Green Synthesis of Quinoline-Based Ionic Liquid

Summary

In the present work, we elucidate the green synthesis of quinoline-based Ionic Liquid (IL), namely, 1-Hexadecylquinolin-1-ium bromide {[C₁₆quin]Br} by mixing quinoline with an excess of 1-Bromohexadecane, along with its detailed characterization using Nuclear Magnetic Resonance and Infrared spectroscopic measurements.

Abstract

The ever-growing menace of Antimicrobial Resistance (AMR) jeopardizes the potency of the prevailing antibiotics against the relentlessly sprouting infections spawned by bacteria, viruses, parasites as well as fungi, posing a great threat to human health and well-being. In this regard, several novel molecules have proved their mettle, with Ionic Liquids (ILs) being one of the most eco-friendly, non-volatile, and thermally stable alternatives to the existing antimicrobials, possessing high solvating potential as well as low vapor pressure. Moreover, the utilization of these entities in both stabilizing as well as destabilizing protein structures and enhancing enzymatic activity has further raised their potential in the biomedical industry. With this in view, we present the green synthesis and characterization of quinoline-based IL, owing to its immense antimicrobial potency, with low cytotoxicity and great artificial chaperone activity. Here, maneuvering the one-pot synthesis approach in solvent-free, greener reaction conditions not only ameliorated the reaction efficiency but also augmented the chemical yield. The purity of the synthesized IL was corroborated using 1H Nuclear Magnetic Resonance (NMR), 13C NMR, and Infrared (IR) spectroscopy. The biological potential of the synthesized compound is further validated by analyzing its Absorption, Distribution, Metabolism, Excretion, and Toxicity (ADMET) properties and authenticated using disc diffusion assay.

Introduction

The monumental growth in the world population accounts for a tremendous increment in the consumption of a vast array of commodities over the past few years, including food, medicaments, as well as other crucial products for the sustenance of mortal organisms. This has invigorated the quest for novel chemical compounds with exceptionally specialized, ecologically sound, and beneficial properties worldwide. Ionic Liquids (ILs) have proved to be felicitous in this regard. The implication of these compounds in the scientific domain has bolstered new ventures in research in contemporary chemical technologies¹. In contrast to the conventional approaches, the utilization of ILs not only facilitates progressive reaction conditions but also promotes a customized strategy to get to grips with various biochemical challenges related to experimental research and development².

Typically, ILs are stable salts constituting cations (organic) and anions (inorganic), possessing a melting point below 100 °C³. Abiding by the 12 principles of green chemistry, empirically, these are convincing substitutes to the customary organic solvents⁴. The astounding properties associated with the utilization of these compounds encompass great intrinsic conductivity, polarity, solvating tendency, thermal stability, non-volatility, acidity/basicity, hydrophilicity/ hydrophobicity, and tunability, making ILs best suited for experimental research⁵.

Apart from the expansive applications of various classes of ILs in modern organic synthesis⁶, catalysis⁷, and various electrochemical processes involving sensors⁸, actuators⁹, batteries¹⁰, and fuel cells¹¹, over the past few years, this class of compounds has been given momentous recognition in the field of biomedicine in light of AMR. Current probations reveal that ILs based on imidazolium, pyridine, choline, and pyrrole are extremely effective as therapeutic agents owing to their high charge and hydrophobicity¹². However, quinoline-based counterparts are still considered to be most potent against the pathogenic microbes¹². Additional biomedical applications accompanying this class of ILs include artificial chaperone activity¹³, cytotoxicity against cancerous cells¹⁴ as well as an excellent drug-carrying capacity¹⁵.

Conventionally, the fabrication of ILs involves the utilization of highly toxic solvent mediums such as dichloromethane, benzene, carbon tetrachloride, dichloroethylene, etc.¹⁶, hindering the biocompatibility and elevating the toxicity of the compound, making them undesirable for biological use. Additionally, the use of harmful solvents in the reaction media not only slows down the reaction time but also increases the unintentional production of waste byproducts released into the environment¹⁷. Moreover, the dissolvent used in the reaction media also influences the pH of the final product; hence, its removal at the end of the reaction is vital, especially when the desired compound is intended to be used for protein-related biological systems. Hence, keeping away from the usage of such solvent is favorable in the realm of green chemistry.

In this study, we report the one-pot synthesis of a biocompatible and non-toxic¹³ IL, namely, 1-Hexadecylquinolin-1-ium bromide, using a greener route. The present strategy omits the utilization of a molecular solvent, leveraging the self-solvating ability of the IL formed within the reaction mixture, promoting high reaction efficiency and chemical yield. Menschutkin reaction¹⁸forms the basis of the current synthesis methodology. The purity of the synthesized compound is probed using NMR and IR spectroscopy. The pharmacokinetic profile of the compound and toxicity were investigated through the ADMET studies. Furthermore, the antimicrobial potential of the synthesized IL against the pathogenic Candida albicans strain has also been demonstrated in the study.

Protocol

NOTE: 1-Hexadecylquinolin-1-ium bromide{[C₁₆quin]Br} was synthesized as described previously by Sharma et al.¹³.

1. Preparation and sterilization of glass apparatus

NOTE: This should be done at least 1 day prior to setting up the reaction for the synthesis of the desired compound.

1. Wash a 24/29, 250 mL, two-neck round bottom flask (RB) thoroughly, along with other glass apparatus such as measuring cylinders, etc., and rinse with distilled water followed by acetone.
2. Dry the washed apparatus in a hot air oven at 60 °C until the apparatus is completely dry for further use.
   NOTE: Usually, washed apparatus should be placed overnight in a drying oven to completely remove the water film and ensure that the reaction system is free from impurities.

2. Setting up the apparatus

NOTE: The apparatus should be clamped properly to ensure uniform heating of the reactants. The schematic diagram of the reaction set-up is demonstrated in Figure 1.

1. Place an oil bath on a hot plate with a magnetic stirrer. Preheat the oil bath to 80 °C before the commencement of the reaction.
2. Allow the RB to stand in an oil bath using a tube retort stand such that it is half immersed in the oil bath placed on a hot plate with magnetic stirrer.
3. Seal the upper mouth of the RB with a cork constituting a purging needle, further connected to a syringe with an N₂ balloon attached to it.
4. Seal the other neck of the RB tight using another rubber cork to avoid the leakage of N₂ from the reaction medium.
5. Preheat the entire medium to 80 °C in an inert atmosphere maintained through N₂ purging before adding the reactants to the RB.
6. Refill the N₂ balloon repeatedly to ensure the reaction system's inertness and maintain the temperature throughout (hence, oil bath heating is preferred).

3. Addition of the reactants to the reaction system

1. Pour 0.1 M Quinoline and 0.105 M 1-bromohexadecane into the reaction system without disturbing the preset reaction environment.
2. Stir the contents continuously at 2500-3000 rpm for 3 days under an inert environment and constant temperature.

4. Purification/Re-crystallization of the compound

NOTE: The entire product should not be subjected to re-crystallization. Instead, batch re-crystallization should be elected to avoid the loss of the product.

1. Dissolve the obtained solid in a 1:2 toluene/ethyl ethanoate mixture. Cool this mixture to -15 °C in a deep freezer (temperature set at -15 °C) and filter under vacuum using a Buchner funnel, attached to a vacuum pump and a filter flask through a tubing. Place a polypropylene filter membrane, constituting a pore size of 0.45 µm, in the Buchner funnel, covering the entire bottom of the filter. Pour a small amount of the solvent mixture through the filter in order to create a proper seal, preventing any sort of air leakage through the setup.
2. Wash the filtered product with cold toluene by pouring the solvent through the funnel gradually, followed by drying at 70 °C in a vacuum oven. Repeat this procedure 2x to ensure the high purity of the desired compound.

5. Validating the compound using NMR spectroscopy

1. Prior to subjecting the compound to ¹H NMR and ¹³C NMR, dissolve it in deuterated chloroform (CDCl₃) by measuring 1-10 mg of the compound and dissolving it in about 1 mL of CDCl_3.
2. Inject this mixture in an NMR tube using a 1 mL syringe for analysis of the sample under an NMR spectrometer. The sample preparation is identical for both ¹H NMR and ¹³C NMR.

6. IR characterization of the synthesized IL

1. No sample preparation is required to obtain the IR spectra of the compound.
2. Subject a few mg of the solid sample to an IR spectrometer to obtain an insight into the different functional groups present in the synthesized compound. The readings were obtained as demonstrated previously¹⁹.

7. Prediction of ADMET properties

1. Enter the canonical SMILES of the desired IL into the free online software ADMETLAB 2.0 and execute the program to obtain various parameters, vindicating the biological potential of the same.

8. Disc diffusion assay demonstrating the biomedical application of the synthesized IL

1. Pre-culture the fungal strain of Candida albicans (ATCC 90028) in yeast peptone dextrose (YPD) broth for nearly 16 h in a BOD shaker incubator at 37 °C.
2. Prepare YPD agar media by mixing 1% yeast extract, 2% peptone, 2% dextrose, and 1.5% agar in 1 L of double distilled water. Pour 25 mL of the freshly formed YPD agar media into a 90 mm Petri plate after autoclaving it for 15 min at 121 °C. Leave the plates undisturbed to allow proper solidification of the media.
3. Once the plate is completely solidified, spread around 100 µL of the freshly prepared fungal inoculum homogeneously over the plate containing the media, using a glass spreader. Leave the plates undisturbed for 5-7 min in the laminar flow hood.
4. Place a sterile circular paper disc of diameter of 5-6 mm in the middle of the same plate, using forceps.
5. Add 50 µL of 0.1 mM aqueous solution of the synthesized IL on the disc, gradually, with the help of a 20-200 µL pipette.
6. Refrigerate the plate for about 30 min to ensure proper diffusion of the IL into the agar. Place the plate in the BOD incubator pre-set at 37 °C for 24 h.
7. Measure the zone of inhibition (inclusive of the disc diameter) using a measuring scale and calculate the area under this zone as per equation 1.
   Area = πr²    (1)

Results

Figure 2 represents the reaction scheme of the Menschutkin reaction involved in bringing about the synthesis process. 1-Hexadecylquinolin-1-ium bromide, thus synthesized, was characterized using NMR and IR spectroscopy. The oily product so acquired is expected to exhibit ¹H NMR (400 MHz, CDCl₃) at δ 9.34 (d, 1H), 8.21 (d, 1H), 7.80 (t, 1H), 7.30-7.35 (m, 3H), 7.20 (d, 1H), 5.00 (t, 2H), 2.00 (p,2H), 1.30-1.35 (m, 26H), 1 (t, 3H), as demonstrated in Figure 3. The compound should show ¹³C NMR (Figure 4; 100 MHz, CDCl₃) at δ 143, 141, 131, 130.27, 130, 127.5, 126.3, 126, 60, 31, 29, 29, 28.5, 27, 23 and 14. Each peak is tagged with the corresponding proton number mentioned in the structure of the compound, responsible for bringing about that signal.

CDCl₃ is transparent to the NMR region (i.e., the radio-frequency region) of the electromagnetic spectra due to the absence of H-atoms that impart prominent signals in this region and is thus used as a solvent for sample preparation in its deuterated form. It does not interfere with the applied magnetic field and is completely inert in nature. However, it is not possible to eliminate the peak corresponding to this solvent (appearing at around 7.26 ppm for 1H NMR spectra) since the deuterization of the NMR solvents cannot reach up to exactly 100%. However, the peak appearing is usually not significant and can be completely removed by running a blank spectrum of CDCl₃ and integrating the required peaks accordingly. These settings can be either made directly within the instrument or by the utilization of certain software (like NMRium, etc.). In case the sample is not perfectly dried, the peak characteristic of water may appear prominently in the spectra. Additionally, CDCl₃ and other deuteriated solvents may also contain some amount of water and thereby impart the peaks attributed to the presence of water. This issue here was resolved by drying the sample properly and keeping the NMR solvent at room temperature, covered using a transparent film. In some cases, certain inert drying agents, such as potassium carbonate or sodium sulfate, are also used to get rid of the water content present in the bottle of CDCl₃.

The FTIR spectra of the IL (Figure 5) is expected to demonstrate vibration bands at 3051 cm^-1, 2917 cm^-1, and 2853 cm^-1, with maximum intensity, corresponding to the C-H stretching vibrations existing in the aromatic ring as well as for alkyl carbons. A broad shoulder at 3440 cm^-1 corresponds to the N-H stretching vibrations. Another shoulder band observed at 1357 cm^-1 is characteristic of stretching vibrations arising due to the C-N bond. Notable bands at 820 cm^-1,770 cm^-1, and 710 cm^-1 are associated with the out-of-plane C-H bending vibrations. The peak at 1155 cm^-1is specific to the in-plane C-H bending vibrations. The C-C stretching vibrations prevailing in the aromatic ring are validated by the peaks visible at 1468 cm^-1, 1520 cm^-1, and 1585 cm^-1. The results thus obtained are comparable with the previously reported data by Sharma et al.¹³ and hence confirm the formation of the desired compound.

The results obtained through the ADMET analysis are tabulated in Table 1. The biomedical potential of [C₁₆quin]Br was investigated using a well diffusion assay against the C. albicans strain (Figure 6). This technique evaluates the antimicrobial potency of IL by measuring the zone of inhibition of growth around the site of application of the compound. It was observed that [C₁₆quin]Br showed a statistically pronounced antimicrobial effect (p < 0.05) against C. albicans, making this class of ILs highly potent and effective in the field of biomedicine.

Percentage yield of the reaction
The theoretical yield of the reaction can be calculated as follows, as per the adopted reaction scheme (Figure 2).

From the scheme, 1 M quinoline leads to the formation of 1 M of 1-Hexadecylquinolin-1-ium bromide, i.e., 129.16 g/mol quinoline = 434 g/mol [C₁₆quin]Br. Hence,

12.916 g (0.1 moles) of quinoline ×12.916= 43.4 g

Thus, the theoretical yield of the reaction is 43.4 g.

However,

% yield= ×100

In the present study, the actual yield of the reaction was found to be 37.84 g after the complete drying of the product obtained.

Hence,

% yield= ×100=87.188% (87%)

Additionally, the longer reaction time, as seen in the present scenario, is pertinent for allowing the reaction system to attain complete thermodynamic equilibrium, where the system's entropy is maximized while minimizing the Gibbs free energy. This eventually maximizes the reaction's yield. However, the reaction conditions are quite mild and thus require an extended reaction time to ensure that the entire reactant has successfully converted into the product. The reaction time, here, is optimized after repeated pragmatism.

Figure 1: Reaction set-up. The figure displays the general schematics of the reaction setup. Please click here to view a larger version of this figure.

Figure 2: Synthesis mechanism. Schematic representation of the reaction scheme involved in the synthesis process. Please click here to view a larger version of this figure.

Figure 3: Expected ¹H NMR. Representation of the ¹H NMR spectra of [C₁₆quin]Br. Please click here to view a larger version of this figure.

Figure 4: Expected ¹³CNMR. Representation of the ¹³C NMR spectra of [C₁₆quin]Br. Please click here to view a larger version of this figure.

Figure 5: Expected IR spectra. Representation of the IR spectra of [C₁₆quin]Br. Please click here to view a larger version of this figure.

Figure 6: Anti-fungal study. Disc diffusion assay was performed by the addition of 50 µL of 0.1 mM of the IL, representing the anti-fungal potential of [C₁₆quin]Br. Please click here to view a larger version of this figure.

Property	Value	Comment
Lipinski rule	Accepted	MW ≤ 500; log P ≤ 5; H-acceptor ≤ 10; H-donor ≤ 5. If two properties are out of range, a poor absorption or permeability is possible, one is acceptable.
Gastrointestinal absorption	High	Enhanced efficacy of oral administration
Water solubility	Moderate	Enhances the drug carrying capacity of the compund
Bioavailability score	0.55 (55%)	Influences the therapeutic potential of the compound

Table 1: ADMET analysis. Table representing the important properties to be considered to validate the biological potential of the synthesized IL.

Discussion

Lately, ILs have divulged various promising implementations in the field of biochemical sciences including protein refolding/ chaperone activity, drug delivery vehicles, and/or catalysts in several organic reactions. Their intriguing physicochemical properties, such as tunability, biocompatibility, solubility, sustainability, stability, etc., have made them potential candidates for the development of novel therapeutic agents²⁰. The proposed research visualizes AMR as a matter of grave concern and one of the greatest threats to global health, food security, and development today, prioritizing the pursuit of the research and development of novel pharmaceutical compounds in the realm of biomedical science. In choosing quinoline-based IL for application in the biomedical industry, we recognize its high potency in the destruction of pathogenic microbes. Apart from the antimicrobial effect of this class of compounds, its artificial chaperone activity raises its merit in the biophysical aspect of the scientific world.

In this study, we successfully synthesized the desired IL using a greener reaction route. In contrast with the conventional methodologies that rely on the utilization of toxic solvents, evolving harmful gaseous byproducts²¹, here, we report a more economical, eco-friendly, solvent-free approach for the synthesis of the same. The protocol involves the utilization of a facile reaction set-up and simple apparatus, facilitating robust laboratory synthesis of the desired compound. The product procured here can be isolated effortlessly through a simple filtration technique. The formation of no substantial byproducts throughout the course of the reaction further promotes the greener aspects of the synthesis. The technique used in this study proved to be efficient both in terms of chemical yield and purity of the desired compound. The otherwise multi-step reaction was brought about in a single pot, with an intent to cut down on time and resources. This technique was adopted in order to get rid of the prolonged separation and purification stages involved in general organic synthesis.

However, the limitations accompanying the above-mentioned protocol involve thorough management of the compatibility of the reaction conditions (especially temperature) and ensuring proper purging of N2 gas in order to perpetuate an inert working environment manually. It is vital to control and monitor the reaction environment in a timely manner for the proper execution of each individual step. Scalability can be another possible impediment to the suggested protocol since scaling up a one-pot synthesis for industrial purposes can be extremely demanding owing to the concerns related to homogeneous mixing as well as control over each reaction parameter.

Conclusively, the reported methodology is a puissant strategy in green chemistry, offering several benefits, with its own set of challenges that need to be meticulously addressed and pondered upon while designing, executing as well and optimizing the reaction routes.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1-bromohexadecane	Merck	CAS no.112-82-3	95% pure (as determined by HPLC analysis)
Ethyl acetate	Merck	CAS no. 205-500-4	95% pure (as determined by HPLC analysis)
Nuclear Magnetic Resonance (NMR) spectrometer	Jeol, Model: JNM-ECZ 400S	Nil	Nil
Quinoline	Merck	CAS no.91-22-5	95% pure (as determined by HPLC analysis)
Toluene	Merck	CAS no. 108-88-3	95% pure (as determined by HPLC analysis)