Sonochemical Decoration of Graphene Oxide with Magnetic Fe3O4@CuO Nanocomposite for Efficient Click Synthesis of Coumarin-Sugar Based Bioconjugates and Their Cytotoxic Activity

Article abstract of DOI:10.1007/s10562-019-02982-6

Abstract: A magnetic nanocomposite of GO-Fe₃O₄@CuO was fabricated via a simple sonochemical technique and successfully utilized for the ultrasound promoted synthesis of regioselective 1,4-disubstituted mono/bis/tris-1,2,3-triazoles (

Full text of DOI:10.1007/s10562-019-02982-6

Catalysis Letters
https://doi.org/10.1007/s10562-019-02982-6
Sonochemical Decoration of Graphene Oxide with Magnetic Fe3O4@
CuO Nanocomposite for Efcient Click Synthesis of Coumarin‑Sugar
Based Bioconjugates and Their Cytotoxic Activity
Yachana Jain1 · Mitlesh Kumari1 · Raman Preet Singh3 · Deepak Kumar3 · Ragini Gupta1,2
Received: 29 May 2019 / Accepted: 27 September 2019
© Springer Science+Business Media, LLC, part of Springer Nature 2019
Abstract
A magnetic nanocomposite of GO-Fe3O4@CuO was fabricated via a simple sonochemical technique and successfully utilized
for the ultrasound promoted synthesis of regioselective 1,4-disubstituted mono/bis/tris-1,2,3-triazoles (3a–k). This catalyst
could be separated conveniently from the reaction mixture using an external magnet and reused up to eight consecutive
runs without noticeable drop in the desired product yield. Other noteworthy features of this green protocol are negligible
metal leaching from the support during reaction, high yield in lesser time, aqueous media and good results with gram scale
synthesis. Representative compounds were also screened for their cytotoxic activity against PC-12 cell line using standard
MTT assay and fow cytometry. Trifuoromethyl group containing triazole derivative (3f) displayed cytotoxic activity (IC50
8.3 µg/mL) comparable to the standard drug cisplatin (IC50 5.8 µg/mL).
Graphic Abstract
Keywords Aqueous click reaction · Graphene oxide-Fe3O4@CuO · Ultrasonication · 1,2,3-Triazoles · Cytotoxic activity ·
PC-12 cell lines
1 Introduction
Click reactions are considered as a magical trick in a syn-
Electronic supplementary material The online version of this
thetic chemist’s tool box since these reactions are character-
article (https://doi.org/10.1007/s10562-019-02982-6) contains
ized by being biorthogonal, modular, wide in scope, show
good functional group tolerance with high atom economy,
ease of procedure with high yield of pure products which
can be fruitfully utilized for creating a combinatorial library
supplementary material, which is available to authorized users.
* Ragini Gupta
guptaragini@yahoo.com
Extended author information available on the last page of the article
Vol.:(0123456789)
1 3

Y. Jain et al.
of biologically important and diverse molecules [17, 28].
The basic requirement for a reaction to be classifed as a
“click reaction” is that it must satisfy the following criteria
such as (i) Show no sensitivity towards moisture or air, (ii)
Generate stereospecifc product in high yield, (iii) Should
furnish desired product even in the absence of a solvent or
in a benign one (iv) Should have easy product isolation pro-
tocol [5, 27].
amount of metal used in the reaction by immobilization and
allow recyclability of the catalyst. Besides this they also
prevent the formation of bistriazoles and diacetylenes which
are formed as byproducts of CuAAC reaction. making these
catalyst magnetically separable induces the advantage of
easy product recovery by simply use an external magnet and
make its reusability feasible in many consecutive cycles [1].
Recently, various carbonaceous nanomaterials like car-
bon nanotubes, nanofbers, graphene, and graphene oxide
have been used as support material in numerous applications
such as sensors [18], cell imaging, adsorbent, drug delivery,
catalysis [8, 33, 46] etc. Inherently they are endowed with
remarkable properties including fexibility, good accessibil-
ity, mechanical strength, large surface area, unique interac-
tion with metal particles, physical and electrical properties
[36]. Graphene oxide has an array of functional groups such
as carboxylic acid, hydroxyl, epoxy (–COOH, –OH, C–O–C)
on its surface which allows for easy chemical modifcation
and fabrication of nanocomposites with metals and metal
oxides [51].
The Huisgen 1,3-dipolar cycloaddition reaction [20, 40]
between azide and its coupling partner alkyne is the epitome
of click reaction resulting in a straightforward creation of
1,2,3-triazoles. This conventional reaction usually needs
high temperature and gives a mixture of 1,4- and 1,5-dis-
ubstituted 1,2,3-triazoles. Later on, Sharpless-Fokin [40]
and Meldal [50], working independently, in 2002 made it
regioselective using Cu(I) catalyst leading to the exclusive
formation of 1,4-disubstituted 1,2,3-triazole derivative.
Triazoles containing three nitrogen atoms is an important
heterocycle which displays, broad spectrum of biological
activities, such as antiviral, antibacterial [34], antifungal,
antituberculour, anticonvulsive, anti-infammatory, antitu-
mor [37], cytostatic [3, 38] etc. to name a few with possible
applications in DNA modifcation, bioconjugation [53], drug
discovery [28], material science, etc. and still their applica-
tions are increasing exponentially. Further, they also play an
impressive role as a linker in increasing biological activity
because they are stable under typical physiological condi-
tions and form hydrogen bonds which can be suitable for sol-
ubility improvement and for binding of biomolecular targets.
Fluorescent triazoles, using sugar scafold as a core with
diferent fuorogenic units (coumarin, anthracene, 1,8-naph-
thalimide) are gaining impetus because of their use in bio-
imaging [16], biosensing, biolabelling, and as chemosen-
sors for detection of ions [14, 25], small molecules and
biomolecules [13]. These fuorophores are easily achieved
by installing an azide or alkyne functional group in either a
sugar moiety or any fuorogenic framework which could be
subsequently “clicked” via CuAAC reaction.
Several improved methods including microwave and
ultrasonic irradiation have been reported to increase the
yield of the product. Ultrasound is a powerful technique in
organic synthesis in terms of enhanced rate of reaction, for-
mation of pure products, and high yield in less time [2, 6,
7, 35, 41].
In continuation to earlier work of on synthesis of dif-
ferent heterocycles via green and sustainable routes [15,
22–24, 29] herein, we have successfully fabricated mag-
netically separable catalyst and efectively applied it for
synthesis of fuorescent sugar-coumarin based 1,4-disub-
stituted 1,2,3-triazole derivatives using well known click
chemistry under ultrasonication. The results reveal that this
magnetically separable catalyst not only exhibits excellent
selectivity and activity, but can also be easily recovered by
using simple permanent magnet from the reaction mixture
and reused eight times without any appreciable drop in its
catalytic activity. In addition, high product yield, mild reac-
tion conditions, simple work-up procedure and stability of
the catalyst are other attractive features of this method. This
work represents the fusion of many pervasive green chemis-
try themes: magnetically recoverable catalysts, recyclability
and aqueous “click reaction”.
Generally, CuAAC reaction proceeds in diferent sol-
vents such as tetrahydrofuran (THF), acetonitrile (CH3CN),
dimethyl sulfoxide (DMSO), or mixed solvents but for bio-
logical application solely aqueous media is preferred. Cu(II)
salts with reducing agent (generally sodium ascorbate) or
Cu(I) salts in the presence of a base are commonly used for
homogeneous catalytic systems but use of coloured toxic
copper salts hinders its practical application and feasibility
on a large scale synthesis due to metal contamination of
end product and recyclability issues. To overcome afore-
mentioned limitations many heterogeneous supports [42, 43]
have been applied as an alternative to homogeneous copper
catalysts for CuAAC such as organic polymers [49], zeolites,
charcoal [44], alumina, silica [45], clay [10], magnetic mate-
rials [19] and polysaccharides [12] which can minimizes the
2 Results and Discussion
2.1 Characterization of GO‑Fe3O4@CuO
Nanocomposite
A semi-heterogeneous nanocatalyst has been developed
using ultrasonic irradiation by combining graphene
oxide, magnetite and copper oxide which can dispersed
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Sonochemical Decoration of Graphene Oxide with Magnetic Fe3O4@CuO Nanocomposite for…
homogeneously in aqueous media for obtaining fuorescent
1,2,3-triazole. Initially, graphene oxide was synthesized
using modifed Hummer’s method [21, 30, 56] which was
then sonochemically decorated by Fe3O4 and CuO nano-
particles on its surface (Fig. 1). Nanocomposite was then
characterized by vibrating sample magnetometer (VSM),
thermogravimetric analysis (TGA), High resolution trans-
mission electron microscopy (HRTEM), energy dispersive
X-ray spectroscopy (EDS), X-ray photoelectron spectros-
copy (XPS), and X-ray difraction (XRD).
Figure 3a shows the X-ray difraction (XRD) pattern of
GO, showing difraction peak at 2 theta=10.5° which cor-
respond to 002 plane of graphene oxide. The XRD pattern
of GO-Fe3O4@CuO (Fig. 3b) nanocatalyst show difrac-
tion peaks at 2 theta 30.1°, 35.6°, 43.1°, 53.9°, 57.0°, 62.7°
correspond to (220), (311), (400), (422), (551) and (440)
difraction planes of magnetic nanoparticles, respectively
(JCPDS card no. 19-0629) [32]. The XRD result for the
prepared catalyst indicates that the crystal structure of the
Fe3O4 core does not change during the immobilization on the
surface of GO. The signals of Copper were not observed in
XRD pattern, indicating that Cu species is highly dispersed.
No characteristic peaks of other impurities were detected.
The TGA thermogram of GO shows a thermal decompo-
sition around 160 °C which is attributed to the vaporization
of oxygen containing functional groups which are present
on the GO surface [9] (Fig. 3c). Figure 3d presents the TGA
curve of graphene oxide GO-Fe3O4@CuO, in which frst
weight loss was 2.3% at 200 °C, which may be assigned to
the loss of absorbed residual or water and/or organic solvents
used during the preparation of catalyst. An obvious weight
loss (8.7 wt%) between 200 and 600 °C is observed, which
can be assigned to the decomposition and vaporization of
Magnetically separation of the catalyst from the reac-
tion mixture depends on the magnetic property of the nan-
oparticles. Magnetic properties of GO-Fe3O4@CuO was
investigated using vibrating sample magnetometer (VSM)
with the feld sweeping from − 20,000 to + 20,000 Oe at
room temperature as shown in Fig. 2. Lower saturation
magnetization (Ms) of Fe3O4 and GO-Fe3O4@CuO nano-
composite are 60 emu g−1 and 50 emu g−1 respectively
which clearly revealed magnetic nature. The Ms value of
prepared nanocomposite was less compared to pure mag-
netite. However, the magnetization is still large enough
and could be easily exploited for removal of the catalyst
from reaction mixture.
Fig. 1 Schematic representation
of synthesis of GO-Fe3O4@
CuO nanocomposite
Fig. 2 VSM magnetization curve of GO-Fe3O4@CuO
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Y. Jain et al.
Fig. 3 XRD analysis of a graphene oxide, b GO-Fe3O4@CuO and TGA analysis of c graphene oxide, d GO-Fe3O4@CuO
various oxygen-containing functional groups at diferent
positions on the surface of the GO-Fe3O4@CuO composite.
Composite shows better thermal stability than GO due to
the presence of Fe3O4@CuO composite on graphene oxide
sheets which does not allow easy degradation of the oxygen
functionalities [54]. The GO-Fe3O4@CuO catalyst possesses
good thermal stabilities (up to 600 °C), which meets the
demands for potential applications in catalysis.
respectively. The HRTEM image in Fig. 4d shows the char-
acteristic lattice fringes of Fe3O4 and CuO nanoparticles
about 0.252 nm and 0.3 nm respectively. Furthermore, ele-
mental mapping was also carried out to illustrate the space
distributions of the component elements. Figure 4e–h exhibit
the individual distribution of elements in prepared nano-
composite. The amount of copper was found 8.33 wt% in
composite by using atomic absorption spectroscopy.
Transmission electron microscopy (TEM) and high res-
olution electron microscopy (HRTEM) analysis were also
performed for Fe3O4 and GO-Fe3O4@CuO nanocomposite.
TEM images clearly revealed that Fe3O4 and CuO nanopar-
ticles were successfully embedded on multilayer graphene
oxide sheets. The TEM image in Fig. 4a shows magnet-
ite nanoparticles which are somewhat spherical in shape,
with some cubic partials and HRTEM image of Fe3O4 (4a,
inset) the characteristic lattice fringes with the d-spacing of
0.23 nm corresponds to the [311] plane of Fe3O4. The TEM
images shows wrinkled sheets of GO which demonstrate
high degree of oxidation [58]. CuO nanoparticles exhibits
spindle like structure and Fe3O4 almost spherical in shape
(Fig. 4b, c). The average width and length of spindle-shaped
CuO nanoparticles were about 20 to 40 nm and 80 to 100 nm
Additionally, XPS was performed to acquire further
insight into the valence information of the GO-Fe3O4@
CuO composite. Full survey spectrum of the sample given
in supplementary information (Fig. S1), which indicates
the presence of Cu, Fe, O and C without obvious impuri-
ties. The C1s spectra can be deconvoluted into three main
components corresponding to carbon atoms in diferent
functional groups, which exhibits the presence of C=C at
284.39 eV, C–O at 285.60 eV and C=C–O at 290.24 eV
(Fig. 5a). These values are in good agreement with pre-
viously reported values [18, 57]. The binding energies of
Cu2p3/2 and Cu 2p1/2 in GO-Fe3O4@CuO found to be around
934.5 eV and 954.20 eV respectively indicating the presence
of Cu2+ (Fig. 5c). The gap between the Cu 2p1/2 and 2p3/2
level is about 20 eV that is in agreement with the standard
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Sonochemical Decoration of Graphene Oxide with Magnetic Fe3O4@CuO Nanocomposite for…
Fig. 4 a TEM image of Fe3O4 (HRTEM image of Fe3O4 inset). b, c TEM images. d HRTEM image and e–h EDS elemental mapping of GO-
Fe3O4@CuO
Fig. 5 XPS analysis. a C1s, b O1s, c Cu2p of the GO-Fe3O4@CuO sample
spectrum of CuO [11, 31]. Figure 5c also shows two addi-
tional shake-up peaks at 943.29 and 963.00 eV, suggesting
the presence of an unflled Cu 3d shell and thus further con-
frming the existence of Cu2+ in the sample which are posi-
tioned at higher binding energies compared to those of the
main peaks. The peaks at 710 eV and 723 eV for Fe2+ and
at 712 eV, 718, 725 characteristic peaks of Fe2p1/2 of Fe3+.
for GO-Fe3O4@CuO mediated triazole synthesis, the efect
of diferent reaction parameters such as amount of catalyst,
reaction time, temperature and solvents have been studied.
Click reaction of azide and alkyne to aford regioselec-
tive 1,4-disubstituted triazoles are usually catalyzed by using
copper salts [52]. To afrm this fact, initially, the model
reaction was performed using sugar azide (1a, 1 mmol) and
coumarin alkyne (2a, 1.1 mmol) in water without any cata-
lyst under ultrasonication at 45 °C for 80 min. No product
formation was observed as revealed by thin-layer chroma-
tography (TLC) monitoring even after 8 h. To ensure that
only copper plays a role as catalyst each component (GO,
Fe3O4, GO-Fe3O4) of the prepared catalyst (GO-Fe3O4@
CuO) under the same conditions cited above were studied
2.2 Catalytic Performance
Catalytic performance of GO-Fe3O4@CuO was assessed
in the synthesis of 1,4-disubstituted mono/bis/tris triazole
derivatives using sugar azide (1a) and coumarin alkyne (2a)
as model substrates. To obtain an optimum reaction profle
1 3

Y. Jain et al.
(Table 1, entries 1–3). It was observed that only trace amount
of product was obtained with GO, Fe3O4, GO-Fe3O4 which
clearly established that copper is essential for CuAAC reac-
tion. This fact was also corroborated when the reaction was
performed with prepared catalyst (GO-Fe3O4@CuO) which
gave 97% yield under identical reaction conditions (Table 1,
entry 5). Catalyst was also compared it with the conventional
catalysts (CuSO4.5H2O/Na ascorbate (1 mol%/5 mol%)) in
water, only 40% product yield was observed in 2 h which
may be due to the low interaction of initial compounds with
water (Table 1, entry 4). Good catalytic activity of prepared
catalyst (GO-Fe3O4@CuO) can be explained on the basis
of hydrophilic nature of GO which has –COOH and –OH
groups on its surface making the reaction feasible in aque-
ous media due to its high dispersity and facile interaction of
Cu with organic substrates and also by “Breslow efect” [4].
The productivity of the click reaction is majorly afected
by the amount of catalyst so a series of reactions were per-
formed using 1a and 2a by varying the catalyst concentration
(2, 4, 6, 8, 10 mg) while keeping other parameters like tem-
perature, solvent, time substrate amount constant. Results
are shown in Fig. 1 which revealed that increase in amount
of catalyst from 2 to 6 mg (2,4, 6 mg) resulted in improve-
ment of the transformation of the initial reactants to the
desired product. A further increase in the catalytic amount
(8, 10 mg) did not lead to any signifcant increase in the
conversion. So, the optimal amount of GO-Fe3O4@Cu was
fxed to 6 mg for the rest of the experiments.
In order to observe the efect of temperature, the model
reaction was carried out at various temperatures (25, 35,
45, 55 °C) for 80 min under ultrasonication using GO-
Fe3O4@CuO nanocomposite and the results are given in
Fig. 6. Detailed examination of the results revealed that
45 °C was optimum temperature for the maximum conver-
sion of the reactants when the reaction was carried out for
80 min under ultrasonication (Fig. 7). For observing the
efect of ultrasonication same reaction was repeated using
simple magnetic stirring at 45 °C for 80 min, only 60%
conversion was observed. So, ultrasonication is used for
rest of the experiments. Excitingly, ultrasonic radiations as
an efcient and convenient process, could shorten reaction
Fig. 6 Optimization of amount of catalyst for model reaction
Table 1 Comparative screening of other catalyst and prepared catalyst for the CuAAC reaction
OAc
OAc
O
O
O
O
N
N
N
catalyst, H2O
O
AcO
AcO
AcO
AcO
N3
OAc
OAc
))))), 45 °C, 80 min
O
1a
2a
3a
O
O
Entry
Catalyst
Amount
Yield (%)a
1
2
3
4
5
GO
Fe3O4
10 mg
10 mg
10 mg
2.49 mg/9.9 mg
10 mg
<10
<10
<15
40b
GO-Fe3O4
CuSO4.5H2O/Na ascorbate
GO-Fe3O4@CuO
97
Reagents and conditions peracetylated glucose azide (1a) (1 mmol), coumarin alkyne (2a) (1.1 mmol), in aqueous medium was ultrasonicated in
open air at 45 °C for 80 min
aIsolated yield of product
bReaction proceeded at high temperature (60 °C) for 2 h
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Sonochemical Decoration of Graphene Oxide with Magnetic Fe3O4@CuO Nanocomposite for…
of water as green solvent at ambient temperature under
ultrasonication.
Mechanism of copper catalyzed synthesis of 1,2,3-tria-
zoles has been recently been reviewed by Fokin who pro-
posed a revisited mechanism whereby a σ-bound Cu(I)-
acetylide bearing a π complexed copper atom reacts with
an organoazide forming a bridging dicopper µ-acetylide
intermediate [40, 55]. Based upon their studies we proposed
following mechanism for the formation of 1,2,3-triazoles
(Fig. S2). In order to investigate the reusability of the cata-
lyst, the model reaction was performed under the optimized
reaction conditions. Upon completion of the reaction, the
catalyst was separated by means of an external magnet
after each run, washed with ethanol and dried and then sub-
jected to the next run under the same conditions. It is obvi-
ous from Fig. S3 that there was no signifcant decrease in
conversion yield % even after eight runs, indicating robust-
ness and reusability of the prepared catalyst. The leaching
test of metal (Copper) after recycling was investigated by
determining content of the metal in reaction solution using
FAAS. Results are revealed that small amount of copper
leached from the support to the reaction solution. The loss
of insignifcant copper metal from the surface of GO may
explain decrease yield of compound 3a during consecutive
runs. To check the heterogeneous nature of catalyst, a hot
fltering experiment was performed for using model reac-
tion under identical reaction conditions. The reaction was
stopped after 30 min, and the catalyst was removed by means
of simple external magnet. The fltrate was further subjected
into reaction vessel and reaction continued for the remaining
period of time (30 min). After the reaction time, the mixture
was cooled and extracted with ethyl acetate to identify the
product and yield. No further conversion was noticed in the
fltrate, which clearly supported that the catalysis is hetero-
geneous in nature. Filtrate was also analyzed by FAAS, it
was found that concentration of copper in the fltrate cor-
responds to negligible catalyst leaching. Subsequently, the
recovered catalyst stability was assessed using XRD and
TEM analyses. The TEM analysis of the recovered catalyst
after the eighth run (Fig. S5) showed that TEM image of
recycled catalyst (after the eighth run) was very similar to
the fresh catalyst image which revealed that the morphology
of the catalyst remains unaltered after the reaction. The XRD
pattern (Figure S6) of the reused catalyst showed similar set
of peaks to those of the fresh one and no additional peaks
were observed, which indicates that the recovered catalyst is
highly stable even after the eight consecutive runs.
Fig. 7 Efect of temperature on model reaction under ultrasonication
Fig. 8 Efect of solvent on click reaction
time. Also, the yields of the obtained desired product were
improved compared with conventional heating.
Subsequently, the activity of the GO supported hetero-
geneous magnetically separable catalyst was evaluated for
the click reaction using diferent solvents in the presence
of the model substrates. The efect of solvents on the syn-
thesis of triazole derivatives is illustrated in Fig. 8. Results
indicated that the best catalytic performance was observed
when water was employed as solvent. The efciency of the
catalyst also calculated by Turnover Number (TON) and
Turnover Frequency methods (TOF). TON can be defned
as number of moles of product per mole catalyst and TOF
is the simply turn over number per unit time. TON and
TOF value were found to be 3200 and 53.33 min−1 respec-
tively (for model reaction).
To further explore the potential of this catalyst for
1,4-disubstituted 1,2,3-triazole synthesis, various alkynes
(2a–k) and sugar azide (1a) were subjected to the click
reaction under the above optimized reaction conditions
using GO-Fe3O4@Cu nanocomposite (Table 2). Analysis
of the results showed that all of the substrates produced
the expected regioselective 1,4-disubstituted triazole
with excellent yield, indicating remarkable catalytic ef-
cacy of the GO-Fe3O4 based copper catalyst. The most
attractive attributes of the present methodology are that
the click reaction is devoid of unwanted products and
the catalyst can be recycled for eight consecutive cycles
without appreciable drop in its catalytic activity and use
2.3 Comparison with the Literature Precedents
The efciency of the GO-Fe3O4@CuO magnetically sepa-
rable catalyst was compared with the previously reported
catalytic system for the synthesis of triazole derivatives.
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Y. Jain et al.
Table 2 Scope and versatility of the prepared catalyst using sugar azide (1a) and various alkynes (2a–k) in aqueous media for CuAAC
OAc
OAc
GO-Fe3O4@Cu (6 mg), H2O
O
N
N
N
O
AcO
AcO
AcO
AcO
N3
OAc
3a-k
OAc
))))), 45 °C, 80 min
2a-k
1a
Entry
Azide
Alkyne
Triazole
Time Yieldb Product
TON/
(1a)
(min)
%
code
3a
TOF min-1
OAc
1.
60
96
3200/53.33
O
N
N
AcO
AcO
N
OAc
OAc
O
O
O
O
O
AcO
AcO
N3
OAc
O
O
2a
OAc
2
60
95
3b
3c
3166/52.76
O
N
N
AcO
AcO
OAc
N
O
O
O
O
OAc
AcO
AcO
O
N3
OAc
CH3
O
O
2b
OAc
3
60
95
3166/52.76
O
N
N
AcO
AcO
OAc
N
O
O
O
OAc
O
AcO
AcO
C2H5
O
N3
OAc
C2H5
O
2c
O
OAc
OAc
4
80
85
3d
2833/35.41
OAc
OAc
O
O
O
AcO
O
O
AcO
AcO
OAc
N
N
N3
OAc
N
O
N
N
O
AcO
AcO
N
OAc
O
O
2d
O
O
OAc
5
6
70
60
90
93
3e
3f
3000/42.85
3100/51.66
O
N
N
AcO
AcO
OAc
N
O
O
O
OAc
O
AcO
AcO
O
N3
O
OAc
O
2e
O
O
OAc
O
N
N
AcO
AcO
OAc
O
O
O
N
O
OAc
AcO
AcO
CF3
O
N3
OAc
CF3
O
2f
O
OAc
OAc
OAc
7
80
85
3g
2833/35.41
O
OAc
AcO
AcO
AcO
AcO
AcO
OAc
N3
O
OAc
O
O
N
N
O
N
AcO
N
O
N
N
N
N
N
N
N
N
O
O
N
O
N
N
O
OAc
AcO
2g
AcO
AcO
1 3

Sonochemical Decoration of Graphene Oxide with Magnetic Fe3O4@CuO Nanocomposite for…
Table 2 (continued)
OAc
OAc
OAc
OAc
CHO
O
8
60
60
60
95
94
92
3h
3i
3166/52.76
3133/52.22
3100/51.66
O
O
AcO
AcO
N
N
AcO
AcO
N3
N3
N3
OAc
N
O
OAc
2h
OHC
OAc
CHO
9
O
O
N
N
AcO
AcO
AcO
AcO
N
NO2
O2N
O
O
OAc
OAc
2i
2j
OHC
OAc
10
3j
O
AcO
AcO
O
N
N
AcO
AcO
OAc
N
OAc
OAc
OAc
11
60
95
3k
3166/52.76
O
O
AcO
AcO
N
N
AcO
AcO
N3
OAc
N
OAc
2k
All reactions were carried out using sugar azide (1 mmol), terminal alkynes (1.1 mmol), GO-Fe3O4@Cu (6 mg) at 45 °C temperature under
ultrasonication
aIsolated yields
From Table S1, it is evident that the newly synthesized cop-
per catalyst is far more profcient in terms of product yield,
time, temperature and cost. Besides, the catalyst can be eas-
ily recovered through a simple magnet without fltration,
centrifugation or sedimentation techniques and reused for
eight consecutive cycles without any signifcant loss in its
catalytic activity making it an interesting alternative for cop-
per catalyzed click promoted triazole synthesis.
0.1 ml/well of MTT dissolving solution (0.01 M HCl in 20%
w/v sodium dodecyl sulphate) and absorbance at 595 nm was
recorded using an ELISA plate reader.
Cytotoxicity of samples 3a-f was determined following
48 h incubation with PC-12 cells. The IC50 values of 3a
and 3e were>500 µg/mL while those of 3b, 3d and 3f were
382.8, 73.9 and 8.3 µg/mL, respectively (Fig. 9). The IC50
value of 3f was comparable to that of cisplatin (5.8 µg/mL)
[26, 47]. Results revealed that compound 3f shows promis-
ing results.
3 Cytotoxic Studies
Forward scatter (FSC) is an indicator of cell size/vol-
ume and an increase in FSC indicates an increase in cell
size/volume. As observed in the above Fig. S4A, the FSC
distribution in 3a, 3d and 3e was comparable to control sug-
gesting no change in cell size/volume. On the other hand,
cells treated with 3b were found to be relatively larger
in size while cells exposed to 3f were relatively smaller.
Side scatter (SSC) is an indicator of cell granularity and
an increase in SSC indicates an increase in granularity
(Fig.S4B). SSC distribution in cells treated with all com-
pounds was shifted towards right indicating an increase in
granularity. The increase in granularity followed the order:
3e < 3d < 3a < 3f < 3b. Figure 10 is FSC-SSC correlation
plots of Control, and sugar coumarin based triazoles (3a,
3b, 3d, 3e, 3f).
Cytotoxicity of representative compounds (3a, 3b, 3d, 3e, 3f)
were tested against rat pheochromocytoma cell line (PC-12)
by using standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-
nyltetrazolium bromide (MTT) assay [48]. Rat pheochro-
mocytoma cell line (PC-12) purchased from National Centre
for Cell Science, Pune, India were maintained in RPMI-1640
supplemented with 10% FBS and antibiotic–antimycotic
solution. The PC-12 cells in the log phase were seeded in
96-well plates at a concentration of 0.1 mL/well and incu-
bated overnight at 37 °C in a 5% CO2 CO2/95%. Working
solutions of the compounds (0.05, 0.5, 5, 50 and 500 µg/
mL) were prepared from a 10 mg/mL stock solution by serial
dilution in culture medium. The working solutions were
added to cell suspension (100 µL/well) and further incubated
for 48 h. The cell viability was determined by MTT assay. A
50 mg/ml MTT stock was prepared in DMSO and diluted to
5 mg/mL in RPMI-1640. The working solution (5 mg/mL)
was added in each well (0.02 ml/well) and incubated for
3 h. Thereafter, the formazon was dissolved by addition of
The results of FSC and SSC were also confrmed by FSC-
SSC correlation plots. Cells treated with 3a constituted of
two types of cell populations: the frst population subset
exhibited FSC-SSC values similar to the control cells while
the second population subset (highlighted in black oval)
exhibited SSC values greater than control cells. Cells treated
1 3

Y. Jain et al.
Fig. 9 Cytotoxic activity of sugar coumarin based triazoles (3a, 3b, 3d, 3e, 3f) against PC-12 cells
with 3b also exhibited a population subset with increase in
SSC along with an increase in SSC (highlighted in black
oval) while a general decrease in FSC along with an increase
in SSC was observed in 3f-treated cells. No signifcant devi-
ations from control cells were observed in cells treated with
3d and 3e.
times without any discernible drop in its catalytic activ-
ity. Easy work-up procedure, mild reaction conditions and
broad substrate scope are additional attributes of the present
methodology. Results of cytotoxic studies of representative
compounds revealed that compound having trifuoromethyl
group 3f showed best results with IC50 (8.3 µg/mL) compa-
rable to that of cisplatin (5.8 µg/mL) against PC-12 cells.
4 Conclusions
5 Experimental Section
Decoration of GO with Fe3O4 and CuO on its surface sono-
chemically, furnishes a semi-heterogeneous nanocomposite
which can be homogeneously dispersed in water, rendering
the hydrophobic organic reactants interact fruitfully to cre-
ate the desired product quantitatively in less time at 45 °C.
AAS results revealed that the catalyst is devoid of leaching
problem. The facile recovery of prepared catalyst by means
of an external magnet allows the catalyst to be recycled eight
5.1 Materials and Method
All chemicals were purchased from commercial sources and
were used as received. The purity of all the compounds was
checked by TLC using silica gel as adsorbent and solvents of
increasing polarity as mobile phase. 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide (MTT), RPMI-1640,
1 3

Sonochemical Decoration of Graphene Oxide with Magnetic Fe3O4@CuO Nanocomposite for…
Fig. 10 FSC-SSC correlation plots of a control, b 3a, c 3b, d 3d, e 3e, f 3f compounds
fetal bovine serum (FBS), antibiotic–antimycotic solution
(100×) were obtained from HiMedia, Mumbai, India. Cis-
platin was a gift sample from Venus Remedies Pvt. Ltd.,
Panchkula, India. Melting points were determined in open
glass capillaries using Gallenkamp melting point apparatus
which was then dispersed in isopropanol solution using an
ultrasonic bath. Some drops of the sample were dropped
onto the copper grid and isopropanol was evaporated at
room temperature. Thermo gravimetric analysis (TGA) was
done on a Mettler thermal analyzer in an inert atmosphere
at a heating rate of 10 °C/min in the range of 10–800 °C to
determine the loading of organic molecules on the surface
of magnetite. Magnetization measurement were performed
on vibrating sample magnetometer (VSM) (quantum design
MPMS) at 300 K. X-ray photoelectron spectroscopy (XPS)
measurements recorded in ESCA+ omicron nanotechnology
oxford instrument. Powder X-ray difraction (XRD) pattern
of the sample was obtained with X-ray Difractometer (Pana-
lytical X Pert Pro) using Cu Kα radiation. Ultrasonication
(Elma S 70 H) with 37 kHz output frequency was used for
synthesis of desired products. Acetylation of glucose and
fructose was carried out by adopting previously reported
procedure [39]. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-
nyltetrazolium bromide (MTT), RPMI-1640, fetal bovine
serum (FBS), antibiotic–antimycotic solution (100×) were
obtained from HiMedia, Mumbai, India. Cisplatin was a gift
sample from Venus Remedies Pvt. Ltd., Panchkula, India.
and are reported uncorrected. 1H NMR (400 MHz) and 13
C
(100 MHz) NMR were recorded on a Jeol ECS 400 MHz
spectrophotometer using CDCl3 as a solvent. TMS was taken
as an internal standard and chemical shifts are reported in δ
ppm. FTIR spectra were recorded on a Perkin Elmer Spec-
trum 2 spectrophotometer using pressed KBr discs in the
region of 4000–400 cm−1. Mass spectra were recorded on a
Xevo G2-S Q-Tof spectrometer (Waters, USA), capable of
recording high resolution mass spectrum (HRMS) in the ESI
(Electrospray Ionization) mode. The visualization of sur-
face morphologies of prepared catalyst (GO-Fe3O4–Cu) was
done by using a feld emission scanning electron microscope
(FESEM) Nova Nano FE-SEM 450 (FEI) operating at 20 kV.
High resolution transmission electron microscope (HRTEM)
images were measured using a Tecnai G2 20 (FEI) S-Twin
transmission electron microscope at 200 kV and equipped
with an energy-dispersive X-ray detector (EDX). Sample
preparation was done by grinding a small amount of sample
1 3

Y. Jain et al.
2. Banerjee B (2017) Ultrason Sonochem 35:1–14
5.2 Synthetic Procedure for Synthesis of GO‑Fe3O4@
CuO
3. Benci K, Mandic L, Suhina T et al (2012) Molecules (Basel, Swit-
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39:5902–5907
GO was synthesized using modifed Hummer’s method [21,
30, 56]. To prepare this composite, Graphene oxide in 25 mL
water was taken in a fat bottom fask and ultrasonicate for
60 min for dispersion. Then, 1 mmol Fe2+, 2 mmol Fe3+ salts
and 2 M NaOH were slowly added into a reaction vessel.
After the mixture ultrasonicate for 1.5 h, CuNO3 and 2 M
NaOH were added simultaneously and slowly into reaction
vessel. After the mixture ultrasonicate for 1.5 h, precipitate
was formed which was then separated, washed with deion-
ized water (3×10) and ethanol (2×10) and dried.
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chem 40:587–593
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5.3 Typical Procedure for the Synthesis
of 1,4‑Disubstituted 1,2,3‑Triazoles Catalyzed
Using the GO‑Fe3O4@CuO Catalyst (3a–k)
13. Ghosh D, Rhodes S, Hawkins K et al (2015) New J Chem
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53:82–90
A fat bottom fask containing coumarin alkyne (1.1 mmol,
0.220 g), sugar azide (1 mmol, 0.373 g), water (10 mL) and
GO-Fe3O4-Cu (6 mg) catalyst was ultrasonicated at 45 °C.
After completion of the reaction as monitored through thin
layer chromatography (TLC), the catalyst was removed by
means of magnet. The reaction mixture was then extracted
with ethyl acetate (10 mL × 3), and dried over anhydrous
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1
Na2SO4. Finally, the H NMR and HRMS analysis of the
products was done to confrm the structures of the resultant
1,4-disubstituted 1,2,3-triazole derivatives.
5.4 Gram scale Synthesis of 1,4‑Disubstituted
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Triazoles (3a)
In a fat bottom fask, to a mixture of sugar azide (3 mmol,
1.119 g) and coumarin alkyne (3.3 mmol, 0.660 g), GO-
Fe3O4-Cu catalyst (18 mg) was added in 10 ml water. The
reaction mixture was ultrasonicated until TLC show com-
pletion of reaction at 45 °C. The catalyst was removed from
reaction mixture using an external magnet. The resulting
reaction mixture was extracted with ethyl acetate and dried
over anhydrous Na2SO4. Product was obtained with 93%
isolated yields as a white solid.
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Acknowledgements Authors are thankful to Materials Research
Centre, MNIT Jaipur for providing spectral facilities. Y. Jain and M.
Kumari are acknowledges to MNIT Jaipur for providing fnancial assis-
tance and DST, New Delhi for awarding INSPIRE fellowship scheme
(IF140506) respectively.
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Afliations
Yachana Jain1 · Mitlesh Kumari1 · Raman Preet Singh3 · Deepak Kumar3 · Ragini Gupta1,2
1
3
Department of Chemistry, Malaviya National Institute
of Technology Jaipur, Jaipur 302017, India
School of Pharmaceutical Sciences, Shoolini University,
Solan 173229, India
2
Materials Research Centre, Malaviya National Institute
of Technology Jaipur, Jaipur 302017, India
1 3