Journal of Hazardous Materials 309 (2016) 97–106

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Journal of Hazardous Materials

journal homepage: www.elsevier.com/locate/jhazmat

Synthesis, characterization and Cu2+ triggered selective fluorescence

quenching of Bis-calix[4]arene tetra-triazole macrocycle
Burhan Khan a , Muhammad Raza Shah a,∗ , Dania Ahmed a , Muhammad Rabnawaz a ,
Itrat Anis b , Saifullah Afridi c , Talat Makhmoor c , Muhammad Nawaz Tahir d
a
H.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Pakistan
b
Department of Chemistry, University of Karachi, Karachi75270, Pakistan
c
Dr.Panjwani Center For Molecular Medicine And Drug Research, International Center for Chemical and Biological Sciences, University of Karachi, Karachi
75270, Pakistan
d
Department of Physics, University of Sargodha, Sargodha, Pakistan

h i g h l i g h t s g r a p h i c a l a b s t r a c t

• The 9 was characterized by Mass,

NMR spectroscopy and X-ray crystal-
lography.
• The 9 lost fluorescence after mixing
with Cu2+. .
• The lower limit of detection for Cu2+
by 9 was found to be 40 nM.
• The chemosensor 9 recognized Cu2+
in living cells selectively

a r t i c l e i n f o a b s t r a c t

Article history: A novel fluorescent bis-calix[4]arene macrocycle 9 incorporating metal-binding pockets was successfully
Received 2 November 2015 prepared. The structure of macrocycle 9 and its precursors were characterized via EI-MS, MALDI-TOF-
Received in revised form 14 January 2016 MS, ESI-MS, 1 H NMR, 13 CNMR, 2D NMR, and X-ray crystallography. The macrocycle 9 displayed selective
Accepted 27 January 2016
fluorescence quenching after interacting with Cu2+ in the presence competing metal cations including
Available online 4 February 2016
Mg2+ , Ca2+ , Ba2+ , Ag+ , Zn2+ , Ti4+ ,Cd2+ , Hg2+ , Pb2+ , In3+ , La3+ , Cr3+ , Ni2+ , Sb3+ , V5+ , Fe3+ , Co2+ , Sn2+ , Sn2+ , and
Tl+ . The Cu2+ limit of detection was found to be 40 nM much lower than its threshold level (∼20 ␮M) in
Keywords:
drinking water permitted by the U.S Environmental Protection Agency (EPA). Furthermore, drinking water
Bis-calix[4]arene
Macrocycle
samples from Karachi University (Pakistan) spiked with Cu2+ were analysed with the sensing system and
Fluorescence the results showed an excellent agreement with the fluorescence quenching phenomenon of macrocycle
Supramolecular 9 examined in deionized water. Importantly, the chemosensor 9 could be used to detect Cu2+ in living
Atomic force microscopy cells
© 2016 Elsevier B.V. All rights reserved.

∗ Corresponding author.
E-mail address: [email protected] (M.R. Shah).

http://dx.doi.org/10.1016/j.jhazmat.2016.01.074
0304-3894/© 2016 Elsevier B.V. All rights reserved.
98 B. Khan et al. / Journal of Hazardous Materials 309 (2016) 97–106

1. Introduction After the reaction reached to completion, the mixture was cooled
down to room temperature. Residual acetone was removed via
Supramolecular fluoro-sensors as selective probes for organic rotary evaporation, and the crude product was then dissolved in
molecules and metal ions is a promising area of applied dichloromethane (50.0 mL) before it was extracted with distilled
supramolecular chemistry [1,2]. A fluorescence probe consists of a water (3 × 20 mL). The organic phase was dried over MgSO4 , and
fluorophore and an ionophore unit. The fluorophore unit serves as was passed through a piece of filter paper. This solution filtrate was
a reporter unit that provides an optical response after the detection concentrated in vacuo to a volume of 2 mL. The crude product was
of a recognition event, while the ionophore unit acts as a binding passed through a silica gel column (∼30 g), using dichloromethane:
site for the analyte [3–6]. Various classes of fluorescence sensors hexane (1:1, v/v) as the mobile phase. The product 3 was obtained
have been reported on the basis of their photophysical properties in 91% yield (1200 mg) as a white solid. mp 79–81 ◦ C; 1 H NMR (in
and signaling processes, such as excimer/exciplex formation [7–9], CDCl3 at 300 MHz at 298 K): ␦ 9.88 (s, aldehyde-H, 1H), 7.84 (dd,
photoinduced electron transfer (PET) [10–12], metal-ligand charge J = 5.1 Hz, Ar-H, 2H), 7.08 (dd, J = 5.4 Hz, Ar-H, 2H), 4.76 (d, J = 2.4 Hz,
transfer (MLCT) [13,14], immine isomerization [15,16], chelation PhOCH2 , 2H), 2.54 (t, J = 2.4 Hz, Acetylene-H, 1H) ppm. EI-MS: found,
enhanced fluorescence (CHEF) [17–19], and fluorescence resonance M+ , m/z = 160.0 and calcd M+ for C10 H8 O2 , m/z = 160.05. Anal. Calcd
energy transfer (FRET) [20,21]. Copper metal has found widespread for C10 H8 O2 ; C, 74.99; H, 5.03. Found: C, 74.87; H, 5.11.
uses in industry for manufacturing various unavoidable and impor-
tant materials, due to which the Cu2+ concentration in many lakes 2.2. Preparation of 1, 3-dialkylbromo calix[4]arene (6)
and rivers is continuously increasing by the common process of cor-
rosion [22]. While the adverse effects of copper on human health are Calix[4]arene 4 (500 mg, 0.77 mmol) was dissolved in 30 mL of
often rather mild at low concentrations, however certain microor- acetonitrile and mixed with K2 CO3 (320 mg, 2.31 mmol, 3 equvi).
ganisms suffer severely from it even at nanomolar concentrations The reaction mixture was stirred for 30 min at 60 ◦ C, followed by
[23,24]. Nevertheless, the uptake of water contaminated with high mixing of 1, 3-dibromopropane 5 (0.19–1.00 mL, 1.92 mmol) and
concentrations of copper also causes human health problems, such further refluxed for 4 h. Acetonitrile was removed via rotary evapo-
as Wilson’s disease, hypoglycemia, dyslexia, gastrointestinal com- ration until the volume of the solution reached ∼2–3 mL. Methanol
plications, catarrhal inflammation, irritation of the nose and throat, (100 mL) was added to the crude solution, thus yielding a white
nausea, vomiting, and diarrhea [24,25]. precipitate. This precipitate was filtered and washed with distilled
Conversely, increased concentrations of Cu2+ in human plasma water (20 mL). The crude product was further purified via silica
have been more specifically associated with cardiovascular risk gel chromatography using chloroform: hexane (20:80, v/v) as the
factors [26] and cancer [27]. The detection of Cu2+ at sub- mobile phase. Compound 6 was obtained in 68% yield (340.0 mg)
micromolar concentrations in water requires an efficient, selective as white powder. mp 301–308 ◦ C; 1 H NMR (in CDCl3 at 300 MHz
and highly sensitive chemosensor [28,29]. Early reported methods at 298 K): ␦ 7.61 (s, OH, 2H), 7.03 (s, Ar-H, 4H), 6.85 (s, Ar-H, 4H),
have suffered from a numerous drawbacks such as low sensitiv- 4.26 (d, Jgem = 12.9 Hz, axial-H of bridged methylene,4H), 4.09 (t,
ity, selectivity and stability. Fluorescent molecules, due to their J = 5.4 Hz, BrCH2, 4H), 4.03 (t, J = 5.7 Hz, NCH2, 4H), 3.98 (t, J = 6.6 Hz,
emissive behavior, easy signal detection, sensitivity, selectivity OCH2 , 4H), 3.34 (d, Jgem = 12.9 Hz, equatorial-H of bridged methy-
and low cost, offer many advantages over other systems in this lene,4H), 2.5 (m, CH2 CH2 CH2 , 4H), 1.20 (s, t-butyl-H, 18H), 0.99 (s,
area of research. Calixarenes, have drawn significant attention as t-butyl-H, 18H) ppm. ESI-MS: found [M + 1] + m/z = 891.29 and calcd
chemosensors, due to their inherent molecular cavities, preorgan- M+ for C50 H66 Br2 O4 , m/z = 890.33. Anal. Calcd for C50 H66 Br2 O4 ; C,
ised binding sites, and the ease of their subsequent modification 67.41; H, 7.47. Found: C, 68.11; H, 7.64.
[30–32]. Literature is rich describing the behavior of calixarene-
based derivatives as fluorosensors for heavy metal ions [33–40]. 2.3. Preparation of propyl azide calix[4]arene (7)
However only few fluorescent Bis-calixarenes macrocycles [41–45]
are reported as receptor. The triazole derivatives [46–49] and Sodium azide (60 mg, 0.92 mmol, 2.5 equvi) and compound 6
Schiff’s base [50–53] moieties have been widely used for the detec- (300 mg, 0.36 mmol in 5.0 mL of DMF) in DMF was stirred at 60 ◦ C
tion and quantification of metal cations. Recently, we have reported for 1 h and the reaction was monitored with TLC (Hexane: Ethyl
[54–59] on a series of supramolecules that have displayed remark- acetate 7:3, v/v). The reaction was quenched treated with distilled
able selectivity in optical discrimination between competing drugs water (10 mL) at room temperature yielding white precipitate. The
and metal ions in aqueous media and real samples. white precipitate was washed with brine solution (100 mL), dried
In continuation of our efforts a novel fluorescent bis-calix [4] in a vacuum oven and compound 7 in 90% yield (270 mg). mp
arene macrocycle 9 bearing triazole moieties, imine functionalities 233–234 ◦ C; 1 H NMR (in CDCl3 at 300 MHz at 298 K): ␦ 7.50 (s, OH,
and phenolic hydroxyl groups in its backbone has been synthesised 2H), 7.23 (s, Ar-H, 4H), 7.03 (s, Ar-H, 4H), 4.22 (d, Jgem = 12.9 Hz,
(Scheme 1). The Cu2+ is known to inherently quench fluorescence, axial-H of bridged methylene,4H), 4.03 (t, J = 5.7 Hz, NCH2 , 4H),
thus facilitating its detection at low concentrations [60–65]. With 3.85 (t, J = 6.6 Hz, OCH2 , 4H), 3.33 (d, Jgem = 12.9 Hz, equatorial-H
this in mind, the highly fluorescent macrocycle 9 was used to detect of bridged methylene,4H), 2.23 (m, CH2 CH2 CH2, 4H), 1.20 (s, t-
and quantify Cu2+ at its trace concentrations in aqueous media. butyl-H, 18H), 0.99 (s, t-butyl-H, 18H) ppm. EI-MS: found M+ , m/z
814.20 and calcd M+ for C50 H66 N6 O4 , m/z = 814.51. Anal. Calcd for
C50 H66 N6 O4 : C, 73.68; H, 8.16; N, 10.31. Found: C, 73.49; H, 8.22;
2. Experimental N, 10.39.

2.1. Preparation of 4-(prop-2-ynyloxy) benzaldehyde (3) 2.4. Preparation of bis-triazole dialdehyde calix[4]arene (8)

4-Hydroxybenzaldehyde 1 (1.0 g, 8.2 mmol) and K2 CO3 (1.69 g, O-propargyl benzaldehyde 3 (60 mg, 0.37 mmol) and diazide
12.3 mmol, 1.5 equvi) were dissolved in 30 mL of acetone derivative 7 (200 mg, 0.25 mmol) were dissolved in 5.0 mL of DMF.
and stirred at 40 ◦ C for 20 min. Propargyl bromide 2 (1.05 mL, The CuSO4 (6.2 mg, 10 mol%, 0.025 equvi) and sodium ascorbate
12.3 mmol, and 1.5eq) was added to this mixture, which was sub- (10 mg, 20 mol% with respect to compound 7) were added in the
sequently stirred for 3 h at 60 ◦ C. The reaction was monitored by reaction. The reaction mixture was allowed to stirrer at 80 ◦ C
TLC using dichloromethane and hexane (7:3, v/v) as the eluent. for 2 h before the reaction was quenched with distilled water
 B. Khan et al. / Journal of Hazardous Materials 309 (2016) 97–106 99

Scheme 1. Synthetic route for compound 9 (i) Acetone, K2 CO3 , 60 ◦ C, 3 h, 91%, (ii), CH3 CN, K2 CO3 , 60 ◦ C, 4 h, 68% (iii) DMF, NaN3 , 70 ◦ C, 1 h, 90% (iv) DMF, sodium-ascorbate,
CuSO4 · 5H2 O, 2 h, 73% (v) EtOH, reflux, 5 h, 53%.

(30 mL), thus yielding a greenish precipitate. This precipitate was 3, 5, 11, 13, 17, 19, 25, 27, 8H), 5.28 (s, PhOCH2 -38,38‘ 4H),
dissolved in dichloromethane and purified via silica gel column 4.83 (t, J = 6.6 Hz, NCH2 -35,35‘, 4H), 4.11 (d, Jgem = 12.6 Hz, axial-
chromatography, using ethyl acetate and dichloromethane (1:1, H of bridged methylene,4H), 3.90 (t, J = 5.8, −OCH2 CH2, 4H), 3.37
v/v) as the eluent. The compound 8 was obtained in 78% yield (d, Jgem = 12.6 Hz, equatorial-H of bridged methylene 4H), 2.56 (t,
(156 mg) as a white solid. mp 180–181 ◦ C; 1 H NMR (DMSO-d6 , J = 6.3 Hz, CH2 CH2 CH2, 4H), 1.17 (s, 18H, t-butyl), 1.11 (s, t-butyl-H,
300 MHz at 298 K):␦ 9.84 (s, aldehyde-H, 2H), 8.52 (s, OH, 2H), 18H). 13 C NMR (DMSO-d6 ): ␦ 191.3 (s, C-45,45‘), 162.91 (s,C-39,
8.35 (s, Triazole-CH-36,36‘, 2H), 7.83 (d, J = 8.6 Hz, CH-41,43,41‘43‘, 39‘), 149.9 (s, C-9.23), 149.3 (s, C-1,15), 147.3 (s, C-37, 37‘), 142.2
4H), 7.18 (d, J = 8.6 Hz, CH-40,44,40‘,44‘, 4H), 7.14, 7.13 (s, CH- (s, C-12, 26), 141.6 (s, C-4, 18), 133.1 (s, C-2, 6, 16, 20), 131.7 (d,
100 B. Khan et al. / Journal of Hazardous Materials 309 (2016) 97–106

C-41, 43, 41‘, 43‘), 129.8 (s, C-8, 10, 22, 24), 127.4 (s, C-42, 42‘), Table 1
Crystal data and structure refinement for 8.
125.3, 125.7 (d, C-3, 5, 11, 13, 17, 19, 25, 27), 124.9 (d, C-36, 36‘),
115.2 (d, C-40, 40‘, 44, 44‘), 72.9 (t, C-33, 33‘), 61.5 (t, C-38, 38‘), Crystal data
46.4 (t, C-35, 35‘), 34.1 (s, C-29, 32), 33.6 (s, C-30, 31), 31.4 (q, C-30‘, C76 H91 N9 O8 F(000) = 2696
30“, 30“‘, 31‘, 31“, 31“‘), 31.2, 30.2 (t, C-7, 14, 21, 28), 30.9 (q, C-29‘, Mr = 1258.58 Dx = 1.181 Mg m−3
29“, 29“‘, 32‘, 32“, 32“‘), 30.4 (t, C-34,34‘). ESI-MS: found [M + H] Monoclinic, C2/c Mo K˛ radiation,  = 0.71073 Å
+ , m/z = 1135.62 and calcu M+ for C H N O , m/z = 1134.62. Anal. a = 30.712 (3) Å Cell parameters from 3263 reflections
70 82 6 8
b = 16.2996 (17) Å  = 2.3–26.0◦
Calcd for C70 H82 N6 O8 : C, 74.05; H, 7.28; N, 7.40 Found: C, 73.98; H,
c = 16.4627 (19) Å  = 0.08 mm−1
7.53; N, 7.34. ˇ = 120.787 (6)◦ T = 296 K
V = 7079.6 (14) Å3 Needle, colorless
Z=4 0.38 × 0.18 × 0.16 mm
2.5. Preparation of the bis-calix[4]arene tetra-trazole macrocycle Data collection
9 Bruker Kappa APEXII CCD 51737 measured reflections
diffractometer 6964 independent reflections
Radiation source: fine-focus sealed 3263 reflections with I > 2(I)
The condensation of dialdehyde 8 and commercially available
tube Rint = 0.101
terephthaloyl dihydrazide yielded bis-calix[4]arene macrocycle 9. Graphite monochromator max = 26.0◦ , min = 2.3◦
Compound 8 (100 mg, 0.09 mmol) was initially dissolved in hot Detector resolution: 8.00 pixels mm-1 h = −37 → 36
ethanol (40 mL), before it was added drop-wise into an ethano- ω scans k = −20 → 20
lic solution of terephthaloyl dihydrazide (17 mg, 0.080 mmol, 1.00 Absorption correction: multi-scan l = −20 → 20
(SADABS; Bruker, 2005)
equi). The reaction mixture was refluxed for ∼5 h, and yielded Tmin = 0.971, Tmax = 0.988
a white precipitate. The reaction mixture was cooled down to Refinement
room temperature, and the precipitate was subsequently gravity Refinement on F2 Hydrogen site location: inferred from
filtered before it was washed with acetone (20 mL), ethyl acetate Least-squares matrix: full neighbouring sites
R[F2 > 2(F2)] = 0.065 H-atom parameters constrained
(20 mL) and acetonitrile (20 mL). The crude precipitate was dis-
wR(F2) = 0.178 w = 1/[2(Fo
solved in dichloromethane and passed through a silica gel column S = 1.00 2) + (0.0747P) 2 + 1.2033P]
chromatography using dichloromethane: methanol (v/v = 6:4) as 6964 reflections where P = (Fo2 + 2Fc2)/3
the eluent. Compound 9 was obtained as a white powder in 53% 437 parameters (/) max < 0.001
yield (53 mg). mp 360–368 ◦ C; 1 H NMR (DMSO-d6 , at 300 MHz at 13 restraints max = 0.22 e Å−3
Primary atom site location: min = −0.20 e Å−3
298 K):␦ 11.84 (s, NH, 1H), 11.70 (s, NH, 3H), 8.54 (s, OH, 4H),
structure-invariant direct Extinction correction: SHELXL,
8.36 (s, CH-45,45‘, 4H), 8.25 (s, Triazole-CH-36,36‘,4H), 7.92 (s, CH- methods Fc* = kFc[1 + 0.001xFc23/sin(2)]-1/4
48,49,51,52, 48‘,49‘,51‘,52‘, 8H) 7.58 (d, J = 8.6 Hz, Ar-H, 8H), 7.16 (s, Secondary atom site location: Extinction coefficient: 0.00123 (19)
Calix-CH, 16H) 6.99 (d, J = 8.6 Hz, Ar-H, 8H), 5.11 (s, PhOCH2 , 8H), difference Fourier map
4.78 (br, t, NCH2, 8H), 4.17 (d, Jgem = 12.6 Hz, axial bridged methy-
lene CH2 ,8H), 4.03 (br, s, −OCH2 CH2 , 8H), 3.45 (d, Jgem = 12.6 Hz,
S4) and displayed characteristic signals corresponding to a bro-
equatorial bridged methylene CH2 , 8H), 2.56 (br, s, CH2 CH2 CH2 ,8H),
mopropoxy chain at ␦ 2.23 (-CH2 -), ␦ 3.83 (-CH2 -Br) and ␦ 4.16
1.18 (s, 36H, t-butyl), 1.11 (s, t-butyl-H, 36H). 13 C NMR ␦, 162.17 (s,
(Ar-OCH2 ). The presence molecular ion peak at 891.29 m/z [M + H]+
C-46,46‘, 53,53‘), 159.6 (s, C-39, 39‘) 150.0 (s, 9, 23), 149.5 (s, C-1,15)
(Fig. S3) in the ESI-MS spectrum further confirmed the formation
148.3 (d, C-45, 45‘), 147.3 (s, C-37, 37‘), 142.6 (s, C-12, 26), 141.6 (s,
bromo-substituted calix[4]arene 6. Nucleophilic substitution reac-
C-4, 18), 135.9 (s, C-47,50, 47‘50‘) 133.2 (s, C-2, 6, 16, 20), 128.8
tion between sodium azide and bromo-substituted calix[4]arene 6
(d, C-41, 43, 41‘, 43‘), 127.6, 127.4 (d, 48,49,51,52, 48‘,49‘,51‘,52‘),
yielded the azide-substituted calix[4]arene derivative 7 (isolated
127.1 (d, C-42, 42‘) 125.7, 125.4 (d, C-3, 5, 11, 13, 17, 19, 25, 27),
yield 90%). The chemical structure of compound 7 was established
124.5 (d, C-36, 36‘), 114.8 (d, C-40, 40‘, 44, 44‘), 73.2 (t, C-33, 33‘),
by NMR spectroscopy, EI-MS (Fig. S5–S8) analysis. Coupling of the
61.3 (t, C-38, 38‘), 46.6 (t, C-35, 35‘), 34.1 (s, C-29, 32), 33.6 (s, C-30,
azide-substituted calix[4]arene 7 with the acetylene substituted
31), 31.3 (t, C-7, 14, 21, 28) 31.4 (q, C-30‘, 30“, 30“‘, 31‘, 31“, 31“‘),
benzaldehyde 3 via click chemistry [66] afforded macromolecule 8
30.9 (q, C-29‘, 29“, 29“‘, 32‘, 32“, 32“‘), 30.4 (t, C-34,34‘), MALDI-TOF-
in a 78% isolated yield. The structure of macromolecule 8 was estab-
MS: found [M + Na] +, m/z = 2608.41 and calc for C156 H176 N20 O16 Na,
lished by NMR and ESI-MS. 1 H NMR and DEPT-135 (Fig. S9–S12).
m/z = 2608.35. Anal. Calcd for C156 H176 N20 O16 : C, 72.42; H, 6.86; N,
The formation of triazole rings in compound 8 through click cou-
10.83, Found: C, 72.58; H, 7.15; N, 10.64.
pling between 3 and 7 was established from the appearance of
aromatic proton singlet at ␦ 8.35 ppm in the 1 HNMR spectrum.
3. Results and discussion Furthermore a molecular ion peak appeared in ESI-MS ([M + H]
+ 1135.62 m/z) spectrum was consistent with the theoretically
,
3.1. Synthesis, characterization of macrocycle 9, and its calculated molecular mass of macromolecule 8. High quality crys-
precursors tals of compound 8 were also developed in acetonitrile, and this
compound was characterized via X-ray crystallography (Fig. 1 and
The synthesis of macrocyle 9 was achieved through the synthetic Table 1). Crystallographic data clearly indicated the cone confor-
route outlined in Scheme 1. Coupling of p-hydroxy benzaldehyde 1 mation of the calix[4]arene moiety with its triazole appended arms
with propargyl bromide 2 afforded O-proparargyl benzaldehyde 3 oriented nearly on the same side. Intramolecular O—H... O between
in 91% isolated yield. EI-MS (Fig. S1) and 1 H NMR (Fig. S2) anal- phenol hydroxy group ethereal O atom was observed. There are
ysis provided evidences for the formation of 3. Notably, the 1 H four acetonitrile moieties for one complete molecule. In the asym-
NMR spectrum exhibited a characteristic signal at 4.76 ppm cor- metric unit, the benzaldehyde ring, triazide moiety and benzene
responding to the methylene protons situated between the ether rings connected through bridging methylene are planar with r.m.s.
linkage and the terminal alkyne group. Treatment of calix[4]arene deviations of 0.0047, 0.0035, 0.0237 and 0.0083 Å, respectively. The
4 with dibromopropane 5 allowed the incorporation of two dibro- four benzene rings of the macrocycle 9 are twisted to the virtual
mopropane moieties at the lower rim of calix[4]arene thus yielding plane defined by four bridging methylene carbon atoms with dihe-
bromo-substituted calix[4]arene 6 in 68% isolated yield. The 1, dral angles ranging from 71.265 (103)◦ to 79.079 (73)◦ , respectively.
3-bromopropoxy calix[4]arene 6 was analysed by 1 H NMR (Fig. The three ending C-atoms of tertiary butyl of hydroxyl containing
 B. Khan et al. / Journal of Hazardous Materials 309 (2016) 97–106 101

Fig. 1. (a) Oak Ridge Thermal Ellipsoid Plot (ORTEP) diagram of 8. Thermal ellipsoids are drawn at a 30% probability level. The disordered and H- atoms are omitted for clarity.
(b) labelled chemical structure of compound 8.

benzene ring are disordered over three set of sites with occu-
pancy ratios of 0.40:0.33:0.27. All disordered atoms were treated
as anisotropic atoms having equal thermal parameters.
The condensation of macromolecule 8 and terephthaloyldihy-
drazide in ethanol under reflux for 5 h yielded the desired
macrocycle 9 in 53% yield. The macrocycle 9 was characterised
by NMR (1H, 13C, DEPT-135, COSY, HSQC, HMBC and NOESY, (Fig.
S13–S20) as well as with MALDI-TOF-MS. The MALDI-TOF-MS (Fig.
S13) of compound 9 exhibited molecular ion peak corresponding
to its sodium adduct [M+ Na] + peak at m/z = 2608.41.
The conformations of calix[4]arene derivatives have attracted
significant attentions since the pioneering work of Gutsche
[67–69]. A number of methods have been used to distinguish the
four main conformations adopted by these macrocyclic tetramers
but those based on the NMR spectroscopy have been the most use-
ful. The protons of the bridging methylenes preferentially exists in
non-identical environments and interconversion between the con-
formations in calix[4]arene derivatives 6, 7, 8 and 9 is restricted so
the bridging methylenes appear as doublets at ␦ 4.20 (axial pro-
ton) and 3.32 (equatorial proton) ppm. It appears that molecule
6–9 adopted flattened cone conformations due to hydrogen bond-
ing between phenol moieties. Additionally the alkyl groups were
sufficiently bulky to inhibit the oxygen-through-the-annulus rota-
tion and therefore result in conformationally immobile 6–9. The
difference in the chemical shift and multiplicities of aromatic pro-
tons and tert-butyl groups between 6 and 7 as well as 8, and 9 can
be attributed to the solvent effect as NMR spectra of 6 and 7 were
recorded in CDCl3 while for 8 and 9 recorded in DMSO. These rigid-
ified calix[4]arene derivatives 6, and 7 may have exciting potentials
as supramolecular hosts.
These features can be employed to influence the self−assembly
and complexation behavior of calix[4]arenes, along with the
reactivity of functional groups introduced at the upper rim.
Fig. 2. (a) Fluorescence emission spectra of 9 (5 ␮M) after mixing with cations (5 ␮M
Additionally, the conformational1y−rigidified calix[4]arenes are
in each case). These spectra were recorded in DMSO: H2 O (1:1, v/v) at an excitation
exceptionally useful in the design of ionophoric cavities with the wavelength of 325 nm. (b) The fluorescence intensity of 9 observed at various pH
desired size and preorganization of ligand groups values in DMSO: H2 O (1:1, v/v, 5 ␮M).

3.2. X−ray structural determination of compound 8: was used. Crystallographic data clearly indicated the cone confor-
mation of the calix[4]arene moiety with its triazole appended arms
Single crystal data was collected at 296 K on a Bruker Kappa oriented nearly on the same side. Intramolecular O—H... O between
APEXII CCD diffractometer equipped with a four-circle goniometer phenol hydroxy group ethereal O atom was observed. There are
and using MoK␣ graphite mono-chromated radiation. The crystal- four acetonitrile moieties for one complete molecule. In the asym-
lographic data was deposited at Cambridge Crystallographic Data metric unit, the benzaldehyde ring, triazide moiety and benzene
Center (CCDC 1017216). The refinement and all further calculations rings connected through bridging methylene are planar with r.m.s.
were carried out using SHELX-97. For molecular graphics PLATON deviations of 0.0047, 0.0035, 0.0237 and 0.0083 Å, respectively. The
102 B. Khan et al. / Journal of Hazardous Materials 309 (2016) 97–106

Fig. 3. (a) Fluorescence emission graph (max,exc = 325 nm) of a 9-Cu2+ complex (5 ␮M) in the presence of competing ions (5 ␮M). (b) Fluorescence emission spectra of 9
(5 ␮M), Cu2+ (5 ␮M), and the 9-Cu2+ (5 ␮M) complex recorded in tap water of Karachi University (Pakistan) (c) Fluorescence titration spectra of fluorophore 9 (5 ␮M) after
gradually increased concentrations (0–12 ␮M) of Cu2+ at the emission wavelength (484 nm) and excitation wavelength as 325 nm.

four benzene rings of the macrocycle 9 are twisted to the virtual emission intensity of macrocycle 9 was monitored after titrating
plane defined by four bridging methylene carbon atoms with dihe- mixture of compound 9 (5 ␮M) and Cu2+ (5 ␮M) against 5 ␮M
dral angles ranging from 71.265 (103)◦ to 79.079 (73)◦ , respectively. aqueous solution of each the metal cations (Fig. 3a). No significant
The three ending C-atoms of tertiary butyl of hydroxyl containing interference due to screened metal cations was observed. The prac-
benzene ring are disordered over three set of sites with occu- tical applicability of the designed fluorescence chemosensor 9 was
pancy ratios of 0.40:0.33:0.27. All disordered atoms were treated also evaluated (Fig. 3b) in tape. Tap water, spiked with 1 × 10−3 M
as anisotropic atoms having equal thermal parameters.Crystal data Cu2+ which was further diluted to 5 ␮M in same tape water and
and details of the data collection are summarized in Table 1. DMSO (1:1, v/v). Furthermore, fluorescence titrations of 9 (5 ␮M)
with Cu2+ (0–12 ␮M) revealed that the fluorescence intensity at
3.3. Cu2+ sensing studies of the macrocycle 11  = 484 nm decreased (Fig. 3c) with the increase in Cu2+ concen-
tration which determined the limit (Fig. S24) of 40 nM (0.04 ␮M),
The binding behavior of 9 (5 ␮M) with Cu2+, Mg2+, Ca2+, Ba2+, in DMSO: Water (v/v, 1:1). The observed reduction in fluorescence
Ag+, Zn2+, Ti4+,Cd2+, Hg2+, Pb2+, In3+, La3+, Cr3+, Ni2+, Sb3+, emission intensity is more sensitive than the Cu2+ concentration
V5+, Fe3+, Co2+, Sn2+ was evaluated through fluorescence spec- range in healthy body plasma samples [73,74]. This can act as diag-
troscopy. A significant quenching in fluorescence intensity of 9 due nostic tool to determine whether the plasma sample is healthy or
to selective interactions with Cu2+ (Fig. 2a) was observed which not.
may be attributed to its selective binding of 9 and its accessibil- The solid 9-Cu2+ host-guest complex was recovered by evap-
ity for Cu2+ ions. The Cu2+ can bind to the chelating functional orating the solvent (DMSO: H2O, 1:1 v/v). An effort was carried
groups of macrocycle 9 or a supramolecular inclusion complex can out to dissolve solid 9-Cu2+ host-guest complex in deuterated
be formed in the preorganized cavity [70–72]. The stability of com- NMR solvents (d6 -DMSO: D2 O, and duterted chloroform, acetoni-
plex formed between macrocycle 9 and Cu2+ was checked at various trile, methanol, pyridine etc) however we were unable to get any
pHs (2.4–12.7), tuning with aqueous solutions of HCl (1.0 M) or appreciable solubility of the complex in order to investigated in
NaOH (1.0 M). The results obtained (Fig. 2b) showed maximum flu- depth the expected Cu2+ perturbation in 1 HNMR signals of the
orescence quenching in alkaline pH range. Similarly, when pH of Bis-calixarene macrocycle 9. Moreover a camera photograph (SI,
the 9-Cu2+ complex (5 ␮M) in DMSO and H2 O (1:1, v/v) was tested Fig. S29) of solidified 9-Cu2+ complex examined under UV-lamp
without the addition of acid or base, the original pH solution was ( = 365 nm) showed a considerable fluorescence quenching as
found to be 7.8. This slightly alkaline pH can be attributed to the compared to the pure solid biscalixarene macrocycle 9.The solidi-
nitrogen moieties of 9 (C156 H176 N20 O16 ). All the succeeding titra- fied non-fluorescent 9-Cu2+ complexex (sparingly soluble in DMSO
tion experiments were therefore performed at the original pH range and HCCA solution) was analyzed for determination of the binding
(8.5–9.0 ± 1.5) and the use of any buffer was therefore excluded. ratio through MALDI-TOF-MS (Fig. 4, S26, S27, and S28). The MALDI-
The selectivity of 9 toward Cu2+ in the presence of aforemen- TOF-MS analysis revealed a 1:3 complex between biscalix[4]arene
tioned competing metal cations was carried out. The fluorescence 9 and Cu2+ by showing a peak at m/z = 2649.29 correspond to [9
 B. Khan et al. / Journal of Hazardous Materials 309 (2016) 97–106 103

Fig. 4. MALDI-TOF-MS spectrum of the 9-Cu2+ complex.

+ Cu(II)]+, a peak at m/z = 2711.22 correspond to [9 + Cu2]+ and a

peak at m/z = 2775.10 correspond to [9 + Cu3]+. The binding ratio
was also detremind by job plot method (Fig. S25). To investigate
the long term stability of our established chemosensing system a
freshly prepared solution (5 ␮M) of Bis-calix[4]arene was mixed
with Cu2+ spiked tap water solution (5 ␮M) and this was subjected
to observed fluorescence quenching phenomenon. The mixed solu-
tion was then left at room temperature for 15 days and again the
fluorescence spectrum was recorded. The day first results and what
we observed after 15 days of storage were found to quite resemble
(Fig. 5). Similarly, the interference due anions was also examined
using Cu2+salts with varying anions. The anions we used in this
study are mostly chlorides, sulphates and bromides. The response
of compound 9 toward CuCl2, CuSO4 and CuBr2 was investigated
and nearly the same results (almost overlapping) were obtained
as shown in Fig. S30. This shows that SO4−2, −Br and Cl are not
interfering between the interactions of Cu(II) and compound 9.
Furthermore, the analytical utility of fluorescent
chemo−sensing system Biscalix[4]arene 9 was established by Fig. 5. Fluorescence emission spectra of 9 (5 ␮M), the 9-Cu2+ (5 ␮M,) complex
titrating fluorescent macrocycle 9 against Cu2+ spiked in tap water recorded in tape water of Karachi University (Pakistan) as a fresh solution and after
15 days of storage at room temperature.
(collected from Karachi, Pakistan). The results obtained before and
after spiking are shown in SI Table 1 revealing a meant recovery of
95.17%. Morphological studies of T-cells with atomic force microscopic
analysis showed that Macrocycle 9 can easily cross the membrane
3.4. Cu2+ sensing by macrocycle 9 in living cells barrier of the T-cells and detect intracellular copper. The copper
accumulation in living organisms accompanied toxicity so T-cell
To elucidate the feasibility of macrocyclic fluorescent probe 9 lines were selected whereupon copper is known to be accumu-
for detection of copper in the cell, we carried out Atomic Force lated after incubation with Macrocycle 9 and Copper. The ability
Microscopic analysis of T-lymphocytes and selected it as test model. of Macrocycle 9 to cross the membrane of T-cells were studied by
104 B. Khan et al. / Journal of Hazardous Materials 309 (2016) 97–106

Fig. 6. (a) Morphological image of controlled T-cell, (b) Melting of T-cell after mixing with 50 ␮M solution of Cu+2 , (c) The T-cells treated with Macrocycle 9 (40 ␮M) and
40 ␮M of Cu+2 , (d) T cells mixed with small concentration (5 ␮M) of copper.

mixing T-cells with multiple doses of Macrocycle 9 in the range of positive) cells was determined using a FACS Calibur. Cytotoxicity
5–50 M for 20 min at 37 ◦ C. The cells were found to be coherently was expressed as the percentage of dead cells based on a histogram
intact with definite nuclear structure, indicating that the Macrocy- of PI-stained cells. Though copper play vital role, is an essential trace
cle 9 is harmless. Morphological changes in T cells were evaluated element, is a cofactor of various proteins and enzymes involved
with AFM after mixing with macrocycle 9 and then further incu- in oxygen metabolism. However excess copper ions are known to
bation with copper (10 M each) at 37 ◦ C for 20 min. Fig. 6a shows lead to necrosis or apoptosis through ROS (Reactive oxygen species)
image of controlled T-cell which is used as control throughout the production and oxidation of lipids or proteins [75] via Fenton or
microscopic studies. The T-cells treated with Macrocycle 9 (40 M) Haber-Weiss reactions [75], the antioxidant properties of copper
remained healthy as indicated by its cell shape and nuclear struc- ions involve it in the defense against ROS [76] It binds to DNA and
ture (Fig. 6c) however when the same T cells were further incubated induces formation of strand breaks and chromosomal aberrations
with copper, melting in the cell were observed (Fig. 6b). It can be by damaging DNA with ROS [77,78]. The results obtained from the
concluded that macrocycle 9 did not exhibit any adverse effect on cytotoxicity are provided in Fig. S31.
cell viability; similar results were obtained when the T cells were
further mixed with small concentration (5 M) of copper (Fig. 6d).
The cell viability reduced drastically after incubation with high 4. Conclusion
concentrated solution of copper, an increased in the viability of
the copper-poisoned cell was observed when the concentration of In conclusion, the synthesis protocol developed for preparation
macrocycle 9 was further increase. It can safely be conclude that the of macrocycle 9 can be used as a rewarding clue in creating numer-
cell viability increased after incubation with macrocycle 9, which ous other important bis-calix[4]arenes of varying intramolecular
was affected by copper. The intensity of copper poisoning of the cell cavities. The synthesized triazol incorporated fluorescent bis-
increased with increasing exposure time. The macrocycle 9 turn out calix[4]arenes 9 exhibited selective fluorescence quenching with
to be handy for the study of cytotoxicity caused by copper in living Cu2+ in the presence of a wide range of metal cations. Importantly,
cells. Furthermore, useful information obtained through AFM and the sensing system displayed the same Cu2+ triggered selective flu-
Surface Plasmon could be useful for sensitive detection of intra- orescence quenching when inspected directly in Cu2+ spiked tap
cellular copper. Matrix effect of the living cells in the FACs assay water, thus enabling the sensing system could be used to detect
was investigated. Significant (p < 0.05) change in SSC shift was not and quantify Cu2+ level in environmental water system. Further-
observed in the presence of compound 9 as compared to control more, the macrocycle 9 showed low Cytotoxicity and have been
cells. elegantly used for detection of Cu2+ in living cells, showing great
potential of detection and quantification of Cu2+ in blood serum
3.5. Cytotoxicity assay of macrocycle 9 and at cellular level.

Cytotoxicity of macrocycle 9 against immortalized human B

cells expressing HLA-DRB1*0101 (15310-LN) was estimated using Acknowledgments
flow cytometry-based PI staining. Briefly, cells were seeded at
7 × 104 cells/well (in 200 ␮L) in the presence of titrated amounts of This research is supported by organization for the Prohibi-
test samples. Cells were incubated for 4 h in an incubator at 37 ◦ C tion of Chemical Weapons based in Netherlands project number
and 5% CO2 , then were centrifuged, rinsed with wash buffer (PBS L/ICA/ICB/194480/14. This work has been conducted at the Interna-
with 5% FBS), and stained with PI (5 ␮L; 0.5 mg/mL) in a total volume tional Centre for Chemical and Biological Sciences (ICCBS), Karachi
of 300 ␮L FC buffer. The frequency of live (PI-negative) and dead (PI- University, Pakistan.
 B. Khan et al. / Journal of Hazardous Materials 309 (2016) 97–106 105

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