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Carbohydrate Polymers 160 (2017) 163–171

Contents lists available at ScienceDirect

Carbohydrate Polymers

journa l homepage: www.e lsev ier .com/ locate /carbpol

ovel 1,2,3-triazolium-functionalized starch derivatives: Synthesis,haracterization, and evaluation of antifungal property

enqiang Tan a,b, Qing Li a, Fang Dong a, Shuai Qiu c, Jingjing Zhang a,b, Zhanyong Guo a,b,∗

Key Laboratory of Coastal Biology and Bioresource Utilization, Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences, Yantai 264003,hinaUniversity of Chinese Academy of Sciences, Beijing 100049, ChinaCollege of Life Sciences, Yantai University, Yantai 264005, China

r t i c l e i n f o

rticle history:eceived 18 October 2016eceived in revised form1 December 2016ccepted 24 December 2016vailable online 26 December 2016

hemical compounds studied in this article:oluble starch (PubChem CID: 439341)-Bromosuccinimide (PubChem CID:7184)riphenylphosphine (PubChem CID: 11776)odium azide (PubChem CID: 33557)-Propyn-1-ol (PubChemCID: 7859)-Butyn-1-ol (PubChemCID: 13566)-Pentyn-1-ol (PubChemCID: 79346)-Hexyn-1-ol (PubChemCID: 70234)

odomethane (PubChemCID: 6328)

a b s t r a c t

Four novel 1,2,3-triazolium-functionalized starch derivatives were synthesized by N-alkylating the pre-cursor starch derivatives with 1,2,3-triazole with iodomethane based on cuprous-catalyzed azide-alkynecycloaddition (CuAAC). The detailed structural characterization was investigated by means of FTIR, UV-vis, 1H NMR, and 13C NMR spectra. The antifungal activities of starch derivatives against Colletotrichumlagenarium, Fusarium oxysporum, and Watermelon fusarium, were then assayed by hypha measure-ment in vitro. The fungicidal assessment revealed that compared with starch and starch derivativeswith 1,2,3-triazole, 1,2,3-triazolium-functionalized starch derivatives displayed tremendously enhancedantifungal activity. Especially, the inhibitory indices of 6-(4-hydroxymethyl-3-methyl-1,2,3-triazolium-1-yl)-6-deoxy starch iodine (2a) with against the tested plant threatening fungi attained 70% above at1.0 mg/mL. It was also found that their antifungal activity profiles were dependent on the variation inalkyl chain length. As novel 1,2,3-triazolium-functionalized starch derivatives could be prepared effi-ciently and exhibited superduper antifungal activity, this synthetic strategy might provide an effectiveway and notion to prepare novel antifungal biomaterials.

© 2016 Elsevier Ltd. All rights reserved.

eywords:tarchhemical modificationntifungal activitylick chemistry,2,3-Triazolium

haracterization

. Introduction

As the principal carbohydrate storage reserve for plants and theajor source of energy in the human diet and animal feed, starch

s composed of anhydroglucose units (AGU) linked together by �-lucosidic bonds (Fuentes et al., 2016). The increasing interest in

tarch-based biomaterials is due to its interesting properties suchs cheap, renewable, biodegradable, and biocompatible (Li et al.,017; Umar et al., 2016), which can facilitate a certain degree

∗ Corresponding author at: Key Laboratory of Coastal Biology and Bioresourcetilization, Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences,antai 264003, China.

E-mail address: zhanyongguo@hotmail.com (Z. Guo).

ttp://dx.doi.org/10.1016/j.carbpol.2016.12.060144-8617/© 2016 Elsevier Ltd. All rights reserved.

of applications of starch in biomedicine, biomaterials, and tex-tile areas (Ahmed, Thomas, Taher, & Joseph, 2016; Nguyen Vu &Lumdubwong, 2016). However, chemical modification via intro-duction of the individual functional moieties to starch backboneis often necessary in order to further improve the bioactivity andbroaden application scope of new valuable biomaterials basedon starch (Adak & Banerjee, 2016; Biduski et al., 2017; Kapelko-Zeberska, Zieba, Spychaj, & Gryszkin, 2015).

1,2,3-Triazole moieties have recently gained an increasing inter-est due to their modular synthesis through cuprous-catalyzedazide-alkyne cycloaddition (CuAAC) (Kraljevic et al., 2016). The

wide range of biological properties, such as antimicrobial, anti-cancer, and antimalarial (Kant, Singh, Nath, Awasthi, & Agarwal,2016), have facilitated the chemical modification of starch with

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,2,3-triazole moieties to achieve the rapid growth of appli-ation scope of starch. Uliniuc et al. (2013) reported that theolycaprolactone-grafted starch copolymers obtained by clickhemistry were able to form micelles in an aqueous solution andad potential to be used in controlled drug delivery and cos-etics. The antibacterial-starch biomaterials could be obtained

y anchoring the strong electron-withdrawing groups onto thetarch backbone by the CuAAC reaction (Tan, Li, Li, Dong, & Guo,016). Very recently, the synthesis of 1,2,3-triazolium groups by N-lkylating 1,2,3-triazole with alkyl halides begins to be paid closettention as they combine the interesting properties of ionic liq-ids and the versatile mechanical and broad-spectrum propertiesnd applications of polymers and catalysis (Abdelhedi-Miladi et al.,014; Mudraboyina et al., 2014; Ohmatsu, Hamajima, & Ooi, 2012).owever, although the 1,2,3-triazole-linked starch derivativesave been reported and described, to date there are surprisinglyery few reports describing the preparation of starch derivativesearing 1,2,3-triazolium. Our group has recently pioneered theynthesis of starch derivative bearing 1,2,3-triazolium and pyri-inium and this novel starch derivative showed strong antifungalctivity because of the combined effect of 1,2,3-triazolium and pyri-inium groups (Tan et al., 2017). However, the single influence ofhe alkylation of 1,2,3-triazole on the bioactivity of starch deriva-ives was still unknown. Although, many studies have reportedhat the length of alkyl groups had important influence on bioac-ivity of compounds (Tang et al., 2015), the effect of the length oflkyl groups in 1,2,3-triazolium moieties on the bioactivity of starcherivatives is still unknown.

The present study attempts to understand the structure-roperty relationships of 1,2,3-triazolium-functionalized starcherivatives with different alkyl chain length by quaternizationf starch derivatives with 1,2,3-triazole issued from CuAAC. The

mpact of the length of alkyl groups in 1,2,3-triazolium moieties onhe antifungal activity was investigated. The chemical structuresf the starch derivatives were characterized using FTIR, UV-vis, 1HMR, and 13C NMR spectra as well as elemental analysis. Threelant-threatening fungi, including Colletotrichum lagenarium (C.

agenarium), Fusarium oxysporum (F. oxysporum), and Watermelonusarium (W. fusarium), were selected to evaluate the antifungalroperty of starch and starch derivatives by hypha measurement

n vitro.

. Experimental

.1. Material

Soluble starch from potato (granules) with weight-averageolecular weight of 9.8 × 104 Da, was purchased from Sinopharm

hemical Reagent Co., Ltd. (Shanghai, China) and used without anyurther purification. N-bromosuccinimide (NBS), triphenylphos-hine (TPP), 2-propyn-1-ol, 3-butyn-1-ol, 4-pentyn-1-ol, 5-hexyn--ol, and methyl iodide were purchased from the Sigma-Aldrichhemical Corp (Shanghai, China). The other reagents were all ana-

ytical grade and used as received.

.2. Structural characterization of starch derivatives

.2.1. Fourier transform infrared (FTIR) spectroscopyAll spectra were recorded on a Jasco-4100 Fourier Transform

nfrared Spectrometer (Japan, provided by JASCO Co., Ltd. Shang-

ai, China) at 25 ◦C using the transmittance mode in the range000–400 cm−1 at a resolution of ± 4.0 cm−1. The tested samplesere pelletized with KBr in the weight ratio of 1/100 for observa-

ions.

mers 160 (2017) 163–171

2.2.2. Ultraviolet-visible (UV-vis) spectroscopyThe UV–vis spectra were carried out at 200–400 nm at 25 ◦C

using a 3–5 mm quartz cuvette using a TU-1810 UV spectrometer(provided by General Instrument Co., Ltd. Beijing, China). For thisanalysis, 5 mL of 40 �g/mL aqueous solution of starch and starchderivatives were put in a cuvette for measurement.

2.2.3. Nuclear magnetic resonance (NMR) spectroscopy1H Nuclear magnetic resonance (1H NMR) and 13C Nuclear mag-

netic resonance (13C NMR) spectra were all recorded on a BrukerAVIII-500 Spectrometer (Switzerland, provided by Bruker Tech. andServ. Co., Ltd. Beijing, China) at 25 ◦C with tetramethylsilane (TMS)as internal standard on ppm scale (�). For analyses, 50 mg of sam-ples were dissolved in 1.0 mL of 99.9% Deuterium Oxide (D2O) orDimethyl Sulfoxide-d6 (DMSO-d6).

2.2.4. Elemental analysisThe elemental analyses by combustion were used to quantita-

tively assess the extent of functionalization (degree of substitution)in starch derivatives. The analyses of elemental carbon, hydrogen,and nitrogen in starch derivatives were performed on a Vario EL III(Elementar, Germany). The degrees of substitution (DS) of starchderivatives were calculated on the basis of the percentages of car-bon and nitrogen according to the following equations (Dos Santos,Caroni, Pereira, da Silva, & Fonseca, 2009; Li, Tan, Zhang, Gu, & Guo,2016).

DS1 = MC × nCMN × nN × WC/N

(1)

DS2 = MN × nN × DS1 × WC/N − MC × nCn × MC

(2)

DS3 = Mn × nN × DS1 × WC/N − MC × nC − n × Mc × DS2Mc

(3)

where DS1, DS2, and DS3 represent the degrees of substitution ofazido in 6-azido-6-deoxy starch, 1,2,3-triazole in starch derivativesbearing 1,2,3-triazole, and 1,2,3-triazolium in starch derivativesbearing 1,2,3-triazolium; MC and MN are the molar mass of carbonand nitrogen, MC = 12, MN = 14; nC and nN are the number of car-bon and nitrogen of 6-azido-6-deoxy starch, nC = 6, nN = 3; n is thenumber of carbon of terminal alkynes, n = 3 − 6; WC/N representsthe mass ratio between carbon and nitrogen.

2.3. Synthesis of starch derivatives

2.3.1. Regioselective bromination of starch (BDST)The method of starch dissolution in DMF/LiBr was adapted from

previous work (Tan, Li, Wang et al., 2016). The synthesis of 6-bromo-6-deoxy-starch (BDST) has been previously published (Tan,Li, Wang et al., 2016). Briefly, soluble starch (3.24 g, 20 mmol AGU)was suspended in anhydrous DMF (80 mL) and stirred at 120 ◦C for1 h. After the slurry was allowed to cool to 90 ◦C, anhydrous LiBr(3.47 g, 40 mmol) was added. The starch was dissolved completelywithin 5 min and formed a transparent solution during cooling toroom temperature under constant stirring. N-bromosuccinimide(NBS, 14.24 g, 80 mmol) and triphenylphosphine (Ph3P, 20.99 g,80 mmol) were added to this solution. The reaction mixture washeated to 80 ◦C for 3 h under an argon atmosphere. The productwas isolated by adding the reaction mixture slowly to 95:5 (v/v%)mixture of absolute ethanol and deionized water (400 mL), fol-lowed by filtration. The unreacted NBS, Ph3P, and other outgrowth(succinimide, triphenylphosphine oxide (Ph3PO)), were extracted

in a Soxhlet apparatus with ethanol and acetone for 48 h, respec-tively. The 6-bromo-6-deoxy starch was obtained by freeze-dryingovernight in vaccum. FTIR: � 3405, 2923, 1029, 682 cm−1. 1H NMR(500 MHz, DMSO-d6): ı 5.85–3.30 (pyranose rings), 3.44 (CH2Br)

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pm. 13C NMR (125 MHz, DMSO-d6): ı 100.22–70.08 (pyranoseings), 34.78 (CH2Br) ppm.

.3.2. Synthesis of 6-azido-6-deoxy starch (ADST)In a 100 mL three-necked round-bottom flask, 6-bromo-6-

eoxy starch (2.25 g, 10 mmol) was weighed and dissolved in DMSO40 mL). Then, NaN3 (1.3 g, 20 mmol) was added to the flask and dis-olved. The solution was heated to 80 ◦C and stirred for 24 h undern argon atmosphere. The product was isolated by pouring the reac-ion solution into absolute ethanol (200 mL). The precipitate wasollected by filtration, and washed with acetone. After being dia-yzed against deionized water for 2 days to remove the probableemained sodium azide, the 6-azido-6-deoxy starch was obtainedy freeze-drying. FTIR: � 3405, 2923, 2105, 1041 cm−1. 1H NMR500 MHz, DMSO-d6): ı 5.72–3.30 (pyranose rings), 3.77 (CH2N3)pm. 13C NMR (125 MHz, DMSO-d6): ı 100.22–70.26 (pyranoseings), 51.69 (CH2N3) ppm.

.3.3. Synthesis of starch derivatives with 1,2,3-triazole (1a, 1b,c, and 1d)

To a solution of 6-azido-6-deoxy starch (187 mg, 1 mmol) inMSO (20 mL) was added cuprous iodide (19 mg, 0.1 mmol), tri-thylamine (0.14 mL, 1 mmol), and terminal alkyne derivatives2-propyn-1-ol, 3-butyn-1-ol, 4-pentyn-1-ol, and 5-hexyn-1-ol)3 mmol), and the reaction solution was kept at 75 ◦C for 24 hnder an argon atmosphere. The mixture was then added slowly tocetone (100 mL), the product was collected by filtration. The prob-ble remained reagents were dialyzed against deionized water for

days, the starch derivatives with 1,2,3-triazole were obtained byyophilization of their aqueous solutions.

.3.3.1. 6-(4-Hydroxymethyl-1,2,3-triazole-1-yl)-6-deoxy starch1a). FTIR: � 3401, 2923, 1554, 1045, 786 cm−1. The UV �max

as 216 nm. 1H NMR (500 MHz, DMSO-d6): � 7.75 (triazole-5-H),.66–3.08 (pyranose rings), 5.12 (CH2OH), 3.94 (NCH2CH) ppm.3C NMR (125 MHz, DMSO-d6): ı 148.01 (triazole-4-C), 124.45triazole-5-C), 100.10–69.68 (pyranose rings), 55.19 (CH2OH),0.27 (NCH2CH) ppm.

.3.3.2. 6-(4-Hydroxyethyl-1,2,3-triazole-1-yl)-6-deoxy starch (1b).TIR: � 3401, 2923, 1554, 1045, 794 cm−1. The UV �max was 220 nm.H NMR (500 MHz, DMSO-d6): � 7.62 (triazole-5-H), 5.68-3.10pyranose rings), 4.69 (CH2OH), 3.87 (NCH2CH), 2.48 (CH2CH2OH)pm. 13C NMR (125 MHz, DMSO-d6): � 144.58 (triazole-4-C),23.99 (triazole-5-C), 99.98-69.76 (pyranose rings), 60.74 (CH2OH),0.44 (NCH2CH), 29.21 (CH2CH2OH) ppm.

.3.3.3. 6-(4-Hydroxypropyl-1,2,3-triazole-1-yl)-6-deoxy starch (1c).TIR: � 3405, 2931, 1554, 1045, 767 cm−1. The UV �max was 220 nm.H NMR (500 MHz, DMSO-d6): � 7.62 (triazole-5-H), 5.68-3.10pyranose rings), 4.48 (CH2OH), 3.87 (NCH2CH), 2.39 (CH2CH2OH),.61 (CH2CH2CH2OH) ppm. 13C NMR (125 MHz, DMSO-d6): ı47.24 (triazole-4-C), 123.32 (triazole-5-C), 99.99-69.75 (pyranoseings), 60.60 (CH2OH), 50.50 (NCH2CH), 32.52 (CH2CH2OH), 25.82CH2CH2CH2OH) ppm.

.3.3.4. 6-(4-Hydroxybutyl-1,2,3-triazole-1-yl)-6-deoxy starch (1d).TIR: � 3405, 2927, 1554, 1045, 763 cm−1. The UV �max was20 nm. 1H NMR (500 MHz, DMSO-d6): ı 7.60 (triazole-5-H),.67-3.09 (pyranose rings), 4.40 (CH2OH), 3.85 (NCH2CH), 2.32

CH2CH2OH), 1.55–1.25 (CH2CH2CH2CH2OH) ppm. 13C NMR125 MHz, DMSO-d6): ı 147.10 (triazole-4-C), 123.51 (triazole-5-), 100.00-69.80 (pyranose rings), 60.58 (CH2OH), 50.44 (NCH2CH),2.48 (CH2CH2OH), 22.17-21.52 (CH2CH2CH2CH2OH) ppm.

mers 160 (2017) 163–171 165

2.3.4. Synthesis of 1,2,3-triazolium-functionalized starchderivatives (2a, 2b, 2c, and 2d)

A solution of starch derivatives with 1,2,3-triazole (1 mmolof 1,2,3-triazole groups) and iodomethane (0.187 mL, 3 mmol) inDMSO (15 mL) was stirred at 60 ◦C for 24 h. Afterwards, the remain-ing iodomethane was evaporated, and the reaction mixture wasprecipitated into acetone (100 mL). The solid product was filtered,and extensively washed with acetone three times. After beingdialyzed against deionized water for 48 h, the 1,2,3-triazolium-functionalized starch derivatives were obtained by lyophilizationof their aqueous solutions.

2.3.4.1. 6-(4-Hydroxymethyl-3-methyl-1,2,3-triazolium-1-yl)-6-deoxy starch iodine (2a). FTIR: � 3405, 2923, 1581, 1041, 794 cm−1.The UV �max was 225 nm. 1H NMR (500 MHz, D2O): ı 8.67(triazolium-5-H), 5.50-3.48 (pyranose rings), 4.90 (CH2OH), 4.33(N+CH3), 4.10 (NCH2CH) ppm. 13C NMR (125 MHz, D2O): ı 143.29(triazolium-4-C), 130.99 (triazolium-5-C), 99.10-68.00 (pyranoserings), 54.42 (NCH2CH), 52.36 (CH2OH), 38.58 (N+CH3) ppm.

2.3.4.2. 6-(4-Hydroxyethyl-3-methyl-1,2,3-triazolium-1-yl)-6-deoxy starch iodine (2b). FTIR: � 3374, 2919, 1581, 1041, 767 cm−1.The UV �max was 225 nm. 1H NMR (500 MHz, D2O): ı 8.60(triazolium-5-H), 5.48-3.50 (pyranose rings), 4.30 (N+CH3), 4.06(NCH2CH), 3.95 (CH2OH), 3.14 (CH2CH2OH) ppm. 13C NMR(125 MHz, D2O): ı 142.74 (triazolium-4-C), 130.78 (triazolium-5-C), 99.22-68.08 (pyranose rings), 58.56 (CH2OH), 54.07 (NCH2CH),38.34 (N+CH3), 26.31 (CH2CH2OH) ppm.

2.3.4.3. 6-(4-Hydroxypropyl-3-methyl-1,2,3-triazolium-1-yl)-6-deoxy starch iodine (2c). FTIR: � 3409, 2923, 1581, 1041, 763 cm−1.The UV �max was 225 nm. 1H NMR (500 MHz, D2O): � 8.57(triazolium-5-H), 5.48-3.42 (pyranose rings), 4.28 (N+CH3), 4.07(NCH2CH), 3.69 (CH2OH), 2.96 (CH2CH2OH), 1.99 (CH2CH2CH2OH)ppm. 13C NMR (125 MHz, D2O): � 144.63 (triazolium-4-C),130.33 (triazolium-5-C), 100.00-67.98 (pyranose rings), 60.29(CH2OH), 54.01 (NCH2CH), 38.01 (N+CH3), 28.93 (CH2CH2OH),19.84 (CH2CH2CH2OH) ppm.

2.3.4.4. 6-(4-Hydroxybutyl-3-methyl-1,2,3-triazolium-1-yl)-6-deoxy starch iodine (2d). FTIR: � 3409, 2931, 1581, 1041, 775 cm−1.The UV �max was 225 nm. 1H NMR (500 MHz, D2O): � 8.56(triazolium-5-H), 5.52-3.26 (pyranose rings), 4.27 (N+CH3),4.06 (NCH2CH), 3.64 (CH2OH), 2.93 (CH2CH2OH), 1.90-1.50(CH2CH2CH2CH2OH) ppm. 13C NMR (125 MHz, D2O): � 144.90(triazolium-4-C), 130.26 (triazolium-5-C), 100.00-67.99 (pyranoserings), 61.05 (CH2OH), 53.95 (NCH2CH), 38.03 (N+CH3), 30.71(CH2CH2OH), 24.60-22.61 (CH2CH2CH2CH2OH) ppm.

2.4. Antifungal assay

Antifungal assay was evaluated against C. lagenarium, F. oxys-porum, and W. fusarium in vitro by mycelium growth rate testaccording to the literatures (Guo et al., 2014). Briefly, stock solu-tions were firstly prepared by adding 50 mg of starch and starchderivatives to 10 mL of distilled water at room temperature. Then,each sample solution was added to sterile PDA medium to give afinal concentration of 0.1, 0.5, and 1.0 mg/mL. The final solutions

were poured into sterilized Petri dishes (9 cm). After solidification,a mycelia disk (diameter: 5 mm) of active fungi was transferred tothe center of the PDA Petri dishes and inoculated at 27 ◦C. When themycelia of fungi reached the edges of the control plate (without the

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resence of samples), the growth inhibition was calculated by theormula:

nhibitoryindex (%) = (1-Da/Db) × 100 (4)

here Da (mm) is the diameter of the growth zone in the test platesnd Db (mm) is the diameter of the growth zone in the control plate.

.5. Statistical analysis

All the experiments were performed in triplicate and the dataere expressed as mean ± the standard deviation (SD, n = 3). Signif-

cant difference analysis was determined using Scheffe’s multipleange test. A level of P < 0.05 was considered statistically significant.

. Results and discussion

.1. Chemical synthesis and characterization

The synthetic strategy for the preparation of 1,2,3-triazolium-unctionalized starch derivatives is outlined in Scheme 1. There arehree hydroxyl groups in anhydroglucose unit (AGU) of starch, a pri-

ary hydroxyl at C-6 and two secondary hydroxyls at C-2 and C-3.hereinto, the primary hydroxyl at C-6 is the highest chemical reac-ive site because of minimal steric hindrance (Zhang & Edgar, 2014).tarch therefore can be brominated with regioselectivity at C-6,hich should be benefit from the steric bulk of three phenyl rings

n alkoxyphosphonium salt intermediate at the primary hydroxylroup, followed by the attacks with bromide anion supplied byhe NBS (Zhang & Edgar, 2014). And the resulting 6-bromo-6-eoxy starch can then serve as a useful intermediate to react withaN3 in DMSO to produce 6-azido-6-deoxy starch by SN2 dis-lacement. Subsequently, the click chemistry could be performedith 6-azido-6-deoxy starch and terminal alkynes with different

hain length to synthesize the starch derivatives bearing 1,2,3-riazole. The preparation of 1,2,3-triazolium-functionalized starcherivatives can be accomplished by alkylation of 1,2,3-triazole with

odomethane. The structures of the newly synthesized compoundsre elucidated by FTIR, UV-vis, 1H NMR, 13C NMR, and elementalnalyses.

.1.1. FTIR analysisComparison of FTIR spectra for starch and starch derivatives is

iven in Fig. 1. The spectrum of the unmodified starch shows a

Scheme 1. Synthetic routes

mers 160 (2017) 163–171

strong broad band at 3428 cm−1, which corresponds to the stretch-ing vibration of O H bonds (Song, Guo, Zhang, Zheng, & Zhao, 2013).The characteristic peaks at 2927 and 1373 cm−1 are respectivelyattributed to the C H stretching and deformation vibration (Nep,Ngwuluka, Kemas, & Ochekpe, 2016). In addition, the characteristicpeaks at 1200–990 cm−1 are assigned to the stretching vibrationsof the C O C stretch in starch (Sukhija, Singh, & Riar, 2016). ForFTIR spectrum of BDST, the new peak at 682 cm−1 is assigned tothe C-Br group (Tan, Li, Wang et al., 2016). The formation of ADST isconfirmed by the peak at 2105 cm−1 in FTIR spectrum which is dueto the vibration absorption of azide group (Qin et al., 2013). Afterthe click reaction of ADST with terminal alkynes, the absorbanceof azide group at 2105 cm−1 disappears completely and new peaksappear at 1554 cm−1 (Tan, Li, Li et al., 2016), which are assignedto the absorbance of C6-1,2,3-triazole rings in the spectra of starchderivatives bearing 1,2,3-triazole. After alkylation of 1,2,3-triazolewith iodomethane, the characteristic bands of the resultant 1,2,3-triazolium-functionalized starch at 1581 cm−1 are assigned to theabsorbance of C6-1,2,3-triazolium moieties.

3.1.2. UV–vis analysisFig. 2 shows the UV–vis spectra of starch and starch derivatives

at a concentration of 40 �g/mL. As far as starch, there are no absorp-tion peaks appeared ranging from 200 to 400 nm because of absenceof chromophore. The UV–vis spectra of starch derivatives bear-ing 1,2,3-triazole indicate broad absorption bands between 200and 250 nm due to the presence of triazole rings and the max-imum absorption values (�max) are observed at 216–220 nm inwater. However, after alkylation with iodomethane, the maximumabsorption peaks shift to 225 nm, which may be ascribed to thechange in electron configuration of 1,2,3-triazole caused by alkyla-tion with iodomethane (Tejero, Lopez, Lopez-Fabal, Gomez-Garces,& Fernandez-Garcia, 2015).

3.1.3. 1H NMR analysisThe 1H NMR spectra of starch and starch derivatives are shown

in Fig. 3 for comparison. The 1H NMR spectrum of starch revealsthe protons in the pyranose ring at around 3.00–5.70 ppm (Tan, Li,Li et al., 2016). Compared with the 1H NMR spectrum of starch,the protons of CH2Br are observed at 3.44 ppm in BDST. After

azidation of BDST, the absorption peak at 3.77 ppm is attributedto hydrogens of CH2N3 (Tan et al., 2017). The presence of theabsorption bands between 7.60 and 7.75 ppm, which are attributedto hydrogens at 5-H position, demonstrates that the 1,2,3-triazole

for starch derivatives.

W. Tan et al. / Carbohydrate Polymers 160 (2017) 163–171 167

Fig. 1. FTIR spectra of starch

Fig. 2. UV–vis spectra of starch and starch derivatives.

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1,2,3-triazole, which demonstrates the high-efficiency of cuprous-catalyzed azide-alkyne cycloaddition. Finally, effective alkylationbetween 1,2,3-triazole and iodomethane has occurred, and the

roups have been successfully introduced to starch backboneSarkar & Rahman, 2017). Moreover, the additional signals at.85-3.94 ppm are assigned to the protons of C6–CH2 in the pyra-ose ring of starch derivatives bearing 1,2,3-triazole and the newhemical shifts at 5.12 ppm (in 1a), 4.69 and 2.48 ppm (in 1b),.48, 2.39, and 1.61 ppm (in 1c), 4.40, 2.32, and 1.55-1.25 ppmin 1d) are assigned to the rest of methylenes (Tan, Li, Li et al.,016). After alkylation with iodomethane, the 1H NMR spectra of,2,3-triazolium-functionalized starch derivatives corroborate thelkylation reaction through the disappearance of the peaks corre-ponding to the protons of the 1,2,3-triazole groups at signaturealues of 7.60–7.75 ppm and the appearance of new signals forhe 1,2,3-triazolium protons at 8.56–8.67 ppm (Mudraboyina et al.,014). In addition, completion of the alkylation reaction is also cor-oborated by the appearance of new signals at 4.27–4.33 ppm forhe pendant methyl groups of 1,2,3-triazolium moieties (Liu et al.,016). Besides, all signals of adjacent methylene groups are shifted

ompared to those initially neighboring the 1,2,3-triazole.

and starch derivatives.

3.1.4. 13C NMR analysisThe formation of the synthesized products is further confirmed

by 13C NMR spectra. As depicted in Fig. 4, the signals between 60and 100 ppm are responded to the chemical shift of 13C NMR ofstarch (Tan, Li, Li et al., 2016). After bromination, new signal atabout 34.78 ppm in highly regioselective BDST is related to thecarbon of C6–Br (Marks, Fox, & Edgar, 2016). However, some dis-turbances of BDST indicate the presence of a low DS of acetateester groups attached to C-6-OH of starch, which can also be foundclearly in the FTIR and 1H NMR. The reasonable interpretation wasthat during the SN2 reaction with starch alkoxyphosphonium saltintermediate, the acetate group was a product of the DMF sol-vent sometimes acting as a nucleophile instead of bromide (Fox& Edgar, 2011). Fortunately, these disturbances are absolutely dis-appeared after proceeding to next step. A new characteristic peak at51.69 ppm appears compared with the spectrum of starch, whichis assigned to the carbon of C6–N3 (Zhang & Edgar, 2015). AfterCuAAC reaction, the 1,2,3-triazole linkers are clearly observed at144.58–148.01 and 123.32–124.45 ppm as new peaks in the 13CNMR spectra of starch derivatives (Uliniuc et al., 2013), and othernew signals for the rest of methylenes are appeared below 65 ppm.13C NMR spectra of 1,2,3-triazolium-functionalized starch deriva-tives also corroborate the alkylation since signals corresponding to1,2,3-triazolium carbons at 142.74-144.90 and 130.26-130.99 ppmand the methyl groups at 38.01-38.58 ppm are observed togetherwith the total disappearance of the 1,2,3-triazole carbons (M’Sahelet al., 2016). In addition, most other carbon signals are gradu-ally shifted downfield after alkylation of the 1,2,3-triazole maybe due to the positive global charge density deshielding effect ofthe cationic macromolecules (Tejero, Lopez, Lopez-Fabal, Gomez-Garces, & Fernandez-Garcia, 2015).

3.1.5. Elemental analysis and degree of substitutionThe yield, elemental analysis, and degree of substitution (DS)

of starch derivatives are shown in Table 1. The degree of sub-stitution of azido in ADST is 0.85, which means that there are85% of hydroxyl groups at C-6 being displaced by azido groups.And then about 80 percent of functional groups at C-6 are1,2,3-triazole groups in the obtained starch derivatives bearing

1,2,3-triazolium has probably accounted for more than 60% of

168 W. Tan et al. / Carbohydrate Polymers 160 (2017) 163–171

Fig. 3. 1H NMR spectra of starc

Table 1The yield, elemental analysis, and degree of substitution of starch derivatives.

Compound Yield (%) Elemental Analysis (%) Degree ofSubstitution

C N H C/N

starch – 42.93 0 7.52 – –ADST 76.52 36.87 18.34 5.22 2.01 0.85a

1a 94.57 39.76 13.48 5.68 2.83 0.81b

1b 92.08 42.20 13.03 6.21 3.12 0.82b

1c 89.56 43.27 12.12 6.54 3.37 0.81b

1d 86.34 45.08 11.62 6.90 3.61 0.79b

2a 68.12 35.22 11.60 4.95 3.04 0.61c

2b 72.59 33.70 10.17 4.75 3.35 0.69c

2c 70.16 36.93 9.87 5.49 3.62 0.72c

2d 65.96 37.69 9.37 5.67 3.86 0.74c

a Degree of Substitution of azido in ADST.b

ftp

and the strongest antifungal activity is observed at 1.0 mg/mL. It is

Degrees of Substitution of 1,2,3-triazole in starch derivatives.c Degrees of Substitution of 1,2,3-triazolium in starch derivatives.

unctional groups at C-6 in the starch derivatives bearing 1,2,3-riazolium. The relatively high yields and degrees of substitutionrovide good evidence that this synthetic strategy for the prepa-

h and starch derivatives.

ration of 1,2,3-triazolium-functionalized starch derivatives is aneffective method.

Based on the analysis of structural characterization above men-tioned, it could demonstrate that 1,2,3-triazolium moieties hadbeen successfully introduced to the starch backbone.

3.2. Antifungal activity

The capabilities of starch and starch derivatives at 0.1, 0.5,and 1.0 mg/mL to inhibit the growth of the tested three plant-threatening fungal strains, including C. lagenarium, F. oxysporum,and W. fusarium, are shown in Figs. 5–7 , respectively. Signifi-cant differences (P < 0.05) are confirmed for the antifungal propertyof starch and starch derivatives against the tested three plant-threatening fungal strains.

Among the pathogenic fungi species, C. lagenarium is the mostsusceptible yeast to the tested compounds. The inhibitory indices ofall the samples mount up with increasing concentration (P < 0.05),

found that starch has less antifungal effect with the inhibitory indexbelow 5%. Whereas, starch derivatives bearing 1,2,3-triazole showslightly higher antifungal property than pristine starch (P < 0.05)

W. Tan et al. / Carbohydrate Polymers 160 (2017) 163–171 169

Fig. 4. 13C NMR spectra of starch and starch derivatives.

F

bbgca

ig. 5. The antifungal activity of starch and starch derivatives against C. lagenarium.

ecause of the introduction of 1,2,3-triazole groups. The hydrogenond interaction formed by 1,2,3-triazole and biomolecular tar-

ets plays key role in antifungal activity (Tang et al., 2015). Theyould inhibit synthesis of the cell membrane and cell wall to exhibitntimicrobial activity (Croce, Conti, Maake, & Patzke, 2016).

Fig. 6. The antifungal activity of starch and starch derivatives against F. oxysporum.

Moreover, after one-step alkylation with iodomethane, 1,2,3-triazolium-functionalized starch derivatives immediately exhibit

tremendously enhanced antifungal property with the inhibitoryindices of over 60% at 1.0 mg/mL (P < 0.05), compared with 1,2,3-triazole-functionalized starch derivatives with the inhibitory

170 W. Tan et al. / Carbohydrate Poly

F

iffwa1pmpF2pogeZmitd

a(2titiaiwGelawampl

4

rwwU

ig. 7. The antifungal activity of starch and starch derivatives against W. fusarium.

ndices of below 10% (P < 0.05). Notably, 1,2,3-triazolium-unctionalized starch derivatives are still active against testedungi even when the dosage is lowered to 0.5 mg/mL (P < 0.05),

hich suggests that 1,2,3-triazolium should be the efficientntifungal function group. The powerful disruptive effect of,2,3-triazolium-functionalized starch on the microorganism wasrobably based on electrostatic interaction of positively chargedoieties of the cationic molecules and negatively charged com-

onents on microbial cell membrane (Hassan, 2015; Jia, Duan,ang, Wang, & Huang, 2016; Khan, Ullah, & Oh, 2016; Ng et al.,014). This electrostatic interaction could facilitate changes ofermeation property of the membrane wall to provoke internalsmotic imbalances and also cause hydrolysis of the peptido-lycans in the microbial cell wall to the leakage of intracellularlectrolytes and nutrients (Cai et al., 2015; Fan et al., 2015; Tawfik,aky, Mohammad, & Attia, 2015). Besides, the greater effect onicrobial of this electrostatic interaction than hydrogen bond

nteraction could cause the higher inhibitory indices of 1,2,3-riazolium-functionalized starch derivatives compared with starcherivatives bearing 1,2,3-triazole.

Many studies from the past had reported that the length of thelkyl groups was an important determinant of antifungal activityChandrika, Shrestha, Ngo, & Garneau-Tsodikova, 2016; Luo et al.,009). Therefore, the effect of the length of the alkyl groups in 1,2,3-riazolium-functionalized starch derivatives on antifungal activitys also evaluated in this paper. The results indicate that increasinghe length of the alkyl chain on the 1,2,3-triazolium rings resultsn a decrease in antifungal activity of starch derivatives (P < 0.05)gainst all the targeted microorganisms and the antifungal activ-ty increases in the order of 2a > 2b > 2c > 2d > 1a ∼ 1d > starch. It

as in accordance with the conclusions of Chandrika et al. (2016),arudachari, Isloor, Satyanarayana, Fun, and Hegde (2014); Yaot al., 2012; while some studies had shown that an increase in chainength of alkyl substituted groups corresponded with an increase inntifungal activity (Zhao et al., 2016). The reasonable interpretationas that the longer alkyl groups had the stronger electron-donating

bility and tended to donate more electrons to 1,2,3-triazoliumoieties accordingly, which inevitably caused the decrease in the

ositive charge density of 1,2,3-triazolium moieties and eventuallyed to reducing in antifungal property (Guo et al., 2007).

. Conclusion

In summary, we have proposed a straightforward synthetic

oute to novel 1,2,3-triazolium-functionalized starch derivativesith alkyl chains of various lengths by associating CuAAC stepith efficient alkylation of 1,2,3-triazole with iodomethane. FTIR,V-vis, 1H NMR, and 13C NMR spectra, and elemental analysis

mers 160 (2017) 163–171

confirmed that 1,2,3-triazolium moieties had been successfullyintroduced to the starch backbone. The antifungal activity againstthree kinds of plant threatening fungal strains was estimated byobserving the percentage inhibition of mycelial growth. The syn-thesized 1,2,3-triazolium-functionalized starch derivatives clearlyshowed stronger antifungal activity than starch derivatives bearing1,2,3-triazole and pristine starch. The obtained findings suggestedthat 1,2,3-triazolium moieties should be excellent antifungal func-tion groups. Moreover, the length of the alkyl groups was animportant determinant of antifungal activity of 1,2,3-triazolium-functionalized starch derivatives, and the antifungal property ofthem against the tested fungi decreased with increasing side-chainlength. And the product described in this paper might serve as anew leading structure for further design of antifungal agents.

Acknowledgements

This work was supported by a grant from the National Natu-ral Science Foundation of China (41576156), Shandong ProvinceScience and Technology Development Plan (2015GSF121045), andYantai Science and Technology Development Plan (2015ZH078),and the Public Science and Technology Research Funds Projects ofOcean (No. 201505022-3).

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