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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 7.2
Prasenjit Mahato, Amrita Ghosh, Sanjiv K. Mishra, Anupama Shrivastav, Sandhya Mishra,*
Amitava Das*
Central Salt and Marine Chemicals Research Institute (CSIR),
Bhavnagar: 364002,
Gujarat, India
E-mail: amitava@csmcri.org
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Table of content
Page Numbers:
1. General synthetic procedure of L and L.Zn
3-4
2. Biological sample preparation
5
3.
6
Solubility experiment
4. ESI-MS specra of L.Zn-ATP and α-CD.L.Zn complex
5.
31
PNMR specral study
7-8
9
6. Spectrophotometric titration of L.Zn with CTP, ADP and α-CD
10 - 11
7. Benesi-Hildebrand plot for titration of L.Zn with varying ATP
And a confirmation for a 1:1 binding stoichiometry
12
8. Interference study
13
9. Absorption spectra of L and L.Zn
14
10. Absorption spectra of L.Zn in presence of ATP/ATP
+ Citratre ion, yeast cells/ yeast cells + Citrate ion
15
11. Absorption spectra of L.Zn at different pH
16
12. Association constant values and methodology
used for calculating binding constant
17 - 18
13. SEM images of yeast cells
19
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1. General synthetic Procedure
1.1 Materials and Methods:
The
chemicals
such
as
1,4,8,11-tetraazacyclotetradecane,
(E)-4-((4-(dimethyl
amino)phenyl)diazenyl)benzene-1-sulfonyl chloride, α-cyclodextrin, tetrabutylammonium
salts of anions, AMP, ADP, ATP, CTP and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic
acid were received from Aldrich and used as such. All the other reagents used were procured
from S. D. fine chemicals, India. Solvents such as THF, methanol, chloroform (AR; Merck,
India), ethanol (Spectrosol; Spectrochem, India) were used as solvents. HPLC grade water
(Merck, India) was used for experiments and spectral studies. Microanalysis(C, H, N) was
performed using a Perkin-Elmer 4100 elemental analyzer. FT—IR spectra were recorded in
KBr pellets using Perkin Elmer Spectra GX 2000 spectrometer. 1H and 31PNMR spectra were
recorded on Bruker 500 MHz FT NMR (model: Advance-DPX 200). Electronic spectra were
recorded with Varian Cary 500 Scan Uv-vis-NIR Spectrophotometer. ESI-MS measurements
were carried out on Waters QTof-Micro instrument. The images of the yeast cells, viewed
under normal light microscope (AXIO IMAGER–Carl Zeiss).
1.2 Synthesis of compound L:
1,4,8,11-tetraazacyclotetradecane (cyclam) (247 mg, 1.23 mmole) was dissolved in dry tetra
hydrofuran (40 ml) in a 250 ml Round Bottomed Flask. A solution (E)-4-((4-(dimethyl
amino)phenyl)diazenyl)benzene-1-sulfonyl chloride (400 mg, 1.23 mmole) and triethyl amine
(Et3N) (1 ml) in tetrahydrofuran (10-15 ml) was added to the above solution in ice cold
condition. Then, the resulting mixture was allowed to stir at room temp (25°C to 30°C) for 10
hr and then it was further refluxed for 1 hr. The orange precipitate thus obtained was filtered
and washed with tetrahydrofuran. Finally the solid residue (L) was dried; Yield: 75%. 1H
NMR (500 MHz, CDCl3, SiMe4, J (Hz), δ (ppm)): 7.97 (2H, d, J = 8.0, Ar-He), 7.9 - 7.87
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(4H, m, Ar-Hc,d), 7.06 (3H, br, -NHmacrocycle), 6.76 (2H, d, J = 8.0, Ar-Hb), 3.15 (16H, br,
Hf,h,i,j,k,m,n,o), 3.11 (6H, s, -N(CH3)2), 2.03 (4H, br , Hg,l). ESI-MS (+ ve mode): m/z; 488.30
(M++H+). Elemental Analysis data: Calc. C: 59.11, H: 7.65, N: 20.11, Expt. C: 59.1, H: 7.62,
N: 20.2.
1.3 Synthesis of compound L.Zn :
A solution of L (200 mg; 0.41 mmole) was dissolved in 25 ml methanol in a 100 ml single
necked flask. To this 148.69 mg of zinc nitrate dissolved in 1.0 ml water (1.2 mole
equivalent) was added dropwise. The resultant solution was stirred at room temperature
(25°C to 30°C) for 24 h. The reaction mixture was kept in refrigerator at 4°C for 5 h and a
violet colored precipitate appeared. This was collected through filtration using Gooch (no. 3)
crucible and was washed several times with CHCl3; Yield: 65%.1HNMR (500 MHz, DMF-d7,
SiMe4, δ (ppm)): 7.92 (2H, d, J = 8.5, Ar-He), 7.87 (2H, d, J = 9, Ar-Hc), 7.80 (2H, d, J =
8.5, Ar-Hd), 6.91 (2H, d, J = 9.5, Ar-Hb), 3.13 (6H, s, -N(CH3)2), 2.92 (8H, br, Hf,g,n,o), 2.75
(12H, br, Hh,i,j,k,l,m). ESI-MS (+ ve mode), m/z: 676.00 (M+). Elemental Analysis: Calc. C:
42.57, H: 5.51, N: 18.62; Expt. C: 42.88, H: 5.56, N: 18.52.
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2. Biological sample preparation
2.1 Saccharomyces cerevisiae cells
Yeast (Saccharomyces cerevisiae) was isolated from curd and then cultured in the Glucose
yeast extract agar (GYE) medium ( Glucose 1g, yeast extract 10g, Distilled water 1000 ml,
pH 7.4). The cells were harvested and vortexed for making the homogenous suspension in
sterile distilled water.
2.2 For SEM Images
The isolates of Saccharomyces sp. were cultured in GYE (glucose yeast extract agar) media,
for 24 h at 37°C. 100 μl of this was taken and was allowed to incubate at 30 to 40°C along
with 100 μl of L.Zn (0.66x10-4 M) solution. The samples were fixed overnight at room
temperature 25-30°C with 2% (v/v) glutaraldehyde in 0.1 M phosphate buffer at pH 7.5. The
samples were then washed with 0.1 M phosphate buffer (pH 7.5) at room temperature 2530°C for 1 h. Post-fixation was carried out in 2% (w/v) osmium tetroxide (OsO4) in the same
buffer and was washed once with 0.1 M phosphate buffer for 20 minutes. Then, the water
was removed by a graded water-ethanol series; 25% ethanol-15 min, 50% ethanol-15min,
75% ethanol-30 min, 90% ethanol-60 min, absolute alcohol-30 min. The specimens were
rinsed in buffer and coated with gold in a sputter coater (Polaron SC7620) prior to
microscopy. The material was examined in a Scanning Electron Microscope (SEM) LEO
1430 VP at an accelerating Voltage of 15 kV.
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3. Solubility Experiment:
Enhancing the solubility of L.Zn in aqueous solution:
Solubility of L.Zn in water is limited (0.045 gL-1). However, this could be enhanced in
presence of α-cyclodextrin (α-CD). 2.3 mg of L.Zn was dissolved in 5 ml of double distilled
water and stirred at room temperature for 1 hr. Then the solution was filtered and the
concentration of the dissolved solid in the filtrate was evaluated based on the equation
O.Dfiltrate / εLZn (O.Dfiltrate is optical density of the filtrate and εLZn is the molar extinction
coefficient of L.Zn) (value was evaluated earlier through an independent experiment) and
was found to be 0.045 gL-1. Similar experiment was repeated in presence of α-CD. In this
case 24.3 mg of α-CD was dissolved in 5 ml of double distilled water containing 2.913 mg of
L.Zn and this solution was stirred at 25°C for 1 hr; while a solution with little suspended
solid was obtained. This was filtered using G-4 gooch crucible. Filtrate was collected and
optical density (O.D.) of the filtrate was monitored at 458 nm. Considering the complete
formation of the inclusion complex (pseudorotaxane form), concentration was evaluated
based on the known εLZn.α-CD value and the O.D. at 458 nm. Calculation revealed that 0.34
gL-1 L.Zn is present in the resultant solution. Hence ~7.5 fold increase in solubility in terms
of L.Zn was achieved and this is also evident in the difference of the visible colour of the
saturated solution of L.Zn in absence and presence of α-CD in aqueous solution (ESI Figure
8). L.Zn present in aqueous solution in the [2]pseudorotaxane form is being represented as
α.CD.L.Zn in Figure 3.
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4. ESI-MS spectra:
4.1 ESI-MS spectra of L.Zn.ATP complex:
L ZNATP R 17 (0.170)
1: TOF MS ES+
8.06
798.59
100
776.63
900.39
736.70
1002.18
878.43
838.53
%
792.59
940.31
1042.09
1103.97
980.22
1082.03
832.53
754.68
714.72
816.54
872.46
856.51
1064.07
934.31
894.42
996.16
918.37
854.44
1036.09
1062.11
956.24
1020.12
1043.09
894.22
0
720
740
760
780
800
820
840
860
880
900
920
940
960
980
1000
1020
1040
1060
1080
1100
m/z
1120
ESI Figure 1: ESI-MS spectra of L.Zn (7.2 μM) in presence of ATP (900 μM) in double
distilled and deionised water. The highlighted peak 1082.03 supports the formation of a 1:1
complex between L.Zn and ATP.
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4.2 ESI-MS spectra of α-CD.L.Zn complex: LZNCD 3 (0.030)
1: TOF MS ES+
2.66
1483.56
100
1481.13
1480.73
1484.15
1474.82
%
1474.19
1485.60
1492.22
1647.22
1492.76
1642.64
1647.94
1494.61
1145.68
1473.03
1648.59
1495.28
1471.97
1131.53
1560.90
1182.72
1448.49
1147.70
1104.17
1187.93
1227.50
1250.06
1296.64
1314.66
1641.25
1495.88
1423.56
1557.73
1618.33
1528.42
1649.43
1649.75
1627.33
1653.60
1663.80
1587.26
0
m/z
1150
1200
1250
1300
1350
1400
1450
1500
1550
1600
1650
ESI Figure 2: ESI-MS spectra of L.Zn (8.6 μM) in presence of α-CD (100 μM) in double
distilled and deionised water. The highlighted peak 1648.59 supports the formation of a 1:1
inclusion complex between L.Zn and α-CD.
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5. Changes in 31PNMR specra of ATP, CTP and ADP in presence of L.Zn: L.Zn+ATP
γ
α
‐10.5
‐11.0
β
ATP
‐11.5
‐22.5
‐23.0
ppm
ESI Figure 3: Partial 31PNMR spectra of ATP (0.066 mM) before and after addition of L.Zn
(1 equivalent).
L.Zn+CTP
γ
-10.5
α
-11.0
β
CTP
-11.5
-22.5
-23.0
-23.5
ppm
ESI Figure 4: Partial 31PNMR spectra of CTP (0.067 mM) before and after addition of L.Zn
(1 equivalent).
ADP + L.Zn
α
β
-7.6
-7.8
-8.0
-8.2
ADP
-8.4
-8.6
ppm
ESI Figure 5: Partial 31PNMR spectra of ADP (0.067 mM) before and after addition of L.Zn
(1 equivalent).
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6. Spectrophotometric titration: 6.1 Spectrophotometric titration of L.Zn with CTP:
Absorbance
0.12
0.09
0.06
0.03
0.00
375
450
525
600
Wavelength(nm)
ESI Figure 6: Absorption spectra of L.Zn (1.79 μM) with varying [CTP] (0–10.17 mM) in
aq. HEPES buffer (10 mM, pH 7.2) at 25°C. 6.2 Spectrophotometric titration of L.Zn with ADP:
Absorbance
0.4
0.3
0.2
0.1
0.0
375
450
525
600
Wavelength (nm)
ESI Figure 7: Absorption spectra of L.Zn (8.86 μM) with varying [ADP] (0–6.55 mM) in
aq. HEPES buffer (10 mM, pH 7.2) at 25°C.
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6.2 Spectrophotometric titration of L.Zn with α-CD:
Absorbance
0.3
0.2
a
0.1
b
0.0
375
450
525
Wavelength(nm)
ESI Figure 8: Absorption spectra of L.Zn (8.3 μM) with varying [α-CD] (0-2.3mM) in
aq.HEPES buffer (10 mM, pH 7.2) at 25°C. Inset is the photograph of the saturated solution
of (a) L.Zn (0.045 gl-1) and (b) L.Zn (0.34 gl-1) in presence of α-CD (4.5 gL-1) in double
distilled and deionised water.
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7. Benesi-Hildebrand plot:
25
1/[A-A0]
20
15
10
5
0
0
4000
8000
12000
1 / [ATP] (M-1)
SI Figure 9: Benesi-Hildebrand plot of 1 / (A-A0) vs. 1 / [ATP] for change in absorption
intensity at 503 nm upon addition of varying [ATP]. Goodness of the straight line fit (R2 =
0.993) confirms 1:1 binding stoichiometry. Binding affinity, thus evaluated, was found to be
1.9 ± 0.15).103 M−1.
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8. Interference Study :
Absorbance Intensity
0.10
0.08
0.06
0.04
0.02
0.00
A B C D E F G H I J K L M N
Anions
SI Figure 10: Plot of absorption intensity of L.Zn (2 μM) with addition of ATP (4 mM) at
503 nm in presence of 5 equivalent excess of other anions in aq. HEPES buffer (0.01 M, pH
7.2). [A: ATP only, B: ATP + CTP, C: ATP + ADP, D: ATP + AMP, E: ATP + H2PO4¯, F:
ATP + F¯, G: ATP + Cl¯, H: ATP + Br¯, I: ATP + I¯, J: ATP + Ac¯, K: ATP + SO42¯, L:
ATP + CN¯, M: ATP + SCN¯, N: ATP + NO3¯].
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9. Absorption spectra of L and L.Zn:
0.4
L
Absorbance
0.3
L.Zn
0.2
0.1
0.0
400
500
600
Wavelength(nm)
ESI Figure 11: Absorption spectra of L (16 μM) and L.Zn (16 μM ) in CH3CN:MeOH (7:3, v/v)
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10. Absorption spectral evidence for the reversible binding of ATP to L.Zn:
0.25
(a)
0.60
(b)
5
1
3
6
0.45
Absorbance
Absorbance
0.20
2
0.15
0.10
4
0.30
0.15
0.05
0.00
0.00
375
450
525
600
375
Wavelength(nm)
450
525
Wavelength(nm)
ESI Figure 12: (a) Absorption spectra of (1) L.Zn (2.97 µM), (2) L.Zn in presence of ATP
(5.0 x 10-4 M), (3) L.Zn in presence of ATP (3.5 x 10-4 M) and sodium citrate solution (5.0 x
10-4 M) in 10 mM HEPES buffer (pH = 7.2); (b) Absorption spectra of (4) L.Zn (7.1 µM),
(5) L.Zn (7.1 µM) in presence of yeast (6) L.Zn (7.1 µM) + in presence of yeast cells and to
this sodium citrate solution (5.0 x 10-4 M) was added 10 mM HEPES buffer (pH = 7.2).
Spectra were recorded at 25oC.
Yeast cells were cultured in the Glucose yeast extract agar (GYE) medium (Glucose 1g, yeast
extract 10g, Distilled water 1000 ml, pH 7.4). The cells were harvested and vortexed for
making the homogenous suspension in sterile distilled water. Solution used for the
spectroscopic measurements with Yeast cells was not as homogeneous as it was the case for
solution of HEPES buffer with ATP and L.Zn. Moreover, other media constituents that were
used for the Yeast culture were also present in the solution for spectroscopic measurements
with
Yeast
cells
and
this
could
also
have
ESI-15 influenced
the
spectral
nature.
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11. Absorption spectra of L.Zn at different pH:
0.20
H
P 2
Absorbance
0.15
H
H
P 3.02
P 7, 9.9, 13
0.10
H
P 4
0.05
0.00
400
500
600
Wavelength(nm)
ESI Figure 13: Absorption spectra of L.Zn (1.79 μM) at different pH.
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12. Methodology used for calculating binding constant:
12.1 Association constant values for ATP, CTP and ADP towards L.Zn:
ESI Table 1. Association constant values calculated from absorption titration method in
aqueous HEPES buffer (pH 7.2).
Anion
ATP
Ka ( M-1)
978 ± 15
CTP
220±15
ADP
142±15
Methodology Used for evaluation of these Association Constants:
General formula used for calculating the binding constants from UV-vis titrations.
L.Zn + A¯ = L.Zn.A¯
Absorption maxima for L.Zn is λL.Zn, while extinction coefficient and OD at this wavelength
are εL.Zn and AL.Zn respectively. Absorption maxima for L.Zn.A¯ is λ L.Zn.A¯ while extinction
coefficient and OD at this wavelength are εL.Zn.A¯ and AL.Zn.A¯, respectively.
Thus, for a given concentration of the receptor molecule ([L.Zn]) and anion ([A¯])
[L.Zn.A¯] = [{ALZn.A¯ - A0 } / εL.Zn.A¯ ]
(1)
Where, A0 is OD value at λ L.Zn.A¯ before addition of externally added A¯.
A L.Zn.A¯ was recorded at λL.Zn.A¯ after addition 100 mole equivalent of A¯.
Therefore, εL.Zn.A¯ = AL.Zn.A¯ / [L.Zn]free
(2)
(Assuming all the receptor molecules is bound to A¯ and A¯ alone does not absorb at this
wavelength)
Thus, once we one can evaluate [L.Zn.A¯], it is possible to calculate concentration of the
uncomplexed receptor molecule, [L.Zn] using equations 1 and 2.
[L.Zn]free = [L.Zn]initial - [L.Zn.A¯]
(3)
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and, [A¯]free = [A¯]initial - [L.Zn.A¯]
(4)
If K is the formation constant for the complex, L.Zn.A¯ for a given concentration of A¯
Binding Constant, K = [L.Zn.A¯] / {[L.Zn]free[A¯]free}
(5)
12.2 Methodology used for calculating binding constant of α-cyclodextrin with L:
General formula that was used for calculating the binding constants from UV-vis titrations is
given below:
L.Zn + α-CD = α-CD.L.Zn
Absorption maxima for L.Zn is λL.Zn, while extinction coefficient and OD at this wavelength
are εL.Zn and AL.Zn respectively. Absorption maxima for α-CD.L.Zn is λα-CD.L.Zn, while
extinction coefficient and OD at this wavelength are εα-CD.L.Zn and Aα-CD.L.Zn respectively.
Thus, for a given concentration of the receptor molecule ([L]) and α-cyclodextrin ([α-CD])
[α-CD.L.Zn] = [{α-CD.L.Zn - A0 } / εα-CD.L.Zn]
(6)
Where, A0 is OD value at λα-CD.L.Zn before addition of externally added α-CD.
Aα-CD.L.Zn was recorded at λα-CD.L.Zn after addition 100 mole equivalent of α-CD.
Therefore, εα-CD.L.Zn = Aα-CD.L.Zn / [L.Zn]free
(7)
(Assuming all the receptor molecules is bound to α-CD and α-CD do not have any absorption
at this wavelength)
Thus, once we one can evaluate [α-CD.L.Zn], it is possible to calculate concentration of the
uncomplexed receptor molecule, [L.Zn] using equation 6 and 7.
[L.Zn]free = [L.Zn]initial - [α-CD.L.Zn]
(8)
and, [α-CD]free = [α-CD]initial - [α-CD.L.Zn]
(9)
If K is formation constant for the inclusion complex, α-CD.L.Zn for a given concentration of
α-CD
Binding Constant, K = [α-CD.L.Zn] / {[L.Zn]free[α-CD]free}
ESI-18 (10)
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13. SEM images:
(B)
(A)
ESI Figure 14: SEM images of (A) blank yeast cells, (B) yeast cells treated with L.Zn
complex, [L.Zn] = 0.066 mM.
ESI-19