1. Introduction
Chitosan is an abundant, renewable polysaccharide derived from chitin that exhibits attractive biopolymer properties for many biomedical applications such as non-toxicity, biocompatibility, and biodegradability (Elsabee & Abdou, 2013; Jayakumar, Prabaharan, Nair, & Tamura, 2010). It has antimicrobial activity (Rabea, Badawy, Stevens, Smagghe, & Steurbaut, 2003; Zheng & Zhu, 2003), and regenerative properties (Dash, Chiellini, Ottenbrite, & Chiellini, 2011). Chitosan is also used in drug delivery applications as an absorption enhancer (Kotz , Lueßen, de Boer, Verhoef, & Junginger, 1999e(´) ), mucoadhesive polymer (He, Davis, & Illum, 1998), to form nanoparticles (Jayakumar, Menon, Manzoor, Nair, & Tamura, 2010; Qi, Xu, Jiang, Hu, & Zou, 2004), and for gene delivery applications (Park, Saravanakumar, Kim, & Kwon, 2010). Chemical modification of chitosan to improve the properties for the intended application or biological activity is also a very active research field (Harish Prashanth & Tharanathan, 2007). The glucosamine monomer in chitin has three nucleophilic functional groups, the C-2 amino group, the C-3 hydroxyl group, and the C-6 hydroxyl groups, which have been targeted for modification.Most commonly, this is done through either Nor Oalkylation or acylation (Ifuku, 2014; Sahariah & Ma´sson, 2017). The primary C-6 has also been replaced with other functional groups such as Br, N(CH3)3(+) or N3 (Gao, Zhang, Chen, Gu, & Li, 2009; Satoh et al., 2006; Zampano, Bertoldo, & Ciardelli, 2010). Chitosan is poorly soluble in most organic solvents, which are often required as the medium for the reactions, and the reported conversion or substitution is only partial with generally less than 50% conversion of targeted groups on the polymer chain. Lack of selectivity is also an issue with many reactions, and a mixed N, O modification is common. One way to address this issue is to use protecting groups in the synthesis of chitosan derivatives. The purpose of the protecting groups is to prevent the reaction of the groups that are not targeted for modification and also to improve the solubility in organic solvents. The tert-butyl dimethyl silyl (TBDMS or TBS) protection of the hydroxyl groups is especially useful in this regard. Di-3,6-O-TBDMS chitosan is well soluble in moderately polar organic solvents, such as dichloromethane and chloroform and has been used for N-selective synthesis of N,N,N-trialkyl and N-acyl derivatives and conjugates with 100% degree of substitution “Click chemistry” is a term that was first introduced by K. B. Sharpless to describe selective reactions that afford carbon-heteroatom bonds in high yield (Kolb, Finn, & Sharpless, 2001). The copper (I) catalyzed azide-alkyne cycloaddition (CuAAC) was proposed to fit these criteria. The “click chemistry” approach is now commonly used to synthesize bio-conjugates, especially for functionalizing peptides and proteins with different moieties (Elchinger et al., 2011; El-Sagheer & Brown, 2010; Hein, Liu, & Wang, 2008) or conjugating them and other functional moieties to nanoparticles (Lu, Shi, & Shoichet, 2009),liposomes (Fritz et al., 2014),solid surfaces (Sun, Stabler, Cazalis, & Chaikof, 2006), and carbohydrates (Nielsen, Wintgens, Amiel, Wimmer, & Larsen, 2010). Thus, a substituent containing a terminal alkyne or azide group is first introduced by acylation or alkylation, and then the functional moieties are introduced by reaction with a corresponding azide or alkyne.
CuAAC modifications of chitosan have been mainly focused on reactions with the azide introduced at the C-6 position and with the C-2 amine protected with phthaloyl groups (Gao et al., 2009; Luan et al., 2018). The 2-amino group has also been modified with acyl moieties carrying terminal alkyne or azide groups that can subsequently be converted to triazole by the CuAAC reaction. This approach has been used for grafting peptides (Barbosa, Vale, Costa, Martins, & Gomes, 2017; Sahariah et al., 2015), poly(ethylene glycols) (Kulbokaite, Ciuta, Netopilik, & Makuska, 2009), drug conjugates, and nanoparticles (Li, Sun, Gu, & Guo, 2018; Li, Tan, Zhang, Gu, & Guo, 2015; Sarwar, Katas, Samsudin, & Zin, 2015).Primary amines, like the 2-amino group of chitosan, can be converted to azide by Cu(II) catalyzed diazo transfer reaction with imidazole-1-sulfonyl azide hydrochloride (Goddard-Borger & Stick, 2007). This approach has been used to convert chitosan prior to CuAAC to introduce PEG moieties (Kulbokaite et al., 2009), or to modify chitosan antimicrobial coatings (Barbosa et al., 2019). This procedure has also been used for the synthesis of insoluble chitosan derivatives (Zhang et al., 2008). The reported grafting ratio for water-soluble derivatives has not been high. For example, a peptide was grafted at a 2 mg/g ratio corresponding to 0.2% degree of substitution (DS) (Barbosa et al., 2017). A previous study found that chitosan could not be converted in more than 40% from amines to triazolevia N-azidated chitosan (Kulbokaite et al., 2009). In the present work, we aimed to use the CuAAC reaction to synthesize new types of water-soluble carbohydrate polymers starting from chitosan. In these structures, all C-2 primary amino groups of chitosan are to be converted to aromatic 1,2,3-triazole, and thus chitotriazolan is the suggested name for these new structures. Herein, the chitotriazolans were synthesized by two different pathways, starting from di-TBDMS protected chitosan or unmodified chitosan. Six of the derivatives could be solubilized in water and were characterized by FTIR, NMR, and SEC-MALS. Five derivatives were insoluble and therefore only analyzed by FT-IR. Antibacterial activity of soluble derivatives was evaluated against S. aureus and E. coli at pH 7.2.
2. Materials and methods
2.1. Material
Chitosan (S160302-1-2-3-4, DA of 17%, and MW 108 kDa) was obtained from Primex ehf Siglufjo(¨)rdur, Iceland. All reagent grade chemicals were purchased from Sigma Aldrich (Germany): Methanesulfonic Epigenetic Reader Domain inhibitor acid, acetic acid, tert-butyldimethylsilyl chloride (TBDMS-Cl), imidazole, sodium azide, sulfuryl chloride, trimethylamine, copper sulfate, sodium ascorbate, acetyl chloride, hydrochloric acid, propargyl Carbohydrate Polymers 267 (2021) 118162 bromide, N-methylpropargylamine, N,N-dimethylpropargylamine, 3butynoic acid, 3-methyl-1-pentyn-3-ol, 2-methyl-3-butyn-2-ol, 3butyn-2-ol, sodium sulfite, N,O-bis(trimethylsilyl)acetamide, tris(trimethylsilyl) phosphite, and 4-bromo-1-butyne. All solvents, including dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), dichloromethane (DCM), acetone, methanol, ethanol, and acetonitrile, were also obtained from Sigma Aldrich. De-ionized water was treated using a Milli-Q™ filtration system. Dialysis membranes (RC, Spectra/Por, MW cutoff 3500 Da 45 mm) were purchased from Spectrum® Laboratories Inc. (Rancho Dominguez, USA).
2.2. Methods and preparations
2.2.1. Preparation of imidazole sulfonyl azide hydrochloride salt
The imidazole sulfonyl azide hydrochloride salt was prepared following a previously published procedure (Goddard-Borger & Stick, 2007). Briefly, sulfuryl chloride (2.48 mL, 30.77 mmol) was added dropwise at 0 ◦ C to the suspension of sodium azide (2.0 g, 30.77 mmol) in anhydrous acetonitrile (40 mL) under nitrogen, and the reaction mixture was stirred at room temperature overnight. Then imidazole (4.19 g, 61.54 mmol) was added portion-wise to the reaction mixture at an ice-cooled condition, and the reaction mixture was stirred at room temperature for 3 h. After that, the reaction mixture was diluted with ethyl acetate (100 mL) and washed with water (3 × 100 mL), and saturated aqueous NaHCO3 solution (2 × 100 mL) in a separatory funnel. The organic phase was dried over Na2SO4 and filtered. A solution of HCl in ethanol [50 mL, dropwise addition of acetyl chloride (12 mL) to ice-cooled ethanol (40 mL)] was added to the filtrate, and the mixture stirred at 0 ◦ C to get a white precipitate. The solids were filtered and washed with ethyl acetate to obtained small white needle crystals as a product. The mother liquors were discarded HAZARD statement: Concentration of mother liquors at this step may result in an explosion (Goddard-Borger & Stick, 2007).
2.2.2. Synthesis of N-propargyl N,N,N-trimethylammonium bromide salt
The title compound was synthesized according to a reported procedure (Nguyen, Fournier, Asseline, Thuong, & Dupret, 1999). Briefly, trimethylamine (1.48 mL, 16.81 mmol) was dissolved in acetonitrile (100 mL) at − 20 ◦ C. Then propargyl bromide (1.27 mL, 16.81 mmol) was added slowly at − 20 ◦ C. The reaction mixture was warmed to room temperature and stirred for 24 h, and then the solvent was removed using rotary evaporation and dried under reduced pressure to provide a white solid as a product. Procedures for the synthesis of propargyl sulfonate and butynyl phosphonate are reported in the supplementary information.
2.2.3. OTBDMS-chitosan amine to azide conversion (A2)
Chitosan OTBDMS (Rathinam, (500 mg, 1.26 mmol) was dissolved in 15 mL of DCM and 15 mL of MeOH. After that,imidazolesulfonyl azide hydrochloride (0.395 g, 1.89 mmol) and Et3N (0.26 mL, 1.89 mmol) were added to the solution. A solution of CuSO45H2O (31 mg, 0.125 mmol dissolved in 1 mL water) was added to the reaction mixture. The color of the reaction mixture changed to a blue tinge, and the product started to precipitate. The reaction was further stirred at room temperature for 60 h under an N2 atmosphere. The material was concentrated under reduced pressure. A precipitate was formed, and this was filtered and washed with ethanol and dried for more than 1 h by suction. The resulting material had a light bluish color, and the product could be confirmed by IR spectroscopy.
2.2.4. OTBDMS-chitosan azide to triazole conversion (A3)
OTBDMS-Chitosan azide 700 mg, (1.75 mmol) was dissolved in DMF (20 mL). Then CuSO4 (56 mg, 0.23 mmol in 2.5 mL water) and sodium ascorbate (174 mg, 0.87 mmol in 2.5 mL water) were added to the reaction mixture, followed by N,N-dimethylamino-1-propyne (0.94 mL, 8.76 mmol) under nitrogen atmosphere. The reaction mixture was stirred at 50 ◦ C for 48 h. Then, the resulting material was dialyzed against water for three days and freeze-dried. Full conversion of starting material to the product was confirmed by the absence of the azide peak in the FT-IR.
2.2.5. OTBDMS-chitosan deprotection
O-TBDMS -Chitosan triazole (A3) (600 mg) was dissolved in methanol (30 mL) and conc. HCl (5 mL was diluted with 10 mL of methanol) was added slowly. The reaction mixture was then stirred at room temperature for 24 h. After that, the reaction mixture was dialyzed against water for three days (first day 5% NaCl, next two days water) and then freeze-dried. Yield: 325 mg, 1H NMR (400 MHz, D2O): 。2.08 (NCOCH3), 2.81 (H6′), 2.95 [N-(CH3)2], 3.14 (H6), 3.52 (H5), 3.77 (H4), 3.94 (H3) 4.40 (H2), 4.56 (triazole CH2), 5.17 (H1), 8.46 (triazole CH).
2.2.6. Chitosan amine to azide conversion (A5)
Chitosan (500 mg, 2.958 mmol) was dissolved in 40 mL of 0.1 M HCl solution, then NaHCO3 (0.248 g, 1.0 equiv) was added to the solution, and the mixture was stirred vigorously for 30 mins. After that, imidazole sulfonyl azide hydrochloride (0.93 g, 4.437 mmol) and NaHCO3 (2.48 g 10.0 equiv) were added slowly in small portions. Then a solution of CuSO4 5H2O (95 mg, 0.384 mmol) in 1 mL of water and 10 mL of methanol solution was added to the reaction mixture. The reaction mixture was turned to bluish color and was stirred at room temperature for 24 h. Finally, the material was precipitated out using acetone. The precipitate was filtered and washed with water five times and acetone. The product was dried, and the presence of the azide group was confirmed by IR spectroscopy.
2.2.7. General procedure for chitosan azide to triazole conversion (derivatives 3-11)
Chitosan azide (1 equiv.) was dissolved in DMSO (15 mL) at 50 ◦ C. Then CuSO4 (0.13 equiv. in 2.5 mL water) and sodium ascorbate (0.5 equiv. in 2.5 mL water) were added to the reaction mixture followed by alkyne (5.0 equiv.) under nitrogen atmosphere. The reaction mixture was stirred at 50 ◦ C for 48 h. Then, the resulting material was dialyzed against water for three days (first day 5% NaCl, next two days water) and freeze-dried. The products were confirmed by FT-IR to show that the azide peak (at 2109 cm− 1) had completely disappeared and by 1H NMR when solutions in D2O could be prepared.
2.2.8. Synthesis of derivative 3
Chitosan azide (200 mg, 1.07 mmol) was dissolved in DMSO (15 mL) at 50 ◦ C. Then CuSO4 (34 mg, 0.139 mmol in 2.5 mL water) and sodium ascorbate (106 mg, 0.534 mmol in 2.5 mL water) were added to the reaction mixture, followed by N-propargyl-N,N,N-trimethylammonium bromide (523 mg, 5.34 mmol). 1H NMR. Yield: 270 mg for 3, 1H NMR (400 MHz, D2O): 。2.08 (N-COCH3), 2.90 (H6′ ), 3.20 [H6, N(CH3)3], 3.52 (H5), 3.78 (H4), 4.44 (H3), 4.58 (H2), 4.77 (triazole CH2 was merging with D2O peak), 5.18 (H1) 8.59 (triazole CH). The procedure for derivatives (4-11) is reported in supporting information.
2.3. Characterization
2.3.1. NMR and FTIR spectroscopy
The chitotriazolan derivatives were characterized by using 1H NMR and 13C NMR spectroscopy. 1H and COSY NMR spectra were recorded on a Bruker Avance 400 spectrophotometer operating at 400 MHz. The 13C NMR and HSQC spectra were recorded on a Bruker 500 MHz spectrometer equipped with a cryoprobe. NMR samples were prepared in CDCl3, D2O, or D2O/DCl in concentrations of 7-15 mg/mL. The Nacetyl peak at 2.08 ppm was used as an internal reference in all proton NMR spectra. The FT-IR spectra of the chitosan (CS) and chitotriazolan derivatives were measured using a Thermo Scientific™ Nicolet™ iS10 FTIR spectrometer in the 500-4000 cm− 1 wavelength region. The set number of scans was 64, and the resolution was 4.0 cm− 1. Few milligrams of the material were used for each IR spectra and all compounds were measured against a blank background.
Scheme 1. A. Synthesis of chitotriazolan via TBDMS (TBS) protection routes and conditions: (i) methane sulfonic acid, deionized water, 10 ◦ C; (ii) imidazole, TBDMS-Cl, DMSO, RT; (iii) imidazolesulfonyl azide HCl salt, triethylamine, CuSO4 5H2O,DCM,methanol, RT; (iv) CuSO4 5H2O, sodium ascorbate, terminal alkyne, DMF 50 ◦ C; (v) Conc. HCl, methanol RT. B. Synthesis of chitotriazolan via without TBDMS protection synthetic routes and conditions: (i) 0.1 M HCl solution, sodium bicarbonate, imidazole sulfonyl azide HCl salt, CuSO4 5H2O, water, methanol, RT; (ii) CuSO4 5H2O, sodium ascorbate, terminal alkyne, DMSO, 50 ◦ C.
Fig. 1. FT-IR spectra for chitosan and chitotriazolan derivatives: CS (A), derivative A5 (B), derivative A2 (C), derivative 3 (D), derivative 5 (E). FT-IR spectra for insoluble chitotriazolan derivative 7 (F), derivative 8 (G), derivative 9 (H), derivative 10 (I).
2.3.2. Gel permeation chromatography
Average Molecular weight (MW) determination was carried out using gel permeation chromatography (GPC). GPC measurements were done using the Polymer Standards Service (PSS) (GmbH, Mainz, Germany), Dionex Ultimate 3000 HPLC system (Thermo Scientific-Dionex Softron GmbH, Germering, Germany), Dionex Ultimate 3000 HPLC pump, and Dionex Ultimate 3000 autosampler (Thermo ScientificDionex Softron GmbH, Germering, Germany), Shodex RI-101 refractive index detector (Shodex/Showa Denko Europe GmbH, Munich, Germany), PSS’s ETA-2010 viscometer and MALLS detector (PPC SLD 7100). WINGPC Unity 7.4 software (PSS GmbH, Mainz, Germany) was used for data collection and processing. A series of three columns [PSS Novema 10 μ guard (50 × 8 mm), PSS Novema 10 μ 30 Å (150 × 8 mm) and PSS Novema 10 μ 1000 Å (300 × 8 mm)] (PSS GmbH, Mainz, Germany) were used in the HPLC system. Ready Cal-Kit Pullulan standards with Mp (180-708,000 Da) from PSS (GmbH, Mainz, Germany) were used for calibration. The eluent used was 0.1 M NaCl/0.1% TFA solution. Each sample was dissolved in the same eluent as mentioned above at a concentration of 1 mg/mL at 25 ◦ C using a flow rate of 1 mL/ min. Each sample had an injection volume of 100 μL, and the time between injections was 30 min.
2.4. Antibacterial assay of the chitosan derivatives
Minimal inhibition concentration (MIC) was measured according to the CLSI standard (CLSI, 2009). The antibacterial activity was tested against two different bacterial species, Gram-positive bacteria Staphylococcus aureus (S. aureus, ATCC 29213) and Gram-negative bacteria Escherichia coli (E. coli, ATCC 25922). Before MIC testing, the bacterial strains were cultured on blood agar at 37 ◦ C for 12-18 h. The bacterial colonies were suspended in saline water and adjusted to 0.5 McFarland and further diluted in Mueller-Hinton broth (MHB) to reach the final concentration of 5 × 105 colony forming units (CFU)/mL in the test wells. The MHB was used forMIC measurement at pH 7.2. Gentamicin, a well-known antibiotic was used as a performance control, MHB without chitosan derivatives or the bacterial solution as a sterility control, and MHB with only the bacterial solution as growth control. The stock solution of compounds was prepared in sterile water at a concentration of 8192 μg/mL, 50 μg/mL of the compounds were added to a microtiter 96well plate, and two-fold dilutions were done in MHB for concentrations 16-8192 μg/mL. Later 50 μL of bacterial 5 × 105 (CFU)/mL suspension was added to each well. The microtiter plates were incubated at 37 ◦ C for 20 to 24 h. The MIC values were observed by the naked eye and determined as the lowest concentrations of the antibacterial agent to completely inhibit the visible growth of microorganisms in the microtiter 96-well plate.
3. Results and discussion
The main aim of the research work was to develop a procedure to quantitatively convert the primary amino groups of chitosan first to azide groups and then to 1,2,3-triazole moieties to enhance solubility in water. Previous investigations have shown that chitosan azides are insoluble in aqueous solutions and organic solvents (Kulbokaite et al.,2009), limiting the conversion of the amino groups (Zhang et al., 2008). We have used di-OTBDMS protected chitosan to address potential issue with the solubility of the product derivatives (Rathinam, lafsd ttirO et al., 2020). It has been shown that O-TBDMS-chitosan and its derivative is soluble, in most cases, insolvents such as dichloromethane and chloroform (Rúnarsson, Malainer, Holappa, Sigurdsson, & M sson,a 2008) (Sahariah, Ma´sson, & Meyer, 2018). Thus, the synthesis was initially attempted starting from O-TBDMS chitosan (Scheme 1A). The conversion to the corresponding azide (A2) could be confirmed by FT-IR (Fig. 1), but to our surprise, it turned out that the O-TBDMS chitosan azide had low solubility inorganic solvents and thus could not be fully characterized by NMR. The O-TBDMS chitosan azide did not dissolve in aqueous and instead of organic solvents such as water, aqueous 0.1 M HCl, 0.1 M NaOH, MeOH, acetonitrile, chloroform, dichloromethane, and NMP. Mixed solvents like 1:1 ratio of MeOH:0.1 M HCl solution and acetonitrile:0.1 M HCl solution could neither be used to solubilize this polymer. The material was partially soluble in DMF, and DMSO (this required the material to be stirred for 12 h at room temperature or 50 ◦ C). Thus the subsequent CuAAC was carried out in DMF to obtain 4(N,N-dimethylaminomethyl)chitotriazolan 1 and 4-(N,N,N-trimethylammoniumethyl)chitotriazolan 2 following the deprotection step.In parallel, an alternative route where chitosan was directly converted to azide without the biomass processing technologies use of protecting groups, was investigated. The conversation to azide could be confirmed with FT-IR, and the aromatictriazole conversion was achieved in near quantitative, which was similar to previous work (Kulbokaite et al., 2009). We found that the material was insoluble in an aqueous solution and organic solvents. However, CuAAC reaction with N-propargyl-N,N,N-trimethylammonium bromide in DMSO proved to be successful, and the resulting product was soluble in H2O and could be purified by dialysis, and the product was freeze-dried. Full conversion to the chitotriazolan product was confirmed by the disappearance of the azide peak in the IR spectra and the appearance of a triazole peak at 8.5 ppm in 1H NMR, corresponding to a 90% degree of substitution for the triazole group.This procedure was also used to synthesize 4-substituted chitotriazolan derivatives with N-methylaminomethyl, carboxymethyl, 2hydroxybut-2-yl, 2-hydroxyprop-2-yl, and 1-hydroxyethyl side groups. Propargylsulfonate and propargylphosphonates were synthesized (see in the supporting information) according to reported procedures (Ouadahi, Allard,Oberleitner, & Larpent, 2012; Wanat et al., 2015) and used to synthesize 4-substituted sulfomethyl, phosphomethyl, and phosphoethyl chitotriazolan derivatives (Scheme 1B).
Fig. 2. 1H NMR spectra for derivative 3 (A) and derivative 4 (B).
Fig. 3. 13C NMR for derivative 1 (A), COSY NMR for biosoluble film derivative 3 (B), HSQC NMR for derivative 3 (C), and derivative 4 (D).
3.1. Characterization by FT-IR spectroscopy
The FT-IR spectra of chitosan, chitosan O-TBDMS azide (A2), chitosan azide (A5), and chitotriazolans 3, 5, and 7-10 are shown in Fig. 1. The characteristic C-(-)O stretching vibration band at 1652 cm− 1 for the N-acetyl group (DA of 17% present in chitosan starting material) was observed in all spectra. New N3 bands appeared at 2109 cm− 1 when the amino group was converted to azide (Fig. 1B and C). The azide band disappeared after the CuAAC reaction to form the 1,2,3-triazole on the chitosan backbone at the C-2 position. In Fig. 1C strong bands at 775 cm− 1 and 831 cm− 1 correspond to Si-C stretching vibrations. A new band at 1475 cm− 1 can be observed in Fig. 1D, which could be assigned to the weak N-CH3 absorbance, and a new band appeared at 795 cm− 1, confirming the P-O bond for the phosphonate group (Fig. 1E). The conversion for insoluble chitotriazolan derivatives were confirmed by the disappearance of the sharp azide peaks (Fig. 1F, G, H, I).
3.2. Characterization by NMR spectroscopy
The 1H NMR spectra of the water-soluble 4-(N,N,N-trimethylammoniummethyl)-chitotriazolan and 4-sulfomethylchitotriazolan are shown in Fig. 2. For derivative 3, the 1,2,3-triazole structure could be confirmed by the aromatic proton peak at 8.59 ppm. The quarternary trimethylammonium group for derivative 3 appeared at 3.2 ppm, and the methylene (CH2) group at 4.8 ppm merged with the HDO peak; however, it was clearly visible in the HSQC spectrum (Fig. 3C). The conversion of the free amino group in the C-2 position on chitosan to the 1,2,3 triazole leads to a dramatic shift in the C-2 proton peak from around 2.8 ppm to 4.58 ppm. Other protons of the chitosan backbone are also shifted significantly. The C-6 protons could be observed at 2.90 ppm and 3.2 ppm (merged with theN(CH3)3 peak) and the C-5, C-4, and C-3 protons at 3.52, 3.78, and 4.44 ppm, respectively. The aromatic triazole proton of derivative 4 was broadened and appeared in a slightly up field position (8.13-8.43 ppm) relative to that of derivative 3. The C-6, C-5, C-4,C-3, and C-2 protons were observed at similar shift values in the two derivatives. The peak for the CH2 adjacent to the sulfonate groups was observed at 4.27-4.42 ppm, merged with the C-3 and C-2 proton peaks.
The aromatic signal for C-4 in the 1,2,3-triazole ring was observed at 137 ppm in the 13C APT NMR spectrum of derivative 1 (Fig. 3A). The chitosan carbon signals for C-2 to C-6 appeared between 60 and 80 ppm and C-1 at 100 ppm. The correlation between 1H NMR and the COSY spectra further confirmed the assignment of the 1,2,3-triazole peak at 8.59 ppm, and the N-acetyl peak at 2.08 ppm (Fig. 3B). The HSQC spectra for derivatives 3 and 4 could be used to confirm the assignment of the proton peaks (Fig. 3C and D). The complete assignment of all peaks also confirmed that the azide had been fully converted to the new structure. The HSQC spectrum clearly shows the trimethylammonium protons at 3.2 ppm for cationic 4-(N,N,N-trimethylammonium methyl) chitotriazolan, whereas this peak was not present in the anionic 4-sulfomethyl chitotriazolan spectrum.
The degree of substitution (DS), degree of acetylation (DA), and molecular weight (MW) of derivatives 1-6, are shown in Table 1. The integration of the NMR peaks in the cationic chitotriazolan derivatives indicated more than 90% conversion from the free amino group in chitosan to the 1,2,3-triazole. However, the peaks were broad, and this could influence the accuracy. Only one peak could be observed for each monomer proton of the chitotriazolan backbone, and this was consistent with 100% conversion. The average molecular weights of derivatives 1 and 2 were more than four times less than the MW of the starting material.This reduction in MW was caused by acid hydrolysis of the polymer chain, which occurs when the chitosan mesylate salt is prepared and in the deprotection reaction to remove TBDMS (Sahariah et al., 2014). The average MW of materials 3 and 5,synthesized without the use of protection groups had
about twice the MW of the starting material, which was consistent with the increase in the MW of the monomer units when chitosan was converted to chitotriazolan derivatives. The MW of 4-sulfomethyl chitotriazolan 4 and 4-phosphoethyl chitotriazolan 6 were found to be around 6 KD which was much less than expected (see SI. MW. chromatogram profile and S.Table 1). This was probably due to low solubility in the mobile phase and that the higher MW material was removed in the filtration of the samples.
3.3. Solubility analysis
Cationic derivatives 1, 2, 3, and sulfonated anionic derivative 4 was completely soluble in water at neutral pH. 4-Phosphoethyl-chitotriazolan 6 was soluble in 0.1 M sodium hydroxide solution, and 4-phosphomethyl-chitotriazolan 5 was partially soluble.The 4-(Nmethylaminomethyl)-chitotriazolan 7, 4-carboxymethyl-chitotriazolan 8, and the 4-(hydroxyalkyl)-chitotriazolan derivatives 9–11 (marked in blue color in the Scheme 1B) were insoluble in all solvents and solvent mixtures tested. Derivatives 2–6 had fully ionized side groups, and this may contribute to better solubility. The low MW of derivative 1 may explain why it had better solubility than derivative 7, which has a similar structure with one less N-methyl group.
3.4. Antibacterial properties for chitotriazolan derivatives
The antibacterial activity of chitosan and chitosan derivatives is influenced by several factors, including the degree of substitution (DS), molecular weight, ionic interactions, and the structure of the substitutents (Kong, Chen, Xing, & Park, 2010; Sahariah & Ma´sson, 2017). The water-soluble chitotriazolan derivatives were studied for antibacterial activity against S. aureus and E. coli bacteria at pH 7.2 (Table 2). The cationic chitotriazolan derivative 3 was most active against the bacteria with MIC equal to 64 μg/mL, whereas the anionic derivative 4 was inactive. The monomer structure of derivative 2 was identical to derivative 3 but the former was more than 30 times less active against the bacteria. Derivative 2 had a markedly lower molecular weight than 3, and there were some residual TBDMS groups (<3% for 1 and <0.4% for 2) left from the deprotection step, which could explain this difference. This is also a consideration for derivative 1, which was inactive and had a similar structure with one less N-methyl group than derivatives 2 and 3. The most active derivative 3 was also tested against E.faecalis (ATCC 29212) and P. aeruginosa (ATCC 27853) and the MIC values found to be 1024 μg/mL and 128 μg/mL, respectively.
4. Conclusion
In the current work, we were successful in obtaining a near-complete conversion of the 2-amino group of chitosan to 1,2,3-triazole and obtain the first water-soluble chitotriazolan derivatives. Eleven chitotriazolan derivatives were synthesized through two routes, and four of the Carbohydrate Polymers 267 (2021) 118162 structures had good water solubility. The derivatives were characterized by FT-IR, 1H, and 2D NMR techniques as well as SEC-MALS to determine the structure and molecular weight. The antibacterial activity was evaluated against S. aureus and E. coli at pH 7.2. The cationic chitotriazolanderivatives had significant antibacterial activity, whereas the anionic chitotriazolans were inactive.Chitoriazolans represent a new class of biopolymers with an aromatic 1,2,3-triazole side group on the 2-deoxyglucopyranose monomer unit. Ionic chitotriazolan derivatives can be water-soluble and theN,N, N-trimethylammoniummethyl derivatives 2 and 3 were active against bacteria. The ease of synthesis and structural modification of this new class of biopolymers should stimulate further research into the biological and other properties and utility for diverse applications.
