Pyridinum-based flexible tripodal cleft:A case of fluorescence sensing of ATP and dihydrogenphosphate under different conditions and cell imaging

Article abstract of DOI:10.1039/c5ra04023j

Pyridinium-based chemosensor 1 built on tris(aminomethyl)amine (tren) has been designed, synthesized and established as a chemosensor for ATP over ADP, AMP and a series of other anions in aqueous CH₃CN at pH 6.5. Compound 1 exhibits a significant change in emission upon complexation of ATP. In CH₃CN, the sensor selectively binds H₂PO₄^- and forms an excimer with significant intensity. Furthermore, the intracellular ATP detection using 1 was possible through fluorescent confocal imaging.

Full text of DOI:10.1039/c5ra04023j

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PAPER
Pyridinum-based flexible tripodal cleft: a case of
fluorescence sensing of ATP and
Cite this: RSC Adv., 2015, 5, 35175
dihydrogenphosphate under different conditions
and cell imaging†
a
a
b
Kumaresh Ghosh,* Debojyoti Tarafdar, Asmita Samadder
b
and Anisur Rahman Khuda-Bukhsh
Pyridinium-based chemosensor 1 built on tris(aminomethyl)amine (tren) has been designed, synthesized
Received 6th March 2015
Accepted 8th April 2015
and established as a chemosensor for ATP over ADP, AMP and a series of other anions in aqueous
CH
3
CN at pH 6.5. Compound 1 exhibits a significant change in emission upon complexation of ATP. In
DOI: 10.1039/c5ra04023j
4ꢀ
CH
3
CN, the sensor selectively binds
H
2
PO
and forms an excimer with significant intensity.
www.rsc.org/advances
Furthermore, the intracellular ATP detection using 1 was possible through fluorescent confocal imaging.
that uorimetrically selectively senses ATP over ADP, AMP and a
series of other anions in CH CN/H O (1 : 1, v/v, pH ¼ 6.5, 10
Introduction
3
2
Anions play a key role in a variety of environmental and bio- mM HEPES buffer). Furthermore, while the sensor was non
1
logical processes. In particular, phosphate-based biomolecules responsive to different inorganic phosphates in CH
CN/H
O
3
2
such as ATP, ADP, AMP and related inorganic phosphates are (1 : 1, v/v, pH ¼ 6.5), it exhibited strong and selective interaction
ꢀ
considered to be important due to their ubiquitous presence in with
H
2
PO
4
3
in pure CH CN. The versatility of 3-
a range of life processes spanning from energy storage and aminopyridinium-based compounds in anion recognition is
2
8
signal transduction to gene construction. ATP is associated recently reviewed by us. Careful scrutiny in this domain, reveals
with the transport of chemical energy within the cells for that pyridinium-based compounds is little explored in nucleo-
6e
metabolism. Apart from its role in intracellular energy transfer, tide binding although we, for the rst time, reported dipodal
ATP is involved in DNA duplication and transcription. De- and tripodal receptors for selective sensing of ATP and AMP,
ciency of ATP results in ischemia, Parkinson's disease and respectively. Compound 1 in the present report is the modi-
hypoglycaemia. Therefore, selective detection of ATP or inor- cation of our earlier reported tripod 2 that showed selective
ganic phosphates released from the hydrolysis of the phosphate sensing of AMP in water. The replacement of the butyryl amide
3
6f
4
6f
chain is considered to be important in anion recognition in 2 by 1-naphthyl acetamide group results in compound 1
chemistry. In detection of these anions, use of uorescent which selectively binds ATP in aqueous CH CN and thus makes
3
receptors draws attention due to the simplicity and high the pyridinium motif-based tripod core versatile in nucleotide
sensitivity of uorescence technique. Considerable effort in this binding.
direction has already been given by different research groups in
5
last few decades. Inspite of reasonable progress, the use of
organic cation in devising uorescent receptors for nucleotides
5,6
is less explored. Survey of the literature reveals that metal–
ligand complexes have been maximally used for phosphate and
5,7
phosphate-based biomolecule recognition.
In this paper, we wish to report a simple tris(aminomethyl)-
amine (tren) coupled pyridinium-based tripodal chemosensor 1
a
Department of Chemistry, University of Kalyani, Kalyani-741235, India. E-mail:
Scheme 1 describes the synthetic route to the synthesis of
ghosh_k2003@yahoo.co.in; Fax: +913325828282; Tel: +913325828750 ext. 306
b
compound 1. The reaction of tris(2-aminoethyl)amine with
Department of Zoology, University of Kalyani, Kalyani-741235, India
6
g
chloroacetyl chloride in CHCl –H O (1 : 1, v/v) gave amide 3.
†
Electronic supplementary information (ESI) available: Change in uorescence
3
2
6e
and UV-vis titrations of receptor 1 with the anions, binding constant curves, Job Amide 3 was next reuxed with pyridine amide 5 which was
1
13
plots, MTT assay, NOESY spectrum, H, C NMR and mass spectra. See DOI:
0.1039/c5ra04023j
obtained from the reaction of 3-aminopyridine with 1-naphthyl
1
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Fig. 2a. Fig. 2b demonstrates the effect of addition of ATP to the
solution of 1 containing other anions. The large change in
emission in the presence of other anions reveals a clear-cut
binding selectivity of 1 towards ATP. This was also true when
the selectivity of 1 towards ATP was tested in presence of both
phosphate and non-phosphate-based anions (ESI, Fig. 2S†). In
ꢀ
this case, only F ion was observed to interfere to some extent
ꢀ
although F – induced change in emission of 1 in CH
3
CN/H
2
O
(
1 : 1, v/v, pH ¼ 6.5, 10 mM HEPES buffer) was considerably
2 3 3 2
Scheme 1 (i) Chloroacetyl chloride, K CO , CHCl –H O, stir 4 h; (ii)
small (ESI, Fig. 2S†).
oxalyl chloride, dry CH
Et N, dry CH Cl , stir 3 h; (iv) 3, CH
MeOH–water, stirring for 1/2 h.
2
Cl
2
, DMF (cat.), stir 8 h; (iii) 3-aminopyridine,
9
3
2
2
3
CN, reflux, 4 days; (v) NH PF
4
6
,
The linearity in Benesi–Hilderband plot in Fig. 3a corre-
sponds to a 1 : 1 stoichiometric interaction between receptor 1
and ATP. This was also true for ADP (ESI, Fig. 3S†). The asso-
ciation constant values were calculated from uorescence
3
ꢀ1
acetyl chloride 4, in dry CH
3
CN–DMF (5 : 1, v/v) mixture solvent titration data and they were found to be 1.16 ꢂ 10 M and
3
ꢀ1
for 6 days to have pure trichloride salt 6. Subsequent anion 1.10 ꢂ 10 M for ATP and ADP, respectively. The detection
ꢀ
6
exchange reaction of 6 with NH PF in MeOH–H O gave the limit for ATP was determined to be 8.44 ꢂ 10
M (ESI,
4
6
2
10
desired compound 1. All the compounds were characterized by Fig. 3S†).
usual spectroscopic techniques.
In UV-vis spectroscopy, the tripodal receptor 1 in CH CN/
3
H O (1 : 1, v/v, pH ¼ 6.5, 10 mM HEPES buffer) showed
2
absorptions at 252 nm and 285 nm. During interaction with
ATP, the band at 285 nm which was attributed to the absorption
Results and discussion
The anion recognition potential of compound 1 was evaluated of naphthalene underwent minor change although the
by uorescence, UV-vis and NMR spectroscopic techniques. In absorption at 252 nm was signicant. This was true for ADP and

uorescence, excitation of 1 in CH
3
CN/H
2
O (1 : 1, v/v, pH ¼ 6.5, AMP also and thus the distinction of these three nucleotides by
10 mM HEPES buffer) at 290 nm introduced the emission at 386 1 was not possible in the ground state (ESI, Fig. 4S†). For other
nm. Upon interaction with some biologically relevant anions the change in absorption of the two peaks (252 nm and
ꢀ
phosphate-based anions such as ATP, ADP, AMP, H PO
,
285 nm) was negligible and thereby suggested the mere inter-
2
4
2
ꢀ
3ꢀ
ꢀ
HPO4
phosphate (G1P) and glucose-6-phosphate (G6P) in CH
O (1 : 1, v/v, pH ¼ 6.5, 10 mM HEPES buffer), the emission in non aqueous solvent, we investigated the interaction of 1 with
,
PO4
and pyrophosphate (P O ), glucose-1- action of the receptor in the ground state (ESI, Fig. 4S†).
2
7
3
CN/
In order to be acquainted with the anion binding behaviour of 1
H
2
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
4
ꢀ
intensity of 1 was changed to the different extents (Fig. 1a). the anions such as F , Cl , Br , I , NO , AcO , ClO , HSO4
,
3
3ꢀ
Among the different phosphate-based anions, only ATP caused HP O
2
and H PO (taken as their tetrabutylammonium salt) in
2
7
4
ꢀ
signicant change in emission (Fig. 1b). On incremental addi- pure CH CN (ESI, Fig. 5S†). Among these anions, only H PO
4
3
2
tion of ATP, a broad emission at 390 nm was intensied. This perturbed the emission of 1 markedly by showing excimer emis-
was also observed in the presence of ADP (ESI, Fig. 1S†). But it sion at 455 nm. We believe that this excimer emission at 455 nm
was much less compared to the case with ATP. During interac- which is retained in the midst of other anions (ESI, Fig. 6S†),
tion, appearance of the peak at ꢁ390 nm is attributed to results in from the complexation induced pulling of the pendant
formation of exciplex between naphthalene and adenine. It is naphthalenes closely. This observation is in accordance with the
6e
11
mentionable that during interaction of 1 with ATP, the inter- ndings observed by us and other researchers.
ference of other anions was negligible. This is evident from
ꢀ
5
Fig. 2 (a) Change in fluorescence ratio of 1 (c ¼ 2.5 ꢂ 10 M) in
Fig. 1 (a) Change in emission ratio of 1 (c ¼ 2.5 ꢂ 10ꢀ M) in the absence and presence of 20 equiv. amounts of ATP in presence of
5
presence of 20 equiv. amounts of the sodium salts of various anions in sodium salts of various anions in CH
3
CN–H
2
O (1 : 1, v/v, pH ¼ 6.5, 10
CH O (1 : 1, v/v, pH ¼ 6.5, 10 mM HEPES buffer); (b) change in mM HEPES buffer); (b) change in fluorescence intensity of 1 in
emission of 1 (c ¼ 2.5 ꢂ 10 M) in CH
mM HEPES buffer) upon gradual addition of 30 equiv. of ATP (c ¼ 1 ꢂ of 20 equiv. amount of ATP in presence of other anions considered in
3
CN–H
2
ꢀ5
3
CN–H
2
3 2
O (1 : 1, v/v, pH ¼ 6.5, 10 CH CN–H O (1 : 1, v/v, pH ¼ 6.5, 10 mM HEPES buffer) upon addition
ꢀ3
1
0
M) [lexc ¼ 290 nm].
the present study [lexc ¼ 290 nm].
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3
ꢀ1
7
.06 ꢂ 10 M by considering the emission titration results,
ꢀ
gathered from addition of 2 equiv. amounts of H PO to the
solution of 1. The detection limit for H PO was determined to
be 1.46 ꢂ 10 M (ESI, Fig. 9S†).
Prior to identify the interacting protons of 1 in the
complexation, we recorded NOESY spectrum of 1 in d -DMSO
Fig. 4). We did not record the NMR spectra in CD CN due to
2
4
ꢀ
4
2
ꢀ
6
10
6
(
3
appearance of non homogeneity of the solution of 1 in NMR
concentration range. As can be seen from Fig. 4, naphthalene
appended pyridinium motifs in the tripod are disposed in
spread out conformation by exhibiting some correlations in the
ꢀ
5
Fig. 3 Benesi–Hilderband plots for receptor 1 (c ¼ 2.5 ꢂ 10 M) with
ꢀ3
(
a) ATP and (b) ADP ([ATP] ¼ [ADP] ¼ 1 ꢂ 10 M) at 390 nm.
individual strand. In the presence of equivalent amount of
ꢀ
H
2
PO
4
6
in d -DMSO some correlations of protons in each pod
Thus, this unique feature of emission can be considered as
ꢀ
as shown in Fig. 4 were destroyed (ESI, Fig. 10S†). Even no
correlation among the pendant naphthalenes was observed.
This nding suggests that the hydrogen bonding interaction of
the diagnostic one for discrimination of H PO from the
2
4
other anions examined in CH
3
CN. In UV-vis study, upon
ꢀ
gradual addition of H
2
PO
4
, the absorption of 1 was changed
ꢀ
ꢀ
H PO occurs into the core involving pyridinium sites in such
2
4
irregularly (ESI, Fig. 7S†). In contrast, gradual addition of F
a way that the naphthalene rings from the different arms are
pulled weakly without showing any characteristic NOESY
correlation which depends on the distance of the nuclei. The
details along this direction including the change in H-NMR of
during complexation of H
ion initially did not bring any marked change in absorption
ꢀ
spectra. But upon increasing the concentration of F , the
absorption at 252 nm was decreased followed by an appear-
ance of a new absorption band at ꢁ300 nm. Such ratiometric
response introduced an isoemissive point at 284 nm. This
1
ꢀ
1
2
PO
4
can be found in the ESI
ꢀ
(Fig. 11S†).
suggests a strong ground state interaction of 1 with F
ATP amplied prominent change of optical behaviour of
receptor 1 in uorescence was understood from H NMR
spectral change. In this experiment receptor 1 was taken in
d -DMSO and to this solution ATP (dissolved in D O) was
6 2
added in equivalent amount. In the presence of equiv.
amount of ATP, amide protons of types ‘o’ and ‘d’ underwent
downeld chemical shis by 0.74 ppm and 0.45 ppm,
respectively. On keeping the solution for ꢁ15 min, the
exchangeable amide protons were not observed due to pres-
although there was no considerable change in emission in
1

uorescence.
4ꢀ
The stoichiometry of the H PO complex as determined
from uorescence Job plot was found to be 1 : 1 (ESI, Fig. 8S†)
and the association constant for H PO was evaluated to be
2
12
9
ꢀ
2
4
ence of D
2
O in the mixture. The pyridinium ring protons
(
types ‘b’ and ‘c’) moved downeld (Fig. 5). These results
indicated that the pyridinium sites in 1 are involved in
complexation of the phosphate chain. The downeld chem-
ical shis of the methylene protons of types ‘n’ and ‘m’ by
0
.57 ppm and 0.27 ppm, respectively also suggested their
participation in hydrogen bonding with the phosphate chain.
Furthermore, during interaction the naphthyl ring protons (i,
j, k and l types) suffered upeld chemical shi by 0.11 ppm
and the adenine ring protons moved upeld by 0.001–0.03
ppm. These changes in chemical shi values may be ascribed
to the stacking of adenine ring with the naphthyl unit (Fig. 6).
1
ꢀ3
Fig. 5 (a) Partial H NMR (d
6
-DMSO, 400 MHz) of 1 (c ¼ 1.5 ꢂ 10 M)
3
Fig. 4 NOESY spectrum of 1 (d
6
-DMSO, 400 MHz) (c ¼ 1.83 ꢂ 10ꢀ M). with equiv. amount of ATP (dissolved in D
2
O).
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Fig. 6 Probable binding mode of 1 with ATP.
Analysis of the NOESY spectrum reveals that in 1$ATP
complex, there is a cross-peak between the signal of adinine
ring proton (d 8.70 ppm) and methylene protons of type ‘m’
(
Fig. 7). This observation means that adenine is located
closely to naphthalene moiety in the suggested mode shown
in Fig. for which naphthalene–adenine–naphthalene
6
p-stacking interaction exists in solution and allows the
formation of exciplex at 390 nm (see Fig. 1b) in uorescence.
Further attempts have been made to understand the bio-
logical application of the compound 1 for detection of ATP
under cellular condition in HEK293T cell line (Human Embry-
onic Kidney cells with T antigen of SV40) using confocal Fig. 8 Confocal microscopy of different sets of normal untreated and
microscopy (Fig. 8). Microscopic images revealed a signicant experimental sets of HEK293T cells: (A) differential interference
contrast (DIC) image and (B) fluorescing image of untreated control
increase in the blue uorescence in HEK293T cells incubated
cells, (C) merge images of (A) + (B), (D) intensity profile of (B) (scale: X
with both ATP and compound 1 (Fig. 8J) when compared to cells
axis ¼ length (mm) [0.32 mm per pixel], Y axis ¼ intensity/pixel), (E) DIC
treated with only compound 1 (Fig. 8F) and/or normal untreated
image and (F) fluorescing image of cells treated with 1 (10 mM) for 1 h at
ꢃ
cells (Fig. 8B). This nding suggests that the compound 1 is 37 C, (G) merge images of (E) + (F), (H) intensity profile of (F) (scale: X
permeable through cellular membrane and is able to sense the axis ¼ length (mm) [0.32 mm per pixel], Y axis ¼ intensity/pixel), (I) DIC
image and (J) fluorescing image of cells which were pre incubated with
presence of intracellular ATP. In MTT assay, there was no
signicant difference in percentage of viable cells of receptor
treated cells when compared to that of solvent treated series of
ꢃ
5
0 mM ATP and then treated with 1 (10 mM) for 30 min at 37 C, (K)
merge images of (I) + (J), (L) intensity profile of one of the represen-
tative fluorescing cells in (J) (scale: X axis ¼ length (mm) [0.32 mm per
cell sets (ESI, Fig. 12S†). This indicates that the receptor is non pixel], Y axis ¼ intensity/pixel).
cytotoxic in nature.
Conclusion
We have thus reported a pyridinium motif coupled tren-
based tripodal receptor 1 which in aqueous CH CN selec-
3
tively recognizes ATP over ADP, AMP, other inorganic phos-
phates and non-phosphate containing anions based on

3
uorescence. In pure CH CN, receptor 1 recognizes tetrabu-
tylammonium dihydrogenphosphate urometrically by
exhibiting diagnostic excimer emission. The recognition
takes place through hydrogen bonding and charge–charge
interactions at the pyridinium sites. Our previous report on
receptor 2 as well as the present observation of 1 rmly
establishes that the tripodal core of 1, comprised of pyr-
idinium motifs, is a unique platform of sensing nucleotides.
While the absence of aromatic surface onto the pyridinium
motif in 2 directs the binding of AMP in pure water, the
presence of naphthalenes in 2 modies the cle to sense
Fig. 7 NOESY spectrum of 1$ATP (d
ꢂ 10 M).
6
2
-DMSO/D O, 400 MHz) (c ¼ 1.83
ꢀ3
3
ATP over ADP and AMP in aqueous CH CN. This versatility of
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the pyridinium motif-based tripodal core will denitely precipitate the compound. Repeated washing of the precipitate
inspire chemists to enquire new ndings in molecular with water and diethyl ether afforded the desired salt 1 in 82%
ꢃ
yields (0.102 g), mp 210 C; H NMR (400 MHz, DMSO-d ) d
6
1
recognition.
1
1.37 (s, 3H), 9.35 (s, 3H), 8.57 (s, 3H), 8.39 (brs, 6H), 8.04–7.86
m, 12H), 7.52–7.49 (m, 12H), 5.31 (s, 6H), 4.26 (s, 6H), 3.39 (6H
overlapped with the signal for d -DMSO water), 3.12 (brm, 6H);
) d 170.5, 164.1, 140.7, 138.4,
(
Experimental
Syntheses
6
13
C NMR (100 MHz, DMSO-d
6
0
00
0
00
N,N ,N -(2,2 ,2 -Nitrilotris(ethane-2,1-diyl))tris(2 chloroa- 135.9, 134.4, 133.2, 131.8, 131.1, 128.4, 128.1, 127.6, 126.2,
6g
cetamide) (3). To a stirred solution of tris-(2-aminoethyl)- 125.7, 125.4, 124.0, 61.9, 52.5, 38.7, 37.3 (one carbon in
ꢀ1
amine (0.6 g, 4.10 mmol) in CHCl
(30 mL), chloroacetyl chlo- aromatic region is unresolved); FTIR (KBr, cm ) 3399, 3098,
3
ride (1.53 g, 13.54 mmol) was added dropwise. Then water (10 1688, 1594, 1553, 1508, 1458; HRMS (TOF MS ES+) calcd for
+
mL) was added to the reaction mixture followed by the addition
of K CO
lammonium hydrogensulphate was added to the reaction
mixture and stirred for 2 h. Aer completion of the reaction, as
monitored by TLC, organic layer was separated and dried over
C
63
H
63
N
) .
10
O
6
$2PF
6
: 1345.4210 (M ꢀ PF
) ; found: 1345.4215 (M
6
+
(1.87 g, 13.54 mmol). Catalytic amount of tetrabuty- ꢀ PF
6
2
3
Cell culture reagents
MTT [3-(4,5-dimethyl-thiazol-2-yl)-2,S-diphenyltetrazolium bro-
mide], and DMSO were of analytical grade and they were
procured from Sigma-Aldrich Inc. (St-Louis, MO, USA); Dul-
becco's modied Eagle's medium (DMEM), fetal bovine
serum, and antibiotics, namely, penicillin, streptomycin, and
neomycin (PSN) were purchased from Gibco BRL (Grand Island,
NY, USA). All organic solvents used were of high performance
liquid chromatography grade.
anhydrous Na
evaporator and puried by silica gel column chromatography
using 1% CH OH in CHCl as eluent to afford pure compound 3
2 4
SO . The solution was concentrated on a rotary
3
3
ꢃ
1
(1.40 g, 90%), mp 102 C, H NMR (CDCl
3
, 400 MHz) d 7.05 (br s,
3
H), 4.08 (s, 6H), 3.38 (q, 6H, J ¼ 4 Hz), 2.65 (t, 6H, J ¼ 4 Hz);
ꢀ1
FTIR (KBr, cm ) 3515, 3273, 2961, 1677, 1660, 1562.
-(Naphthalen-1-yl)acetyl chloride (4). To a stirred solution
of 1-napthylacetic acid (0.700 g, 3.76 mmol) in dry CH Cl (20
2
2
2
mL), oxalyl chloride (0.365 mL, 4.51 mmol) was added followed
by addition of one drop of dry DMF. Aer stirring at room
Cell culture procedure
temperature for 8 h under nitrogen atmosphere, excess oxalyl HEK293T cells (Human embryonic kidney cells with T antigen
chloride was completely removed under vacuum to give acid of SV40), were obtained from National Centre for Cell Science,
ꢃ
chloride 2 (0.720 g, yield 93%). This was directly used in the next Pune, India. Cells were grown in 5% carbon dioxide, at 37 C in
step without any purication.
-(Naphthalen-1-yl)-N-(pyridin-3-yl)acetamide (5).
Dulbecco's modied Eagle's Medium (DMEM) supplemented
with 10% foetal bovine serum and 1% antibiotic (PSN). For
6e
2
To
a
stirred solution of 3-aminopyridine (0.390 g, 4.14 mmol) in dry experimental studies, the cells were allowed to grow to 70–80%
CH Cl (20 mL), 2-(naphthalen-1-yl) acetyl chloride 4 (0.706 g, conuence, aer which they were harvested in ice-cold buffer
.45 mmol) was added followed by addition of Et N (0.58 mL, saline (PBS) and plated at desired density. The cells were
.14 mmol). Aer stirring the reaction mixture for 8 h at room allowed to re-equilibrate for 24 h before any treatment.
2
2
3
4
3
13
temperature under nitrogen atmosphere, the reaction mixture
was poured into water and extracted with CH Cl
(3 ꢂ 40 mL).
Organic layer was dried over anhydrous Na SO and concen-
trated in vacuo. The crude product was puried by chromatog-
raphy using 50% ethyl acetate in petroleum ether to give 5 in
2
2
Cytotoxicity assessment of receptor in living cells
To check whether the receptor render any cytotoxicity to the
2
4
14
cells, the MTT assay was conducted by following the standard
technique of Mossman. Different concentrations of the
receptor, ranging from 1 mL through 5 mL of stock solution, were
ꢃ
1
8
8
7
8% yield (0.800 g); mp 125 C; H NMR (400 MHz, CDCl ) d
3
.27–8.24 (m, 2H), 8.02–7.99 (m, 2H), 7.92–7.87 (m, 2H), 7.59–
.55 (m, 2H), 7.53–7.49 (m, 2H), 7.20–7.17 (m, 2H), 4.20 (s, 2H);
C NMR (100 MHz, CDCl ) d 169.9, 145.1, 141.1, 134.6, 134.0,
3
6
added to the cultured cells (10 cells per mL) in 96-well micro
plates and incubated for 24 h. A set of cells, not exposed to any
of the concentrations of the receptors, were kept as untreated
control. At the termination of incubation period, MTT was
added into each well. Aer 4 h, the formazon crystals thus
formed were dissolved with dimethyl sulfoxide (DMSO) and the
absorbance of the solution was measured at 595 nm using
ELISA reader (Thermo scientic, Multiskan ELISA, USA). The
percentage of cell survival was calculated as: (mean experi-
mental absorbance/mean control absorbance) ꢂ 100%.
1
3
1
1
1
32.0, 130.3, 128.9, 128.8, 128.4, 127.9, 127.0, 126.3, 125.6,
23.6, 123.5, 42.33; FTIR (KBr) 3281, 3122, 2850, 1742, 1658,
ꢀ
1
606, 1549, 1481 cm
Receptor 1. To a solution of tris amide 3 (0.150 g, 0.399
mmol) in CH CN (10 mL), compound 5 (0.419 g, 1.60 mmol) in
CH CN (25 mL) was added. The reaction mixture was reuxed
.
3
3
with stirring for 4 days under nitrogen atmosphere. On cooling
the reaction mixture, precipitate appeared. The precipitate was

ltered off and washed with CH
pure trichloride salt 6 (0.374 g, 80.7%). The pure trichloride salt
(0.100 g, 0.07 mmol) was dissolved in 2 mL hot MeOH, and
NH PF
stirring the reaction mixture for 20 min, water was added to (PBS). They were then incubated in 20 mL of ATP solution
3
CN for several times to have
Evaluation of potential efficacy of the receptor to detect the
presence of ATP in cultured cell line
5
4
6
(0.053 g, 0.32 mmol) was added in one portion. Aer At rst the cells were washed with phosphate buffered saline
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50 mM). Aer 30 min of incubation at 37 C, the cells were again
Paper
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(
S. Biagini, A. Bencini, E. Faggi, C. Giorgi, I. Matera and
B. Valtancoli, Chem. Commun., 2006, 4087; (d) J. Kaur and
P. Singh, Chem. Commun., 2011, 47, 4472; (e) D. Amilan
Jose, S. Mishra, A. Ghosh, A. Shrivastav, S. K. Mishra and
A. Das, Org. Lett., 2007, 9, 1979; (f) F. Sancenon,
A. B. Descalzo, R. Martinez-Manez, M. A. Miranda and
J. Sato, Angew. Chem., Int. Ed., 2001, 40, 2640; (g) Y. Zhou,
Z. Xu and J. Yoon, Chem. Soc. Rev., 2011, 40, 2222; (h)
K. Ghosh and I. Saha, Org. Biomol. Chem., 2012, 10, 9383.
(a) C. Schmuck and M. Heller, Org. Biomol. Chem., 2007, 5,
washed in PBS and then further treated with 30 mL of receptor
stock solution (10 mM) made in PBS (maintained at pH 6.5) and
ꢃ
kept for another 30 min at 37 C. Washing excess of receptor
with PBS the cells were then photographed under Andor Spin-
ning Disk Confocal Microscope and evaluated against untreated
control set of cells. The uorescence intensity was quantied
using the Analysis soware, NIS Elements AR Version 4.00.
6
Acknowledgements
7
87 and references cited therein; (b) Z. Xu, N. R. Song,
We thank DST and UGC, New Delhi, India for providing facili-
ties in the department under FIST and SAP programs, respec-
tively. DT thanks CSIR, New Delhi, India for a fellowship.
J. H. Moon, J. Y. Lee and J. Yoon, Org. Biomol. Chem., 2011,
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