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Novel cationic chitosan derivative bearing 1,2,3-triazolium and pyridinium: Synthesis, characterization, and antifungal property
Carbohydrate Polymers 182 (2018) 180–187
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Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol
Research Paper
Novel cationic chitosan derivative bearing 1,2,3-triazolium and pyridinium: Synthesis, characterization, and antifungal property
MARK
Wenqiang Tana,b, Qing Lia, Fang Donga, Jingjing Zhanga,b, Fang Luana,b, Lijie Weia, Yuan Chena,b, ⁎ Zhanyong Guoa,b, a b
Key Laboratory of Coastal Biology and Bioresource Utilization, Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences, Yantai 264003, China University of Chinese Academy of Sciences, Beijing 100049, China
A R T I C L E I N F O
A B S T R A C T
Chemical compounds studied in this article: Chitosan (PubChem CID: 439341) Iodomethane (PubChemCID: 6328) Propargyl bromide (PubChem CID: 7842) 3-Aminopyridine (PubChem CID: 10009) Sodium azide (PubChem CID: 33557) Triethylamine (PubChemCID: 8471) Cuprous iodide (PubChemCID: 24350)
In this paper, novel cationic chitosan derivative possessing 1,2,3-triazolium and pyridinium groups was synthesized conveniently via cuprous-catalyzed azide-alkyne cycloaddition (CuAAC) and methylation. FTIR, 1H NMR, and elemental analysis examined the structural characteristics of the synthesized derivatives. The antifungal efficiencies of chitosan derivatives against three plant-threatening fungi were assayed by hypha measurement in vitro. The determination showed that chitosan derivative bearing 1,2,3-triazolium and pyridinium displayed tremendously enhanced antifungal activity as compared with chitosan and chitosan derivative bearing 1,2,3-triazole and pyridine. Notably, the inhibitory indices of it against Colletotrichum lagenarium attained 98% above at 1.0 mg/mL. The results showed that N-methylation of 1,2,3-triazole and pyridine could effectively enhance antifungal activity of the synthesized chitosan derivatives. Besides, the prepared chitosan derivatives showed non-toxic effect on cucumber seedlings. This synthetic strategy might provide an effective way and notion to prepare novel cationic chitosan antifungal biomaterials.
Keywords: Chitosan Chemical modification Click chemistry 1,2,3-Triazolium Pyridinium Antifungal activity
1. Introduction Chitosan, a deacetylated derivative from chitin, is the only readily available basic amino-polysaccharide in the nature (de Oliveira Pedro, Schmitt & Neumann, 2016; Wu et al., 2016). As one of the most promising biomaterials with good biocompatibility and biodegradability (Li, Duan, Huang & Zheng, 2016; Qian, Xu, Shen, Li & Guo, 2013), chitosan has wide application as wastewater treatment agent, a drug delivery system, and bactericidal or antibacterial agent in the medical industry (Cruz, Garcia-Uriostegui, Ortega, Isoshima & Burillo, 2017; Khan, Ullah & Oh, 2016). However, applications of chitosan are considerably limited by poor solubility in both organic and aqueous solvents (Chen et al., 2016; Qin et al., 2012). Chemical modification of chitosan can overcome this problem to some extent (Jia, Duan, Fang, Wang & Huang, 2016; Sun, Shi, Wang, Fang & Huang, 2017). Derivatization by introducing small functional groups to chitosan backbone can drastically increase the solubility of chitosan at neutral and alkaline pH values as well as strengthen its original bioactivities to broaden the industrial applications (Li, Duan, Huang & Zheng, 2016; Liu, Meng, Liu,
⁎
Kan & Jin, 2017). Cationic chitosan is one of the most important group of chitosan derivatives, which has many important physicochemical features such as water solubility and chemical stability as well as antimicrobial properties, due to the electrostatic interactions with anionic biomolecules at the cell surface (Moreno-Vasquez et al., 2017; Sun et al., 2017). Currently, cationic chitosan derivatives can be successfully produced by transforming amino groups into quaternary ammonium salts or by introducing cationic functional groups, such as ammonium, phosphonium, or sulfonium, to chitosan backbones via chemical reactions with primary amino and hydroxyl groups (Chen et al., 2016). The cuprous-catalyzed azide-alkyne cycloaddition (CuAAC) leading to regioselective 1,4-disubstituted-1,2,3-triazoles (Saravanakumar, Ramkumar & Sankararaman, 2011) has received widespread attention in polysaccharide modification due to the merits of CuAAC, including wide in scope, high yield, modularity, tolerant to other functional groups, ready available starting materials, and be stereospecific (Su et al., 2017; Wang et al., 2017). And the application of CuAAC in polysaccharide modification has brought substantial progress
Corresponding author at: University of Chinese Academy of Sciences, Beijing 100049, China. E-mail address: [email protected] (Z. Guo).
https://doi.org/10.1016/j.carbpol.2017.11.023 Received 7 May 2017; Received in revised form 24 October 2017; Accepted 5 November 2017 Available online 07 November 2017 0144-8617/ © 2017 Elsevier Ltd. All rights reserved.
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million (ppm) downfield from the tetramethylsilane resonance which was used as the internal standard.
(Elchinger et al., 2011; Meng & Edgar, 2016; Sahariah et al., 2015). Very recently, 1,2,3-triazolium cations prepared by N-alkylation of 1,2,3-triazoles with alkyl halides begin to gain attention because of their interesting properties and versatile applications in ionic liquids, polymers, and catalysis (Aizpurua et al., 2014; Mudraboyina, Obadia, Abdelhedi-Miladi, Allaoua, & Drockenmuller, 2015; Ohmatsu, Hamajima & Ooi, 2012; Sood et al., 2014). However, at the best of our knowledge, to date surprisingly there are no reports describing the preparation of chitosan derivatives containing 1,2,3-triazolium. Besides, 1,2,3-triazole or 1,2,3-triazolium can be also regarded as attractive bridge groups, which can effectively connect pharmacophores to give an innovative bioactive compound (Ruddarraju et al., 2016; Tang et al., 2016). Moreover, pyridine group can be also considered as an excellent reactive precursor to synthesize pyridinium cation by the Nalkylation reaction (Jia et al., 2016; Sajomsang, Ruktanonchai, Gonil & Warin, 2010). However, the effect of N-alkylation of 1,2,3-triazole and pyridine moieties on the bioactivity of cationic chitosan derivatives was still unknown. The aim of our project was to synthesize the 1,2,3-triazolium and pyridinium functionalized chitosan derivative and investigate the effect of 1,2,3-triazolium and pyridinium charged units on biological activity of cationic chitosan derivatives. In this paper, we report the preparation and antifungal property of a functionalized chitosan bearing N-methyl1,2,3-triazolium and N-methyl-pyridinium installed via efficient CuAAC reaction. These novel cationic chitosan derivatives were characterized in details by FTIR and 1H NMR spectroscopy. The quantitative data on degree of substitution, thermal stability, and water solubility of the synthesized chitosan derivatives were also calculated. Three plantthreatening fungi, Colletotrichum lagenarium (C. lagenarium), Watermelon fusarium (W. fusarium), and Fusarium oxysporum (F. oxysporum), were selected to evaluate the antifungal property by hypha measurement in vitro.
2.2.3. Thermogravimetric analysis (TGA) TGA measurements of samples were performed on a TA instrument Mettler 5MP (Mettler-Toledo, Switzerland) by heating the samples at a rate of 10 °C min−1 from 25 °C to 800 °C under nitrogen atmosphere. 2.2.4. Elemental analysis The C, H, and N proportions in the native and derived chitosan derivatives were determined on a Vario EL III (Elementar, Germany). The degrees of substitution (DS) of chitosan derivatives were defined as the molar number of grafted functionalized groups per mol of monomeric unit of original chitosan and were calculated on the basis of the percentages of carbon and nitrogen according to the following formulas (Tan, Li and Dong et al., 2017):
DS1 =
n1 × MC−MN × W1 n2 × MC
(1)
DS2 =
MN × (W2 − W1 ) n3 × MC
(2)
DS3 =
MN × (W3 − W2 ) n4 × MC
(3)
DS4 =
MN × (W4 − W3 ) n5 × MC − n6 × MN × W4
(4)
DS5 =
(MN + n6 × MN × DS4 ) × (W5 − W4 ) n7 × MC
(5)
where DS1, DS2, DS3, DS4, and DS5 represent the deacetylation degree of chitosan, the degrees of substitution of N,N,N-trimethyl in chitosan derivatives, propargyl in chitosan derivative a, 1,2,3-triazole groups in chitosan derivatives b, and 1,2,3-triazolium groups in chitosan derivatives c; MC and MN are the molar mass of carbon and nitrogen, MC = 12, MN = 14; n1, n2, n3, n4, n5, n6, and n7 are the number of carbon of chitin, carbon of acetamido group, carbon of trimethyl, carbon of propargyl group, carbon and nitrogen of 3-azidopyridine, and carbon of dimethyl groups, n1 = 8, n2 = 2, n3 = 3, n4 = 3, n5 = 5, n6 = 4, n7 = 2; W1, W2, W3, W4, and W5 represent the mass ratios between carbon and nitrogen in chitosan derivatives.
2. Experimental 2.1. Material Chitosan with molecular weight of 200 kDa was purchased from Qingdao Baicheng Biochemical Corp. (Qingdao, China) and the degree of deacetylation of it was 0.83 calculated by elemental analysis (C: 43.42%, N: 7.98%, H: 6.30%, C/N: 5.44). Iodomethane, propargyl bromide, and 3-aminopyridine were purchased from the Sigma-Aldrich Chemical Corp (Shanghai, China). Hydrochloric acid, sodium nitrite, urea, sodium azide, diethyl ether, magnesium sulfate, N-methyl-2-pyrrolidone, sodium iodide, sodium hydroxide, dimethylsulfoxide, triethylamine, cuprous iodide, absolute ethanol, and acetone were purchased from Sinopharm Chemical Reagent Co.,Ltd (Shanghai, China).
2.2.5. Inductively coupled plasma mass spectrometry (ICP-MS) analysis In consideration of the biotoxicity of cuprum, the determination of cuprum content of chitosan derivatives b and c by ICP-MS were performed with an Elan DRC II instrument (America, provided by PerkinElmer) with dual detector mode. External Calibration was done as standard addition with commercial available cuprum standard solutions. The analytical curve was prepared using seven points at concentrations in the range of 0–200 μg L−1. Results of ICP-MS analysis were received in μg L−1 and were converted into μg g−1 by taking the initial sample weight and the volume of used acid for the etching steps into account. The determination of Cu by ICP-MS was performed in triplicate.
2.2. Structural characterization of chitosan derivatives 2.2.1. Fourier transform infrared (FTIR) spectroscopy The FTIR spectra of compounds powder in transmission mode were recorded using a Jasco-4100 Fourier Transform Infrared Spectrometer (Japan, provided by JASCO Co., Ltd. Shanghai, China) under ambient conditions. The samples were prepared in the pellet form by mixing the powder with KBr by the ratio 1:100 and analyzed in the mid-infrared range (from 4000 to 400 cm−1) at a resolution of 4.0 cm−1.
2.3. Synthesis of chitosan derivatives 2.3.1. Synthesis of 3-azidopyridine 3-Aminopyridine (1.88 g, 20 mmol) was added to 24 mL of 2 N aqueous solution of HCl before the solution was heated to 55 °C while stirring until a clear solution was obtained and then chilled to 0 °C using an ice bath. A solution of sodium nitrite (1.67 g, 24 mmol) in 14 mL of deionized water was subsequently added dropwise. The reaction mixture was allowed to stir for 20 min at an ice bath, followed by the addition of urea (0.24 g, 4 mmol). Then a solution of sodium azide (1.56 g, 24 mmol) in 15 mL of deionized water was added dropwise
2.2.2. 1H nuclear magnetic resonance (NMR) spectroscopy 1 H NMR spectra of compounds were all collected on a Bruker AVIII500 Spectrometer (Switzerland, provided by Bruker Tech. and Serv. Co., Ltd. Beijing, China) at room temperature operated at a resonance frequency of 500 MHz using 99.9% Deuterium Oxide (D2O) or Dimethyl Sulfoxide-d6 (DMSO-d6) as the solvent. The concentration of the polymer solution was about 40–50 mg mL−1 and data were processed using MestReNova software. Chemical shifts were reported in parts per 181
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2.4. Estimation of water solubility
with accompanied by the formation of the colorless gas. The mixture was allowed to warm up to the ambient temperature (20 °C) and stirred for an additional 1.5 h. The mixture was then extracted with diethyl ether, dried using MgSO4, filtrated and the solvent evaporated in vacuo. The resulting crimson oil with a yield of 87.08% was obtained without further purification. FTIR: ν 3046, 2129, 1573, 1477, 802 cm−1. 1H NMR (500 MHz, DMSO-d6): δ 8.4 (d, 1H, Py-2-H), 8.39 (d, 1H, Py-6-H), 7.62 (ddd, 1H, Py-5-H), 7.45 (ddd, 1H, Py-4-H) ppm.
The water solubility of chitosan and chitosan derivatives at various pH values was determined by a turbidity measurement (Li et al., 2012). Briefly, 0.1 g of chitosan was dissolved in 100 mL of 1% HOAc aqueous solution and subsequently the transmittance of the solution at different pH values was recorded with the stepwise addition of 1 M NaOH on a TU-1810 UV spectrometer (General Instrument Co., Ltd., China) at 600 nm.
2.3.2. Synthesis of cationic propargyl chitosan derivative a Chitosan (0.322 g, 2 mmol of glucosamine) was dispersed in 15 mL of N-methyl-2-pyrrolidone (NMP) and stirred at room temperature for 1 h. Then, NaI (0.90 g, 6 mmol), 15% aqueous solution of NaOH (3 mL, 11 mmol), and CH3I (3 mL, 48 mmol) were subsequently added. The mixture was allowed to warm up to 60 °C and stirred under reflux for an additional 1 h. The reaction solution was poured into 150 mL of absolute ethanol to afford some flavescent precipitate (Elemental analysis: C: 31.43%, N: 4.52%, H: 5.49%, C/N: 6.95, DS trimethyl: 0.59). The precipitate collected by filtration was then dissolved in 30 mL of NMP before 5% aqueous solution of NaOH (7.2 mL, 9 mmol) was added dropwise and stirred at room temperature for 1 h. To this mixture, propargyl bromide (0.70 mL, 9 mmol) was added dropwise. After stirred continuously at 60 °C for 48 h, the solution was poured into 200 mL of absolute ethanol to produce some yellowish precipitate. The precipitate was collected by filtration and then washed with ethanol for three times carefully. The resultant product was obtained by freezedrying overnight in vaccum. Chitosan derivative a: Yield: 76.35%; Elemental analysis: C: 38.94%, N: 4.18%, H: 6.69%, C/N: 9.32, DS −1 . propargyl: 0.92. FTIR: ν 3428, 3274, 2927, 2121, 1473, 1076, 644 cm 1 H NMR (500 MHz, D2O): δ 5.60-2.00 (pyranose rings), 4.32 (CH2C^CH), 3.39 (N+(CH3)3), 2.71 (CH2C^CH) ppm.
2.5. Antifungal assay Mycelium growth rate method was used to perform assays for antifungal activity according to the method described in previous literature (Tan, Li and Gao et al., 2017). Different concentrations (0.1, 0.5, and 1.0 mg/mL) of various chitosan derivatives in aqueous solution were used in antifungal activity test against three fungus species viz. C. lagenarium, W. fusarium, and F. oxysporum in vitro. Potato dextrose agar (PDA) medium was prepared and poured in Petri dishes (90 mm × 15 mm) with above mentioned aqueous solution and then allowed to solidify. After solidification, a mycelia disk (diameter: 5 mm) of 5-day-old culture of active fungi was placed to the center of the above Petri plates. The plates were incubated at 27 °C until the mycelia colonies of the control group covered full growth (90 mm), then mycelia growth with PDA containing chitosan derivatives were determined by measuring the colony diameter using decussating method and the growth inhibition was calculated by the formula: Inhibitory indox (%) = (1 − Da/Db) × 100
(6)
where Da (mm) is the diameter of the growth zone in the test plates and Db(mm) is the diameter of the growth zone in the control plate (without the presence of samples).
2.3.3. Synthesis of cationic chitosan derivative bearing 1,2,3-triazole and pyridine b In a 100 mL three-necked round-bottom flask, cationic propargyl chitosan derivative a (0.369 g) was weighed and dissolved in 20 mL of DMSO. Then, 3-azidopyridine (0.36 g, 3 mmol), triethylamine (0.14 mL, 1 mmol), and cuprous iodide (19 mg, 0.1 mmol) were added to the flask and dissolved. The solution was heated to 75 °C and stirred for 48 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 ethanol. After being dialyzed against deionized water for 2 days, the cationic chitosan derivative b was obtained by freeze-drying. Chitosan derivative b: Yield: 80.12%; Elemental analysis: C: 43.09%, N: 14.22%, H: 6.58%, C/ N: 3.03, DS 1,2,3-triazole: 0.80. FTIR: ν 3428, 3081, 2927, 1604, 1558, 1484, 1056, 809 cm−1. 1H NMR (500 MHz, DMSO-d6): δ 9.30–8.00 (Pyridine-4H), 7.65 (1,2,3-triazole-5-H), 5.50-2.00 (pyranose rings), 3.35 (N+(CH3)3) ppm. Average content of Cu in the sample is 255 μg g−1.
2.6. Effect of chitosan derivatives on seed germination Cucumber seeds were used to determine the effect of chitosan derivatives on seedling growth using standard method (Saharan et al., 2015; Sathiyabama & Parthasarathy, 2016). Briefly, the seeds of cucumber were surface sterilized by immersing in 10% sodium hypochlorite solution for 10 min and then washed thoroughly with deionized water. The sterilized seeds were placed in sterile Petri plates (90 × 15 mm, AMA Co., Ltd., Qingdao, China) lined with filter paper. The filter paper was wet with 5.0 mL of different concentrations (0.1, 0.5, and 1.0 mg/mL) of chitosan derivatives. Seeds incubated with sterile distilled water served as control. Each treatment was performed intriplicates with 10 seeds in each plate. The Petri plates were maintained at 28 ± 2 °C in a dark growth room. Data were recorded for seed germination percentage, mean seedling length, and fresh and dry weight after 10-days. Seedling vigor index (SVI) was calculated by the formula:
2.3.4. Synthesis of cationic chitosan derivative bearing 1,2,3-triazolium and pyridinium c To a solution of cationic chitosan derivative b (0.489 g) in 20 mL of DMSO, iodomethane (0.187 mL, 3 mmol) was stirred at 60 °C for 24 h. Afterwards, the remaining iodomethane was evaporated, and the reaction mixture was precipitated into 200 mL of acetone. The solid product was filtered, extensively washed with acetone for three times. After being dialyzed against and deionized water for 2 days, the cationic chitosan derivative c were obtained by lyophilization of their aqueous solutions. Chitosan derivative c: Yield: 64.58%; Elemental analysis: C: 31.28%, N: 9.36%, H: 4.81%, C/N: 3.34, DS 1,2,3-triazolium: 0.76. FTIR: ν 3424, 3058, 2927, 1581, 1519, 1477, 1060, 809 cm−1. 1H NMR (500 MHz, D2O): δ 9.75-8.70 (Pyridinium-4H), 8.30 (1,2,3-triazolium-5-H), 5.60-2.10 (pyranose rings), 4.54 (N+CH3), 3.36 (N+(CH3)3) ppm. Average content of Cu in the sample is 17 μg g−1.
Seed Vigor Index = (germination%)×seedling length
(7)
2.7. Statistical analysis All the experiments were performed in triplicate and the data were expressed as mean ± the standard deviation (SD, n = 3). Significant difference analysis was determined using Scheffe’s multiple range test. A level of P < 0.05 was considered statistically significant. 3. Results and discussion 3.1. Chemical synthesis and characterization The synthesis of cationic chitosan derivatives is conducted in a 182
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Fig. 2. 1H NMR spectra of chitosan and chitosan derivatives. Scheme 1. Synthetic routes for chitosan derivatives. Reagents and conditions: (i) NaNO2, HCl (2N), 0 °C; NaN3, H2O, rt, 1.5 h; (ii) 1) NaOH (15%), NaI, CH3I, NMP, rt to 60 °C, 1 h; 2) NaOH (5%), propargyl bromide, NMP, rt to 60 °C, 48 h; (iii) 3-azidopyridine, TEA, CuI, DMSO, 75 °C, 48 h; (iv) CH3I, DMSO, 60 °C, 24 h.
3.1.1. FTIR analysis Comparison of FTIR spectra for chitosan and chitosan derivatives is given in Fig. 1. The characteristic absorbance bands of the unmodified chitosan are observed, such as the NeH and OeH stretching vibrations at 3417 cm−1 (Subhapradha & Shanmugam, 2017), the CeH stretching vibration of methylene at 2877 cm−1 (Baghbani, Chegeni, Moztarzadeh, Hadian-Ghazvini & Raz, 2017), the NeH bending vibration at 1600 cm−1 (Moreno-Vasquez et al., 2017; Rahmani et al., 2016), and the stretching vibration of the CeO bond of the glucosamine ring at 1072 cm−1 (Sukamporn et al., 2017). For the cationic chitosan derivatives a, b, and c, the region 1473–1484 cm−1 is characteristic of the quaternary ammonium salts and these bands are attributed to the stretching vibrations of the eN+–(CH3)3 in FTIR spectra (Badawy, Rabea & Taktak, 2014). The propargylation for cationic chitosan derivative a is confirmed by the absorption peaks at 3274, 2121, and 644 cm−1 in FTIR spectrum which are attributed to the vibration absorptions of eC^CH (Tan, Li and Gao et al., 2017). After CuAAC reaction, the comparison of the FTIR spectra between the chitosan derivatives a and b shows that the absorptions at 3274, 2121, and 644 cm−1 disappear completely and a new peak appears at 1558 cm−1, which is assigned to the absorbance of 1,2,3-triazole ring in the spectra of cationic chitosan derivative b (Tan, Li and Dong et al., 2017). Besides, the success of CuAAC reaction is also proved by the appearance of new peaks at 3081, 1604, and 809 cm−1, which are ascribed to the ring stretching vibrations of 3-substituted pyridine. After N-methylation of 1,2,3-triazole and pyridine with iodomethane, the characteristic peak considered as 1,2,3-triazolium moiety of cationic chitosan derivative c is observed at 1581 cm−1 (Tan, Li and Dong et al., 2017). Besides, other new peaks for pyridinium at 3058, 1519, and 809 cm−1 are also clearly detected (Rúnarsson et al., 2010). 3.1.2. 1H NMR analysis To further confirm the inclusion of the 1,2,3-triazolium and pyridinium moieties in the chitosan backbones, 1H NMR spectra is conducted shown in Fig. 2 and main characteristic groups of peaks have been identified and assigned. The peaks between 3.05 and 5.00 ppm are ascribed to the protons of glucosamine unit and the peak at 2.04 ppm is assigned to the methyl protons of the N-acetyl group in the 1H NMR spectrum of chitosan (Badawy, Rabea & Taktak, 2014; Wu et al., 2016). Compared with the peaks of chitosan, the 1H NMR spectrum of chitosan derivative a shows the prominent peak of −N+–(CH3)3 at 3.39 ppm (a in the spectrum) (Patrulea et al., 2016; Rahmani et al., 2016) along with the resonance peaks at 4.33 and 2.70 ppm (b and c in the spectrum) corresponding to the hydrogen atoms on the methylene and terminal alkynyl of propargyl group (Uliniuc et al., 2013). When terminal alkyne is transformed to 1,2,3-triazole through the click reaction of chitosan derivative a with 3-azidopyridine, one of the key peaks confirming the success of reaction is the downfield signal at
Fig. 1. FTIR spectra of chitosan and chitosan derivatives.
sequential approach, as shown in Scheme 1. The poor solubility of pristine chitosan in water and organic solvents could inevitably lead to inefficient heterogeneous reaction in the chemical step. Fortunately, the N,N,N-trimethylation of chitosan as one of the most simple and efficient methods could greatly overcome this obstacle (Patrulea et al., 2016). Without any purification, N,N,N-trimethyl chitosan reacted directly with propargyl bromide in the alkaline condition to obtain the reaction intermediate, cationic propargyl chitosan derivative a. Subsequently, the click chemistry was performed with 3-azidopyridine to synthesize the cationic chitosan derivative bearing 1,2,3-triazole and pyridine b. Then, the targeted cationic chitosan derivative c was synthesized by Nmethylation, reacting cationic chitosan derivative b with iodomethane. The chemical structures of the resultant chitosan derivatives were confirmed by FTIR (Fig. 1) and 1H NMR (Fig. 2) spectroscopy. 183
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Fig. 3. TGA (a) and DTG (b) curves of chitosan and chitosan derivatives.
7.65 ppm (d in the spectrum), which is assigned to hydrogen atoms at 5-H position of 1,2,3-triazole ring (Tan, Li, Li, Dong & Guo, 2016). Moreover, the additional signals at 7.80–9.26 ppm (e in the spectrum) are assigned to the protons of pyridine group of chitosan derivative b (Qin, Liu, Xing, Li, Yu & Li, 2013). After N-alkylation with iodomethane, the signals corresponding to the hydrogen atoms on 1,2,3triazole and pyridine groups at 7.65 and 7.80–9.26 ppm (d and e in the spectrum) completely disappear, and instead, the new peaks at around 8.30 and 8.70–9.75 ppm (f and g in the spectrum) emerge due to the protons of 1,2,3-triazolium and pyridinium (Jia, Duan, Fang, Wang & Huang, 2016; Sood et al., 2014). In addition, completion of the alkylation reaction is also corroborated by the appearance of new signals at 4.54 ppm (h and i in the spectrum) for the pendant methyl groups attached to the nitrogen of 1,2,3-triazolium and pyridinium moieties (Abdelhedi-Miladi et al., 2014). 3.2. Thermal stability Fig. 4. pH dependence of water solubility of chitosan and chitosan derivatives.
Fig. 3 shows the thermogravimetric analyses (TGA) and derivative thermogravimetric (DTG) analyses curves of chitosan and chitosan derivatives. The pyrolysis process of chitosan could be accomplished by mainly two steps under nitrogen atmosphere. The first step observed at below 150 °C may be due to the evaporation of the water molecules which trapped between the carbohydrate polymer chains (Tan, Li and Wang et al., 2016). At the second stage, the degradation of chitosan skeleton occurs from 280 °C to 450 °C with 64% of mass loss. In comparison, chitosan derivatives undergo three-step pyrolysis process: one water loss and two thermal degradations. The first stage ranges from 25 to 150 °C, with a 8% miss loss of water molecules. The second thermal event occurs in the temperature range 150–220 °C, which is due to the elimination of the functional groups. The more intense degradation and carbonization of chitosan chain in the third stage start earlier from about 220 °C and continue until 400 °C and reach the highest rate in the range of 238–261 °C, corresponding to mass loss with 65–70% up to 800 °C. The lower thermal stability of the modified chitosan may be attributed to the disruption of the intramolecular and intermolecular hydrogen bonds from chemical modification (Chivangkul et al., 2014).
with iodomethane further improves water solubility of chitosan derivative, especially at alkaline pH. Current finding suggests that the quaternization reaction is an effective mean for improving water solubility of chitosan, which is favorable to further applications. 3.4. Antifungal activity Three destructive phytopathogenic fungi, C. lagenarium, W. fusarium, and F. oxysporum, were applied to test the potential antifungal efficiency of the prepared compounds in vitro by measuring mycelial inhibition of radial growth. In the case of pristine chitosan with the poor water solubility, it was unfeasible to assess the antifungal property of it by mycelium growth rate method in aqueous systems, so watersoluble low-molecular chitosan (LCS) was chosen in antifungal activity test. The cuprum content in chitosan derivatives b and c measured by ICP-MS was below 0.3 mg·g−1. That is to say, when the sample solution was added to sterile PDA medium to give a final concentration of 0.1, 0.5, and 1.0 mg/mL, the actual concentration of cuprum in sterile PDA medium is below 0.3 ppm, at which it had almost no antifungal effect against the tested plant pathogenic fungi. Therefore, in this paper, the influence of residual copper catalyst on the antifungal activity of chitosan derivatives could be ignored. The antifungal activities of chitosan and chitosan derivatives have been assessed at 0.1, 0.5, and 1.0 mg/mL and the results are summarized in Fig. 5. Among the three tested plant pathogenic fungi, all of the compounds reveal the relatively strong antifungal effect against C. lagenarium but the relatively weak inhibition towards F. oxysporum with dose independence. The radial growth inhibition percentages of chitosan against these tested plant pathogenic fungi ranges from 11.80 to 25.00% at the maximum concentration of 1.0 mg/mL. All of the synthesized derivatives show better ability of inhibiting the growth of the tested phytopathogenic fungi than chitosan. It is found that compared with chitosan and compound a, chitosan derivative bearing 1,2,3-
3.3. Water solubility Fig. 4 illustrates the water solubility of chitosan and chitosan derivatives at different pH values. It is well established that chitosan has poor solubility in water due to the strong intramolecular and intermolecular hydrogen bonds (Chen et al., 2016). It is highly soluble in acidic solutions (pH < 6.5) due to the protonation of the primary amine (Patrulea et al., 2016). After trimethylation of chitosan, chitosan derivative a shows higher water solubility because of the high hydrophilia of quaternary ammonium groups (Chivangkul et al., 2014). However, after CuAAC reaction with 3-azidopyridine, chitosan derivative b exhibits relatively weak water solubility because of the formation of amide bond between 1,2,3-triazole and pyridine groups in chitosan backbone. Fortunately, the N-methylation of 1,2,3-triazole and pyridine 184
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Fig. 6. Effect of chitosan derivatives on cucumber seedling growth.
Moreover, cationic chitosan derivative bearing 1,2,3-triazolium and pyridinium c exhibits greatly enhanced antifungal action by one-step Nmethylation of chitosan derivatives b with iodomethane. A maximum of 98.44% inhibition rate is recorded for C. lagenarium, followed by 79.16% and 67.56% for W. fusarium and F. oxysporum at 1.0 mg/mL. Notably, cationic chitosan derivative c is still active against tested fungi even when the dosage is lowered to 0.5 mg/mL. It is reasonably hypothesized that the chemical modification of chitosan molecule by introducing 1,2,3-triazolium and pyridinium improves significantly the antimicrobial activity of chitosan. Many studies from the past have reported that quaternary ammonium salts possessed strong antimicrobial activity based on electrostatic interactions (Chang, Yang & Liang, 2010; Sajomsang, Gonil & Saesoo, 2009; Sajomsang, Gonil & Tantayanon, 2009). As novel types of quaternary ammonium salts, 1,2,3-triazolium and pyridinium groups would interact electrostatically with the electronegative charged components (i.e., proteins, phospholipids) on fungal cell walls or cytomembranes (Guo et al., 2007; Tan, Li and Dong et al., 2016). This adherence of polycations to the outer membranes of the fungi could lead to a change of permeation property of the membrane wall, which could provoke internal osmotic imbalances and in turn cause the leakage of intracellular electrolytes and proteinaceous constituents as well as the obstruction of nutrient intake (Li, Yang & Yang, 2015; Tejero, Lopez, Lopez-Fabal, GomezGarces & Fernandez-Garcia, 2015). 3.5. Effect of chitosan derivatives on seed germination In order to evaluate the effect of chitosan derivatives on seedling growth, the cucumber seeds was treated with chitosan and chitosan derivatives and the results show the positive morphological effects of chitosan derivatives with slightly increased germination rate, seedling length, fresh and dry weight, and seed vigor index (Fig. 6 and Table 1). That is to say, the chitosan derivatives synthesized in this paper exhibit non-toxicity on the cucumber at the range of the testing concentration. 4. Conclusion
Fig. 5. The antifungal activity of chitosan and chitosan derivatives against C. lagenarium (a), W. fusarium (b), and F. oxysporum (c). * (P < 0.05), ** (P < 0.01) for comparisons between compound c and LCS, a, b.
In summary, we have recently proposed a convenient and robust method to prepare novel cationic chitosan derivative bearing 1,2,3triazolium and pyridinium charged units by associating CuAAC step with efficient N-alkylation of 1,2,3-triazole and pyridine with iodomethane. FTIR, 1H NMR spectra, and elemental analysis confirmed that 1,2,3-triazolium and pyridinium moieties had been successfully introduced to chitosan backbone. The thermal stability and water solubility of the synthesized chitosan derivatives were also measured. The antifungal activity against three kinds of plant threatening fungal strains was estimated by observing the percentage inhibition of mycelial growth. The synthesized chitosan derivative bearing 1,2,3-triazolium and pyridinium moieties clearly showed stronger antifungal action than chitosan and chitosan derivative bearing 1,2,3-triazole and pyridine groups. The obtained findings suggested that the N-methylation of
triazole and pyridine b shows slightly higher antifungal property and the highest growth inhibitions (32.64–45.94%) against three tested plant pathogenic fungi are observed at the maximum concentration of 1.0 mg/mL. The above results confirm the contribution of the 1,2,3triazole and pyridine moieties to antifungal activity. Like other nitrogen-containing heterocyclic compounds, it was reported that 1,2,3triazole and pyridine were capable of inhibiting the growth of the tested phytopathogenic fungi by interrupting the synthesis of the cell membrane and cell wall by hydrogen bond interaction (Kant, Singh, Nath, Awasthi & Agarwal, 2016; Tan, Li and Gao et al., 2017; Vijai Kumar Reddy et al., 2016) (Fig. 6). 185
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Table 1 Effect of chitosan derivatives on seedling growth of cucumber (data recorded after 10 days of growth). Treatment Control LCS
a
b
c
0.1 mg/mL 0.5 mg/mL 1.0 mg/mL 0.1 mg/mL 0.5 mg/mL 1.0 mg/mL 0.1 mg/mL 0.5 mg/mL 1.0 mg/mL 0.1 mg/mL 0.5 mg/mL 1.0 mg/mL
Germination (%)
Seedling length (cm)
Fresh weight (gm)
Dry weight (gm)
SVI
86.67 90 ± 90 ± 93.33 90 ± 90 ± 86.67 90 ± 86.67 90 ± 93.33 90 ± 86.67
12.23 12.65 13.24 14.74 13.36 14.99 14.88 14.63 15.23 15.02 13.89 14.56 14.68
0.30 0.31 0.32 0.34 0.32 0.35 0.33 0.34 0.39 0.36 0.32 0.34 0.33
0.018 0.019 0.020 0.020 0.019 0.021 0.020 0.020 0.025 0.023 0.020 0.021 0.020
1060 1139 1192 1376 1202 1349 1290 1317 1320 1352 1296 1310 1272
± 4.71 0 0 ± 4.71 8.16 0 ± 4.71 0 ± 4.71 0 ± 4.71 0 ± 4.71
± ± ± ± ± ± ± ± ± ± ± ± ±
0.40 0.20 0.39 0.75 0.52 0.25 0.32 0.61 1.01 0.82 0.25 0.79 0.87
1,2,3-triazole and pyridine could contribute a lot to the water solubility and the antifungal efficiency of synthesized chitosan derivatives. Meanwhile, the synthesized chitosan derivatives exhibited non-toxicity on the cucumber by recording the germination rate, seedling length, fresh and dry weight, and seed vigor index in the seedling bioassay. The product described in this paper might serve as a new leading structure for further design of novel antifungal agents.
± ± ± ± ± ± ± ± ± ± ± ± ±
0.01 0.01 0.01 0.02 0.01 0.01 0.02 0.01 0.02 0.01 0.01 0.01 0.02
± ± ± ± ± ± ± ± ± ± ± ± ±
0.001 0.001 0.001 0.001 0.001 0.001 0.002 0.002 0.002 0.001 0.001 0.001 0.001
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Research Paper
Novel cationic chitosan derivative bearing 1,2,3-triazolium and pyridinium: Synthesis, characterization, and antifungal property
MARK
Wenqiang Tana,b, Qing Lia, Fang Donga, Jingjing Zhanga,b, Fang Luana,b, Lijie Weia, Yuan Chena,b, ⁎ Zhanyong Guoa,b, a b
Key Laboratory of Coastal Biology and Bioresource Utilization, Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences, Yantai 264003, China University of Chinese Academy of Sciences, Beijing 100049, China
A R T I C L E I N F O
A B S T R A C T
Chemical compounds studied in this article: Chitosan (PubChem CID: 439341) Iodomethane (PubChemCID: 6328) Propargyl bromide (PubChem CID: 7842) 3-Aminopyridine (PubChem CID: 10009) Sodium azide (PubChem CID: 33557) Triethylamine (PubChemCID: 8471) Cuprous iodide (PubChemCID: 24350)
In this paper, novel cationic chitosan derivative possessing 1,2,3-triazolium and pyridinium groups was synthesized conveniently via cuprous-catalyzed azide-alkyne cycloaddition (CuAAC) and methylation. FTIR, 1H NMR, and elemental analysis examined the structural characteristics of the synthesized derivatives. The antifungal efficiencies of chitosan derivatives against three plant-threatening fungi were assayed by hypha measurement in vitro. The determination showed that chitosan derivative bearing 1,2,3-triazolium and pyridinium displayed tremendously enhanced antifungal activity as compared with chitosan and chitosan derivative bearing 1,2,3-triazole and pyridine. Notably, the inhibitory indices of it against Colletotrichum lagenarium attained 98% above at 1.0 mg/mL. The results showed that N-methylation of 1,2,3-triazole and pyridine could effectively enhance antifungal activity of the synthesized chitosan derivatives. Besides, the prepared chitosan derivatives showed non-toxic effect on cucumber seedlings. This synthetic strategy might provide an effective way and notion to prepare novel cationic chitosan antifungal biomaterials.
Keywords: Chitosan Chemical modification Click chemistry 1,2,3-Triazolium Pyridinium Antifungal activity
1. Introduction Chitosan, a deacetylated derivative from chitin, is the only readily available basic amino-polysaccharide in the nature (de Oliveira Pedro, Schmitt & Neumann, 2016; Wu et al., 2016). As one of the most promising biomaterials with good biocompatibility and biodegradability (Li, Duan, Huang & Zheng, 2016; Qian, Xu, Shen, Li & Guo, 2013), chitosan has wide application as wastewater treatment agent, a drug delivery system, and bactericidal or antibacterial agent in the medical industry (Cruz, Garcia-Uriostegui, Ortega, Isoshima & Burillo, 2017; Khan, Ullah & Oh, 2016). However, applications of chitosan are considerably limited by poor solubility in both organic and aqueous solvents (Chen et al., 2016; Qin et al., 2012). Chemical modification of chitosan can overcome this problem to some extent (Jia, Duan, Fang, Wang & Huang, 2016; Sun, Shi, Wang, Fang & Huang, 2017). Derivatization by introducing small functional groups to chitosan backbone can drastically increase the solubility of chitosan at neutral and alkaline pH values as well as strengthen its original bioactivities to broaden the industrial applications (Li, Duan, Huang & Zheng, 2016; Liu, Meng, Liu,
⁎
Kan & Jin, 2017). Cationic chitosan is one of the most important group of chitosan derivatives, which has many important physicochemical features such as water solubility and chemical stability as well as antimicrobial properties, due to the electrostatic interactions with anionic biomolecules at the cell surface (Moreno-Vasquez et al., 2017; Sun et al., 2017). Currently, cationic chitosan derivatives can be successfully produced by transforming amino groups into quaternary ammonium salts or by introducing cationic functional groups, such as ammonium, phosphonium, or sulfonium, to chitosan backbones via chemical reactions with primary amino and hydroxyl groups (Chen et al., 2016). The cuprous-catalyzed azide-alkyne cycloaddition (CuAAC) leading to regioselective 1,4-disubstituted-1,2,3-triazoles (Saravanakumar, Ramkumar & Sankararaman, 2011) has received widespread attention in polysaccharide modification due to the merits of CuAAC, including wide in scope, high yield, modularity, tolerant to other functional groups, ready available starting materials, and be stereospecific (Su et al., 2017; Wang et al., 2017). And the application of CuAAC in polysaccharide modification has brought substantial progress
Corresponding author at: University of Chinese Academy of Sciences, Beijing 100049, China. E-mail address: [email protected] (Z. Guo).
https://doi.org/10.1016/j.carbpol.2017.11.023 Received 7 May 2017; Received in revised form 24 October 2017; Accepted 5 November 2017 Available online 07 November 2017 0144-8617/ © 2017 Elsevier Ltd. All rights reserved.
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million (ppm) downfield from the tetramethylsilane resonance which was used as the internal standard.
(Elchinger et al., 2011; Meng & Edgar, 2016; Sahariah et al., 2015). Very recently, 1,2,3-triazolium cations prepared by N-alkylation of 1,2,3-triazoles with alkyl halides begin to gain attention because of their interesting properties and versatile applications in ionic liquids, polymers, and catalysis (Aizpurua et al., 2014; Mudraboyina, Obadia, Abdelhedi-Miladi, Allaoua, & Drockenmuller, 2015; Ohmatsu, Hamajima & Ooi, 2012; Sood et al., 2014). However, at the best of our knowledge, to date surprisingly there are no reports describing the preparation of chitosan derivatives containing 1,2,3-triazolium. Besides, 1,2,3-triazole or 1,2,3-triazolium can be also regarded as attractive bridge groups, which can effectively connect pharmacophores to give an innovative bioactive compound (Ruddarraju et al., 2016; Tang et al., 2016). Moreover, pyridine group can be also considered as an excellent reactive precursor to synthesize pyridinium cation by the Nalkylation reaction (Jia et al., 2016; Sajomsang, Ruktanonchai, Gonil & Warin, 2010). However, the effect of N-alkylation of 1,2,3-triazole and pyridine moieties on the bioactivity of cationic chitosan derivatives was still unknown. The aim of our project was to synthesize the 1,2,3-triazolium and pyridinium functionalized chitosan derivative and investigate the effect of 1,2,3-triazolium and pyridinium charged units on biological activity of cationic chitosan derivatives. In this paper, we report the preparation and antifungal property of a functionalized chitosan bearing N-methyl1,2,3-triazolium and N-methyl-pyridinium installed via efficient CuAAC reaction. These novel cationic chitosan derivatives were characterized in details by FTIR and 1H NMR spectroscopy. The quantitative data on degree of substitution, thermal stability, and water solubility of the synthesized chitosan derivatives were also calculated. Three plantthreatening fungi, Colletotrichum lagenarium (C. lagenarium), Watermelon fusarium (W. fusarium), and Fusarium oxysporum (F. oxysporum), were selected to evaluate the antifungal property by hypha measurement in vitro.
2.2.3. Thermogravimetric analysis (TGA) TGA measurements of samples were performed on a TA instrument Mettler 5MP (Mettler-Toledo, Switzerland) by heating the samples at a rate of 10 °C min−1 from 25 °C to 800 °C under nitrogen atmosphere. 2.2.4. Elemental analysis The C, H, and N proportions in the native and derived chitosan derivatives were determined on a Vario EL III (Elementar, Germany). The degrees of substitution (DS) of chitosan derivatives were defined as the molar number of grafted functionalized groups per mol of monomeric unit of original chitosan and were calculated on the basis of the percentages of carbon and nitrogen according to the following formulas (Tan, Li and Dong et al., 2017):
DS1 =
n1 × MC−MN × W1 n2 × MC
(1)
DS2 =
MN × (W2 − W1 ) n3 × MC
(2)
DS3 =
MN × (W3 − W2 ) n4 × MC
(3)
DS4 =
MN × (W4 − W3 ) n5 × MC − n6 × MN × W4
(4)
DS5 =
(MN + n6 × MN × DS4 ) × (W5 − W4 ) n7 × MC
(5)
where DS1, DS2, DS3, DS4, and DS5 represent the deacetylation degree of chitosan, the degrees of substitution of N,N,N-trimethyl in chitosan derivatives, propargyl in chitosan derivative a, 1,2,3-triazole groups in chitosan derivatives b, and 1,2,3-triazolium groups in chitosan derivatives c; MC and MN are the molar mass of carbon and nitrogen, MC = 12, MN = 14; n1, n2, n3, n4, n5, n6, and n7 are the number of carbon of chitin, carbon of acetamido group, carbon of trimethyl, carbon of propargyl group, carbon and nitrogen of 3-azidopyridine, and carbon of dimethyl groups, n1 = 8, n2 = 2, n3 = 3, n4 = 3, n5 = 5, n6 = 4, n7 = 2; W1, W2, W3, W4, and W5 represent the mass ratios between carbon and nitrogen in chitosan derivatives.
2. Experimental 2.1. Material Chitosan with molecular weight of 200 kDa was purchased from Qingdao Baicheng Biochemical Corp. (Qingdao, China) and the degree of deacetylation of it was 0.83 calculated by elemental analysis (C: 43.42%, N: 7.98%, H: 6.30%, C/N: 5.44). Iodomethane, propargyl bromide, and 3-aminopyridine were purchased from the Sigma-Aldrich Chemical Corp (Shanghai, China). Hydrochloric acid, sodium nitrite, urea, sodium azide, diethyl ether, magnesium sulfate, N-methyl-2-pyrrolidone, sodium iodide, sodium hydroxide, dimethylsulfoxide, triethylamine, cuprous iodide, absolute ethanol, and acetone were purchased from Sinopharm Chemical Reagent Co.,Ltd (Shanghai, China).
2.2.5. Inductively coupled plasma mass spectrometry (ICP-MS) analysis In consideration of the biotoxicity of cuprum, the determination of cuprum content of chitosan derivatives b and c by ICP-MS were performed with an Elan DRC II instrument (America, provided by PerkinElmer) with dual detector mode. External Calibration was done as standard addition with commercial available cuprum standard solutions. The analytical curve was prepared using seven points at concentrations in the range of 0–200 μg L−1. Results of ICP-MS analysis were received in μg L−1 and were converted into μg g−1 by taking the initial sample weight and the volume of used acid for the etching steps into account. The determination of Cu by ICP-MS was performed in triplicate.
2.2. Structural characterization of chitosan derivatives 2.2.1. Fourier transform infrared (FTIR) spectroscopy The FTIR spectra of compounds powder in transmission mode were recorded using a Jasco-4100 Fourier Transform Infrared Spectrometer (Japan, provided by JASCO Co., Ltd. Shanghai, China) under ambient conditions. The samples were prepared in the pellet form by mixing the powder with KBr by the ratio 1:100 and analyzed in the mid-infrared range (from 4000 to 400 cm−1) at a resolution of 4.0 cm−1.
2.3. Synthesis of chitosan derivatives 2.3.1. Synthesis of 3-azidopyridine 3-Aminopyridine (1.88 g, 20 mmol) was added to 24 mL of 2 N aqueous solution of HCl before the solution was heated to 55 °C while stirring until a clear solution was obtained and then chilled to 0 °C using an ice bath. A solution of sodium nitrite (1.67 g, 24 mmol) in 14 mL of deionized water was subsequently added dropwise. The reaction mixture was allowed to stir for 20 min at an ice bath, followed by the addition of urea (0.24 g, 4 mmol). Then a solution of sodium azide (1.56 g, 24 mmol) in 15 mL of deionized water was added dropwise
2.2.2. 1H nuclear magnetic resonance (NMR) spectroscopy 1 H NMR spectra of compounds were all collected on a Bruker AVIII500 Spectrometer (Switzerland, provided by Bruker Tech. and Serv. Co., Ltd. Beijing, China) at room temperature operated at a resonance frequency of 500 MHz using 99.9% Deuterium Oxide (D2O) or Dimethyl Sulfoxide-d6 (DMSO-d6) as the solvent. The concentration of the polymer solution was about 40–50 mg mL−1 and data were processed using MestReNova software. Chemical shifts were reported in parts per 181
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2.4. Estimation of water solubility
with accompanied by the formation of the colorless gas. The mixture was allowed to warm up to the ambient temperature (20 °C) and stirred for an additional 1.5 h. The mixture was then extracted with diethyl ether, dried using MgSO4, filtrated and the solvent evaporated in vacuo. The resulting crimson oil with a yield of 87.08% was obtained without further purification. FTIR: ν 3046, 2129, 1573, 1477, 802 cm−1. 1H NMR (500 MHz, DMSO-d6): δ 8.4 (d, 1H, Py-2-H), 8.39 (d, 1H, Py-6-H), 7.62 (ddd, 1H, Py-5-H), 7.45 (ddd, 1H, Py-4-H) ppm.
The water solubility of chitosan and chitosan derivatives at various pH values was determined by a turbidity measurement (Li et al., 2012). Briefly, 0.1 g of chitosan was dissolved in 100 mL of 1% HOAc aqueous solution and subsequently the transmittance of the solution at different pH values was recorded with the stepwise addition of 1 M NaOH on a TU-1810 UV spectrometer (General Instrument Co., Ltd., China) at 600 nm.
2.3.2. Synthesis of cationic propargyl chitosan derivative a Chitosan (0.322 g, 2 mmol of glucosamine) was dispersed in 15 mL of N-methyl-2-pyrrolidone (NMP) and stirred at room temperature for 1 h. Then, NaI (0.90 g, 6 mmol), 15% aqueous solution of NaOH (3 mL, 11 mmol), and CH3I (3 mL, 48 mmol) were subsequently added. The mixture was allowed to warm up to 60 °C and stirred under reflux for an additional 1 h. The reaction solution was poured into 150 mL of absolute ethanol to afford some flavescent precipitate (Elemental analysis: C: 31.43%, N: 4.52%, H: 5.49%, C/N: 6.95, DS trimethyl: 0.59). The precipitate collected by filtration was then dissolved in 30 mL of NMP before 5% aqueous solution of NaOH (7.2 mL, 9 mmol) was added dropwise and stirred at room temperature for 1 h. To this mixture, propargyl bromide (0.70 mL, 9 mmol) was added dropwise. After stirred continuously at 60 °C for 48 h, the solution was poured into 200 mL of absolute ethanol to produce some yellowish precipitate. The precipitate was collected by filtration and then washed with ethanol for three times carefully. The resultant product was obtained by freezedrying overnight in vaccum. Chitosan derivative a: Yield: 76.35%; Elemental analysis: C: 38.94%, N: 4.18%, H: 6.69%, C/N: 9.32, DS −1 . propargyl: 0.92. FTIR: ν 3428, 3274, 2927, 2121, 1473, 1076, 644 cm 1 H NMR (500 MHz, D2O): δ 5.60-2.00 (pyranose rings), 4.32 (CH2C^CH), 3.39 (N+(CH3)3), 2.71 (CH2C^CH) ppm.
2.5. Antifungal assay Mycelium growth rate method was used to perform assays for antifungal activity according to the method described in previous literature (Tan, Li and Gao et al., 2017). Different concentrations (0.1, 0.5, and 1.0 mg/mL) of various chitosan derivatives in aqueous solution were used in antifungal activity test against three fungus species viz. C. lagenarium, W. fusarium, and F. oxysporum in vitro. Potato dextrose agar (PDA) medium was prepared and poured in Petri dishes (90 mm × 15 mm) with above mentioned aqueous solution and then allowed to solidify. After solidification, a mycelia disk (diameter: 5 mm) of 5-day-old culture of active fungi was placed to the center of the above Petri plates. The plates were incubated at 27 °C until the mycelia colonies of the control group covered full growth (90 mm), then mycelia growth with PDA containing chitosan derivatives were determined by measuring the colony diameter using decussating method and the growth inhibition was calculated by the formula: Inhibitory indox (%) = (1 − Da/Db) × 100
(6)
where Da (mm) is the diameter of the growth zone in the test plates and Db(mm) is the diameter of the growth zone in the control plate (without the presence of samples).
2.3.3. Synthesis of cationic chitosan derivative bearing 1,2,3-triazole and pyridine b In a 100 mL three-necked round-bottom flask, cationic propargyl chitosan derivative a (0.369 g) was weighed and dissolved in 20 mL of DMSO. Then, 3-azidopyridine (0.36 g, 3 mmol), triethylamine (0.14 mL, 1 mmol), and cuprous iodide (19 mg, 0.1 mmol) were added to the flask and dissolved. The solution was heated to 75 °C and stirred for 48 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 ethanol. After being dialyzed against deionized water for 2 days, the cationic chitosan derivative b was obtained by freeze-drying. Chitosan derivative b: Yield: 80.12%; Elemental analysis: C: 43.09%, N: 14.22%, H: 6.58%, C/ N: 3.03, DS 1,2,3-triazole: 0.80. FTIR: ν 3428, 3081, 2927, 1604, 1558, 1484, 1056, 809 cm−1. 1H NMR (500 MHz, DMSO-d6): δ 9.30–8.00 (Pyridine-4H), 7.65 (1,2,3-triazole-5-H), 5.50-2.00 (pyranose rings), 3.35 (N+(CH3)3) ppm. Average content of Cu in the sample is 255 μg g−1.
2.6. Effect of chitosan derivatives on seed germination Cucumber seeds were used to determine the effect of chitosan derivatives on seedling growth using standard method (Saharan et al., 2015; Sathiyabama & Parthasarathy, 2016). Briefly, the seeds of cucumber were surface sterilized by immersing in 10% sodium hypochlorite solution for 10 min and then washed thoroughly with deionized water. The sterilized seeds were placed in sterile Petri plates (90 × 15 mm, AMA Co., Ltd., Qingdao, China) lined with filter paper. The filter paper was wet with 5.0 mL of different concentrations (0.1, 0.5, and 1.0 mg/mL) of chitosan derivatives. Seeds incubated with sterile distilled water served as control. Each treatment was performed intriplicates with 10 seeds in each plate. The Petri plates were maintained at 28 ± 2 °C in a dark growth room. Data were recorded for seed germination percentage, mean seedling length, and fresh and dry weight after 10-days. Seedling vigor index (SVI) was calculated by the formula:
2.3.4. Synthesis of cationic chitosan derivative bearing 1,2,3-triazolium and pyridinium c To a solution of cationic chitosan derivative b (0.489 g) in 20 mL of DMSO, iodomethane (0.187 mL, 3 mmol) was stirred at 60 °C for 24 h. Afterwards, the remaining iodomethane was evaporated, and the reaction mixture was precipitated into 200 mL of acetone. The solid product was filtered, extensively washed with acetone for three times. After being dialyzed against and deionized water for 2 days, the cationic chitosan derivative c were obtained by lyophilization of their aqueous solutions. Chitosan derivative c: Yield: 64.58%; Elemental analysis: C: 31.28%, N: 9.36%, H: 4.81%, C/N: 3.34, DS 1,2,3-triazolium: 0.76. FTIR: ν 3424, 3058, 2927, 1581, 1519, 1477, 1060, 809 cm−1. 1H NMR (500 MHz, D2O): δ 9.75-8.70 (Pyridinium-4H), 8.30 (1,2,3-triazolium-5-H), 5.60-2.10 (pyranose rings), 4.54 (N+CH3), 3.36 (N+(CH3)3) ppm. Average content of Cu in the sample is 17 μg g−1.
Seed Vigor Index = (germination%)×seedling length
(7)
2.7. Statistical analysis All the experiments were performed in triplicate and the data were expressed as mean ± the standard deviation (SD, n = 3). Significant difference analysis was determined using Scheffe’s multiple range test. A level of P < 0.05 was considered statistically significant. 3. Results and discussion 3.1. Chemical synthesis and characterization The synthesis of cationic chitosan derivatives is conducted in a 182
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Fig. 2. 1H NMR spectra of chitosan and chitosan derivatives. Scheme 1. Synthetic routes for chitosan derivatives. Reagents and conditions: (i) NaNO2, HCl (2N), 0 °C; NaN3, H2O, rt, 1.5 h; (ii) 1) NaOH (15%), NaI, CH3I, NMP, rt to 60 °C, 1 h; 2) NaOH (5%), propargyl bromide, NMP, rt to 60 °C, 48 h; (iii) 3-azidopyridine, TEA, CuI, DMSO, 75 °C, 48 h; (iv) CH3I, DMSO, 60 °C, 24 h.
3.1.1. FTIR analysis Comparison of FTIR spectra for chitosan and chitosan derivatives is given in Fig. 1. The characteristic absorbance bands of the unmodified chitosan are observed, such as the NeH and OeH stretching vibrations at 3417 cm−1 (Subhapradha & Shanmugam, 2017), the CeH stretching vibration of methylene at 2877 cm−1 (Baghbani, Chegeni, Moztarzadeh, Hadian-Ghazvini & Raz, 2017), the NeH bending vibration at 1600 cm−1 (Moreno-Vasquez et al., 2017; Rahmani et al., 2016), and the stretching vibration of the CeO bond of the glucosamine ring at 1072 cm−1 (Sukamporn et al., 2017). For the cationic chitosan derivatives a, b, and c, the region 1473–1484 cm−1 is characteristic of the quaternary ammonium salts and these bands are attributed to the stretching vibrations of the eN+–(CH3)3 in FTIR spectra (Badawy, Rabea & Taktak, 2014). The propargylation for cationic chitosan derivative a is confirmed by the absorption peaks at 3274, 2121, and 644 cm−1 in FTIR spectrum which are attributed to the vibration absorptions of eC^CH (Tan, Li and Gao et al., 2017). After CuAAC reaction, the comparison of the FTIR spectra between the chitosan derivatives a and b shows that the absorptions at 3274, 2121, and 644 cm−1 disappear completely and a new peak appears at 1558 cm−1, which is assigned to the absorbance of 1,2,3-triazole ring in the spectra of cationic chitosan derivative b (Tan, Li and Dong et al., 2017). Besides, the success of CuAAC reaction is also proved by the appearance of new peaks at 3081, 1604, and 809 cm−1, which are ascribed to the ring stretching vibrations of 3-substituted pyridine. After N-methylation of 1,2,3-triazole and pyridine with iodomethane, the characteristic peak considered as 1,2,3-triazolium moiety of cationic chitosan derivative c is observed at 1581 cm−1 (Tan, Li and Dong et al., 2017). Besides, other new peaks for pyridinium at 3058, 1519, and 809 cm−1 are also clearly detected (Rúnarsson et al., 2010). 3.1.2. 1H NMR analysis To further confirm the inclusion of the 1,2,3-triazolium and pyridinium moieties in the chitosan backbones, 1H NMR spectra is conducted shown in Fig. 2 and main characteristic groups of peaks have been identified and assigned. The peaks between 3.05 and 5.00 ppm are ascribed to the protons of glucosamine unit and the peak at 2.04 ppm is assigned to the methyl protons of the N-acetyl group in the 1H NMR spectrum of chitosan (Badawy, Rabea & Taktak, 2014; Wu et al., 2016). Compared with the peaks of chitosan, the 1H NMR spectrum of chitosan derivative a shows the prominent peak of −N+–(CH3)3 at 3.39 ppm (a in the spectrum) (Patrulea et al., 2016; Rahmani et al., 2016) along with the resonance peaks at 4.33 and 2.70 ppm (b and c in the spectrum) corresponding to the hydrogen atoms on the methylene and terminal alkynyl of propargyl group (Uliniuc et al., 2013). When terminal alkyne is transformed to 1,2,3-triazole through the click reaction of chitosan derivative a with 3-azidopyridine, one of the key peaks confirming the success of reaction is the downfield signal at
Fig. 1. FTIR spectra of chitosan and chitosan derivatives.
sequential approach, as shown in Scheme 1. The poor solubility of pristine chitosan in water and organic solvents could inevitably lead to inefficient heterogeneous reaction in the chemical step. Fortunately, the N,N,N-trimethylation of chitosan as one of the most simple and efficient methods could greatly overcome this obstacle (Patrulea et al., 2016). Without any purification, N,N,N-trimethyl chitosan reacted directly with propargyl bromide in the alkaline condition to obtain the reaction intermediate, cationic propargyl chitosan derivative a. Subsequently, the click chemistry was performed with 3-azidopyridine to synthesize the cationic chitosan derivative bearing 1,2,3-triazole and pyridine b. Then, the targeted cationic chitosan derivative c was synthesized by Nmethylation, reacting cationic chitosan derivative b with iodomethane. The chemical structures of the resultant chitosan derivatives were confirmed by FTIR (Fig. 1) and 1H NMR (Fig. 2) spectroscopy. 183
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Fig. 3. TGA (a) and DTG (b) curves of chitosan and chitosan derivatives.
7.65 ppm (d in the spectrum), which is assigned to hydrogen atoms at 5-H position of 1,2,3-triazole ring (Tan, Li, Li, Dong & Guo, 2016). Moreover, the additional signals at 7.80–9.26 ppm (e in the spectrum) are assigned to the protons of pyridine group of chitosan derivative b (Qin, Liu, Xing, Li, Yu & Li, 2013). After N-alkylation with iodomethane, the signals corresponding to the hydrogen atoms on 1,2,3triazole and pyridine groups at 7.65 and 7.80–9.26 ppm (d and e in the spectrum) completely disappear, and instead, the new peaks at around 8.30 and 8.70–9.75 ppm (f and g in the spectrum) emerge due to the protons of 1,2,3-triazolium and pyridinium (Jia, Duan, Fang, Wang & Huang, 2016; Sood et al., 2014). In addition, completion of the alkylation reaction is also corroborated by the appearance of new signals at 4.54 ppm (h and i in the spectrum) for the pendant methyl groups attached to the nitrogen of 1,2,3-triazolium and pyridinium moieties (Abdelhedi-Miladi et al., 2014). 3.2. Thermal stability Fig. 4. pH dependence of water solubility of chitosan and chitosan derivatives.
Fig. 3 shows the thermogravimetric analyses (TGA) and derivative thermogravimetric (DTG) analyses curves of chitosan and chitosan derivatives. The pyrolysis process of chitosan could be accomplished by mainly two steps under nitrogen atmosphere. The first step observed at below 150 °C may be due to the evaporation of the water molecules which trapped between the carbohydrate polymer chains (Tan, Li and Wang et al., 2016). At the second stage, the degradation of chitosan skeleton occurs from 280 °C to 450 °C with 64% of mass loss. In comparison, chitosan derivatives undergo three-step pyrolysis process: one water loss and two thermal degradations. The first stage ranges from 25 to 150 °C, with a 8% miss loss of water molecules. The second thermal event occurs in the temperature range 150–220 °C, which is due to the elimination of the functional groups. The more intense degradation and carbonization of chitosan chain in the third stage start earlier from about 220 °C and continue until 400 °C and reach the highest rate in the range of 238–261 °C, corresponding to mass loss with 65–70% up to 800 °C. The lower thermal stability of the modified chitosan may be attributed to the disruption of the intramolecular and intermolecular hydrogen bonds from chemical modification (Chivangkul et al., 2014).
with iodomethane further improves water solubility of chitosan derivative, especially at alkaline pH. Current finding suggests that the quaternization reaction is an effective mean for improving water solubility of chitosan, which is favorable to further applications. 3.4. Antifungal activity Three destructive phytopathogenic fungi, C. lagenarium, W. fusarium, and F. oxysporum, were applied to test the potential antifungal efficiency of the prepared compounds in vitro by measuring mycelial inhibition of radial growth. In the case of pristine chitosan with the poor water solubility, it was unfeasible to assess the antifungal property of it by mycelium growth rate method in aqueous systems, so watersoluble low-molecular chitosan (LCS) was chosen in antifungal activity test. The cuprum content in chitosan derivatives b and c measured by ICP-MS was below 0.3 mg·g−1. That is to say, when the sample solution was added to sterile PDA medium to give a final concentration of 0.1, 0.5, and 1.0 mg/mL, the actual concentration of cuprum in sterile PDA medium is below 0.3 ppm, at which it had almost no antifungal effect against the tested plant pathogenic fungi. Therefore, in this paper, the influence of residual copper catalyst on the antifungal activity of chitosan derivatives could be ignored. The antifungal activities of chitosan and chitosan derivatives have been assessed at 0.1, 0.5, and 1.0 mg/mL and the results are summarized in Fig. 5. Among the three tested plant pathogenic fungi, all of the compounds reveal the relatively strong antifungal effect against C. lagenarium but the relatively weak inhibition towards F. oxysporum with dose independence. The radial growth inhibition percentages of chitosan against these tested plant pathogenic fungi ranges from 11.80 to 25.00% at the maximum concentration of 1.0 mg/mL. All of the synthesized derivatives show better ability of inhibiting the growth of the tested phytopathogenic fungi than chitosan. It is found that compared with chitosan and compound a, chitosan derivative bearing 1,2,3-
3.3. Water solubility Fig. 4 illustrates the water solubility of chitosan and chitosan derivatives at different pH values. It is well established that chitosan has poor solubility in water due to the strong intramolecular and intermolecular hydrogen bonds (Chen et al., 2016). It is highly soluble in acidic solutions (pH < 6.5) due to the protonation of the primary amine (Patrulea et al., 2016). After trimethylation of chitosan, chitosan derivative a shows higher water solubility because of the high hydrophilia of quaternary ammonium groups (Chivangkul et al., 2014). However, after CuAAC reaction with 3-azidopyridine, chitosan derivative b exhibits relatively weak water solubility because of the formation of amide bond between 1,2,3-triazole and pyridine groups in chitosan backbone. Fortunately, the N-methylation of 1,2,3-triazole and pyridine 184
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Fig. 6. Effect of chitosan derivatives on cucumber seedling growth.
Moreover, cationic chitosan derivative bearing 1,2,3-triazolium and pyridinium c exhibits greatly enhanced antifungal action by one-step Nmethylation of chitosan derivatives b with iodomethane. A maximum of 98.44% inhibition rate is recorded for C. lagenarium, followed by 79.16% and 67.56% for W. fusarium and F. oxysporum at 1.0 mg/mL. Notably, cationic chitosan derivative c is still active against tested fungi even when the dosage is lowered to 0.5 mg/mL. It is reasonably hypothesized that the chemical modification of chitosan molecule by introducing 1,2,3-triazolium and pyridinium improves significantly the antimicrobial activity of chitosan. Many studies from the past have reported that quaternary ammonium salts possessed strong antimicrobial activity based on electrostatic interactions (Chang, Yang & Liang, 2010; Sajomsang, Gonil & Saesoo, 2009; Sajomsang, Gonil & Tantayanon, 2009). As novel types of quaternary ammonium salts, 1,2,3-triazolium and pyridinium groups would interact electrostatically with the electronegative charged components (i.e., proteins, phospholipids) on fungal cell walls or cytomembranes (Guo et al., 2007; Tan, Li and Dong et al., 2016). This adherence of polycations to the outer membranes of the fungi could lead to a change of permeation property of the membrane wall, which could provoke internal osmotic imbalances and in turn cause the leakage of intracellular electrolytes and proteinaceous constituents as well as the obstruction of nutrient intake (Li, Yang & Yang, 2015; Tejero, Lopez, Lopez-Fabal, GomezGarces & Fernandez-Garcia, 2015). 3.5. Effect of chitosan derivatives on seed germination In order to evaluate the effect of chitosan derivatives on seedling growth, the cucumber seeds was treated with chitosan and chitosan derivatives and the results show the positive morphological effects of chitosan derivatives with slightly increased germination rate, seedling length, fresh and dry weight, and seed vigor index (Fig. 6 and Table 1). That is to say, the chitosan derivatives synthesized in this paper exhibit non-toxicity on the cucumber at the range of the testing concentration. 4. Conclusion
Fig. 5. The antifungal activity of chitosan and chitosan derivatives against C. lagenarium (a), W. fusarium (b), and F. oxysporum (c). * (P < 0.05), ** (P < 0.01) for comparisons between compound c and LCS, a, b.
In summary, we have recently proposed a convenient and robust method to prepare novel cationic chitosan derivative bearing 1,2,3triazolium and pyridinium charged units by associating CuAAC step with efficient N-alkylation of 1,2,3-triazole and pyridine with iodomethane. FTIR, 1H NMR spectra, and elemental analysis confirmed that 1,2,3-triazolium and pyridinium moieties had been successfully introduced to chitosan backbone. The thermal stability and water solubility of the synthesized chitosan derivatives were also measured. The antifungal activity against three kinds of plant threatening fungal strains was estimated by observing the percentage inhibition of mycelial growth. The synthesized chitosan derivative bearing 1,2,3-triazolium and pyridinium moieties clearly showed stronger antifungal action than chitosan and chitosan derivative bearing 1,2,3-triazole and pyridine groups. The obtained findings suggested that the N-methylation of
triazole and pyridine b shows slightly higher antifungal property and the highest growth inhibitions (32.64–45.94%) against three tested plant pathogenic fungi are observed at the maximum concentration of 1.0 mg/mL. The above results confirm the contribution of the 1,2,3triazole and pyridine moieties to antifungal activity. Like other nitrogen-containing heterocyclic compounds, it was reported that 1,2,3triazole and pyridine were capable of inhibiting the growth of the tested phytopathogenic fungi by interrupting the synthesis of the cell membrane and cell wall by hydrogen bond interaction (Kant, Singh, Nath, Awasthi & Agarwal, 2016; Tan, Li and Gao et al., 2017; Vijai Kumar Reddy et al., 2016) (Fig. 6). 185
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Table 1 Effect of chitosan derivatives on seedling growth of cucumber (data recorded after 10 days of growth). Treatment Control LCS
a
b
c
0.1 mg/mL 0.5 mg/mL 1.0 mg/mL 0.1 mg/mL 0.5 mg/mL 1.0 mg/mL 0.1 mg/mL 0.5 mg/mL 1.0 mg/mL 0.1 mg/mL 0.5 mg/mL 1.0 mg/mL
Germination (%)
Seedling length (cm)
Fresh weight (gm)
Dry weight (gm)
SVI
86.67 90 ± 90 ± 93.33 90 ± 90 ± 86.67 90 ± 86.67 90 ± 93.33 90 ± 86.67
12.23 12.65 13.24 14.74 13.36 14.99 14.88 14.63 15.23 15.02 13.89 14.56 14.68
0.30 0.31 0.32 0.34 0.32 0.35 0.33 0.34 0.39 0.36 0.32 0.34 0.33
0.018 0.019 0.020 0.020 0.019 0.021 0.020 0.020 0.025 0.023 0.020 0.021 0.020
1060 1139 1192 1376 1202 1349 1290 1317 1320 1352 1296 1310 1272
± 4.71 0 0 ± 4.71 8.16 0 ± 4.71 0 ± 4.71 0 ± 4.71 0 ± 4.71
± ± ± ± ± ± ± ± ± ± ± ± ±
0.40 0.20 0.39 0.75 0.52 0.25 0.32 0.61 1.01 0.82 0.25 0.79 0.87
1,2,3-triazole and pyridine could contribute a lot to the water solubility and the antifungal efficiency of synthesized chitosan derivatives. Meanwhile, the synthesized chitosan derivatives exhibited non-toxicity on the cucumber by recording the germination rate, seedling length, fresh and dry weight, and seed vigor index in the seedling bioassay. The product described in this paper might serve as a new leading structure for further design of novel antifungal agents.
± ± ± ± ± ± ± ± ± ± ± ± ±
0.01 0.01 0.01 0.02 0.01 0.01 0.02 0.01 0.02 0.01 0.01 0.01 0.02
± ± ± ± ± ± ± ± ± ± ± ± ±
0.001 0.001 0.001 0.001 0.001 0.001 0.002 0.002 0.002 0.001 0.001 0.001 0.001
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