Fluorometric sensing of Cu2+ ion with smart fluorescence light-up probe, triazolylpyrene (TNDMBPy)

Article abstract of DOI:10.1016/j.tetlet.2012.08.074

We report the detection of Cu²⁺ ion with fluorescence light-up probe, triazolylpyrene (^TNDMBPy). Thus, the probe showed a remarkable fluorescence enhancement of both of the monomer and excimer emissions and a 2:1 probe-Cu²⁺ complexation with high selectivity, high stability constant and low detection limit. Formation of the excimer was also rationalized on the basis of DFT calculation. Our probe is also capable of sensing SDS micelle-encapsulated-Cu²⁺ ion in aqueous media with high association constant, thus, showing its practical utility. Thus, our probe could be used as an efficient Cu²⁺ ion selective fluorescence light-up probe, and may find applications in chemical and biological systems.

Full text of DOI:10.1016/j.tetlet.2012.08.074

Fluorometric sensing of Cu2+ ion with smart fluorescence light-
up probe, triazolylpyrene (TNDMBPy)
Subhendu Sekhar Bag,* Rajen Kundu and Sangita Talukdar
Bioorganic Chemistry Laboratory, Department of Chemistry, Indian Institute of
Technology Guwahati, Guwahati-781039, Assam, India. Fax: +91-361-258-2349; Tel:
+91-361-258-2324; E-mail: ssbag75@iitg.ernet.in
Contents
Page No.
S2-S4
S2
1. Chemical synthesis
1.1.
1.2.
General Procedure for the Preparation of Aryl Azides
General Procedure for the [3+2] Cycloaddition of Azides and Terminal
S3-S4
Alkynes and the spectral data
2. Photophysical study
S4-S7
S5
S5
S6
2.1.
2.2.
2.3.
Emission spectra of 1 (TNDMBPy) in different solvents.
Emission spectra of 1 (TNDMBPy) titrated with Cu2+ in CH3CN.
Fluorescence intensity change of 1 (TNDMBPy) in presence of various
metal ions
2.4.
Plot of intensity ratio of dual band titrated with Cu2+
S7
S7-S8
S8
3. Job’s plot of 1 (TNDMBPy) with Cu2+ ion and the ESI-MS of 1.Cu2+ complex
4. Benesi-Hildebrand Plot
5. Photophysical summary table of 1 (TNDMBPy) titrated with Cu2+ ion
6. Detection limit determination of 1 (TNDMBPy) towards Cu2+ ion
7. Cu2+ Selectivity in presence of various background interfering metal cations
8. Fluorescence Studies in SDS Micelle Media
S9
S9
S10
S10-S11
S11-S16
S17
9. UV-Vis and emission spectra of 1 (TNDMBPy) in presence of different metal ions
10. Comparison among the three fluorophores toward their efficiencies as selective
fluorophores
10.1. UV-Vis and emission spectra of fluorophores 2-3 in presence of different
S18-S21
metal ions
10.2. Test for Selectivity in Sensing of Cu2+ Ion
11. Gaussian optimized geometry of 1.Cu2+ complex
S22-S23
S24-S26
S1

1. Chemical synthesis
The fluorescent probes 1-3 were synthesized via our previously published method.
Briefly, 1-aminopyrene (4) was converted to its corresponding 1-azidepyrene (5) via
reaction with NaNO2/aq. H2SO4 to diazonium salt followed by reaction with NaN3 at 0
oC. Then, 5 react with terminal alkynes under click condition in presence of sodium
ascorbate and CuSO4 in a mixture of CH2Cl2-EtOH solvent at room temperature to form
the product 1-3.
N
N
N
N
N
N
Sodium ascorbate,
CuSO4,
CH2Cl2/EtOH, RT
NH2
N
1 (TNDMBPy)
(1) aq. NaNO2 / aq. H2SO4
(2) aq. NaN3, 0 to 5 o
Hexane
C
N
N
CN
R
N
4
5
2 (TCNPPy)
R = -NMe2 or -CN or -H
N
N
N
3 (TPPy)
Scheme S1: Synthesis of fluorescent probe 1 (TNDMBPy).
1.1.
General Procedure for the Preparation of Aryl Azides
To an ice cold solution of sodium nitrite (3 eqv.) in water was added dropwise to a
cold solution of aryl amine 1 (1 eqv.) in water and concentrated hydrochloric acid at 0oC
over 7 – 10 min. The reaction mixture was slowly stirred for 1 - 2 min before an ice cold
o
solution of sodium azide (6 eqv.) in water was added dropwise at 0 C over 10 min. The
o
mixture was stirred for 15 min at 0 C. The resulting mixture was extracted with ether.
The organic layer was washed with water, followed by a brine solution, dried over
anhydrous Na2SO4 and then concentrated to yield pure aryl azides. Formation of the
product azide was confirmed from IR study (1-azidopyrene: Yield: 75%; IR: 2133 cm-1) and
the produced azide was then immediately used for the next step without further purification.
S2

1.2.
General Procedure for the [3+2] Cycloaddition of Azides and Terminal
Alkynes and the spectral data
In a round bottom flask fitted with a septum, 1-azidopyrene (1.0 eqv.) was dissolved
by dry 1:1 dichloromethane and ethanol and was degassed for 5 min with N2. Then,
alkyne (1.5 eqv.) was added and both the stirring and degassing were continued for next 5
min. After that a 6 mol % sodium ascorbate dissolved in water was added. The reaction
mixture was stirred for 5 min and then 1 mol % powdered CuSO4 dissolved in water was
added. Degassing was continued for next 10 min. The reaction was allowed to proceed to
room temperature and monitored by TLC. After total consumption of the starting azide,
the reaction mixture was evaporated completely to a solid material and partitioned
between water and ethyl acetate. The organic layer was washed with water followed by
brine solution, dried over Na2SO4 and then concentrated. The title triazolyl-compounds
were separated by column chromatography and characterized.
Synthesis of N, N-dimethyl-4-(1-(pyren-1-yl)-1H-1,2,3-triazol-4-yl)benzenamine
(1): Using the general procedure, starting from 0.025 g of 1-azidopyrene (5) and 0.022 g
of 4-N,N-dimethylaminophenylacetylene, 0.024 g of the title compound 1 was isolated as
1
a light pink solid (68% yield). IR (KBr): 1617, 1567, 1503, 1460, 1351, 819 cm-1; H
NMR (CDCl3; 400 MHz) δ 3.03 (6H, s), 6.84 (2H, d, J = 8.0 Hz), 7.87 (2H, d, J = 8.4
13
Hz), 7.97 (1H, d, J = 9.2 Hz), 8.06 – 8.20 (6H, m), 8.24 – 8.29 (3H, m); C NMR
(CDCl3; 100 MHz) δ 40.6, 112.7, 118.6, 121.4, 123.5, 124.3, 124.9, 125.2, 126.1, 126.5,
126.9, 127.1, 128.9, 129.7, 130.8, 131.3, 132.3, 148.6, 150.8; HRMS calcd. for C26H21N4
([M+H]+) 389.1766, found 389.1768.
Synthesis of 4-(1-(pyren-1-yl)-1H-1,2,3-triazol-4-yl)benzonitrile (2)
: Using the
general procedure, starting from 0.09g of 1-azidopyrene (5) and 0.071 g of 4-
ethynylbenzonitrile, 0.068 g of the title compound 2 was isolated as a red solid (50%
1
yield). IR (KBr): 2222, 1613, 1489, 1459, 848 cm-1; H NMR (CDCl3; 400MHz) δ 7.77
(2H, d, J = 8.0 Hz), 7.88 (1H, d, J = 8.8 Hz), 8.08 – 8.20 (6H, m), 8.23 – 8.32 (4H, m),
13
8.36 (1H, s); C NMR (CDCl3; 100 MHz) δ 112.0, 118.9, 121.0, 123.5, 124.0, 124.3,
S3

124.9, 125.3, 126.5, 126.9,127.1, 129.4, 130.2, 130.8, 131.3, 132.7, 133.1, 134.9,146.3;
ESI-TOF-MS m/z 371[M+H]+; HRMS calcd. for C25H15N4 ([M+H]+) 371.1297, found
371.1302.
Synthesis of 1-(Pyren-1-yl)-1H-1,2,3-traizol-4-yl-benzene (3): Using the general
procedure, starting from 0.1 g of 1-azidopyrene (5) and 0.063 g of 1-phenylacetylene,
0.085 g of the title compound 3 was isolated as a white solid (60% yield). IR (KBr):
1510, 1461, 1037, 836.8 cm-1; 1H NMR (CDCl3, 400MHz) δ 7.40 (1H, t, J = 7.6
Hz),7.50 (2H, t, J = 7.6 Hz),7.93 (1H, d, J = 8.8 Hz), 8.00 (1H, d, J = 7.2 Hz), 8.06 – 8.24
13
(7H, m), 8.27 (1H, s), 8.29 (1H, s); C NMR (CDCl3, 100 MHz) δ 121.3, 122.9, 123.5,
124.3, 124.9, 125.2, 126.1, 126.3, 126.4, 126.6, 126.9, 127., 128.6, 129.2, 129.9, 130.6,
130.9, 131.3, 132.5, 148.1; ESI-TOF-MS m/z 346 [M+H]+.
2. Photophysical study
All the UV –visible spectra of the compounds (10 µM) in presence of various metal
ions with different concentration were measured using a UV-Visible spectrophotometer
with a cell of 1 cm path length.
All the sample solutions were prepared as described in UV measurement experiments.
Fluorescence spectra were obtained using a fluorescence spectrophotometer at 25 °C
using 1 cm path length cell. The fluorescence quantum yields (ꢀf) were determined using
quinine sulphate as a reference with the known ꢀf (0.54) in 0.1 molar solution in
sulphuric acid.
S4

2.1. Emission spectra of 1 (TNDMBPy) in different solvents.
600000
Diox
10% wt in Diox
CH3CN
10% wt in CH3CN
50% wt in CH3CN
400000
pH 7.0 (Buffer)
200000
0
350
450
550
650
750
Wavelength (nm)
Figure S1: Emission spectra of 1 (TNDMBPy) in different solvents.
2.2. Emission spectra of 1 (TNDMBPy) titrated with Cu2+ ion
400000
400000
300000
300000
200000
200000
100000
100000
0
0
350
450
550
650
350
450
550
650
Wavelength (nm)
Wavelength (nm)
Figure S2. Emission spectra of 1 (TNDMBPy) titrated with Cu2+ ion and the colour change
indicating the complexation via N-coordination in day light and the emission image under
transilluminator (254 nm).
S5

2.3. Fluorescence intensity change of 1 (TNDMBPy) in presence of various metal ions
400000
300000
200000
100000
0
Free probe
Na(I)
K(I)
Mg(II)
Ca(II)
Co(II)
Ni(II)
Cu(I)
Cu(II)
Zn(II)
Ag(I)
350
450
550
650
Wavelength (nm)
1800
1400
1000
600
200
-200
Na(I) K(I) Mg(II) Ca(II) Co(II) Ni(II) Cu(I) Cu(II) Zn(II) Ag(I)
Metal ions
Figure S3: Fluorescence intensity changes ((I – I0)/I0 x 100%) of 1 (TNDMBPy) (10 µM) at
298 K upon addition of various metal ions (2.5 equiv). Excitation wavelength was at 343
nm. I0 is fluorescent emission intensity at 383 nm of free host and I is the fluorescence
intensity after adding metal ions.
S6

2.4. Plot of intensity ratio of dual band titrated with Cu2+
ꢃꢀ
ꢂꢆ
ꢂꢀ
ꢆ
ꢀꢁꢅ
ꢀꢁꢄ
ꢀꢁꢃ
ꢀꢁꢂ
ꢀ
(a)
(b)
ꢀ
Figure S4: Plot of IExcimer to IMonomer (a) and IMonomer to IExcimer (b) ratio of 1 (TNDMBPy)
titrated with Cu2+ in CH3CN
3. Job’s plot of 1 (TNDMBPy) with Cu2+ ion and the ESI-MS of 1.Cu2+ complex
Job’s plot showed the 2:1 ligand-to-metal complexation which is also supported by an
ESI-MS mass peak at m/z [(TNDMBPy)2Cu-2H]+ 841.65.
Job's Plot
0.8
0.6
0.4
0.2
0.0
300000
250000
200000
150000
100000
(a)
(b)
0.2
0.4
0.6
0.8
1.0
0.0
0.4
0.8
1.2
[H]/([H] + [Cu2+])
[H]/([H]+[Cu2+])
Figure S5: (a) Absorption and (b) fluorescence Job’s plot of 1 (TNDMBPy) in presence of
Cu2+ ion.
S7

Figure S6: The ESI-MS of 2:1 ligand-to-metal complex of [(TNDMBPy)2Cu-2H]+.
4. Benesi-Hildebrand Plot
The association constant of Cu2+ with 1 (TNDMBPy) was determined by Benesi-
Hildebrand plot using the following equation, which come as 2.23 x 105 M-1.
1
I − I
1
1
=
+
I − I
(I∞ − I0 )K[Cu2+ ]
(
)
(
)
0
∞
0
7
8
6
4
Equation, Y = A + B*X
R2 = 0.97375
6
5
4
3
2
0.0
0.1
0.2
0.0
0.2
0.4
1/[Cu2+]
1/[Cu2+]
Figure S7: Benesi-Hildebrand plot of the monomer band of 1 (TNDMBPy) with Cu2+, (a)
full region, (b) linear region.
S8

5. Photophysical summary table of 1 (TNDMBPy) titrated with Cu2+ ion
Table S1: Absorption, emission and quantum yield summary of 1 (TNDMBPy) titrated
with Cu2+ in CH3CN
Cu2+ (eqv.)
Abs
abs
max
fl
fl
exci
ꢀf
0.003
λ
λ
/
λ
mono
0.00
0.25
0.50
0.75
1.00
1.50
2.00
2.50
276/343 0.287/0.191
276/343 0.286/0.194
276/343 0.286/0.197
276/343 0.279/0.193
276/343 0.276/0.195
276/343 0.267/0.197
276/343 0.267/0.202
276/343 0.281/0.208
382
382
382
382
382/466
382/466
382/466
382/466
0.020
0.026
0.026
0.035
0.038
0.049
0.073
6. Detection limit determination of 1 (TNDMBPy) towards Cu2+ ion
We have calculated the detection limit of the fluorescent probe 1 towards Cu2+ ion by
plotting (Imin - I)/(Imin - Imax) vs. log[Cu2+]. The linear fit was drawn by taking the five
points (2.5 µM to 15 µM) of the linear region. The extended line where it crossed the
ordinate axis is the detection limit which comes in the region of 177 nM.
R2 = 0.99387
1.0
0.8
0.6
0.4
0.2
0.0
-7.0 -6.5 -6.0 -5.5 -5.0 -4.5 -4.0
log[Cu2+]
Figure S8: Plot of (Imin - I)/(Imin - Imax) vs. log[Cu2+] of 1 for monomer band.
S9

7. Cu2+ Selectivity in presence of various background interfering metal cations
0.3
6
0.2
4
0.1
2
0
0
Mixed metal ions
Mixed metal ions
Figure S9: Ratio of fluorescence intensity at 466 and 384 nm of 1 (10µM) with Cu2+ (2.0
equiv.) in the absence and presence of different interfering metal ions (50 equiv.).
8. Fluorescence Studies in SDS Micelle Media
Surfactant (SDS) solution was prepared in MQ water. The concentration of SDS was
10 mM, above micellar concentration to make sure the formation of micelle in the
experimental conditions. pH of the solutions was adjusted by dilute HCl and or dilute
NaOH. The probe’s solutions in SDS surfactant (10 mM) were mixed well and then Cu2+
ion (solution of [Cu(ClO4)2.6H2O] in MQ water) of different concentrations were added
maintaining the final volume 3 mL of the mixtures (10 µM probe in 10 mM SDS solution
and 0, 10, 25, 50, 75, 100, 125 etc.µM Cu2+ ions respectively). Then again the whole
solutions were mixed well before taking the fluorescence spectra. All the experiments
were carried out at 25 °C.
S10

(a)
(b)
250000
200000
150000
100000
50000
0
240000
180000
120000
60000
0
1.25
1.20
1.15
1.10
1.05
2.8
2.4
2.0
1.6
1.2
0.8
+
KSV = 1.1 x 104
KSV = 3.8 x 102
2
0
1
2
3
4
5
[Cu2+] x 10-4M
0 to 450 µM
0
50 100 150 200
[Cu2+] (
µM)
of Cu2+
350
450
550
650
750
350
425
500
575
Wavelength (nm)
Wavelength (nm)
(c)
500000
0 to 275 µM of Cu2+
Binding constant,
K = 3.65 X 103
45
400000
300000
200000
100000
0
30
15
0
0.000
0.015
0.030
0.045
1/[Cu2+] x 106 M-1
360
435
510
585
Wavelength (nm)
Figure 10: Emission behavior of triazolyl pyrene (10 µM) in presence of increasing
concentration of Cu2+ ion in SDS micelle at (a) pH 2.0, (b) pH 7.0, and at (b) pH 8.0.
S11

9. UV-Vis and emission spectra of 1 (TNDMBPy) in presence of different metal ions
0.4
400000
300000
200000
100000
0
Free probe
Na(I)
K(I)
Mg(II)
Ca(II)
Co(II)
Ni(II)
Free probe
Na(I)
K(I)
Mg(II)
Ca(II)
Co(II)
Ni(II)
0.3
0.2
0.1
0
Cu(I)
Cu(I)
Cu(II)
Zn(II)
Ag(I)
Cu(II)
Zn(II)
Ag(I)
250
300
350
400
450
350
450
550
650
Wavelength (nm)
Wavelength (nm)
Figure S11: (a) UV-Vis, (b) Emission spectra of 1 (TNDMBPy) (10 µM) in presence of
various metal ions (2.5 equiv.).
500000
400000
300000
200000
100000
0
500000
400000
300000
200000
100000
0
2.5 Eq Cu
3 Eq Cu
3.5 Eq Cu
4 Eq Cu
5 Eq Cu
7 Eq Cu
10 Eq Cu
12 Eq Cu
15 Eq Cu
20 Eq Cu
Free probe
0.25 Eq Cu
0.5 Eq Cu
0.75 Eq Cu
1 Eq Cu
1.5 Eq Cu
2 Eq Cu
2.5 Eq Cu
250
300
350
400
450
250
300
350
400
450
Wavelength (nm)
Wavelength (nm)
Figure S12: (a) UV-Vis, (b-c) excitation spectra of 1 (10µM) in presence of Cu2+ ion.
S12

400000
300000
200000
100000
0
400000
300000
200000
100000
0
Free
2.5 Eq Cu(II)
3 Eq Cu(II)
3.5 Eq Cu(II)
4 Eq Cu(II)
5 Eq Cu(II)
7 Eq Cu(II)
10 Eq Cu(II)
12 Eq Cu(II)
15 Eq Cu(II)
20 Eq Cu(II)
0.25 Eq Cu(II)
0.5 Eq Cu(II)
0.75 Eq Cu(II)
1 Eq Cu(II)
1.5 Eq Cu(II)
2 Eq Cu(II)
2.5 Eq Cu(II)
350
450
550
650
350
450
550
650
Wavelength (nm)
Wavelength (nm)
Figure S13: Emission spectra of 1 (10µM) in presence of Cu2+ ion in CH3CN.
(a)
(b)
Figure S14: (a) UV-Vis, (b) emission spectra of 1 (10µM) in presence of Na+ ion.
S13

Figure S15: (a) UV-Vis, (b) emission spectra of 1 (10µM) in presence of K+ ion.
Figure S16: (a) UV-Vis, (b) emission spectra of 1 (10µM) in presence of Mg2+ ion.
S14

Figure S17: (a) UV-Vis, (b) emission spectra of 1 (10µM) in presence of Ca2+ ion.
Figure S18: (a) UV-Vis, (b) emission spectra of 1 (10µM) in presence of Co2+ ion.
(a)
(b)
Figure S19: (a) UV-Vis, (b) emission spectra of 1 (10µM) in presence of Ni2+ ion.
S15

Figure S20: (a) UV-Vis, (b) emission spectra of 1 (10µM) in presence of Cu+ ion.
(a)
(b)
Figure S21: (a) UV-Vis, (b) emission spectra of 1 (10µM) in presence of Zn2+ ion.
Figure S22: (a) UV-Vis, (b) emission spectra of 1 (10µM) in presence of Ag+ ion.
S16

10. Comparison among the three fluorophores toward their efficiencies as selective
fluorophores
The role of the donor dimethylamino phenyl substituent in selective sensing of
Cu2+ was established by synthesizing other substituted phenyls, such as, fluorophore 2
(
TCNPPy) containing an electron withdrawing substituents (-CN) and 3 (TPPy) with no
substitution at phenyl ring and studied their specificity/selectivity in sensing the same
metal ion. These two fluorophores show only a single emission band at around 380
nm which are characteristics of pyrene vibronic structures of the monomer emission
both in presence or in absence of any metal ions in acetonitrile.
We have examined the optical sensor property of our probes 2-3 for metal ions,
the tested metal Na+, Mg2+, Ni2+, and Cu2+ ions were added to the solution of 2-3,
respectively, and photophysical properties were studied. Thus, from the UV-visible
absorption it was observed that the including Cu2+ ion no metal ions induced any
change in absorption spectra, which implied that no metal ion interact significantly in
a well-defined manner with both the probes 2-3 under the experimental condition.
The addition of various concentrations of metal ions including Cu2+ion, only a
single monomer emission with no significant change in fluorescence emission were
observed (Figure S23 to Figure S30). However, in presence of large excess of Cu2+
ion (>50 µM), quenching of the monomer emission was observed. Therefore, from
the metal titration experiments in presence and/or absence of Cu2+ ions and other
metal ions, it was clear that both of the latter synthesized fluorophores (2-3) did not
induce any selective change in absorbance or fluorescence intensity of the probes.
Also in a competitive scenario, in presence of other background metal ions, addition
of Cu2+ ions to any of the probe’s solution does not bring any change in fluorescence
intensity of the probes. This observation suggests that probes 2-3 are inefficient to
interact significantly with any metal ions tested. These observations are totally
opposite of that observed in case of probe 1 that showed a clear selectivity in sensing
of Cu2+ ion. The results can easily be explained if we consider the probable role of
donating and/or acceptor substituent. Thus, in case of probe 1 the donor N,N-
dimethylaminophenyl group is donating the electron which is accumulated within the
nitrogens of triazole ring yielding to better coordinating nitrogens for Cu2+ ions.
However, in case of probe 2, the acceptor unit (-CN) is withdrawing the electron
density from the triazole’s nitrogens, therefore, the less electron dense triazole’s
nitrogens are unable to coordinate with metal ions. In case of probe 3, the
intermediate situation, also we observed no interaction with metal ions. Therefore, it
is clear that the presence of donor N,N-dimethylaminophenyl unit is mainly
responsible for Cu2+ ion coordination and detection with high selectivity and large
fluorescence enhancement.
S17

10.1. UV-Vis and emission spectra of fluorophores 2-3 in presence of different
metal ions
3.E+07
2.E+07
2.E+07
1.E+07
5.E+06
0.E+00
0.8
0.6
0.4
0.2
0
Probe
Probe
0.5 eq Cu(II)
1.0 eq Cu(II)
1.5 eq Cu(II)
2.0 eq Cu(II)
2.5 eq Cu(II)
0.5 Eq Cu(II)
1.0 Eq Cu(II)
1.5 Eq Cu(II)
2.0 Eq Cu(II)
2.5 Eq Cu(II)
250
300
350
400
350
450
550
650
Wavelength(nm)
Wavelength(nm)
Figure S23: (a) UV-visible and (b) fluorescence spectra of 4-(1-(Pyren-1-yl)-1H-1,2,3-
traizol-4-yl)benzonitrile (2) in acetonitrile in presence of Cu2+ ion. [10 ꢁM, r.t.; ꢂex = 342
nm].
0.6
0.4
0.2
0
3.E+07
2.E+07
2.E+07
1.E+07
5.E+06
0.E+00
Probe
Probe
0.5 eq Mg(II)
1.0 eq Mg(II)
1.5 eq Mg(II)
2.0 eq Mg(II)
2.5 eq Mg(II)
0.5 Eq Mg(II)
1.0 Eq Mg(II)
1.5 Eq Mg(II)
2.0 Eq Mg(II)
2.5 Eq Mg(II)
250
300
350
400
450
350
450
550
650
Wavelength(nm)
Wavelength (nm)
Figure S24: (a) UV-visible and (b) fluorescence spectra of 4-(1-(Pyren-1-yl)-1H-1,2,3-
traizol-4-yl)benzonitrile (2) in acetonitrile in presence of Mg2+ ion [10 ꢁM, r.t.; ꢂex = 342
nm].
S18

0.6
0.4
0.2
0
3.E+07
2.E+07
2.E+07
1.E+07
5.E+06
0.E+00
Probe
Probe
0.5 Eq Ni(II)
1.0 Eq Ni(II)
1.5 Eq Ni(II)
2.0 Eq Ni(II)
2.5 Eq Ni(II)
0.5 Eq Ni(II)
1.0 Eq Ni(II)
1.5 Eq Ni(II)
2.0 Eq Ni(II)
2.5 Eq Ni(II)
350
450
550
650
250
300
350
400
450
Wavelength (nm)
Wavelength(nm)
Figure S25: (a) UV-visible and (b) fluorescence spectra of 4-(1-(Pyren-1-yl)-1H-1,2,3-
traizol-4-yl)benzonitrile (2) in acetonitrile in presence of Ni2+ ion [10 ꢁM, r.t.; ꢂex = 342
nm].
3.E+07
2.E+07
2.E+07
1.E+07
5.E+06
0.E+00
0.8
0.6
0.4
0.2
0
Probe
Probe
0.5 Eq Na(I)
1.0 Eq Na(I)
1.5 Eq Na(I)
2.0 Eq Na(I)
2.5 Eq Na(I)
0.5 Eq Na(I)
1.0 Eq Na(I)
1.5 Eq Na(I)
2.0 Eq Na(I)
2.5 Eq Na(I)
250
300
350
400
350
450
550
650
Wavelength(nm)
Wavelength (nm)
Figure S26: (a) UV-visible and (b) fluorescence spectra of 4-(1-(Pyren-1-yl)-1H-1,2,3-
traizol-4-yl)benzonitrile (2) in acetonitrile in presence of Na+ ion [10 ꢁM, r.t.; ꢂex = 342
nm].
S19

0.8
0.6
0.4
0.2
0
8.E+06
6.E+06
4.E+06
2.E+06
0.E+00
Probe
Probe
0.5 Eq Cu(II)
1.0 Eq Cu(II)
1.5 Eq Cu(II)
2.0 Eq Cu(II)
2.5 Eq Cu(II)
7.5 Eq Cu(II)
Excess Cu(II)
0.5 Eq Cu(II)
1.0 Eq Cu(II)
1.5 Eq Cu(II)
2.0 Eq Cu(II)
2.5 Eq Cu(II)
250
300
350
400
450
360
410
460
510
560
Wavelength(nm)
Wavelength (nm)
Figure S27: (a) UV-visible and (b) fluorescence spectra of 4-(1-(Pyren-1-yl)-1H-1,2,3-
traizol-4-yl)benzene (3) in acetonitrile in presence of Cu2+ ion [10 ꢁM, r.t.; ꢂex = 342 nm].
0.8
0.6
0.4
0.2
0
1.E+07
9.E+06
6.E+06
3.E+06
0.E+00
Probe
0.5 Eq Mg(II)
1.0 Eq Mg(II)
1.5 Eq Mg(II)
2.0 Eq Mg(II)
2.5 Eq Mg(II)
0.5 Eq Mg(II)
1.0 Eq Mg(II)
1.5 Eq Mg(II)
2.0 Eq Mg(II)
2.5 Eq Mg(II)
250
300
350
400
450
360
410
460
510
560
Wavelength(nm)
Wavelength(nm)
Figure S28: (a) UV-visible and (b) fluorescence spectra of 4-(1-(Pyren-1-yl)-1H-1,2,3-
traizol-4-yl)benzene (3) in acetonitrile in presence of Mg2+ ion [10 ꢁM, r.t.; ꢂex = 342
nm].
S20

0.8
0.6
0.4
0.2
0
9.E+06
6.E+06
3.E+06
0.E+00
(a)
(b)
0.5 Eq Ni(II)
1.0 Eq Ni(II)
1.5 Eq Ni(II)
2.0 Eq Ni(II)
2.5 Eq Ni(II)
Probe
0.5 Eq Ni(II)
1.0 Eq Ni(II)
1.5 Eq Ni(II)
2.0 Eq Ni(II)
2.5 Eq Ni(II)
200
250
300
350
400
360
410
460
510
560
Wavelength(nm)
Wavelength (nm)
Figure S29: (a) UV-visible and (b) fluorescence spectra of 4-(1-(Pyren-1-yl)-1H-1,2,3-
traizol-4-yl)benzene in acetonitrile in presence of Ni2+ ion [10 ꢁM, r.t.; ꢂex = 342 nm].
9.E+06
0.8
Probe
Probe
0.5 Eq Na(I)
1.0 Eq Na(I)
1.5 Eq Na(I)
2.0 Eq Na(I)
2.5 Eq Na(I)
0.5 Eq Na(I)
1.0 Eq Na(I)
1.5 Eq Na(I)
2.0 Eq Na(I)
2.5 Eq Na(I)
0.6
0.4
0.2
0
6.E+06
3.E+06
0.E+00
360
410
460
510
560
250
300
350
400
Wavelength(nm)
Wavelength (nm)
Figure S30: (a) UV-visible and (b) fluorescence spectra of 4-(1-(Pyren-1-yl)-1H-1,2,3-
traizol-4-yl)benzene in acetonitrile in presence of Na+ ion [10 ꢁM, r.t.; ꢂex = 342 nm].
S21

10.2. Test for Selectivity in Sensing of Cu2+ Ion
Competition experiments were carried out to examine ion-selective fluorescence
emission if all is shown by the fluorophores 2-3, though they did not show any specicity
in sensing any of the metal ions tested. Thus, to do so, the fluorescence spectra (Figure
S31-32) of the probe (10 ꢁM) were taken in presence of Cu2+ ion (20 ꢁM) along with
other background metal ions in 50 equiv. excess concentration each. Although the
fluorescence property of the probe with Cu2+ ion in presence of other background metal
ions did not change. Thus, this result indicated that the probes are not all selective for
Cu2+ or for any other metal ions tested.
1
0.5
0
7.E+06
6.E+06
5.E+06
4.E+06
3.E+06
2.E+06
1.E+06
0.E+00
Probe
Probe
Cu(II)
Cu(II)
Mg(II)+Cu(II)
Ni(II)+Cu(II)
Na(I)+Cu(II)
Cu(II)+Mg(II)
Cu(II)+Ni(II)
Cu(II)+Na(I)
250
300
350
400
450
360
410
460
510
560
Wavelength(nm)
Wavelength(nm)
Figure S31a: (a) UV-visible and (b) fluorescence spectra (ꢂex = 343 nm) of 4-(1-(Pyren-
1-yl)-1H-1,2,3-traizol-4-yl)benzonitrile (2) (10ꢁM) with Cu2+ ion (2 equiv.) in absence
and presence of other metal ions (50 equiv.).
1.E+07
5.E+06
0.E+00
Probe
Cu(II)
Mg(II)
Ni(II)
Na(I)
Mixed metal ions
S22

Figure S31b: Change in Fluorescence intensity at 390 nm (I390) of 4-(1-(Pyren-1-yl)-1H-
1,2,3-traizol-4-yl)benzonitrile(10 ꢁM) with Cu2+ ion (2 equiv.) in absence and presence
of other metal ions (50 equiv.).
1
0.5
0
9.E+06
6.E+06
3.E+06
0.E+00
Probe
Probe
Cu(II)
Cu(II)
Cu(II)+Mg(II)
Cu(II)+Ni(II)
Cu(II)+Na(I)
Mg(II)+Cu(II)
Ni(II)+Cu(II)
Na(I)+Cu(II)
360
410
460
510
560
250
300
350
400
Wavelength(nm)
Wavelength(nm)
Figure S32a: (a) UV-visible and (b) fluorescence spectra (ꢂex = 343 nm) of 4-(1-(Pyren-
1-yl)-1H-1,2,3-traizol-4-yl) benzene (3) (10ꢁM) with Cu2+ ion (2 equiv.) in absence and
presence of other metal ions (50 equiv.).
1.E+07
5.E+06
0.E+00
Probe Cu(II)
Mg(II)
Ni(II)
Na(I)
Mixed metal ions
Figure 32b: Change in Fluorescence intensity at 386 nm (I386) ratio of fluorescence
intensity of 4-(1-(Pyren-1-yl)-1H-1,2,3-traizol-4-yl)benzene (10 ꢁM) with Cu2+ ion (2
equiv.) in absence and presence of other metal ions (50 equiv.).
S23

11. Gaussian optimized geometry of 1-Cu2+ complex
Figure S33: B3LYP/3-21G* energy minimized structure of 1.Cu2+ complex by DFT
calculation. The LANL2DZ basis set was used for the Cu2+ cation.
Cartesian Co-ordinate:
E(UB+HF-LYP) = -2625.96196719 a.u.
2 2
N
N
N
N
C
C
C
C
H
C
C
H
C
C
C
C
H
0.56908000 1.14276400 -0.70243700
2.66532400 1.10615400 -1.30839800
1.47729900 0.37796800 -1.37849000
-1.50230000 6.44281900 2.60668200
3.82905600 0.62970800 -1.99517700
1.15404900 2.30480700 -0.22157800
0.45480900 3.34291200 0.51878200
2.48291100 2.27431700 -0.62126800
3.25828000 3.00971100 -0.51538500
5.08465400 0.58530400 -1.34433300
3.67825000 0.23139600 -3.32568200
2.69637200 0.27116800 -3.77731400
6.06290100 -0.20258100 -3.48941600
6.21852900 0.17727900 -2.11915400
7.51284000 0.14350900 -1.52042300
1.14499000 4.13776800 1.45729200
2.19131200 3.94101800 1.66099500
S24

C
H
C
H
C
H
C
C
H
C
H
C
C
C
H
C
H
C
H
C
H
C
H
C
H
C
H
H
H
C
H
H
H
N
N
N
N
C
C
C
C
H
C
C
H
C
0.50877700 5.15023700 2.14737300
1.07481400 5.73032800 2.86120200
-1.56087600 4.62201900 0.98993400
-2.60471900 4.80605400 0.78183800
-0.91176200 3.61216000 0.30483000
-1.46100800 3.04775800 -0.44105300
-0.87092500 5.43310700 1.93571000
5.27847600 0.89187600 0.04523800
4.41951900 1.13437200 0.65723100
4.77631000 -0.18352400 -4.06289800
4.65082700 -0.47720400 -5.09793400
8.64952200 -0.24348900 -2.29483900
7.68008500 0.49213200 -0.14686100
9.91906400 -0.25986000 -1.68566600
10.78239700 -0.54882100 -2.27403100
8.46045700 -0.60699300 -3.66939200
9.32796300 -0.89971100 -4.24974000
6.51895700 0.84657200 0.61365400
6.64246200 1.07108400 1.66726000
10.07281100 0.08817500 -0.34421700
11.05730300 0.06889800 0.10599000
7.22051400 -0.59234300 -4.24089100
7.08984300 -0.87409700 -5.27910800
8.96854900 0.45739700 0.42107200
9.09421900 0.72136100 1.46498500
-2.93224800 6.72876600 2.36306200
-3.10489500 6.99374600 1.31346200
-3.23131100 7.57005400 2.98692500
-3.55386600 5.86353800 2.62140200
-0.76091600 7.28448300 3.57200300
-0.35440100 6.67700400 4.38858700
-1.44632300 8.01843500 3.99393600
0.06010200 7.81623000 3.07756500
-4.00631300 -0.03771700 -0.90068100
-3.01554500 -1.92142200 -0.22760000
-2.82041800 -0.55276500 -0.53667500
-10.36559500 -0.10542500 -2.20928000
-1.91466000 -2.76350900 0.15850700
-4.95192000 -1.04084400 -0.84141000
-6.34396000 -0.81530800 -1.18344800
-4.32140100 -2.21691500 -0.41673600
-4.71266200 -3.20038100 -0.23189800
-1.31770000 -2.58711200 1.42489200
-1.46760900 -3.73343000 -0.74344100
-1.94999100 -3.82753400 -1.70866800
0.23737300 -4.43370300 0.83909600
S25

C
C
C
H
C
H
C
H
C
H
C
C
H
C
H
C
C
C
H
C
H
C
H
C
H
C
H
C
H
C
H
H
H
C
H
H
H
Cu
-0.21946800 -3.44076100 1.76350200
0.41237300 -3.31017400 3.03587000
-7.30848900 -1.84018200 -1.08641100
-7.02296100 -2.82771200 -0.74404800
-8.62759100 -1.61356100 -1.41918600
-9.33526400 -2.42442400 -1.33013600
-8.07849200 0.69526200 -1.96925900
-8.35934500 1.67998400 -2.31285600
-6.76390900 0.45572000 -1.63153500
-6.03169200 1.24849500 -1.71321000
-9.06219400 -0.33386200 -1.87537300
-1.77063600 -1.62034400 2.39038400
-2.62035800 -0.99435200 2.14880700
-0.40551200 -4.56067000 -0.40656200
-0.06255100 -5.31089600 -1.10856300
1.50287000 -4.16543100 3.38252300
-0.04877800 -2.33494700 3.97210200
2.10403400 -4.02632000 4.64506300
2.92816100 -4.67785600 4.91231700
1.94224000 -5.14810600 2.42874200
2.76843000 -5.79488900 2.70166900
-1.16001200 -1.50314500 3.60371800
-1.52085000 -0.77918100 4.32620500
1.64673200 -3.07082500 5.55239600
2.11863800 -2.98426700 6.52327300
1.33872500 -5.27746800 1.21476300
1.67607600 -6.02667500 0.50811900
0.58241600 -2.23292300 5.22365300
0.22735000 -1.49881300 5.93810400
-10.80216800 1.22782700 -2.68774500
-10.61458100 1.99188000 -1.92555400
-11.87094400 1.19236000 -2.89246500
-10.27540900 1.49875300 -3.60924800
-11.37561200 -1.18616800 -2.10850600
-11.11333900 -2.02262000 -2.76543300
-12.34236800 -0.79005400 -2.41497800
-11.45375700 -1.54651300 -1.07704800
-1.13209600 0.31647400 -0.54791700
S26