Synthesis of two new naphthalene-containing compounds and their bindings to human serum albumin

Article abstract of DOI:10.1080/07391102.2020.1764867

Two naphthalene-containing compounds, 4-hydroxy-6,7-dimethoxy-1-(4-(trifluoromethyl)phenyl)-2-naphthoic acid (A) and 4-hydroxy-6,7-dimethoxy-1-phenyl-2-naphthoic acid (B), were prepared by several steps. Their bindings to human serum albumin (HSA) were st

Full text of DOI:10.1080/07391102.2020.1764867

Journal of Biomolecular Structure and Dynamics
ISSN: 0739-1102 (Print) 1538-0254 (Online) Journal homepage: https://www.tandfonline.com/loi/tbsd20
Synthesis of two new naphthalene-containing
compounds and their bindings to human serum
albumin
Xia Wang, Yue Xing, Jing Su, Changsheng Wang, Zishi Wang, Yinghui Yu,
Hong-liang Xu & DongSheng Ma
To cite this article: Xia Wang, Yue Xing, Jing Su, Changsheng Wang, Zishi Wang, Yinghui Yu,
Hong-liang Xu & DongSheng Ma (2020): Synthesis of two new naphthalene-containing compounds
and their bindings to human serum albumin, Journal of Biomolecular Structure and Dynamics, DOI:
10.1080/07391102.2020.1764867
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Synthesis of two new naphthalene-containing compounds and their
bindings to human serum albumin
a,*
b,*
b
a
b
Xia Wang , Yue Xing , Jing Su , Changsheng Wang , Zishi Wang ,
a
b, #
a, #
Yinghui Yu , Hong-liang Xu , DongSheng Ma
a
School of Chemistry and Materials Science, Heilongjiang University, 150080,
Harbin, China
b
School of Agricultural Resources and Environment, Heilongjiang University,
1
50080, Harbin, China
*
*
These authors contributed equally to this work.
Corresponding author. E-mail address: xuhongliang@hlju.edu.cn (H. Xu); E-mail
address: madongsheng@hlju.edu.cn (D. Ma).
Abstract
Two naphthalene-containing compounds, methyl 4-hydroxy-6,
7
4
-dimethoxy-1-(4-(trifluoromethyl)phenyl)-2-naphthoate (A) and methyl
-hydroxy-6,7-dimethoxy-1-phenyl-2-naphthoate (B), were prepared by several
steps. Their bindings to human serum albumin (HSA) were studied by
ultraviolet-visible (UV-Vis) absorption, fluorescence, synchronous
1

fluorescence, three-dimensional fluorescence, circular dichroism (CD)
spectroscopies and molecular docking. The crystal of these compounds shows
the structure of the compounds. The results show that the mechanism of the
interaction between A and HSA is mixed-quenching (both dynamic and static
quenching), while that of B and HSA is static quenching. The number of
binding sites, binding constants, and binding distance (r) were obtained. The
interaction processes are spontaneous. A mainly interacts with HSA through
hydrogen bonds and van der Waals forces, and B binds to HSA mainly by the
typical hydrophobic interaction. The conformation of HSA changes slightly
after the addition of the compounds. Besides, molecular docking method was
used to study the interaction details between the compounds and HSA. This is
helpful to understand the absorption and metabolism of these two compounds
in the body, and provides a basis for designing naphthalene-containing drugs.
Key Words: naphthalene; synthesis; interaction; human serum albumin;
spectroscopy
Abbreviations:
HSA - human serum albumin
A - methyl 4-hydroxy-6, 7-
dimethoxy-1-(4-(trifluoromethyl)phenyl)-2-naphthoate
2

B - methyl 4-hydroxy-6,7-dimethoxy-1-phenyl-2-naphthoate
UV-Vis - ultraviolet-visible
CD - circular dichroism
K - Stern-Volmer constant
sv
K – binding constant
a
1
. Introduction
In recent decades, research on naphthalene-containing drugs has attracted widespread
attention. They many naphthalene-containing compounds have also been approved by
FDA and are being marketed as therapeutics, such as Rifampicin, Justiprocumin A, B,
Patentiflorin A, Naphyrone, Tolnaftate, Naftifine, Nafcillin, Terbinafine, Propranolol,
Nabumetone, Nafimidone, Naproxen, Duloxetine, Lasofoxifene, Bedaquiline etc.
(Pietro, M. E. D., et al., 2015; Stiufiuc, R., et al., 2015; Makar, S., et al., 2019). In the
other words, Naphthalenes show various antagonistic activities including anticancer,
antimicrobial, anti-inflammatory, antiviral, antihypertensive, antidiabetic,
anti-neurodegenerative, antipsychotic anticonvulsant, antidepressant( Makar, S., et al.,
2
019; Groot, F. M. D., et al., 2001; Valente, D., et al., 2014; Chen, Y. Y., et al., 2017;
Goudie, A. C., et al., 1978; Debnath, A. K., et al., 1999; Atwal, K. S., et al. 1987;
Bokor, É., et al. 2017).
3

Human serum albumin (HSA) , an important carrier of transporters, is one of the
most abundant proteins in plasma, accounting for about 60% of total serum protein
(
Leila, R., et al. 2019). Approximately 80% of the blood osmotic pressure is provided
by HSA. Many endogenous and exogenous substances that enter the body bind to
HSA through reversible non-covalent binding and are transported to various parts of
the body after binding (Salanee, K., et al., 2019; Vennila, K. N., et al., 2018). HSA
also is a single-chain line protein and consists of 585 amino acid sequences which
divided into three functional domains (I, II, III). These domains are further divided
into a pair of subdomains (A, B) who existing common structural motifs. And the
main drug binding sites of HSA are lies in hydrophobic pockets in subdomains IIA
and IIIA, such as Trp-214 and Tyr-411 residues that named as sites I and II (Aseman,
M. D., et al., 2019; Rabbani, G., et al., 2018). In addition, have revealed a new
binding site in the subdomain IB (Handing, K. B., et al., 2016) . The binding of drug
molecules to HSA could affect its structure and function. So it is important to study
the interaction of the compounds with HSA for understanding metabolic and
biological effects in vivo.
In this paper, we designed and synthesized two new naphthalene-containing
compounds A and B. We have studied the effects of compounds on HSA by
multispectral method and molecular docking. It provides a theoretical basis for the
study of naphthalene-containing drugs.
2
. Materials and methods
4

2
.1. Chemical reagents
All chemical reagents in experiment were purchased commercially and used as
received. HSA (purity ≥ 96%), kept in the dark at 4 ℃, was purchased from Solarbio
Company and dissolved in 0.02 M phosphate buffer saline (PBS) of pH = 7.4.
2
.2. Instrumentation
The IR spectra were recorded from KBr pellets on a Perkin-Elmer Spectrum 100
-1
FT-IR spectrometer (PerkinElmer, USA) in the range of 4000-500 cm . Perkin-Elmer
2
400 elemental analyzer (PerkinElmer, USA) was used for perform elemental
analysis. Fluorescence, synchronous fluorescence, the three-dimensional fluorescence
spectroscopies was taken from by employing the Perkin-Elmer LS-55fluorescence
spectrophotometer (PerkinElmer, USA). The CD spectrum was recorded on a
Chirascan qCD spectrometer (Applied Photophysics, the UK) with a cell of 1 mm
path length. The UV-Vis absorption spectroscopy was recorded on a UV-1780
spectrophotometer (Shimadzu, Japan) equipped with 1 cm quartz cells. The pH
measurements were carried out on a Delta 320 pH meter (Mettler Toledo, USA). The
experimental data were analyzed by origin 8.0 (OriginLab Corporation, USA).
2
.3. General procedure for synthesis of the compounds
2
.3.1. Synthesis of compound A
The first step: adding the polyphosphoric to the reaction flask acid and softening it.
After stirring uniformly, 4-trifluoromet hylbenzoic acid and phthalic acid were added,
5

then raised the temperature to 90 ℃, and the reaction was monitored until the reaction
was completed.
Cooling a while, added the product to an appropriate amount of water, and stirred
overnight, a yellow solid was obtained by centrifugation. Next, the yellow solid was
added to the reaction flask, methanol and water were added at the same time, the
temperature was refluxed for 30 minutes, the solid was precipitated by cooling, and
the mixture was centrifuged to obtain a1.
The second step: the product a1 that obtained in the previous step, diethyl
succinate, xylene and tert-butanol were added to the reaction flask, heated to reflux
for 3 hours, until the solution turned to a viscous liquid, the reaction was monitored.
The reaction solution was cooled, stirred for 10 minutes, and allowed to stand for
separation. The aqueous layer was separated into a reaction flask, toluene was added
to adjust the pH to about 2, and the aqueous layer was separated, and the toluene was
distilled under reduced pressure until substantially no more, a2 was obtained.
The third step: the reaction liquid (a2) obtained in the previous step and acetic
anhydride, anhydrous sodium acetate were added to the reaction flask and heated to
1
20-125℃ for 3.5 hours. The reaction liquid was introduced into a cooling vessel
containing water while stirring, and the mixture was stirred and frozen overnight to
precipitate a yellow solid. Centrifugation, rinse with methanol to obtain a3.
The fourth step: a3, anhydrous potassium carbonate, and methanol were added to
the reaction bottle in turn. Then the reaction was about 3 hours after raising the
6

temperature to 35-40℃ until the reaction was monitored. Cooling the temperature
down, add water to precipitate solids, centrifuge to obtain product a4.
The fifth step: a4 and methanol, water was added to the reaction flask, and the
temperature was refluxed for 4 hours. Cool down to room temperature, adjust the pH
to about 2, precipitate a light grayish white solid, churn for 30 minutes, set aside to
obtain a gray solid, and dry in an oven at 50℃ to obtain product A, which was
purified with ethyl acetate (Scheme 1).
Scheme 1. The synthetic route of A
7

1
13
FTIR spectra and the H and C NMR spectral images of all the compounds
were given as Fig. S1, Fig. S2 and Fig. S3 in supporting information.
Elemental Analysis C H O F (392): Theory, C, 61.22%; H, 3.83%; actual, C,
2
0
15
5 3
-
1
6
1
1.17%; H, 3.89%. FT-IR (KBr, cm ): 3479(-OH), 3352(ph-OH), 1687(-COOH).
1
1
H NMR (DMSO-d , 400MHz, ppm): H NMR (400 MHz, DMSO) δ 10.46 (s,
6
H), 7.80 (d, J = 8.0 Hz, 1H), 7.52 (s, 1H), 7.46 (d, J = 7.9 Hz, 1H), 7.22 (s, 1H), 6.58
(s, 1H), 3.91 (s, 1H), 3.55 (s, 1H).
A (0.05 mmol, 0.0230 g), distilled water (10 ml) was added into a 15 ml
Teflon-lined stainless steel vessel. The sealed system was placed in an electric blast
drying oven for three days at the 130℃ after the magnetizer was add and stirred at
room temperature for 30 minutes. After three days, the temperature was lowered to
room temperature at a rate of 10℃ per hour to obtain a light yellow crystal.
2
.3.2. Synthesis of compound B
The first step of acylation: softening the polyphosphoric acid into the reaction flask,
stirring evenly, adding benzoic acid and phthalic acid, heating to 80-85℃, monitoring
the reaction until the reaction was completed.
After the reaction mixture was slightly cooled, it was added to an appropriate
amount of water, stirred for 1.5 hours, and a white solid was precipitated. The mixture
was washed with water and filtered to give a white solid, which was washed with 25%
NaOH solution and water. Add white solid, ethanol to the reaction flask stir evenly,
8

and add water, stir at room temperature for 1 hour, filter and rinse, dry in a 50℃ oven
to obtain b1.
The second step: the product b1 obtained in the previous step, diethyl succinate,
xylene, potassium t-butoxide and tert-butanol were added to the reaction flask, heated
under reflux for 3 hours, the solution became a viscous liquid with a milky white solid
produce. The liquid phase monitoring reaction was completed.
Cool the reaction solution to an appropriate amount of water, adjust the pH of the
solution to about 10 for 10 minutes, then let stand for stratification, separate the
toluene layer, add toluene to the solution, adjust the pH to about 2, separate the water
layer, and reduce the toluene. Pressurized distillation until it was almost no longer
produced, and the reaction liquid b2 was obtained.
The third step: the reaction liquid b2 obtained in the previous step and acetic
anhydride, anhydrous sodium acetate were added to the reaction flask and heated to
1
20-125℃ for 3.5 hours. The reaction solution was cooled to about 50℃ while hot,
poured into lignin-added water, and stirred for 1 hour to obtain a flesh-colored solid.
Filter by suction and rinse with water to obtain b3.
The fourth step: b3, ethanol KOH was sequentially added to the reaction flask,
and the reaction was monitored after the temperature was raised to reflux about 3.5
hours. The reaction solution was cooled to room temperature, water and toluene were
added and stirred for 10 min, and the toluene layer was separated. Adjust the pH of
the aqueous phase to 2, precipitate with an off-white solid, continue stirring for 30
9

minutes, filter to obtain a gray solid, and dry in a 50℃ oven to obtain product B
(Scheme 2).
Scheme 2. The synthetic route of B
Elemental Analysis C H O (324): Theory, C, 70.37%; H, 4.94%; actual, C,
1
9
16
5
-1
7
0.40%; H, 4.89%. FT-IR (KBr, cm ): 3451(-OH), 3352(ph-OH), 1687(-COOH).
1
H NMR (DMSO-d , 400MHz, ppm) δ 10.46 (s, 1H), 7.80 (d, J = 8.0 Hz, 1H),
6
7
1
.52 (s, 1H), 7.46 (d, J = 7.9 Hz, 1H), 7.22 (s, 1H), 6.58 (s, 1H), 3.91 (s, 1H), 3.55 (s,
H).
B (0.1 mmol, 0.0382 g), distilled water (10 ml) was added into a 15 ml
Teflon-lined stainless steel vessel. The sealed system was placed in an electric blast
drying oven for three days at the 130℃ after the magnetizer was add and stirred at
room temperature for 30 minutes. After three days, the temperature was lowered to
room temperature at a rate of 10℃ per hour to obtain a light yellow crystal.
1
0

2
.4. UV-Vis absorption
The UV-Vis absorption spectra of HSA were measured in a wavelength from 200 to
00 nm in the PBS solution (pH = 7.4) in the absence and presence of the compounds
4
-6
at 290 K. The concentration of a series of samples is 5 × 10 M HSA in the presence
-6
of 0, 5, 10, 15, 20, 25, 30 and 35 × 10 M compounds (A or B).
2
.5. Fluorescence measurements
With the excitation and emission slit width of 15 nm, the emission spectra was
recorded between 300-500 nm when excitation wavelength is 280 nm. The
fluorescence signal of a series of assay samples were recorded after preheating for 3
-7
min at 290 K, 300 K, and 310 K, respectively. The concentration of HSA is 4 × 10
-7
M, followed by the addition of the compounds (from 0 to 0.5 × 10 M). Fluorescence
signal were corrected for inner filter effect according to the following equation (Li, X.,
et al., 2015)
Fcor  Fobs exp[( Aex  Aem ) / 2]
(1)
Where, Fcor and Fobs are the corrected and observed fluorescence signal,
respectively; and A and A are the absorbance at excitation and emission
ex
em
wavelengths of the system, respectively.
.5.1. Synchronous fluorescence spectra
2
Synchronous fluorescence spectra of only HSA and in the presence of different
concentration the compounds were recorded by scanning the excitation and emission
monochromator simultaneously at range from 200 to 400 nm. The spectroscopic
1
1

behavior of tyrosine and tryptophan residues of HSA was displayed at the wavelength
-7
interval (∆λ) was 15 nm and 60 nm. The concentration of HSA is 4 × 10 M with the
ratio of compound A to human serum albumin is 0 : 1, 0.5 : 1, 1 : 1, 1.5 : 1, 2 : 1, and
2
1
2
.5 : 1, the ratio of compound B to human serum albumin is 0 : 1, 0.2 : 1, 0.4 : 1, 0.6:
, 0.8 : 1, and 1: 1.
.5.2. The three-dimensional fluorescence spectra
-7
-7
The three-dimensional fluorescence spectra of 4 × 10 M HSA and HSA with 2 × 10
M the compounds (A or B) were recorded at 290 K. The excitation interval was 20
nm, the spectral bandwidth of emission and excitation was 5 nm. Meanwhile, the
excitation wavelength scanning range was set from 200 to 380 nm, emission
wavelength was set from 300 nm to 500 nm.
2
.6. CD studies
The CD spectra of HSA was measured in a wavelength from 200 to 500 nm in the
circumstances in absence or presence the compounds and corrected by PBS buffer
-
5
(
pH = 7.4) at 310 K. When the concentration of HSA was 1.0 × 10 M, the radios of
the compounds to HSA were set to 0: 1, 3: 1 and 9: 1.
.7. Molecular modeling and docking simulation
2
The three-dimensional structure of HSA at 3. 23 Å resolution was downloaded from
the Protein Data Bank (PDB ID: 4K2C) (Wang, Y., et al., 2013). The protein was
pre-processed by removing water molecules, hydrogen atoms and other operations,
and using small molecule 3D structures constructed by ourselves to simulate. The
1
2

optimal conformation was determined by combining relevant experiments after
searching the conformation alignment with docking calculations of 100 times by
Lamarck genetic algorithm (LGA).
3
. Result and discussion
3
.1. X-ray Crystallography
Single crystal X-ray diffraction measurements of the compounds were carried out on a
Rigaku R-AXIS RAPID imaging plate diffractometer with graphite-monochromated Mo
Kα radiation (k = 0.71073 Å) at the room temperature. Empirical absorption correction of
data was executed by SADABS program. OLEX and SHELXTL software package was
used to parsing the structure of crystal. And the crystallographic data and structural
improvement parameters of the compounds were shown in Table. 1 and Figure 1.
Two naphthopyrones were synthesis, and characterized by NMR, and FTIR. We
not only tested for their binding with transporter protein HSA by these techniques
such as UV-Vis, fluorescence, and CD spectrometry, revealed the binding site of the
test compounds and their interactions with amino acid residues of HSA, but also
discussed the result.
Table 1. Crystallographic data for A and B.
Parameter
A
B
Empirical formula
Fw
C H F O
C H O
4
0
24
6
8
38 26
8
746.59
Triclinic
P-1
610.59
Crystal system
Space group
Triclinic
P-1
1
3

a (Å)
8.6714(4)
8.7600(4)
11.9929(6)
103.239(2)
107.006(2)
94.257(2)
838.20(7)
1
6.9966(2)
9.7146(4)
10.3277(4)
90.910(2)
95.9400(10)
93.5710(10)
696.66(4)
1
b (Å)
c (Å)
α (deg)
β (deg)
γ (deg)
3
V (Å )
Z
-3
D (gcm )
1.479
1.455
calc
-1
μ(mm )
0.125
0.102
F(000)
382
318
Collected/ unique
R(int)
25255/3871
0.0678
14699/3447
0.0210
2
GOF on F
1.052
1.025
a
R [I>2σ(I)]
0.0850
0.0434
l
b
wR [I>σ(I)]
0.2384
0.1180
2
a
R (all)
0.1405
0.0562
l
b
wR (all)
0.2836
0.1366
2
a
b
2
2 2
c
2 2 1/2
R = Σ||Fo| - |Fc|| / Σ|Fo|; wR = {Σ[w(F - F ) / Σw(F ) ]} .
1
2
o
o
1
4

(a)
(b)
Figure 1. Crystal structures of complexes (a) Asymmetric units of compound A. (b)
Asymmetric units of compound B.
3
.2. Absorption spectral studies
It can be known from the Figure 2 that HSA shows two peaks at around 214 nm and
73 nm. The strong absorption peak near 214 nm reflects the absorption of HSA
2
peptide framework structure, whereas the weak absorption peak of the aromatic amino
acids (Trp, Tyr and Phe) near 273 nm. The absorption signal at around 214 nm
decreased after the addition of the compounds (A or B), accompanied by the red shift
phenomenon of maximum absorption wavelength, indicating that the polarity in the
chromophore microenvironment increased. The absorption signal at around 273 nm
increased, but not obvious, which shows that the microenvironment near the aromatic
amino acid has not changed significantly. UV-Vis absorption spectroscopy is one of
the methods that detecting the changes in the structural conformation of proteins up
1
5

on binding to ligands. The UV-Vis absorption spectroscopy is used to distinguish the
dynamic quenching and static quenching. Dynamic quenching is not affects the
absorption spectrum of the fluorescence but the excited state of the fluorescence. And
the change of the absorption spectra is owing to form the ground state complex, which
determined whether static quenching (Ma, J. Y., et al., 2007). All of these shows that
the quenching mechanism of the interaction between the compounds (A or B) and
HSA should be static quenching.
1
6

Figure 2. Ultraviolet-Visible spectra of human serum albumin in the present of
various concentrations of compounds.
-6
-6
(
a) 5 × 10 M HSA only; (b - h) 5× 10 M HSA in the presence of 5, 10, 15, 20, 25,
-6
-6
3
0 and 35 × 10 M compounds (A or B); (i) 5 × 10 M compounds (A or B) only, pH
=
7.4, T = 290 K.
3
.3. Fluorescence quenching of HSA
Tryptophan residues, tyrosine residues and phenylalanine residues are the only amino
acid residues that emit fluorescence. Then tryptophan, located in the 214 position, is
the intrinsic fluorescence of HSA to a large extent. In the absence and presence of
these compounds, the fluorescence spectrum of HSA was recorded. It was suggested
that the fluorescence signal of HSA was decreased after the addition of the
compounds from the figure of the fluorescence effect of the compounds on HSA at
1
7

3
10 K (Figure 3). And the quenching of the fluorescence of HSA by the compounds
was suggested through this. The fluorescence data were analysed by Stern-Volmer
equation in order to describe the quenching mechanism (Zolfagharzade, M., et al.,
2
014; Karami, K., et al., 2019)
1
8

Figure 3. Fluorescence emission spectra of human serum albumin in the presence of
various concentrations of compounds.
-7
(
a - f) 4 × 10 M human serum albumin in the presence of 0, 0.1, 0.2, 0.3, 0.4 and 0.5
-7
-7
×
10 M compounds (A or B), (g) 0.5 × 10 M compounds (A or B), pH = 7.4, T =
3
10 K.
F0 / F  1  K sv [Q ]  1  K τ [Q ]
(2)
q
0
In this formula, F and F are the fluorescence signal in the absence and presence
0
of the quencher, the quenching rate constant of the biomolecule is K ; τ is the
q
0
-8
average lifetime of the biomolecule without quencher (about 10 s) (Dezhampanah,
H., et al., 2016). Q is the concentration of quencher; K is the Stern-Volmer
sv
quenching rate constant, which measures the efficiency of quenching (Salci, A., et al.,
2
017). The maximum dynamic quenching rate constant often as an evidence to
1
9

determine whether the type of action is static quenching or dynamic quenching. It
1
0
-1
-1
belongs to static quenching unless K is not bigger than 2.0 × 10 L· mol · s
q
(Yousuf, I., et al., 2018).
2
0

Figure 4. Stern-Volmer pots for quenching of human serum albumin fluorescence by
compounds at different temperatures.
[
Q] - the concentration of quencher (A or B); F - fluorescence signal in the absence of
0
quencher; F - fluorescence signal in the presence of quencher, pH = 7.4.
Table 2. Stern-Volmer quenching constants and quenching rate constants of
compounds (A or B) on human serum albumin at different temperatures.
A
1
3
K (10
q
5
−1
pH
T (K)
K (10 M )
R
sv
−
1 -1
M s )
2
90
300
10
4.651±0.0935
6.557±0.1317
7.769±0.1309
4.651±0.0935
6.557±0.1317
7.769±0.1309
0.9984
0.9984
0.9989
7
.4
3
B
6
−1
14 −1 -1
pH
T (K)
90
300
10
K (10 M ) K (10 M s )
R
sv
q
2
2.916±0.0040
2.904±0.0163
2.916±0.0040
2.904±0.0163
0.9999
0.9999
0.9995
7
.4
3
2.658±0.0300
2.658±0.0300
T - absolute temperature; K - quenching constant; K - quenching rate constants; R -
sv
q
correlation coefficient for K values. pH = 7.4.
sv
2
1

Stern-Volmer plots were shown in Figure 4, and K was calculated in Table 2.
sv
Not only was the Stern-Volmer quenching rate constant inversely correlated with
temperature should be known by the calculated of K from the table of Stern-Volmer
sv
plots, but also the K values was larger than the maximum fractional collision
q
1
0
-1
-1
quenching constant (2.0 × 10 L· mol · s ) of biological macromolecules at all three
temperatures. And this indicated that the quenching mechanism of the binding of B to
HSA is due to the formation of the compounds. In the other words, the quenching
mechanism of the binding of B to HSA belongs to static quenching. And it is agreed
with the conclusion in UV-Vis. But for A, the K values are larger than the maximum
q
1
0
-1
-1
fractional collision quenching constant (2.0 × 10 L· mol · s ) of biological
macromolecules at all three temperatures, which indicates that quenching is partially
static in nature. On the other hand, the calculated K values increase with increasing
sv
temperatures suggesting that quenching of HSA by A is also dynamic partly (Lin, J.
L., et al., 2019). Taken together, the values of K and K obtained from Stern-Volmer
sv
q
plots implying that HSA is quenched by A through a mixed-quenching procedure of
both static and dynamic mechanisms.
3
.4. Binding studies
3
.4.1. Determination of binding constants and the number of binding sites
The binding constants K and the number of binding sites between HSA and the
a
compounds (A or B), can be used to calculate as following equation (Tanzadehpanah,
H., et al., 2019; Molaee, H., et al., 2019)
2
2

log[(F  F) / F]  log K  n log[Q ]
(3)
0
a
Where relative fluorescence signal of HSA is the F , the relative fluorescence
0
signal of HSA with compounds is F, K is the binding constant, the number of binding
a
sites is n, and the concentration of compounds is [Q].
2
3

Figure 5. Plots of log [(F - F) / F] versus log [Q] for the interaction between the
0
compounds and human serum albumin at different temperatures.
[
Q] - the concentration of quencher (compounds); F - fluorescence signal in the
0
absence of quencher; F - fluorescence signal in the presence of quencher, pH = 7.4.
Table 3. Binding constants and the number binding sites of compounds (A or B) on
human serum albumin at different temperatures.
A
−
1
pH
T (K)
90
300
10
K ( M )
n
R
a
3
4
6
2
（1.935±0.0165）×10
（6.302±0.0585）×10
（2.157±0.1359）×10
0.631±0.0013
0.846±0.0009
1.066±0.0033
0.9997
0.9954
0.9996
7
.4
3
2
4

B
−
1
pH
T (K)
90
300
10
K ( M )
n
R
a
7
7
6
2
（4.563±0.0074）×10
（1.072±0.0029）×10
（3.735±0.0085）×10
1.157±0.0001
1.066±0.0002
1.002±0.0004
0.9999
0.9994
0.9998
7
.4
3
T - absolute temperature; K - binding constant; n - the number of binding site; R -
a
correlation coefficient for K values. pH = 7.4.
a
The binding constants and binding site number at different temperatures were
obtained by the intercept and slope of log [(F -F) / F] - log [Q] curve according to the
0
equation (Figure 5). The the number of binding sites of the compounds on HSA is 1
and the binding constants at different temperature are shown in Table 3. For A, it is
indicated that K value increases with increasing temperature, which suggests that the
a
interaction between HSA and A is enhanced by increasing temperature. But for B,
with increasing temperature, K value decreases which indicated that the interaction
a
between HSA and B is weakened.
3
.4.2. Thermodynamic parameter and binding force study
The intrinsic fluorescence of HSA upon A and B were measured at 290, 300, and 310
K. The thermodynamic parameters were calculated for ascertain the mechanism of the
quenching phenomena (Ansari, S. S. et al., 2018; Bagoji, A. M. et al., 2017).
0
0
L nK    H / R T   S / R
(4)
2
5

0
0
0
Δ G   H  T  S
(5)
The constant K is analogous to the binding constant at the corresponding
temperature in this formula (Aseman, M. D. et al., 2019). Plots of Ln K versus 1 / T
according to the Van’t Hoff equation was shown in Figure 6. According to the rule
of judging the binding force between biomacromolecules and small molecules in
0
0
0
thermodynamic (Gao, X., et al., 2017). For B, ∆G < 0, ∆H < 0, ∆S < 0, which
showed that as Table 4, it could be revealed that the binding of B to HSA is
spontaneous and mainly through hydrogen bond and van der Waals force. At the same
time, we can know that the binding force of HSA and A is a typical hydrophobic.
2
6

Figure 6. Plots of LnK versus 1 / T according to the Van’ t Hoff equation.
K - the binding constant; T - absolute temperature, pH = 7.4.
Table 4. Thermodynamic parameters for the interaction between compounds (A or B)
and human serum albumin at different temperatures.
A
0
-1)
0
-1
0
-1
-1
pH T (K)
90
300
10
ΔG (KJ · mol
ΔH (KJ · mol )
ΔS (J · mol · K )
2
-18.136
-27.800
-37.463
7
.4
262.101
966.335
3
B
0
-1)
0
-1
0
-1
-1
pH T (K)
ΔG (KJ · mol
ΔH (KJ · mol )
ΔS (J · mol · K )
2
7

2
90
300
10
-42.401
-40.633
-38.866
7
.4
-93.659
-176.753
3
ΔG - Gibbs’ free-energy change; ΔH - enthalpy change; ∆S - entropy change. pH =
7
.4.
3
.4.3. Non-radiative energy transfer and binding distance between HSA and the
compounds
The Förster energy transfer theory, which can explain the non-radiative energy
transfers between the compounds (A or B) and HSA, was used to measure the binding
distance between the compounds and HSA (Rabbani, G., et al., 2017). There is a
relationship among the energy transfer efficiency E, the distance r and the energy
transfer distance R between the energy donor and the acceptor. It is given by the
0
formula (Matei, I., et al., 2011)
6
6
6
E  1  F / F  R0 / ( R0  r )
(6)
0
Where E is the energy transfer efficiency, r is the binding distance of the donor
and acceptor, R is the critical distance when the energy transfer efficiency is 50%.
0
6
-2 5
2
-4
R 0  8.79  10
K
N
 J
(7)
2
2
Where K is the spatial orientation factor of the dipole, K = 2 / 3; N is the
average refracted index of medium in the wavelength range where spectra overlap is
significant, and the average value of the refractive index of water and organic matter
2
8

is 1.336; φ is the fluorescence quantum yield of the donor, and the quantum yield of
tryptophan in HSA is approximately 0.118; J is the spectral overlap that could be
calculated by the following equation between the emission spectrum of the donor and
the absorption spectrum of the acceptor.
4
J 
 F ( ) ( )     F（  ）  
(8)
Where F (λ) is the corrected fluorescence signal of the donor in the wavelength
range, from λ to λ + Δλ, and ε (λ) is the extinction coefficient of the acceptor at λ.
2
9