Nickel-L-leucine framework (MOF): synthesis, structural characterization, and potential applications in biological studies

The microbial resistance to antibiotics and the generation of free radicals inside as a result of different oxidative processes are modern global challenges for researchers. The exploration of MOFs as an antibacterial agent against pathogenic bacteria and as an antioxidant agent to scavenge free radicals as countermeasures to alleviate these problems. For this purpose, the metal organic framework (MOF), composed of L-leucine as a linker and nickel as a metal, was synthesized via a convenient, one-pot process under reflux conditions. The products formed were characterized through different techniques, including N2 adsorption experiments, Fourier transform infrared spectroscopy (FTIR), transmission electron microscopy (TEM), scanning electron microscopy (SEM), and X-ray diffraction (XRD). The physicochemical analysis shows that the MOF has a crystalline nature with a surface area of 129 (±5) m2/g and a pore size of 1.95 nm. The synthesized MOF was then subjected to antibacterial activity, and the high activity was recorded against S. aureus. The dose-dependent antioxidant study shows the activity increases with increasing the concentration of the MOF. However, both the antibacterial and antioxidant activities were found to be less than those of the standard drugs (clindamycin and ascorbic acid).


Introduction
Drug-resistant strains of bacteria, viruses, and other pathogens develop through a process known as natural selection and genetic mutation.This process occurs when microorganisms are exposed to drugs (such as antibiotics or antiviral medications) that are designed to kill or inhibit their growth [1].Antibiotic resistance poses a significant threat to public health, rendering many existing treatments ineffective against bacterial infections [2,3].Oxidative stress is a physiological condition that occurs when there is an imbalance between the production of reactive oxygen species (ROS) and the body's ability to detoxify and repair the resulting damage.ROS are highly reactive molecules that contain oxygen and are generated as natural byproducts of various metabolic processes within cells.While ROS play essential roles in cell signaling and defense against pathogens, an excess of these molecules can lead to oxidative stress, which can damage cells, proteins, lipids, and DNA [4][5][6].
To address these challenges by exploring innovative solutions through the synthesis of a MOF using L-leucine as a linker and nickel as a metal.
Metal-organic frameworks (MOFs) are a class of porous materials consisting of metal ions or clusters connected by organic linkers [7].These materials have a highly regular, three-dimensional structure with a high surface area, making them useful in a variety of applications such as gas storage and separation, catalysis, sensing, and drug delivery [8,9].The unique properties of MOFs arise from their precise control over pore size and shape, as well as their tunable functionality through the choice of metal ions and organic ligands [10].The porous nature of MOFs allows for the storage of gases such as hydrogen, methane, and carbon dioxide, and their separation from other gases [11,12].MOFs also have applications in catalysis, where the metal ions act as active sites for chemical reactions, and in drug delivery, where the pore size and shape can be tailored to allow for the controlled release of drugs [13].MOFs have gained significant attention in recent years due to their potential in addressing environmental, energy-related challenges and their use in carbon capture and storage, water purification, and solar energy conversion also been explored [14,15].Furthermore, the MOFs have been used in a variety of other applications such as chemical sensing, electrochemistry, and even in the fabrication of electronic devices [16].
Amino acids (AAs) as bioligands provide wide range of functionalities and coordination sites [17].AAs are organic compounds containing an amine group, a carboxylic acid group, and a specific side-chain that is R group.The role of AAs in living system is indispensable to run normal functioning as AAs are part of proteins and proteins make nearly 75 percent of our body.They play their role in almost all body functions for instance, growth, development, healing and repairing, food digestions and many more [18].AAs also have therapeutic applications primarily, improve cardiovascular health and immunity.Some amino acids are synthesized in our body while some AAs are obtained from outside sources.Based on these prerequisites, AAs are classified nonessential and essential.Additionally, some AAs are required conditionally and are regarded as semi essential AAs.AAs are part of our food and are taken in food on daily basis [19].The organic ligand, L-leucine is α-amino acid and human body cannot synthesize it hence it is an essential amino acid.In body, leucine together with other essential and non-essential AAs, is utilized during effort and exertion to supply immediate energy [20].The originality lies in the unique combination of L-leucine and nickel within a MOF, synthesized using a convenient one-pot reflux process.This innovative approach yields a MOF with exceptional structural characteristics, paving the way for novel applications in biological studies.Furthermore, the assessment of antibacterial and antioxidant activities provides original insights into the versatile potential of this MOF, underlining its distinctiveness in addressing contemporary global challenges.
This article reports the synthesis of nickel-L-leucine framework (MOF) by simple stirring under reflux conditions.The prepared MOF was characterized by XRD, FTIR, TEM, SEM, and N 2 adsorption techniques.The biological potentials, i.e., antibacterial and antioxidant activities, were examined against the selected bacteria and free radicals.This study aims to contribute to the development of multifunctional materials that can combat antibiotic resistance and mitigate oxidative damage, thereby offering new avenues for biological applications and potential advancements in medicine.

Synthesis of nickel-L-leucine framework
A previously publicised method, one-pot synthesis [21], was used to synthesize the nickel-L-leucine framework; 5 mmol of NiCl 2 and 20 mmol of L-leucine are separately dissolved in DMSO.Then both solutions were mixed in a 100 ml round-bottom flask and subjected to reflux with continuous mechanical stirring for 14 h.A solid product was formed, and the reaction mixture was allowed to cool to room temperature.The product was separated and washed twice with distilled water and once with methanol.After washing, a greenish-grey product was dried in the oven at 60 °C.

Instrumentation
The surface area and pore size distribution were studied using Gemini model 2390t, whereas the BET and BJH equations were applied to N 2 adsorption data to determine the texture parameters.To study the crystalline nature and other crystallographic parameters, XRD (model: Panalytical X'pert Pro) was run.The surface topology was examined through SEM (model JSM 5910) and TEM (model HITACHI H-7700), whereas the chemical composition of MOF was studied via FTIR (model Nicolet 6700) [22,23].

Bactericidal assay
The agar-well diffusion method was employed to investigate the bactericidal potential of the nickel-L-leucine framework against the selected bacterial species.Bacterial cultures were spread onto agar plates using swabs, and sterile borer was used to create wells.The nickel-L-leucine framework dispersion was prepared by ultrasonication of 1 mg of MOF in 1 ml of distilled water.A specific volume of the prepared suspension was added to each well of the plates and incubated at 37 °C.The diameter of the clear zone around the wells was measured in millimetres and used to determine the activity of the nickel-L-leucine framework.

Free radical scavenging assay
The antioxidant activity of the framework was evaluated using both ABTS and DPPH free radical scavenging assays.
For the ABTS assay, a stock solution of ABTS • + was prepared by mixing ABTS and potassium persulfate in water and allowing it to react for 16 h in the dark.The solution was then diluted with ethanol to an absorbance of 0.70 ± 0.02 at 734 nm.The nickel-L-leucine framework was dissolved in DMSO and added to the diluted ABTS solution.The absorbance was measured after 10 min at 734 nm and the percentage of ABTS • + scavenging was calculated.
For the DPPH assay, the nickel-L-leucine framework was dissolved in DMSO and added to a DPPH solution in ethanol.The absorbance was measured after 30 min at 517 nm and the percentage of DPPH scavenging was calculated.

Surface area and pore distribution analysis
The surface area and pore size distribution analysis of the synthesized MOF was carried out using the BET and BJH equations applied to N 2 adsorption data, and the obtained plots are shown in figure 1.The plot (a) shows that with increasing the relative pressure, the adsorption gradually increases, and the maximum adsorption of 39.91 cm 3 /g is obtained at 0.25 p/p°.Up to 0.15 p/p°, the adsorption rapidly increased and then slowed gradually up to 0.25 p/p°.The specific surface area (S BET ) determined via the multiple points BET equation is 129 (±5) m 2 /g, assuming that the particles are solid with a smooth surface.The BJH adsorption accumulative volume of pores is 0.010118 cm 3 /g, whereas the BHJ adsorption average pore size is 1.95 nm.

XRD analysis
The XRD pattern of the MOF (which comprises L-leucine and nickel) is shown in figure 2, which possesses several diffraction peaks for l-leucine at 12.17, 18 and 2.87, respectively.All these peaks and the d-spacing values are matched with reference card no.00-005-0274, attributed to the formation of an unknown crystal system.The crystallite size determined is 5.14 nm with a lattice strain of 0.383 percent.The other diffraction peaks, along with the hkl values at 15.43 (003) and 36.96(104), correspond to JCPDS card no.00-001-1134, which confirm the presence of nickel chloride in the sample with a rhombohedra crystal system with a space group of R-3m and a crystallite size of 67.9 nm with a 0.377 percent imperfection.

FTIR analysis
The FTIR spectrum of Ni-L-leucine framework (figure 3) ranging from 4000-400 cm −1 exhibits several peaks corresponding to the functional groups and vibrations of both the nickel ions and the L-leucine ligands.A broad peak at around 3300-3400 cm −1 , corresponding to the stretching vibration of the N-H and O-H bonds in the L-leucine ligand [24].A peak at around 2934.63 cm −1 was attributed to the stretching vibration of the C-H bond in the L-leucine ligand.The peak at 1899.57cm −1 was due to the stretching vibration of the C=O bond in the carboxylic acid group of the L-leucine ligand [25].A strong peak at around 1629 cm −1 was attributed to the bending vibration of the N-H bond in the L-leucine ligand [26].A weak peak at around 1380 cm −1 , because of the stretching vibration of the C-N bond in the L-leucine ligand [24].The vibrational frequency at 1103 cm −1 indicates the vibration of O-Ni-O [27].The band 615 cm −1 specifies the formation of metal linker bond [28].

SEM analysis
The low-and high-magnification SEM micrographs shown in figure 4 reveal the porous nature of the MOF.The low-magnification SEM image shows that the particles are irregularly arranged, forming a porous network.The majority of the particles appeared to have their individual bounders; however, in some regions, the particles are combined together, forming a flat surface, where they lose their individuality.For the closed surface  examination, the SEM analysis was carried out at high magnification, as shown in figure 4(b).The irregularly shaped (few elongated) particles are closely attached to each other, forming a bunch-like structure, and the size of that larger structure depends upon the number of particles present in each cluster.The particle size estimated from the SEM image ranges from 87.29 to 120 nm, with an average size of 105.74 nm.

TEM analysis
The TEM image presented in figure 5 offers a more detailed understanding of the morphology of the synthesized MOF, revealing a diverse range of structures within the sample.The TEM image shows the mixed morphology of the synthesized MOF.In the lower region, the particles appear to be fused together due to the high surface energy, resulting in the disappearance of boundaries between adjacent particles.This fusion process leads to the formation of a sheet-like structure where the individual particles lose their separate identities and merge into a continuous sheet.Whereas in the upper region, particles exhibit a closer connection with each other, but the boundaries between the particles can still be observed upon closer examination.This indicates that the particles  in this region have retained their individual morphological identities, even though they lack a specific, defined shape.Within this region, most of the particles exhibit a nearly spherical shape, while others possess a polyhedral morphology.The distribution of these particles appears to be random throughout the sample.The TEM image also provides insights into the size distribution of the MOF.It reveals a wide range of particle sizes, ranging from 25 to 60 nm.Notably, the lower range of sizes is particularly close to the crystallite size of the MOF, which was determined during the XRD study.Comparing the TEM image to the SEM images, it is evident that the grains observed in the SEM images appear larger than the particles seen in the TEM image.In fact, the grains observed in the SEM images are approximately 2 to 3 times larger than the particles resolved in the TEM image.

Antibacterial activity
Two strains of bacteria were taken as test specimens, namely, E. coli (Gram negative) and S. aureus (Gram positive), and the agar-well diffusion method was used to test antibacterial activity.In conventional solvents, all the synthesized products were insoluble at room temperature.To test the activity against Gram positive and Gram-negative bacteria, 1 mg of MOF was added to 1 ml of DMSO and sonicated to form a suspension.The standard antibiotic clindamycin was taken as a positive control.The pictorial representation of the antibacterial activity is given in figure 6, where three wells are present in each plate.The antibiotic showed the highest activity, followed by the synthesized MOF, whereas the solvent showed no activity.That means the activity is just due to the antibiotic or MOF, and the solvent has not contributed to the activity.The results also shows that the activity of the synthesized MOF is less than that of the standard drug.The S. aureus shows less resistance toward the antibiotic and synthesized MOF as compared E. coli.This difference in the activity could be attributed to the specific mode of action of the antibacterial agent and also to the variation in the cell wall composition of both types of bacteria [29,30].
Antibacterial agents like antibiotics and MOF have specific mechanisms of action that target bacterial cells; they might disrupt bacterial cell membranes or interfere with essential metabolic processes.The effectiveness of antibiotic and MOF can vary depending on the susceptibility of the target bacteria to these mechanisms.This specific mode of action may be more effective against Gram-positive bacteria due to differences in their cell wall composition.S. aureus has a thick peptidoglycan layer in their cell walls that provides structural support and plays an essential role in protecting the bacteria from environmental stresses.However, it also makes them more susceptible to agents that target cell wall synthesis or disrupt the integrity of the cell wall.E. coli has a thinner peptidoglycan layer and an additional outer membrane composed of lipopolysaccharides.This outer membrane acts as a barrier and can make it more challenging for certain antibacterial agents to penetrate and reach the cell's interior.Moreover, E. coli often has efflux pumps that can actively remove antibiotics from the cell, contributing to resistance.The combined effect of these factors can lead to the observed difference in antibacterial activity, with the MOF and antibiotics being more effective against S. aureus compared to E. coli [31].

Antioxidant activity
The ABTS free radical cation scavenging potential of the Ni-L-leucine framework was evaluated at different concentration and the results are shown in table 1.It has been observe that the percent scavenging potential of the MOF increases with increasing the concentration of the MOF in the reaction and a maximum of 90.86 percent of the radicals has been stabilized when a concentration 40 mg ml −1 was used.The activity of the MOF was compared with activity of ascorbic acid which was used as standard.The determined IC 50 value for MOF is 17.81 mg ml −1 , which is found slightly higher than that determined for ascorbic acid (16.14 mg ml −1 ).This suggest the higher efficacy of the ascorbic acid as compared to MOF.The chemical reaction for scavenging ABTS free radical cation using nickel-L-leucine framework is represented as follows:

MOF ABTS MOF ABTS
• 2 In this reaction, the ABTS free radical cation (ABTS •+ ) reacts with the nickel-L-leucine framework (MOF), resulting in the formation of a reduced form of ABTS and a Ni(II) complex (Ni-L-leucine +2 ).The Ni(II) complex is formed as a result of the electron transfer from the nickel ion in the framework to the ABTS •+ radical, neutralizing it and preventing it from causing oxidative damage.The reduced form of ABTS is colorless, while the ABTS •+ radical is blue-green in color.The scavenging of ABTS •+ radical cation by nickel-L-leucine framework indicates its potential as an antioxidant agent [32,33] The scavenging potential of the MOF towards DPPH free radical cation was assessed at varying concentrations, and the findings are presented in table 2. The results demonstrated that the percentage of scavenged radicals by the MOF increased with an increase in MOF concentration in the reaction.The highest observed percent scavenging potential was 88.82%, achieved with a concentration of 40 mg ml −1 , however the percent activity of the ascorbic acid was found higher than the MOF at each concentration.The IC50 values for both MOF and ascorbic acid are 14.85 mg ml −1 and 8.56 mg ml −1 also suggest the higher efficacy of the ascorbic acid at lower concentration.The chemical reaction for scavenging DPPH free radical cation using nickel-Lleucine framework can be represented as follows: In this reaction, the DPPH free radical cation (DPPH•) reacts with the MOF, resulting in the donation of an electron from the nickel ion to the DPPH• radical.This results in the neutralization of the DPPH• radical and the formation of a reduced form of DPPH (DPPH-H).The nickel ion in the framework is oxidized to a Ni(II) complex, which forms as a result of the electron transfer from the nickel ion to the DPPH• radical.The leucine ligands in the framework may also contribute to the scavenging of DPPH free radical cation by acting as radical scavengers [32,34]  To compare the antioxidant activity of the present study with those already reported in the literature, as shown in table 3. The collected data shows that the synthesized MOF exhibits significantly enhanced antioxidant activity against both ABTS and DPPH free radicals compared to the MOF reported in the literature.The optimized synthesis conditions, unique ligands and metal ions interaction, structural modifications, composition, purity, and surface reactivity are the factors that can affect the antioxidant activity of MOF [35,36].

Conclusion
The goal of this research is to synthesized biologically active novel compounds and test them for various biological applications.For the purpose, L-leucine-nickel framework synthesized via very simple one spot synthesis under reflux conditions.The high surface of the MOF determined via N 2 adsorption experiment signify the potential of the MOF for various applications.The crystallographic study shows that the all the three MOF exhibits both crystalline and amorphous phase, which is evident from the sharp diffraction bands and the noisy XRD pattern.It has been experience that the majority of metal contents is present in amorphous phase whereas the organic portion of the MOF are well crystalline.The morphological study shows that the molecules are combine together form larger aggregates, due to which the exact morphological shape of the MOF is difficult to determine.The chemical composition of the MOF were investigated through FTIR and slight shifted peaks for the organic moieties is due to the insertion of the metal (Ni) in the organic network.The antibacterial results demonstrate that the activity of the MOFs were higher against S. aureus as compared to E. coli.This increase in the activity is may be due to the difference in the cell wall composition and surface charges of the both bacterial cell.The antioxidant nature of the synthesized MOFs was explored against ABTS and DPPH free radicals.The IC 50 values shows that the activity of the MOFs was higher against DPPH free and as compared to ABTS free radicals.However, in case of the performed biological activities, the efficacy of the MOF was found less than the standard drug.

Figure 1 .
Figure 1.BET plots for N 2 adsorption data of Ni-L-leucine framework.

Table 3 .
Comparison of the antioxidant activity of synthesized MOF with the MOF reported in the literature.