M. Budri, R. Vadavi, P. Kadolkar et al.
Polyhedron 197 (2021) 115046
((CH3)3), 29.21 (C-(CH3)3), 34.71 (C-(CH3)3), 112.18 (C6), 158.32
(C7), 167.01 (C8), 136.54 (C17), 128.24 (C16), 139.69 (C11),
+
121.15 (C10), 110–140 (C1-C4, aromatic). EI-MS: m/z – 384 [M]
.
2.2. Synthesis of zinc complex
A 4% aqueous acetonitrile solution of Zn(NO3)2ꢂ6H2O (0.77 g,
2.6 mmol) was slowly added to the 4% aqueous acetonitrile solu-
tion of L (1 g, 2.6 mmol) with constant stirring for 10 h. The yellow
solid obtained was filtered, washed with cold 4% aqueous acetoni-
trile and dried in air.
Color: Yellow, yield: 75%, m.p: 340 °C, Anal. Calcd. for C22H28N2-
O5Zn (%): C, 56.72; H, 6.06; N, 6.01; Zn 14.04. Found (%): C, 56.80;
H, 6.14; N, 6.05; Zn 12.40. IR (cmꢁ1): 3489 (OAH), 1575(C@N),
1617 (C@N, new), 1128 (CAO). 1H NMR (400 MHz, DMSO d6,
ppm): 1.29 (9H, s, tert-Bu, C14H, C15H, C16H), 1.39 (9H, s, tert-
Bu, C20H, C21H, C22H), 8.54 (1H, s, C7H), 14.13 (1H, s, O4-H),
6.00–8.00 (Ar-H), ESI-MS (m/z): 467 [M + H]+. Molar conductance
Fig. 2. Relative absorbance of probe at 372 nm upon interaction with various metal
ions.
(X
-1cm2 molꢁ1): 3.35.
2.3. Cytotoxicity assay and bioimaging experiment
The cytotoxic effect of probe and its Zinc complex on HeLa cells
was ascertained by MTT assay. HeLa cells were grown in tissue cul-
ture flask in Dulbecco’s modified Eagle medium (DMEM) supple-
mented with 10% (v/v) fetal bovine serum (FBS) + penicillin
(100 g/mL) + streptomycin (100 lg/mL) at 37 °C in a CO2 incubator.
HeLa cells were plated (approximately 104 cells per well) into a 96-
well flat-bottom micro plate and grown at 37 °C in 95% humidity
and 5% CO2 for overnight. After that, HeLa cells were incubated
with different concentrations of L and its Zinc complex in DMEM
solution for period 24 h. The cells were washed with phosphate
buffer solution and 20
lL of the MTT staining solution was added
to each well and incubated at 37 °C for 5 h. 100
lL of dimethyl sul-
foxide was added to each well to dissolve formazan crystals formed
and absorbance was recorded at 570 nm using micro plate reader
(ELISA) [43].
For cell imaging experiment, cultured HeLa cells were treated
with the 10
37 °C in dark. HeLa cells were washed with PBS buffer and corre-
sponding images were taken. Later on, 10 M of Zinc was incu-
lM of probe and incubated separately for 30 min at
Fig. 3. Electronic spectral changes of L (1 ꢀ 10ꢁ5 M) in the presence of different
concentrations of Zn2+ ions in 4% aq. CH3CN. Inset: UV–Vis absorbance at 372 nm
versus the number of equiv. of Zn2+ added.
l
bated for 4 h and respective photographs were captured [44].
2. Experimental section
3. Results and discussion
2.1. Synthesis of optical probe
3.1. UV–Vis absorption titration of Zinc (II) ion
Synthetic path for fluorophore is shown in Scheme 1.
The electronic spectrum of probe (1ꢀ 10-5 M) in 4% aqueous
acetonitrile exhibits three absorption maxima at 340, 299 and
290 nm. The electronic properties of L with various metal ions
3, 5-di-tert-butyl-2-hydroxybenzohydrazide was prepared by
the reported method [42]. Methanolic solution of 2, 4-dihydroxy-
benzaldehyde (2 g, 14.5 mmol) was slowly added to a methanolic
solution of 3, 5-di-tert-butyl-2-hydroxybenzohydrazide (3.8 g,
14.5 mmol) and stirred for 7 h at room temperature. Completion
of the reaction was monitored by TLC. The precipitate obtained
was filtered, washed with cold methanol and dried in air.
Color: Light yellow: 89%, m.p: 265 °C, Anal. Calcd for
(viz.; Al3+ (M(NO3)3)), Ca2+, Ba2+, Mg2+, Mn2+, Ni2+, Zn2+, Cu2+
,
Co2+, Cd2+, Pb2+, Hg2+ (M(NO3)2) and K+, Li+, Na+ (M(NO3) was pur-
sued in 4% aqueous acetonitrile. Amongst various metal ions (10
equivalent of each) tested, only Zn2+ ion induces significant change
in the electronic spectrum of L showing its higher selectivity for
Zn2+ ion (Fig. S9). Colorless solution immediately turns to intense
yellow (Figs. 1–2). Upon the addition of Zn2+ ion, the intensity of
bands at 340, 299 and 290 nm have decreased and at the same
time three new bands at 429, 390 and 372 nm have appeared.
The formation of equilibrium points at 310 and 353 nm confirms
the transformation of free L into its Zinc complex (Fig. 3) [44].
Upon slowly titrating Zinc ion with L, intensity of bands at 429,
390 and 372 nm increases while that of 340, 299 and 290 nm
decreases till saturation point is reached. A band at 340 nm exhi-
bits 15 nm blue shift due to increased ICT character of L due to for-
C
22H28N2O4 (%): C, 68.73.; H, 7.34; N, 7.29. Found (%): C, 68.69;
H, 7.30; N, 7.32. IR (cmꢁ1): 3470 (OAH), 3187 (NAH), 1631
(C@O), 1616 (C@N). 1H NMR (400 MHz, DMSO d6, ppm): 1.30
(9H, s, tert-Bu, C13H, C14H, C15H), 1.37 (9H, s, tert-Bu, C19H,
C20H, C21H), 7.71 (1H, d, C1H, J = 2 Hz), 6.33 (1H, d, C4H,
J = 2.4 Hz), 7.43 (1H, d, C16H, J = 2 Hz), 11.99 (1H, s, N2H), 13.08
(1H, s, O4H), 12.25 (1H, s, O2H), 10.00 (1H, s, O1H), 7.42 (1H, d,
C10H, J = 2 Hz), 8.64 (1H, s, C7H), 6.33 (1H, d, C4H, J = 2.4 Hz);
13C NMR (100 MHz, DMSO d6, ppm): 31.30 ((CH3)3), 34.22
5