Nucleophilic Displacement Reaction on Tosyl Cellulose by L- Methionine to the Synthesis of Novel Water-Soluble Cellulose Derivative and Its Antibacterial Activity

A novel ampholytic cellulose derivative, cellulose-L-methionine, has been synthesized by means of an esterification reaction of microcrystalline cellulose with tosyl chloride (p-TsCl) in DMAc/LiCl (8%) at 8°C that was followed by nucleophilic displacement (SN) of the tosyl group by the L-methionine amino acid. The resulting structure of cellulose-L-methionine has been characterized by elemental analysis (CHNSO), Fourier-transform infrared spectroscopy (FTIR), proton nuclear magnetic resonance (H-NMR), and scanning electron microscopy (SEM). The antibacterial activity of the synthesized product was screened against Gram-positive and Gram-negative microbial strains such as Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa, by the agar well diffusion method, and compared with commercial antibiotics such as ampicillin and chloramphenicol. It was found that antibacterial experiment revealed excellent antibacterial activity of the cellulose-methionine with respect to a minimal inhibitory concentration (MIC) reference.


Introduction
Cellulose and its derivatives are of huge importance for various applications areas, food medical products, pharmaceutical industries, packaging, textile, etc., because of their very interesting chemical and physical properties, such as biodegradability, biocompatibility, bioactivity, and nontoxicity property [1,2]. Cellulose is the most abundant biopolymer in nature. Cellulose is insoluble in common organic solvents and in water [3]. This is due to the fact that the hydroxyl groups are responsible for the extensive hydrogen bonding network forming both intra-and intermolecular hydrogen bonding. However, special solvents were developed for the dissolved cellulose and for preparing a broad variety of cellulose derivatives by homogeneous chemical modification of the biopolymer [4][5][6][7][8]. By a chemical functionalization of cellulose, a wide variety of new macromolecules have been obtained [9][10][11]. For several years, great effort has been devoted to the synthesis of water-soluble cellulose derivative as promising biomaterials in high-end applications for drug delivery such as potential antibacterial activity [12,13]. For example, the synthesis of tosyl cellulose derivative intermediates through the tosylation of the primary hydroxyl group at C-6 of cellulose has been reported since the tosyl group is a good electrophile and leaving group in nucleophilic displacement (S N ) reactions or as protecting group in further reactions of the remaining free hydroxyl (-OH) groups [13][14][15]. In our previous work, we were able to synthesize tosyl cellulose with different degrees of substitution (DS) by reacting microcrystalline cellulose with p-tosyl chloride, and we have studied the effects of reaction parameters on DS by response surface methodology (RSM) [16]. Ampholytic biomaterials are semisynthesized biomacromolecular carrying both the ionic and cationic groups; they offer beneficial applications in drug delivery, protein separation, surfactants, and chelation with metal ions because of their structure specificity [17][18][19][20]. Several routs have been reported for the preparation of ampholytic cellulose such as the synthesis of cellulose sulfates functionalized with quaternary ammonium salt [21], synthesis of 6-deoxy N-sulfonated and N-carboxymethylated cellulose [22], and cellulose zwitterions [23].
Up to now, although researchers have synthesized some cellulose derivatives that are functionalized with the amino groups [12,13,24,25], there are no published studies related to the synthesis of cellulose-L-methionine and its antibacterial activities to our knowledge.
In our concept, the key reaction is the nucleophilic displacement (S N ) reaction of the tosyl cellulose by Lmethionine to synthesize new water-soluble ampholytic cellulose derivative with interesting molecular structure and properties, cellulose-L-methionine, and evaluated its antibacterial activities against a Gram-positive and Gram-negative microbial strains such as Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Streptococcus fasciens. The tosyl cellulose with DS ≈ 1 was chosen as an intermediate to yield new water-soluble ampholytic cellulose by S N . The reaction proceeds homogeneously in DMF at 80°C for 24 h. The chemical structure and properties of the synthesized product were characterized by elemental analyses, Fouriertransform infrared spectroscopy (FTIR), proton nuclear magnetic resonance ( 1 H-NMR), and scanning electron microscopy (SEM).
All pathogenic microorganisms isolated from patients were stored at the culture collection of the Biology Department (Microtech Unity) at the Faculty of Science, Rabat, Morocco. They were maintained in brain heart infusion (BHI) at -80°C. Prior to the experiment, cultures were prepared by subculturing 1 mL of each culture stock in 9 mL of BHI broth.

Methods of Material Analysis
2.3.1. Spectroscopic Measurements. The apparatus used for infrared spectroscopy characterization is a Bruker Tensor 70, which operates in transmittance mode. This apparatus is equipped with a Globar source that emits radiation in the region of midinfrared and of a DLaTGS detector. The acqui-sition is between 4000 and 400 cm -1 in wavenumber. The number of scans is of 20 with a resolution of 4 cm -1 . Infrared spectrum was recorded from a sample in solid form prepared as pellets to 1% by weight of product dispersed in KBr.
Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Advance 300 MHz spectrometer with 16 scans for 1 H-NMR at 25°C in D 2 O or DMSO and a sample concentration of 30 mg/mL. The chemical shifts (δ) were expressed as part per million (ppm) against tetramethylsilane (TMS) as internal reference.

Scanning Electron Microscopy (SEM)
Analysis. The surface morphology of the microcrystalline cellulose and the tosyl cellulose was analyzed by scanning electron microscopy (SEM), using a FEI Quanta 200 microscope. The samples were air dried for 24 h before imaging and were coated with a carbon layer to increase their conductivity. The images of samples were obtained using an accelerating voltage of 30 kV.

Elemental Analysis.
Elemental analysis was performed by Euro EA -CHNSO Elemental Analyzer.

Typical Cellulose Dissolution Process in DMAc/LiCl
Solvent System. In brief, 0.5 g of oven-dried microcrystalline cellulose (3.08mmol) was added to 20 mL of DMAc and the suspension was heated at 130°C for 2 h. The resulted slurry was cooled down to 100°C, and 1.6 g of anhydrous LiCl (8%) was added to the mixture with stirring while cooling down the mixture at room temperature. The stirring was continued until the complete dissolution of cellulose within a few hours.

Agar Disc Diffusion
Method. The agar disc diffusion method (ADD) was employed for the determination of antibacterial activities of the tested products as described previously. The principle of this technique is to estimate the bacteriostatic activity of antibacterial agents by measuring the growth inhibition zone of germs around wells. It is mostly used in a preliminary step to further study because it provides access to essentially qualitative results. The test samples were first dissolved in distilled water (DW), which did not affect the microbial growth.
Briefly, the test was performed in sterile petri plates containing medium agar. 30 mL of sterilized medium was poured into sterile petri plates. After solidification, 100 μL of fresh cultures of bacteria species (one microorganism per petri plate). Sterile filter paper disc (6 mm in diameter) was impregnated with 6 μL of the test samples (40 mg/mL). All plates were sealed with sterile laboratory films to avoid eventual evaporation of the test samples and then incubated at 37°C for 24 h. The diameter of inhi-bition zone was measured in millimeters. In addition, the antibacterial activity of the cellulose-L-methionine sample on Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Streptococcus fasciens bacteria was compared with the commercially available antibiotics. The antibiotic discs such as ampicillin and chloramphenicol were placed on the surface of the plates. Distilled water was used as a negative control. The plates were incubated at 37°C for 24 h after incubation. The diameter of inhibition zone was measured in mm and was recorded [28,29].

Determination of the Minimum Inhibition Concentration
(MIC). We tested six (6) serial concentrations of the high active products at concentrations 40, 20, 10, 5, 2.5, and 1.25 mg/mL, diluted in BHI broth. For MIC assessed, 5 mL of culture medium was inoculated with 0.1 mL of bacteria species. The MIC is the lowest concentration of samples, for which no growth was detected for 24 h at 37°C.   International Journal of Polymer Science glucose ring against nucleophilic attack of the tosyl group. The reaction process for the synthesis of cellulose-Lmethionine is shown in Figure 3.   [15]. Meanwhile, the 1 H-NMR spectrum of cellulose-L-methionine revealed the presence of new signals related to the L-methionine amino acid moieties substituted C-6 of cellulose, such as the signals at δ 2.06, 2.51, and 2.67 ppm for CH 3 , CH 2 , and CH, respectively. It is evident that the complete replacement of the tosyl group was confirmed by the disappearance of the signals ascribed to the tosyl group. Figure 4 displays the morphological structure of MCC (a), tosyl cellulose (b), and cellulose-L-methionine derivative (c). The SEM images of the surface of (a), (b), and (c) show clear differences between them. The MCC is mainly composed of platelet-like cellulose microfibrils, shaped into a spherical agglomeration. However, the surface structure of tosyl cellulose (b) is compact, the cellulose-L-methionine (c) had much greater porosity, and the surface roughness of cellulose derivative increased more than unmodified MCC. Interruption of the backbone of tosyl cellulose polymer can explain this observation as a result of reaction of the hydroxyl group (-OH) of MCC with p-tosyl chloride and probably due to breaking of hydrogen bonds present in the MCC and interaction between the newly introduced hydrophobic phenyl groups. Thus, nucleophilic substitution reaction of tosyl cellulose with Lmethionine amino acid leads to very significant changes in the morphology of the unmodified MCC.

3.3.
In Vitro Antibacterial Activity. The development of generations of new strains of bacteria is potentially harmful and stresses the importance of developing materials to fight human pandemics; for that, the natural antibacterial agent has been one of the hot topics in scientific research to develop  International Journal of Polymer Science antibacterial agent from naturally abundant biopolymers such as cellulose and chitosan. It is expected in the field of biomedical and antibacterial films, cosmetics, textile industries, pharmaceutical, and other fields. To compare and analyze the antibacterial behavior contributed from cellulose-L-methionine and commercial antibiotics such as chloramphenicol and ampicillin, we have tested the cellulose-L-methionine against the bacteria: Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Streptococcus fasciens using the diffusion method disk for evaluating the applicability of cellulose-L-methionine product as an antibacterial agent [30,31]. Figure 5 summarizes the diameter of inhibition (mm) values of cellulose-L-methionine and commercial antibiotics chloramphenicol and ampicillin. The minimal inhibitory concentration (MIC) values of the cellulose-L-methionine against the bacteria are presented in Table 1. The cellulose-L-methionine product showed high antibacterial activity and different degrees of growth inhibition against the four bacteria tested; the maximum effect of the product tested was recorded against Streptococcus fasciens (diameter of inhibition 21. 2 mm), when compared with other bacteria, with MIC value of 5 mg/mL. It is clear that there is a significant enhancement and a strong antibacterial activity associated with cellulose-L-methionine, as compared to commercial antibiotic. This biologic effect is due to the chemical structure and chemical functionality of cellulose-L-methionine. No zone inhibition was observed with water. In our previous work, we have synthesized the celluloseacetanilide ether with different degrees of substitution (DS); it was found that the cellulose-acetanilide product was found to be more active against the chosen pathogenic bacteria strain Rhodococcus sp. GK1, and that the antibacterial activity of this product increased with DS [32]. Indeed, several studies have been focused on antibacterial properties of cellulose derivatives and proved effects against various bacterial strains [33][34][35][36][37]. Similarly, El-Sayed et al. [13] synthesized new water-soluble cellulose derivatives such as 4(celluloseamino) butyric acid and 2(celluloseamino) succinic acid; they have found that its cytotoxicity profile is negligible. The mechanism of antibacterial action of our compound synthesized is not attributable to one specific mechanism because there are several target sites in the bacterial cell such as the cell wall, cytoplasmic membrane and cytoplasm, leakage of cell contents, and coagulation of the cytoplasm; however, the structures of the bacterial outer envelope are more interesting as they differ between microorganisms. For example, the outer membrane of Gram-negative bacteria [38,39] and the cell wall of mycobacteria [40] act as permeability barriers and are responsible for the intrinsic resistance of these microorganisms to antimicrobial compounds. A study is needed to understand the susceptibility of bacteria to our antimicrobial compounds; thus, anti-inflammatory, antifungal, antiparasitic, and anticancer activities are necessary, and some other types of Gram-negative and Gram-positive bacteria may possibly be tested by employing the same method. The literature gives a lot of interesting results on these topics [29,41].

Conclusion
The tosyl cellulose with degree of substitution DS = 1 was successfully synthesized by an esterification reaction of microcrystalline cellulose in DMAc/LiCl (8%) solvent system in the presence of triethylamine (TEA) at 8°C for 24 h. The nucleophilic displacement (S N ) reaction of the tosyl group with L-methionine amino acid could be efficiently carried out leading to water-soluble methionine-substituted cellulose derivative. The characterization by means of NMR and FTIR spectroscopy, elemental analysis, and SEM indicates a uniform structure of the tosyl cellulose and cellulose-Lmethionine. The antibacterial test of cellulose-L-methionine shows significant activity against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Streptococcus fasciens strains of bacteria. In our future research, we intend to focus on explaining the exact mechanism of cellulose-Lmethionine that induces bacterial cytotoxicity. This work encourages us to develop cellulose-based antibacterial materials for use in different biomedical and pharmaceutical applications.

Data Availability
The data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
The authors declare that there is no conflict of interest.