S1
Supporting Information
New family of fluorogenic azacrown probes with identical cavity size but different
electronic environment outside the macrocycle: Effects on sensitivity of Cu2+ detection
Anita D. Pandeya, Raghuvir R. S. Pissurlenkarb and Anil V. Karnik*,a
a
Department of Chemistry, University of Mumbai, Vidyanagari, Santacruz (East), Mumbai 400098, India.
b
Department of Pharmaceutical Chemistry, Molecular Simulations Group, Bombay College of Pharmacy,
Kalina, Santacruz (East), Mumbai-400098, India.
Email: avkarnik@chem.mu.ac.in
Table of Contents
General
Synthetic procedures
Thermal analyses (DTA and TGA) of azacrowns 3 & 4 and their Cu(II) complexes 3.Cu and
4.Cu. & Table-1
Figure SI-1A: FTIR(KBr) spectrum of 3
Figure SI-1B: CO-FTIR(KBr) spectrum of 3 and Cu2+ complex of 3
Figure SI-2: 1H NMR spectrum of 3
Figure SI-3: 13C NMR spectrum of 3
Figure SI-4: 1H-1H COSY spectrum of 3
Figure SI-5: NOESY spectrum of 3
Figure SI-6: HMQC spectrum of 3
Figure SI-7A: GC-MS spectrum of 3
Figure SI-7B: TGA spectrum of azacrown 3
Figure SI-7C: TGA spectrum of Cu2+ complex of azacrown 3
Figure SI-8A: FTIR(KBr) spectrum of 4
Figure SI-8B: CO-FTIR(KBr) spectrum of 4 and Cu2+ complex of 4
Figure SI-9: 1H NMR spectrum of 4
Figure SI-10: 13C NMR spectrum of 4
Figure SI-11: 1H-1H COSY spectrum of 4
Figure SI-12: NOESY spectrum of 4
Figure SI-13: HMQC spectrum of 4
Figure SI-14A: GC-MS spectrum of 4
Figure SI-14B: TGA spectrum of azacrown 4
Figure SI-14C: TGA spectrum of Cu2+ complex of azacrown 4
Fluorescence studies
Figure SI-15: Stern-Volmer Plots for receptors 3 interactions with various metal ions
Figure SI-16: Stern-Volmer Plots for receptors 4 interactions with various metal ions
Figure SI-17: Comparitive fluoresence quenching of 3 & 4 by Cu2+
Figure SI-18: Determination of Limit of detection for Cu2+ by receptors 3
Figure SI-19: Determination of Limit of detection for Cu2+ by receptors 4
Figure SI-20: Double logrithmic plot according to Modified Stern-Volmer equation to
determine association constant of 3 with Cu2+
Figure SI-21: Double logrithmic plot according to Modified Stern-Volmer equation to
determine association constant of 4 with Cu2+
Figure SI-22: Job’s plot of receptor 3 with Cu2+ in buffer
Figure SI-23: Job’s plot of receptor 4 with Cu2+ in buffer
Figure SI-24: Single crystal XRD of receptor 3
Figure SI-25: Semirigid nature of receptor 3 based on crystal structure.
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General information
Reagents were of AR grade and used without further purificaion. Perchlorate
salts of Li+, Na+, Ca2+, Mg2+, Cd2+, Co2+, Cu2+, Ba2+, Zn2+ and Ni2+ were
purchased from Sigma-Aldrich and were used directly without further
purification. Column chromatography was carried out using silica-gel 60-120
mesh (S.D.Fine Chemicals, India). Boiling point of petroleum-ether used was in
the range of 60–80oC. Melting points reported are uncorrected. Infrared (IR) spectra
were recorded on PerkinElmer Spectrum Version 10.03.07 spectrometer. The
samples were prepared in KBr disks. 1H NMR spectra were recorded on Bruker,
AV II 300 (300 MHz) spectrometer using TMS as internal standard. 13C NMR
spectra were recorded on 75 MHz Bruker, AV II 300 spectrometer.
Thermogravimetric analysis (TGA) and Differential thermal analysis (DTA
measurements of complexes were recorded on a Shimadzu DT-50 and TG-50
thermal analyzers with heating rate of 10 °C/min from ambient temperature up
to 1000oC under nitrogen. The weight of the sample was in the range 2-10 mg.
Elemental analyses were performed on Carlo Enra instrument EA-1108
Elemental analyzer. GC-MS spectrum were recorded on Thermoelectron
spectrometer. UV were recorded on Shimadzu UV–visible Spectrophotometer
UV-2100. Fluorescence spectra were recorded on a Perkin-Elmer LS-55
spectrofluorimeter. Spectroscopy grade Acetonitrile (ACN), Water (H2O), 2-[4(2-hydroxyethyl)-1-piperazinyl] ethanesulfonic acid (HEPES) were purchased
from S.D. fine Chemical Company, India.
CAUTION: Metal perchlorate salts are potentially explosive in certain
conditions. All due precautions should be taken while handling perchlorate
salts!!!
Synthetic procedures for the preparation of receptors 3 and 4:
Synthesis of 2-Methyl-[3.6.9.12 tetraoxa benzimidazolo] monoaza-15-crown-5 (3)
S3
H
N
CH3
N
O
O
O
O
In a Nitrogen flushed, dry 250 ml 3-necked round bottomed flask fitted with a dropping
funnel, a reflux condenser and Nitrogen inlet was charged with sodium hydride (240 mg, 6
mmol) and was washed with hexane (2 x 20 mL). After decanting Hexane, dry THF (50 mL)
was added and the mixture was refluxed under nitrogen and for 30 mins. To the refluxing
solution was added a dropwise solution of (S)-(-)-2-α-(hydroxyethyl)benzimidazole (1) (324
mg, 1 mmol) in 75 ml of dry THF in 1 hour and the mixture was stirred and refluxed at 70°C
for 2 hours. To this reaction mixture was added a dropwise solution of tetraethylene glycol
ditosylate (0.502 gm, 1 mmol) in 75 ml dry THF in 1hr and the reaction mixture refluxed
further for 8 hours till the starting materials were consumed (monitored by TLC). THF was
concentrated under reduced pressure to afford a light yellow oily residue, to which was added
to 100 gm of ice. The yellow semi solid residue was extracted with CHCl3(3x50 mL) and the
organic layer washed with water (2x75 mL), concentrated in-vacuo to provide a light yellow
viscous oil which was purified by column chromatography on silica gel using Petroleum
ether: Chloroform (40:60) as eluent to afford white crystalline solid of monoaza-15-crown-5
(2) in 48% yield. M.p. 65°C, FTIR(KBr): 3392, 2947, 2867, 2349, 1672, 1616, 1517, 1466,
1452, 1323, 1123, 1096, 749,735 cm-1. 1H NMR (300 MHz, CDCl3) δ: 1.66(d, 3H, J=6.6Hz),
3.5-3.6(m, 10H), 4.0(t, 2H, J=6.6 Hz), 4.3(m,1H), 4.5(m, 1H), 5.0 (q, 1H, J=6.6 Hz), 7.2-7.7
(Ar, 4H).13C NMR (75 MHz, CDCl3) δ: 154.2, 141.6, 135.6, 122.8, 122.1, 119.6, 109.9, 71.6,
71, 70.9, 70.1, 70, 69.1, 67.9, 63.6, 44.2, 18.9(Fig. SI-3).GC-MS: 319.6 [M+1]+.
Synthesis of 2-Phenyl-[3.6.9.12 tetraoxa benzimidazolo] monoaza-15-crown-5 (4)
H
N
N
O
O
O
O
In a dry 250 mL 3-necked round bottomed flask, flushed with Nitrogen and fitted with
a dropping funnel, a reflux condenser and Nitrogen inlet was charged Sodium hydride
(0.480 g, 12 mmol) and was washed twice with hexane (2 x 20 mL). After decanting
Hexane, dry THF (50 mL) was added and the mixture was refluxed under nitrogen
and for 30 mins. To the refluxing solution was added a solution of 2-(αhydroxybenzyl)benzimidazole (2) (0.448 g, 2 mmol) in 100 mL of dry THF through
dropping funnel in 1 hour and the mixture was stirred and refluxed at 70°C for 2
hours. To this reaction mixture was added a dropwise solution of tetraethylene glycol
ditosylate (1.0 gm, 2 mmol) in 100 ml dry THF in 1hr and the reaction mixture
refluxed further for 7-8 hours till the starting materials were consumed (monitored by
S4
TLC). THF was concentrated under reduced pressure to afford a light yellow oily
residue, to which was added to 100 gm of ice. The yellow semi solid residue was
extracted with CHCl3(3x50 mL) and the organic layer washed with water (2x75 mL),
concentrated in-vacuo to provide a light yellow viscous oil which was purified by
column chromatography on silica gel using Chloroform:Petroleum ether (60:40) as
eluent to afford azacrown (4) as a pale yellow oil. 0.382gm (50%) yield. IR (Oil):
3052, 2866, 1614, 1463, 1408, 1112, 771, 748 cm-1. 1H NMR (300 MHz, CDCl3,
ppm): δ 6.117 (s, 1H; CH), δ 3.54-4.07 (m, 16 H; CH2 crown part), δ 7.2 to 7.8 (m,
9H; Ar). 13C NMR (75 MHz, CDCl3, ppm) (Figure S3): δ : 153.2, 142, 138, 130,
128.7, 128.5, 128.09, 127.08, 126.52, 122.79, 122.39, 122.02, 119.8, 109.9, 71.2,
71.06, 70.14, 69.95, 69.27, 69.16, 44.19, 38.71, 31.9, 29.68, 29.34, 22.68, 14.13,
14.06, 10.9. ESI MS: m/z (Figure S4): [M + 1]+ 383.
Synthesis of copper azacrown complexes 3-Cu2+ {[Cu-3 (H2O)](ClO4)2} and 4-Cu2+
{[Cu-4 (H2O)](ClO4)2}
The copper complexes of azacrowns 3 and 4 were synthesized by adding a 0.785 mM
(20.61 mg) anhydrous copper (II) perchlorate solution in dry acetonitrile (500L) to a
colourless solution of 0.785 mM of azacrown-3 in dry acetonitrile (300 L). The
resulting yellowish green solution was stirred for 5-10 mins and concentrated to half
the volume (~350 mL) and cooled to room temperature, following which light green
coloured solid was obtained. This copper complex was crystallized using Pet ether:
chloroform mixture (6:4) to afford green crystalline solid (85-90%) yields which was
filtered, dried and kept in dessiccator.
Thermal analyses (DTA and TGA) of azacrowns 3 & 4 and their Cu(II) complexes 3.Cu
and 4.Cu.
In the TGA curves for the samples in all the cases (Figure SI 7A-7B & 14A-14B), at about
110-120oC occurs a transition that can be attributed to the loss of water of crystallization. The
temperatures of dehydration T1 are presented in the second column of Table-1. From the
weight loss in TGA, it was established that the samples were actually monohydrates of the
corresponding Cu2+ salts of the azacrowns 3 & 4. Afterwards, samples begin losing weight
again, reflecting the decomposition o fthe anhydrous salts. With an increase in the aliphatic
chain length of the salts, the temperature at which the decomposition begins diminishes. For
Cu2+complex decomposition starts immidiately after the sample has lost the water. The TGA
confirms the formation of CuO as the end products.
Table 1. TGA and DTA data of azacrowns 3 & 4 and their Cu(II) complexes 3.Cu and 4.Cu.
Compound Step
TGA
Temperature
% Weight
Assignment
(thermal process)
DTA
Temperature
Peaks
S5
1
range oC
75-80
loss
3.99
3
2nd
1st
225-310
100-150
95.52
11.45
3.Cu
2nd
155-400
64.27
3rd
455-590
98.6
1st
80-165
4.06
2nd
3rd
1st
220-410
410-520
100-150
84.54
2nd
3rd
4th
155-275
400-520
415-600
60.48
82.73
86.60
st
4
4.Cu
9.353
range oC
solvent 48-64
Endo.
240-325
30-160
Exo.
Endo.
200-275
Exo.
485-545
Exo.
60-125
Endo.
190-410
400-530
50-120
Exo.
Endo.
Endo.
165-270
390-450
410-580
Exo.
Endo.
Exo.
Elimination
of
molecule (Hexane)
complete decomposition of 3
Elimination of the coordinated water molecule
Partial decomposition of the
complex
Complete decomposition of
the complex and formation of
CuO as a final product
Elimination
of
the
coordinated water molecule
Partial decomposition of 4
complete decomposition of 4
Elimination of the coordinated water molecule
Partial decomposition of 4
Partial decomposition of 4
Complete decomposition of
the complex and formation of
CuO as a final product
The Thermogravimetric analyses showed that Cu(II) complexes of azacrowns 3 & 4
decompose mainly in three steps. The first step appeared within the temperature range 75170ºC was due to the elimination of coordinated water molecule. This appeared as an
endothermic peak in the DTA curves within the temperature range 30-170ºC. The second step
within the temperature range 170-430ºC was associated with an exothermic DTA peak at
160-450ºC range. This peak was due to the partial decomposition of the ligand. The final
decomposition step appeared above 400ºC corresponding to the complete thermal
decomposition of the complexes and the loss of their organic portion resulting in the
formation of CuO as final products. This last step appeared as an exothermic peak in the
DTA curves at 250-790ºC range, confirming the data obtained from the TGA curves.
S6
Fig SI-1A FTIR(KBr) spectrum of azacrown (3)
Fig SI-1B CO-FTIR(KBr) spectrum of azacrown 3 & 3.Cu2+ complex
S7
H
N
CH3
N
O
O
O
O
Fig. SI-2 1H NMR (300 MHz, CDCl3) spectra of (3)
H
N
CH3
N
O
O
O
O
Fig. SI-3 13C NMR (75 MHz, CDCl3) spectra of (3)
S8
Fig. SI-4 COSY (300 MHz, CDCl3) spectra of (3)
Fig. SI-5: NOESY spectra (CDCl3) of (3)
S9
H
N
CH3
N
O
O
O
O
Fig. SI-6 HMQC spectra (CDCl3) of (3)
Fig SI-7A GC-MS spetrum of compound (3)
S 10
Fig SI-7B: TGA spectrum of azacrown (3)
Fig SI-7C: TGA spectrum of compound Cu2+ complex of azacrown (3)
S 11
Fig SI-8A: FTIR(KBr) spectrum of compound (4)
Fig SI-8B: CO-FTIR(KBr) spectrum of azacrown 4 & 4.Cu2+ complex
S 12
Fig. SI-9 1H NMR (300 MHz, CDCl3) spectrum of (4)
Fig. SI-10 13C NMR (75 MHz, CDCl3) spectrum of (4)
S 13
Fig. SI-11 1H-1H COSY (CDCl3) spectrum of (4)
Fig. SI-12 NOESY (CDCl3) spectrum of (4)
S 14
Fig. SI-13 HMQC (CDCl3) spectrum of (4)
Fig. SI-14A ESI MS of spectrum of (4)
S 15
Fig. SI-14B: TGA spectrum of azacrown (4)
Fig. SI-14C TGA spectrum of compound Cu2+ complex of azacrown (4)
S 16
Fluorescence studies
Fluorescence spectra was recorded on a Perkin-Elmer LS-55 spectrofluorimeter and 1cm cell
was used for titrations. Stock solutions of receptors 3 and 4 (1x10-4 M) were freshly prepared
in buffer. The stock solutions of metal perchlorates (Li+, Na+, Ba2+, Ca2+, Cd2+, Co2+, Cu2+,
Mg2+, Ni2+ and Zn2+) were prepared in acetonitrile in the order of 10-3 M. Working solutions
of 3 and 4 and metal perchlorates were prepared from the stock solutions. Spectrofluorimetric
titration was performed by an incremental addition of solutions of different metal
perchlorates individually and change in emission intensities for each set was analyzed.
Samples were excited at =254 nm with all excitation and emission slit widths at 5/5 nm
respectively, with a scan speed of 500 nm/min.
1.
Determination of Binding constants
Binding constant values of metal perchlorates with receptors 3 and 4 has been determined by
Stern-Volmer equation.R [3,4]
F0 / F =1 + Ksv[G]
F0
F
[G]
Ksv
=
=
=
=
fluorescence intensity of the sensor in the absence of of guests;
fluorescence intensity of the sensor in the presence of the guest / quencher;
Concentration of the guest species and
Stern-Volmer constant.
Thus Ksv can be calculated from the graph of F0/F against [G], where F0 and F are found out
experimentally. The Stern-volmer equation does not contain a variable for the host or sensor
concentration [H], suggesting that the quenching is independent of host concentration. The
change in fluorescence behaviour during complexation is dependent only on concentration of
the guest.
S 17
2. Stern-Volmer Plots for receptors 3 and 4 interactions with various metal ions
Fig. SI-15 Stern-Volmer plots for spectrofluorimetric titration of receptor 3 with Cu2+ and
Li+, Na+, Ba2+, Ca2+,Cd2+, Co2+,Mg2+, Ni2+ and Zn2+ metal perchlorates
Fig. SI-16 Stern-Volmer plots for spectrofluorimetric titration of receptor 4 with Cu2+ and
Li+, Na+, Ba2+, Ca2+,Cd2+, Co2+,Mg2+, Ni2+ and Zn2+ metal perchlorates
S 18
Fig. SI-17 Comparative % quenching of fluorescence intensity of receptors 3 (dark blue) and
4 (light blue) in company of Cu2+ Li+, Na+, Ba2+, Ca2+, Cd2+, Co2+, Cu2+, Mg2+, Ni2+, Zn2+
cations, in buffer (pH 7.2 ±0.1) solution.
3.
Determination of Limit of detection for Cu2+ by receptors 3 and 4
S 19
Fig. SI-18 Semilogarithmic plot for determination of detection limit of 3 towards
Cu2+ in buffer
Fig. SI-19 Semilogarithmic plot for determination of detection limit of 4 towards
Cu2+ in buffer
4. Determination of number of binding sites by double logarithmic plot
(modified Stern-Volmer Equation)
S 20
Fig. SI-20 Double logarithmic plot for determination of association constant (Ka) of
receptor 3 towards Cu2+ in buffer
Fig. SI-21 Double logarithmic plot for determination of association constant (Ka) of
receptor 4 towards Cu2+ in buffer
5.
6. Job plot by Fluorescence method for determination of binding stoichiometry
Stock solution of same concentration of the receptors 2 and the guest were prepared in the
order of ca. (1.0 X 10-5 M) buffer. The absorbance in each case with different host–guest ratio
(0-0.9) but equal in volume was recorded. Job plots were drawn by plotting (F0-F/F0) x  guest
vs guest. Here A is the change in absorbance of receptor in the presence and absence of
guest, added cation, [Mn+]
(F= change of intensity of the absorbance spectrum during titration and host is the mole
fraction of the host in each case, respectively)
S 21
Fig. SI-22 Job’s plot of receptor 3 with Cu2+ in buffer, to determine binding stoichiometry
Fig. SI-23 Job’s plot of receptor 4 with Cu2+ in buffer, to determine binding stoichiometry
S 22
Figure SI- 24: ORTEP diagram of X-ray crystal structure of (3)
Figure SI- 25: Semirigid nature of azacrown 3 indicated by the X-ray crystal structure