Magnetic chitosan-copper nanocomposite: A plant assembled catalyst for the synthesis of amino- and N-sulfonyl tetrazoles in eco-friendly media

Article abstract of DOI:10.1016/j.carbpol.2019.115819

A greener, cost efficient and simple method is described to prepare copper nanoparticles (NPs) immobilized on the magnetic chitosan (one of the more versatile polysaccharides) using Euphorbia falcata leaf extract as reducing/stabilizing agent. The prepare

Full text of DOI:10.1016/j.carbpol.2019.115819

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Magnetic chitosan-copper nanocomposite: A plant assembled catalyst for
the synthesis of amino- and N-sulfonyl tetrazoles in eco-friendly media
Narjes Motahharifar, Mahmoud Nasrollahzadeh, Asghar
Taheri-Kafrani, Rajender S. Varma, Mohammadreza Shokouhimehr
PII:
S0144-8617(19)31487-0
DOI:
https://doi.org/10.1016/j.carbpol.2019.115819
CARP 115819
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Carbohydrate Polymers
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7 October 2019
25 December 2019
30 December 2019
Please cite this article as: Motahharifar N, Nasrollahzadeh M, Taheri-Kafrani A, Varma RS,
Shokouhimehr M, Magnetic chitosan-copper nanocomposite: A plant assembled catalyst for
the synthesis of amino- and N-sulfonyl tetrazoles in eco-friendly media, Carbohydrate
Polymers (2019), doi: https://doi.org/10.1016/j.carbpol.2019.115819
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Magnetic chitosan-copper nanocomposite: A plant assembled catalyst for the
synthesis of amino- and N-sulfonyl tetrazoles in eco-friendly media
Narjes Motahharifar,a Mahmoud Nasrollahzadeha,* Asghar Taheri-Kafranib, Rajender S. Varmac and Mohammadreza
Shokouhimehrd,*
aDepartment of Chemistry, Faculty of Science, University of Qom, Qom 3716146611, Iran
bDepartment of Biotechnology, Faculty of Advanced Sciences and Technologies, University of Isfahan, Isfahan 81746-73441, Iran
cRegional Centre of Advanced Technologies and Materials, Department of Physical Chemistry, Faculty of Science, Palacky University, Šlechtitelů
27, 783 71 Olomouc, Czech Republic
dDepartment of Materials Science and Engineering, Research Institute of Advanced Materials, Seoul National University, Seoul 08826, Republic
of Korea
*Corresponding authors e-mail addresses: mahmoudnasr81@gmail.com (M. Nasrollahzadeh), and mrsh2@snu.ac.kr (M. Shokouhimehr).
Graphical abstract
1

Highlights

Synthesis of the magnetic chitosan-copper nanocomposite, Cu NPs@Fe3O4-chitosan.

Cu NPs@Fe3O4-chitosan was characterized by FT-IR, XRD, TG/DTG, FESEM, TEM, HRTEM, VSM,
elemental mapping and EDS.


Synthesis of amino- and N-sulfonyl tetrazoles in eco-friendly media using Cu NPs@Fe3O4-chitosan.
The catalyst can be easily recycled and reused five times without much loss in its catalytic efficiency.
ABSTRACT
A greener, cost efficient and simple method is described to prepare copper nanoparticles (NPs) immobilized on the
magnetic chitosan (one of the more versatile polysaccharides) using Euphorbia falcata leaf extract as
reducing/stabilizing agent. The prepared catalyst (Cu NPs@Fe3O4-chitosan) was authenticated by field emission
scanning electron microscope (FESEM), X-ray diffraction (XRD), transmission electron microscopy (TEM),
Scanning transmission electron microscopy (STEM), energy-dispersive X-ray spectroscopy (EDS),
thermogravimetry/derivative thermogravimetry (TG/DTG), Vibrating sample magnetometer (VSM), and elemental
mapping. TEM analysis indicates that Cu NPs with average sizes in 5-10 nm range is formed on magnetic chitosan
with the spherical morphology. The Cu NPs@Fe3O4-chitosan was employed as a new catalyst for the synthesis of
different tetrazoles by the reaction of various secondary or tertiary cyanamides with sodium azide in water under
reflux conditions. Easy separation by external magnetic field, mild reaction conditions, low cost and the reusability
are some of the beneficial features of this catalyst.
Keywords: Magnetic chitosan; Polysaccharide; Cu NPs@Fe3O4-chitosan; Nanocatalyst; Tetrazoles; Eco-friendly
1. Introduction
Tetrazoles are a versatile member of five-membered nitrogen heterocycles with unique properties such as highest
acidity and number of N atoms, high formation enthalpy and lowest basicity with good stability. Among the azole
heterocycles, tetrazoles manifest extensive abilities in diverse research areas as it has been applied in the medicinal
chemistry as lipophilic spacers, material science as analytical reagents, propellants, and particularly as ligands for the
2

preparation of complexes (Frija, Cristiano, Gómez-Zavaglia, Reva, & Fausto, 2014; Frija, Ismael, & Cristiano, 2010;
Ichinari, Nagaki, & Yoshida, 2017; Koldobskii, 2006; Nasrollahzadeh, Sajjadi, & Khonakdar, 2018; Ostrovskii,
Popova, & Trifonov, 2017; Pino-Gonzalez, Romero-Carrasco, Calvo-Losada, Oña-Bernal, Quirante, & Sarabia, 2017;
Wei, Bian, & Gong, 2015). Besides that, tetrazole derivatives have been effectively utilized as starting materials for
synthesis of N-containing compounds (Burger, 1991; Frija, Ismael, & Cristiano, 2010; Gutmann, Glasnov, Razzaq,
Goessler, Roberge, & Kappe, 2011). The importance of tetrazole-containing compounds as effective antitumor agents,
antihypertensive drugs, and peptidase inhibitor has been also highlighted in the documented literature (Arshad, Bhat,
Pokharel, Kim, Lee, Athar, & Choi, 2014; ElMonaem, Abdel-Aziz, Morsy, Badria, ElSenduny, El-Ashmawy, &
Moustafa, 2016; Sajjadi, Nasrollahzadeh, & Sajadi, 2017; Schmidt, & Schieffer, 2003; Tanaka, Okuda, Yamamoto,
2004; Wexler, Greenlee, Irvin, Goldberg, Prendergast, Smith, & Timmermans, 1996).
Owing to widespread application, the research on their catalytic synthesis has been of huge interest, and
particularly, arylaminotetrazoles have been a hot topic of fundamental investigation in view of their pharmaceutical
and biological applications (Henry, Finnegan, & Lieber, 1954; Herr, 2002; Katritzky, Rees, & Scriven, 1996; Peet,
Baugh, Sundler, Lewis, Matthews, Olberding, & Shah, 1986; Singh, Chawla, Kapoor, Paul, & Malhotra, 1980). The
replacement of N atom affords N-substituted arylaminotetrazoles such as N-alkyl-, N-acyl-, and N-sulfonyl-
arylaminotetrazoles and based on their unique structures, properties and applications, their synthesis should receive
immense attentions, but for the lack of tenable procedures and inadequate synthetic methods (Miller, Feeney, Ma,
Zarcone, Aziz, & Magnuson, 1990; Nasrollahzadeh, Sajjadi, Komber, Khonakdar, & Sajadi, 2019).
Traditional synthetic procedures have been reported for synthesis of aminotetrazole which entail (i) the
condensation of carbodiimides, cyanamides, imidoyl chlorides, aminoiminomethanesulfonic acids, and/or thioureas
with sodium azide (NaN3), (ii) reaction of aminoguanidine with sodium nitrite (NaNO2), (iii) reaction of tetrazoles-
comprising a leaving group in 5-position with suitable amines, (iv) conversion of 1-aryltetrazoles to 1-
arylaminotetrazoles via an one step consecutive ring-opening procedure by using NaN3 and organolithium reagents
under harsh conditions at -70 °C, (v) the reaction of cyanamide derivatives with hydrazoic acid (HN3), and (vi) 1-
substituted tetrazoles isomerization in the melt state or boiling ethylene glycol (Scheme 1) (Congreve, 1996;
Garbrecht, & Herbst, 1953; Habibi, Nasrollahzadeh, Faraji, & Bayat, 2010; Henry, Finnegan, & Lieber, 1955; Sajjadi,
Nasrollahzadeh, & Sajadi, 2017; Nasrollahzadeh, Habibi, Shahkarami, & Bayat, 2009; Vorobiey, Gaponik, Petrov, &
Ivashkevich, 2006). Among the published protocols, most of them have several disadvantages e.g. the use of highly
3

toxic azide source, expensive/toxic reagents, high boiling solvents (N,N-dimethylformamide), homogeneous catalysts,
tedious work-up, lower yields, harsh reaction conditions, and also formation of a mixture of isomers that restrict their
applications.
Although arylaminotetrazoles have been recently accessed via [2 + 3] cycloaddition of monosubstituted
cyanamides with NaN3 using various catalysts like zeolite (Nasrollahzadeh, Habibi, Shahkarami, & Bayat, 2009),
ZnCl2 (Habibi, Nasrollahzadeh, Faraji, & Bayat, 2010), HOAc (Modarresi-Alam & Nasrollahzadeh, 2009), FeCl3-
SiO2 (Habibi, & Nasrollahzadeh, 2010), Ag/Fe3O4 (Sajjadi, Nasrollahzadeh, & Sajadi, 2017) and Cu(II) complex
(Nasrollahzadeh, Sajjadi, & Khonakdar, 2018), thus far, there is no report for the synthesis of N‐sulfonyl tetrazoles.
Hence, the advancement of straightforward approaches and eco-friendly catalytic procedures for the N‐substituted
tetrazoles is warranted to circumvent the abovementioned drawbacks and limitations.
Scheme 1. Synthesis of aminotetrazoles using different methods.
Additionally, homogeneous catalytic systems (Yella, Khatun, Rout, & Patel, 2011) in various processes need to be
substituted with heterogeneous systems due to inherent advantages of their recoverability, reusability, regenerability,
and ease of handling (Baig, Nadagouda, & Varma, 2014; Li, & Guan, 2017; Nasrollahzadeh, Sajjadi, Komber,
4

Khonakdar, & Sajadi, 2019; Polshettiwar, & Varma, 2009; Rathi, Gawande, Pechousek, Tucek, Aparicio, Petr, &
Zboril, 2016; Wang, Li, Zhang, O'Connor, Varma, Yu, & Hou, 2018). Over the decades, metal/metal oxide NPs like
Pd, Ag, Au, Cu, Fe3O4, and TiO2 have been investigated (Gawande, Branco, Varma, 2013; Hu, Liu, Wang, Liu, Liu,
Jing, & Zhang, 2013; Nasrollahzadeh, Sajjadi, & Khonakdar, 2018; Oi, Choo, Lee, Ong, Hamid, & Juan, 2016;
Sharma, Dutta, Sharma, Zboril, Varma, & Gawande, 2016; Titirici, White, Brun, Budarin, Su, del Monte, &
MacLachlan, 2015; Varma, 2016; Xu & Wang, 2012), but the clustering of various NPs is generally inescapable. Due
to environmental and economic significance, the catalysts heterogenization on the novel supports has been
investigated especially magnetic NPs on polymer materials have garnered maximum attention as proficient
nanocatalysts because of their propitious properties e.g. high surface energy, thermal stability, small size, high
catalytic activity, and high magnetic permeability, among others (Hu, Liu, Wang, Liu, Liu, Jing, & Zhang, 2013;
Nasrollahzadeh, Sajjadi, & Khonakdar, 2018; Nasrollahzadeh, Sajjadi, Komber, Khonakdar, & Sajadi, 2019; Sharma,
Dutta, Sharma, Zboril, Varma, & Gawande, 2016).
The selection of renewable biopolymers such as alginate, egg-shell membrane, cellulose, starch, chitin, and
especially chitosan (CS) comprise a pivotal aspect in the promotion of biodegradable catalysts. CS is one of the
distinctive and most versatile biopolymers encompassing linear polysaccharides with reactive amino groups (N-
deacetylated chitin), with relatively biocompatible, and inexpensive abundance compared to other biopolymers
(Krajewska, 2004; Lee, Chen, & Den, 2015). The fusion of biopolymers and magnetic NPs can afford fascinating
nanomagnetic support for easy recovery of nanocatalysts; the surface functionalities of CS are easily accessible for
immobilization of metal/metal oxide NPs.
Several physicochemical approaches have been used for the synthesis of metal NPs but mostly suffer from
limitations that present potential environmental risks which has prompted search for alternative eco-friendly and
sustainable procedures. Nowadays, a variety of greener resources have been deployed to fabricate NPs which avoid
the use of toxic reagents and organic solvents, harmful stabilizers and/or capping agents, longer reaction times, and
high temperature calcinations (Banerjee, Satapathy, Mukhopahayay, & Das, 2004; Kou & Varma, 2012; Nadagouda
& Varma, 2008; Nasrollahzadeh, Sajjadi, & Khonakdar, 2018; Nasrollahzadeh, Sajjadi, Komber, Khonakdar, &
Sajadi, 2019; Plachtovꢀ, Medꢁꢂkovꢀ, Zboꢁil, Tuꢃek, Varma, & Marꢄꢀlek, 2018; Varma, 2012; Varma, 2014). Among
these biological methods, the processes based on plant extracts or gums have been described in literature (Banerjee,
Satapathy, Mukhopahayay, & Das, 2004; Kou & Varma, 2012; Nadagouda & Varma, 2008; Narayanan & Sakthivel,
5

2011; Plachtovꢀ, Medꢁꢂkovꢀ, Zboꢁil, Tuꢃek, Varma, & Marꢄꢀlek, 2018), the main benefits being an easy reaction
system operating under soft conditions, application of safe solvents like water, absence of noxious and hazardous
materials as well as compatibility for pharmaceutical and biomedical utilization (Abdel-Halim, El-Rafie, & Al-Deyab,
2011; Qazi, Hussain & Tahir, 2016).
The genus Euphorbia (spurge family) comprise several species as herbs, shrubs and trees with a wide distribution
in the world; Euphorbiaceae family plants contain latex with unique flower structures (Citoğlu & Özbilgin, 2012;
Irshad, Rubina, Wahib, Rukhsana, & Choudhary, 2009; Noori, Chehreghani, & Kaveh, 2009; El Bribri, Tabyaoui,
Tabyaoui, El Attari, & Bentiss, 2013).
Herein, we report the production of 1-aryl-5-amino-1H-tetrazoles (B isomer), 5-arylamino-1H-tetrazoles (A
isomer) and N‐ sulfonyl‐ N‐ aryl‐ 5‐ amino‐ 1H‐ tetrazoles via the reaction of secondary or tertiary cyanamides
with NaN3 in refluxed water using Cu NPs@Fe3O4-chitosan as a recyclable magnetic nanocatalyst with extraordinary
catalytic prowess (Scheme 2) without the use of toxic reagents such as HN3. The Cu NPs@Fe3O4-chitosan was formed
via decoration of Cu NPs onto Fe3O4-chitosan nanostructure by Euphorbia falcate extract as a reducing and stabilizing
agent. Green chemistry is reflected in the entire process of synthesis. 1-Aryl-5-amino-1H-tetrazoles (B isomer) and 5-
arylamino-1H-tetrazoles (A isomer) were prepared in DMF in our research group, previously (Nasrollahzadeh, Habibi,
Shahkarami, & Bayat, 2009). In the present work, tetrazoles were prepared in water as a green solvent. Water is cheap,
non-combustible, non-toxic, non-explosive, and environmentally acceptable. The use of water as a solvent has several
advantages, including avoiding the use of hazardous substances, preventing the generation of waste and minimization
of energy requirements. The water-insolubility of tetrazoles further facilitates their isolation. Results show that the Cu
NPs loaded on Fe3O4-chitosan exhibit excellent catalytic activity and good stability. On the other hand, to the best of
our knowledge, this is the first report for the synthesis of N‐ sulfonyl‐ N‐ aryl‐ 5‐ amino‐ 1H‐ tetrazoles.
6

Scheme 2. Synthesis of arylaminotetrazoles and N-sulfonyl-N-aryl-5-amino-1H-tetrazoles using Cu NPs@Fe3O4-
chitosan nanocatalyst.
2. Experimental section
2.1 Instruments and reagents
All materials were procured from the Merck/Aldrich chemical company. Chitosan (De-acetylation degree: 85%,
molecular weight: 50000-80000 Da (medium), particle dimension: powder with mesh size of 80, dissolvable in acetic
acid 1%) was purchased from Nano Novin Polymer (NNP) Co., prepared by Shrimp shell waste. The structure of the
products was confirmed by FT-IR, 1HNMR, 13CNMR, XRD, UV-visible, TEM, FESEM, EDS, and VSM. FT-IR, UV-
Vis and NMR spectra were conducted by Perkin-Elmer 781, Hitachi-U-2900 and Bruker spectrometer Avance DRX-
400 and 600 MHz, respectively. The Cu NPs@Fe3O4-chitosan size and morphology were characterized applying a
TEM, JEM-F200 JEOL and FESEM, Hitachi S-4700. Next analysis, VSM and XRD measurements that were validated
by Lake shore VSM-7410 magnetometer at 298 K and Philips PW 1373 diffractometer, respectively. The Cu
NPs@Fe3O4-chitosan chemical composition was analyzed by EDS equipped in FESEM. TG/DTG analysis was
performed by STA 1500 Rheometric-Scientific.
2.2 Preparation of extract of Euphorbia falcata
To achieve a suitable concentration of antioxidant phenolics (e.g; flavonoids) as highly soluble compounds in water,
50 g of dried plant in 300 mL double distilled water was heated for 0.5 h at 70 ºC. Then after cooling to room
7

temperature, the produced extract was centrifuged at 7000 rpm, then filtered and filtrate was kept in the refrigerator
for the synthesis of Cu NPs.
2.3 Synthesis of Cu NPs
10 mL of 5 M CuCl2⋅2H2O solution was combined with 100 mL of aqueous extract at 70 °C. After 7 min, the mixture
color was changed to dark due to Cu NPs formation. Then, the biosynthesized nanoparticles were separated via
centrifugation at 7000 rpm for 30 min. Finally, the obtained precipitation washed with absolute alcohol to remove
possible impurities.
2.4 Preparation of magnetic chitosan (MCS)
Fe3O4 NPs were prepared according to the literature by using co-precipitation method in a basic media (Sohrabi,
Rasouli, & Torkzadeh, 2014). The chitosan has not appropriate functional groups to graft directly onto Fe3O4 NPs,
thus it should be carboxymethylated and then covalently bound on Fe3O4 NPs with carbodiimide activation. This step
is as follows:
Solution of chitosan was prepared via dissolving 0.25 g CS in 50 mL of solution of acetic acid 1% (V/V). Then, 2.0 g
of Fe3O4 NPs that prepared in previous step was added to the CS solution at ambient temperature and stirred for 0.5
h, and then NaOH solution (50 mL, 1.0 M) was added slowly. Finally, the obtained Fe3O4-chitosan was collected
using external supper magnet and washed by ethanol, distilled water and acetone and kept under vacuum drying.
2.5 Synthesis of the Cu NPs@Fe3O4-chitosan
To prepare of the Cu NPs@Fe3O4-chitosan, 1.0 g MCS and 0.5 g CuCl2.2H2O was mixed with 100 mL aqueous extract
and stirred at 80 ºC for 4 h. The produced precipitation was removed by decantation using an external magnet, washed
with deionized water and finally dried.
2.6 Synthesis of N‐substituted‐N‐aryl‐5‐amino‐1H‐tetrazoles in water
An appropriate cyanamide (1.0 mmol), Cu NPs@Fe3O4-chitosan (0.08 g), NaN3 (1.1 mmol), and water (15 mL) were
stirred under reflux conditions. After affirming the end of process by TLC, the catalyst was removed using external
supper magnet and 25 mL ethyl acetate and 25 mL HCl (2 M) were added to the mixture reaction. The organic phase
was isolated by a separator, and then the resulting aqueous phase was extracted twice with ethyl acetate. The products
were obtained via concentration of organic phases and then recrystallization from aqueous ethanol to give pure
tetrazoles.
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2.7 Spectroscopic data for new compounds
5-(2,4-Dichlorophenyl)amino-1H-tetrazole
M.p. 218-220 °C; FT-IR (KBr, cm-1): 3299, 3136, 1658, 1589, 1489, 1392, 1317, 1243, 1135, 1089, 1030, 878, 824,
1
748, 695, 662, 593, 557, 510, 449; H NMR (400 MHz, DMSO-d6): δ 11.97 (s, 1H), 7.94 (d, J = 2.4 Hz, 1H), 7.87
H
(d, J = 8.8 Hz, 1H), 7.61 (d, J = 8.8 Hz, 1H), 7.05 (s, 1H); 13C NMR (100 MHz, DMSO-d6): δ 155.4, 133.5, 132.6,
C
132.5, 132.1, 126.9, 125.3; Anal. Calcd for C7H5N5Cl2: C, 36.55; H, 2.19; N, 30.44. Found: C, 36.66; H, 2.21; N,
30.55.
N-(4-Chlorophenyl)-4-methyl-N-(1H-tetrazol-5-yl)benzenesulfonamide
FT-IR (KBr, cm-1): 3471, 3098, 3067, 2981, 1628, 1596, 1487, 1398, 1358, 1307, 1294, 1274, 1244, 1211, 1187,
1
1162, 1121, 1088, 1039, 1016, 960, 931, 812, 717, 664, 634, 617, 583, 549, 518, 490, 444, 441, 425; H NMR (600
MHz, DMSO-d6): δ 7.67 (d, J = 8.2 Hz, 2H), 7.37 (d, J = 8.5 Hz, 2H), 7.35 (d, J = 8.5 Hz, 2H), 7.18 (d, J = 8.2 Hz,
H
2H), 2.37 (s, 3H); 13C NMR (150 MHz, DMSO-d6): δ 159.9, 144.0, 138.4, 135.1, 132.0, 129.5, 129.0, 128.2, 21.1;
C
Anal. Calcd for C14H12ClN5O2S: C, 48.07; H, 3.46; N, 20.02. Found: C, 48.15; H, 3.57; N, 20.13.
4-Bromo-N-(4-bromophenyl)-N-(1H-tetrazol-5-yl)benzenesulfonamide
FT-IR (KBr, cm-1): 3437, 2940, 2921, 1627, 1572, 1481, 1364, 1171, 1068, 1012, 965, 928, 821, 744, 707; 1H NMR
(600 MHz, DMSO-d6): δ 7.82 (d, J = 8.6 Hz, 2H), 7.72 (d, J = 8.6 Hz, 2H), 7.52 (d, J = 8.7 Hz, 2H), 7.14 (d, J = 8.7
H
Hz, 2H); 13C NMR (150 MHz, DMSO-d6): δ 159.3, 138.7, 137.4, 132.13, 132.10, 130.2, 129.4, 127.5, 120.6; Anal.
C
Calcd for C13H9Br2N5O2S: C, 34.01; H, 1.98; N, 15.25. Found: C, 34.10; H, 2.09; N, 15.34.
4-Bromo-N-(4-chlorophenyl)-N-(1H-tetrazol-5-yl)benzenesulfonamide
FT-IR (KBr, cm-1): 3105, 3063, 1645, 1591, 1575, 1516, 1485, 1471, 1411, 1390, 1375, 1353, 1339, 1304, 1286,
1275, 1239, 1203, 1180, 1172, 1111, 1087, 1068, 1062, 1038, 1016, 1010, 987, 965, 929, 827, 818, 784, 744, 722,
710, 699, 670, 633, 612, 566, 520, 497, 490, 454, 440; 1H NMR (600 MHz, DMSO-d6): δ 7.89 (d, J = 8.7 Hz, 2H),
H
7.70 (d, J = 8.7 Hz, 2H), 7.28 (d, J = 8.7 Hz, 2H), 7.52 (d, J = 8.7 Hz, 2H); 13C NMR (150 MHz, DMSO-d6): δ 157.7,
C
136.2, 135.9, 133.9, 132.7, 130.3, 130.2, 129.8, 128.7; Anal. Calcd for C13H9BrClN5O2S: C, 37.66; H, 2.19; N, 16.89.
Found: C, 37.78; H, 2.22; N, 17.01.
3. Results and discussion
9

In this study, a simple process for the preparation of the Cu NPs@Fe3O4-chitosan is presented via in situ decoration
of Cu NPs on the Fe3O4-chitosan substrate biosupport in presence of E. falcate leaf extract (Scheme 3). The easy
synthetic procedure afforded catalyst and its catalytic prowess was investigated for the synthesis of a variety of
tetrazoles.
Scheme 3. Procedure for the preparation of Cu NPs@Fe3O4-chitosan.
3.1 Characterization of extract and the catalyst
The phytochemical study of E. falcata extract was previously described (Sulyok, Vasas, Rédei, Forgo, Kele, Pinke, &
Hohmann, 2011). E. falcata phytochemical constituents include antioxidant phenolics such as quercetin, kaempferol
and rutin as aglycones or bearing sugar moieties and can be used for biological synthesis of Cu NPs. Four diterpenes
were isolated via the method introduced by Sulyok et al. from the E. falcata extract by a combination of different
chromatographic methods. First, the synthesis of the Cu NPs (Scheme S1) was performed using polyphenolics of
extract as reducing/stabilizing agents.
Figure S1 shows peaks at 235 and 351 nm owing to benzoyl and cinnamoyl systems, respectively (Bhat,
Nagasampagi, & Sivakumar, 2005). Moreover, Figure S2 described the changes in the maximum absorbance of Cu2+
10

ions undergo SPR phenomenon and Cu NPs production. Further, the stability of synthesized NPs was investigated to
15 days using UV-Vis spectroscopy. The FT-IR analysis of the crude extract (Figure S3) showed signals in 3400,
1715 and 1445 cm-1 which represent stretching vibrations of OH, C=O and C=C, respectively as the functional groups
of phenolics. The FT-IR analysis of Cu NPs depicted the signals around 3500, 1725, 1428, 1300 to1000 cm-1 represent
the stretching vibrations of OH, C=O, C=C and C-OH, respectively (Figure S4). The nanoparticles stability refers to
the adsorbed polyphenolics on the Cu NPs surface. Cu NPs structure was ascertained by XRD (Figure S5). The peaks
located at 43.4° (111), 50.8° (200) and 74.5° (220) are assigned to Cu NPs.
3.2 Characterization of Cu NPs@Fe3O4-chitosan
The structure of Cu NPs@Fe3O4-chitosan was thoroughly characterized using powder XRD, FESEM, EDS, TEM,
STEM, elemental mapping, VSM, TG/DTG and FT-IR spectroscopy. Figure S6 depicts the Cu NPs@Fe3O4-chitosan
FT-IR. The broad peak at 3400 cm-1 could be related to the stretching vibrations of -OH and/or -NH in chitosan, while
weak peaks around 2935 cm-1 could be pertinent to the asymmetric stretch of -CH group (Liu, Wang, Liu, Jiang, Yu,
Mu, & Wang, 2012). The signals at 1623, 1564 and 1261 cm-1 related to stretching vibrations of C=O in the amide I,
-NH bending and C-N stretching in amide II and N-H bending and the C-N stretching bands in the amide III,
respectively. The signals at 1385 and 1069 cm-1 corresponded to CH2OH of C-6 position and O-H bending of the sugar
piece of chitosan, respectively (Mansur, Mansur, Curti, & De Almeida, 2013). The peak related to C-O-C
nonsymmetric stretching and glycoside linkage of saccharide structure, appeared in order at 1151 and 892 cm-1 (Zhang,
Jiang, Ma, Cai, Zhou, & Wang, 2011). The signal at 634 cm-1 attributed to the Fe-O bond vibrating of iron oxide
nanoparticles.
The presence of Cu and Fe3O4 was verified with XRD analysis. Six separately Bragg peaks are depicted in Figure
1 at 30.2, 35.8, 43.5, 53.7, 57.2, 62.8 corresponding to 220, 311, 400, 511, 440, and 533 Bragg reflection planes
(JCPDS file, PDF No. 65-3107). It indicates that the magnetic Fe3O4 nanoparticles has been successfully combined
with CS. On the other hand, the diffraction peaks for Cu NPs appeared at 43.4, 50.8 and 74.5 assigning to the 111,
200 and 220 planes, respectively. Figure 2 shows morphology of the Cu NPs@Fe3O4-chitosan according to SEM
analysis. FESEM images were confirmed that the Cu NPs were attached to the substrate of the magnetic chitosan.
The chemical composition and Cu NPs distribution in the green synthesized Cu NPs@Fe3O4-chitosan was
characterized by EDS and elemental mapping (Figure 3). In EDS spectrum, Cu, Fe, O, N and C peaks were observed,
11

indicating the authenticity of Cu NPs@Fe3O4-chitosan. As shown in Figure 3, the Cu NPs@Fe3O4-chitosan elemental
mapping was verified the Cu NPs distribution on the magnetic CS surface.
The Cu NPs@Fe3O4-chitosan TEM analysis (Figure 4) displayed the decoration of the Cu NPs on the magnetic CS
surface, comprising Cu NPs and magnetic CS with the average size of 5-10 and ~50 nm, respectively with spherical
morphology.
The HRTEM and fast Fourier transform (FFT) image of the nanocomposite catalyst show that both Fe3O4 and Cu
nanoparticles are highly crystalline (Figure 5a-c). STEM image confirms a homogeneously assembled nanostructured
catalyst (Figure 5d).
TG/DTG analysis was applied to examine the Cu NPs@Fe3O4-chitosan thermal stability (Figure S7). The mass
loss happens up to 200 °C is ascribed to the loss of molecules moisture and physically adsorbed solvent. The weight
loss high 200°C is associate to polysaccharide chain thermal decomposition. So, the above temperature for chitosan
decomposition shown the elevated thermal stability of the catalyst. The magnetic property of the nanocomposite
catalyst was studied using a VSM magnetometer (Figure S8) demonstrating an outstanding magnetic separation for
effective recycling of the catalyst.
Figure 1. XRD pattern of the Cu NPs@Fe3O4-chitosan.
12

Figure 2. FESEM analysis of the Cu NPs@Fe3O4-chitosan.
O K
3000
2500
2000
FeK
1500
1000
500
0
FeL
N K
C K
CuL
FeK
CuK
CuK
keV
0
5
10
13

Figure 3. EDS spectrum and elemental mapping of the Cu NPs@Fe3O4-chitosan.
14

Figure 4. TEM images of the Cu NPs@Fe3O4-chitosan.
15

Figure 5. (a, b) HRTEM, (c) FFT and STEM images of the Cu NPs@Fe3O4-chitosan.
3.3 Preparation various tetrazoles using Cu NPs@Fe3O4-chitosan catalyst
2,4-Dimethylphenylcyanamide and NaN3 were selected for the initial exploratory investigation (Table 1). Notably,
the reaction led to clean conversion when conducted in water as a green solvent and catalyzed by Cu NPs@Fe3O4-
chitosan. The efficiency of catalyst loading on the production of product was studied by different quantities of catalyst.
As shown in entry 1, the product was not obtained in the absence of the Cu NPs@Fe3O4-chitosan. When 0.03 g of the
catalyst is used as the catalyst, 78% yield of product is attained (entry 2). This high yield indicates that Cu NPs@Fe3O4-
chitosan acts as an effective catalyst for the synthesis of tetrazole; 0.04, 0.06 and 0.08 g of catalyst, afforded 81, 84
and 88 %, respectively. No further improvement in the yield of reaction discerned by increasing the amount to 0.1 g
(entry 7).
Based on the optimized conditions, we investigated the effect of various substituents on the cyanamides aromatic
ring. Essentially, the Cu NPs@Fe3O4-chitosan displayed high catalytic activity for the reaction of various N-
arylcyanamides possessing diverse functionalities with NaN3 in water and corresponding tetrazoles were prepared in
16

high yields (Table 2). There is an excellent interrelationship between substituent type and outcome for the product.
According to the obtained results, B and A isomers can be produced from N-arylcyanamides carrying electron-
donating and electron-withdrawing groups, respectively via the formation of guanidine azide intermediate (Scheme
S2). According to the documented results in the literature, in most cases, isomeric mixtures was observed in the
synthesis of tetrazoles from cyanamides, while the present method was completely regiospecific (Scheme S3). The
regiospecificity of cyanamides reaction with NaN3 provides a novel process for the synthesis of specific type of
tetrazole (A or B).
Commonly, when the substitution on the cyanamides ring in ortho position is Cl (Table 2, entries 9-12), the
formation of A isomer is favoured owing to the intramolecular hydrogen bonding between Cl and NH amine group
(Scheme S4).
We have also assessed the catalytic prowess of Cu NPs@Fe3O4-chitosan for the synthesis of N-sulfonyl-N-aryl-5-
amino-1H-tetrazoles for the first time from corresponding cyanamides in refluxing water conditions (Table 2, entries
17-19). As shown in Table 2, compared to B and A isomers, N-sulfonyl-N-aryl-5-amino-1H-tetrazoles preparation
required longer reaction times owing to sulfonyl groups presence on the nitrogen atom in N-sulfonyl-N-
arylcyanamides.
To indicate the comparative efficacy of our method, Scheme S5 show comparison of designed Cu NPs@Fe3O4-
chitosan with reported catalysts in the literature for the [2+3] cycloaddition between 2,4-dichlorophenylcyanamide
and NaN3 (Henry, Finnegan, & Lieber, 1954; Herr, 2002; Katritzky, Rees, & Scriven, 1996; Nasrollahzadeh, Sajjadi,
Komber, Khonakdar, & Sajadi, 2019; Peet, Baugh, Sundler, Lewis, Matthews, Olberding, & Shah, 1986; Singh,
Chawla, Kapoor, Paul, & Malhotra, 1980). As shown in Scheme S5, Cu NPs@Fe3O4-chitosan catalyst is a more
efficient catalyst than previously reported ones from the view point of product yield, regiospecificity, reaction time,
and used solvent. The following disadvantages, observed in reported protocols in the literature for the synthesis of
tetrazoles, were overcome; none of the listed drawbacks are found in our protocol for this regiospecific reaction:





The employment of harmful solvents and dangerous HN3
In situ preparation of HN3
Low regiospecificity of reaction (production of isomers mixture)
Low product yields
Great reaction times
17



The use of high amounts of homogeneous catalysts without recyclability
Difficulties in availability of the natural zeolite
Table 1. Optimization of synthesis of 5-(2,4-dimethylphenyl)amino-1H-tetrazole.a
Entry Cu NPs@Fe3O4-chitosan (g) Time (h) ld (%)b
1
2
3
4
5
6
0.0
24
10
8.5
7
0.0
78
81
84
88
88
0.03
0.04
0.06
0.08
0.1
5.5
5.5
aReaction conditions: 2,4-dimethylphenylcyanamide (1.0 mmol); NaN3 (1.1 mmol) and H2O (15 mL), reflux.
bIsolated yield.
Table 2. Synthesis of tetrazoles by Cu NPs@Fe3O4-chitosan.a
Entr Substrate
y
Product
Time (h)
5.5
Yield%a
88
1
2
3
4
5.5
5.5
5.5
87
88
83
18

5
5.5
86
6
7
6
6
90
87
8
9
6
6
87
85
10
11
12
13
14
15
6
86
87
82
81
82
85
6
7
7
7
4.5
19

16
17
6
86
83
22
18
19
22
22
82
82
a Reaction conditions: Cyanamide derivative (1.0 mmol), NaN3 (1.1 mmol), H2O (15 mL), catalyst (0.08 g), reflux.
bIsolated yield of the pure product.
The structure of tetrazoles were confirmed by 1HNMR, 13CNMR, elemental (CHN) analysis and FT-IR analyses.
The conversion of cyanamides to tetrazoles could be monitored using FT-IR spectroscopy. After the tetrazole
formation, CN stretching band in cyanamide structure disappeared with the appearance of two stretching bands
corresponding to NH groups which confirmed the formation of tetrazole (Figure S9 and S10). The 1HNMR spectrum
of A isomer shows two NH signals corresponding to tetrazole ring (NHT) and amine group bonded with aryl ring
1
(NHA)) (Figure S11), while the HNMR spectrum of B isomer was characterized via appearance of one NH2 peak.
The 13CNMR spectrum confirmed the tetrazole formation via appearance of peak at δ = 154-159 ppm concerning
carbon of tetrazole ring (Figure S12).
3.4 Catalyst recyclability
20

The Cu NPs@Fe3O4-chitosan reusability for the synthesis of tetrazoles was studied using the reaction of 2-
chlorophenylcyanamide with NaN3; the Cu NPs@Fe3O4-chitosan catalyst could be simply removed from the reaction
deploying an external magnetic field, washed twice by ethanol, dried at 100 °C for 5 h and finally was used again for
the next catalytic run. Interestingly, this nanocatalyst can be reused five times without significant decrease in the
catalytic ability (Figure S13). Moreover, the integrity of the recycled Cu NPs@Fe3O4-chitosan in terms of size, shape
and chemical composition after the 5th run was verified by FESEM (Figure S14), EDS (Figure S15) analysis and
elemental mapping (Figure S16) and TEM (Figure S17). The experimental results display that the magnetic chitosan
play an essential role in the stabilization of Cu NPs and prevent their agglomeration, thus minimizing the leaching of
Cu species during the reaction. According to the studies conducted using ICP-MS analysis in the present work, less
than 0.1% of the Cu was observed in the solution during cycloaddition.
4. Conclusions
In this research, a proficient magnetic NPs-supported Cu adorned on the surface of chitosan modified with Fe3O4 NPs.
Cu NPs were simply synthesized and immobilized on the magnetic chitosan using E. falcata leaf extract as stabilizing
and reducing agent for the preparation of Cu NPs@Fe3O4-chitosan without of use of poisonous reducing agents.
FESEM, XRD, EDS, TEM, VSM, TG/DTG, STEM, elemental mapping and FT-IR techniques were applied for the
confirmation of the Cu NPs@Fe3O4-chitosan. FT-IR analysis of extract indicated the presence of flavonoids or
polyphenols in the E. falcata which are responsible for Cu2+ ions reduction. The Cu NPs@Fe3O4-chitosan showed
excellent catalytic efficiency for the synthesis of various tetrazoles in high yields. This synthetic procedure has
numerous benefits of high yields, avoidance of the preparation and use of hazardous and harmful hydrazoic acid, easy
work‐up procedure, use of water as an eco-friendly solvent instead of toxic solvents such as DMF with the added
advantage of catalyst stability and reusability.
Acknowledgement
The support of this work by the University of Qom and the Iranian Nano Council is greatly appreciated.
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