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

Article abstract of DOI:10.1016/j.saa.2015.09.023

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

Full text of DOI:10.1016/j.saa.2015.09.023

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 153 (2016) 688–703
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
journal homepage: www.elsevier.com/locate/saa
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
a b s t r a c t
Article history:
Received 11 March 2015
Received in revised form 10 August 2015
Accepted 26 September 2015
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 de-
scribed 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 con-
stants, and the thermodynamic parameters were also measured; the raw data indicated that imidazole deriva-
tives 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 loca-
tion of binding. Furthermore, alteration of the secondary structure of HSA in the presence of the imidazole deriv-
atives was tested.
Keywords:
Imidazole derivatives
Human serum albumin
Quenching mechanisms
Molecular modeling
© 2015 Elsevier B.V. All rights reserved.
1. Introduction
The knowledge on the interaction between drug and protein will con-
tribute to the design and modification of drugs, and is also an important
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, an-
tifungal and antibacterial [1,2]. Consequently, considerable efforts
have been made to constituting an important synthetic route to synthe-
sis 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 de-
rivatives is highly desirable. In this article, we reported a series of the
imidazole derivatives to find their behavior toward proteins (Fig. 1,
Scheme 1).
way of understanding the biological effect of proteins, which will be of
great significance on pharmacodynamics, pharmacokinetics, pharma-
cology 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 com-
posed of 585 amino acid residues which are linearly arranged in the ter-
tiary 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 sci-
ence, medicine, toxicology, chemistry, and agriculture. However, the na-
ture of HSA-imidazole derivatives' interactions remains unclear. The
binding characteristics of imidazole derivatives on HSA were systemat-
ically investigated by multispectroscopic techniques including UV–vis
absorption, fluorescence (quenching, synchronous, time-resolved fluo-
rescence, and three-dimensional) and Fourier transform infrared (FT-
IR) 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 informa-
tion for drug design and pharmaceutical research.
Serum albumin is the major protein of the circulatory and lymphatic
system. When entering the blood, drugs will generally bind to serum al-
bumin with varying degrees, which will affect the absorbance, metabo-
lism, efficiency, pharmacological and toxicological effects of drugs [3].
⁎
Corresponding authors.
E-mail address: yueyuan-8047@163.com (Y. Yue).
http://dx.doi.org/10.1016/j.saa.2015.09.023
1386-1425/© 2015 Elsevier B.V. All rights reserved.

Y. Yue et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 153 (2016) 688–703
689
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
fluoro-Benzaldehyde, 1-naphthaldehyde, p-polualdehyde, p-
chlorobenzaldehyde, 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
2.1. Chemicals
Benzothiophene, benzoin, 4-bromoaniline, ammonium ace-
tate and various aldehydes (benzaldehyde, 2-thenaldehyde, 4-

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691
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
KSV
K
n
ra
ΔG0
ΔH0
ΔS0
( Lmol−1 s−1
)
(L mol−1
)
(L/mol)
(kJ mol−1
)
(kJ mol−1
)
(J 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
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
−19.94
−21.56
−8.575
−19.34
−26.44
−17.15
28.81
24.28
18.49
56.31
26.99
−1.887
30.95
PFDI
PDTI
PDPI
PNDI
PCDI
PMDI
1.074
0.9618
0.9962
0.9427
0.9861
0.9575
0.9957
1.013
0.9502
a
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. Character-
ization data for the products were shown in Supplementary
information.
The time-resolved fluorescence decays were measured by a FL 980
spectrofluorimeter (Edinburgh Instruments) using time correlated sin-
gle 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
τ1
−t
τ2
IðtÞ ¼ α1 exp
þ α2 exp
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 ana-
lyzed using the impulse response function (IBH DAS6 software). Mean
fluorescence lifetimes bτN for bi-exponential iterative fittings were cal-
culated from the decay times and the pre-exponential factors using the
following relation:
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 im-
idazole derivatives were shown in Supplementary information.
X
αiτ2
X
hτi ¼
:
ð2Þ
αiτ
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 res-
olution 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 dif-
ference spectrum was obtained by subtracting the sample-free spec-
trum 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].
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 pro-
tein in the presence of different imidazole derivatives concentrations
were corrected for any possible inner filter effect.
For synchronous fluorescence measurements, the spectra were re-
corded 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 iden-
tical to those used for steady state fluorescence as mentioned above.

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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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Table 2
Fluorescence quenching was measured quantitatively with the Stern–
Volmer equation.
Fluorescence lifetime data of HSA as a function of imidazole derivatives.
Samples
τ1 (ns)
τ2 (ns)
A1
A2
τ (ns)
χ2
F0
F
Free HSA
HSA + PTI
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
¼ 1 þ Kqτ0½Qꢀ ¼ 1 þ Ksv½Qꢀ
ð3Þ
HSA + PFDI
HSA + PDTI
HSA + PDPI
HSA + PNDI
HSA + PCDI
HAS + PMDI
In Eq. (3), the quencher concentration is [Q], the Stern–Volmer con-
stant 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 out-
comes 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].
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 collect-
ed 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.
In drug-protein binding studies, the Scatchard Eq. (4) is frequently
used for calculating the affinity constant of a ligand with a protein:
r=Df ¼ nK−rK
ð4Þ
3. Results and discussion
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 deriva-
tives–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 de-
crease 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 compari-
son 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-ex-
ponential 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 ob-
viously, 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
3.1. Fluorescence quenching of HSA upon addition of imidazole derivatives
Fluorescence spectroscopy is an effective method to study the inter-
actions between small molecule and biomacromolecules. The fluores-
cence 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 fluores-
cence 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 benzimid-
azole and benzothiophene, which is an important structural motif pres-
ent 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-donat-
ing group at 2-substituent imidazole. The different structures of imidaz-
ole 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 de-
rivative addition, it was noted that there were clear isosbestic point for-
mation 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].

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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 re-
lationship
Table 3
Effect of site maker probe for the interaction of HSA and imidazole derivatives.
K (×105 M−1
)
FA (×105 M−1
)
PB (×105 M−1
)
ΔG0 ¼ ΔH0−TΔS0:
ð6Þ
PTI + HSA
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
PFDI + HSA
PDTI + HSA
PDPI + HSA
PNDI + HSA
PCDI + HSA
PMDI + HSA
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 bind-
ing 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 in-
teraction was hydrophobic contact [26,27]. At the same time, the nega-
tive ΔH0 value indicated that there was hydrogen bonding in the
interaction between imidazole derivatives and HSA. These results sug-
gested that the main forces of PTI/PFDI/PDTI/PDPI/PNDI/PMDI–HSA in-
teraction 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.
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 ther-
modynamic functions are related by using the van't Hoff equation:
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 fluo-
rescence 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 influ-
ence in the presence of FA. The result indicated that the binding site of
imidazole derivatives was mainly located within site I of HSA.
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 ab-
solute 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
3.5. Molecular modeling
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
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 repre-
sented using a dashed line (yellow).

Y. Yue et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 153 (2016) 688–703
699
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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Y. Yue et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 153 (2016) 688–703
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, re-
spectively, which perhaps implied that the presence of weak hydro-
gen 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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701
tryptophan and tyrosine residues, buried more amino acids in the hy-
drophobic pocket. Compared with other imidazole derivatives, the fluo-
rescence 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 conforma-
tional changes in HSA.
FT-IR spectroscopy can also provide information about conforma-
tional 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 pro-
tein. However, the amide I band is more sensitive to the change of pro-
tein 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 red-
shift. The change of the peak position indicated that imidazole deriva-
tives interacted with C=O groups in HSA, which caused the rearrange-
ment 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 struc-
tural changes of proteins and to investigate protein–ligand complex for-
mation [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 aromat-
ic 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 in-
creased 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 re-
ported 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 conforma-
tional 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 α-
helix based on the following equation. The secondary structure compo-
nents were calculated on the basis of raw CD data to quantitatively an-
alyze 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 bio-
logical activity of proteins, and these results indicated that imidazole
derivatives probably adopt a looser conformation.
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 testi-
mony of structural changes of HSA after binding with imidazole deriva-
tives were represented by synchronous fluorescence spectra [34,35].
The synchronous fluorescence spectra are frequently used to character-
ize 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 simulta-
neously 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, charac-
teristic 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 ob-
served. 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 hydropho-
bicity was decreased in the presence of imidazole derivatives. However,
there was not any shift in the maximum fluorescence emission wave-
lengths 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 be-
tween HSA and imidazole derivatives.
The three-dimensional fluorescence spectra can provide total infor-
mation regarding the fluorescence characteristics by changing excita-
tion and emission wavelength simultaneously [39]. The three-
dimensional fluorescence spectra of HSA before and after imidazole de-
rivatives 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 in-
tensities of peak 1 and peak 2 were decreased, indicating that the bind-
ing of imidazole derivatives with HSA decreased the polarity of
4. Conclusions
In summary, the binding mechanism of imidazole derivatives
interacting with HSA was investigated using several spectroscopic tech-
niques and molecular docking under simulated physiological condi-
tions. Fluorescence quenching analysis had proved the formation of

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Y. Yue et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 153 (2016) 688–703
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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Acknowledgments
We gratefully acknowledge the financial support of the
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Appendix A. Supplementary data
Supplementary data to this article can be found online at http://dx.
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