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Antibacterial film from chlorinated polypropylene via CuAAC click chemistry
Progress in Organic Coatings 125 (2018) 73–78
Contents lists available at ScienceDirect
Progress in Organic Coatings journal homepage: www.elsevier.com/locate/porgcoat
Antibacterial film from chlorinated polypropylene via CuAAC click chemistry Gokhan Acika,b, Cagatay Altinkokc, Hulya Olmezd, Mehmet Atilla Tasdelena,
T
⁎
a
Department of Polymer Engineering, Faculty of Engineering, Yalova University, TR-77100 Yalova, Turkey Department of Chemistry, Faculty of Sciences and Letters, Piri Reis University, Tuzla, 34940, Istanbul, Turkey c Department of Chemistry, Faculty of Science, Trakya University, Merkez, Edirne, Turkey d TUBITAK Marmara Research Center, Material Institute, 41470 Gebze, Kocaeli, Turkey b
A R T I C LE I N FO
A B S T R A C T
Keywords: Antibacterial activity Chlorinated polypropylene Copper (I)-catalyzed azide- alkyne cycloaddition Polypropylene
Polypropylene possessing quaternary ammonium salt (PP-QAS) is synthesized by copper (I)-catalyzed azidealkyne cycloaddition “click” reaction (CuAAC) starting from chlorinated polypropylene (PP-Cl). The antibacterial properties of PP-QAS have been investigated on gram-positive (Staphylococcus aureus) and gram-negative (Escherichia coli) bacteria. For this purpose, clickable azide functionality has been introduced into PP-Cl backbone by using azidotrimethylsilane (TMS-N3) - tetrabutylammonium fluoride solution (TBAF) system in tetrahydrofuran (THF) (PP-N3). Independently clickable alkyne functionalized QAS is prepared by the reaction between 3-dimethylamino-1-propyne and benzyl bromide in the presence of triethyl amine (TEA). Finally, the polypropylene film containing quaternary ammonium salt (PP-QAS) is prepared by the successive CuAAC click reaction and solution casting method. The preparation of PP-QAS is monitored by fourier transform infrared (FTIR) spectroscopy, proton nuclear magnetic resonance (1H-NMR), differential scanning calorimetry (DSC) and scanning electron microscope (SEM) analyses at various stages. Based on antibacterial activity experiments, the PP-QAS coupons significantly inhibit both the growth of S. aureus and E. coli (p < 0.05) compared to neat PP-Cl and control samples.
1. Introduction The polyolefins are the most commonly used polymers in the world due to their low cost, good recyclability and mechanical properties. Over the past years, there have been many studies to improve their properties and expand their usages in industrial applications [1]. Due to their chemical structures not having reactive functional or polar groups, they could not easily modified with traditional organic reactions [2]. There are two distinct modification routes including direct polymerization which is not simple because of deficient sensitivity to Ziegler-Natta catalysts or post-polymerization in the literature [3,4]. Among the post-polymerization techniques, the chlorination of polyolefins is the most feasible way to improve their polarity, adhesion, wettability and chemical resistance by introduction of –Cl groups into backbone with chemical or physical treatments [5]. Thus, the chlorinated polyolefins including chlorinated polypropylene are one of the most preferred functional polyolefins and they can be applied for industrial applications in various forms. In addition to above-mentioned modification techniques, some new approaches using click chemistry
⁎
reactions have been recently reported for the modification of polyolefins [6–11]. During the recent years the click chemistry tools including CuAAC [12–16], Diels-Alder [17], thiol-ene [18,19] and thiolhalogen [20] click reactions have been extensively utilized for the modification of polyolefins. Although the polyolefins are one of the most widely used polymers in many areas, they can be also utilized for specific applications such as food packaging, medical device, clothing and textiles that required additional antibacterial properties. In an effort to obtain desirable antibacterial activity for the polymers against to microorganisms, three main compounds including quaternary ammonium salts [21–23], pyridinium salts [24–27], quaternary phosphonium salts [28–30] should be introduced on their structures. Among them, quaternary ammonium salts that covalently bonded to active sides of polymer chains have more antibacterial activity and deficient leaking of biocidal infections in the environment [31–33]. In the light of above information, synthesis of quaternary ammonium salt containing polypropylene via CuAAC click reaction was reported and its antibacterial activity evaluated against Staphylococcus
Corresponding author. E-mail address: [email protected] (M.A. Tasdelen).
https://doi.org/10.1016/j.porgcoat.2018.08.029 Received 1 January 2018; Received in revised form 4 August 2018; Accepted 28 August 2018 0300-9440/ © 2018 Published by Elsevier B.V.
Progress in Organic Coatings 125 (2018) 73–78
G. Acik et al.
methanol (HPLC grade, ≥99.9%, Sigma Aldrich) were used without distillation.
aureus and Escherichia coli. Before the CuAAC click reaction, clickable azide functional polypropylene was synthesized by using TMS-N3 / TBAF system, whereas clickable alkyne functional quaternary ammonium salt was prepared by quaternization reaction of benzyl bromide and 3-dimethylamino-1-propyne. The CuAAC click reaction of these clickable products in the presence of CuCl / PMDETA catalyst system led to obtain of polypropylene having antibacterial properties.
2.2. Instrumentation Fourier transform infrared (FT-IR) analyses were conducted by Perkin-Elmer brand instrument (Waltham, USA) FT-IR Spectrum Two Spectrometer equipped with a diamond ATR device to verify specific groups of intermediates and final products. 1H-NMR (Palo Alto, California, USA) measurements were recorded by Varian 400 MHz NMR spectrometer in chloroform-d with tetramethylsilane as internal standard. Differential scanning calorimeter (DSC) measurements were performed to determine glass transition temperatures using PerkinElmer brand device (Waltham, USA) with diamond equipment under nitrogen flow (20 mL/min.) with a heating rate of 10 °C/min.
2. Experimental part 2.1. Materials Chlorinated polypropylene (PP-Cl, Mn = 50.000 g/mol, chlorine mass fraction: 29–32% (m/m) was purchased from Mark Zhang Shanghai Sunking Industry Incorporation. Azidotrimethylsilane (TMSN3, 95%), tetrabutylammonium fluoride solution (TBAF, 1.0 M in THF), 3-dimethylamino-1-propyne (97%), benzyl bromide (98%), triethylamine (TEA, ≥99.9%), N,N,N',N”,N”-pentamethyldiethylenetriamine (PMDETA, 99%), and copper(I)chloride (CuCl, 99.99%) were purchased from Sigma Aldrich and used as received. Solvents such as tetrahydrofuran (anhydrous, ≥99.9%, inhibitor-free, Sigma Aldrich) and
2.3. General procedure for azidation reaction of chlorinated polypropylene (PP-N3) According to typical azidation procedure [34]: In a 25 mL one necked round bottomed flask, equipped with a magnetic stirrer bar, PP-
Fig. 1. Photograph of PP-Cl (a) and PP-QAS (b) samples. The films were prepared by solvent casting method from their solutions in THF.
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Scheme 1. Synthesis of PP-QAS from PP-N3 and QAS-Alkyne via CuAAC click chemistry.
concentrations were prepared in THF using PP-QAS and PP-Cl samples. Then the solutions were casted in petri dish to remove the residual solvent for two days at room temperature and polymeric films were scraped from petri dish for the antibacterial activity test. Fig. 1 shows a visual appearances of the PP-Cl and PP-QAS films.
Cl (5.0 g, 0.1 mmol) was dissolved in THF (25 mL). After the preparation of this solution, TBAF (2.5 mL, 7.5 mmol) and TMS-N3 (1 mL, 8.5 mmol) were added by drop-wise, respectively. (Caution: TMS-N3 is flammable chemical.) Subsequently, the formulation was purged with nitrogen gas for 10 min and it was left under vigorous stirring for 24 h heated up to 50 °C in oil bath. After the given time, the obtained PP-N3 solution was precipitated with excess methanol. Then, the precipitate was filtered and dried under vacuum for overnight.
2.7. Antibacterial test of PP-Cl and PP-QAS The bacterial culture of Staphylococcus aureus ATCC 25,923 (Grampositive) and Escherichia coli ATCC 25,922 (Gram-negative) were grown on nutrient agar slants and stored at 4 °C A loop full of bacteria from the agar slant was transferred into 15 ml tryptic soy broth (TSB) in a sterile centrifuge tube and incubated at 35 °C for 24 h. The bacterial cultures for the antibacterial activity tests were diluted to a concentration of 106 CFU/ml to obtain the test suspension. The polypropylene-based samples were cut into 1 x 1 cm coupons to be used in the antibacterial activity test. Then the coupons were transferred into 15 ml sterile tubes and 2 ml of the test suspension was added on each tube and incubated at 35 °C for 24 h in a shaking incubator. The test suspensions of S. aureus and E.coli without any coupons were used as the control sample. After 24 h of contact time, serial dilutions from each sample were spread inoculated on plate count agar (PCA) plates. The PCA plates were incubated at 35 °C for 24 h and colonies were counted. The experiments were replicated twice.
2.4. Quaternization of 3-dimethylamino-1-propyne (QAS-Alkyne) The 3-dimethylamino-1-propyne (2 mL, 18.5 mmol) and benzyl bromide (6 mL, 50 mmol) in 20 mL toluene were added in to a 50 mL one necked round bottomed flask, flamed previously and equipped with a magnetic stirrer bar. This mixture was heated up to 50 °C in an oil bath and left under vigorous stirring for 72 h. After the given time, the quaternized ammonium salt was filtered and dried under vacuum for 24 h, respectively. 2.5. Synthesis of antibacterial polypropylene via CuAAC click chemistry (PP-QAS) In a 50 mL flask with a magnetic stirrer, PP-N3 (3 g, 0.06 mmol), QAS-Alkyne (0.21 g, 1.8 mmol), PMDETA (50 μL, 0.25 mmol) and CuCl (3 mg, 0.03 mmol) were dissolved in the mixture of 15 mL THF/DMF (1:1). Then, the mixture was degassed by vacuum and flushed with nitrogen for 10 min. This mixture was heated up to 50 °C in an oil bath and left under vigorous stirring for 24 h. At the end of given time, the mixture was diluted with THF and passed from neutral alumina to remove metal salts. This eluent was concentrated by evaporation and then it precipitated in to methanol before dried under vacuum for 24 h at room temperature. Afterwards, solutions with 100 mg/ml concentrations were prepared in THF using PP-QAS and PP-Cl. Then the solutions were casted in petri dish to remove the residual solvent for two days at room temperature and polymeric films were scraped from petri dish for the antibacterial activity test.
2.8. Results and discussion Synthesis of quaternary ammonium salt containing polypropylene was achieved from commercial chlorinated polypropylene by taking advantages of CuAAC click reaction including efficiency under ambient conditions, speed and chemoselectivity. As illustrated Scheme 1, firstly, the chlorine groups of PP-Cl (29–32% (m/m)) were converted to azide functionalities in the presence of TBAF and TMS-N3 at 60 °C. In addition, the desired alkyne-functional quaternary ammonium salt was independently prepared by the reaction of 3-dimethylamino-1-propyne with benzyl bromide in the presence of triethylamine at 50 °C. In the final step, the CuAAC click reaction between clickable PP-N3 and QASAlkyne allowed to obtain the PP-QAS having antibacterial properties (Scheme 1). The chemical structures of desired PP-QAS and its precursors were firstly confirmed by FT-IR spectroscopy (Fig. 2). After the azidation, the
2.6. Preparation of polypropylene-based antibacterial film Solution-casting method was used to the preparation of PP-Cl or PP-QAS based films. For this purpose, solutions with 100 mg/ml 75
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Fig. 4. DSC thermograms of PP-Cl and PP-QAS.
triazole ring, aromatic and quaternary ammonium bands demonstrated the successful formation of desired PP-QAS. The 1H-NMR studies were also performed for further spectral confirmations of the PP-QAS and it precursors (Fig. 3). Mainly, there were three types of protons (namely methyl (eCH3), methylene (eCH2) and methine (eCH) protons) for the PPeCl [36]. The peaks appeared between 1.71 and 2.81 ppm were assigned to eCH2 and eCH protons (1–3 and 5–10). In addition, the protons located at 3.55 and 3.68 ppm were assigned to eCH2eCl and eCHeCl (4 and 11) [36]. After the azidation reaction, new peaks belonging to the protons of carbon atom next to eN3 group were obviously appeared at 2.24 and 3.22 ppm (a and b). The characteristic alkyne (c and d) and benzyl (f and g) protons of QAS-Alkyne were also visibly detected in Fig. 3. The disappearance of specific alkyne (c and d) protons at 3.08 ppm and the appearance of triazole (d) proton at 7.75 ppm were great evidence of formation clicked product PP-QAS [31,36–39]. Furthermore, the benzyl protons (f and g) of QAS-Alkyne and the aliphatic protons (1–11) of the PP-Cl were still observed in the Fig. 3.
Fig. 2. FT-IR spectra of PP-Cl, PP-N3, QAS-Alkyne and PP-QAS.
characteristic C-Cl band of PP-Cl at around 730 cm−1 was disappeared and a sharp peak corresponding to -N3 group at around 2095 cm−1 was obviously appeared. In addition, the characteristic bands of alkyne at 3315 and 2125 cm−1, aromatic band at 3105 cm−1 and quaternary ammonium groups at 1040 and 1175 cm−1 were clearly detected in the spectrum of QAS-Alkyne [35]. Upon the CuAAC click reaction, the disappearance of azide and alkyne bands and the presence of N]N of
Fig. 3. 1H-NMR spectra of PP-Cl, PP-N3, QAS-Alkyne and PP-QAS. 76
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Fig. 5. Antibacterial activity against (a) S. aureus, (b) E. coli after 24 h of incubation at 35 °C. (Initial; initial bacterial count, PP-QAS; quaternary ammonium salt containing polypropylene, PP-Cl; chlorinated polypropylene, Control; inocubated medium without PP-Cl).
Fig. 6. Fig. 5 Plate counts of S.aureus inoculated samples after 24 h of incubation at 35 °C; (a), PP-QAS, (b) PP-Cl, (c) Control.
Fig. 7. Plate counts of E.coli inoculated samples after 24 h of incubation at 35 °C; (a), PP-g-QAS, (b) PP-Cl, (c) Control.
The thermal properties of PP-QAS and initial polymers (PP-Cl and PP-N3) were investigated by differential scanning calorimeter (DSC) between room temperature and 150 °C (Fig. 4). Both PP-Cl and PP-N3 samples displayed a glass transition temperature (Tg) at 39 and 36 °C, whereas the Tg of obtained PP-QAS was detected at 47 °C [40]. This increase could be due to the polarity of quaternary ammonium salts or rigidity of benzyl group. The S. aureus and E.coli were used to investigate the potential antibacterial property of neat PP-Cl and PP-QAS discs by direct contact test under identical conditions (incubation for 24 h at 35 °C). The PPQAS coupons inhibited both the growth of S. aureus and E.coli significantly (p < 0.05) compared to the PP-Cl and control samples (Fig. 5a and b). More than 4 log reduction was observed on the S. aureus counts of PP-QAS samples. On the other hand, no reduction in S. aureus and E.coli counts of PP-Cl samples was observed compared to the control. The inhibitory effect of PP-QAS was more pronounced for S.aureus than for E.coli (Figs. 6 and 7).
and QAS-Alkyne) were obtained by classical azidation and quaternization reactions. The subsequent CuAAC click reaction of these compounds yielded to the desired PP-QAS. The spectral (FT-IR and 1HNMR) and thermal (DSC) analyses confirmed the successful synthesis of the PP-QAS. Bacterial studies revealed that the PP-QAS significantly inhibited both the growth of the S. aureus and E.coli bacteria. It is envisaged that the PP-QAS displaying high antibacterial activity will find various potential applications in medical, textile and packaging industries. Further studies in this line are now in progress. Conflicts of interest There are no conflicts to declare. Acknowlesdgments One of the authors (G.A.) would like to acknowledge and thank Turkish Scientific and Technical Research Council; 1002-Short Term R& D Funding Program (Project No: 216Z060) for financial support.
3. Conclusion
References
In conclusion, the quaternary ammonium salt containing polypropylene film was prepared from chlorinated polypropylene via CuAAC click reaction. The clickable azide and alkyne precursors (PP-N3
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Contents lists available at ScienceDirect
Progress in Organic Coatings journal homepage: www.elsevier.com/locate/porgcoat
Antibacterial film from chlorinated polypropylene via CuAAC click chemistry Gokhan Acika,b, Cagatay Altinkokc, Hulya Olmezd, Mehmet Atilla Tasdelena,
T
⁎
a
Department of Polymer Engineering, Faculty of Engineering, Yalova University, TR-77100 Yalova, Turkey Department of Chemistry, Faculty of Sciences and Letters, Piri Reis University, Tuzla, 34940, Istanbul, Turkey c Department of Chemistry, Faculty of Science, Trakya University, Merkez, Edirne, Turkey d TUBITAK Marmara Research Center, Material Institute, 41470 Gebze, Kocaeli, Turkey b
A R T I C LE I N FO
A B S T R A C T
Keywords: Antibacterial activity Chlorinated polypropylene Copper (I)-catalyzed azide- alkyne cycloaddition Polypropylene
Polypropylene possessing quaternary ammonium salt (PP-QAS) is synthesized by copper (I)-catalyzed azidealkyne cycloaddition “click” reaction (CuAAC) starting from chlorinated polypropylene (PP-Cl). The antibacterial properties of PP-QAS have been investigated on gram-positive (Staphylococcus aureus) and gram-negative (Escherichia coli) bacteria. For this purpose, clickable azide functionality has been introduced into PP-Cl backbone by using azidotrimethylsilane (TMS-N3) - tetrabutylammonium fluoride solution (TBAF) system in tetrahydrofuran (THF) (PP-N3). Independently clickable alkyne functionalized QAS is prepared by the reaction between 3-dimethylamino-1-propyne and benzyl bromide in the presence of triethyl amine (TEA). Finally, the polypropylene film containing quaternary ammonium salt (PP-QAS) is prepared by the successive CuAAC click reaction and solution casting method. The preparation of PP-QAS is monitored by fourier transform infrared (FTIR) spectroscopy, proton nuclear magnetic resonance (1H-NMR), differential scanning calorimetry (DSC) and scanning electron microscope (SEM) analyses at various stages. Based on antibacterial activity experiments, the PP-QAS coupons significantly inhibit both the growth of S. aureus and E. coli (p < 0.05) compared to neat PP-Cl and control samples.
1. Introduction The polyolefins are the most commonly used polymers in the world due to their low cost, good recyclability and mechanical properties. Over the past years, there have been many studies to improve their properties and expand their usages in industrial applications [1]. Due to their chemical structures not having reactive functional or polar groups, they could not easily modified with traditional organic reactions [2]. There are two distinct modification routes including direct polymerization which is not simple because of deficient sensitivity to Ziegler-Natta catalysts or post-polymerization in the literature [3,4]. Among the post-polymerization techniques, the chlorination of polyolefins is the most feasible way to improve their polarity, adhesion, wettability and chemical resistance by introduction of –Cl groups into backbone with chemical or physical treatments [5]. Thus, the chlorinated polyolefins including chlorinated polypropylene are one of the most preferred functional polyolefins and they can be applied for industrial applications in various forms. In addition to above-mentioned modification techniques, some new approaches using click chemistry
⁎
reactions have been recently reported for the modification of polyolefins [6–11]. During the recent years the click chemistry tools including CuAAC [12–16], Diels-Alder [17], thiol-ene [18,19] and thiolhalogen [20] click reactions have been extensively utilized for the modification of polyolefins. Although the polyolefins are one of the most widely used polymers in many areas, they can be also utilized for specific applications such as food packaging, medical device, clothing and textiles that required additional antibacterial properties. In an effort to obtain desirable antibacterial activity for the polymers against to microorganisms, three main compounds including quaternary ammonium salts [21–23], pyridinium salts [24–27], quaternary phosphonium salts [28–30] should be introduced on their structures. Among them, quaternary ammonium salts that covalently bonded to active sides of polymer chains have more antibacterial activity and deficient leaking of biocidal infections in the environment [31–33]. In the light of above information, synthesis of quaternary ammonium salt containing polypropylene via CuAAC click reaction was reported and its antibacterial activity evaluated against Staphylococcus
Corresponding author. E-mail address: [email protected] (M.A. Tasdelen).
https://doi.org/10.1016/j.porgcoat.2018.08.029 Received 1 January 2018; Received in revised form 4 August 2018; Accepted 28 August 2018 0300-9440/ © 2018 Published by Elsevier B.V.
Progress in Organic Coatings 125 (2018) 73–78
G. Acik et al.
methanol (HPLC grade, ≥99.9%, Sigma Aldrich) were used without distillation.
aureus and Escherichia coli. Before the CuAAC click reaction, clickable azide functional polypropylene was synthesized by using TMS-N3 / TBAF system, whereas clickable alkyne functional quaternary ammonium salt was prepared by quaternization reaction of benzyl bromide and 3-dimethylamino-1-propyne. The CuAAC click reaction of these clickable products in the presence of CuCl / PMDETA catalyst system led to obtain of polypropylene having antibacterial properties.
2.2. Instrumentation Fourier transform infrared (FT-IR) analyses were conducted by Perkin-Elmer brand instrument (Waltham, USA) FT-IR Spectrum Two Spectrometer equipped with a diamond ATR device to verify specific groups of intermediates and final products. 1H-NMR (Palo Alto, California, USA) measurements were recorded by Varian 400 MHz NMR spectrometer in chloroform-d with tetramethylsilane as internal standard. Differential scanning calorimeter (DSC) measurements were performed to determine glass transition temperatures using PerkinElmer brand device (Waltham, USA) with diamond equipment under nitrogen flow (20 mL/min.) with a heating rate of 10 °C/min.
2. Experimental part 2.1. Materials Chlorinated polypropylene (PP-Cl, Mn = 50.000 g/mol, chlorine mass fraction: 29–32% (m/m) was purchased from Mark Zhang Shanghai Sunking Industry Incorporation. Azidotrimethylsilane (TMSN3, 95%), tetrabutylammonium fluoride solution (TBAF, 1.0 M in THF), 3-dimethylamino-1-propyne (97%), benzyl bromide (98%), triethylamine (TEA, ≥99.9%), N,N,N',N”,N”-pentamethyldiethylenetriamine (PMDETA, 99%), and copper(I)chloride (CuCl, 99.99%) were purchased from Sigma Aldrich and used as received. Solvents such as tetrahydrofuran (anhydrous, ≥99.9%, inhibitor-free, Sigma Aldrich) and
2.3. General procedure for azidation reaction of chlorinated polypropylene (PP-N3) According to typical azidation procedure [34]: In a 25 mL one necked round bottomed flask, equipped with a magnetic stirrer bar, PP-
Fig. 1. Photograph of PP-Cl (a) and PP-QAS (b) samples. The films were prepared by solvent casting method from their solutions in THF.
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Scheme 1. Synthesis of PP-QAS from PP-N3 and QAS-Alkyne via CuAAC click chemistry.
concentrations were prepared in THF using PP-QAS and PP-Cl samples. Then the solutions were casted in petri dish to remove the residual solvent for two days at room temperature and polymeric films were scraped from petri dish for the antibacterial activity test. Fig. 1 shows a visual appearances of the PP-Cl and PP-QAS films.
Cl (5.0 g, 0.1 mmol) was dissolved in THF (25 mL). After the preparation of this solution, TBAF (2.5 mL, 7.5 mmol) and TMS-N3 (1 mL, 8.5 mmol) were added by drop-wise, respectively. (Caution: TMS-N3 is flammable chemical.) Subsequently, the formulation was purged with nitrogen gas for 10 min and it was left under vigorous stirring for 24 h heated up to 50 °C in oil bath. After the given time, the obtained PP-N3 solution was precipitated with excess methanol. Then, the precipitate was filtered and dried under vacuum for overnight.
2.7. Antibacterial test of PP-Cl and PP-QAS The bacterial culture of Staphylococcus aureus ATCC 25,923 (Grampositive) and Escherichia coli ATCC 25,922 (Gram-negative) were grown on nutrient agar slants and stored at 4 °C A loop full of bacteria from the agar slant was transferred into 15 ml tryptic soy broth (TSB) in a sterile centrifuge tube and incubated at 35 °C for 24 h. The bacterial cultures for the antibacterial activity tests were diluted to a concentration of 106 CFU/ml to obtain the test suspension. The polypropylene-based samples were cut into 1 x 1 cm coupons to be used in the antibacterial activity test. Then the coupons were transferred into 15 ml sterile tubes and 2 ml of the test suspension was added on each tube and incubated at 35 °C for 24 h in a shaking incubator. The test suspensions of S. aureus and E.coli without any coupons were used as the control sample. After 24 h of contact time, serial dilutions from each sample were spread inoculated on plate count agar (PCA) plates. The PCA plates were incubated at 35 °C for 24 h and colonies were counted. The experiments were replicated twice.
2.4. Quaternization of 3-dimethylamino-1-propyne (QAS-Alkyne) The 3-dimethylamino-1-propyne (2 mL, 18.5 mmol) and benzyl bromide (6 mL, 50 mmol) in 20 mL toluene were added in to a 50 mL one necked round bottomed flask, flamed previously and equipped with a magnetic stirrer bar. This mixture was heated up to 50 °C in an oil bath and left under vigorous stirring for 72 h. After the given time, the quaternized ammonium salt was filtered and dried under vacuum for 24 h, respectively. 2.5. Synthesis of antibacterial polypropylene via CuAAC click chemistry (PP-QAS) In a 50 mL flask with a magnetic stirrer, PP-N3 (3 g, 0.06 mmol), QAS-Alkyne (0.21 g, 1.8 mmol), PMDETA (50 μL, 0.25 mmol) and CuCl (3 mg, 0.03 mmol) were dissolved in the mixture of 15 mL THF/DMF (1:1). Then, the mixture was degassed by vacuum and flushed with nitrogen for 10 min. This mixture was heated up to 50 °C in an oil bath and left under vigorous stirring for 24 h. At the end of given time, the mixture was diluted with THF and passed from neutral alumina to remove metal salts. This eluent was concentrated by evaporation and then it precipitated in to methanol before dried under vacuum for 24 h at room temperature. Afterwards, solutions with 100 mg/ml concentrations were prepared in THF using PP-QAS and PP-Cl. Then the solutions were casted in petri dish to remove the residual solvent for two days at room temperature and polymeric films were scraped from petri dish for the antibacterial activity test.
2.8. Results and discussion Synthesis of quaternary ammonium salt containing polypropylene was achieved from commercial chlorinated polypropylene by taking advantages of CuAAC click reaction including efficiency under ambient conditions, speed and chemoselectivity. As illustrated Scheme 1, firstly, the chlorine groups of PP-Cl (29–32% (m/m)) were converted to azide functionalities in the presence of TBAF and TMS-N3 at 60 °C. In addition, the desired alkyne-functional quaternary ammonium salt was independently prepared by the reaction of 3-dimethylamino-1-propyne with benzyl bromide in the presence of triethylamine at 50 °C. In the final step, the CuAAC click reaction between clickable PP-N3 and QASAlkyne allowed to obtain the PP-QAS having antibacterial properties (Scheme 1). The chemical structures of desired PP-QAS and its precursors were firstly confirmed by FT-IR spectroscopy (Fig. 2). After the azidation, the
2.6. Preparation of polypropylene-based antibacterial film Solution-casting method was used to the preparation of PP-Cl or PP-QAS based films. For this purpose, solutions with 100 mg/ml 75
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Fig. 4. DSC thermograms of PP-Cl and PP-QAS.
triazole ring, aromatic and quaternary ammonium bands demonstrated the successful formation of desired PP-QAS. The 1H-NMR studies were also performed for further spectral confirmations of the PP-QAS and it precursors (Fig. 3). Mainly, there were three types of protons (namely methyl (eCH3), methylene (eCH2) and methine (eCH) protons) for the PPeCl [36]. The peaks appeared between 1.71 and 2.81 ppm were assigned to eCH2 and eCH protons (1–3 and 5–10). In addition, the protons located at 3.55 and 3.68 ppm were assigned to eCH2eCl and eCHeCl (4 and 11) [36]. After the azidation reaction, new peaks belonging to the protons of carbon atom next to eN3 group were obviously appeared at 2.24 and 3.22 ppm (a and b). The characteristic alkyne (c and d) and benzyl (f and g) protons of QAS-Alkyne were also visibly detected in Fig. 3. The disappearance of specific alkyne (c and d) protons at 3.08 ppm and the appearance of triazole (d) proton at 7.75 ppm were great evidence of formation clicked product PP-QAS [31,36–39]. Furthermore, the benzyl protons (f and g) of QAS-Alkyne and the aliphatic protons (1–11) of the PP-Cl were still observed in the Fig. 3.
Fig. 2. FT-IR spectra of PP-Cl, PP-N3, QAS-Alkyne and PP-QAS.
characteristic C-Cl band of PP-Cl at around 730 cm−1 was disappeared and a sharp peak corresponding to -N3 group at around 2095 cm−1 was obviously appeared. In addition, the characteristic bands of alkyne at 3315 and 2125 cm−1, aromatic band at 3105 cm−1 and quaternary ammonium groups at 1040 and 1175 cm−1 were clearly detected in the spectrum of QAS-Alkyne [35]. Upon the CuAAC click reaction, the disappearance of azide and alkyne bands and the presence of N]N of
Fig. 3. 1H-NMR spectra of PP-Cl, PP-N3, QAS-Alkyne and PP-QAS. 76
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Fig. 5. Antibacterial activity against (a) S. aureus, (b) E. coli after 24 h of incubation at 35 °C. (Initial; initial bacterial count, PP-QAS; quaternary ammonium salt containing polypropylene, PP-Cl; chlorinated polypropylene, Control; inocubated medium without PP-Cl).
Fig. 6. Fig. 5 Plate counts of S.aureus inoculated samples after 24 h of incubation at 35 °C; (a), PP-QAS, (b) PP-Cl, (c) Control.
Fig. 7. Plate counts of E.coli inoculated samples after 24 h of incubation at 35 °C; (a), PP-g-QAS, (b) PP-Cl, (c) Control.
The thermal properties of PP-QAS and initial polymers (PP-Cl and PP-N3) were investigated by differential scanning calorimeter (DSC) between room temperature and 150 °C (Fig. 4). Both PP-Cl and PP-N3 samples displayed a glass transition temperature (Tg) at 39 and 36 °C, whereas the Tg of obtained PP-QAS was detected at 47 °C [40]. This increase could be due to the polarity of quaternary ammonium salts or rigidity of benzyl group. The S. aureus and E.coli were used to investigate the potential antibacterial property of neat PP-Cl and PP-QAS discs by direct contact test under identical conditions (incubation for 24 h at 35 °C). The PPQAS coupons inhibited both the growth of S. aureus and E.coli significantly (p < 0.05) compared to the PP-Cl and control samples (Fig. 5a and b). More than 4 log reduction was observed on the S. aureus counts of PP-QAS samples. On the other hand, no reduction in S. aureus and E.coli counts of PP-Cl samples was observed compared to the control. The inhibitory effect of PP-QAS was more pronounced for S.aureus than for E.coli (Figs. 6 and 7).
and QAS-Alkyne) were obtained by classical azidation and quaternization reactions. The subsequent CuAAC click reaction of these compounds yielded to the desired PP-QAS. The spectral (FT-IR and 1HNMR) and thermal (DSC) analyses confirmed the successful synthesis of the PP-QAS. Bacterial studies revealed that the PP-QAS significantly inhibited both the growth of the S. aureus and E.coli bacteria. It is envisaged that the PP-QAS displaying high antibacterial activity will find various potential applications in medical, textile and packaging industries. Further studies in this line are now in progress. Conflicts of interest There are no conflicts to declare. Acknowlesdgments One of the authors (G.A.) would like to acknowledge and thank Turkish Scientific and Technical Research Council; 1002-Short Term R& D Funding Program (Project No: 216Z060) for financial support.
3. Conclusion
References
In conclusion, the quaternary ammonium salt containing polypropylene film was prepared from chlorinated polypropylene via CuAAC click reaction. The clickable azide and alkyne precursors (PP-N3
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