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International Journal of Biological Macromolecules 91 (2016) 623–629

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules

j ourna l h o mepa ge: www.elsev ier .com/ locate / i jb iomac

ynthesis of water soluble chitosan derivatives withalogeno-1,2,3-triazole and their antifungal activity

ing Lia, Wenqiang Tana,b, Caili Zhangc, Guodong Gud, Zhanyong Guoa,∗

Key Laboratory of Coastal Biology and Bioresource Utilization, Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences, Yantai, Shangdong64003, ChinaGraduate School of Chinese Academy of Sciences, Beijing 100039, ChinaSchool of Chemistry and Chemical Engineering, Yantai University, Yantai 264005, ChinaAlliance Pharma, Inc. 17 Lee Boulevard Malvern, PA 19355, USA

r t i c l e i n f o

rticle history:eceived 19 April 2016eceived in revised form 16 May 2016ccepted 2 June 2016vailable online 3 June 2016

eywords:

a b s t r a c t

Chitosan is an abundant and renewable polysaccharide, which exhibits attractive bioactivities and natu-ral properties. Improvement such as chemical modification of chitosan is often performed prior to furtherutilization. Three novel water soluble chitosan derivatives containing 1,2,3- triazole with or without halo-gen was designed and synthesized. Their antifungal activity against three kinds of phytopathogens wasestimated by hyphal measurement in vitro. The inhibitory property and water solubility of the synthe-sized chitosan derivatives exhibited a remarkable improvement over chitosan. It is hypothesized that

hitosan derivatives,2,3-Triazolentifungal activityalogenslectron-withdrawing capacity

thiazolyl groups enable the synthesized chitosan to possess obviously better antifungal activity. More-over, CTCTS and BTCTS, which have halogens at the periphery of polymers, inhibited the growth of testedphytopathogens more effectively with inhibitory indices of 81–93% at 1.0 mg/mL. The halogens couldhave a synergistic effect with triazole as they exhibited antifungal activity and electron-withdrawingcapacity, which improve the antifungal activity of chitosan derivatives.

© 2016 Elsevier B.V. All rights reserved.

. Introduction

Chitosan is one of the most abundant natural polysaccharidesnd has attracted broad attention due to its antifungal activitygainst various groups of pathogenic fungi [1–6]. The antifungalbility, coupled with its non-toxicity, biodegradability, and bio-ompatibility, facilitates emerging applications of chitosan in foodcience, agriculture, medicine, and textile areas [7]. Another advan-age of chitosan, which makes it highly desirable, is the capabilityf being chemically modified [8]. Through chemical modification,

great deal of chitosan derivatives have been prepared to pro-ote their biological activity [9–13]. However, the application of

hitosan is restricted to only acidic conditions where the NH2roup becomes protonated [14,15]. The further enhancement ofhe antimicrobial activity of chitosan over a broader pH range willromote the better application of chitosan in many areas.

Triazole derivatives represent an interesting class of hetero-yclic compounds; they possess many biological activities such asntimicrobial, anti-tubercular, anti-inflammatory and anticancer

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

ttp://dx.doi.org/10.1016/j.ijbiomac.2016.06.006141-8130/© 2016 Elsevier B.V. All rights reserved.

activities [16–20]. As an important type of nitrogen-containingaromatic heterocyclic compounds, 1,2,3-triazole was regarded asthe bioisosteric replacement of itsbioisoster 1,2,4-triazole, amide,or even other nitrogen-containingaromatic heterocycles [21,22].1,2,3-Triazole was stable to metabolic degradation and capa-ble of forming hydrogen bonds, which could favor binding tobiomolecular targets and improve solubility [23]. The team of Ifukuprepared new chitosan derivatives containing triazolyl moieties atthe C6 position of glucosamine units by coupling between azideand propargyl groups of chitosan via a 1,3-dipolar cycloaddition[24–26]. Novel 1,2,3-triazole-linked starch derivatives synthesizedvia ‘click chemistry’ exhibited improved antibacterial property andantioxidant property [21,27]. These observations inspired us tomodify chitosan with triazole as a substituent to improve anti-fungal activity of chitosan derivatives. Moreover, 1,2,3-triazole asan attractive bridge group could connect pharmacophores to givean innovative bioactive compound. This synthetic strategy enablesfurther chemical modification at the residual positions of chitosan,which leads to the creation of finely designed novel chitosan deriva-

tives having several different functional groups at each position.

In this paper, we reported the synthesis and antifungal prop-erties of a group of chitosan derivatives with 1,2,3- triazole assubstituent including TCTS, CTCTS, and BTCTS (Scheme 1). After the

624 Q. Li et al. / International Journal of Biological Macromolecules 91 (2016) 623–629

O

CH2OH

OH

NH2

On

O

CH2OH

OH

N

On

OO

O

OH

N

On

OO

Br

O

OH

N

On

OO

N3

O

OH

NH2

On

N3

O

OH

N

On

N3

I

O

OH

N

On

N

I

NN

R

O

OH

NH2

On

Br

O

OH

N

On

Br

I

O

OH

N

On

N

I

NN

HNNN

R

1 2 3 4

5 6 7

TCTS

CTCTS: R=Cl

Br

the pr

pp6ogsotCawtat6thgwt(Fsh

2

2

itpCbSss

Scheme 1. Synthetic scheme for

rotection of C2 NH2 by phthaloyl, we activated C6 OH and pre-ared a 6-azido-6-deoxy-N-phthaloyl-chitosan firstly. [24] Then-azido-6-deoxy-N-phthaloyl-chitosan was treated with aque-us hydrazine monohydrate to remove the phthaloyl protectingroup and the C2 NH2 was modified as quaternary ammoniumalt. That quaternary ammonium salt was selected by virtuef water solubility, which can enlarge the application of chi-osan as a food preservative or bioactive matrix. Subsequently,TCTS and BTCTS were synthesized via “click reaction” using

6-azido-6-deoxy-N-quaternary ammonium chitosan derivativeith two halogenated terminal alkynes. For comparison, a chi-

osan derivative without halogen, TCTS was also synthesizednd studied under identical conditions. TCTS was synthesizedhrough the nucleophile substitution reaction between 6-bromo--deoxy-N-quaternary ammonium chitosan and 1,2,3-triazole. Thearget chitosan derivatives designed in this way were expected toave advantageous features, namely high antifungal activity andood water solubility. The chemical structures of the derivativesere characterized by FT-IR and 13C NMR. Three common plant-

hreatening fungi, Colletotrichum lagenarium (Pass) Ell.et halstATCC30016), Fusarium oxysporum f.sp.niveum (ATCC36116), andusarium oxysporum.f.sp.cucumebrium Owen (ATCC42357) wereelected to evaluate the antifungal properties of the derivatives byyphal measurement in vitro.

. Experimental

.1. Materials

Chitosan was purchased from Qingdao Baicheng biochem-cal Corp. (China). Its degree of deacetylation is 97% andhe viscosity-average molecular weight is 7.0 × 104 Da. 3-yridinecarboxaldehyde was purchased from Aladin Chemicalorp. Halogenated terminal alkynes (3-chloropropyn and 3-

romopropyne) and 1,2,3-1H-triazole were purchased fromigma-Aldrich with a minimum purity of 98%. The other reagentsuch as hydrazine monohydrate, iodomethane, sodium iodide,odium hydroxide, cuprous iodide, potassium iodide and solvents

BTCTS: R=

eparation of chitosan derivatives.

are analytical grade and are supplied by Sinopharm ChemicalReagent Co., Ltd., Shanghai, China.

2.2. Analytical methods

FT-IR spectra were measured on a Jasco-4100 Fourier TransformInfrared Spectroscopy (Japan, provided by JASCO Co., Ltd. Shanghai,China) with KBr disks. 13C Nuclear Magnetic Resonance (13C NMR)spectra were measured with a Bruker AVIII-850 Spectroscopy withTCI CryoProbe (Switzerland, provided by Bruker Tech. and Serv. Co.,Ltd. Beijing, China.). The elemental analyses (C, H, and N) were per-formed on a Vario Micro Elemental Analyzer (Elementar, Germany).The Degree of Substitution (DS) was calculated based on elementalanalysis results. [28]

DSx = 100nC,R − nC,P

(nC,R − MN

MC× nN,R × wC/N

)

where nC,R and nN,R are the number of carbon and nitrogen molesper mole of reactant unit respectively; nC,P is the number of nitro-gen moles per mole of product unit; MC and MN are the molar massof carbon and nitrogen; wC/N is the mass ratio between carbon andnitrogen.

2.3. The synthesis of chitosan derivatives

2.3.1. Synthesis of 6-azido-6-deoxy-N-trimethyl quaternaryammonium chitosan (7)

6-Azido-6-deoxy-N-phthaloyl-chitosan (5) was preparedaccording to the methods reported by Ifuku [24]. DSazide 0.94;13C NMR/DMSO: ı172.8 ppm (carbon of C O in phthaloyl group);ı139.8, 136.7, and 128.3 ppm (phthaloyl group); ı102.4–55.2 ppm(pyranose rings); FT-IR (thin film): v 3463 (NH2 and OH), v 2105(C-6-azido), v 1774, 1716 (C O in phthaloyl group), v 721 (arom).

6-Azido-6-deoxy-chitosan (6) and compound 7 were prepared

according to the methods of literatures [29,30].

Compound 6: 6-azido-6-deoxy-N-phthaloyl-chitosan (5) (2 g,6.3 mmol) was dissolved in 60 mL N-methyl-2-pyrrolidone, 50 mLof 4 M aqueous hydrazine monohydrate was added afterward, and

logical Macromolecules 91 (2016) 623–629 625

tptfrv

swweb8n((

2

hiapuad

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aı3(

2

adao

3

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2

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3500 300 0 250 0 2000 150 0 100 0

350 0 300 0 250 0 200 0 150 0 100 0

1473

1473

342 4 802

802TCTS

BTCTS

4

CTCTS

34321473

802342 4

7CTS

5

6

Wavelengh

2105

7213463

1716

1774

1592

1477

3378

3428

3424

Q. Li et al. / International Journal of Bio

he mixture was stirred at 100 ◦C for 4 h under Ar. The mixture wasrecipitated into ethanol, and the precipitate was collected by fil-ration and washed by ethanol. The products were dried at 60 ◦Cor 24 h, yield: 67.0%; 13C NMR/DMSO: ı107.7–56.6 ppm (pyranoseings); FT-IR (thin film): v 3378 (NH2 and OH), v 2109 (C-6-azido),

1592 (NH2).Compound 7: a mixture of compound 6 (0.72 g, 3.6 mmol), 2.16 g

odium iodide, 4 mL aqueous sodium hydroxide solution (15%,/v), and 4 mL iodomethane in 30 mL N-methyl-2-pyrrolidoneas stirred at 60 ◦C for 1 h. The mixture was precipitated into

thanol, and the precipitate was collected by filtration and washedy ethanol. The products were dried at 60 ◦C for 24 h, yield:7.0%; DSquaternary 0.91; 13C NMR/DMSO: ı96.8–57.4 ppm (pyra-ose rings); ı54.4 ppm (carbon of quaternary ammonium); FT-IRthin film): v 3428 (NH2 and OH), v 2105 (C N of azido), v 1477C H of quaternary ammonium).

.3.2. Synthesis of CTCTS and BTCTSA mixture of compound 7 (1.0 mmol), 12 mg cuprous iodide,

alogenated terminal alkynes (2.0 mmol), and 3 mL triethylaminen 20 mL dimethyl sulfoxide was stirred at 75 ◦C for 48 h underrgon. The mixture was filtered and the filtrate was collected andrecipitated into ethanol. The precipitate was washed with sat-rated solution of potassium iodide and filtered. The unreactedlkyne was extracted in a Soxhlet apparatus with ethanol for twoays. The products were dried at 60 ◦C for 24 h.

CTCTS: yield: 72.6%; DStriazole 0.78; 13C NMR/DMSO: ı135.3nd 129.5 ppm (triazole rings); ı97.0–50.9 ppm (pyranose rings);54.0 ppm (carbon of quaternary ammonium); FT-IR (thin film): v424 (NH2 and OH), v 1473 (C H of quaternary ammonium), v 802C H of triazole).

BTCTS: yield: 51.9%; DStriazole 0.83; 13C NMR/DMSO: ı134.9nd 129.3 ppm (triazole rings); ı96.8–51.0 ppm (pyranose rings);53.7 ppm (carbon of quaternary ammonium); FT-IR (thin film): v424 (NH2 and OH), v 1473 (C H of quaternary ammonium), v 802C H of triazole).

.3.3. Synthesis of TCTS6-Bromo-6-deoxy-N-phthaloyl-chitosan (2) was prepared

ccording to the methods reported by Ifuku [24]. For 6-bromo-6-eoxy-chitosan (3) and 6-bromo-6-deoxy-N-trimethyl quaternarymmonium chitosan (4), the synthesis method is the same as thatf compound 7 and 8, respectively.

6-Bromo-6-deoxy-chitosan (3), yield: 50.0%; FT-IR (thin film): v370 (NH2 and OH), v 1599 (NH2).

6-Bromo-6-deoxy-N-trimethyl quaternary ammonium chi-osan (4), yield: 87.0%; 13C NMR/DMSO: ı96.6–58.0 ppm (pyranoseings); ı53.9 ppm (carbon of quaternary ammonium); FT-IR (thinlm): v 3424 (NH2 and OH), v 1473 (C H of quaternary ammonium).

TCTS: a mixture of compound 4 (0.6 g, 1.5 mmol), 1,2,3-1H-riazole(207 mg, 3.0 mmol), and 66 mg sodium hydroxide in 10 mLMSO was stirred at 50 ◦C for 2 h. The mixture was precipitated

nto ethanol, and the precipitate was collected by filtration andashed by ethanol. The products were dried at 60 ◦C for 24 h, yield:

6.0%; DStriazole 0.86; 13C NMR/DMSO: ı130.1 and 128.8 ppm (tri-zole rings); ı96.2–58.0 ppm (pyranose rings); ı53.4 ppm (carbonf quaternary ammonium); FT-IR (thin film): v 3424 (NH2 and OH),

802 (C H of triazole), v 1473 (C H of quaternary ammonium).

.4. Antifungal assays

Antifungal assays were performed by following the plate growth

ate method described by Guo et al. [31]. Briefly, the compoundsere dispersed in distilled water at a concentration of 5.0 mg/mL.

hen, each sample (chitosan, TCTS, CTCTS, and BTCTS) solution wasdded to sterilized potato dextrose agar to give a final concentra-

Waveleng h

Fig. 1. FT-IR spectra of intermediate products, TCTS, CTCTS, and BTCTS.

tion of 0.1, 0.5, and 1.0 mg/mL. After the mixture was cooled in theplate (6.0 cm diameter), 5.0 mm diameter of fungi mycelium wastransferred to the test plate and incubated at 27 ◦C for 2–3 days.When fungi mycelium reached the edges of control plate (withoutthe presence of samples), the inhibitory index was calculated asfollows:

Inhibitoryindex(%) = (1 − Da/Db) × 100

where Da is the diameter of growth zone in test plate and Db isthe diameter of the growth zone in control plate. Each experimentwas performed in three replicates, and the data were shown asmean ± SD. The Scheffe’s method was used to evaluate the differ-ences of inhibitory index in the antifungal tests. Data were analyzedby an analysis of variance (P < 0.05) to guarantee statistical sig-nificance. The results were processed by the computer programs:Origin and Statistic software SPSS.

3. Results and discussion

3.1. Chemical syntheses and characterization

Each step of synthesis was followed by FT-IR and 13C NMRspectroscopy measurements. The FT-IR and 13C NMR spectra ofintermediate products, TCTS, CTCTS, and BTCTS are shown inFigs. 1 and 2 respectively.

6-Azido-6-deoxy-N-phthaloyl-chitosan was selected as inter-mediate to protect the amino group of chitosan, as it was reported

that chemical modification of chitosan with phthaloyl groups couldlead to 2-N regioselective substitution [24,32]. In spectrum of6-Azido-6-deoxy-N-phthaloyl-chitosan (5), characteristic peak ofC-6-azido is observed at 2105 cm−1, and three absorption bands

626 Q. Li et al. / International Journal of Biologica

Fig. 2. 13C NMR spectra of intermediate products, TCTS, CTCTS, and BTCTS.

l Macromolecules 91 (2016) 623–629

at 721, 1716, and 1774 cm−1 are attributed to phthalimide group(Fig. 1) [33]. As shown in Fig. 2, the stretching vibration of the car-bonyl groups C O at 172.8 ppm and of the aromatic ring at 139.8,136.7, and 128.3 ppm can be obviously observed. The peak of C-6 carbon shifts downfield to 55 ppm as a single signal, supportingregioselective and complete C-6 substitution of the azide moiety[24].

Subsequently, deprotection of compound 5 was conducted toremove the pathaloyl protecting group, and the amino groupcould be exposed for further chemical modification. Comparedwith compound 5, three peaks at 721, 1716, and 1774 cm−1 arecompletely absent from the FT-IR spectrum of 6-azido-6-deoxy-chitosan, which is assigned to the success of deprotection ofpathaloyl group (Fig. 1). Meanwhile, the peaks at 172.8, 139.8,136.7, and 128.3 in 13C NMR spectrum disappear, confirming thesuccess of the preparation (Fig. 2).

When 6-azido-6-deoxy-chitosan was transformed to 6-azido-6-deoxy-N-trimethyl quaternary ammonium chitosan (7), new peaksappear at about 1623 cm−1, which are assigned to quaternaryammonium salts. The peak at 1477 cm−1 is ascribed to the char-acteristic absorption of N CH3 [34]. In the 13C NMR spectrumof compound 7 (Fig. 2), carbon of N CH3 is clearly observed at54.3 ppm. 6-Bromo-6-deoxy-N-trimethyl quaternary ammoniumchitosan (4) has similar spectra with compound 7. Peaks at 1658and 1473 cm−1 in FT-IR spectrum and peak at 53.9 ppm in 13C NMRspectrum could prove the existence of quaternary ammonium salt(Figs. 1 and 2). The quaternary ammonium salt was chosen as poly-mer part by virtue of its water solubility in neutral and alkalineaqueous solutions.

As long as we got 6-azido-6-deoxy-N-trimethyl quaternaryammonium chitosan, the ‘click chemistry’ could be performed inan elegant way with propynyl amine to synthesize the aimedamphiphilic aminated chitosan [33]. The peak at 2105 cm−1 in com-pound 7 spectrum disappear when the C-6-azido in compound 7was transformed to 1,2,3-triazoles and a new absorption band atabout 802 cm−1 appear (Fig. 1) [35]. The 1,4-triazole linker is clearlyobserved at 129.3–135.3 ppm as two new peaks in the 13C NMRspectrum of CTCTS and BTCTS [33,36]. In the spectra of TCTS, newpeak at 802 cm−1 in FT-IR spectrum and 130.1 and 128.8 ppm in 13CNMR spectrum are well attributed to the structure of TCTS.

3.2. Water solubility and antifungal activity

Fig. 3 shows the aqueous solution of the synthesized chitosanderivatives (neutral water, 1.0 mg/mL) and chitosan (neutral waterand 1% HAc). It is obvious that chitosan is soluble in 1% HAc due toprotonation of the amino groups (NH3

+). CTCTS, BTCTS, and TCTShave favorable water solubility, and can be prepared to aqueoussolution (0.1–1.0 mg/mL) at room temperature. The good watersolubility of the synthesized chitosan derivatives was due to both1,2,3-triazoles and the quaternary ammonium salt at the C2 posi-tion of chitosan. As hydrophilic moiety at the end of the molecularchains, triazole can enable chitosan with better water solubility.The large dipole moment of 1,2,3-triazole could make it func-tionalized as a weak hydrogen bond donor [27]. Moreover, thequaternization of chitosan is also an important means for improv-ing its solubility.

Chitosan has poor solubility in water, so we used water-solublelow molecular chitosan in antifungal activity test. Here, we testedthe antifungal activity of the starting chitosan, TCTS, CTCTS, andBTCTS against three common plant-threatening fungi F. oxysporum

f.sp.niveum, C. lagenarium, and F. oxysporum.sp.cucumebrium Owen.The results are shown in Figs. 4–6.

As seen in Fig. 4, the inhibitory indices of all the samples enhancewith increasing concentration. The antifungal activity of chitosan

Q. Li et al. / International Journal of Biological Macromolecules 91 (2016) 623–629 627

Fig. 3. Solution of the synthesized chitosan derivatives (neutral water, 1.0 mg/mL) and chitosan (neutral water and 1% HAc).

chitosan TCTS BTCTS CTCTS0

10

20

30

40

50

60

70

80

900.1mg/ml 0.5mg/ml 1.0mg/ml

Inhi

bito

ry In

dex(

%)

Fp

iici8addosmg

chitosan TCTS BTCTS CTCTS0

20

40

60

80

100

Inhi

bito

ry In

dex(

%)

0.1mg/ml 0.5mg/ml 1.0mg/ml

starch against the E. coli and S. aureus [27].

ig. 4. The antifungal activity of chitosan, TCTS, CTCTS, and BTCTS against F. oxys-orum f.sp.niveum.

s weak against F. oxysporum f.sp.niveum and the inhibitory indexs 24.2% at 1.0 mg/mL. Compared with the antifungal activity ofhitosan, the chitosan derivatives have better activity, and thenhibitory indices of TCTS, CTCTS, and BTCTS are 58.5%, 84.0%, and1.8% at 1.0 mg/mL, respectively. It is obvious that TCTS, CTCTS,nd BTCTS show much better antifungal activity due to the intro-uction of functional groups—thiazolyl groups. As illustrated by theata, the antifungal activity of CTCTS and BTCTS are better than thatf TCTS. Comparing the difference of compound structures, we can

peculate that the increased antifungal activity of CTCTS and BTCTSay benefit from halogens grafted in the compounds. The chloro-

roup and bromo-group were used in many fungicides such as

Fig. 5. The antifungal activity of chitosan, TCTS, CTCTS, and BTCTS against C. lage-narium.

pentachloronitrobenzene, chlorothalonil, tektamer, and bromopol[37]. It was reported that the introduction of halogen such aschlorine was important for improving the fungicidal activity of het-erocyclics [38]. Meanwhile, the above results suggested that thechitosan derivatives with stronger electron-withdrawing capacityshowed better antifungal activity. The observation is in agree-ment with the results obtained by Tan, who found that the starchderivatives contained 1,2,3-triazoles with electron-withdrawingsubstitutions had enhanced antibacterial activity compared with

Figs. 5 and 6 show the antifungal activity of chitosan and all thederivatives against C. lagenarium and F. oxysporum.sp.cucumebriumOwen. All the samples show antifungal activity against C. lagenar-

628 Q. Li et al. / International Journal of Biologica

chitosan TCTS BTCTS CTCTS0

20

40

60

80

1000.1mg/ml 0.5mg/ml 1.0mg/ml

Inhi

bito

ry In

dex(

%)

Fp

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ig. 6. The antifungal activity of chitosan, TCTS, CTCTS, and BTCTS against F. oxys-orum.sp.cucumebrium Owen.

um and F. oxysporum.sp.cucumebrium Owen, and the inhibitoryndices of them mount up with increasing concentration. Thenhibitory indices of chitosan, TCTS, CTCTS, and BTCTS against. lagenarium are 30.0%, 85.1%, 93.2%, and 88.1% at 1.0 mg/mL,espectively. And the inhibitory indices of chitosan, TCTS, CTCTS,nd BTCTS against F. oxysporum.sp.cucumebrium Owen are 14.7%,6.1%, 91.7%, and 91.7% at 1.0 mg/mL, respectively. Similar to thentifungal activity against F. oxysporum f.sp.niveum, three chitosanerivatives show much stronger antifungal activity than unmod-

fied chitosan. The results further confirmed that triazole groupsrafted into the synthesized chitosan derivatives contribute a loto the antifungal action and consequently increase the antifungalctivity of them. The triazole groups could inhibit synthesis of theell membrane and cell wall to exhibit antimicrobial activity [39].t the same time, the inhibitory index of TCTS with DS of 0.86%as slightly inferior to that of CTCTS with DS of 0.78 and BTCTSith DS of 0.82, suggesting that a higher inhibitory index of CTCTS

nd BTCTS should be possible if TCTS, CTCTS, and BTCTS have theame DS. The halogens could have a synergistic effect as they exhib-ted a variety of biological activities including antifungal activitynd electron-withdrawing capacity. The chitosan derivatives withubstituted groups own stronger electron withdrawing ability rel-tively possessed greater antifungal activity [27]. Moreover, manyungicides containing halogens have pronounced toxicities andheir residues in the environment have already aroused seriousnvironment problems. When these groups are grafted onto chi-osan, they could be released slowly which will largely avail thenvironmental issue [37].

. Conclusion

In this paper, a group of novel water soluble chitosan derivativesontaining 1,2,3-triazole with or without halogen were designednd synthesized. We modified chitosan at its primary hydroxyl via

click chemistry’ to get 1,2,3-triazole and at its amino to get quater-ary ammonium salt. The antifungal activity against three kinds ofhytopathogens was estimated by hyphal measurement in vitro. Allhe chitosan derivatives have good solubility in water, and exhibitigher inhibition indices than chitosan. These data demonstratehat the 1,2,3-triazole functional groups contribute to the biologi-al activity against some plant pathogenic fungi because triazole isffective antimicrobial functional group. Moreover, the antifungalctivity of CTCTS and BTCTS, which have halogens at the periph-

ry of polymers, are better than that of TCTS. This suggests that theynergistic effect of halogens and triazole will improve the antifun-al activity of chitosan derivatives. The halogens could influencehe antifungal activity of chitosan derivatives as they exhibited

[

[

l Macromolecules 91 (2016) 623–629

a variety of biological activities including antifungal activity andelectron-withdrawing capacity. Further study will be carried outto ascertain this hypothesis. These findings mentioned above bringfurther evidence that chitosan derivatives are active and have thepotential of becoming alternatives of some harmful pesticides fordisease control.

Acknowledgments

We thank the National Natural Science Foundation of China(41576156), Shandong Province Science and Technology Develop-ment Plan (2015GSF121045), and Yantai Science and TechnologyDevelopment Plan (2015ZH078), and Technology Research FundsProjects of Ocean (No. 2015418022-3) for financial support of thiswork.

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