ROMP polymer-based antimicrobial films repeatedly chargeable with silver ions

Reactive & Functional Polymers 71 (2011) 195–203

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ROMP polymer-based antimicrobial films repeatedly chargeable with silver ions Shigenaga Takano, Hideyuki Tamegai, Toshihiro Itoh, Seitaro Ogata, Hiroki Fujimori, Shoujiro Ogawa, Takashi Iida, Yasuo Wakatsuki ⇑ Department of Chemistry, College of Humanities and Sciences, Nihon University, Sakurajosui 3-25-40, Setagaya, Tokyo 156-8550, Japan

a r t i c l e

i n f o

Article history: Received 19 October 2010 Received in revised form 1 December 2010 Accepted 1 December 2010 Available online 5 December 2010 Keywords: ROMP Ag–polymer complex Bactericidal film

a b s t r a c t Two norbornene derivatives bearing a pendant pyridyl group were prepared: the 3-(pyridin-2-yl)propyl ester of 5-norbornene-endo-2-carboxylic acid (1) and N-(pyridin-2-ylmethyl)-5-norbornene-endo-2,3dicarboxyimide (2). Both of these compounds produced high yields of ROMP polymers, poly(1) and poly(2), with the 2nd generation Grubbs catalyst. When Ag+ ions were added to these polymer solutions, the polymer–Ag+ composites, poly(1-Ag) and poly(2-Ag), were formed quantitatively. Poly(2) and poly(2Ag) produced films from their DMF solutions, and the latter film showed strong antimicrobial properties against Gram-positive Bacillus subtilis and Gram-negative Escherichia coli. Alternatively, when the film of poly(2) was immersed in a solution of Ag+, it was able to trap the ion to give a surface-modified film [Ag/ poly(2)]. The antimicrobial efficacy of [Ag/poly(2)] was the same as that of films made of poly(2-Ag), which indicated that the solid-state reaction of the film surfaces toward Ag+ ions in solution was quantitative. When the [Ag/poly(2)] film lost its biocidal effect after repeated use, the Ag+ ions could be reloaded by immersing the film in a silver ion solution, which fully restored original activity. Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction The use of silver as an antimicrobial agent has been known for centuries, and silver still plays an important role in a variety of medical and health care systems [1,2]. As synthetic polymer materials, e.g., plastics and textiles, are becoming ubiquitous in our modern life, the protection of polymer surfaces from microbes (fungi, bacteria, and viruses), which not only cause staining and odors but also could mediate infection, has become an important issue. The need for biocidal additives to polymers has been predicted to continue to increase, driven primarily by consumer awareness regarding hygienic surfaces. Most silver-containing antimicrobial polymer materials consist of dispersed nanoparticles of elemental silver. Typically, they are prepared by the in situ reduction of silver ions in a polymer solution and successive evaporation of the solvent to obtain a solid or film of the composite [3]. Sparingly soluble colloidal silver bromide/polymer composite has also been proposed to realize steady and improved bioactivity [4,5]. A problem with polymer–silver hybrid solids of this type is that only the particles exposed on the polymer–solid surfaces can be effective, while those within the solid bulk can have no effect unless the polymer matrix degrades. To localize silver nanoparticles only onto solid surfaces, their direct deposition on preformed polymer solids has been studied [6–8]. ⇑ Corresponding author. Tel./fax: +81 3 5317 9740. E-mail address: [email protected] (Y. Wakatsuki). 1381-5148/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.reactfunctpolym.2010.12.001

In some cases, porous polymer surfaces have been used to mechanically hold the silver particles [9–11]. The antimicrobial efficiency should depend on the size of the silver particle [3a,4], which is often not uniform and is governed by various factors, such as the reduction/aggregation rate of silver and the chemical nature of the polymer matrix. Another, albeit less common, strategy is to release Ag+ ions from polymer-bound complexes of otherwise very water-soluble silver ions. Examples of polymer side chains that can coordinate to Ag+ ion include sulfides [12], triethylenetetramine [13], and sulfadiazines [14]. All polymer matrices in this category described so far are prepared from monomers that are polymerizable via radical initiators because those monomers with a pendant strongly coordinating Lewis base are generally difficult to polymerize by transition metal-catalyzed coordination polymerization. In general, the antibacterial efficiency of Ag+ ion-containing polymers is lost more readily than that of polymers containing Ag metal nanoparticles due to the faster leaching out of Ag+. It may be possible to overcome this drawback if the polymer solid can repeatedly reload Ag+ ions. Recently, transition metal-catalyzed ring-opening metathesis polymerization (ROMP) has emerged as a new type of industrially applicable process to produce materials for a variety of products such as bathroom fixtures, ballistic panels, and large equipment body parts [15]. Therefore, it is highly desirable to develop methodologies that would make it possible to fix silver on ROMP polymer matrices in a controllable manner. We report here that ROMP

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ene-endo-2-carbonyl chloride, respectively, according to the conventional Diels–Alder reaction with cyclopentadiene [17]. 1 H and 13C NMR spectra were recorded on a JEOL JNM-ECA500 spectrometer at 500 and 125.77 MHz, respectively. The NMR signals for monomers and their Ag+ complexes were fully assigned using DEPT, COSY, HMQC, and HMBC techniques. For the NMRnumbering scheme of the atoms in the monomers, see Scheme 1. IR spectra were recorded on a Jasco FT/IR-4100 spectrometer. The molecular weights of the polymers were measured by TOSOH HLC-8220 GPC using an HZM-H column at 40 °C with THF eluent. Differential scanning calorimetry (DSC) curves were obtained with a SII NanoTechnology DSC-120 cell with an aluminum crucible under a dynamic nitrogen atmosphere. Elemental analyses were performed by Chemical Analysis Team, D & S Center, RIKEN.

is possible for NB (NB = norbornene or bicyclo[2,2,1]hept-2-ene) monomers with pendant 2-pyridyl groups. A related ROMP polymer of a NB derivative functionalized with N,N-di-2-pyridylamide was prepared using Schrock’s catalyst and the resulting polymer beads were used for the selective extraction of mercury and palladium ions [16]. The polymer with a 2-pyridyl group reported here could form a film, and its surface could fully trap Ag+ when the film was fully immersed in a solution of silver ions. The extent of this trapping was confirmed chemically and assessed biologically on a molecular basis. The antimicrobial efficiencies of the resulting films were evaluated by the use of both agar plate tests and LB media using Bacillus subtilis and Escherichia coli. Furthermore, complete leaching and full recharging cycles of Ag+ ion on the polymer film surfaces were demonstrated.

2.2. Preparation of norbornene derivative 1 2. Experimental To a 2-methylpyridine solution (35 ml) of 3-(pyridin-2-yl)propan-1-ol (2.12 g, 15.5 mmol), 5-norbornene-endo-2-carbonyl chloride (2.26 g, 14.4 mmol) was added dropwise at 0 °C with stirring. The mixture was stirred at room temperature for 24 h. 2-Methylpyridine was evaporated under reduced pressure. The residue was dissolved in a minimum amount of dichloromethane and chromatographed on a silica-gel column (3  20 cm). A yellow band was eluted with dichloromethane followed by a mixture of ethyl acetate and dichloromethane (1:2). The eluate was concentrated almost to dryness under reduced pressure and redissolved in a minimum amount of dichloromethane. The solution was subjected to column chromatography on alumina (3  20 cm, deactivated beforehand with 10 wt.% of H2O). A yellow band, eluted with

2.1. General procedures and materials All manipulations that involved air-sensitive compounds were performed under an atmosphere of argon or purified nitrogen using standard Schlenk or dry box techniques. Solvents were dried and deoxygenated by refluxing on sodium or CaH2 under argon and distilled before use. Grubbs catalyst (G2), 3-(pyridin-2-yl)propan1-ol, 3-(pyridin-4-yl)propan-1-ol, and 2-(aminomethyl)pyridine were purchased from Sigma–Aldrich Co. and used as received. Dicyclopentadiene, maleic anhydride, and 2-propenoyl chloride were also obtained from Sigma–Aldrich Co. and used to prepare 5-norbornene-endo-2.3-dicarboxylic anhydride and 5-norborn-

5

HO-(CH2)3

N

6

14

7

4 3

COCl

14

1

13

G2

n

10

8

2

15

CO

O

N

11 12

9

CO-(CH2)3

16

O

poly(1)

1 5

H2N-CH2

6

4

N

1

7 2

3

O O O

O

8

N

O

12

N H2C

15

N

4

1

N 10

8

O 13

no polymer

14

N

12 11

2'

G2 3

16

15

20

Ag+ -O

3S 21

20'

19

Cl

17

18 19'

CH3

N

N

Ag(PTS)

G2

Ru

Cy3P

Cl

Scheme 1. Synthesis of ROMP polymers with a pendant pyridyl group.

14 15

N

6 7 2

O 9

13

poly(2)

16

5

H2N-(CH2)2

O

14

10

N

O

9

13

2

n

G2

Ph

15

13

16

N

16

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dichloromethane followed by a mixture of ethyl acetate and dichloromethane (1:2), was collected. From the eluate solution, solvent was evaporated under high vacuum to leave 1 as a colorless oil (1.63 g, 44% yield). Elemental analysis calc. for C16H19NO2: C, 74.68; H, 7.44; N, 5.44. Found: C, 74.06; H, 7.41; N, 5.33%. IR (neat) vmax/cm1: 1731 (C@O). 1H NMR (DMF d-7) d: 1.31 (2H, m, 7-H), 1.86 (2H, m, 3-H), 2.01 (2H, m, 10-H), 2.83 (1H, t, J = 7.4 Hz, 4-H), 2.99–3.02 (3H, m, 1-H, 11-H), 3.14 (1H, s, 2-H), 4.03 (2H, m, 9-H), 5.92 (1H, dd, J1 = 5.2, J2 = 2.9 Hz, 6-H), 6.14 (1H, dd, J1 = 5.2, J2 = 2.9 Hz, 5-H), 7.19 (1H, t, J = 6.0 Hz, 15-H), 7.28 (1H, d, J = 7.4 Hz, 13-H), 7.70 (1H, t, J = 7.4 Hz, 14-H), 8.50 (1H, d, J = 4.0 Hz, 16-H). 13C NMR (DMF d-7) d: 28.5 (C-3), 28.8 (C-10), 34.3 (C-11), 42.5 (C-4), 43.0 (C-1), 45.7 (C-2), 49.4 (C-7), 63.5 (C9), 121.4 (C-15), 122.9 (C-13), 132.6 (C-6), 136.5 (C-5), 137.7 (C14), 149.4 (C-16), 161.2 (C-12), 174.0 (C-8). 2.3. Preparation of norbornene derivative 2 2-(Aminomethyl)pyridine (6.7 g, 62 mmol) was slowly added to a solution of 5-norbornene-endo-2.3-dicarboxylic anhydride (10.3 g, 62 mmol) in 1,2-dichloroethane (150 ml). A white precipitate formed immediately. The suspension was heated at 60 °C with stirring for two days, during which time the precipitate gradually disappeared. The solvent was evaporated and the remaining red oily residue was chromatographed on a silica-gel column (3  15 cm). A colorless fraction eluted with dichloromethane/ THF (1:1) was collected. Evaporation of the solvent gave 2 as colorless crystals (13 g, 82% yield) [18]. M.p. 121–122 °C; elemental analysis calc. for C15H14N2O2: C, 70.85; H, 5.55; N, 11.02. Found: C, 70.72; H, 5.59; N, 11.02. IR(KBr) vmax/cm1: 1699 (C@O); 1H NMR (DMF d-7) d: 1.61 (2H, t, J = 1.72 Hz, 7-H), 3.28 (2H, m, 1-H, 4-H), 3.49 (2H, dd, J1 = 2.87, J2 = 1.72 Hz, 2-H, 3-H), 4.56 (2H, s, 10-H), 6.09 (2H, t, J = 1.72 Hz, 5-H, 6-H), 7.21–7.26 (2H, m, 13-H, 15-H), 7.74 (1H, td, J1 = 7.7, J2 = 1.1 Hz, 14-H), 8.46 (1H, d, J = 4.0 Hz, 16-H); 13C NMR (DMF d-7) d: 43.1 (C-10), 44.9 (C-1, C-4), 46.0 (C-2, C-3), 52.1 (C-7), 121.3 (C-13), 122.5 (C-15), 134.8 (C-5, C-6), 136.8 (C-14), 149.2 (C-16), 155.8 (C-12), 177.4 (C-8, C-9). Using a similar reaction of 4-(2-aminoethyl)pyridine with 5-norbornene-endo-2.3-dicarboxylic anhydride, 20 was obtained as colorless crystals. M. p. 148–152 °C; elemental analysis calc. for C16H16N2O2: C, 71.62; H, 6.01; N, 10.44. Found: C, 71.52; H, 6.08; N, 11.68. IR(KBr) vmax/cm1: 1687 (C@O); 1H NMR (DMF d-7) d: 1.54 (2H, m, 7-H), 2.76 (2H, t, J = 7.16 Hz, 11-H), 3.20 (2H, m, 1-H, 4-H), 3.33 (2H, dd, J1 = 2.87, J2 = 1.72 Hz, 2-H, 3-H), 3.57 (2H, t, J = 7.4 Hz, 10-H), 5.89 (2H, t, J = 1.72 Hz, 5-H, 6-H), 7.23 (2H, d, J = 5.2 Hz, 13-H, 16-H), 8.47 (2H, d, J = 5.2 Hz, 14-H, 15-H); 13C NMR (DMF d-7) d: 32.6 (C-11), 37.9 (C-10), 44.7 (C-1,4), 45.6 (C-2,3), 51.8 (C-7), 124.4 (C-13,16), 134.5 (C-5,6), 147.4 (C-12), 149.9 (C-14,15), 177.4 (C-8,9). 2.4. Reaction of 1 with Ag(PTS) A DMF solution (4 ml) containing 1 (0.38 g, 1.47 mmol) and a solution of Ag(PTS) (silver p-toluenesulfonate, 0.49 g, 1.76 mmol) in DMF (10 ml) were mixed and stirred at room temperature. After 18 h, about half of the solvent was evaporated and the white precipitate of the resulting 1-Ag was separated by filtration (0.312 g, 40% yield). M.p. 138–146 °C (decomp.); elemental analysis calc. for C23H26AgNSO5: C, 51.50; H, 4.85; N, 2.61. Found: C, 50.95; H, 4.86; N, 2.89. IR(KBr) vmax/cm1: 1732 (C@O); 1H NMR (DMF d-7) d: 1.28 (2H, dd, J1 = 17.8, J2 = 7.4 Hz, 7-H), 1.85 (2H, m, 3-H), 2.06 (2H, m, 10-H), 2.28 (3H, s, 17-H), 2.84 (1H, s, 4-H), 2.98–3.01 (3H, m, 1-H, 11-H), 3.12 (1H, s, 2-H), 4.05 (2H, ddd, J1 = 23.1, J2 = 10.7, J3 = 6.4 Hz, 9-H), 5.90 (1H, dd, J1 = 5.2, J2 = 2.9 Hz, 6-H), 6.15 (1H, dd, J1 = 5.2, J2 = 2.9 Hz, 5-H), 7.12 (2H,

d, J = 8.0 Hz, 19-H, 19’-H), 7.39 (1H, t, J = 6.0 Hz, 15-H), 7.52 (1H, d, J = 8.0 Hz, 13-H), 7.65 (2H, d, J = 8.0 Hz, 20-H, 20’-H), 7.92 (1H, t, J = 7.2 Hz, 14-H), 8.66 (1H, d, J = 4.6 Hz, 16-H); 13C NMR (DMF d-7) d: 20.5 (C-17), 28.8 (C-3), 29.0 (C-10), 36.1 (C-11), 42.5 (C-4), 43.0 (C-1), 49.3 (C-7), 63.4 (C-9), 122.4 (C-15), 124.1 (C-13), 126.0 (C-20,20’), 128.2 (C-19,190 ), 132.3 (C-6), 137.4 (C-5), 138.2 (C-18), 138.4(C-14), 146.2 (C-21), 150.8 (C-16), 161.5 (C12), 174.0 (C-8). 2.5. Reaction of 2 with Ag(PTS) Monomer 2 (0.11 g, 0.43 mmol) in DMF (1 ml) and Ag(PTS) (0.12 g, 0.43 mmol) in DMF (2 ml) were mixed. After the mixture was stirred at room temperature overnight, the solvent was removed under high vacuum and the remaining heavy oil of 2-Ag was subjected to spectral characterization. IR(KBr) vmax/cm1: 1694 (C@O); 1H NMR (DMF d-7) d: 1.61 (2H, m, 7-H), 2.28 (3H, s, 17-H), 3.29 (2H, s, 1-H, 4-H), 3.50 (2H, dd, J1 = 2.87, J2 = 1.72 Hz, 2-H, 3-H), 4.68 (2H, s, 10-H), 6.07 (2H, t, J = 2.29 Hz, 5-H, 6-H), 7.12 (2H, d, J = 7.4 Hz, 19-H, 190 -H), 7.32 (1H, d, J = 7.4 Hz, 13-H), 7.37 (1H, t, J = 6.0 Hz, 15-H), 7.65 (2H, d, J = 8.0 Hz, 20-H, 20’-H), 7.87 (1H, t, J = 7.2 Hz, 14-H), 8.59 (1H, d, J = 4.6 Hz, 16-H); 13C NMR (DMF d-7) d: 20.5 (C-17), 43.9 (C-10), 44.9 (C-1,4), 46.0 (C2, 3), 52.1 (C-7), 122.3 (C-13), 123.2 (C-15), 126.0 (C-20, 200 ), 128.2 (C-19, 190 ), 134.8 (C-5, 6), 138.0 (C-14), 138.2 (C-18), 146.3, (C-21), 150.3 (C-16), 155.9 (C-12), 177.5 (C-8, 9). 2.6. Polymerization of 1 To a stirred solution of monomer 1 (0.92 g, 3.58 mmol)) in DMF (7 ml), complex G2 (3.5 lmol in 3 ml DMF) at room temperature was added. After 1 h, ethyl vinyl ether (1 ml) was added and the stirring was continued for 1 h. The reaction mixture was concentrated under reduced pressure to ca. 1/3 of the original volume and poured into a large amount of methanol to precipitate the polymer (poly(1)) in 64% yield. IR(THF) vmax/cm1: (C@O); 1H NMR (DMF d-7) d: 1.10–1.46 (1H, br-m), 1.58–2.17 (5H, br-m), 2.38–3.08 (5H, br-m, partly overlapped by DMF peak), 3.97–4.26 (2H, br-s, –CO2CH2–), 5.13–5.61 (2H, br-m, HAC'), 7.15 (1H, s, py-H), 7.23 (1H, s, py-H), 7.65 (1H, s, py-H), 8.48 (1H, s, py-H); 13 C NMR (DMF d-7) d: 28.5, 63.5, 121.3, 122.8, 136.4, 149.3, 161.2 (py-ipso), 174.1(–CO2–). Other polymerizations of 1 shown in Table 1 were performed similarly. 2.7. Polymerization of 2 and film preparation To a stirred solution of monomer 2 (0.50 g, 2.0 mmol) in 1,2dichloroethane (4 ml), a 1,2-dichloroethane solution (1 ml) containing complex G2 (0.66 lmol) was added, and the mixture was stirred at room temperature. After 5 h, ethyl vinyl ether was added and the mixture was stirred for 1 h. The reaction mixture was poured into a large volume of methanol to precipitate the polymer

Table 1 Typical ROMP of 1 catalyzed by the ruthenium complex G2.a Run

Reaction time (m)

Yield (%)

Mn (GPC)b

Mw/Mnb

1 2 3 4

2 30 60 300

28 40 64 77

31,500 54,200 56,300 40,700

2.41 2.11 2.72 2.37

a Polymerizations were performed in DMF (10 ml) at room temperature with initial [1]0/[G2] ratio of 1000. [1]0 = 1.0 g (3.6 mmol). b Mn(GPC) and Mw/Mn are the relative number-averaged molecular weight and the polydispersity index, respectively, determined by GPC and calibrated with polystyrene standards.

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(poly(2), 0.31 g, 62% yield). IR(film) vmax/cm1: 1688 (C@O); 1H NMR (DMF d-7) d: 1.21–1.93 (2H, br-m), 3.22–3.74 (4H, br-m), 4.54–5.00 (2H, br-m, -CH2-py), 5.34–5.83 (2H, br-m, HAC@), 7.11–7.44 (2H, br-m, py-H), 7.58–7.84 (1H, br-m, py-H), 8.33– 8.54 (1H, br-m, py-H); 13C NMR (DMF d-7) d: 39.8, 43.0, 45.0, 49.2, 121.5, 122.6, 129.5, 136.8, 149.2, 155.4 (py-ipso), 176.6 (–CO2–). Other polymerizations shown in Table 2 were performed similarly. For the preparation of films, poly(2) (0.08 g) was dissolved in 1,2-dichloroethane (2 ml) and the solution was casted in a petri dish (4-cm diameter) and allowed to stand overnight. The resulting film could be easily detached from the dish. 2.8. Reaction of poly(1) with Ag(PTS) A DMF solution of poly(1) (0.188 g) was mixed with a DMF solution (5 ml) of Ag(PTS) (0.20 g) at room temperature. After being stirred for 22 h, the mixture was concentrated to ca. half of the original volume and dichloromethane was slowly added until a precipitate of poly(1-Ag) was formed as a heavy oil (0.317 g, 80% yield). 1H NMR (DMF d-7) d: 1.20–1.27 (1H, br-s), 1.54–2.23 (5H, br-m), 2.28 (3H, s, CH3), 2.83–3.13 (5H, br-m, partly overlapped by DMF peak), 3.98–4.25 (2H, br-s, -CO2CH2-), 5.11–5.63 (2H, brm, HAC@), 7.09 (2H, d, J = 7.4 Hz, Ar–H), 7.29–7.61 (2H, br-m, py-H), 7.64 (2H, d, J = 7.4 Hz, Ar–H), 7.78–7.91 (1H, br-m, py-H), 8.59–8.92 (1H, br-m, py-H); 13C NMR (DMF d-7) d: 20.5 (CH3), 63.4, 126.0, 128.1, 138.1, 146.4, 174.0 (–CO2–).

(18 ml) containing Ag(PTS) (0.15 g), and the mixture was gently stirred at room temperature under Ar for two days. The film ([Ag/poly(2)]) was washed with methanol and dried. A poly(NB) film was subjected to the same procedure for comparison. 2.11. Antimicrobial test of the Ag–polymer composite films Pre-grown cells of B. subtilis or E. coli (approximately 5  107 CFU) were added to 20 ml of sterilized 1.5% agar containing 0.5% peptone, and the solution was poured into a dish. The film was placed on the solidified agar. The dish was then incubated at 37 °C for 16 h and examined visually to determine whether the bacterial growth made the agar cloudy. For the estimation of antimicrobial activity in a liquid medium, the film was placed in 10 ml sterilized LB medium in a 50-ml flask. Pre-grown cells of E. coli were seeded on the medium (approximately 5  105 CFU/ml), and the flask was incubated with shaking at 37 °C. After 16 h of incubation, the turbidity of the medium was checked. If the medium was still clear, it was carefully decanted without removing the film from inside the flask. Subsequently, 10 ml of freshly sterilized LB medium was added to the flask and pre-grown cells of E. coli were seeded on the medium (approximately 5  105 CFU/ml). The procedure was repeated until the LB medium became turbid after incubation. 3. Results and discussion

2.9. Reaction of poly(2) with Ag(PTS) and preparation of a composite film To a DMF solution (4 ml) of poly(2) (0.42 g), Ag(PTS) (0.46 g) in DMF (8 ml) was added. A white precipitate of poly(2-Ag) formed immediately and was isolated by filtration and dried in vacuo, yielding 0.72 g (82%) of the solid composite. IR(film) vmax/cm1: 1707 (C@O); 1H NMR (DMF d-7) d: 0.99–1.78 (2H, br-m), 2.26 (3H, s, CH3), 4.56–5.21 (2H, br-s, -CH2-py), 5.38–5.91 (2H, br-s, HAC@), 7.09 (2H, d, J = 8.0 Hz, py-H), 7.29–7.55 (2H, br-s, py-H), 7.63 (2H, d, J = 7.4 Hz, py-H), 7.79–7.96 (1H, br-s, py-H), 8.50– 8.97 (1H, br-s, py-H); 13C NMR (DMF d-7) d: 20.6 (CH3), 126.0, 128.3, 138.3, 146.1, 155.8. 177.0 (–CO2–). For film preparation, poly(2-Ag) (0.05 g) was dissolved in DMF (4 ml) at 90–100 °C. The solution was casted into a petri dish (4cm diameter) and heated at 90 °C until the solvent was completely evaporated, leaving a yellow film of the polymer containing Ag(PTS). 2.10. Preparation of poly(2) film holding Ag(PTS) on its surface Four pieces (10  10 mm each) of poly(2) film prepared as described in Section 2.7 were immersed in a methanol solution

Table 2 ROMP of 2 catalyzed by the ruthenium complex G2.a Run

[2]0/[G2]b

Reaction time (h)

Yield (%)c

5 6 7 8 9

500 1000 3000 3000 3000

5 5 1 5 20

92 97 5 62 60

a Polymerization was performed in 1,2-dichloroethane (5 ml) at room temperature. b [2]0 = 0.5 g (2 mmol). c The resulting polymers were insoluble in THF and toluene: molecular weights were not measured.

3.1. Monomers bearing a pendant pyridyl group In this study, pyridine derivatives were chosen as Lewis bases for the trapping of Ag+ ions to the polymer chain. The isolated complex of Ag+ with pyridine (Py) is known to have the composition AgPyþ 2 , but an equilibrium study showed the presence of a small amount of AgPy+ species in solution [19]. While isolation of the 1:1 complex species appears to be quite difficult in the case of pyridine molecule, we assumed that such species might be stable with pyridine derivatives that have a sterically demanding substituent. N-donor ligands, including pyridine, have been reported to inhibit the activity of Grubbs’ catalyst for ROMP reactions. The addition of 1–5 equivalents (relative to Ru catalyst) of free pyridine decreases the rate of ROMP of cyclooctene by a factor of >100 [20]. Indeed, when we examined ROMP of NB using Grubbs’ catalyst (G2 in Scheme 1) in the presence of pyridine in an amount equiv to NB ([NB]/[G2] = 1000; 70 °C, in 1,2-dichloroethane, 24 h), no polymer was obtained. In contrast, when 2-methylpyridine was used in place of pyridine under the same reaction conditions, poly(NB) was formed within 1 min in a quantitative yield. Apparently, the a-methyl group sterically prevents the neighboring nitrogen from interacting with the crowded ruthenium center. This result, together with the fact that free 2-methylpyridine easily coordinates to naked Ag+ ion to form a 2:1 complex, prompted us to prepare monomers 1 and 2 according to the reactions shown in Scheme 1. Monomer 1 was a pale-yellow oil obtained in 44% yield while crystalline 2 was isolated in 82% yield. For comparison, an analog of 2 in which the pyridine ring was connected through its 4-position (20 ) was also prepared by the same method. When monomer 1 and an equivalent molar amount of Ag(PTS) (PTS = p-toluene sulfonate) were mixed in DMF, a white precipitate (1-Ag) formed immediately. Similarly, 2 in DMF solution reacted with an equivalent amount of Ag(PTS) to give a heavy oil (2-Ag). Fig. 1 compares the alkenic and aromatic regions of the 1H NMR spectra (in DMF d-7) of monomers 1 and 2 with those of their respective silver adducts, 1-Ag and 2-Ag. The shifts of the peaks and their integrations for the pyridyl and PTS protons clearly

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13-H 16-H

14-H 5-H 15-H

6-H

1

(a)

DMF

PTS PTS

1-Ag

(b)

5-H + 6-H DMF

2

15-H 16-H

14-H

(c)

13-H

PTS

PTS 2-Ag

(d)

Fig. 1. 1H NMR spectra (DMF-d7) of monomers and their Ag(PTS) complexes: (a) monomer 1, (b) 1:1 complex of 1 with Ag(PTS), (c) monomer 2, (d) 1:1 complex of 2 with Ag(PTS). For the numbering scheme of atoms, see Scheme 1.

indicated that pure 1:1 complex was formed in both cases. Elemental analysis of 1-Ag was also consistent with the isolation of a 1:1 complex. 3.2. Polymerization In accordance with the above observation that the polymerization of NB is not retarded in the presence of a large excess of 2-

methylpyridine, the ROMP of monomers 1 and 2 was successfully performed by using complex G2 as a catalyst precursor. In contrast, the 4-pyridyl analog 20 did not polymerize at all under similar conditions (Scheme 1). The 4-pyridyl isomer of 1, which was not fully characterized because it is a heavy oil, also showed no activity toward G2. DMF was chosen as the solvent for the polymerization of 1 because of the rather high solubility of the polymers in this solvent, while 1,2-dichloroethane could be used to polymerize 2. In

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typical ROMPs. In some ROMPs involving sterically demanding monomers, rather broad PDIs were reported and were attributed to intermolecular secondary metathesis (chain-transfer) and/or intramolecular cyclo-depolymerization (back-biting) [21–25]. A film was not obtained from a solution of poly(1), and a thin gummy solid was obtained upon evaporation of the solvent

the polymerization of 1, the amount of solvent was an important factor: use of a smaller amount of DMF solvent resulted in the formation of a gelled polymer ball in a short period of time, which was very difficult to dissolve. The results are summarized in Tables 1 and 2. The polydispersity indices (PDI = Mw/Mn) for the polymers shown in Table 1 were broader than those generally expected for

DMF

poly(1)

16-H

13-H

14-H

15-H

(a)

PTS

vinyl-H (cis+tr)

PTS

(b)

poly(1-Ag)

DMF poly(2) vinyl-H 13-H

16-H 14-H

15-H

(c)

tr cis

PTS PTS

poly(2-Ag)

(d)

Fig. 2. 1H NMR spectra (DMF-d7) of polymers and their Ag(PTS) complexes: (a) poly(1), (b) complex of poly(1) with Ag(PTS), (c) poly(2), (d) complex of poly(2) with Ag(PTS). For the numbering scheme of atoms, see Scheme 1.

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(DMF). However, a thin film of poly(2) was obtained by casting its 1,2-dichloroethane solution. The poly(2) sample obtained in run 5 in Table 2 was used for the subsequent reactions with the Ag+ ion and the formation of composites.

(c)

3.3. Polymer/silver complexes

3.4. Antimicrobial activity of the polymer–Ag film composite The antibacterial properties of the composite films were tested against Gram-positive Bacillus subtilis and Gram-negative Escherichia coli. Fig. 4 shows bacteria-inoculated agar plate tests of the poly(2) film alone, poly(2-Ag) film, and poly(2) film treated with Ag(PTS) solution, i.e., [Ag/poly(2)]. The antibacterial activity against both bacteria was evident in the case of poly(2-Ag) and [Ag/poly(2)] films due to the presence of a clear zone around each film placed on the agar plates, which demonstrated that the bacteria could not grow near the film. Since the film of poly(2) did not show a similar zone of inhibition, the clear zone was apparently the result of leaching of Ag+ ions from poly(2-Ag) and [Ag/poly(2)] films into the agar media. The PTS anion has been shown to have no bioactivity [4]. The antimicrobial activity in solution was examined using LB media (10 ml) containing a piece of the film and E. Coli (0.1%, approximately 5  105 CFU/ml) at an incubation temperature of 37 °C. The activity was assessed after 16 h of incubation by visually examining whether the LB media, which was initially clear, became cloudy due to growth of the bacterium. Under these condi-

(b) Heatflow

To a polymer solution of DMF, Ag(PTS) in a molar amount equivalent to that of the pyridyl group present in the solution was added. In the case of poly(1), the adduct (poly(1-Ag)) was forced to precipitate by the addition of dichloromethane to the reaction mixture, while the adduct poly(2-Ag) immediately precipitated when DMF solutions of Ag(PTS) and poly(2) were mixed. In Fig. 2, proton NMR spectra of poly(1) and poly(2) are compared to those of their Ag(PTS) complexes, poly(1-Ag) and poly(2-Ag), respectively. As in the case of the monomer complexes of Ag(PTS), the pyridyl protons shifted to a lower field upon coordination of the pyridyl nitrogen in the polymer to Ag+. Clearly, the entire polymer-tethered pyridyl group served to trap Ag+ ions. The poly(1-Ag) complex gave readily breakable brittle films upon the evaporation of DMF solvent, while the evaporation of DMF at 90 °C from the poly(2-Ag) composite solution casted into a petri dish gave palebrown flexible films with a thickness of ca. 30 lm, which could easily be detached from the glass surface. Though the poly(2-Ag) composite provides a rare example of a silver–polymer complex with film-forming ability, only the silver ions exposed on the film surface can contribute to the expected antimicrobial activity, and those within the film should remain intact. To use the silver ions more efficiently, we examined the preparation of another composite that only contains silver ions on its surface. Thus, a piece of film of poly(2) was immersed in a methanol solution containing excess Ag(PTS) for two days, washed briefly with methanol and dried. The film sample prepared in this way, which we denote as [Ag/poly(2)], is expected to hold Ag+ ions only on its surface. As a control, a film made of ROMP polymer of the parent NB (poly(NB)) was treated in exactly the same way. The thermal properties of the composite films were studied by differential scanning calorimetry (DSC). The DSC curves of poly(2), poly(2-Ag), and [Ag/poly(2)] gave Tg values of 482 K, 450 K, and 478 K, respectively. As the Ag(PTS) content increases, the Tg value decreases gradually. For films containing Ag+ ions, an exothermic event was observed in the temperature range of 480–570 K (Figs. 3b and c) and was attributed to curing of the polymer, in which the Ag+ ion could act as a cure-promoting agent.

(a)

0.5 mW

400

450

500

550

Temperature / K Fig. 3. DSC curves of the films obtained under a nitrogen flow (60 ml/min) at a heating rate of 10 K/min: (a) 1.9-mg poly(2) film, (b) 1.9-mg [Ag/Poly(2)] film, and (c) 1.5-mg poly(2-Ag) film.

tions, the minimum area of the film, i.e., minimum area of inhibition (MAI) necessary to keep the solution clear after 16 h, was found to be 3–4 mm2/ml for both the poly(2-Ag) and [Ag/ poly(2)] films. As expected from the agar experiment above, the MAI for poly(2) alone was larger than 300 mm2/ml, and, thus, poly(2) alone was virtually ineffective against E. Coli in solution. The observation that films of poly(2-Ag) and [Ag/poly(2)] have the same MAI value is significant. All of the 2-pyridyl units in poly(2) serve to bind Ag+ ions in solution, as observed by 1H NMR (Fig. 2). However, when in the form of a film, its MAI should be governed by the number of (2-pyridyl-Ag+) units present at the surface of the film. Therefore, the 2-pyridyl units present on the surface of the [Ag/poly(2)] film must also be completely filled with Ag+ ions because its biocidal efficacy was similar to that of the poly(2-Ag) film. This result suggests that when a solid film of poly(2) is immersed into a solution containing Ag+ ions, all of the 2-pyridyl groups at the solid film-surface capture Ag+ ions from the solution (Fig. 5). Physical absorption of the ion must be negligible, since the same treatment of a poly(NB) film as a control with the Ag+ ion solution gave a sample with MAI of up to 150 mm2/ml. To examine the durability of the film’s bioactivity, a piece of [Ag/poly(2)] film was placed in aqueous LB broth (10 ml, containing 0.1% E. Coli, 5  105 CFU/ml) similar to that used for the determination of MAI (vide supra). After the first 16 h of incubation at 37 °C, the LB medium, which was clear due to killing the bacterium, was replaced by careful decantation with fresh LB media which was inoculated with new bacterium. This procedure was repeated until growth of the bacterium was finally noted by turbidity. With a 100-mm2 piece of [Ag/poly(2)] film, three cycles of decantation/incubation proceeded with clear broth, but growth of the bacterium was observed during the fourth incubation. 3.5. Rechargeability of the polymer film with silver ions In most cases, silver-based antimicrobial activity is due to the leaching of Ag+ ions from the polymer layer, regardless of whether the silver is originally contained in the polymer as Ag+ ions or in the form of Ag metal nanoparticles. The depletion of Ag+ ions from the polymer surface eventually leads to a loss of antibacterial

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B. subtilis (a)

(b)

(c)

(e)

(f)

E. coli (d)

Fig. 4. Films placed on an LB agar plate inoculated with B. subtilis (upper three windows) and E. Coli (lower three windows); (a) and (d): poly(2) film alone showing no inhibition, (b) and (e): poly(2-Ag) film showing a zone of inhibition, and (c) and (f): [Ag/Poly(2)] showing a zone of inhibition similar to (b) and (e).

Ag+ Ag+N N + N +Ag N Ag

Ag+ N

N

Ag+

Ag+ Ag+N N Ag+ N

N N

poly(2-Ag) film

N

N

[Ag/poly(2)] film

-Ag+

1) +Ag+ 2) film formation

+Ag+

N

N

N

poly(2)

Ag+ N

N N

N

N

solution poly(2) film Fig. 5. Schematic drawing of a poly(2) film and its composite with Ag+. PTS- anion is omitted for clarity.

activity. Therefore, a simple strategy that can be used to renew the bactericidal activity on the surface is highly desirable. Since the bioactivity of [Ag/poly(2)] is due to Lewis base coordination/decoordination on the polymer surface with Ag+, it should be easy to reintroduce new Ag+ ions to the freed Lewis base on the surface. Indeed, the film of [Ag/poly(2)], which was used for the durability test in the LB broth solution and deactivated by the loss of Ag+ ions (see Section 3.4), could be reactivated by immersing the used film in a solution of Ag(PTS) by the same procedure as that used for its original preparation (see Experimental section). The renewed film had the same biocidal activity as before, which was confirmed by a bioactive-durability experiment. The deactivation/activation cycle of the film was tested four consecutive times, and the results confirmed that the bioactivity was perfectly renewed each time.

poly(2) formed a flexible film on casting its solution. Antibacterial ROMP polymer films bearing chemically complexed Ag+ ions on their surfaces were prepared simply by immersing this polymer film in an Ag(PTS) solution to give [Ag/poly(2)]. In this reaction, all of the 2-pyridyl units exposed at the poly(2) film surface were used to trap Ag+ ions through the solid–liquid interface. To demonstrate this phenomenon, the reaction of poly(2) with Ag(PTS) in solution was performed to give poly(2-Ag), followed by film formation. The results confirmed that [Ag/poly(2)] and poly(2-Ag) films had the same antimicrobial efficacy. The antibacterial film prepared in this study can be used many times. When it loses activity due to the leaching of Ag+ from the surface, it can be simply immersed in Ag+ solution, which restores the original antimicrobial potency. The simple method to prepare antibacterial ROMP films reported here might have potential applications in the food storage and personal hygiene industries.

4. Conclusions

Acknowledgements

Norbornene derivatives with pendant 2-pyridyl groups smoothly polymerized with a typical ROMP catalyst. Among them,

The authors are grateful for the financial support from the Strategic Research Base Development Program for Private Universities

S. Takano et al. / Reactive & Functional Polymers 71 (2011) 195–203

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