Cell permeable fluorescent receptor for detection of H2PO 4- in aqueous solvent

Article abstract of DOI:10.1039/c2ob27201f

A new colorimetric fluorescent receptor for H₂PO ₄^- is reported in this communication. The receptor can detect dihydrogen phosphates optically by developing a color change from yellow to green. Acute spectral responses to

Full text of DOI:10.1039/c2ob27201f

Organic &
Biomolecular Chemistry
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Cell permeable fluorescent receptor for detection of
H2PO4− in aqueous solvent†
Cite this: Org. Biomol. Chem., 2013, 11,
1537
Supriti Sen,a Manjira Mukherjee,a Kuheli Chakrabarty,b Ipsit Hauli,c
Subhra Kanti Mukhopadhyayc and Pabitra Chattopadhyay*a
−
A new colorimetric fluorescent receptor for H2PO4 is reported in this communication. The receptor can
detect dihydrogen phosphates optically by developing a color change from yellow to green. Acute spec-
tral responses to H2PO4− in HEPES buffer (DMSO–HEPES 1 : 9) have been observed. The selectivity zone in
Received 19th October 2012,
Accepted 19th December 2012
−
terms of pH of the receptor for H2PO4 is attributed to the fitness in the acidity (pKa) of receptor with
H2PO4−. Hydrogen bonding plays the key role here which is confirmed by 1H NMR titration. The receptor
also has good potential for bio-imaging. The mode of interaction has also been established by ab initio
calculation.
DOI: 10.1039/c2ob27201f
www.rsc.org/obc
components. Furthermore, phosphates are industrially impor-
tant components of both medicinal drugs and fertilizers. Pol-
Introduction
The development of simple receptors for selective sensing of lution from phosphate and phosphorylated compounds is in
anionic substrates is an active area of research today.1 Many part responsible for the eutrophication of natural water
chemosensors and receptors for metal ions and pH sensors sources, leading to a dangerous increase in toxic algal
have been developed.2 But now there is an increasing interest blooms.8 The adverse effect of excess phosphate in blood
in anion recognisation3a–c and anion sensing3d,e due to their causes hyperphosphatemia, hyperparathyroidism, soft tissue
intensive role in biology, pharmacy and environmental calcification, kidney failure, cardiovascular complications
science.4 Among the reported methods,5 fluorescent receptors resulting from the development of metastatic calcifications at
possess innate advantages over receptors of other types the cardiac level, and increased morbidity and mortality.9
because of their high sensitivity, specificity, simplicity of
For the last few decades different types of fluorescent
implementation and fast response times, offering application sensor such as neutral amide type sensor derived from urea,
methods not only for in vitro assays but also for in vivo thiourea, imidazole, indole, amine, pyrrole and sulphonamide
imaging studies.6
moieties with hydrogen bond donor functionalities10 and cat-
Among the interest in biologically functional anions, phos- ionic receptors derived from ammonium, guanidium, quinoli-
phate and its derivatives are of the particular importance nium, phosphonium protonated quinoxaline salts, with
owing to their established role in transduction, energy storage, electrostatic interaction11 between the anionic guest and cat-
and gene construction in biological systems. Moreover phos- ionic receptors have been used for anion sensing studies.
phate is the key intermediate for many biochemical reactions More recently metal complexes have also been used for detec-
and is the main component of biomolecules. It is a component tion of anionic analyte.12,2b
of adenosine triphosphate (ATP), a fundamental energy source
Several synthetic receptors including metal complexes have
in living things and is deeply involved in DNA duplication and been reported for sensing phosphate,3c,13a–d and pyrophospha-
transcription.7 Phosphate is also an essential ingredient of te13e,f but only a few for dihydrogen phosphate have been
bone matrix and teeth. In addition, chemotherapeutic and reported.3a,13g However among them some receptors involve a flu-
antiviral drugs have phosphates as integral structural orescence “turn-off” response as the readout mechanism.13h
Limitations of the currently available “turn-on” phosphate sensor
include lack of selectivity,13i low sensitivity and/or excitation pro-
files in the ultraviolet region.13j Recently Zn-complexes13a have
been shown to have a tendency to sense dihydrogen phosphate
but to prepare the probe in this way is time consuming and
tedious and they are not practicable for intracellular imaging.
To overcome this problem we have designed and syn-
aDepartment of Chemistry, The University of Burdwan, Golapbag, Burdwan-713104,
India. E-mail: pabitracc@yahoo.com; Tel: +91-9434473741
bDepartment of Chemistry, Visva-Bharati, Santiniketan, WB, India
cDepartment of Microbiology, The University of Burdwan, Golapbag, Burdwan-
713104, India
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
c2ob27201f
thesized
a
new small Schiff-base compound that
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fluorometrically recognizes one of the most important anions cooled, the reaction mixture was filtered and a red colored
dihydrogen phosphate by hydrogen bonding with bio-friendly crystalline solid (1) was obtained after evaporation with 90%
visible region excitation and intracellular imaging.
yield. The purity was checked by 1H NMR, 13C NMR, ESI-MS
and FTIR study (see ESI†).
C23H18N2O7: Anal. Found: C, 63.59; H, 4.18; N, 6.45; Calc.:
C, 63.67; H, 4.25; N, 6.52%. EI-MS: [M + H]+, m/z, 435.04 [M +
H]+; IR (KBr, cm−1): νCvN, 1618, νC–H, 2858; νO–H, (phenolic
Experimental
Instruments and reagents
1
–OH), 3366; νO–H, (carboxylic –OH) 2913. H NMR (δ, ppm in
The elemental analyses (C, H and N) were performed on a
Perkin Elmer 2400 CHN elemental analyzer. Electronic absorp-
tion spectra were recorded on a JASCO UV-Vis spectropho-
tometer model V-570. IR spectra were recorded on a JASCO
FTIR spectrophotometer (model: FTIR-H20) using KBr disks.
1H NMR spectra were recorded on a Bruker 400 MHz spec-
trometer using TMS as an internal standard in DMSO-d6. 13C
NMR spectra were recorded on a Bruker 400 MHz spectrometer
using TMS as an internal standard in DMSO-d6. Electron spray
ionization (ESI) mass spectra were recorded on a Qtof Micro
YA263 mass spectrometer. The measurement of pH was done
with the help of a digital pH meter, Equip-Tronics (ER-614A)
model. The fluorescence spectra of the titration of phosphate
with the receptor were recorded using the fluorometer Hitachi-
4500.
All reactions were carried out under nitrogen atmosphere
while workup procedures were done in air. All the solvents uti-
lized in this work were obtained from Merck with analytical
reagent grade and have been used without further purification.
Milli-Q 18 Ω water was used throughout all the experiments. 4-
Amino-3-hydroxybenzoic acid and all reagents were purchased
from Sigma-Aldrich and Merck, India respectively. 4-Methyl-
2,6-diformylphenol was prepared according to literature proce-
dures.2b The selectivity study of the receptor towards different
anions was carried out using tetrabutylammonium salts of flu-
oride, chloride, bromide, iodide, acetate, dihydrogen phos-
phate and sodium salt of monohydrogen phosphate, sulphate,
azide, thiocyanate, nitrate.
DMSO-d6, 400 MHz): 10.434 (s, 2H); 9.913 (br, 3H); 9.058 (s,
2H); 7.757–7.459 (m, 8H); 2.321 (s, 3H). 13C NMR (400 MHz, δ,
ppm in DMSO-d6): 189.2, 168.0, 163.9, 162.5, 151.2, 150.9,
134.6, 132.6, 124.1, 123.0, 115.3, 112.9, 30.2. Yield: 90%.
Synthesis of compound 2 (1-dihydrogen phosphate
compound)
The preparation of the solid complex of dihydrogen phosphate
was carried out as follows (viz. Scheme S1†). 2 mL DMSO solu-
tion of Bu4N[H2PO4] (74.68 mg, 0.22 mmol) was added slowly
to a stirred 8 mL DMSO solution of 1 (100 mg; 0.22 mmol) and
stirred for 10.0 h. The orange–red colored reaction mixture was
filtered and left to allow slow evaporation. After a few days, the
red colored dihydrogen phosphate complex was obtained by
filtration and dried in vacuo for performing the
characterization.
C23H20N2PO11: Anal. Found: C, 52.38; H, 3.06; N, 5.31;
Calc.: C, 52.43; H, 3.11; N, 5.37%. EI-MS: [M + H]+, m/z, 435.04
[M + H]+; IR (KBr, cm−1): νCvN, 1626, νC–H, 2862; νO–H, 3369
(phenolic –OH), νO–H, 2913 (carboxylic –OH), νO–H, 3052
(chelate compound) (see ESI†).
General procedure for fluorescence and absorption study
The fluorescence properties of the sensor were investigated in
HEPES buffer (100 mM) (DMSO–HEPES 1 : 9) co-solvent at
25 °C temperature. The pH of the solvent mixture was adjusted
to ∼7.2. We use such a polar co-solvent mixture to diminish
the scope of intramolecular H-bonding of 1. The selectivity
study of 1 towards different anions was carried out with the
previously mentioned anions in HEPES buffer (DMSO–HEPES
1 : 9) solution. During the selectivity study the anion concen-
tration is 10 times more than that of receptor. The pH study
was done in 100 mM HEPES buffer solution by adjusting pH
with HCl or NaOH. An in vivo study was also completed at bio-
logical pH ∼ 7.2. The absorption property of the receptor was
studied in HEPES buffer (DMSO–HEPES 1 : 9) solution. The
fluorescence quantum yields (Φ) were estimated by integrating
the area under the fluorescence curves with the equation:
Synthesis of 4-methyl-2,6-bis((3-hydroxy-4-carboxyphenyl)-
imino)phenol (1)
To synthesize (1) firstly 4-methyl-2,6-diformylphenol was syn-
thesized starting from p-cresol by following a published proce-
dure.2b Then 3-hydroxy-4-aminobenzoic acid (0.306 g; 2 mmol
in 10 mL methanol) was added to the methanolic solution of
4-methyl-2,6-diformylphenol (0.164 g; 1 mmol in 25 mL) as
shown in Scheme 1. Then the reaction mixture was refluxed at
80 °C for 12.0 h in nitrogen atmosphere. After the solution was
ODstandard ꢀ Asample
Φsample
¼
ꢀ Φstandard
ODsample ꢀ Astandard
where A is the area under the fluorescence spectral curve and
OD is the optical density of the compound at the excitation
wavelength. The standard used for the measurement of fluor-
escence quantum yield was anthracene (Φ = 0.29 in ethanol).
The binding constant values were determined from the emis-
sion intensity data following the modified Benesi–Hildebrand
Scheme 1 Synthetic scheme for 1.
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equation:14,15
(LEICA DM 1000 LED) using UV filter. Cells without ligand
were used as control.
1=ΔF ¼ 1=ΔFmax þ ð1=K½CꢁÞð1=ΔFmaxÞ; ΔF ¼ Fx ꢂ F0; ΔFmax
¼ F1 ꢂ F0
Imaging system
where F0, Fx, and F∞ are the emission intensities of the
organic moiety considered in the absence of anion, at an inter- An inverted fluorescence microscope (Leica DM 1000 LED) was
mediate anion concentration, and at a concentration of com- used for cellular imaging. The microscope was equipped with
plete interaction, respectively, and where K is the binding a digital compact camera (Leica DFC 420C), an image pro-
constant and [C] is the anion concentration.
cessor (Leica Application Suite v3.3.0) and a mercury 50 watt
lamp.
Geometry optimization
Trial geometries of the host and host–guest complex were con-
structed using Molden software.16 Geometry optimization of
the trial structures were performed by the ab initio method
using the 6-31G* basis set (HF/6-31G*). The GAMESS17
quantum mechanical package was used for geometry optimiz-
ation. Analysis of the vibrational modes of the optimized geo-
metries revealed that there was no negative frequency, which
confirmed the optimized geometries as minimum energy
structures.
Results and discussion
Design and synthesis
The design of synthetic receptors in anion sensing is the most
vital work. One of the important interactions between sensor
and anion is related to hydrogen bonding. Keeping this in
mind the receptor (1) was synthesized. The receptor 4-methyl-
2,6-bis((3-hydroxy-4-carboxyphenyl)imino) phenol was syn-
thesized by a facile one step condensation of 3-hydroxy-4-
aminobenzoic acid and 4-methyl-2,6-diformylphenol. It was
Preparation of cells
characterized by analytical data: H NMR, 13C NMR, ESI-mass
1
Grains of Tecoma stans are collected by gently rupturing pollen
sacs of fresh flower buds on a sterile Petri plate and suspend-
ing them in HEPES buffer (0.1 M, pH ∼ 7.2). Debris of pollen
sacs was removed by filtering the suspension through a thin
layer of nonabsorbent cotton and then pollen was precipitated
by centrifugation at 5000 rpm for 5 min. The supernatant was
discarded and the pollen pellet was re-suspended in the same
buffer containing the receptor (50 μM) and 0.01% Triton X100
as permeability enhancing agent. After 30 minutes of incu-
bation the pollen grains were washed twice in HEPES buffer
(0.1 M, pH ∼ 7.2) by centrifugation as mentioned above.
Exponentially growing cells of Candida albicans (IMTECH
No. 3018) in a yeast extract glucose broth medium (pH 6.0,
incubation temperature, and 37 °C) were precipitated by cen-
trifugation at 5000 rpm for 10 minutes and then washed twice
by suspending them in HEPES buffer (0.1 M, pH ∼ 7.2) fol-
lowed by centrifugation at 5000 rpm for 10 minutes to remove
trace of the growth medium used. Then cells were treated with
the (5 μM) solution for 30 minutes. After incubation, the cells
were again washed with the same HEPES buffer and 1 treated
cells were mounted on a grease free glass slide.
spectra and IR study (ESI, Fig. S1–S5†). The spectroscopic ESI-
mass spectra (Fig. S4†), IR study (Fig. S5†) and also the 1H
NMR titration data also support the formation of the 1–H2PO4
(2) assembly in solid state.
Absorption study
To get an excellent anion sensing the photophysical property
of the sensor was investigated in protic solvent (DMSO : buffer
9 : 1). The receptor was yellow color (λmax = 440 nm) in the
above medium. Addition of dihydrogen phosphate to the
yellow colored receptor solution causes instantaneous develop-
ment of green color (λmax = 525 nm) (Fig. 1). The peak at
440 nm in the absorption spectra gradually decreases with
−
addition of H2PO4 salt and a new peak generates with an
85 nm red shift at 525 nm (Fig. 2) through an isobestic point
at 464 nm. This suggested a hydrogen bond mediated strong
interaction of H2PO4− with the receptor (Scheme S1†). This red
The cells of bacterium used in the present experiment were
Bacillus sp. which has long been used as an excellent bio-pesti-
cide for controlling looper caterpillars at tea plantations. Bac-
terial cells from an exponentially growing overnight culture in
nutrient broth medium were centrifuged at 6000 rpm for
10 minutes, the supernatant was discarded and bacterial
pellets were washed using HEPES buffer (0.1 M, pH ∼ 7.2) by
centrifugation. Washed cells were suspended in the same
buffer containing the 1 (5 μM) for 30 minutes and then centri-
fuged at 6000 rpm for 10 minutes and the supernatant was
discarded.
Fig. 1 Photographic images of the receptor in the presence of different anions
(A) visual color change and (B) fluorescence color change. The concentration of
Then all cells prepared above were photographed under
high power magnification (100×) of a florescence microscope anions is 100 times greater than the receptor.
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Fig. 2 Absorption spectra of 6.25 μM of 1 in the presence of 0, 2.50, 5.0, 7.5,
−
8.75, 10.0, 12.5, 20.0, 25.0, 50.0, 75.0, 125.0, 200.0 μM of H2PO4 in HEPES
buffer (DMSO–HEPES 1 : 9, v/v) at 25 °C (inset is the image of UV-Vis titration).
Fig. 3 Excitation and emission spectra of 12.5 μM of
(DMSO–HEPES 1 : 9) at 25 °C.
1 in HEPES buffer
shift induces a colour change from yellow to green after
addition of dihydrogen phosphate which can be recognized by
the naked eye. This receptor is capable of optically sensing
changes in H2PO4− concentration at a level of 10−4 mol dm−3
.
Emission study
The signaling mechanism operating here is through intramole-
cular H-bonding (IHB) of three hydrogen atoms with three
oxygen atoms (–OH⋯O) which is also a negative factor which
weakens the fluorescence intensity of the sensor. But in pres-
ence of the competitive anions the scope of the IHB decreases
and intermolecular H-bonding between anion and sensor dra-
matically enhances the emission intensity of the receptor
Scheme S1.†
On excitation at 480 nm the receptor gives emission at
550 nm in the fluorescence spectrum in DMSO : HEPES buffer
9 : 1 (Fig. 3). The change of fluorescence intensity of 1 in the
presence 10 equiv. amounts of different anions was studied
and the result has been summarized in Fig. 4. Here only
Fig. 4 Emission enhancement spectra of 1 (10 μM) in the presence of different
anions (100 μM) in HEPES buffer (DMSO–HEPES 1 : 9) at 25 °C, λex = 480 nm.
−
H2PO4 gives a high signal but other anions HPO42−, F−, Cl−,
Br−, I−, N3−, HSO4−, NO3−, AcO− remain entirely silent (taking
Bu4N+ salts for halogens and Na+ salts for the others). This
result demonstrated that receptor 1 exhibited high selectivity
−
towards H2PO4 in such a polar solvent mixture. This selectiv-
ity observed is due to the charge density of monobasic anions.
−
From Fig. 5 it was observed that detection of H2PO4 was not
affected by the other mentioned anions. In the presence of
various anions the fluorescence intensity enhancement of 1 is
−
almost the same as that shown by H2PO4 alone. The addition
−
of H2PO4 offers a dramatic change in emission intensity of 1
with a more than 30 fold increase in quantum yield from 1 (Φ1
= 4.51 × 10−3 and Φcomplex = 1.4 × 10−1) and also in emission
spectra. The emission intensity increases with gradual
−
addition of the H2PO4 and is recorded in Fig. 6. The pKa
value of dihydrogen phosphate is ∼7.2 which means that pro-
tonation of the guest occurs above 7.2. The plot of emission
−
intensity vs. H2PO4 concentration (Fig. 7) indicates that the
fluorescence intensity increases with increasing analyte con-
Fig. 5 Interference plot of different anions (100 μM) in the presence of 1
centration. There is a good linearity of fluorescence intensity (10 μM) and [H2PO4−] = 100 μM in HEPES buffer (DMSO–HEPES 1 : 9) at 25 °C.
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Fig. 9 Jobs plot showing maxima at 1 : 1 (1 + H2PO4− = 100 μM).
Fig. 6 Emission spectra of 6.25 μM of 1 in the presence of 0, 0.63, 1.25, 1.88,
2.50, 3.75, 5.0, 6.25, 7.5, 8.75, 10.0, 12.5, 20.0, 25.0, 50.0, 125.0 μM of H2PO4
−
in HEPES buffer (DMSO–HEPES 1 : 9, v/v) at 25 °C.
−
Fig. 10 Double reciprocal plot for 1–H2PO4 assembly (ka = 0.2 × 104 M−1) in
−
the lower concentration region of H2PO4 in HEPES buffer (DMSO–HEPES 1 : 9,
v/v) at 25 °C.
Fig. 7 Fluorescence intensity as a function of [H2PO4−] concentration. Inset:
linearity up to 4.5 × 10−5 M.
H2PO4− assembly was established as 1 : 1 from Job’s plot analy-
sis (Fig. 9). To observe the binding interaction the binding
constant value has been determined from the modified
Benesi–Hildebrand equation and the Ka value is 0.2 × 104 M−1
−
for H2PO4 binding (Fig. 10). The effects of pH on the fluor-
escence property of 1 also investigated. The receptor exhibits
high fluorescence intensity in the basic region since the phe-
nolic –OH groups of 1 remain as O− in the basic region with
no possibility of IHB. Therefore, 1 senses the anions with high
intensity in the basic region (Fig. 11). It is noteworthy to
mention that the fluorescence emission of 1 is also high in the
basic region in the presence of H2PO4−. The detection limit
was determined from the fluorescence titration data18 (Fig. 12)
and we could detect H2PO4 in the limits of 3.5 × 10−6
which is significantly low.
M
−
Fig. 8 Fluorescence response to pH of 1 in 100 mM HEPES buffer at 25 °C, pKa
In order to know more about the pattern of interaction
between receptor 1 and tetrabutylammonium phosphate, 1H
NMR titration was performed in DMSO-d6 solvent. As shown
= 6.85237 0.05366.
of 1 as a function of [H2PO4−] concentration in the low region in Fig. 13 the broad phenolic protons (H2) peak (equivalent to
up to 4.5 × 10−5 M. The pKa value of our receptor is 6.85 3H) vanish and become a sharp peak (equivalent to 1H) after
(Fig. 8). This value suggests strong H-bonding of the host– addition of H2PO4−, indicating the hydrogen bonding inter-
guest near neutral pH. The stoichiometry of the receptor– actions between 1 and H2PO4−. This signal of one H2 proton
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Fig. 11 Emission response of probe 10 μM of 1 in the absence and presence of
−
H2PO4 (1 equivalent) at different pH in HEPES buffer (DMSO–HEPES buffer:
1 : 9).
Fig. 14 Optimised geometry of (a) 1 and (b) 1–phosphate assembly by ab
initio method.
Fig. 12 Detection limit of H2PO4− in HEPES buffer (DMSO–HEPES 1 : 9, v/v).
Fig. 15 Fluorescence image of (a) pollen grains of Tecoma stanswi without any
receptor addition. (b) Pollen grains were incubated with 5 μM 1 for 30 minutes.
(c) Candida albicans cells were incubated with 5 μM 1 for 30 minutes. (d) Bacillus
sp. cells strained with 5 μM receptor after 30 min incubation. All the samples
were excited at ∼480 nm with emission ca. ∼530 nm by using [40×] objective.
performed. The energy of HOMO and LUMO of receptor and
receptor–H2PO4− complex are −8.09 eV, 1.21 eV, −5.53 eV, 3.74
eV. The difference in energies are 9.30 eV and 9.27 eV for 1
and 2. This shows that these two compounds are equally
stabilized and it demonstrates the facile conversion of 1 to
−
H2PO4 complexation (Fig. 14). This is why the red shift was
−
observed in the UV-Vis spectra due to the addition of H2PO4
.
Due to the significant interaction of the receptors with the
dihydrogen phosphates in both organic and aqueous organic
systems, we planned to investigate their interactions with
living cells. A cell consists of a higher order complex entity of
dihydrogen phosphate ions. Consequently the receptor will be
Fig. 13 Partial 1H NMR titration (DMSO-d6, 400 MHz) (a) of 1; (conc. = 1.16 ×
10−3 M) (b) 1 + 1.0 equivalent of tetrabutylammonium dihydrogen phosphate.
remains intact in Fig. 13b because it is involved in intramole- a good staining agent to visualize cells by interaction with in
cular H-bonding.
vivo dihydrogen phosphates. For this study three types of cells
To clarify the configurations and H-bonding features of the viz. pollen grains of Tecoma stans, Bacillus sp., Candida albicans
guest and guest–host complex ab initio calculations were were taken. Apart from the pollen the other two cells were not
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visualised (black) through the fluorescence microscope. Only
pollen has a slight blue fluorescence at the edge of the cell
under the fluorescence microscope. But after incubation with
5 μM 1 for only 30 minutes all the cells sparkled with green
fluorescence (Fig. 15). The distribution of the probe within the
cells was observed by fluorescence microscopy following exci-
tation at ∼480 nm. These results indicate that 1 is an efficient
staining agent and we can monitor the intracellular dihydro-
gen phosphate.
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Conclusion
In conclusion, a colorimetric and fluorescent dual receptor for
−
H2PO4 was synthesized by a facile one-step process. The for-
mulation and detailed structural characterisations have been
established using physico-chemical and spectroscopic tools.
1
The mode of interaction was verified by H NMR titration and
theoretical calculation. This water soluble compound is a
−
potent probe for H2PO4 in biological applications as both
wavelengths (λex = 480 nm and λem = 530 nm) are not in the UV
region but in the bio-friendly visible region. The fluorescence
intensities are also almost unaffected by the biologically rele-
vant anions and the tolerable limit of different interfering
anions is significantly high (up to 10 equivalent). There is a
good linearity between fluorescence intensity of
1 and
[H2PO4−] at low region and detection limit is 3.5 × 10−6 M. The
study of the distribution of the probe in the living cells showed
that the receptor (1) is significantly efficient to detect dihydro-
gen phosphate in vitro in aqueous medium at biological pH by
developing a good image and has potential for bio-medical
applications.
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Acknowledgements
Financial assistance from DST, New Delhi (vide project no.
SR/S1/IC-37/2008) is gratefully acknowledged.
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