cary 100 UV-vis spectrophotometer.
136.8, 142.1, 144.1, 144.7, 148.9, 148.8, 155.1, 158.5. IR (KBr,
cm−1): 3439, 1592, 1569, 1473, 1435, 1299, 1157, 1075, 996,
950, 861, 546. MS (ESI): 695.3 (M + H+), 717.1 (M + Na+).
+
HRMS (ESI): m/z calcd for C38H35N10O4 [M + H]+ 695.2837,
found 695.2868.
Synthesis of 4. To a 100 mL round-bottom flask was added
4-hydroxyacetanilide (3, 302 mg, 2.0 mmol), ethanol (50 mL),
paraformaldehyde (150 mg, 5.0 mmol) and bis-(2-pyridylmethyl)-
amine (877 mg, 4.4 mmol). After the reaction mixture was
refluxed for over 30 h, the solvent was evaporated under reduced
pressure. To the residue was added dichloromethane (25 mL),
and the organic phase was washed with water (5 mL × 3), and
then dried in anhydrous Na2SO4. The crude product was purified
by silica gel column (eluent: dichloromethane–methanol = 80 : 1
Synthesis of 1·2Zn. To a solution of 1 (26 mg, 0.037 mmol)
in 5 mL of MeOH, was added Zn(NO3)2·6H2O (22.5 mg,
0.076 mmol), and the mixture was stirred for 30 min at rt. After
concentrating under reduced pressure, the obtained solid was
recrystallised from MeOH to give sensor 1·2Zn. Mp: >300 °C.
1H NMR (600 MHz, DMSO-d6): δ 3.67 (4H, br, 2CH2), 4.24
(8H, br, 4CH2), 5.86 (1H, s, CH), 6.79 (2H, s, 2CH), 7.56 (8H,
br, PyH), 8.01 (4H, br, PyH), 8.66 (1H, d, CH), 8.80 (4H, br,
PyH), 10.72 (1H, s, NH). IR (KBr, cm−1): 3443, 3133, 1608 (s),
1659, 1471, 1442, 1384 (s), 1297 (s), 1260, 1124, 1507, 997,
957, 769, 564, 519. UV-vis spectra: 35 μM in 50 mM HEPES
buffer solution, pH = 7.4, Abs = 0.344 (370 nm), 0.795
(501 nm, λmax). MS (ESI): 1034.45 (M − NO3− + OH− + Na+)+.
Elemental analysis calcd for C38H34N10O4Zn2·(NO3)4·5H2O: C
39.22, H 3.81, N 16.85; found: C 39.21, H 3.47, N 16.63.
1
(v/v)) to afford 4 (631 mg, 55% yield). H NMR (400 MHz,
CDCl3): δ 2.14 (3H, s, CH3), 3.78 (4H, s, 2CH2), 3.86 (8H, s,
4CH2), 7.12 (1H, m, PyH), 7.41 (2H, s, ArH), 7.48 (4H, d, J =
5.2 Hz), 7.56 (1H, br, NH), 7.60 (4H, m, PyH), 8.51 (4H, d, J =
2.8 Hz, PyH). 13C NMR (150 MHz, CDCl3): δ 24.2, 54.4, 59.4,
121.4, 122.0, 123.1, 123.8, 129.6, 136.6, 148.7, 152.4, 158.7,
168.3. IR (KBr, cm−1): 3256, 3065, 2924, 1677, 1592, 1569,
1478, 1433, 1373, 1151, 762. MS (ESI): 574.2 (M + H+), 596.1
+
(M + Na+). HRMS (ESI): m/z calcd for C34H36N7O2 [M + H]+
Synthesis of 6. The compound 6 (400 mg, 35%) was syn-
thesized from 4-hydroxyacetanilide 3 (302 mg, 2.0 mmol) in
ethanol with paraformaldehyde (150 mg, 5.0 mmol) and bis-(2-
pyridylmethyl)amine (399 mg, 2.0 mmol) by the same method
described for the preparation of compound 4. 1H NMR
(400 MHz, CDCl3): δ 2.13 (3H, s, CH3), 3.76 (2H, s, CH2), 3.87
(4H, s, 2CH2), 6.85 (1H, d, J = 6.0 Hz, ArH), 7.15–7.17 (4H,
m), 7.32–7.34 (3H, m), 7.63 (2H, t, J = 4.4 Hz, PyH), 8.56 (2H,
t, J = 2.8 Hz, PyH). 13C NMR (150 MHz, CDCl3): δ 24.1, 56.6,
58.7, 116.5, 121.4, 122.2, 122.4, 122.7, 122.8, 123.2, 136.8,
148.7, 154.1, 157.9, 168.5. IR (KBr, cm−1): 3417, 3289, 2961,
1652, 1592, 1566, 1499, 1434, 1371, 1256, 1154, 1084, 756.
MS (ESI): 363.1 (M + H+), 385.0 (M + Na+). HRMS (ESI): m/z
calcd for C21H23N4O2+ [M + H]+ 363.1816, found 363.1826.
574.2925, found 574.2964.
Synthesis of 5. A solution of 4 (574 mg, 1.0 m mol) in 10 mL
of 6.3 M aq. HCl was heated to reflux for 3 h. After cooling, the
solution was basified with 2 M aq. NaOH solution to pH =
9. The neutralized product was extracted into dichloromethane
(10 mL × 3), dried with anhydrous Na2SO4, and the solvent was
evaporated under reduced pressure. The crude product was
purified by silica gel column (eluent: dichloromethane–methanol
= 100 : 1 (v/v)) to give a dark brown oil (186 mg, 35% yield).
1H NMR (400 MHz, CDCl3): δ 3.74 (4H, s, 2CH2), 3.86 (8H, s,
4CH2), 6.64 (2H, s, ArH), 7.13 (4H, m, PyH), 7.50 (4H, m,
PyH), 7.60 (4H, m, PyH), 8.53 (4H, m, PyH), 10.38 (2H, br,
NH2). 13C NMR (100 MHz, CDCl3): δ 54.8, 59.6, 116.8, 122.0,
123.1, 124.3, 136.6, 137.6, 148.8, 158.7. IR (KBr, cm−1): 3381
(br), 2923, 2823, 1591, 1477, 1434, 1367, 1260, 1151, 1000,
860, 766. MS (ESI): 532.8 (M + H+), 554.8 (M + Na+). UV-vis
spectra: 50 μM in CH3CN, Abs = 0.594 (256 nm), 0.161
(302 nm), 0.039 (395 nm, λmax). This compound was used
immediately in the next reaction.
Synthesis of 7. The compound 7 (132 mg, 78%) was syn-
thesized from 6 (190 mg, 0.52 mmol) by the same method
described for the preparation of compound 5. 1H NMR
(400 MHz, CDCl3): δ 3.70 (2H, s, CH2), 3.85 (4H, s, 2CH2),
6.47 (1H, d, J = 2.4 Hz), 6.56 (1H, dd, J = 2.8, 2.8 Hz, ArH),
6.75 (1H, dd, J = 2.0, 2.0 Hz, ArH), 7.15 (2H, t, J = 3.2 Hz,
PyH), 7.35 (2H, m, PyH), 7.59–7.64 (2H, m, PyH), 8.56 (2H, d,
J = 2.8 Hz, PyH). 13C NMR (150 MHz, CDCl3): δ 57.0, 59.1,
116.3, 116.9, 117.5, 122.2, 123.2, 136.7, 138.1, 148.8, 150.2,
158.2. IR (KBr, cm−1): 3209, 2923, 2826, 1591, 1498, 1433,
1372, 1255, 1152, 764. MS (ESI): 321.1 (M + H+), 343.0 (M +
Na+). HRMS (ESI): m/z calcd for C19H21N4O+ [M + H]+
321.1710, found 321.1727.
Synthesis
of
1. 4-Chloro-7-nitro-2,1,3-benzoxadiazole
(NBD-Cl, 50 mg, 0.25 mmol) in 5 mL of methanol was added
dropwise to a solution of compound 5 (133 mg, 0.25 mmol) and
NaHCO3 (21 mg, 0.25 mmol) in 5 mL methanol. After stirring
at room temperature for 4 h, the mixture was poured into 40 mL
water and extracted with ethyl acetate (10 mL × 5). The organic
phase was washed with brine (10 mL), dried over anhydrous
Na2SO4, and then evaporated under reduced pressure. The crude
solid product was recrystallised from dichloromethane/petroleum
Synthesis of 2. By the same procedure described for the syn-
thesis of 1, the product 2 (190 mg, 98% yield) was synthesized
from 7 (132 mg, 0.41 mmol) with NBD-Cl (82 mg, 0.41 mmol).
1H NMR (400 MHz, DMSO-d6): δ 3.74 (2H, s, CH2), 3.84 (4H,
s, 2CH2), 6.53 (1H, d, J = 8.8 Hz), 6.95 (1H, d, J = 8.8 Hz),
7.20 (1H, d, J = 2.4 Hz), 7.23 (1H, d, J = 2.4 Hz), 7.28 (2H, t, J
= 6.0 Hz, PyH), 7.41 (1H, d, J = 2.4 Hz), 7.48 (2H, d, J = 7.2
Hz, PyH), 7.76 (2H, t, J = 8.4 Hz, PyH), 8.38 (1H, t, J = 8.8
Hz), 8.52 (2H, d, J = 5.2 Hz, PyH), 10.87 (1H, br, NH).
1
ether to give the desired product 1 (136 mg, 78%). H NMR
(600 MHz, CDCl3): δ 3.87 (4H, s, 2CH2), 3.92 (8H, s, 4CH2),
6.51 (1H, d, J = 8.4 Hz, CH), 7.17 (4H, t, J = 5.4 Hz, PyH),
7.33 (2H, s, ArH)), 7.46 (4H, d, J = 7.8 Hz, PyH), 7.62 (4H, t,
J = 7.2 Hz, PyH), 8.24 (1H, d, J = 9.0 Hz), 8.43 (1H, br, NH),
8.55 (4H, d, J = 3.6 Hz, PyH). 13C NMR (150 MHz, CDCl3):
δ 54.2, 59.5, 100.3, 122.2, 122.9, 123.8, 124.5, 125.5, 127.2,
5610 | Org. Biomol. Chem., 2012, 10, 5606–5612
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13C NMR (150 MHz, DMSO-d6): δ 53.7, 58.6, 101.2, 116.6,
121.6, 122.3, 122.7, 124.5, 124.8, 125.9, 128.6, 136.8, 137.4,
143.3, 144.2, 144.7, 148.7, 156.0, 158.3. IR (KBr, cm−1): 3442,
1570, 1497, 1430, 1299, 1159, 1070, 861, 547. MS (ESI): 484.2
(M + H+), 506.0 (M + Na+). HRMS (ESI): m/z calcd for
C25H22N7O4+ [M + H]+ 484.1728, found 484.1738.
6 K. A. Lomashvili, W. Khawandi and W. C. O’Weill, J. Am. Soc.
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10 D. H. Vance and A. W. Czarnik, J. Am. Chem. Soc., 1994, 116, 9397.
11 Some recent examples of fluorescent sensors for PPi in water:
(a) S. Mizukami, T. Nagano, Y. Urano, A. Odani and K. Kikuchi, J. Am.
Chem. Soc., 2002, 124, 3920; (b) D. H. Lee, S. Y. Kim and J.-I. Hong,
Angew. Chem., Int. Ed., 2004, 43, 4777; (c) J. H. Lee, A. R. Jeong,
J.-H. Jung, C.-M. Park and J.-I. Hong, J. Org. Chem., 2011, 76, 417;
(d) Y. J. Jang, E. J. Jun, Y. J. Lee, Y. S. Kim, J. S. Kim and J. Yoon,
J. Org. Chem., 2005, 70, 9603; (e) H. N. Lee, K. M. K. Swamy,
S. K. Kim, J.-Y. Kwon, Y. Kim, S.-J. Kim, Y. J. Yoon and J. Yoon, Org.
Lett., 2007, 9, 243; (f) H. K. Cho, D. H. Lee and J.-I. Hong, Chem.
Commun., 2005, 1690; (g) H. N. Lee, Z. Xu, S. K. Kim,
K. M. K. Swamy, Y. Kim, S.-J. Kim and J. Yoon, J. Am. Chem. Soc.,
2007, 129, 3828; (h) I.-S. Shin, S. W. Bae, H. Kim and J.-I. Hong, Anal.
Chem., 2010, 82, 8259; (i) W.-H. Chen, Y. Xing and Y. Pang, Org. Lett.,
2011, 13, 1362; ( j) R. Villamil-Ramos and A. K. Yatsimirsky, Chem.
Commun., 2011, 47, 2694; (k) X. Zhao and K. S. Schanze, Chem.
Commun., 2010, 46, 6075; (l) G. Su, Z. Liu, Z. Xie, F. Qian, W. He and
Z. Guo, Dalton Trans., 2009, 7888; (m) C. Bazzicalupi, A. Bencini,
S. Puccioni, B. Valtancoli, P. Gratteri, A. Garau and V. Lippolis, Chem.
Commun., 2012, 48, 139; (n) W. Zhu, X. Huang, Z. Guo, X. Wu, H. Yu
and H. Tian, Chem. Commun., 2012, 48, 1784; (o) H. J. Kim, J. H. Lee
and J.-I. Hong, Tetrahedron Lett., 2011, 52, 4944; (p) I. Ravikumar and
P. Ghosh, Inorg. Chem., 2011, 50, 4229.
12 Some recent examples of fluorescent recognition of PPi in organic con-
taining solvent: (a) D. Aldakov and P. Anzenbacher Jr., J. Am. Chem.
Soc., 2004, 126, 4752; (b) Z. Zeng, A. Torriero, A. Bond and L. Spiccia,
Chem.–Eur. J., 2010, 16, 9154; (c) M. J. Kim, K. M. K. Swamy,
K. M. Lee, A. R. Jagdale, Y. Kim, S.-J. Kim, K. H. Yoo and J. Yoon,
Chem. Commun., 2009, 7215; (d) J. Gao, T. Riis-Johannessen,
R. Scopelliti, X. Qian and K. Severin, Dalton Trans., 2010, 39, 7114;
(e) N. Shao, H. Wang, X. Gao, R. Yang and W. Chan, Anal. Chem.,
2010, 82, 4628; (f) X. Huang, Z. Guo, W. Zhu, Y. Xie and H. Tian,
Chem. Commun., 2008, 5143; (g) J. F. Zhang, S. Kim, J. H. Han,
S.-J. Lee, T. Pradhan, Q. Y. Cao, S. J. Lee, C. Kang and J. S. Kim, Org.
Lett., 2011, 13, 5294; (h) T. Cheng, T. Wang, W. Zhu, X. Chen, Y. Yang,
Y. Xu and X. Qian, Org. Lett., 2011, 13, 3656; (i) B. Roy, A. S. Rao and
K. H. Ahn, Org. Biomol. Chem., 2011, 9, 7774; ( j) C. Park and
J.-I. Hong, Tetrahedron Lett., 2010, 51, 1960; (k) L. J. Liang, X. J. Zhao
and C. Z. Huang, Analyst, 2012, 137, 953; (l) Z. Guo, W. Zhu and
H. Tian, Macromolecules, 2010, 43, 739.
Synthesis of 2·Zn. 2·Zn was prepared in 71% yield using the
same method described for the preparation of 1·2Zn. Mp:
232–235 °C. H NMR (600 MHz, DMSO-d6): δ 3.68 (2H, s,
1
CH2), 4.08–4.24 (4H, br, 2CH2), 6.47 (1H, d, CH), 6.84 (1H, s,
CH), 7.17 (2H, s, 2CH), 7.55 (2H, s, PyH), 7.64 (2H, s, PyH),
8.07 (2H, s, PyH), 8.59 (1H, s, CH), 8.91 (2H, s, PyH), 10.48
(1H, br, s), 10.93 (1H, s, NH). IR (KBr, cm−1): 3441, 3133,
1608, 1659 (s), 1442, 1304, 1262, 1122 (s), 1071 (s), 995, 957,
865, 617, 539, 518. UV-vis spectra: 50 μM in 50 mM HEPES
buffer solution, pH = 7.4, Abs = 0.147 (366 nm), 0.371
(503 nm, λmax). MS (ESI): 546.6 (M − NO3−)+, 587.6 (M −
NO3− + OH− + Na+)+.
Conclusions
In summary, we have developed a new NBD-phenol based col-
orimetric sensor, which shows a high selective coloration for PPi
in pure aqueous solution over a wide pH range. This sensor
shows high affinity (Ka ≈ 3 × 108 M−1) and selectivity for PPi
over other anions, which enables the detection of PPi in the pres-
ence of a large excess of ATP and inorganic phosphate Pi, and
provides a convenient way of assaying pyrophosphatase in real
time. In addition, it’s worth noting that this is the first use of the
NBD-phenol as the chromophore in a colorimetric sensor. The
combination of its convenient synthetic incorporation and its
absorption at long wavelength (∼500 nm) mean that it should be
applicable to a variety of other colorimetric sensors.
We acknowledge the National Natural Science Foundation of
China (Grants No. 20902033, 21032001 and 21172086) and the
Natural Science Foundation of Hubei Province (No.
2009CBD013) for financial support. This work was also spon-
sored by the Scientific Research Foundation for the Returned
Overseas Chinese Scholars, State Education Ministry, and in part
by the PCSIRT (No. IRT0953).
13 Some recent examples of IDA for the detection of PPi: J. H. Lee,
J. Park, M. S. Lah, J. Chin and J.-I. Hong, Org. Lett., 2007, 9, 3729;
L. Fabbrizzi, N. Marcotte, F. Stomeo and A. Taglietti, Angew. Chem.,
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G. A. Galdamez and R. C. Smith, Eur. J. Org. Chem., 2009, 343;
D. J. Oh, K. M. Kim and K. H. Ahn, Chem.–Asian J., 2011, 6, 2034;
L. Tang, Y. Li, H. Zhang, Z. Guo and J. Qian, Tetrahedron Lett., 2009,
50, 6844.
Notes and references
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This journal is © The Royal Society of Chemistry 2012