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Supplementary information
Zn(II) and Cd(II) based complexes for probing the enzymatic
hydrolysis of Na4P2O7 by Alkaline phosphatase in physiological
condition
Priyadip Das, Sourish Bhattacharya, Sandhya Mishra and Amitava Das*
Central Salt and Marine Chemicals Research Institute (CSIR),
G.B. Marg, Bhavnagar: 364002, Gujarat, India.
Email address: [email protected]
Telephone number: +91 278 2567760 [672]; Fax number: +91 278 2567562/6970
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Table of Contents:Materials and method
S3
Synthetic scheme
S3
Syntesis and characterisation of (L)
S4
Synthesis and characterisation of (L.Zn)
S4
Synthesis and characterisation of (L.Cd)
S5
UV spectra of L
S5
UV spectra of L.Zn
S6
UV spectra of L.Cd
S6
Luminescence response of L.Zn towards different anions
S7
Luminescence response of L.Cd towards different anions
S7
Benesi Hildebrand plot for the titration of L.Zn with sodium pyrophosphate
S8
Benesi Hildebrand plot for the titration of L.Cd with sodium pyrophosphate
S8
31
S9
P NMR of PPi in absence and presence of L.Zn, L.Cd
A plot of Relative intensity of L.Zn with varying concentration of ALP
S9
A plot of Relative intensity of L.Zn with varying concentration of ALP
S10
Emission spectra of L.Zn in absence and presence of ALP
S10
Emission spectra of L.Cd in absence and presence of ALP
S11
Interference Study for the binding of NaPPi with L.Zn
S12
Interference Study for the binding of NaPPi with L.Cd
S12
Mass spectra of L.Cd complex in presence of NaPPi
S13
Mass spectra of L.Zn complex in presence of NaPPi
S14
Relative Quantum yield values for L.Zn L.Cd
S14
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Materials and method:
The chemical such as 3-nitro-4-choloro coumarin, di picolyl amine, Hg(ClO4)2, Cd(ClO4)2,
Zn(ClO4)2, Ni(ClO4)2, Co(ClO4)2, Pb(ClO4)2, Fe(ClO4)2, Cu(ClO4)2, Cr(ClO4)2, and different
nucleotides (adenosine 5’-monophosphate monohydrate, adenosine 5’-diphosphate sodium
salt, adenosine 5’-triphosphate disodium hydrate, cytidine 5’-triphosphate disodium salt
hydrate) were obtained from Sigma-Aldrich and were used as received without any further
purification. Other salts like, NaF, NaI, NaBr, NaOAc, NaCl, NaH2PO4, Na4P2O7, Na2SO4,
NaNO3 and all the other reagents used were of reagent grade (S. D. Fine chemical, India) and
were used as received. Various analytical and spectroscopic data obtained for L, LZn and
LCd agreed well with the proposed formulation and required purity. HPLC grade water
(Fisher scientific) was used as a solvent. Ethyl acetate and methanol, which were used for
different synthetic procedures, were purified through distillation following standard
procedures, prior to use. Microanalysis (C, H, N) were performed using a Perkin-Elmer 4100
elemental analyzer. FTIR spectra were recorded as KBr pellets using Perkin Elmer Spectra
GX 2000 spectrometer. 1H and
31
P NMR spectra were recorded on Bruker 200 MHz
(Avance-DPX 200)/ 500 MHz (Bruker Avance II 500) FT NMR. Electronic spectra were
recorded with Cary-Varian UV-VIS NIR spectrophotometer. Fluorescence spectra were
recorded using Fluorolog (Horiba Jobin Yvon) or Edinburgh F920 (Edinburgh Instrument)
fluorescence spectrometer.
Synthetic scheame:
SI Figure 1: Methodology adopted for synthesis of L, L.Zn, L.Cd.
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Synthesis of L (4-(bis(pyridin-2-ylmethyl)amino)-3-nitro-2H-chromen-2-one):
Di-(2-picolyl) amine (398.5mg, 0.45 mL 2.5 mmol) was dissolved in 30 mL ethyl acetate.
This solution was taken in a 100 mL two necked round bottom flask and cooled to 0◦C. 3nitro-4-choloro coumarin (564 mg, 2.5 mmol) dissolved in 20 mL of ethyl acetate was added
to this solution in a dropwise manner. The reaction mixture was stirred at 0◦C for 1 hr. The
reaction mixture was further stirred for another 2 hrs to ensure completion. Subsequently a
yellow coloured precipitate was appeared, which was filtered off, this residue was purified by
column chromatography (neutral Al2O3, hexane-ethyl acetate as eluent to get the L as a pure
product (551mg, 71%). 1H NMR (500 MHz, CD3CN, 25 °C, TMS) δ (ppm): 8.60 (d, J = 5Hz,
2H; ArH), 8.15 (d, J = 8 Hz, 1H; ArH), 8.08 (t, J = 7.5Hz, 2H; ArH), 7.739 (t, J = 7.5Hz
1H; ArH), 7.65 (d, J = 8Hz, 2H; ArH), 7.60-7.59 (m, 2H; ArH), 7.489-7.416 (m, 2H; ArH),
4.708 (s, 4H; Ar-CH2), 13C NMR (500 MHz, CD3CN, 25 °C, TMS) δ (ppm): 58.165 (s, Ar CH2), 153.23, 150.632, 149.48, 140.89, 140.83, 139.76, 135.72, 134.182, 130.06, 127.69,
126.71, 125.76, 125.05, 124.84. IR (KBr) νmax/cm-1: 3434, 3167, 2364, 1716, 1602, 1553,
1516, 1460, 1402, 1280, 1049, 988, 767, 627. ESI-MS (m/z): 388.31 ((M+), 100%) 411.38
((M+ + Na+). Elemental analysis: C21H16N4O4: calculated C (64.94), H (4.15) N (14.43);
found C (64.7), H (4.2) N (14.25).
Synthesis of L.Zn
L (102mg, 0.261mmol) was dissolved in 20 mL of methanol. To this, Zn(ClO4)2, xH2O (146
mg, 0.391 mmol) solution in 5 mL HPLC water was added in a drop wise manner. The
resultant solution mixture was allowed to stir for 4 h at room temperature. A white coloured
precipitate appeared, which was filtered off and dried in air to get the desired complex, LZn
in pure form (Yield: 85.5 mg, 51%). 1H NMR (200 MHz, dmso-d6, 25 °C, TMS) δ (ppm):
8.69 (d, J = 4.8Hz, 2H; ArH), 8.18 (t, J = 8.2 Hz, 2H; ArH), 7.92 (d, J = 8.2Hz, 1H; ArH),
7.74 - 7.65 (m, 5H; ArH), 7.36 (t, J = 6.2Hz, 2H; ArH), 4.585 (s, 4H; Ar-CH2).
13
C NMR
(500 MHz, dmso-d6, 25 °C, TMS) δ (ppm): 57.867(s, Ar -CH2), 157.38, 156.01, 153.68,
148.08, 143.38, 140.72, 137.38, 135.79, 130.54, 127.11, 126.92, 126.68, 119.49 IR (KBr)
νmax/cm-1: 3442, 3049, 2685, 2361, 1656, 1549, 1503, 1457, 1340, 1285, 1229, 1093, 909,
772, 621. ESI-MS (m/z): 527.81 (M+ + 2H2O + K+, 50%), 674.76 (M+ + 2ClO4+ + H2O, 15%),
692.81 (M+ + 2ClO4+ + 2H2O, 20%). Elemental analysis: C21H20Cl2N4O14Zn: Calculated C
(36.62), H (2.93), N (8.13); found C (36.8), H (2.76), N (8.08).
4
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Synthesis of L.Cd
L (100 mg, 0.257 mmol) was dissolved in 20 mL of methanol and then a solution of
Cd(ClO4)2.xH2O (161 mg, 0.385 mmol) in 5 mL HPLC water was added in a drop wise
manner into it. The resultant solution was allowed to stir for 4h at room temperature. A white
coloured precipitate was appeared, which was filtered off and dried in air to get the pure
metal complex, Cd.L (Yield: 87.3mg, 46.2%). 1H NMR (200 MHz, dmso-d6, 25°C, TMS) δ
(ppm): 8.65 (d, J = 4.8Hz, 2H; ArH), 8.11 (t, J = 8.2Hz, 2H; ArH), 7.95 (d, J = 8.2Hz, 1H;
ArH), 7.67 - 7.58 (m, 5H; ArH), 7.38 (t, J = 6.0Hz, 2H; ArH), 4.585 (s, 4H; Ar-CH2).
13
C
NMR (500 MHz, dmso-d6, 25 °C, TMS) δ (ppm): 57.903(s, Ar -CH2), 157.35, 155.82,
153.43, 147.38, 147.67, 147.57, 143.85, 135.73, 130.55, 127.10, 126.89, 119.44. IR (KBr)
νmax/cm-1: 3437, 3049, 2984, 2684, 2362, 1655, 1549, 1503, 1457, 1340, 1285, 1229, 1186,
1093, 909, 861, 772, 621. ESI-MS (m/z): 519.34 (M+ + H2O, 41%), 537.35 (M+ + 2H2O,
52%) 601.37 (M+ + 2ClO4+ + 2H2O, 20%). Elemental analysis: C21H20Cl2N4O14Cd:
calculated C (34.28), H (2.74) N (7.62); found C (34.13), H (2.7) N (7.57).
UV- visible spectra of L
0.4
Absorbance
0.3
0.2
0.1
0.0
300
400
500
Wavelength(nm)
SI Figure 1: Absorption spectra of L (2.12 x10-5 M) in aqueous 0.01mM HEPES buffer medium of pH 7.4.
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UV- visible spectra of L.Zn
Absorbance
0.5
0.4
0.3
0.2
0.1
240
320
400
W avelength
SI Figure 2: Absorption spectra of L.Zn (2.0 x10-5 M) in aqueous 0.01mM HEPES buffer of pH 7.4, molar
extinction coefficient 1.1129 x 104 M-1cm-1 at wavelength 326nm
UV- visible spectra of LCd
Absorbance
0.4
0.3
0.2
0.1
0.0
250
300
350
400
wavelength(nm)
SI Figure 3: Absorption spectra of L.Cd (2.0 x10-5 M) in aqueous 0.01mM HEPES buffer of pH 7.4, molar
extinction coefficient 1.278 x 104 M-1cm-1 at wavelength 326nm.
6
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Luminescence response Of LZn towards different Anions.
6
Intensity(CPS)
2.0x10
6
1.5x10
6
1.0x10
5
5.0x10
0.0
1
2
3
4
5
6
7
8
9 10 11 12 13
different anions
SI Figure 4: Luminescence response of L.Zn (2.0 x10-5 M) in aqueous 0.01mmol HEPES buffer (pH7.4) medium on addition of the solution of sodium salt of various anions and nucleotides: 1. L.Zn, 2.
NO3−, 3. ATP, 4. H2PO4−, 5. I−, 6. AMP, 7. SO4− , 8. PPi, 9. Cl−, 10. Br−, 11. CH3CO2−, 12. ADP, 13.
CTP. (2.0 x 10-4 M) in 0.01mmol HEPES buffer (pH-7.4) with λmon = 428 nm and λext = 328 nm.
Luminescence response Of LCd towards different Anions.
5
Intensity(CPS)
3.0x10
5
2.5x10
5
2.0x10
5
1.5x10
1
2
3
4
5
6
7
8
9
10 11 12 13
different anions
SI Figure 5: Luminescence response of L.Cd (2.0 x10-5 M) in aqueous 0.01mmol HEPES buffer
(pH-7.4) medium on addition of the solution of sodium salt of various anions and nucleotides: 1.
L.Cd, 2. NO3−, 3. ATP, 4. H2PO4−, 5. I−, 6. AMP, 7. SO4− , 8. PPi, 9. Cl−, 10. Br−, 11. CH3CO2−, 12.
ADP, 13. CTP. (2.0 x10-4 ) with λmon=414 nm, λext = 328 nm.
7
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Benesi-Hildebrand plot for binding studies of NaPPI towards LZn:
-6
R=0.99865
2.0x10
1/(F0-Fx)
-6
1.6x10
-6
1.2x10
-7
8.0x10
-7
4.0x10
0.00
5
5
5
1.50x10 3.00x10 4.50x10
1/[NaPPi]
SI Figure 5: Benesi-Hildebrand plot of for evaluation of binding constant and stoichiometry for the
formation of L.Zn-PPi complex in aqueous 0.01mmol HEPES buffer (pH-7.4) medium. λext = 328 nm
and λmon = 428 nm were used for emission studies. Goodness of the fit of the plot confirms the 1:1
binding stochiometry.
Benesi-Hildebrand plot of L.Cd with NaPPi
-5
2.8x10
R=0.99822
1/(Fx-F0)
-5
2.1x10
-5
1.4x10
-6
7.0x10
4
2.0x10
4
4.0x10
4
6.0x10
1/[NaPPi]
SI Figure 6: Benesi-Hildebrand plot of for evaluation of binding constant and stoichiometry for the
formation of L.Cd-PPi complex in aqueous 0.01mmol HEPES buffer (pH-7.4) medium. λext = 328
nm and λmon = 414 nm were used for emission studies. Goodness of the fit of the plot confirms the 1:1
binding stochiometry.
8
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31
P NMR of PPi in absence and presence of L.Zn, L .Cd
O-
O
O
P
O
-
O
P
O
O-
-5
-6
δ(ppm)
-7
-8
SI Figure 7: 31P NMR spectra of L.Zn and L.Cd (15 mM) before and after addition of Na4P2O7 (150
mM) in D2O.
5
M
n
75
M
4
nM
50
3
nM
25
10
5
100
80
60
2
nM
1
nM
nM
0
Relative
0n
10
intensity
A Ribbon diagram plot of Relative change in emission intensity of L.Zn with varying [ALP].
B
[AL 40 20
P]
0
SI Figure 8: A ribbon diagram plot of relative change in emission intensity of L.Zn with varying
[ALP] in aqueous 0.01mmol HEPES buffer (pH-7.4) medium. For each measurement of emission
intensity, a time interval of 900 sec was allowed. λext = 328 nm and λmon = 428 nm were used for
emission studies.
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1.0
0
0.9
nM
5
nM
nM
25
nM
50
nM
75
nM
0.7
0.6
0.5
10
0.8
100
i n te
Relative
ns i t y
A Ribbon diagram plot of Relative intensity of L.cd with varying concentration of ALP
nM
B
0
40 20
60
100 80
[ALP]
SI Figure 9: A ribbon diagram plot of relative change in emission intensity of L.Cd with varying
[ALP] in aqueous 0.01mmol HEPES buffer (pH-7.4) medium. For each measurement of emission
intensity, a time interval of 900 sec was allowed. λext = 328 nm and λmon = 414 nm were used for
emission studies.
Emission spectra of L.Zn in absence and presence of Alkaline phosphatase
6
Intensity(CPS)
2.1x10
L.Zn
L.Zn + ALP
6
1.4x10
5
7.0x10
400
500
600
wavelength (nm)
SI Figure 10: Emission spectra of L.Zn (2.0 X10-5) in absence and presence of 100 nM ALP in
aqueous 0.01mmol HEPES buffer (pH-7.4) medium.
10
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Intensity (CPS)
Emission spectra of LCd in absence and presence of Alkaline phosphatase
4
9.0x10
L.Cd
L.Cd + ALP
4
6.0x10
4
3.0x10
400
450
500
550
600
Wavelength(nm)
SI Figure 11: Emission spectra of L.Cd (2.0 X10-5) in absence and presence of 100 nM ALP in
aqueous 0.01mmol HEPES buffer (pH-7.4) medium.
ESI figure 10, 11, reveals that there is no interaction of Alkaline phosphatase (ALP) with
L.Zn and L.Cd in absence of Na4P2O7 (NaPPi) in aqueous 0.01mmol HEPES buffer (pH-7.4)
n+
2+
2+
medium. We had made two different types assay. First one has (i) L.M (M = Zn , Cd ) 2.0
x10-5M; while the second one has (ii) L.Mn+ (M = Zn2+, Cd2+) 2.0 x10-5M + ALP (100 nM) in
0.01M aqueous HEPES buffer (pH-7.4) medium. The emission spectra were recorded for the
first aliquot then the emission intensity of the second aliquot was measured after 900 sec of
mixing ALP. No change in the emission spectral pattern was observed after addition of ALP
to both the metal complexes (L.Zn and L.Cd) in absence of Na4P2O7 (NaPPi), which
confirms that there is no interaction between L.Zn or L.Cd and ALP. Again the significant
change in the emission intensity of L.Zn and L.Cd in presence both Na4P2O7 and ALP
implied that enzymatic hydrolysis of PPi by ALP reduces the effective concentration of PPi
in the medium and thus effective concentration of L.Zn-PPi or L.Cd-PPi in the medium and
resulted overall changes in the emission intensity.
11
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6
2.0x10
L.Zn
Interference Study for the binding of NaPPi with L.Zn in aqueous 0.01mmol HEPES
buffer (pH-7.4) medium:
6
L.Zn + PPi
L.Zn + PPi +AMP
L.Zn + PPi +ADP
L.Zn + PPi +CTP
L.Zn + PPi + ATP
L.Zn + PPi + Pi
Intensity(CPS)
1.5x10
2
3
4
5
6
7
6
1.0x10
5
5.0x10
0.0
1
Wavelength(nm)
SI Figure 12: Emission intensity at 428 nm of L.Zn (2.0 x10-5 M) with Na4P2O7 (1.0 x 10-4 M) in the
absence and presence of 5.0 x 10-3 M of different coexisting phosphate anions and nucleotides in in
aqueous 0.01mmol HEPES buffer (pH-7.4) medium (λext = 328 nm).
.
Interference Study for the binding of NaPPi with L.Cd
5
L.Cd + PPi + Pi
L.Cd + PPi + ADP
5
1.0x10
L.Cd + PPi + AMP
5
1.5x10
L.Cd + PPi + CTP
L.Cd + PPi
5
2.0x10
L.Cd + PPi + ATP
5
2.5x10
3
4
5
6
7
L.Cd
Intensity(CPS)
3.0x10
1
2
Wavelength(nm)
SI Figure 13: Emission intensity at 414 nm of L.Cd (2.0 x10-5 M) with Na4P2O7 (1.0 x 10-4 M) in the
absence and presence of 5.0 x 10-3 M of different coexisting phosphate anions and nucleotides in in
aqueous 0.01mmol HEPES buffer (pH-7.4) medium (λext = 328 nm).
12
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Mass spectra of L.Cd complex in presence of NaPPi
CD PPI 14 (0.140)
1: TOF MS ES+
12.9
L.Cd + Na+ + PPi
694.51
100
L.Cd + Na+ +
ClO4 + PPi
797.59
638.39
%
629.41
L.Cd + Na+ +
2ClO4 + PPi
695.52
798.61
823.83
824.82
639.39
897.97
691.50
691.39
879.91
697.52
642.41
712.59
8
722.50
607.40
610
620
630
640
650
660
670
680
690
700
710
720
730
768.57
750.53
754.57 769.51 782.63
883.89
799.61
815.82
740.54
740
750
760
770
780
790
800
810
820
825.73
830
884.94
840
850
860
870
880
890
m/z
900
SI Figure 14: ESI-MS spectra of L.Cd in presence of 10 mole equivalent of NaPPi in aqueous medium.
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Mass spectra of L.Zn complex in presence of NaPPi
ZN PPI 1 (0.010)
1: TOF MS ES+
6.56
560.21
100
L.Zn +2ClO4 + PPi
+ 2K+
903.79
%
904.78
561.22
904.84
520.11
905.70
531.17
532.16
398.27
0
455.64
518.12
486.04
557.17
584.22
607.20
638.39
669.94
694.49 709.60
798.56 816.78
727.52 766.49
824.76 847.48
903.38 906.84
977.80
939.50960.92 993.03
380 400 420 440 460 480 500 520 540 560 580 600 620 640 660 680 700 720 740 760 780 800 820 840 860 880 900 920 940 960 980
m/z
SI Figure 15: ESI-MS spectra of L.Zn in presence of 10 mole equivalent of NaPPi in aqueous medium.
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L.Zn + PPi +
H2O + 2Na+
L.Zn + PPi
ZN PPI 8 (0.080)
100
1: TOF MS ES+
1
694.45
629.39
694.40
638.27
L.Zn + PPi +
ClO4 + Na+
638.36
754.54
630.40
639.37
%
687.46
740.52
755.53
713.40
686.19
678.16
699.32
620
740.42
729.77
749.17
745.81
767.48
785.52
708.64
642.38 650.47
630
757.34
752.66
732.56
631.35
616.69
610
723.55
724.34
663.18
621.15
0
600
711.95
670.55
650.34
603.34
717.47
685.43
677.73
621.22
754.32
640
650
759.36
739.68
660
670
680
690
700
710
720
730
740
750
760
771.45 778.79
770
780
788.63
m/z
790
SI Figure 16: ESI-MS spectra of L.Zn in presence of 10 mole equivalent of NaPPi in aqueous medium.
Relative Quantum yield (Φ) values for L.Zn L.Cd with respect to quinine sulphate as
standard.
[Φ]L.Zn =0.01559 (with respect to quinine sulphate ([Φ]Std = 0.54 in 0.1(M) H2SO4))
[Φ]L.Cd =0.003274 (with respect to quinine sulphate ([Φ]Std = 0.54 in 0.1(M) H2SO4)).
15