A comparative evaluation of antibacterial activities of imidazolium-, pyridinium-, and phosphonium-based ionic liquids containing octyl side chains

Antibacterial activity is an essential property of ionic liquids. In this work, a comprehensive study has been performed on the antibacterial activity of ionic liquids to be utilized for further research and applications. Eighteen ionic liquids viz. Octyl Imidazolium, octyl Pyridinium, quaternary phosphonium-based cations containing bromide, sodium methane sulphonates, bis(trifluoromethane sulfonyl) imide, dichloroacetate, tetrafluoroborate, hydrogen sulfate were prepared and characterized with the help of different spectroscopic techniques. All these samples of ionic liquids were tested for their antibacterial activity against the most commonly occurring bacteria in the environment, i.e., Enterobacter aerogenes (E. aerogenes), Proteus vulgaris (P. vulgaris), Klebsiella pneumoniae (K. pneumoniae), Pseudomonas aeruginosa (P. aeruginosa), Escherichia coli (E. coli), and Streptococcus pyogenes (S. pyogenes). Most of the ionic liquids show good antibacterial properties, and imidazolium-based ionic liquids were even more antibacterial as compared to positive control. It was observed that a unique combination of cation and anion is essential to achieve desired antibacterial properties. The mechanism of antibacterial activity was further investigated using density functional theory calculations. A good correlation was found between experimental and theoretical studies.


Introduction
The Healthcare community faces the major challenge of an enhanced number of deaths in hospitalized patients due to microbial infections [1]. These increasing numbers of microbial infections have now gained the attention of researchers worldwide, as evident from the growing number of publications on microbial resistance and its effects on the health of the human population [2]. Bacterial infections can be controlled by sanitization [3], sterilization [4], and by the use of biocides, but the bacteria have developed resistance against them. For example, E. coli is responsible for both complicated and uncomplicated UTIs. But, some German populations have developed resistance against cotrimoxazole, which was once used as the first line of defense against UTIs [5].
Ionic liquids (ILs) with inherent antimicrobic properties and tunable nature have now emerged as active pharmaceutical ingredients (API) [6,7,8]. ILs mainly consist of an organic cation like imidazolium, pyridinium, quaternary ammonium, and phosphonium which can be combined with various anions to form a diverse set of molecules with distinct properties which can be further changed by the functionalization of either cation or anion [9]. The length of alkyl side chains of ILs directly affects the cytotoxicity, interaction with bovine serum albumin, and toxicity for microbial growth [10]. ILs possess unique physicochemical properties like high viscosity and density as compared to water [11], reduced biodegradability [12], low melting point (<100 C) [9,13], high thermal, chemical, and electrochemical stability [11,14,15,16], non-volatile [17], low toxicity [6] solubility in most of the solvents and negligible vapor pressure [18].
This work reported the synthesis and characterization of pyridinium, phosphonium, and imidazolium-based ionic liquids with different anions. This study compares the effect of different cations containing octyl side chains on antibacterial activity. These ionic liquids were tested for antibacterial activity against S. pyogenes, E. coli, K. pneumoniae, E. aerogenes, P. vulgaris, P. aeruginosa. The ILs based on imidazolium show better antibacterial activity than all other tested cations.
FTIR stirred at room temperature for 12 h (Scheme 1). The LiBr formed during the metathesis reaction was removed by solvent extraction followed by filtration. The solvent was evaporated to obtain the pure product (S1-03) and dried overnight under a vacuum at 40 C. The yield of obtained product is~75%.
FTIR  CO 2 ]. The [C 8 mim][Br] (0.5 g, 12 mmol) was added to 60 mL acetonitrile and mixed with sodium dichloroacetate (0.3 g, 2 mmol), followed by overnight stirring at room temperature (Scheme 1). The NaBr formed was removed by solvent extraction followed by filtration. The solvent was evaporated to obtain the pure product (S1-04) with a 96% yield.
FTIR 72 mmol) solution was slowly added into solution of NaBF 4 (0.3 g, 2.7 mmol) in 60 mL acetonitrile. The reaction mixture was stirred overnight under an inert atmosphere at room temperature (Scheme 1). The NaBr formed was separated through filtration and solvent extraction. The solvent was evaporated with a rotary evaporator and dried under a vacuum to obtain the pure product (S1-05) with a 70% yield.
FTIR KBr formed was separated through solvent extraction followed by filtration. The product was dried under a vacuum. . The trioctyl phosphine (5 g, 13.5 mmol, and 6.02 mL) was mixed with butyl bromide (1.85 g, 13.5 mmol) in a round bottom flask containing acetonitrile and evacuated to create an inert atmosphere and refluxed at 120 C. When TLC confirmed the completion of the reaction, the solvents were removed by evaporation to obtain the product (S3-01) as a dark yellow oily liquid. It was dried in a vacuum oven at 50

Antibacterial activity
The samples of equal density and potency were prepared using distilled water. Each sample was tested in triplicate. Eighteen samples were used to check their antibacterial activity against pathogenic bacteria. New bacterial culture of 24 h was used to make the lawn in Petri dishes. About 100 μl of each equalized bacterial sample was spread on Petri dishes. Using a sterilized borer, 3 mm wells were made at equal distances. Positive and negative controls were used in equal v/v ratio on every plate. About 25 μl of sample solutions were poured into each well and then incubated for 24 h at 37 C. The distilled water was used as negative control and levofloxacin as the positive control [43].

Density functional theory studies
All the ILs were modeled, and their geometry optimizations were performed at the B3LYP/6-31g level of Density Functional Theory methods [44] using the Gaussian 09 program. The geometry optimization was performed in the vacuum keeping the overall charge zero for all the ILs. For validation purposes, frequency calculations were performed on the optimized geometries using the same level of the DFT method used for optimization. As the application of these ILs was to act as inhibitors for the protein, single-point energy on all the optimized geometries was calculated in the aqueous medium with the same method and basis set enhanced by polarization functions for all atoms except hydrogen.

Molecular docking studies
Molecular docking was performed to understand the binding affinity between the target protein, β-lactamase, and the designed ILs [45,46,47,48]. As the β-lactamase enzymes are involved in the antibiotic resistance by hydrolyzing the peptide bond of the β-lactam ring, therefore the   [52], and S. pneumoniae (PDB ID: 1RPS) [53] are used for molecular docking. For docking purposes, optimized geometry of all the ILs geometries was used. Molecular docking studies were performed using PyRx software where the optimized ILs and the proteins (PDB structure) were loaded, and molecular docking was performed after the selection of the predicted protein chains. Before docking, all the protein structure's energy was minimised where protonation and water molecules were removed. Default settings were maintained for all parameters during the docking process. As a result of docking, nine different conformations were generated for every pair of protein and ligand. The conformation with the highest Binding Affinity was selected for evaluation.

Result and discussion
ILs based on imidazolium, pyridinium, and phosphonium with different anions (bromide, hydrogen sulfate, dichloroacetate, bis(trifluoromethane sulfonyl)imide, tetrafluoroborate, and methanesulphonate, etc.) were prepared and characterized with the help of characterization techniques (TLC, ATR-FTIR, NMR, etc.) at different levels during and after the synthesis.
FTIR indicated the formation of S1-02 due to band appearance at 1040 cm À1 responsible for S¼O stretch. This characteristic band appears due to the exchange of anions. The formation of S1-03 was confirmed by FTIR due to the appearance of peaks at 1358 cm À1 , indicating asymmetric stretching vibration of S¼O and at 1180 cm À1 symmetric stretching vibration of S¼O. The C¼O and C-O stretching bands at 1644 cm À1 and 1358 cm À1 , respectively, indicate the formation of S1-04. The formation of S1-05 was confirmed by the presence strong band at 1049 cm À1 due to [BF 4 ] anion. A peek at 1047 cm À1 due to S¼O shows the formation of S1-06.
FTIR for S2-02 shows S¼O stretching vibration at 1042 cm À1, indicating the exchange of methane sulphonate anion. In FTIR for S2-03, the bands at 1345 cm À1 and 1052 cm À1 are due to S¼O stretching. The C¼O and C-O stretching bands appear at 1633 cm À1 and 1368cm À1 , respectively, indicating the formation of S2-04. The formation of S2-05 was confirmed by the presence of a strong band at 1033 cm À1 due to [BF 4 ] anion. The peak at 1046 cm À1 appears due to S¼O stretching, indicating S2-06 formation.

Phosphonium based ionic liquids
The tri-octyl phosphine (3) was reacted with butyl bromide under an inert atmosphere at 120 C for 48 h to give S3-01 as the product. 1 H-NMR confirmed the quaternization of (3) as the -CH 2 peak shifts from 3.35 ppm in butyl bromide to 2.508-2.525 ppm in product S3-01. FTIR of S3-01 also confirms the quaternarization of tri-octyl phosphine with butyl bromide. Following bands appeared in FTIR of S3-01; 2960 cm À1 and 2854 cm À1 (CH stretch), 1463 cm À1 (CH 2 bend), and 746 cm À1 (longchain CH 2 in butyl and octyl groups attached to phosphorous).
The S¼O stretching vibration at 1347 cm À1 and 1056 cm À1 in FTIR of S3-03 confirms its formation by replacing bromide anion. The C¼O and C-O stretching vibration at 1651 cm À1 and 1376 cm À1 , respectively, indicate the formation of S3-04 by replacing dichloroacetate with bromide anion. The formation of S3-05 was confirmed by the presence of a band at 1049 cm À1 due to [BF 4 ] anion. The S¼O stretching vibrations at 1236 cm À1 and 1161 cm À1 indicate the formation of S3-06.

Antibacterial activity
Compared to other living organisms, the short generation time of bacteria had indirectly led the researchers to investigate the antibacterial activity of ILs [54]. The toxicity of pyridinium, phosphonium, and imidazolium-based ILs increases with alkyl chain length in their quaternary ammonium salts [55,56]. In this study, 18 different ILs were synthesized to check their potential toxicity and effectiveness as antibacterial agents against gram-positive and gram-negative bacterial  The antibacterial activity of these synthesized ILs was evaluated by the diameter of the inhibition zone on agar plates. This zone is defined as an area on the plate where the growth of the bacteria was prevented.
The ILs based on octyl-imidazolium showed the highest antibacterial activity against all strains of the bacteria; some ILs were even more effective than positive control levofloxacin. Table 1 and Figure 1 show that the ILs based on octyl imidazolium were effective against all bacterial strains. The IL S1-02 showed the highest inhibition values of 27 mm for E. aerogenes and 24 mm against P. aeruginosa, while IL S1-06 showed no inhibition against S. pneumoniae. The ILs based on quaternary salts of pyridinium showed antibacterial activity against E. aerogenes and K. pneumoniae. It is clear from Figure 2 and Table 2 that ILs S2-04 and S2-05 show the highest inhibition against K. pneumoniae with an IZ value of 20 mm Table 3 and Figure 3 show the bacterial inhibition for ILs based on quaternary salts of phosphonium with the highest IZ value of 14 mm against E. aerogenes. All phosphonium-based ILs also showed effectiveness against E. coli. Overall, all 18 synthesized ILs showed inhibition effectiveness, but ILs based on imidazolium are more effective than others. The order of effectiveness is as follows: ILs (Imidazolium) > ILs (phosphonium)~ILs (Pyridinium).

In-silico antibacterial activity
The antibacterial activity can also be analyzed using in-silico methods like Molecular Docking studies. The strong binding energy of ligand (drug or inhibitors) with the protein shows the maximum inhibition or higher antibacterial activity. Therefore, in this research, molecular docking studies were performed on all the 18 ionic liquids with the β-Lactamase protein of six bacteria; E. coli (PDB ID: 5A92), E. aerogenes (PDB ID: 5KID), K. pneumonia (PDB ID: 6MGX), P. vulgaris (PDB ID: 1HZO), P. aeruginosa (PDB ID: 4GZB), and S. pneumoniae (PDB ID: 1RPS). Details of the docking protocol are mentioned in the Computational details; however, docking results are presented in Table 4.
Molecular docking results for the imidazolium-based ionic liquids show that the IL S1-01 shows potent inhibition with all the gram-positive and gram-negative bacteria's ranging binding affinity values from -6.3 to -7.7 kcal/mol. All the binding pockets and their interactions are presented in Table 5 and Figure 4.    The proposed reaction mechanism for inhibiting β-lactamase with IL S1-03 is presented in Figure 5, showing the maximum inhibition capacity. It has been observed in Figure 4 (S1-03-ILP3) that Ser and Lys are present at the binding sight showing maximum interaction. According to the proposed reaction mechanism, the simultaneous transfer of protons occurs ( Figure 5), resulting in the formation of positively charged Lys and direct bonding of Ser with the anion part of ionic liquid.
On comparing the docking results for the pyridinium-based ILs, the same behavior was observed as for imidazolium-based ILs. The IL S2-03 shows the maximum inhibition with all the six bacteria with the binding affinity values ranging from -6.4 to -8.4 ( Figure 6).
On the other hand, when the molecular docking results for phosphonium-based ionic liquids were analyzed, slightly different behavior was observed; the IL S3-03 shows maximum inhibition for E. coli, K. pneumonia, P. vulgaris, and P. aeruginosa, however, S3-06 shows maximum inhibition for E. aerogenes and S3-02 shows maximum inhibition for S. pneumonia (Figure 7).
Analyzing the molecular docking results, it was also observed that the anionic part of imidazolium and pyridinium-based ILs interacts with the amino acids present at the active site; however, in the case of phosphonium based ILs, the cationic part of ILs shows interaction at the binding site. On comparing all the molecular docking results of the three schemes of imidazolium, pyridinium, and phosphonium-based ILs, it was observed that the pyridinium-based ILs would show strong binding affinity values and has maximum inhibition capacity relative to the other two schemes of ILs.

Conclusions
In brief, 18 ILs based on octyl pyridinium, octyl imidazolium, and mono-butyl-tri-octyl phosphonium cations and anions like bromide, sodium methane sulphonate, lithium bis(trifluoro-methane-sulfonyl)imide, sodium dichloroacetate, and sodium tetrafluoroborate, and potassium hydrogen sulfate were prepared as effective antibacterial agents. All the synthesized ILs were characterized by FTIR and NMR. The antibacterial activity of these ILs was tested by the agar well diffusion method against both gram-positive and gram-negative bacteria like E. aerogenes, P. vulgaris, K. pneumoniae, P. aeruginosa, E. coli, and S. pyogenes.
The ILs based on imidazolium showed the highest antibacterial activity against all tested bacteria and are more antibacterial than positive control levofloxacin. The IL S1-02 showed the highest inhibition with an IZ value of 27 mm for E. aerogenes and 24 mm against P. aeruginosa, while IL S1-06 showed no inhibition against S. pneumoniae. ILs based on pyridinium showed activity against E. aerogenes and K. pneumoniae, with ILs S2-04 and S2-05 showing the highest inhibition against K. pneumoniae with an IZ value of 20 mm. In contrast, the ILs based on phosphonium showed activity against E. coli and E. aerogenes, with the highest IZ value of 14 mm against E. aerogenes. The order of effectiveness is as follows: ILs (Imidazolium) > ILs (phosphonium)~ILs (Pyridinium). The molecular docking results show that IL S1-03 shows potent inhibition with all bacteria with binding affinity values ranging from -6.3 to -7.7 kcal mol À1 . From pyridinium-based ILs, the IL S2-03 shows the maximum inhibition with all the six bacteria with the binding affinity values ranging from -6.4 to -8.4 kcal mol À1 . While in the case of phosphonium-based ILs, the IL S3-03 shows maximum inhibition for E. coli, K. pneumonia, P. vulgaris, and P. aeruginosa, however, S3-06 shows maximum inhibition for E. aerogenes and S3-02 shows maximum inhibition for S. pneumonia.

Declarations
Author contribution statement Rabia Hassan: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.
Mudassir Iqbal: Conceived and designed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.
Uzma Habib: Conceived and designed the experiments; Contributed reagents, materials, analysis tools or data; Wrote the paper.
Bashir Ahmad: Performed the experiments; Analyzed and interpreted the data.

Funding statement
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors and proceed further with the article.

Data availability statement
Data included in article/supplementary material/referenced in article.

Declaration of interests statement
The authors declare no conflict of interest.

Additional information
No additional information is available for this paper.