Molecular interactions of bovine serum albumin (BSA) with pyridine derivatives as candidates for non-covalent protein probes: a spectroscopic investigation
Graphical abstract
Introduction
Human proteins are recognized as some of the most significant bioactive molecules owing its involvement in nutritional, metabolic, and immune processes [1]. Furthermore, the composition of biomacromolecules in body fluid has the potential to be employed in clinical diagnostics as vital indicators [2]. Hence, the determination of blood protein concentrations is considered of such prominence in the fields of life and environmental sciences and clinical diagnostics [3], [4], [5]. The liver-produced and most dominant, albumin is a plasma protein, accounting for 55–65 % of all blood proteins [6]. Also known as a key transport protein, it has the ability to bind ligands, both acidic and neutral, such as ions, lipids, drugs, and toxins [7]. Albumins also influence the maintenance of normal blood volume and pH conditions while providing significant antioxidant potential [3], [8], [9], [10], [11], [12], [13], [14], [15]. Among serum albumins, Bovine Serum Albumin (BSA) is widely applied as a model protein for evaluation of the interactions for active compounds with albumin due to its low cost, good availability, and structural homology to Human Serum Albumin (HSA) [16]. Structurally, BSA is a globular protein (66,4 kDa) consisting of 583 amino acids arranged in the single-chain polypeptide. The primary structure of BSA consists of 17 disulphide bonds holding nine loops, which create three homologous domains (I, II and III), each consisting of two sub-domains (A, B) [17]. This molecular structure of albumin means that compound binding should be considered at two levels. The structural backbone of albumin determines the binding site of the compound, while the structural elements of the molecule affect the strength of binding [10]. The secondary structure consists of approximately 67 % of α-helix. There are no β-harmonic structures in the albumin structure, which provides the protein with high conformational flexibility. As a result, albumin has multiple hydrophobic binding sites and binds both exogenous and endogenous compounds, which has been confirmed by using, among others, X-ray crystallography studies [18]. Six major binding sites have been identified in the albumin molecule that shows high specificity for ligands [10]. Due to differences in the structure of these sites, which are characterized by distinct size and polarity, it is possible to simultaneously bind compounds with different structures and sizes. Two binding sites that show high affinity for drugs and endogenous ligands have been characterized [19]. These sites are located in subdomain IIA and IIIA and tend to bind acidic and lipophilic compounds [14], [15], [20], [21]. BSA shares 76% of sequence with HSA and contains two tryptophan (Trp) amino acid residues, Trp-134 and Trp-212, located respectively in domains I and II [22], [23], [24]. In comparison to BSA, HSA houses only one tryptophan (Trp) amino acid residue (Trp-214). The Trp-134 residue is located in a hydrophilic environment, near the surface of sub-domain IB, while Trp-212 residue is located within a hydrophobic protein pocket of sub-domain IIA [25], [26], [27], [28]. Very different types of interactions can occur between albumin and ligand, such as van der Waals forces, electrostatic forces, hydrogen bonds, hydrophobic interactions and irreversible covalent bonds [29]. The long half-life, high concentration and number of binding sites explain the fact that so many metabolic compounds and therapeutic drugs are transported by albumin [20], [29].
A change in albumin concentration may be due to several reasons indicating potential inflammation or disease [30]. A low level of albumin in the blood is called hypoalbuminemia. For example, the presence of inflammation in the body causes a reduction in plasma albumin concentration by up to 30–60% from the physiological value [31]. The reduced concentration of this protein alters the concentration of the free fraction of exogenous and endogenous substances with which it is associated [32]. Another reason may be renal insufficiency due to changes in blood pH and accumulation of compounds competing with drugs for binding sites, which may affect the binding of drugs with albumins [33]. Moreover, in the course of renal failure excessive loss of albumin by damaged glomeruli occurs, which results in decreased serum albumin concentration [32], [34]. Therefore, it is important to develop efficient and sensitive sensors to determine albumin levels quickly and accurately.
Functional properties of 2-amino-4,6-diphenyl-pyridine-3-carbonitrile derivatives to determine albumin concentration has been performed following fluorescence emission of the Trp residues or extrinsic fluorophores bound to the protein [35], [36]. Pyridine derivatives have been widely used in medicinal chemistry because of their well-described properties such as stability, basicity, and ability to form hydrogen bonds [37], [38], [39], [40]. Additionally, due to their properties, they have been used in the development of new drugs, for example, against Alzheimer's disease or anticancer drugs [41]. However, a major obstacle to fully exploiting the potential of pyridines is their insufficient solubility in water. One of the most popular methods to cope with poor solubility of the tested compounds is an admixture of aqueous solutions with an organic solvent (for example, dimethyl sulfoxide) or the use of cyclodextrins [42], [43], [44]. In a wide range of methods used to determine the interaction of drugs with protein, fluorescence techniques are the most popular due to their high sensitivity, rapidity, and ease of implementation.
The aim of the investigation was to study the interaction and affinity of new 2-amino-4,6-diphenyl-pyridine-3-carbonitrile derivatives Figure 1 for BSA using UV–Vis and fluorescence spectroscopy to determine applicability of derivatives as a fluorescent probe for quantitative and qualitative determination of albumin. Moreover, the binding parameters such as Stern-Volmer constant, binding constant, number of binding site and Gibbs free energy were calculated to clarify mechanism of quenching. Förster distance and FRET efficiency were calculated based on the FRET experiment. Additionally limits of detection and quantitation was calculated as well as the influence of various ions and pH on complex formation.
Section snippets
Synthesis
The target compounds were synthesized following previously published procedure [45], [46]. Structural formulas of the synthesized 2-amino-4,6-diphenyl-pyridine-3-carbonitrile derivatives are presented in Table S1.
All reagents and solvents were analytical grade and used as received. Structure and purity of obtained products were confirmed by NMR and LC–MS analysis. 1H and 13CNMR spectra were recorded in DMSO–D6 on Avance III HD 400 MHz (Bruker, USA) spectrometer (Figure S1-S12). Chemical shifts
Absorbance and fluorescence spectra
In this study, a series of pyridine derivatives was synthetized and their solubility in water was examined. The best results were obtained for the derivatives containing a (4-dimethylamino)phenyl group and a 4-cyanophenyl group at positions 4 and 6, respectively. These substituents provided sufficiently high solubility in water that the addition of DMSO was not necessary. A small addition of DMSO was necessary for derivative A-64A-PC containing a 4-(dimethylamino)phenyl group at position 6 and
Conclusion
A series of new derivatives of 2-amino-4,6-diphenyl-pyridine-3-carbonitrile was studied as fluorescence probes for BSA determination. The interaction on investigated probes and BSA leads to 12–176-fold enhancement of emission intensity. Calculated binding parameters allow to conclude that those probes binds with BSA in a 1:1 stoichiometry with a moderate strength and the quenching for probes A-64A-PC and A-64A-44CN-PC is due to static interaction. For probe A-46bisA-PC mixed mechanism of
CRediT authorship contribution statement
Patryk Szymaszek: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Data curation, Writing – original draft, Writing – review & editing, Visualization, Project administration, Funding acquisition. Paweł Fiedor: Conceptualization, Software, Validation, Formal analysis. Anna Chachaj-Brekiesz: Formal analysis, Investigation, Data curation. Małgorzata Tyszka-Czochara: Methodology, Investigation. Tomasz Świergosz: Methodology, Software, Validation, Resources, Writing –
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
The authors are grateful to the Foundation for Polish Science (Warsaw, Poland) - Project POWROTY (Contract No. POIR.04.04.00-00-1E42/16-00 - POWROTY/2016-1/4 – „Synthesis and photochemistry/photophysics studies of the intelligent luminescent molecular sensors for selective detection in biochemistry and chemistry”) for financing part of the synthetic research. Moreover, authors are also grateful to the Ministry of Science and Higher Education (MNiSW) under the Diamond Grant project, contract
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