Synthesis of imidazole derivatives and the spectral characterization of the binding properties towards human serum albumin

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 153 (2016) 688–703

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Synthesis of imidazole derivatives and the spectral characterization of the binding properties towards human serum albumin Yuanyuan Yue ⁎, Qiao Dong, Yajie Zhang, Xiaoge Li, Xuyang Yan, Yahui Sun, Jianming Liu ⁎ Collaborative Innovation Center of Henan Province for Green Manufacturing of Fine Chemicals, School of Chemistry and Chemical Engineering, Henan Normal University, Xinxiang, Henan 453007, PR China

a r t i c l e

i n f o

Article history: Received 11 March 2015 Received in revised form 10 August 2015 Accepted 26 September 2015 Keywords: Imidazole derivatives Human serum albumin Quenching mechanisms Molecular modeling

a b s t r a c t Small molecular drugs that can combine with target proteins specifically, and then block relative signal pathway, finally obtain the purpose of treatment. For this reason, the synthesis of novel imidazole derivatives was described and this study explored the details of imidazole derivatives binding to human serum albumin (HSA). The data of steady-state and time-resolved fluorescence showed that the conjugation of imidazole derivatives with HSA yielded quenching by a static mechanism. Meanwhile, the number of binding sites, the binding constants, and the thermodynamic parameters were also measured; the raw data indicated that imidazole derivatives could spontaneously bind with HSA through hydrophobic interactions and hydrogen bonds which agreed well with the results from the molecular modeling study. Competitive binding experiments confirmed the location of binding. Furthermore, alteration of the secondary structure of HSA in the presence of the imidazole derivatives was tested. © 2015 Elsevier B.V. All rights reserved.

1. Introduction The synthesis and applications of imidazole derivatives have attracted much attention because they are widely present in bioactive compounds, synthetic intermediates and pharmaceuticals. Imidazole represents an important structural motif found frequently in the main structure of some well-known components of human organisms, i.e., the amino acid histidine, a component of DNA base structure, histamine and biotin. Moreover, imidazole derivatives also show many significant biological properties, such as anti-plasmodium, anti-inflammatory, antifungal and antibacterial [1,2]. Consequently, considerable efforts have been made to constituting an important synthetic route to synthesis the imidazole derivatives. As designed to be the desired molecular, our target compounds should have the basic structure of imidazole ring, benzothiazole ring, and appropriate bonding strength to protein. In addition, a facile route of antimicrobial and antioxidant imidazole derivatives is highly desirable. In this article, we reported a series of the imidazole derivatives to find their behavior toward proteins (Fig. 1, Scheme 1). Serum albumin is the major protein of the circulatory and lymphatic system. When entering the blood, drugs will generally bind to serum albumin with varying degrees, which will affect the absorbance, metabolism, efficiency, pharmacological and toxicological effects of drugs [3]. ⁎ Corresponding authors. E-mail address: [email protected] (Y. Yue).

http://dx.doi.org/10.1016/j.saa.2015.09.023 1386-1425/© 2015 Elsevier B.V. All rights reserved.

The knowledge on the interaction between drug and protein will contribute to the design and modification of drugs, and is also an important way of understanding the biological effect of proteins, which will be of great significance on pharmacodynamics, pharmacokinetics, pharmacology and toxicology [4–8]. As a kind of serum albumin, human serum albumin (HSA) is one of the most extensively studied [9,10]. HSA, a heart-shaped globular protein, is a single polypeptide chain composed of 585 amino acid residues which are linearly arranged in the tertiary structure in three structurally distinct and evolutionarily related domains (I–III), and each domain consists of two subdomains (A and B). There is one tryptophan residues (Trp-214) located in subdomains IIA [11]. The endogenous and exogenous ligand binding sites of HSA may be located in subdomains IIA, called Sudlow I and II, where the drug binding site is usually located. The imidazole derivatives have the ability to interact with proteins that make them worthy of attention by diverse areas such as food science, medicine, toxicology, chemistry, and agriculture. However, the nature of HSA-imidazole derivatives' interactions remains unclear. The binding characteristics of imidazole derivatives on HSA were systematically investigated by multispectroscopic techniques including UV–vis absorption, fluorescence (quenching, synchronous, time-resolved fluorescence, and three-dimensional) and Fourier transform infrared (FTIR) spectroscopy. We also drew support from molecular modeling. It has been shown that these methods tend to complement each other. The results presented here will help to provide some valuable information for drug design and pharmaceutical research.

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Fig. 1. The chemical structures of PTI, PFDI, PDTI, PDPI, PNDI, PCDI, and PMDI.

Scheme 1. Synthesis of PTI, PFDI, PDTI, PDPI, PNDI, PCDI, and PMDI.

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Fig. 2. The fluorescence spectra of the imidazole derivatives–HSA system. [HSA] = 3.0 × 10−6 M. [imidazole derivatives] = (a–g) (0, 0.66, 1.33, 2.00, 2.66, 3.32, 3.98) × 10−6 M. (A: PTI; B: PFDI; C: PDTI; D: PDPI; E: PNDI; F: PCDI; G: PMDI).

2. Materials and methods 2.1. Chemicals Benzothiophene, benzoin, 4-bromoaniline, ammonium acetate and various aldehydes (benzaldehyde, 2-thenaldehyde, 4-

fluoro-Benzaldehyde, 1-naphthaldehyde, p-polualdehyde, pchlorobenzaldehyde, and anisaldehyde,) were purchased from Aladdin China Ltd. (Shanghai, China) and directly used without purification. Phenylbutazone (PB) and flufenamic acid (FA) were of analytical grade and purchased from J&K Scientific Ltd. (China). All other reagents were of analytical grade and used

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Fig. 3. The Stern–Volmer plot of the imidazole derivatives–HSA system at different temperatures. [HSA] = 3.0 × 10−6 M. [imidazole derivatives] = (0.66, 1.33, 2.00, 2.66, 3.32, 3.98, 4.64, 5.31) × 10−6 M (A: PTI; B: PFDI; C: PDTI; D: PDPI; E: PNDI; F: PCDI; G: PMDI).

as received. HSA was purchased from Sigma-Aldrich. HSA was dissolved in Tris (0.1 mol/L)–HCl (0.1 mol/L) buffer (pH = 7.40) solution to form an aqueous protein solution and then preserved at 4 °C for later use.

2.2. The synthesis procedure for imidazole derivatives 5 in Scheme 1 [12] In a typical experiment, benzoin 1 (5.0 mmol), 4-bromoaniline 2 (5.0 mmol), ammonium acetate 3 (10 mmol), aromatic aldehydes 4

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Table 1 Binding parameters of HSA with imidazole derivatives at different temperatures. Quencher

T (K)

Kq ( Lmol−1 s−1)

KSV (L mol−1)

K (L/mol)

n

ra

ΔG0 (kJ mol−1)

ΔH0 (kJ mol−1)

PTI

303 296 289 303 296 289 303 296 289 303 296 289 303 296 289 303 296 289 303 296 289

6.353 × 1012 6.902 × 1012 7.084 × 1012 6.235 × 1012 6.820 × 1012 7.033 × 1012 6.046 × 1012 6.729 × 1012 6.927 × 1012 2.606 × 1012 2.778 × 1012 3.192 × 1012 6.142 × 1012 6.695 × 1012 8.001 × 1012 2.815 × 1012 3.322 × 1012 4.678 × 1012 4.839 × 1012 4.440 × 1012 3.689 × 1012

6.353 × 104 6.902 × 104 7.084 × 104 6.235 × 104 6.820 × 104 7.033 × 104 6.046 × 104 6.729 × 104 6.927 × 104 2.606 × 104 2.778 × 104 3.192 × 104 6.142 × 104 6.695 × 104 8.001 × 104 2.815 × 104 3.322 × 104 4.678 × 104 4.839 × 104 4.440 × 104 3.689 × 104

5.457 × 104 6.584 × 104 7.828 × 104 5.040 × 104 6.238 × 104 7.399 × 104 4.772 × 104 6.018 × 104 7.227 × 104 2.649 × 104 2.811 × 104 3.123 × 104 5.574 × 104 6.672 × 104 8.086 × 104 2.963 × 104 3.477 × 104 4.920 × 104 3.791 × 104 4.319 × 104 5.270 × 104

1.049 1.016 0.9123 1.243 1.066 0.9840 1.228 1.071 0.9903 0.9946 0.9892 1.018 1.074 0.9618 0.9962 0.9427 0.9861 0.9575 0.9957 1.013 0.9502

0.9993 0.9986 0.9995 0.9990 0.9988 0.9991 0.9975 0.9984 0.9990 0.9992 0.9993 0.9991 0.9981 0.9993 0.9986 0.9991 0.9990 0.9993 0.9994 0.9991 0.995

−27.49 −27.28 −27.08 −27.30 −27.13 −26.96 −27.16 −27.04 −26.91 −25.64 −25.24 −24.84 −27.52 −27.33 −27.14 −25.87 −25.88 −25.90 −26.53 −26.31 −26.10

−18.76

28.81

−19.94

24.28

−21.56

18.49

PFDI

PDTI

PDPI

PNDI

PCDI

PMDI

a

−8.575

ΔS0 (J mol−1)

56.31

−19.34

26.99

−26.44

−1.887

−17.15

30.95

r is the correlation coefficient for Scatchard plots.

(5.0 mmol), iodine (10 mol%) and acetic acid (5.0 mL) were introduced into a Schlenk tube equipped with a magnetic stirring bar. The Schlenk was placed in an oil bath preheated to 110 °C, and the mixture was stirred for 16 h. After completion of the reaction, it was quenched by aqueous Na2S2O3 solution and extracted with ethyl acetate. The organic layers were combined and dried over sodium sulfate. The pure product was obtained by flash column chromatography on silica gel. Characterization data for the products were shown in Supplementary information. 2.3. The synthesis procedure for imidazole derivatives Benzothiophene (0.50 mmol), K2CO3 (0.75 mmol), Pd (OAc)2 (4.0 mol%), PCy3·HBF4 (8.0 mol%), pivalic acid (30 mol%) and benzimidazol derivatives (0.50 mmol) were placed in a Schlenk. After the Schlenk was sealed and flushed with N2 atmosphere, DMAC (2.0 mL) was added to the reaction mixture. Then the content was stirred for 24 h at 100 °C. After the completion, the reaction was cooled to room temperature. It was quenched by aqueous Na2S2O3 solution and extracted with ethyl acetate. The resulting mixture filtered through column chromatography on silica gel to obtain the targeted product. Characterization data for imidazole derivatives were shown in Supplementary information. 2.4. Spectroscopic experiments The fluorescence measurements were performed on an FP-6500 spectrofluorimeter (JASCO, Japan) equipped with a thermostat bath. The HSA concentration was kept at 3 × 10− 6 mol/L. The excitation and emission slit widths were fixed at 5 nm. The excitation wavelength was set at 280 nm, and the emission spectra were read at 290–550 nm at a scan rate of 1000 nm min−1. Each fluorescence spectrum of the protein in the presence of different imidazole derivatives concentrations were corrected for any possible inner filter effect. For synchronous fluorescence measurements, the spectra were recorded by adjusting the excitation and emission wavelength interval (Δλ) at 15 and 60 nm, respectively. Three-dimensional fluorescence spectra were obtained under the following conditions: the emission wavelength was recorded between 240 and 500 nm, the initial excitation wavelength was set to 220 nm with increment of 5 nm, and the other scanning parameters were identical to those used for steady state fluorescence as mentioned above.

The time-resolved fluorescence decays were measured by a FL 980 spectrofluorimeter (Edinburgh Instruments) using time correlated single photon counting method. The goodness of fit was judged in terms of both chi-squared (χ2) values and weighted residuals. The data was analyzed by the fluorescence response function:

 −t −t IðtÞ ¼ α 1 exp þ α 2 exp τ1 τ2

ð1Þ

where I (t) is the time-dependent fluorescence intensity; α1 and α2 are constants and τ1 and τ2 are the lifetimes for the bound and free states of the ligand, respectively. Time-resolved fluorescence decays were analyzed using the impulse response function (IBH DAS6 software). Mean fluorescence lifetimes b τN for bi-exponential iterative fittings were calculated from the decay times and the pre-exponential factors using the following relation: X

α i τ2 : hτi ¼ X αi τ

ð2Þ

Binding location studies between PTI/PFDI/PDTI and HSA in the presence of two site markers (PB and FA) were measured as follows: PB (3.0 μM) and FA (3.0 μM) were incubated with HSA (3.0 μM), pH = 7.4, at 298 K for 30 min. Then a 3.0 mL sample was added to a 1 cm quartz cuvette, followed by titration of sample. An excitation wavelength of 280 nm was selected, and the fluorescence spectra were recorded over a wavelength range of 290–550 nm. The absorption spectra were recorded on Shimadzu double beam spectrophotometer (Model UV 1700) using a cuvette of 1 cm path length. For FT-IR measurements, a solution of PTI/PFDI/PDTI was added to the HSA solution and to have ligands concentration of 6.0 μM with a final HSA concentration of 3.0 μM. Spectra were acquired at 4 cm−1 resolution and 60 scans. The spectra of the buffer solution were collected at the same condition. Then, the absorbance of the buffer solution was subtracted from the spectra of the sample solution by accompanying software to obtain the FT-IR spectra of the protein. Finally, the HSA difference spectrum was obtained by subtracting the sample-free spectrum from the sample-HSA spectrum. The subtraction criterion was that the original spectrum of protein solution between 2200 and 1800 cm−1 was featureless [13].

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Fig. 4. The Scatchard curves for imidazole derivatives quenching the fluorescence of HSA (A: PTI; B: PFDI; C: PDTI; D: PDPI; E: PNDI; F: PCDI; G: PMDI).

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Fig. 5. The time-resolved fluorescence decay of imidazole derivatives–HSA system. [HSA] = 3.0 × 10−5 M; [imidazole derivatives] = 6.0 × 10−3 M (A: PTI; B: PFDI; C: PDTI; D: PDPI; E: PNDI; F: PCDI; G: PMDI); the instrument response is represented by a red line.

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Fluorescence quenching was measured quantitatively with the Stern– Volmer equation.

Table 2 Fluorescence lifetime data of HSA as a function of imidazole derivatives. Samples

τ1 (ns)

τ2 (ns)

A1

A2

τ (ns)

χ2

Free HSA HSA + PTI HSA + PFDI HSA + PDTI HSA + PDPI HSA + PNDI HSA + PCDI HAS + PMDI

3.105 2.706 2.494 2.787 2.819 2.504 2.169 2.193

6.466 6.340 6.309 6.428 6.378 6.296 6.286 6.267

0.1932 0.1349 0.1233 0.1519 0.1654 0.1115 0.1255 0.1077

0.8068 0.8651 0.8767 0.8481 0.8346 0.8885 0.8745 0.8923

5.817 5.849 5.838 5.874 5.789 5.873 5.769 5.828

1.023 1.043 1.100 1.072 1.184 1.060 1.006 1.007

CD measurements were carried out on an Olis DSM 1000 (USA) in the range of 200–260 nm at 1 nm intervals and CD spectra were collected with the scan speed of 20 nm/min. Furthermore, each CD spectrum was the average of 3 scans. 2.5. Molecule docking investigation Molecular docking was carried out using docking software AutoDock 4.2 and AutoDock Tools (ADT). The crystal structure of HSA was taken from the Brookhaven Protein Data Bank (entry codes 1H9Z). All water molecules were removed, and the potential 3D structure of HSA was assigned according to the Amber 4.0 force field with Kollman-all-atom charges. A grid size of 90 × 90 × 90 points with a grid spacing of 0.4 Å was applied.

F0 ¼ 1 þ Kq τ 0 ½Q  ¼ 1 þ Ksv ½Q  F

ð3Þ

In Eq. (3), the quencher concentration is [Q], the Stern–Volmer constant is Ksv, F0 is the measured fluorescence intensity without quencher present, and F is the measured fluorescence intensity with [Q] present. After plotting (F0/F) against [Q], the slope can be determined to give the value of Ksv, the Stern–Volmer constant. Kq is the bimolecular quenching constant, τ0 is the lifetime of the fluorophore in the absence of quencher and is equal to 10−8 s [18]. The F0/F–[Q] curves of HSA with the imidazole derivatives at different temperatures (289, 296 and 303 K) were shown in Fig. 3. The Stern–Volmer plots were apparently linear, which hinted that just one type of quenching reaction existed (dynamic or static). Simultaneously, the corresponding results fitted from Stern–Volmer plots (Fig. 3) were summarized in Table 1. The outcomes indicated that the Stern–Volmer quenching constants KSV were the counter correlated with temperature, and the value of Kq was 10 times higher than the maximum value for diffusion-controlled quenching in water (1010 M−1 s−1), signifying that the quenching mechanism for HSA fluorescence by imidazole derivatives was a static type [19]. In drug-protein binding studies, the Scatchard Eq. (4) is frequently used for calculating the affinity constant of a ligand with a protein: r=D f ¼ nK−rK

ð4Þ

3. Results and discussion 3.1. Fluorescence quenching of HSA upon addition of imidazole derivatives Fluorescence spectroscopy is an effective method to study the interactions between small molecule and biomacromolecules. The fluorescence emission spectra of HSA in the presence of imidazole derivatives at different concentrations were shown in Fig. 2. It could be obviously seen that the fluorescence intensity of HSA at 340 nm decreased with an increase in imidazole derivative concentration, indicating that the binding of imidazole derivatives to HSA quenched the intrinsic fluorescence of HSA [14]. In addition, the imidazole derivatives with different functional groups caused varying degrees of fluorescence quenching. The imidazole derivatives contained the functional group of benzimidazole and benzothiophene, which is an important structural motif present in several natural products and pharmacologically relevant structures. Of the seven imidazole derivatives, PDPI induced the smallest degree of fluorescence quenching of HSA compared to other six imidazole derivatives because PDPI would have the electron-donating group at 2-substituent imidazole. The different structures of imidazole derivatives resulted in the change of their binding abilities with HSA. Moreover, the visible bathochromic shift of the maximum emission wavelength of imidazole derivatives–HSA complex indicated that the altered microenvironment of the tryptophan residues and the residues of HSA were placed in a more hydrophilic environment by interactions of imidazole derivatives with HSA [15]. Further, upon the imidazole derivative addition, it was noted that there were clear isosbestic point formation at 369, 371, 378, 374, 370, 373, and 379 nm, respectively. This phenomenon might also indicate the existence of bound and imidazole derivatives in equilibrium [16]. 3.2. Fluorescence quenching mechanisms Fluorescence quenching typically occurs through the mechanisms of either static (formation of a non-fluorescent ground state complex) or dynamic quenching (a collision between the quencher and fluorophore takes place during the excitation lifetime of the fluorophore) [17].

where r is the number of moles of drug bound per mole of protein, Df is the concentration of free drug, K is the binding constant, and n is the number of binding sites. The Scatchard plots for imidazole derivatives–HSA systems at different temperatures were shown in Fig. 4. The values of K and n were listed in Table 1. Binding parameters calculating from Eq. (4) have shown that imidazole derivatives bound to HSA with the binding affinities of the order 104 L mol−1 and the binding sites n approximately equal to 1. The linearity of the Scatchard plots indicated that imidazole derivatives bound to a single class of binding sites on HSA, which were consistent with the number of binding sites n. The K decreased while the temperature increased, resulting in a decrease in the stability of imidazole derivatives–HSA systems [20]. Many articles have appeared in the literature about the binding affinity of small molecular to SA. Liu et al. have observed that the binding constant Ka of chrysoidine to BSA is (2.73 ± 0.34) × 105 L mol−1 at 298 K [21]. Wu et al. have shown that the binding constant Ka for allura red AC and HSA was (1.075 ± 0.006) × 106 L mol−1 at 298 K [22]. In comparison with available data published, the results obtained show that the binding between imidazole derivatives and HSA were not strong. One consideration to bear in mind was that static and dynamic quenching could be distinguished by their different dependence on temperature, but preferably by fluorescence lifetime measurements, which can directly discriminate between static and dynamic process [23]. To further acquire direct proof of the nature of the HSA–imidazole recognition, the fluorescence decay figures of HSA in the absence and presence of imidazole derivatives in Tris–HCl buffer (pH = 7.4) were displayed in Fig. 5, and the values of parameters (τi, αi, bτ N and χ2) were listed in Table 2. The results showed that the addition of imidazole derivatives had no effect in the lifetime of HSA, which reveals that the quenching mentioned above is not initiated by dynamic collision but from static quenching. The decay profiles were well fitted into a bi-exponential function, and the fluorescence lifetime of free HSA was τ1 = 3.105 ns, τ2 = 6.466 ns and amplitude were α1 = 0.1932 and α2 = 0.8068. The mean fluorescence lifetime of HSA did not change obviously, just from 5.817 ns to 5.849/5.838/5.874/5.789/5.873/5.769/ 5.828 ns, which was further proved that the fluorescence quenching

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Fig. 6. Van't Hoff plot for the molecular recognition of imidazole derivatives by HSA (A: PTI; B: PFDI; C: PDTI; D: PDPI; E: PNDI; F: PCDI; G: PMDI).

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Fig. 7. Effect of the site marker probe on the fluorescence of the imidazole derivatives–HSA system. [HSA] = 3.0 × 10−6 M. [imidazole derivatives] = (0.66, 1.33, 2.00, 2.66, 3.32, 3.98, 4.64, 5.31) × 10−6 M (A: PTI; B: PFDI; C: PDTI; D: PDPI; E: PNDI; F: PCDI; G: PMDI).

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and ΔS0, and the free energy change (ΔG0) can be obtained from the relationship

Table 3 Effect of site maker probe for the interaction of HSA and imidazole derivatives.

PTI + HSA PFDI + HSA PDTI + HSA PDPI + HSA PNDI + HSA PCDI + HSA PMDI + HSA

K (×105 M−1)

FA (×105 M−1)

PB (×105 M−1)

0.6584 0.6238 0.6018 0.2811 0.6672 0.3477 0.4319

0.7227 0.6661 0.6106 0.4690 0.6523 0.4329 0.4787

0.2959 0.3792 0.4279 0.1650 0.3002 0.1432 0.3250

was truly static mechanism [24]. These results were in good agreement with the results of steady state fluorescence. 3.3. Thermodynamic analysis There are primarily four types of noncovalent interactions that can play an important role in ligand binding to protein, such as hydrogen bond, van der Waals force, electrostatic force, hydrophobic interaction force, etc. [25]. The thermodynamic parameters, enthalpy change (ΔH0) and entropy change (ΔS0), of binding reaction provide evidence for confirming binding modes. To obtain the information about the forces persisting in the present binding process, supposing the enthalpy ΔH0 does not vary clearly over the temperature range studied, the thermodynamic functions are related by using the van't Hoff equation: InK ¼ −ΔH0 =RT þ ΔS0 =R

ð5Þ

where K is the affinity constant for a given association reaction under a definite set of experimental conditions, R is the gas constant, T is the absolute temperature, ΔH0 and ΔS0 are enthalpy change and entropy change, respectively. According to the binding constants of imidazole derivatives–HSA system obtained at the three temperatures (289, 298 and 303 K), a linear plot (Fig. 6) of lnK versus 1/T can produce ΔH0

ΔG0 ¼ ΔH0 −TΔS0 :

ð6Þ

The results of the thermodynamical parameters (ΔH0, ΔS0, and ΔG0) were listed in Table 1. From Table 1, it can be seen that ΔH0 was a small negative value, whereas ΔS0 was a positive value for PTI/PFDI/PDTI/ PDPI/PNDI/PMDI. The negative sign for ΔG0 indicated that the formation of the imidazoles–HSA complex was entropically driven, and the binding processes were spontaneous. In this study, ΔH0 was a small negative value, so the main source of the negative ΔG0 value was derived from a larger contribution of a positive ΔS0 value, indicating that the main interaction was hydrophobic contact [26,27]. At the same time, the negative ΔH0 value indicated that there was hydrogen bonding in the interaction between imidazole derivatives and HSA. These results suggested that the main forces of PTI/PFDI/PDTI/PDPI/PNDI/PMDI–HSA interaction were hydrophobic interaction, and the hydrogen bond also stabilized the formation of the PTI/PFDI/PDTI/PDPI/PNDI/PMDI–HSA complex. For PCDI, both ΔH0 and ΔS0 were negative, which contributed from H-bonding or van der Waals forces. Meanwhile, the above results were in good agreement with the information coming from molecular modeling. 3.4. Site-selective binding of imidazole derivatives on HSA As mentioned above, HSA has two ligand-specific binding sites which are called Sudlow's binding sites I (in subdomain IIA) and II (in subdomain IIIA) [28,29]. In order to identify the imidazole derivative binding site on HSA, site marker competitive experiments were carried out, using drugs which specifically bind to a known site or region on HSA. Warfarin, phenylbutazone, etc. have been demonstrated to bind to the sub-domain IIA (Sudlow's site I) while ibuprofen, flufenamic acid, etc. are known to be a sub-domain IIIA binder (Sudlow's site II) [30,31]. In the site marker competitive experiment, information about the site selective binding interaction of imidazole derivatives with HSA in the absence and presence of the two site markers (PB, FA) were obtained [32]. With the addition of site marker into HSA, the fluorescence intensity was lower than that of without site marker. The Stern–Volmer equation yielded a linear plot indicating the existence of possible competition between imidazole derivatives and PB or FA. Moreover, the binding constants in the presence of site markers were analyzed by using the Scatchard equation and the results were listed in Fig. 7 and Table 3. From the analysis of Table 3, the binding constants were surprisingly variable in the presence of PB, while a smaller influence in the presence of FA. The result indicated that the binding site of imidazole derivatives was mainly located within site I of HSA. 3.5. Molecular modeling

Fig. 8. Molecular modeling of the interaction between imidazole derivatives and HSA (A: PTI; B: PFDI; C: PDTI). The hydrogen bond between imidazole derivatives and HSA represented using a dashed line (yellow).

In order to further attest the binding site of HSA on which imidazole derivatives were located, the complementary applications of molecule modeling also had been employed by computer methods. The best docked result between imidazole derivatives and HSA were shown in Fig. 8 (other data are provided in the Supplementary information, Fig. S1). As displayed in Figs. 8 and S1, imidazole derivatives were found to be deep inside the subdomain IIA (site I). In Fig. 8a, PTI was surrounded by subdomain IIA hydrophobic cavity lined by the following amino acid residues Trp214, Lys199, Arg218, Arg212, etc.; Arg 212, Arg 218, Trp214, etc. for PFDI; and Trp214, Arg 218, Ser202, etc. for PDTI. For PDPI, the optimal binding sites were Trp214, Arg218, His242, Lys199, and Ser202. For PNDI, the optimal binding sites were Trp214, His242, and Lys199. For PCDI, the optimal binding sites were Glu153, His288, and His242. For PMDI, the optimal binding sites were Trp214, Arg218, and Ser192. The most remarkable of these was Trp214, which was in subdomain IIA of HSA, suggesting the existence of hydrophobic

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Fig. 9. Synchronous fluorescence spectra (Δλ = 60 nm) of HSA (3.0 × 10−6 M) in the presence of 0, 0.66, 1.33, 2.00, 2.66, 3.32 and 3.98 × 10−6 M imidazole derivatives (A: PTI; B: PFDI; C: PDTI; D: PDPI; E: PNDI; F: PCDI; G: PMDI).

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Fig. 10. The three-dimensional fluorescence spectra of HSA (A) and PTI; PFDI; PDTI; PDPI; PNDI; PCDI; PMDI-HSA (B, C, D, E, F, G and H). [HSA] = 3 × 10−5 M, [imidazole derivatives] = 6 × 10−5 M.

interaction between imidazole derivatives and HSA [33]. In addition, hydrogen bond was predicted from the conformation, with the bond lengths of 2.136, 2.208, 2.217, 2.008, 2.157, 2.306, and 2.412 Å, respectively, which perhaps implied that the presence of weak hydrogen bond interaction between HSA and imidazole derivatives. And the results also enunciated the existence of prominent π–π

interactions between HSA and imidazole derivatives. The binding energy were − 27.01 kJ/mol (PTI), − 27.69 kJ/mol (PFDI), − 26.84 kJ/mol (PDTI), − 25.91 kJ/mol (PDPI), − 27.89 kJ/mol (PNDI), − 25.04 kJ/mol (PCDI), and − 26.33 kJ/mol (PMDI), respectively, which were extremely close to the thermodynamic experiment data at 296 K.

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Fig. 11. FT-IR spectra of the imidazole derivatives–HSA system. [HSA] = 3.0 × 10−5 M, [imidazole derivatives] = 6.0 × 10−5 M.

3.6. The effect of imidazole derivatives on HSA conformation In order to investigate the conformational changes of HSA, the testimony of structural changes of HSA after binding with imidazole derivatives were represented by synchronous fluorescence spectra [34,35]. The synchronous fluorescence spectra are frequently used to characterize the interaction between fluorescence probe and proteins because it can provide information about the molecular microenvironment in the vicinity of the fluorescent molecules. Besides, it involves simultaneously scanning of the excitation and emission monochromators while retaining a stable wavelength interval (Δλ) or fixed increment of energy (Δv) between them [36]. When the wavelength intervals (Δλ) are stabilized at 60 nm, the synchronous fluorescence gives the characteristic information of Trp, respectively [37,38]. Similarly, characteristic knowledge of tyrosine is obtained by adjusting the Δλ value at 15 nm. Fig. 9 displayed the synchronous fluorescence of HSA in Tris– HCl buffer in the presence of different concentration of these imidazole derivatives. It could be seen from Fig. 9 that the maximum emission wavelength of tryptophan in HSA had a slight red shift at Δλ = 60 nm from 345 nm to 347 nm for PTI, 345 nm to 347 nm for PFDI, whereas in the case of PDTI/PDPI/PNDI/PCDI/PMDI a similar red shift was observed. These differences may be aroused due to structural peculiarities (the substituent of the benzene ring). This phenomenon revealed that the polarity around Trp residues was increased, and thus the hydrophobicity was decreased in the presence of imidazole derivatives. However, there was not any shift in the maximum fluorescence emission wavelengths of tyrosine residues (Supplementary information, Fig. S2), which indicated that the microenvironments around tyrosine residues in HSA did not have a discernable change during the binding process between HSA and imidazole derivatives. The three-dimensional fluorescence spectra can provide total information regarding the fluorescence characteristics by changing excitation and emission wavelength simultaneously [39]. The threedimensional fluorescence spectra of HSA before and after imidazole derivatives addition were depicted in Fig. 10. Peak 1(F = 205, λex = 230 nm, λem = 340 nm) referred to the fluorescence characteristic of polypeptide backbone structures, which was caused by the transition of π–π* of HSA's characteristic polypeptide backbone structure C = O and peak 2 (F = 219, λex = 280 nm, λem = 340 nm) mainly revealed the spectral characteristic of Trp and Tyr residues [40,41]. As shown in Fig. 10, with the addition of imidazole derivatives, the fluorescence intensities of peak 1 and peak 2 were decreased, indicating that the binding of imidazole derivatives with HSA decreased the polarity of

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tryptophan and tyrosine residues, buried more amino acids in the hydrophobic pocket. Compared with other imidazole derivatives, the fluorescence intensities of PDPI decreased significantly. The results may be due to structural peculiarities (methyl groups in benzene rings) [6]. These changes resulted in the slight folding of the polypeptide chain of the protein and induced some micro-environmental and conformational changes in HSA. FT-IR spectroscopy can also provide information about conformational changes of proteins, if the protein secondary structure changed in the ligand–HSA complex. Among the amide bands of the protein, the amide I band (1700–1600 cm−1, mainly C=O stretch) and amide II band (1600–1500 cm− 1, C–N stretch coupled with N–H bending mode) both have a relationship with the secondary structure of the protein. However, the amide I band is more sensitive to the change of protein secondary structure than the amide II band [42]. To explore the changes of the HSA secondary structure, the FT-IR spectra of free HSA and the difference spectra after binding with imidazole derivatives in Tris–HCl buffer solution were recorded. As seen from Fig. 11, the peak position of amide I band significantly shifted from 1638 to 1640 cm−1 for PDPI, to 1648 cm−1 for PCDI, to 1653 cm− 1 for PMDI/PDTI/PFDI/ PTI/PNDI. The functional group might be responsible for the slight redshift. The change of the peak position indicated that imidazole derivatives interacted with C=O groups in HSA, which caused the rearrangement of the polypeptide carbonyl hydrogen bonding pattern and changed the secondary structure of HSA. It was important to note that the decrease in the intensity of the amide I band was due to the decrease of the proportion of protein α-helix structure, the result also suggested that HSA conformational changed upon addition of derivatives. The UV–vis absorption technique can be used to explore the structural changes of proteins and to investigate protein–ligand complex formation [43]. HSA has two absorption peaks, the strong absorption peak at about 213 nm reflects the framework conformation of the protein, the weak absorption peak at about 279 nm appears to be due to the aromatic amino acids (Trp, Tyr, and Phe). From Fig. 12, with gradual addition of imidazole derivatives, the intensity of HSA of the peak at 213 nm increased with no obvious shift. This enhancement of absorption of HSA in the presence of imidazole derivatives may be due to the formation of ground state complex from the intermolecular interactions. It was reported that the difference in the spectral peak of 213 nm was due to changes in the conformation of the peptide backbone associated with helix-coil transformation [44]. Hence, an increase in the absorption peak around 213 with the addition of imidazole derivatives indicated that the binding of imidazole derivatives to HSA induced the conformational change of HSA. We also conducted CD analyses to determine the structural changes in HSA induced by the binding of imidazole derivatives. Fig. 13 showed that the CD spectra of free HSA have two negative bands at 208 and 222 nm, which were both attributed to n–π* transfers of the peptide bonds in the a-helix structure. With addition of imidazole derivatives, the CD signal of HSA decreased, indicating that the secondary structure of protein had changed. The CD results were expressed in terms of αhelix based on the following equation. The secondary structure components were calculated on the basis of raw CD data to quantitatively analyze the conformational changes. The calculated results exhibited a decrease in the α-helical content from 50.3% in free HSA to 49.3% (PDPI), 49.2% (PCDI), 46.5% (PMDI), 44.1% (PDTI), 39.7% (PFDI), 39.6% (PTI), 38.6% (PNDI). Secondary structure was closely related to the biological activity of proteins, and these results indicated that imidazole derivatives probably adopt a looser conformation. 4. Conclusions In summary, the binding mechanism of imidazole derivatives interacting with HSA was investigated using several spectroscopic techniques and molecular docking under simulated physiological conditions. Fluorescence quenching analysis had proved the formation of

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Fig. 12. The absorption spectra of the imidazole derivatives–HSA system. [HSA] = 3.0 × 10−6 M. [imidazole derivatives] = (a–f) (0, 0.66, 1.33, 2.00, 2.66, 3.32) × 10−6 M (a: PTI; b: PFDI; c: PDTI; d: PDPI; e: PNDI; f: PCDI; g: PMDI).

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Fig. 13. CD spectra of HSA (3.5 × 10−6 M) in the absence and presence of imidazole derivatives.[imidazole derivatives] = 3.5 × 10−6 M.

the imidazole derivatives–HSA complex, and it was confirmed to be static quenching mechanism in multiple angles. Results of thermodynamic analysis had shown that hydrophobic interactions play a vital role during the interaction, this occurrence provoked the conformational change of HSA with a noticeable decline of α-helix evoking perturbation of the protein, as stemmed from synchronous fluorescence, and three-dimensional fluorescence measurements. We anticipated that the complexation of imidazole derivatives with protein delineated here may be exploited as the contribution to the design and modification of drugs and the examination of imidazole derivatives properties. It should lend important information for pharmaceutical research or biomedical research. Acknowledgments We gratefully acknowledge the financial support of the National Natural Science Foundation of China (No. 21205029) and the National Undergraduates Innovating Experimentation Project (No. 201310476024). Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.saa.2015.09.023. References [1] M. Doble, A. Puratchikody, Bioorg. Med. Chem. 15 (2007) 1083–1090.

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