PROBING INTERACTIONS BETWEEN IONIC LIQUIDS DERIVED FROM ESSENTIAL OIL-INSPIRED ANIONS AND HUMAN SERUM ALBUMIN VIA STD-NMR

Proteins are currently described as one of the most important molecules of life, responsible for many different functions in the human body. Although, especially in the pharmaceutical field, proteins are very sensible to work with, presenting some challenges to the industry, such as: stability, administration, production and long-term storage (1). Many approaches to enhance protein stability have been studied during the years. Recently, ionic liquids (ILs) have been explored as an alternative to enhance proteins and enzymes stability, as well as its progress of application as property modulating agents in the pharmaceutical industry. Ionic liquids are organic salts composed by an organic cation and an organic or inorganic anion. ILs are versatile compounds and have adjustable physicochemical properties (2), which are responsible for the interest and diverse applications of these materials. Saturation Transference Difference-Nuclear Magnetic Resonance (STD-NMR) it is one of the methods to investigate ligand–protein interactions (3). STD is an NMR technique used to study interactions between small ligand and large molecules, that works by selective saturating irradiation in a specific signal and observation how this saturation can be transfered to the ligand through intermolecular interactions, providing information about binding affinity and the ligand's binding site on the protein (4). In this study, we intend to show the interactions between two ILs ([DEC(MIM)][EUG]; [DEC(MIM)][CIN]) and human serum albumin (HSA), a protein responsible for maintaining the blood’s osmotic pressure and substance’s transport in human body, using STD-NMR. In previous studies, the selected ILs demonstrated strong antioxidant capacity against reactive species, such as free radicals. The ILs are derived from imidazolium cation with a long alkyl side chain and an organic anion inspired in the main components of clove and cinnamon essential oils: eugenol and cinnamic acid and, were chosen due the great antimicrobial activities. The ILs were synthesized and characterized from structural (¹H and ¹³C NMR) and thermal (DSC and TGA) analysis. The synthesis involved an S_N2 reaction of methylimidazole with the respective alkyl halide, followed by anion exchange (from halide to hydroxide) using resin and subsequent neutralization with acid of the anion of interest. STD-NMR experiments were performed at 25°C on a Bruker 600 MHz spectrometer. The [DEC(MIM)][EUG] and [DEC(MIM)][CIN] samples were solubilized at 50 µM in a solution containing 5 µM HSA and D₂O. The on-resonance irradiation was performed at 1.60, 2.15 and 2.84 ppm, off-resonance irradiation was applied at 40.0 ppm. The irradiation frequencies were determined based on a standard ¹H NMR spectrum of the HSA protein, following the methodology of Walpole et al. (2019). A saturation pulse power of 7.3 mW was applied. The saturation time was set at: 0.5; 0.75; 1.0; 2.0; 4.0; 6.0 and 8.0 seconds. Subsequently, an STD build-up curve was generated, representing the STD intensity as a function of saturation time. This was followed by the fitting of the exponential curve using STD equation. The results made possible the construction of an epitope map, based on STD% of the signals monitored, which identifies the regions of closest proximity and strongest interaction between the protein and the ligand nuclei. To avoid accounting for direct irradiation, only signals with chemical shifts greater than 1 ppm from the irradiation frequency were considered. STD-NMR experiments showed that HSA interacts with the cation alkyl chain as well as the anion of both ionic liquids. Additionally, when the IL cation is irradiated, it was observed that the anion receives irradiation due to spatial proximity. These observations suggested that the ILs are found as ion-pair. Based on the epitope mapping, a stronger interaction was observed for HSA and [DEC(MIM)][EUG], indicating a closer proximity between the IL anion and the protein surface. In the case of [DEC(MIM)][CIN], a stronger interaction was observed between HSA and the alkyl chain. The findings of this study demonstrate that although both ILs can interacts with human serum albumin the structural characteristics of the ILs were shown to influence their proximity and interactions with the protein. Understanding the role of these ligands of distinct characteristics in the stabilization of human serum albumin is of great importance, given their previously demonstrated strong antioxidant capacity and their potential to protect HSA against the action of radical species.

REFERENCES

(1) GUNCHEVA, M. Role of ionic liquids on stabilization of therapeutic proteins and model proteins. The protein journal, v. 41, n. 3, p. 369–380, 2022.

(2) MARTINS, M. A. P. et al. Chem. Rev. 108 (2014), 6, 2015.
(3) WALPOLE, S. et al. STD NMR as a technique for ligand screening and structural studies. Methods in enzymology, v. 615, p. 423–451, 2019.

(4) MONACO, S. et al. Differential Epitope mapping by STD NMR spectroscopy to reveal the nature of protein–ligand contacts. Angewandte Chemie: v. 129, n. 48, p. 15491–15495, 2017.

Acknowledgements: The authors would like to express their sincere gratitude to the funding agencies CNPq, CAPES and FAPERGS as well as to the institution UFSM, for their valuable support and assistance in the development of this work.