Design, synthesis of novel starch derivative bearing 1,2,3-triazolium and pyridinium and evaluation of its antifungal activity

Article abstract of DOI:10.1016/j.carbpol.2016.09.093

Based on cuprous-catalyzed azide-alkyne cycloaddition (CuAAC), starch derivative bearing 1,2,3-triazole and pyridine (II) was prepared and subsequently followed by alkylation with iodomethane to synthesize starch derivative bearing 1,2,3-triazolium and py

Full text of DOI:10.1016/j.carbpol.2016.09.093

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Carbohydrate Polymers 157 (2017) 236–243
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Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol
Design, synthesis of novel starch derivative bearing 1,2,3-triazolium
and pyridinium and evaluation of its antifungal activity
Wenqiang Tana,b, Qing Lia, Zhenpeng Gaoc, Shuai Qiud, Fang Donga, Zhanyong Guoa,∗
a Key Laboratory of Coastal Biology and Bioresource Utilization, Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences, Yantai 264003,
China
b University of Chinese Academy of Sciences, Beijing 100049, China
c School of Marine Science and Technology, Harbin Institute of Technology at Weihai, Weihai 264209, China
d College of Life Sciences, Yantai University, Yantai 264005, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Based on cuprous-catalyzed azide-alkyne cycloaddition (CuAAC), starch derivative bearing 1,2,3-triazole
and pyridine (II) was prepared and subsequently followed by alkylation with iodomethane to synthesize
starch derivative bearing 1,2,3-triazolium and pyridinium (III). The antifungal activities of starch deriva-
tives against Colletotrichum lagenarium, Watermelon fusarium, and Phomopsis asparagi, were then assayed
by hypha measurement in vitro. Apparently, starch derivatives showed enhanced antifungal activity
against three fungi at the tested concentrations compared with starch. Especially, the best inhibitory index
of starch derivative (III) against Colletotrichum lagenarium attained 97% above at 1.0 mg/mL. Meanwhile,
starch derivative (III) had stronger antifungal activity than starch derivative (II), which was reasonable to
propose that the alkylation of 1,2,3-triazole and pyridine was significant for enhanced antifungal activ-
ity. As this novel starch derivative bearing 1,2,3-triazolium and pyridinium could be prepared efficiently
and exhibited superduper antifungal activity, this material might provide an effective way and notion to
prepare novel antifungal agents.
Received 14 July 2016
Received in revised form
28 September 2016
Accepted 30 September 2016
Available online 1 October 2016
Chemical compounds studied in this article:
Soluble starch (PubChem CID: 439341)
N-bromosuccinimide (PubChem CID:
67184)
Triphenylphosphine (PubChem CID: 11776)
Sodium azide (PubChem CID: 33557)
Propargyl amine (PubChem CID: 239041)
Nicotinoyl chloride hydrochloride
(PubChem CID: 88438)
© 2016 Elsevier Ltd. All rights reserved.
Iodomethane (PubChem CID: 6328)
Keywords:
Starch derivative
Antifungal activity
1,2,3-Triazolium
Pyridinium
Click chemistry
1. Introduction
such as carboxyl, sulfate ester, and amino (Tan, Li, Wang et al.,
2016). In order to effectively broaden the industrial applications
Starch, the natural polysaccharide derived from a large variety of
higher green plants, such as cereals, legumes, and tubers (Fan et al.,
2016), is composed of anhydroglucose units (AGU) linked together
by ␣-glucosidic bonds (Nep, Ngwuluka, Kemas, & Ochekpe, 2016).
Due to its interesting properties such as abundant, cheap, nontoxic,
biodegradable, and biocompatible (Ubeyitogullari & Ciftci, 2016;
Verma, Le Bras, Jain, & Muzart, 2013), starch has been developed
and used in some fields, including in the food, pharmaceutical, bev-
erages, papermaking, packaging, and textiles (Li et al., 2016; Shi &
Gao, 2016). However, native starch can not meet the demands for
further industrial applications on account of lacking active groups
of new valuable products based on starch in sustainable chemi-
cal research field and bioactive material, one valid solution is the
chemical modification via introduction of the individual functional
moieties to native starch molecules (Kumar, Verma, & Jain, 2015;
Sukhija, Singh, & Riar, 2016; Verma, Jain & Sain, 2011; Verma, Jain,
& Sain, 2011; Verma, Le Bras, Jain, & Muzart, 2013; Verma et al.,
2013c).
The outstanding features of the 1,3-dipolar cycloaddition of
azide and alkyne using catalytic amounts of Cu(I), such as ver-
satility, high efficiency, and robustness (Singh et al., 2015; Sood
et al., 2014), have promoted the development of an extremely
broad palette of polysaccharide materials containing 1,2,3-triazole
units. The many interesting biological properties of 1,2,3-triazoles,
such as anti-HIV (Pribut, Veale, Basson, van Otterlo, & Pelly, 2016),
antimicrobial (Garudachari, Isloor, Satyanarayana, Fun, & Hegde,
∗
Corresponding author.
E-mail address: zhanyongguo@hotmail.com (Z. Guo).
http://dx.doi.org/10.1016/j.carbpol.2016.09.093
0144-8617/© 2016 Elsevier Ltd. All rights reserved.

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W. Tan et al. / Carbohydrate Polymers 157 (2017) 236–243
237
2014; Zhang, Wei, Vijaya Kumar, Rasheed, & Zhou, 2014), anti-
cancer (Kumar et al., 2011), antimalarial (Pereira et al., 2014),
bon and nitrogen. X-ray diffraction (XRD) analyses of the samples
were performed using an X-ray diffractometer (Rigaku Ultima IV,
Rigaku Corporation, Japan) using Cu K␣ radiation (␭ = 1.5418 Å) set
at 40 kV and 30 mA. All samples were scanned at diffraction angle
(2␪) from 5 to 50◦ at a rate of 1.20◦/min and with a step size of
0.02◦. The morphology of the samples was examined through a
Scanning electron microscope (SEM) (S-4800, Hitachi, Japan). Each
sample was coated with gold in an ion sputter (E-1045, Hitachi,
Japan) before being scanned and photographed at the magnifica-
tions (1000×). An accelerating potential of 3 kV was used during
image acquisition.
and antioxidant (Tan, Li, Li, Dong
& Guo, 2016), have also
facilitated the chemical modification of polysaccharide with 1,2,3-
triazoles. Meanwhile, alkylation of 1,2,3-triazoles can provide the
1,2,3-triazolium cations, which have been prepared for novel
ionic liquids (Mudraboyina, Obadia, Abdelhedi-Miladi, Allaoua,
& Drockenmuller, 2015; Obadia, Crépet, Serghei, Montarnal, &
Drockenmuller, 2015; Obadia et al., 2014) and catalysts (Aizpurua
et al., 2014; Jha
& Jain, 2013; Ohmatsu, Hamajima, & Ooi,
2012) because of high thermal stability, tunable solubility, and
low flammability (Liu et al., 2016). However, although the
1,2,3-triazole-linked starch derivatives have been reported and
described, to our knowledge there are no reports on synthesis and
bioactivity of starch derivatives bearing 1,2,3-triazolium cations so
far. Moreover, pyridine group has been also regarded as an excel-
lent reactive precursor, which can synthesize pyridinium group by
the alkylation reaction (Jia, Duan, Fang, Wang, & Huang, 2016). But
the effect of alkylation of 1,2,3-triazole and pyridine groups on the
bioactivity of starch derivatives was still unknown.
This study aimed to investigate the effect of 1,2,3-triazolium
and pyridinium groups on biological activity of starch derivative.
Herein, we presented the synthesis, characterization, and anti-
fungal activity of starch derivative bearing 1,2,3-triazolium and
pyridinium (III) obtained by alkylation of starch derivative bear-
ing 1,2,3-triazole and pyridine (II) issued from CuAAC reaction. The
chemical structures of the starch derivatives were characterized by
FTIR, 1H NMR, and 13C NMR. Three plant-threatening fungi, includ-
ing Colletotrichum lagenarium (C. lagenarium), Watermelon fusarium
(W. fusarium), and Phomopsis asparagi (P. asparagi), were selected
to evaluate the antifungal property of starch and starch derivatives
(II) and (III) by hypha measurement in vitro.
2.3. The synthesis of the starch derivatives
2.3.1. Synthesis of N-prop-2-ynylnicotinamide (I)
A stirred solution of propargyl amine (0.65 mL, 10 mmol), tri-
ethyl amine (1.4 mL, 10 mmol), and DMAP (24 mg, 0.2 mmol)
in 20 mL of CH2Cl2 was cooled to 0 ◦C. The nicotinoyl chloride
hydrochloride (1.78 g, 10 mmol) was then added in batches. The
reaction mixture was then stirred at 0 ◦C for 0.5 h and overnight
at room temperature. The mixture was then extracted with
0.1 M aqueous solutions of HCl (2 × 10 mL) and NaOH (2 × 10 mL),
washed with water (3 × 20 mL), dried over MgSO4, filtrated and
the solvent evaporated under vacuum. The resulting N-prop-2-
ynylnicotinamide (I) was sufficiently pure to be used without
further purification. N-prop-2-ynylnicotinamide (I), Yield: 35.89%.
FTIR: ␯ 3224, 3046, 2958, 2113, 1658, 1596, 1550, 713, 640 cm−1. 1H
NMR (500 MHz, DMSO-d6): ı 9.02 (m, 1H, Py-2-H), 8.73 (m, 1H, Py-
6-H), 8.21 (m, 1H, Py-4-H), 8.19 (m, 1H, NHCH2C CH), 7.53 (m, 1H,
Py-5-H), 4.09 (dd, J = 1.5, 3.9 Hz, 2H, NHCH2C CH), 2.51 (dt, J = 1.8,
3.6 Hz, 1H, NHCH2C CH) ppm. 13C NMR (125 MHz, DMSO-d6): ı
164.99 (1C, C O), 152.57 (1C, Py-2-C), 148.88 (1C, Py-6-C), 135.49
(1C, Py-4-C), 129.75 (1C, Py-3-C), 123.98 (1C, Py-5-C), 81.41 (1C,
NHCH2C CH), 73.60 (1C, NHCH2C CH), 28.92 (1C, NHCH2C CH)
ppm. MS [ESI]: m/z [M + H]+ calcd for C9H8N2O 161.06; found
161.17.
2. Experimental
2.1. Materials
2.3.2. The dissolution of starch
Soluble starch from potato (granules) with weight-average
molecular weight of 9.8 × 104 Da, was purchased from Sinopharm
Chemical Reagent Co., Ltd. (Shanghai, China). N-bromosuccinimide
(NBS), triphenylphosphine (TPP), nicotinoyl chloride hydrochlo-
ride, propargyl amine, and iodomethane were purchased from the
Sigma-Aldrich Chemical Corp (Shanghai, China). Triadimefon (20%
emulsifiable concentrates) was obtained from Hebei Shenhua Phar-
maceutical Co., Ltd. (Hebei, China). The other reagents were all
analytical grade and used as received.
Soluble starch (3.24 g, 20 mmol) was stirred in 80 mL of anhy-
drous N,N-dimethylformamide (DMF), while the mixture was
heated to 120 ◦C for 1 h. The slurry was then allowed to cool to
90 ◦C, at which point LiBr (3.47 g, 40 mmol) was added. The starch
could dissolve within 5 min to form a transparent solution. The con-
tents of the flask were allowed to cool further to room temperature
while stirring.
2.3.3. Synthesis of 6-bromo-6-deoxy starch (BDST)
When transparent solution above-mentioned was cooled to 0 ◦C,
N-bromosuccinimide (NBS) (14.24 g, 80 mmol) and triphenylphos-
phine (TPP) (20.99 g, 80 mmol) were added. The reaction solution
was heated to 80 ◦C for 3 h under an argon atmosphere. The prod-
uct was isolated by adding the reaction mixture slowly to 400 mL
of 95:5 (v/v) mixture of absolute ethanol and deionized water, fol-
lowed by filtration. The unreacted NBS, TPP, and other outgrowth,
were extracted in a Soxhlet apparatus with ethanol and acetone for
48 h, respectively. The 6-bromo-6-deoxy starch was obtained by
freeze-drying overnight in vaccum. Yield: 89.31%. FTIR: ␯ 3405.67,
2.2. Analytical methods
Fourier transform infrared (FTIR) spectra were recorded on
a Jasco-4100 Fourier Transform Infrared Spectrometer (Japan,
provided by JASCO Co., Ltd. Shanghai, China) at 25 ◦C in the trans-
mittance mode. About 1 mg of sample with 100 mg of KBr was fully
grinded and mixed. The mixed samples were pressed into pills with
a compressor and prepared pellets were used for studies. All spec-
tra were scanned against a blank KBr pellet back-ground in the
range of 4000–400 cm−1 with resolution of 4.0 cm−1
.
13C Nuclear
2923.56, 1029.80, 682.68 cm−1 1H NMR (500 MHz, DMSO-d6): ı
.
magnetic resonance (13C NMR) and 1H Nuclear magnetic resonance
(1H NMR) spectra were all recorded on a Bruker AVIII-500 Spec-
trometer (Switzerland, provided by Bruker Tech. and Serv. Co., Ltd.,
Beijing, China) at 25 ◦C using DMSO-d6 or D2O as solvent. Chemi-
cal shifts (␦ ppm) were referenced to tetramethylsilane (TMS). The
elemental analyses (C, H, and N) were performed on a Vario EL III
(Elementar, Germany). The degrees of substitution (DS) of starch
derivatives were calculated on the basis of the percentages of car-
5.85-3.30 (pyranose rings), 3.44 (CH2Br) ppm. 13C NMR (125 MHz,
DMSO-d6): ı 100.22–70.08 (pyranose rings), 34.78 (CH2Br) ppm.
2.3.4. Synthesis of 6-azido-6-deoxy starch (ADST)
In a 100 mL three-necked round-bottom flask, 6-bromo-6-
deoxy starch (2.25 g, 10 mmol) was weighed and dissolved in
40 mL of anhydrous dimethylsulfoxide (DMSO). Then, NaN3 (1.3 g,
20 mmol) was added to the flask and dissolved. The solution was

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W. Tan et al. / Carbohydrate Polymers 157 (2017) 236–243
heated to 80 ◦C and stirred for 24 h under an argon atmosphere. The
product was isolated by pouring the reaction solution into 200 mL
of absolute ethanol. The precipitate was collected by filtration, and
washed with acetone. After being extracting in a Soxhlet apparatus
with ethanol for 48 h and being dialyzed against deionized water for
48 h to remove the probable remained sodium azide, the 6-azido-6-
deoxy starch was obtained by freeze-drying. Yield: 71.10%. DSazido
:
95.57%. FTIR: ␯ 3405.67, 2923.56, 2105.89, 1041.37 cm−1 1H NMR
.
(500 MHz, DMSO-d6): ı 5.72–3.30 (pyranose rings), 3.77 (CH2N3)
ppm. 13C NMR (125 MHz, DMSO-d6): ı 100.22–70.26 (pyranose
rings), 51.69 (CH2N3) ppm.
2.3.5. Synthesis of starch derivative bearing 1,2,3-triazole and
pyridine (II)
6-Azido-6-deoxy starch (187 mg, 1 mmol) was dissolved in
20 mL of DMSO, cuprous iodide (19 mg, 0.1 mmol), triethylamine
(0.14 mL, 1 mmol), and N-prop-2-ynylnicotinamide (I) (322 mg,
2 mmol) were added, and the solution was stirred at 75 ◦C for
24 h under an argon atmosphere. The mixture was precipitated
in 100 mL of acetone, and collected by filtration. The probable
remained reagents were extracted in a Soxhlet apparatus with ace-
tone for 48 h. After being dialyzed against deionized water for 48 h,
the starch derivative (II) was obtained by lyophilization of their
aqueous solutions. Yield: 90.48%. DS triazole: 97.46%. FTIR: ␯ 3351.68,
3081.69, 2923.56, 1650.77, 1596.77, 1542.77, 1469.49, 1041.37,
809.96 cm−1 1H NMR (500 MHz, DMSO-d6): ı 9.13 (s, 1H, Py-2-H),
.
Fig. 1. FTIR spectra of starch and starch derivatives.
8.76 (s, 1H, Py-6-H), 8.08 (2H, Py-4-H and CONHCH2), 7.75 (m, 1H,
Py-5-H), 7.49 (m, 1H, triazole-5-H), 5.72-3.02 (pyranose rings), 4.31
(s, NHCH2C), 3.84 (s, 1H, NCH2CH) ppm. 13C NMR (125 MHz, DMSO-
d6): ı 165.35 (1C, C O), 152.17 (1C, Py-2-C), 148.74 (1C, Py-6-C),
144.45 (triazole-4-C), 124.47 (triazole-5-C), 135.45 (1C, Py-4-C),
129.95 (1C, Py-3-C), 124.47 (1C, Py-5-C), 99.90-69.66 (pyranose
rings), 50.19 (1C, NCH2CH), 35.06 (1C, NHCH2C) ppm.
(without the presence of samples), the inhibitory index was calcu-
lated as follows:
Inhibitoryindex(%) = (1 − Da/Db) × 100
where Da is the diameter of the growth zone in the test plates and
Db is the diameter of the growth zone in the control plate.
2.3.6. Synthesis of starch derivative bearing 1,2,3-triazolium and
pyridinium (III)
2.5. Statistical analysis
A solution of 1,2,3-triazole-functionalized starch derivative
(II) (347 mg, 1 mmol of 1,2,3-triazole groups) and iodomethane
(0.187 mL, 3 mmol) in 15 mL of DMSO was stirred at 60 ◦C for 24 h.
Afterwards, the remaining iodomethane was evaporated, and the
reaction mixture was precipitated into 100 mL of acetone. The solid
product was filtered, washed with acetone three times. After being
dialyzed against deionized water for 48 h, the starch derivative (III)
was obtained by lyophilization of their aqueous solutions. Yield:
76.35%. DS quaternization: 93.02%. FTIR: ␯ 3397.96, 3062.41, 2919.70,
1670.05, 1592.91, 1542.77, 1500.35, 1041.37, 794.53 cm−1. 1H NMR
(500 MHz, D2O): ı 9.24 (d, J = 77.3 Hz, 1H, Py-2-H), 8.88 (m, 4H,
Py-4,5,6-H and triazole-5-H), 8.20 (s, 1H, CONHCH2), 5.52-3.14
(pyranose rings), 4.94 (s, NHCH2C), 4.42 (d, J = 41.3 Hz, 6H, N+CH3),
4.09 (s, 1H, NCH2CH) ppm. 13C NMR (125 MHz, D2O): ı 164.02 (1C,
Each experiment was performed three times. All data were
expressed as means SD. Data were analyzed by an analysis of vari-
ance (P < 0.05) and the Scheffe’s method was used to evaluate the
differences in inhibitory indices in antifungal tests. The results were
processed by the computer programs: Origin and SPSS.
3. Results and discussion
3.1. Chemical synthesis and characterization
The synthetic strategy for the preparation of starch derivatives
via CuAAC reaction is shown in Scheme 1. Each step of synthesis
is followed by FTIR, 1H NMR, and 13C NMR spectroscopy shown in
Figs. 1–3, respectively.
C
O), 147.63 (1C, Py-4-C), 145.45 (1C, Py-2-C), 143.97 (1C, Py-6-C),
140.06 (triazolium-4-C), 128.30 (triazolium-5-C), 133.09 (2C, Py-
3,5-C), 99.90-62.55 (pyranose rings), 49.11 (1C, NCH2CH), 39.27,
32.93 (2C, N+CH3), 36.73 (1C, NHCH2C) ppm.
Firstly, 6-bromo-6-deoxy starch was first regioselectivity
synthesized in N,N-dimethylformamide (DMF)/LiBr as a useful
intermediate for synthesis of 6-azido-6-deoxy starch by SN2 dis-
placement of the bromide. This highly regioselectivity should be
given the credit to alkoxyphosphonium salt intermediate at the
primary hydroxyl group attached to the 6-carbon due to the steric
bulk of three phenyl rings (Fox & Edgar, 2011). A bromide anion,
supplied by the NBS, then attacked C-6 via SN2 mechanism with
triphenylphosphine oxide as the leaving group (Fox & Edgar, 2012).
In FTIR spectrum of BDST, a new peak at 682.68 cm−1 is assigned
to C-6-Br group (Tan, Li, Wang et al., 2016). In 1H NMR spectrum
of BDST, hydrogens of CH2Br are observed at 3.44 ppm. Moreover,
carbon of CH2Br is clearly observed at 34.78 ppm in 13C NMR spec-
trum (Zhang & Edgar, 2015). However, there are many disturbances
of BDST, which indicate the presence of a low DS of acetate ester
2.4. Antifungal assay
Antifungal assay was performed based on the method of Guo
et al. with minor modification (Guo et al., 2014). Briefly, the com-
pounds (starch and starch derivatives (II) and (III)) were dissolved
in distilled water containing 2% (w/w) DMSO at a concentration of
5 mg/mL. Then, to each solution, sterilized potato dextrose agar was
added to give a final concentration of 0.1, 0.5, and 1.0 mg/mL. After
the mixture was cooled in the plate, the mycelium of fungi was
transferred to the test plate and incubated at 27 ◦C for 2–3 days.
When the mycelium of fungi reached the edges of the control plate

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239
Scheme 1. Synthetic routes for starch derivatives.
Fig. 2. 1H NMR spectra of starch and starch derivatives.
groups attached to C-6-OH of starch. The reasonable interpretation
was that during the SN2 reaction with starch alkoxyphosphonium
salt intermediate, the acetate group was a product of the DMF sol-
vent sometimes acting as a nucleophile instead of bromide (Fox
& Edgar, 2011). Fortunately, after azidation of BDST, these dis-
turbances are disappeared absolutely and characteristic peak of
C-6-N3 is observed at 2105 cm−1 in FTIR spectrum (Li, Tan, Zhang,
Gu, & Guo, 2015). As shown in Figs. 2 and 3, the peak of CH2N3
shifts to 3.77 and 51.69 ppm in 1H NMR and 13C NMR respectively