Scheme 3.1. Schematic representation of protein-ligand binding models
3.2. Some Recent Small Molecule Probes of BSA
To gain insight into structure, functions and binding interactions of BSA with drugs and small fluorescent molecules, fluorescence-based techniques have been frequently utilized. The protein binding ability of a variety of small molecules, drugs, chromophores has been studied by utilizing their fluorescence photophysical properties.35-40 In 2015, Li et al. reported a 1,2, 5- triphenylpyrole (TPP) derivative, 3.01 (Figure 3.2) and utilized it for the quantification of BSA and HSA without isolation from serum.41 The compound showed aggregation induced emission property (AIE) and found to correlate linearly with both BSA and HSA over a wide range of concentration (2.18-70 µg/mL for BSA) with an enhancement of fluorescence signal. It was reported that the interactions occurred between the compound and BSA are mainly hydrophobic and hydrogen bonding. Malathi et al. designed and synthesized a benzimidazoquinoline derivative, 3.02 for sensing of BSA and metal ions.42 It was reported that this compound also showed AIE properties and strong binding interaction with BSA due to a FRET process from the BSA to the probe. An enhancement of AIE (AIEE) of the probe was observed in the presence of BSA. Cell viability assay for both of the compounds (3.01, 3.02) indicated low cytotoxicity and in-vivo experiments suggested their future application in cell imaging. Yang et al. reported two red-NIR (red to near-infrared) probes (3.03 and 3.04) for the quantitative and qualitative detection of both BSA and HSA.43 These two probes showed differential recognition towards BSA and HSA in terms of fluorescence intensity even though
HSA
these two proteins have structural homology. The compound 3.03 showed strong fluorescence in the presence of HSA, while 3.04 being highly selective towards BSA showed a strong fluorescence upon interaction with BSA. Docking study revealed that the active binding site of 3.04 was located in between subdomains II and IIA of BSA and binding interaction mainly depend upon hydrogen bonding interaction. On the other hand, the binding site of 3.03 was located within the site I of HSA and binding interactions were mainly hydrophobic and π-π stacking.
Figure 3.2. Examples of various molecular probes for BSA.
Polyaromatic hydrocarbons (PAH) constitute a class of highly fluorescent molecules with strong photophysical properties. Thus, these molecules and their derivatives often utilized as fluorescent probes in various investigations related to proteins. For example, Wang et al.
studied diketopyrrolopyrrole (DPP)-anthracenone conjugates for sensing of BSA.44, 45 One representative example of their work is the compound 3.05 (Figure 3.3) which exhibited enhanced fluorescence emission upon binding with BSA.44 It was reported that this compound exhibits amphiphilic character due to its structure and undergoes complexation with BSA via electrostatic and hydrophobic interactions. Densil et al. reported three anthracene-derived Schiff base compounds, 3.06-3.08 as BSA sensing probes.46 It was reported that these compounds are low toxic and AIE active and exhibit strong emission upon binding with BSA with an enhancement in their fluorescence signal. The binding interactions of all the three compounds with BSA were found to be mainly hydrogen bonding and hydrophobic interaction.
Ali et al. reported a phenanthrene-pyrene fluorescent conjugate (3.09) for probing BSA.
Association of this compound with BSA caused both static and dynamic quenching of BSA fluorescence.47 Experimental and molecular docking study revealed the location of the binding site within the subdomain IIA of BSA. Singh et al. reported a perylenediimide- benzimidazolium derived fluorescent probe (3.10), which showed selective detection of BSA
and HSA among other proteins and inorganic ions.48 This compound exhibited AIE characteristics and underwent complex formation with BSA with an enhancement in its fluorescence signal. This compound is reported as low cytotoxic and finds applications in detection of HSA in blood serum, urine samples and cell imaging in HeLa cells.
Figure 3.3. PAH derived fluorescent probes for BSA.
Synthetic dyes find widespread applications in the field proteins analysis such as characterization of protein folding, protein surface-hydrophobicity sensing, and detection of protein aggregation etc.49-55 Superior fluorescence emission properties of synthetic and natural dyes established themselves as powerful components of fluorescent tools. As for an example, Jurek et al. investigated binding interactions of a few amino-substituted squaraine dyes with BSA.56 Two representative examples of their work are 3.11 and 3.12 (Figure 3.4). Both of the compounds showed efficient binding interaction with binding constants 45.5 × 104 and 37.3 × 104, respectively. Reis et al. also reported several squaraine dye derivatives for the detection of BSA and HSA.57 A few examples (3.13-3.15) of their work are shown in Figure 3.4. Authors reported low solubility of these compounds in aqueous media which led to poor fluorescence emission. However, significant enhancement in fluorescence intensity was observed in the presence of BSA and HSA, which indicated a strong interaction between these compounds and BSA/HSA. Onganer et al. studied the photophysical and biophysical impact of coumarin 35 dye (3.16) on BSA.58, 59 A strong interaction between the coumarin 35 dye with BSA was reported leading to a significant enhancement in the fluorescence signal the probe. The authors also studied the influence of certain metal cations on the binding interactions between the
Coumarin 35 dye and BSA. The binding constant of the BSA-coumarin35 complex was found to be increased in the presence of metal ions and was highest in the presence of Sn4+ ions. Qian et al. investigated boron-dipyrromethene (BODIPY)-triphenylamine derivatives (3.17 and 3.18) for sensing of BSA.60 These fluorescent compounds with AIE properties showed efficient association with BSA with an enhancement in their fluorescence signal. Vodyanova et al. also reported several BODIPY derivatives which can be utilized as fluorescence light-up probes for the detection of BSA.61 Some representative examples of their work are shown in Figure 3.4 (3.19-3.21).
Figure 3.4. Fluorescent probes derived from various dyes for BSA detection.
Our research group also contributed towards the detection and sensing of BSA.62, 63 As representative examples, two recent fluorescent molecules (3.23 and 3.24) reported by our research group are shown in Figure 3.5. Both the compounds exhibited strong fluorescence signals upon binding with BSA and therefore can be utilized as light-up fluorescent probes for the detection of BSA. Experimental results and molecular docking study indicated that the
binding interactions in both cases are mainly due to hydrophobic and electrostatic interactions in the hydrophobic pocket of BSA.
Figure 3.5. Fluorescent molecules reported by our research group for sensing of BSA.