Synthesis and antibacterial activities of novel polymerizable Gemini quaternary ammonium monomers

Abstract

In order to get durable antibacterial polymer materials, two series of novel polymerizable Gemini quaternary ammonium salts (QAS) were synthesized via a facile approach. The first series of monomers which have a completely symmetrical structure contain two unsaturated double bonds that allow it to polymerize as cross-linker. The second series of monomers are asymmetric Gemini QAS and contain only one polymerizable double bond. All the QAS monomers were characterized by ¹H NMR and IR spectra. The six kinds of QAS monomers were bonded to dental restoration resin system. To evaluate the bactericidal activity of these compounds against Escherichia coli and Staphylococcus aureus (S. aureus), the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration were tested by using broth dilution test. Among the six monomers, four compounds were found to have MIC values (for S. aureus) less than 10 μg/mL. The asymmetric Gemini QAS which have a long alkyl chain length of 12 carbons on the nitrogen showed stronger antibacterial activity than other monomers. The inhibitory effect of polymeric resins incorporated with QAS against the growth of S. aureus was determined by counting the number of viable cells. The result showed that the antibacterial activities of cured resins were increased as the concentration of QAS monomers increased.

Keywords:

• Gemini quaternary ammonium salts
• polymerizable
• antibacterial activity

1. Introduction

With increasing concerns of micro-organism contaminations and infections, there is a growing interest in the development of new antimicrobial compounds.[1–8] Quaternary ammonium salts (QAS) are well known and effective antibiotics due to their broad spectrum efficacy, low toxicity, non-irritation, and chemically stable properties.[9–14] However, there are some irreconcilable disadvantages of conventional quaternary ammonium compounds, for example, more additive amount, not sensitive to Gram-negative bacteria and so on.[15–18] Gemini QAS, which have been studied by many chemists worldwide, can avoid the disadvantage of conventional QAS compounds. These Gemini biocides possess at least two hydrophobic chains and two cationic groups, and exhibit more excellent antibacterial activities than comparable conventional ones.[19–23]

Several reports have described experiments in which an antibacterial agent was incorporated into dental filling materials, in order to inhibit bacterial attachment and plaque accumulation on their surfaces. However, the antibacterial activity of these materials is dependent upon release of the agent, which is associated with some disadvantages including influence on mechanical properties, toxic effects, and disruption of microbial homeostasis.[24–26] Meanwhile, QAS polymeric materials that can kill bacteria without releasing any low-molecular-weight biocides have been developed.

Introduction of biocide monomers during the process of polymerization is one of the promising approaches in the development of non-leaching biocide polymers.[27–31] In this perspective, polymerizable Gemini monomers, with quaternary ammonium groups as polar heads and methyl acrylic functions as the polymerizable units, were synthesized to build antibacterial materials.[32] Antimicrobial activity of these materials likely results from the positively charged ammonium group (N⁺) interaction with negatively charged bacterial cell membrane.[6,10,13–17] Long alkyl-chained QAS can utilize additional antibacterial activity by inserting into the bacterial membrane.[18–23] According to the action mechanism of antibacterial materials containing QAS, it can be proposed that the antibacterial activity of polymeric QAS materials can be increased by increasing the degree of freedom of N⁺ groups. For building dental resin with improved antibacterial activity, novel polymerizable Gemini monomers, with a long spacer between the polymerizable units and N⁺ groups, were specifically designed. In this article, we describe the synthesis of six polymerizable Gemini quaternary ammonium monomers: N,N,N′,N′-tetramethyl-dimethylene-bis(1-undecanoate-2-(2-methacryloyl-oxy) ethyl) bromide (MEBU-TMEDA-MEBU), N,N,N′,N′-tetramethyl-hexamthy-lene-bis(1-undecanoate-2-(2-methacryloyloxy) ethyl) bromide (MEBU-TMHDA-MEBU), N,N,N′,N′-tetramethyl-dimethylene-dodecyl-1-undecanoate-2-(2-methacry-loyloxy)ethyl) bromide (MEBU-TMEDA-C₁₂), N,N,N′,N′-tetramethyl-dimethylene-tetradecyl-1-undecanoate-2-(2-methacry-loyloxy)ethyl) bromide (MEBU-TMEDA-C₁₄), N,N,N′,N′-tetramethyl-hexamthylene-dodecyl-1-undecanoate-2-(2-methacryloyl-oxy)ethyl) bromide (MEBU-TMHDA-C₁₂), and N,N,N′,N′-tetramethyl-hexamthylene-tetradecyl-1-undecanoate-2-(2-methacryloyloxy) ethyl) bromide (MEBU-TMHDA-C₁₄). These monomers were investigated on the bactericidal efficacy against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus).[33] The six monomers were bonded to dental resin system by light curing method which is a common method of clinical dental restoration. The inhibitory effect of cured resin incorporated with QAS against the growth of S. aureus on its surface was determined by counting the number of viable cells. The effect of monomer structure on the minimal inhibitory concentration (MIC), minimum bactericidal concentration (MBC) values of monomers, and the inhibitory effect of antibacterial resin was also carefully discussed.

2. Experimental

2.1. Materials and bacterial strains

11-bromoundecanoic acid, 2-hydroxyethyl methacrylate, N,N,N′,N′-tetramethyle-thylenediamine, TMHDA, 1-bromododecane, 1-bromotetradecane, p-toluenesulfonic acid (PTSA), and hydroquinone were obtained from Aladdin Chemical Reagent CO. Ltd. Bisphenol glycidyl methacrylate (Bis-GMA) was obtained from Sigma-aldrich Reagent CO. Ltd. Triethylene glycol dimethacrylate (TEGDMA), camphorquinone (CQ), and N, N-dimethylaminoethyl methacrylate (DMAEMA) were obtained from Acros Organics Reagent CO. Ltd. Toluene, isopropanol, acetone, acetonitrile, and anhydrous diethyl ether were obtained from Shanghai Lingfeng Chemical Reagent CO. Ltd. All reagents were used without any purification. Yeast extract, peptone, agar were got from Sangon Biotech CO. Ltd and used as received. Bacterial strains used for antibacterial activity tests included S. aureus RN4220 and E. coli TOP10 strain.

2.2. Characterization

¹H NMR spectra were recorded on a Varian Gemini 500 MHz spectrometer using tetramethylsilane as an internal standard in CDCl₃ and DMSO-d₆. Fourier-transform infrared (FT-IR) spectra were obtained with a Mattson 5000 FT-IR spectrometer using NaCl windows.

2.3. Synthesis of symmetrical Gemini quaternary ammonium monomers

2.3.1. Intermediates MEBU

Equimolar amounts of 2-hydroxyethyl methacrylate (6.5 g, 0.05 mol) and 11-bromoncanoic acid (13.25 g, 0.05 mol), 0.005 g hydroquinone, 0.077 g PTSA, and 40 mL toluene were added to a 100 mL round-bottom flask. The mixture was stirred for 24 h at 110 °C using magnetic stirrer. After evaporation of the solvent with a rotary evaporator under vacuum, the crude product was purified with column chromatography using ethyl acetate and petroleum ether as eluent. The pure product was isolated as clear, colorless oil.

¹H NMR (CDCl₃): δ = 1.29 (t, 10H, CH₂), 1.43 (m, 2H, O=C–C–C–CH₂), 1.63 (m, 2H, O=C–C–CH₂), 1.86 (m, 2H, CH₂–CBr), 1.96 (s, 3H, C=C–CH₃), 2.32 (t, 2H, O=C–CH₂), 3.43 (t, 2H, CH₂Br), 4.35 (m, 4H, ester–CH₂–CH₂–ester), 5.60–6.14 (s, 2H, C=CH₂) ppm.

FT-IR: σ = 2921 cm⁻¹(ν C–H), 1718 cm⁻¹(ν C=O), 1632 cm⁻¹(ν C=C), 1161 cm⁻¹(ν (O=)C–O), 1328 and 940 cm⁻¹(ν (C=)C–H).

2.3.2. Monomer MEBU-TMEDA-MEBU and MEBU-TMHDA-MEBU

The monomers MEBU-TMEDA-MEBU and MEBU-TMHDA-MEBU were made using the same procedure. About 0.01 mol TMEDA (or TMHDA), 0.02 mol MEBU, 0.001 g hydroquinone, and 16 mL isopropanol were added to a 50 mL round-bottom flask, and the mixture was stirred for 25 h at 95 °C using magnetic stirrer. After evaporation of the solvent, the crude product was dissolved by acetone. The supernatant was collected after centrifugation. After acetone was removed on a rotary evaporator, the crude product was purified with diethyletheranhydrous as eluent. All the final products were obtained as yellow viscous liquids.

MEBU-TMEDA-MEBU

¹H NMR (DMSO-d6): δ = 1.24 (m, 24H, CH₂), 1.5 (s, 4H, O=C–C–CH₂), 1.72 (s, 4H, CH₂–C–N), 1.87 (s, 6H, C=C–CH₃), 2.39 (t, 4H,O=C–CH₂), 3.15 (s, 12H, CH₃–N–CH₃), 3.36–3.37 (t, 8H, CH₂–N–CH₂), 4.30 (m, 8H, O=C–CH₂–CH₂–C=O), 5.71–6.02 (s, 4H, C=CH₂) ppm.

FT-IR: σ = 3388 cm⁻¹(ν C–N), 2921 cm⁻¹(ν C–H), 1718 cm⁻¹(ν C=O), 1632 cm⁻¹(ν C=C), 1161 cm⁻¹(ν (O=)C–O), 1328 and 940 cm⁻¹(ν (C=)C–H).

MEBU-TMHDA-MEBU

¹H NMR (DMSO-d6): δ = 1.24 (m, 28H, CH₂), 1.5 (s, 4H, O=C–C–CH₂), 1.72 (s, 8H, H₂C–C–N), 1.87 (s, 6H, C=C–CH₃), 2.39 (t, 4H, O=C–CH₂), 3.15 (s, 12H, CH₃–N–CH₃), 3.36–3.37 (t, 8H, CH₂–N–CH₂), 4.30 (m, 8H, O=C–CH₂–CH₂–C=O), 5.71–6.02 (s, 4H, C=CH₂) ppm.

FT-IR: σ = 3388 cm⁻¹(ν C–N), 2921 cm⁻¹(ν C–H), 1718 cm⁻¹(ν C=O), 1632 cm⁻¹(ν C=C), 1161 cm⁻¹(ν (O=)C–O), 1328 and 940 cm⁻¹(ν (C=)C–H).

2.4. Synthesis of asymmetric Gemini quaternary ammonium monomers

2.4.1. Intermediates MEBU-TMEDA and MEBU-TMHDA

The intermediates MEBU-TMEDA and MEBU-TMHDA were made with the same process. In 50 mL round bottom flask, 0.02 mol TMEDA (or TMHDA) was reacted with 0.02 mol MEBU. After 0.001 g hydroquinone was added, the reaction was conducted in 15 mL acetone for 25 h at 70 °C with magnetic stirring. The supernatant was collected after centrifugation. After acetone was evaporated with a rotary evaporator under vacuum, the intermediates were obtained as yellow viscous liquids.

MEBU-TMEDA

¹H NMR (DMSO-d6): δ:1.24 (m, 12H, CH₂), 1.5 (m, 2H, O=C–C–CH₂), 1.68 (m, 2H, H₂C–C–N⁺), 1.87 (s, 3H, C=C–CH₃), 2.2 (s, 6H, C–N(CH₃)₂), 2.39 (t, 2H, O=C–CH₂), 2.61 (t, 2H, N–CH₂), 3.15 (s, 6H, CH₃–N⁺–CH₃), 3.36–3.46 (t, 4H, CH₂–N⁺–CH₂), 4.30 (m, 4H, O=C–CH₂–CH₂–C=O), 5.71–6.02 (s, 2H, C=CH₂).

FT-IR: σ: 3388 cm⁻¹(ν C–N), 2921 cm⁻¹(ν C–H), 1718 cm⁻¹(ν C=O), 1632 cm⁻¹(ν C=C), 1161 cm⁻¹(ν (O=)C–O), 1328 and 940 cm⁻¹(ν (C=)C–H).

MEBU-TMHDA

¹H NMR (DMSO-D6): δ:1.24 (m, 16H, CH₂), 1.4 (m, 2H, CH₂–C–N) 1.5 (m, 2H, O=C–C–CH₂), 1.68 (m, 4H, H₂C–C–N⁺), 1.87 (s, 3H, C=C–CH₃), 2.08 (t, 2H, O=C–CH₂), 2.15 (s, 6H, C–N(CH₃)₂), 2.27 (m, 2H, CH₂–N), 3.02 (s, 6H, CH₃–N⁺–CH₃), 3.36 (t, 4H, CH₂–N⁺–CH₂), 4.30 (m, 4H, O=C–CH₂–CH₂–C=O), 5.71–6.02 (s, 2H, C=CH₂).

FT-IR: σ: 3388 cm⁻¹(ν C–N), 2921 cm⁻¹(ν C–H), 1718 cm⁻¹(ν C=O), 1632 cm⁻¹(ν C=C), 1161 cm⁻¹(ν (O=)C–O), 1328 and 940 cm⁻¹(ν (C=)C–H).

2.4.2. Monomer MEBU-TMEDA-C₁₂ and MEBU-TMEDA-C₁₄

About 0.01 mol MEBU-TMEDA, 0.01 mol 1-bromododecane (or 1-bromotetradecane), 0.001 g hydroquinone, and 15 mL acetonitrile were added to 50 mL round-bottom flask, and the mixture was heated for 25 h at 95 °C with magnetic stirring. After evaporation of the solvent, the crude product was dissolved by acetone, and the supernatant was collected after centrifugation. Acetone was removed using rotary evaporator. Then the products were purified with anhydrous diethyl ether. The pure products were obtained as yellow viscous liquids.

MEBU-TMEDA-C₁₂

¹H NMR (DMSO-d6): δ = 0.9 (t, 3H, C–C–CH₃), 1.24 (m, 30H, CH₂), 1.5 (s, 2H, O=C–C–CH₂), 1.72 (m, 4H, H₂C–C–N⁺), 1.87 (s, 3H, C=C–CH₃), 2.39 (t, 2H, O=C–CH₂), 3.15 (s, 12H, CH₃–N⁺–CH₃), 3.36–3.37 (t, 8H, CH₂–N⁺–CH₂), 4.30 (m, 4H, O=C–CH₂–CH₂–C=O), 5.71–6.02 (s, 2H, C=CH₂) ppm.

FT-IR: σ: 3388 cm⁻¹(ν C–N), 2921 cm⁻¹(ν C–H), 1718 cm⁻¹(ν C=O), 1632 cm⁻¹(ν C=C), 1161 cm⁻¹(ν (O=)C–O), 1328 and 940 cm⁻¹(ν (C=)C–H).

MEBU-TMEDA-C₁₄

¹H NMR (DMSO-d6): δ = 0.9 (t, 3H, C–C–CH₃), 1.24 (m, 34H, CH₂), 1.5 (s, 2H, O=C–C–CH₂), 1.72 (m, 4H, H₂C–C–N⁺), 1.87 (s, 3H, C=C–CH₃), 2.39 (t, 2H, O=C–CH₂), 3.15 (s, 12H, CH₃–N⁺–CH₃), 3.36–3.37 (t, 8H, CH₂–N⁺–CH₂), 4.30 (m, 4H, O=C–CH₂–CH₂–C=O), 5.71–6.02 (s, 2H, C=CH₂) ppm.

FT-IR: σ: 3388 cm⁻¹(ν C–N), 2921 cm⁻¹(ν C–H), 1718 cm⁻¹(ν C=O), 1632 cm⁻¹(ν C=C), 1161 cm⁻¹(ν (O=)C–O), 1328 and 940 cm⁻¹(ν (C=)C–H).

2.4.3. Monomer MEBU-TMHDA-C₁₂ and MEBU-TMHDA-C₁₄

Via a procedure similar to that for monomer MEBU-TMEDA-C₁₂ and MEBU-TMEDA-C₁₄, 0.01 mol MEBU-TMHDA, 0.01 mol 1-bromododecane (or 1-bromotetradecane), 0.001 g hydroquinone, and 16 mL acetonitrile were heated for 25 h at 95 °C in 50 mL round-bottom flask. The products were purified with anhydrous diethyl ether. MEBU-TMHDA-C₁₂ and MEBU-TMHDA-C₁₄ were obtained as yellow viscous liquids.

MEBU-TMHDA-C₁₂

¹H NMR (DMSO-d6): δ = 0.9 (t, 3H, C–C-CH₃), 1.24 (m, 34H, CH₂), 1.5 (s, 2H, O=C–C–CH₂), 1.72 (m, 8H, H₂C–C–N⁺), 1.87 (s, 3H, C=C–CH₃), 2.39 (t, 2H, O=C–CH₂), 3.15 (s, 12H, CH₃–N⁺–CH₃), 3.36–3.37 (t, 8H, CH₂–N⁺–CH₂), 4.30 (m, 4H, O=C–CH₂–CH₂–C=O), 5.71–6.02 (s, 2H, C=CH₂) ppm.

FT-IR: σ = 3388 cm⁻¹(ν C–N), 2921 cm⁻¹(ν C–H), 1718 cm⁻¹(ν C=O), 1632 cm⁻¹(ν C=C), 1161 cm⁻¹(ν (O=)C–O), 1328 and 940 cm⁻¹(ν (C=)C–H).

MEBU-TMHDA-C₁₄

¹H NMR (DMSO-d6): δ = 0.9 (t, 3H, C–C–CH₃), 1.24 (m, 38H, CH₂), 1.5 (s, 2H, O=C–C–CH₂), 1.72 (m, 8H, H₂C–C–N⁺), 1.87 (s, 3H, C=C–CH₃), 2.39 (t, 2H, O=C–CH₂), 3.15 (s, 12H, CH₃–N⁺–CH₃), 3.36–3.37 (t, 8H, CH₂–N⁺–CH₂), 4.30 (m, 4H, O=C–CH₂–CH₂–C=O), 5.71–6.02 (s, 2H, C=CH₂) ppm.

FT-IR: σ = 3388 cm⁻¹(ν C–N), 2921 cm⁻¹(ν C–H), 1718 cm⁻¹(ν C=O), 1632 cm⁻¹(ν C=C), 1161 cm⁻¹(ν (O=)C–O), 1328 and 940 cm⁻¹(ν (C=)C–H).

2.5. Synthesis of QAS polymeric materials

Bis-GMA, TEGDMA, CQ, DMAEMA, and monomers (Table 1, for example, the resin incorporated with MEBU-TMEDA-MEBU) were added to a 5 mL beaker, stirred, and bubbles were removed by ultrasound. The above solutions were added dropwise to a diameter of 10 mm, a height of 2 mm mold, then cured for 100S with a dental curing light. Ultrasonic cleaning of the resin sheet to remove the unpolymerized monomer was done and it was air dried.

Table 1. The composition of polymeric materials.

Groups	Bis-GMA (g)	TEGDMA (g)	CQ (g)	DMAEMA (g)	MEBU-TMEDA-MEBU (g)
1	1.5	0.5	0.012	0.024	0
2	1.5	0.5	0.012	0.024	0.02
3	1.5	0.5	0.012	0.024	0.06
4	1.5	0.5	0.012	0.024	0.10
5	1.5	0.5	0.012	0.024	0.14

2.6. Antibacterial assessment

The antibacterial assessments of polymerizable Gemini monomers were carried out using the MIC and the MBC method. Two micro-organisms were used including S. aureus and E. coli, which represent Gram-positive and gram-negative bacteria, respectively.[34] The MIC is defined as the lowest concentration of an antimicrobial agent that will inhibit the growth of a micro-organism after incubation. The MIC values were determined by the broth dilution method using geometric twofold dilutions in LB. Solutions of biocide monomers in the series were mixed with 10⁷ CFU/mL of the organism in test tubes. The test tubes were incubated at 37 °C for 24 h. The MIC value is the concentration of the first clear test tube.[35,36] The MBC is defined as the lowest concentration of antimicrobial agent that will kill micro-organims and consequently will prevent growth after subculture onto antibiotic-free media. The MBC values were determined by agar dilution method.[37] The inoculating needle was put into the clear test tubes with a little amount of solution, then lines were drawn on the agar plates. These plates were incubated at 37 °C for 24 h. The MBC value is the minimum concentration of the clear solution where plate has no colonies. The inhibitory effect of resin incorporated with QAS against the growth of S. aureus on its surface was determined by counting the number of viable cells.[1–4] The cured resins were placed in test tubes with 10⁴ CFU/mL of initial S. aureus suspension in PBS. These tubes were shaken in incubators at 37 °C for 24 h. About 0.2 mL bacteria suspensions were pipetted out from the tubes, and plated onto the triplicate solid agar using the spread plate method. After incubating for 24 h, the number of viable bacteria was counted.

3. Results and discussion

3.1. Synthesis of Gemini biocide monomers

Two series of novel polymerizable Gemini QAS were synthesized via a facile method. Gemini biocide monomers contain polymerizable moieties which can polymerize during the synthesis; we used mild reaction temperature and appropriate solvent to get high yield and avoid any polymerization reaction.

3.1.1. Symmetrical monomer synthesis

The symmetrical Gemini quaternary ammonium monomers were synthesized via a two-step route (Schemes 1 and 2). The synthesis of intermediate MEBU was carried out via esterification reaction according to the JP56156237 [38] in an 80% yield. The synthesis route is shown in Scheme 1. MEBU, as a bromo compound, is known as very efficient quaternization agents leading very easily to the quaternary ammonium compounds when reacting with a tertiary amine. In this work, monomers MEBU-TMEDA-MEBU and MEBU-TMHDA-MEBU were prepared, in a 70% yield, via quaternization reaction of MEBU with TMEDA or TMHDA with isopropanol as solvent. Scheme 2 outlines the general synthesis of symmetrical biocide monomers. In this reaction procedure, we controlled the proportion of MEBU and diamine (2:1) and used isopropanol as solvent.

3.1.2. Asymmetric monomer synthesis

Unlike the symmetrical Gemini monomers, the other four polymerizable Gemini QAS which have asymmetric structure were synthesized via a three-step route (Schemes 1, 3, and 4). MEBU was also prepared by reaction as shown in Scheme 1. Then we used MEBU as the first alkyl bromide to react with TMEDA or TMHDA, and got intermediate MEBU-TMEDA or MEBU-TMHDA in about 75% yield. In this quaternization reaction procedure, we controlled the proportion of MEBU and diamine at equimolar ratio and used isopropanol as solvent. The general route of synthesis is shown in Scheme 3. As the second alkyl bromide, 1-bromododecanes (1-bromotetradecanes) were added to MEBU-TMEDA or MEBU-TMHDA at equimolar ratio and refluxed in acetonitrile. At last, we got monomers MEBU-TMEDA-C₁₂, MEBU-TMEDA-C₁₄, MEBU-TMHDA-C₁₂, and MEBU-TMEDA-C₁₄ in about 70% yield, the general synthesis route is shown in Scheme 4.

3.2. Characterization of monomers

All the intermediates and Gemini QAS monomers were recorded using NMR and FT-IR to confirm their structures. Figure 1 shows the ¹H NMR spectrum of intermediate MEBU. The peaks at 5.60–6.14 ppm are assigned to the methacrylate double bond protons, and the chemical shift of methyl is at 1.96 ppm. Peak at 4.35 ppm is ascribed to methylenes between the two ester groups. Besides, the peaks at 1.29, 1.43, 1.63, 1.86, 2.32, and 3.43 ppm are from the methylenes of 11-Bromo-ncanoic acid. From the FT-IR spectra, the vibration of C=O appears at 1718 cm⁻¹. The C=C appears at 1632 cm⁻¹. The vibration of C–O of ester group appears at about 1162 cm⁻¹. The C–H of alkene appears at 1328 and 940 cm⁻¹. The ¹H NMR and FT-IR results indicated that intermediate MEBU was successfully prepared.

Figure 1. ¹H NMR spectrum of MEBU.

Figure 2 shows the ¹H NMR spectrum of symmetrical monomer MEBU-TMEDA-MEBU. Methylenes between the two N⁺ ionic groups appear at about 4.30 ppm, and the signal at 3.15 ppm is assigned to methyls attached to the N⁺, which means the MEBU has already reacted with TMEDA to form the quaternary ammonium structure. Absorption peaks around 1720 and 1640 cm⁻¹ in FT-IR spectra, chemical shifts at 5.71–6.02(C=CH₂), 1.87(C=C–CH₃), and 4.30(O=C–CH₂–CH₂–C=O) ppm revealed that there were still methacrylate groups in the products. Similarly, MEBU-TMHDA-MEBU was also successfully synthesized, as demonstrated by¹H NMR and FT-IR.

Figure 2. ¹H NMR spectrum of MEBU-TMEDA-MEBU.

Intermediate MEBU-TMEDA was prepared via quaternization reaction of MEBU and TMEDA. All characteristic peaks of MEBU-TMEDA could be observed from Figure 3. The protons of alkene appear at 5.71–6.02 ppm. Methyls attached to the N atom and N⁺ ionic group appear at 2.2 and 3.15 ppm, respectively. The peaks at 3.36–3.46 ppm is ascribed to methylenes attached to the N⁺ ionic group, and the chemical shift of methylene attached to the N atom is at 2.61 ppm. The results revealed that MEBU-TMEDA was successfully synthesized. Similarly, intermediate MEBU-TMEDA was also prepared and demonstrated.

Figure 3. ¹H NMR spectrum of MEBU-TMEDA.

¹H NMR spectra of asymmetric monomer MEBU-TMEDA-C₁₂ are shown in Figure 4. The protons of alkene appear at 5.71–6.02 ppm. Methyls attached to the N⁺ ionic groups appear at 3.15 ppm. The peaks at 3.36–3.46 ppm are ascribed to methylenes attached to the N⁺ ionic group. Besides, the peaks at 0.90, 1.24, and 1.72 ppm are from the methylenes of 1-Bromododecane alkyl chain. The other asymmetric monomer MEBU-TMEDA-C₁₄, MEBU-TMHDA-C₁₂, and MEBU-TMEDA-C₁₄ were also characterized with the similar methods.

Figure 4. ¹H NMR spectra of MEBU-TMEDA-C₁₂.

short-legendScheme 1.

short-legendScheme 2.

short-legendScheme 3.

short-legendScheme 4.

3.3. Antibacterial properties of six monomers

The antibacterial activities of these Gemini monomers were investigated against S. aureus and E. coli, which are representative of Gram-negative and Gram-positive bacteria. The MIC values and the MBC values of biocide monomers were tested by using a broth dilution test.

As can be seen from MIC and MBC values presented in Table 2, all the synthesized Gemini monomers have excellent antibacterial activities for both Gram-negative and Gram-positive bacteria. Among the six compounds, four compounds were found to have MIC values (for S. aureus) less than 10 μg/mL. The asymmetric Gemini quaternary ammonium monomers have a lower MIC values than the monomers which have a completely symmetrical structure. Among the asymmetric Gemini QAS, the monomers which have a long alkyl chain length of 12 carbon atoms have better antibacterial activity. The spacer length between the two N⁺ ionic groups seems having teeny effect on the antibacterial ability for asymmetric Gemini quaternary ammonium monomers. On the contrary, it is an important factor that affects the MIC values and MBC values of the symmetrical Gemini QAS.

Table 2. The MIC values and MBC values of QAS monomers.

Samples	MIC (μg/mL)		MBC (μg/mL)
	S. aureus	E. coli	S. aureus	E. coli
MEBU-TMEDA-C12	6	11	23	23
MEBU-TMEDA-C14	8	12	15	30
MEBU-TMEDA-MEBU	15	15	30	31
MEBU-TMHDA-C12	7	10	12	16
MEBU-TMHDA-C14	9	12	19	19
MEBU-TMHDA-MEBU	10	11	16	20

3.4. Antibacterial properties of QAS polymeric materials

The antibacterial properties of polymeric materials were investigated against S. aureus and determined by counting the number of viable cells.

The data of Table 3 presented that the concertration of bacteria on the surface of antibacrerial modification composite resin was less than the control group. It showed that the resin incorporated with monomers have antibacterial activities, and the activities increased with the concentration of QAS monomers increased. The antibacterial activities of resin incorporated with asymmetric Gemini quaternary ammonium monomers (MEBU-TMEDA-C₁₂, MEBU-TMEDA-C₁₄, MEBU-TMHDA-C₁₂, MEBU-TMHDA-C₁₄) were better than the resin incorporated with symmetric Gemini quaternary ammonium monomers (MEBU-TMHDA-MEBU, MEBU-TMHDA-MEBU). The reason of poor peformance of the latter is that the symmetric Gemini QAS monomers have two unsaturated double bonds. When monomers were bonded to resin, both ends of the molecule were fixed, which reduced the freed degree of N⁺. The resin incorporated with MEBU-TMEDA-C₁₂ and MEBU-TMEDA-C₁₄ showed better antibacterial activities than the resin incorporated with MEBU-TMHDA-C₁₂ and MEBU-TMHDA-C_14. The long spacer between the two N⁺ of MEBU-TMHDA-C₁₂ and MEBU-TMHDA-C₁₄ reduced the charge density. The resin incorporated with MEBU-TMEDA-C₁₂ had the best antibacterial activities.

Table 3. Antibacterial properties of QAS polymeric materials.

Monomer	Added amount of monomer （% w.t.）	Inoculum concentration（CFU/mL）	Concentration of bacteria on material surface after 24 h（CFU/mL）
Control group	0	3.6 × 10^4	1.8 × 10^8
MEBU-TMEDA-C12	1	3.6 × 10^4	3.6 × 10^5
	3		2.3 × 10^5
	5		8.6 × 10^4
	7		4.5 × 10^4
MEBU-TMEDA-C14	1	3.6 × 10^4	4.5 × 10^5
	3		3.1 × 10^5
	5		9.1 × 10^4
	7		5.8 × 10^4
MEBU-TMHDA-C12	1	3.6 × 10^4	4.3 × 10^5
	3		2.9 × 10^5
	5		8.7 × 10^4
	7		5.7 × 10^4
MEBU-TMHDA-C14	1	3.6 × 10^4	4.1 × 10^5
	3		3.2 × 10^5
	5		1.5 × 10^5
	7		6.8 × 10^4
MEBU-TMEDA-MEBU	1	3.6 × 10^4	1.3 × 10^7
	3		8.1 × 10^6
	5		3.3 × 10^6
	7		6.5 × 10^5
MEBU-TMHDA-MEBU	1	3.6 × 10^4	3.8 × 10^7
	3		1.3 × 10^7
	5		4.1 × 10^6
	7		7.3 × 10^5

4. Conclusion

Two series of novel polymerizable Gemini quaternary ammonium monomers were synthesized with a facile method and characterized by ¹H NMR and IR to confirm their expected structures. The antibacterial activities of monomers were investigated against S. aureus and E. coli bacteria. All novel biocide monomers came out as broad spectrum biocides against both Gram positive and negative bacteria. The six kinds of QAS monomers were bonded to the resin system by light-curing method to get QAS polymeric materials for dental resin. The inhibitory effect of resin against the growth of S. aureus was determined. The result showed that the resin incorporated with the monomers had antibacterial activities, and the antibacterial activities were increased with increasing the concentration of QAS monomers.

Funding

This research was supported by the grant from the Natural Science Foundation of China [grant number 81130078]; NUST Research Funding [grant number 2011YBXM54].