Zn(II) based colorimetric sensor for ATP and its use as a viable staining agent in pure aqueous media of pH 7.2

Article abstract of DOI:10.1039/c0cc01996h

Selective colorimetric detection of ATP in physiological conditions by a Zn(II)-based receptor is reported. This reagent was found to be non-toxic to the living cells and could be used for studying the growth of the yeast cells.

Full text of DOI:10.1039/c0cc01996h

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Zn(II) based colorimetric sensor for ATP and its use as a viable staining
agent in pure aqueous media of pH 7.2w
Prasenjit Mahato, Amrita Ghosh, Sanjiv K. Mishra, Anupama Shrivastav, Sandhya Mishra*
and Amitava Das*
Received 21st June 2010, Accepted 6th October 2010
DOI: 10.1039/c0cc01996h
Selective colorimetric detection of ATP in physiological
conditions by a Zn(II)-based receptor is reported. This reagent
was found to be non-toxic to the living cells and could be used for
studying the growth of the yeast cells.
phosphate.z Also, we have discussed the possibility of using
this reagent as a viable staining agent for cell growth studies.
The intermediate ligand (E)-4-((4-(1,4,8,11-tetraazacyclo-
tetradecan-1-ylsulfonyl)phenyl)diazenyl)-N,N-dimethylaniline
(L) was synthesized by reacting 1,4,8,11-tetraazacyclotetra-
decane with (E)-4-((4-(dimethylamino)phenyl)diazenyl)benzene-
1-sulfonyl chloride (Scheme 1).w This was purified prior to
further use for reaction with Zn(NO3)2ꢀ6H2O for synthesis of
the corresponding Zn(II)-complex (L.Zn).w
Phosphates are among the most significant anions in living cells
and play crucial roles in numerous biological processes; like
universal energy storage, ion-channel regulation, intercellular
signalling mediation, protein phosphorylation, enzymatic
reactions, DNA replication, etc.1–4 Among these, adenosine-
50-triphosphate (ATP) is the one which is studied most due to
its role as a molecular unit of currency for intercellular energy
transfer.2 The production of energy is achieved through the
cleavage of one or two phosphate groups from ATP resulting
in adenosine-50-diphosphate (ADP) and adenosine-50-
monophosphate (AMP), respectively. Pyrophosphate (PPi) is
also the product of ATP hydrolysis under cellular conditions.3
More importantly, deficiency in ATP results in ischemia,
Parkinson’s disease and hypoglycaemia.4 Thus, the
monitoring of the ATP concentration level is crucial for the
study of different cellular mechanisms, enzymatic processes
and even cell apoptosis. Though, there are many examples of
artificial receptors that are efficient in recognition of
phosphates, reports on their bioanalytical application in
physiological conditions are rather limited.5–8 Such reports
on specific recognition of ATP and its probable in vivo
application are extremely rare and this led us to develop a
reagent that is non-toxic to the living cells.7,8
Uv-vis spectra recorded for L and L.Zn (Fig. 1) show a
broad absorption band with lmax at 422 and 453 nm,
respectively in CH3OH–CH3CN medium (3 : 7, v/v). A band
maximum at 422 nm for
L was attributed to the
intercomponent (p–p*) charge transfer (CT) band. This CT
transition was further red shifted on coordination to the cationic
Zn(II)-centre in L.Zn.w This CT band for L.Zn in 10 mM
HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)
buffer medium appeared at 463 nm.
In order to examine the binding behaviour of the reagent,
L.Zn towards various anionic analytes, like ATP, CTP
2ꢁ
(cytidine triphosphate), ADP, AMP, PPi, H2PO4ꢁ, SO4
,
,
CH3CO2ꢁ, Iꢁ, Brꢁ, Clꢁ, Fꢁ, CNꢁ, SCNꢁ, NO3 and NO2
ꢁ
ꢁ
Uv-vis spectra for L.Zn were recorded in absence and presence
of large excess (2000 mole equivalent) of respective anions in
10 mM HEPES buffer medium (pH 7.2). As shown in Fig. 1, a
red shifted CT absorption band for L.Zn with a maximum at
503 nm was observed on addition of excess sodium salt of
ATP. An insignificant shift of 9 nm was observed when CTP of
comparable concentration was added (Fig. 1A). In contrast,
the change for ADP was even less significant and other anionic
analytes failed to induce any detectable change in electronic
spectra (Fig. 1A). A visually detectable change in solution
colour was observed on addition of ATP to L.Zn solution
(10 mM HEPES buffer medium), whereas this change was
barely detectable for CTP and ADP. For all other anionic
analytes used in this study, solution colour remained
unchanged. No change in spectra on addition of these anions
suggests either no or a very weak binding of these anions to the
Zn(II)-centre of L.Zn. Systematic spectrophotometric
The most widely studied receptor fragment that has been
explored to date for recognition of ATP and other biological
phosphates is derived from Zn(II)-complex of 2,20-dipicolyl
amine (dpa).7,8 Among various Zn(II)-dpa-based receptors,6,7
including ours,8 use of a certain amount of non-aqueous
solvent is essential in most cases due to the limited solubility
of these receptors in water. As a part of our ongoing effort to
develop a sensor molecule that is soluble in pure water and can
be used for in vivo detection of ATP in living cells, we have
synthesized
a
new Zn(II)-complex using
a
1,4,8,11-
tetraazacyclotetradecane derivative. We report herein its
synthesis, characterization, specificity and relative binding
affinity towards ATP as compared to other biologically
important phosphates like CTP, ADP, AMP, PPi and
Central Salt & Marine Chemicals Research Institute (CSIR),
Bhavnagar, 364002, Gujarat, India. E-mail: amitava@csmcri.org,
smishra@csmcri.org
w Electronic supplementary information (ESI) available: Synthesis,
characterization and biological studies. See DOI: 10.1039/c0cc01996h
Scheme 1 Methodology adopted for synthesis of L and L.Zn.
c
9134 Chem. Commun., 2010, 46, 9134–9136
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M) to L.Zn-ATP. The original spectrum of L.Zn (with lmax
=
463 nm) was restored when excess aqueous solution of sodium
citrate was added to the solution of L.Zn-ATP.w
More interestingly, the solubility of the L.Zn (0.045 g Lꢁ1) in
water was found to be substantially enhanced (0.34 g Lꢁ1) in
presence of excess a-CD (4.5 g Lꢁ1). Favoured non-bonded
interactions between the azo functionality of L.Zn and the
hydrophobic interior of the a-CD on inclusion into the a-CD
cavity could account for the observed enhanced solubility of
L.Zn in water (Fig. 3);w such inclusion of analogous (E)-1,2-
di(pyridin-4-yl)ethene and (E)-1,2-di(pyridin-4-yl)diazene in
a-CD was reported earlier.11
Spectral studies reveal that the absorption maximum for
L.Zn (463 nm) shifts to shorter wavelength (458 nm) on
addition of aqueous solution of a-CD.w Such inclusion in the
a-CD cavity is expected to influence the HOMO–LUMO
energy gap and thereby the absorption spectral
bands—though the perturbation of the energy levels is not
expected to be an appreciable one.11,12 Thus, in this present
study, the observed shift of 5 nm could be attributed to
Fig. 1 (A) Absorption spectra of L.Zn (5.32 mM) in presence of
various anions (B10.6 mM); 1: ATP; 2: CTP; 3: ADP; 4: blank, Fꢁ,
Clꢁ, Brꢁ, Iꢁ, CH3COOꢁ, H2PO4ꢁ, PPi, SO42ꢁ, NO3ꢁ, NO2ꢁ, CNꢁ,
AMP, SCNꢁ. (B) Absorption spectra of L.Zn (9.46 mM) in presence of
varying [ATP] (0–17.28 mM). Inset: represents change in colour of
L.Zn (8.87 mM) in absence and presence of various phosphate ions.
From left to right: Blank, with added H2PO4ꢁ, PPi, AMP, ADP, ATP,
CTP (anion concentration B500 mM). For all experiments pH of 7.2
was maintained (10 mM HEPES buffer solution) at 25 1C.
titrations were carried out in 10 mM HEPES buffer medium in
order to evaluate the relative binding affinities of ATP
(Fig. 1B), CTP and ADP towards L.Zn and respective
binding constants were found to be 978 ꢂ 4 Mꢁ1, 220 ꢂ
15 Mꢁ1 and 142 ꢂ 15 Mꢁ1 at 25 1C.w The binding constant
was evaluated based on the absorbance changes at 503 nm.
Binding of ATP with L.Zn was also confirmed by 31P NMR
spectral studies. Upfield shifts for the 31P signals for the
a- (0.07 ppm), b- (0.09 ppm) and g- (0.05 ppm) phosphorus
atoms of the ATP bound to L.Zn were observed (Fig. 2). The
shifts in 31P NMR signals for a-, b- and g-P atoms signify the
binding to Zn-atom of L.Zn through an oxygen atom bearing
the negative charge of the respective phosphate unit. Relatively
weaker interaction of the O[g-PO ]ꢁ and O[a-PO ]ꢁ relative to the
the inclusion phenomena and the formation of
a
[2]pseudorotaxane complex, a-CD.L.Zn. Presumably,
formation of the more polar excited state for a-CD.L.Zn is
less favored in the apolar interior of a-CD than in the
surrounding solvent for a non-included reagent L.Zn.12
Although small, this may have some contribution in
increasing the energy of activation for optical electron
transfer and thus the observed blue shift. Spectrophotometric
titration with varying [a-CD] revealed a systematic shift in the
spectral pattern with the appearance of two simultaneous
isosbestic points at 420 and 446 nm.w The association
constant (255 ꢂ 15 Mꢁ1, 25 1C) for this inclusion process
was evaluated from the spectrophotometric titration data.w
This value is close to those reported earlier for related
systems.11,12 Thus, in presence of excess of a-CD (10 fold),
the [2]pseudorotaxane form, i.e., a-CD.L.Zn (Fig. 3) is
expected to be the major component ( Z 96%) in the
aqueous solution. Higher solubility of L.Zn in water in
presence of a-CD helped to achieve higher concentration of
L.Zn (0.5 mM) in solution and thus a more intense colour
change on binding to ATP (5 mM) (Fig. 3). Formation of the
[2]pseudorotaxane was also confirmed by ESI-MS studies; a
molecular ion peak for a-CD.L.Zn was observed at an m/z
value of 1648.6.w
4
4
ꢁ
O[b-PO ] unit accounts for the smaller Dd shift in 31P NMR
4
spectra. This observation also confirms the formation of a
heptacoordinated Zn(II)-centre in L.Zn in presence of ATP.
Examples of heptacoordinated Zn(II)-centres are available in
the literature.9 A very insignificant shift in 31P signals was
observed when similar experiments were repeated for CTP.w
The 31P NMR spectral data also confirm the observed trend in
binding affinity (ATP c CTP > ADP c AMP).w Presumably,
the formation of a higher number of chelate rings between ATP
or CTP and L.Zn is crucial for efficient L.Zn–O[phosphate]
binding, as compared to those for ADP and AMP. Lack of any
binding of PPi to L.Zn perhaps could be better explained based
on the much higher solvation energy for PPi than that for
ATP.10 The 1 : 1 complex formation, i.e., L.Zn-ATP was
further confirmed by ESI-MS spectral studies.w Signals at
1103.9 and 1082 correspond to m/z for [[L.Zn-ATP] ꢁ
2(NO3ꢁ)] (A) and [(A–Na+) + H+],w respectively with
anticipated isotope distribution.
Preferential binding to ATP and the visual change in colour
for L.Zn or a-CD.L.Zn on binding to ATP led us to consider
the possible use of these reagents for staining of the live yeast
cells, as maximum concentration of negatively charged ATP is
released through mitochondria in the periplasmic space and
Reversible binding of L.Zn to ATP could be demonstrated
by adding an aqueous solution of sodium citrate (5.0 ꢃ 10ꢁ4
Fig. 3 Schematic representation of the inclusion process of L.Zn with
a-CD and changes in solution colour for L.Zn and a-CD.L.Zn in
presence of ATP.
Fig. 2 31P NMR spectra recorded in D2O; only (A) ATP and (B) ATP
with Zn(II)-complex L.Zn.ATP, at 25 1C.
c
This journal is The Royal Society of Chemistry 2010
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staining agent for living cells in physiological conditions.
Further, the use of biologically benign a-CD enhances the
solubility in water and HEPES buffer (pH = 7.2) and thereby
the staining efficiency. The stained microbial cell could be seen
distinctly through a light microscope. Examples of such a non-
toxic colorimetric staining agent that works completely in
physiological conditions are extremely rare in the literature.
DST and CSIR have supported this work. PM, AG, SKM
and AS acknowledge CSIR for fellowships.
Fig. 4 Images of S. cerevisiae cells: Light microscopic images of (A)
unstained cells, (B) cells stained with L.Zn (0.66 ꢃ 10ꢁ4 M) and (C)
a-CD.L.Zn [L.Zn (5.0 ꢃ 10ꢁ4 M) and a-CD (5.0 ꢃ 10ꢁ3 M)] at 25 1C in
10 mM HEPES buffer solution (pH = 7.2); SEM images of (D) yeast
cells, (E) yeast cells treated with L.Zn (0.66 ꢃ 10ꢁ4 M).
Notes and references
then gets excreted to the cell surface.13 We have used the yeast
cells, Saccharomyces cerevisiae (S. cerevisiae) in 10 mM
HEPES buffer solution (pH = 7.2) for our studies. The
images of the colorless yeast cells, viewed under a normal
light microscope (AXIO IMAGER-Carl Zeiss), are shown in
Fig. 4A; while images of the yeast cells exposed to the reagents
L.Zn (for B5 min) and a-CD.L.Zn (for B2 min) are shown in
Fig. 4B and C. Control experiments with yeast cells exposed
separately to Zn(NO3)2 and L did not produce any change in
colour. These observations together revealed that S. cerevisiae
cells could be stained with L.Zn or a-CD.L.Zn, and the colour
of the cells changed to pink-red. Further, for a-CD.L.Zn, the
higher solubility allows a higher local concentration of the
L.Zn and thus a more effective binding to the ATP, produced
in situ in the cell surface and thereby better staining of the
yeast cells.
z Patent application details: 0093NF2009 [1242DEL2010], 24.05.2010.
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Finally, the cell growth, cell division, as well as the viability
of the yeast cells in presence of these two staining agents were
observed when the stained cell suspension was viewed on a
concavity slide by light microscope (Fig. 5). Cell division and
growth in presence of L.Zn and a-CD.L.Zn confirms the
possibility of using these reagents as viable staining agents
for studies on cell growth dynamics, which has significance in
terms of the application potential in environmental studies and
food industries.
In conclusion, we have successfully demonstrated that an
easily synthesizable Zn(II)-reagent could be used as a viable
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Fig. 5 Light microscopic image of S. cerevisiae in presence of L.Zn
and a-CD.L.Zn in 10 mM HEPES buffer at different time intervals.
c
9136 Chem. Commun., 2010, 46, 9134–9136
This journal is The Royal Society of Chemistry 2010