Spectroscopic investigation of the interaction of water-soluble azocalix[4]arenes with bovine serum albumin

Article abstract of DOI:10.1016/j.bioorg.2014.12.002

In this work, three hydrosoluble azocalix[4]arene derivatives, 5-(o-methylphenylazo)-25,26,27-tris(carboxymethoxy)-28-hydroxycalix[4]arene (o-MAC-Calix), 5-(m-methylphenylazo)-25,26,27-tris(carboxymethoxy)-28-hydroxycalix[4]arene (m-MAC-Calix) and 5-(p-methylphenylazo)-25,26,27-tris(carboxymethoxy)-28-hydroxycalix[4]arene (p-MAC-Calix) were synthesized. Their structures were characterized by infrared spectrum (IR), nuclear magnetic resonance spectrum (¹H NMR and ¹³C NMR) and mass spectrum (MS). The interactions between these compounds and bovine serum albumin (BSA) were studied by fluorescence spectroscopy, UV-vis spectrophotometry and circular dichroic spectroscopy. According to experimental results, three azocalix[4]arene derivatives can efficiently bind to BSA molecules and the o-MAC-Calix displays more efficient interactions with BSA molecules than m-MAC-Calix and p-MAC-Calix. Molecular docking showed that the o-MAC-Calix was embedded in the hydrophobic cavity of helical structure of BSA molecular and the tryptophan (Trp) residue of BSA molecular had strong interaction with o-MAC-Calix. The fluorescence quenching of BSA caused by azocalix[4]arene derivatives is attributed to the static quenching process. In addition, the synchronous fluorescence spectroscopy indicates that these azocalix[4]arene derivatives are more accessible to Trp residues of BSA molecules than the tyrosine (Tyr) residues. The circular dichroic spectroscopy further verified the binding of azocalix[4]arene derivatives and BSA.

Full text of DOI:10.1016/j.bioorg.2014.12.002

Bioorganic Chemistry 58 (2015) 88–95
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Spectroscopic investigation of the interaction of water-soluble
azocalix[4]arenes with bovine serum albumin
Ping Fan a, , Lu Wan , Yunshan Shang , Jun Wang , Yulong Liu , Xiaoyu Sun , Chen Chen
⇑
a
b
a
a
a
a
a
College of Chemistry, Liaoning University, Shenyang 110036, PR China
State Key Laboratory of Heavy Oil Processing, The Key Laboratory of Catalysis of CNPC, China University of Petroleum, Changping, Beijing 102249, PR China
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 3 July 2014
Available online 16 December 2014
In this work, three hydrosoluble azocalix[4]arene derivatives, 5-(o-methylphenylazo)-25,26,27-tris(carb-
oxymethoxy)-28-hydroxycalix[4]arene (o-MAC-Calix), 5-(m-methylphenylazo)-25,26,27-tris(carboxy-
methoxy)-28-hydroxycalix[4]arene (m-MAC-Calix) and 5-(p-methylphenylazo)-25,26,27-tris(carboxy
methoxy)-28-hydroxycalix[4]arene (p-MAC-Calix) were synthesized. Their structures were characterized
Keywords:
1
13
by infrared spectrum (IR), nuclear magnetic resonance spectrum ( H NMR and C NMR) and mass spec-
trum (MS). The interactions between these compounds and bovine serum albumin (BSA) were studied by
fluorescence spectroscopy, UV–vis spectrophotometry and circular dichroic spectroscopy. According to
experimental results, three azocalix[4]arene derivatives can efficiently bind to BSA molecules and the
o-MAC-Calix displays more efficient interactions with BSA molecules than m-MAC-Calix and p-MAC-
Calix. Molecular docking showed that the o-MAC-Calix was embedded in the hydrophobic cavity of heli-
cal structure of BSA molecular and the tryptophan (Trp) residue of BSA molecular had strong interaction
with o-MAC-Calix. The fluorescence quenching of BSA caused by azocalix[4]arene derivatives is attrib-
uted to the static quenching process. In addition, the synchronous fluorescence spectroscopy indicates
that these azocalix[4]arene derivatives are more accessible to Trp residues of BSA molecules than the
tyrosine (Tyr) residues. The circular dichroic spectroscopy further verified the binding of azocalix[4]arene
derivatives and BSA.
Interaction
Azocalix[4]arene derivatives
Bovine serum albumin (BSA)
Fluorescence spectroscopy
Ó 2014 Elsevier Inc. All rights reserved.
1
. Introduction
Calixarene is made up of phenol linked via methylene groups,
In recent years, several reported water-solution calix[4]arene
derivatives, had showed lots of applications in biological aspects,
such as drug delivery, inhibitor of infection, antibacterial agents,
identification and detection of the organisms protein [14–21].
However, the azocalix[4]arene derivatives were few used in biol-
ogy. Some biological studies related to plasmid DNA binding were
reported by Ungaro and co-workers [22–24]. A series of calix[4]ar-
ene derivatives with modified guanidinium groups in the upper
and lower rims were synthesized. These calix[4]arenes were used
to investigate DNA condensation, toxicity and cell transfection
properties, etc. [22,23]. Some of hydrophilic derivatives had also
showed interesting levels of bioactivity against bacteria, fungi, can-
cerous cells, and enveloped viruses [24].
As we all known, the protein is a kind of important biological
macromolecules, which is the most abundant in the function mate-
rial of biological systems. Compared with other biological mole-
cules, the serum albumins (SA) are the most sufficient proteins in
the plasma, and also show many physiological functions [25–27].
What is more, the structure of BSA molecule is similar to human
serum albumins (HSA) molecule in 76%, therefore, BSA molecules
are used as the study model [28,29]. Koh and his coworkers used
which can exhibit weak fluorescence. Calixarene is a flexible
molecular scaffold for its unique cavity structure, excellent ion
sensing properties, and easy modifications [1–5]. In many cases,
the azo moiety is introduced into the upper rim of calixarenes to
monitor the binding process upon complexation with ions and
molecules. Many studies on azocalixarene derivatives also
involved the functionalization of the lower rim with chelating
groups, such as carboxylic acid, ester and other groups. As a result,
the modified azocalix[4]arene derivatives can form chromogenic
sensor and fluorescent sensor [6–11]. Recently, the triazole linked
anthracenyl appended calix[4]arene that used for recognition of
Co(II) had been reported by Rao and co-workers [12]. They had also
designed and synthesized the 1,3-di-amidoquinoline conjugate of
calix[4]arene as a ratiometric and colorimetric sensor for Zn(II)
[
13].
⇑
Corresponding author. Fax: +86 024 62202053.
E-mail address: pingfan_bft@126.com (P. Fan).
http://dx.doi.org/10.1016/j.bioorg.2014.12.002
045-2068/Ó 2014 Elsevier Inc. All rights reserved.
0

P. Fan et al. / Bioorganic Chemistry 58 (2015) 88–95
89
self-assembled calix[4]arene carboxylic acid derivative monolayers
for protein immobilization with BSA as a model. Shahgaldian and
co-workers synthesized three amphiphilic dodecanoyl calix[4]ar-
ene derivatives and investigated the interactions of their solid lipid
nanoparticles with BSA [30,31]. It is expected that calix[4]arene
derivatives should have a variety of potential functions in biologi-
cal research.
o-MAC-Calix, m-MAC-Calix and p-MAC-Calix were synthesized
in a similar manner to that reported in a previous paper with
raw materials of compound 5a, 5b and 5c, yield 94%, 95% and
95% as yellow solids [35]. Spectroscopy data are as follows
respectively.
o-MAC-Calix: Mp 257–260 °C. ESI–MS (m/z): 739.22 (M + Na);
717.24 (M + H). IR (KBr pellet, cm ): 3394, 2926, 1721, 1343. 1
ꢀ
1
H
For exploring further systematic applications of calix[4]arene
derivatives in biological systems, three novel calix[4]arene
derivatives, containing the hydrophilic groups-carboxyl groups to
interact with biological protein and the azobenzene moiety for a
better visual observant were synthesized. In this research, BSA
was used as a model protein. The maximum value of intrinsic
fluorescence and synchronous fluorescence spectrum were used
to determine the interaction pattern and the binding sites of the
azocalix[4]arene derivatives to BSA molecules, respectively. This
research supplied original reference of hydrosoluble azoca-
lix[4]arene derivatives interact with protein molecules.
6
NMR (300 MHz, DMSO-d ), d (ppm): d 7.73 (s, 2H), 7.48 (d,
J = 8.0 Hz, 1H), 7.38 (d, J = 6.3 Hz, 2H), 7.32–7.26 (m, 1H), 7.18 (d,
J = 7.6 Hz, 2H), 6.85 (q, J = 6.1 Hz, 5H), 6.66 (t, J = 7.5 Hz, 2H), 4.83
(s, 2H), 4.75 (d, J = 11.2 Hz, 4H), 4.47 (d, J = 14.6 Hz, 4H), 3.36 (d,
1
3
6
J = 13.0 Hz, 4H), 2.66 (s, 3H). C NMR (75 MHz, DMSO-d ) d
171.08, 170.73, 156.28, 154.66, 154.02, 150.37, 145.40, 136.72,
135.30, 133.98, 132.61, 131.34, 130.29, 129.40, 129.33, 129.00,
128.86, 128.71, 126.61, 124.43, 123.79, 115.17, 72.12, 70.76,
30.92, 30.56, 17.30.
m-MAC-Calix: Mp 269–273 °C decompose. ESI–MS (m/z): 715.0
ꢀ1
(M–H), 752.9 (M–H + K). IR (KBr pellet, cm ): 3390, 2925, 1724,
1
1
343. H NMR (300 MHz, DMSO-d
J = 6.3 Hz, 2H), 7.44 (t, J = 8.0 Hz, 1H), 7.31 (d, J = 7.5 Hz, 1H), 7.19
d, J = 7.5 Hz, 2H), 6.92–6.80 (m, 5H), 6.71–6.63 (m, 2H), 4.87–
.68 (m, 6H), 4.56–4.40 (m, 4H), 3.55 (d, J = 12.8 Hz, 2H), 3.36 (d,
6
) d 7.75 (s, 2H), 7.63 (d,
2
. Experimental section
(
4
2.1. Reagents
13
J = 9.9 Hz, 2H), 2.39 (s, 3H).
C NMR (75 MHz, DMSO-d
6
) d
1
1
1
2
71.15, 170.84, 156.48, 154.70, 154.08, 152.51, 145.00, 138.94,
Commercially prepared bovine serum albumin (BSA, purity
99.0%) was purchased from Beijing Abxing Biological Technology
35.38, 134.08, 132.67, 131.19, 129.44, 129.31, 129.11, 128.97,
28.86, 124.60, 124.10, 123.83, 119.97, 72.22, 70.91, 31.03, 30.72,
1.16.
>
Company and stored in refrigerator at 4.0 °C. The Tris (hydroxyl-
methyl) aminomethane (Tris), HCl and NaCl were all of analytical
reagent grade, and double distilled water was used for all solution
preparation. The solvents for synthesis of azocalix[4]arene deriva-
tives were purchased from Bodi (Tianjin, China) and were treated
with molecular sieves to remove water before use.
p-MAC-Calix: Mp 255–257 °C. ESI–MS (m/z): 739.22 (M + Na);
17.24 (M + H). IR (KBr pellet, cm ): 3392, 2926, 1714, 1343. 1
ꢀ1
7
H
NMR (300 MHz, DMSO-d
J = 8.3 Hz, 2H), 7.17 (d, J = 7.6 Hz, 2H), 6.87 (d, J = 7.3 Hz, 5H),
6
) d 7.78–7.67 (m, 4H), 7.35 (d,
6
4
.66 (t, J = 7.5 Hz, 2H), 4.82 (s, 2H), 4.74 (d, J = 9.7 Hz, 4H), 4.54–
.40 (m, 4H), 3.36 (d, J = 12.9 Hz, 4H), 2.38 (s, 3H). 13C NMR
(
6
75 MHz, DMSO-d ) d 171.09, 170.81, 156.26, 154.65, 154.06,
2
.2. Apparatus
1
1
7
50.44, 144.88, 140.50, 135.32, 134.04, 132.67, 130.13, 129.49,
29.37, 129.04, 128.91, 128.80, 124.51, 124.08, 123.64, 122.21,
2.21, 70.85, 40.15, 30.95, 30.65, 21.11.
The IR spectra were determined by Fourier transform infrared
spectrophotometer (Spectrum 100, Perkin-Elmer Company, USA).
1
13
The H NMR and C NMR spectra were recorded with nuclear
magnetic double resonance spectrometer (Mercury 300, Varian
Company, USA). The mass spectra were measured on the OTOF-Q
Micromass instrument (Bruker Varian, Germany) and LC–MSD-
Trap/SL (Agilent 1100 Varian Company, USA) using electrospray
ionization methods. The fluorescence measurements were per-
formed with fluorophotometer (Cary 300, Varian Company, USA)
and the UV–vis absorption spectra were recorded with UV–vis
spectrophotometer (Cary 50, Varian Company, USA). Circular
dichroism (CD) measurements were performed by a J-810 spectro
polarimeter (JASCO, Japan) using a 1.0 cm quartz cell.
2
.4. Measurement of binding parameters
The binding parameters of BSA and three azocalix[4]arene deriv-
atives (o-MAC-Calix, m-MAC-Calix and p-MAC-Calix) were mea-
sured by fluorescence spectroscopy method. BSA solution was
prepared in 0.05 mol/L Tris–HCl–NaCl buffer solution (pH = 7.40
and containing 0.05 mol/L NaCl) for keeping the solution acidity
ꢀ
5
and ionic strength. The concentration was 2.00 ꢁ 10 mol/L. Azoc-
alix[4]arene derivatives were prepared in 0.05 mol/L Tris–HCl–NaCl
ꢀ
5
buffer solution and its concentration was 5.00 ꢁ 10 mol/L. The
1
2.50 mL BSA stock solution and the appropriate volume of azoca-
lix[4]arene derivatives stock solution were added into a 25.00 mL
volumetric flask and diluted with Tris–HCl–NaCl buffer solution.
2
.3. Syntheses of calixarene derivatives
ꢀ5
The final BSA concentration was 1.00 ꢁ 10 mol/L, and the concen-
ꢀ5
Compounds 1–3 were synthesized according to previously
tration of azocalix[4]arene derivatives vary from 0.00 ꢁ 10 mol/L
ꢀ5 ꢀ5
described methods [32,33]. The synthesis of the other compounds
, 5a, 5b, 5c, 6a (o-MAC-Calix), 6b (m-MAC-Calix) and 6c (p-MAC-
to 2.50 ꢁ 10 mol/L at an increment of 0.50 ꢁ 10 mol/L. The
fluorescence spectra of BSA solutions along with the increase of
azocalix[4]arene derivatives were recorded in the wavelength of
250–500 nm upon excitation wavelength at 278 nm using a slit
width of 5.0 nm. The tested solutions were incubated for 10 min
before the measurement and the curves of fluorescence spectrum
were got at 37.00 ± 0.02 °C. For the calculation of quenching param-
eters, the maximal intensities of BSA intrinsic fluorescence were
recorded at 345 nm. According to the fluorescent data, the calcu-
lated quenching rate constants, apparent binding association con-
stants, average numbers of binding sites and the binding distances
were showed in Fig. 1. The corresponding parameters were offered
in Table 1.
4
Calix) were first reported in this study (Scheme 1).
Under a nitrogen stream, calix[4]arene 3 and ethyl bromoace-
tate in a 1:1.5 M ratio were added to dry acetonitrile using CaH
2
as catalyst, the mixture was refluxed for 24 h. The suspension
was allowed to cool at room temperature. Then the inorganic salts
were removed by filtration and the volatiles were removed under
reduced pressure to give an oil mixture. The oil was washed with
dichloromethane and concentrated to dryness. The compound 4
was obtained after recrystallization. The compounds 5a, 5b and
5
c were also synthesized according to previously described
methods [34].

9
0
P. Fan et al. / Bioorganic Chemistry 58 (2015) 88–95
Scheme 1. Synthesis of compounds 4, 5a, 5b, 5c, 6a (o-MAC-Calix), 6b (m-MAC-Calix) and 6c (p-MAC-Calix). Reagents and conditions: (i) toluene, aluminum trichloride, rt,
3
5
h; (ii) K
h; (v) 15%NaOH, THF, CH
2
CO
3
, BrCH
2
COOEt, CH
3 2 2 3 2
CN(S) , refluxed 16 h; (iii) CaH , BrCH COOEt, dryCH CN(S), refluxed 24 h; (iv) (2) aromatic amine/acetone, NaNO /4 N HCl, pyridine, 0 °C,
3
CH OH, refluxed 24 h.
2
Fig. 1. Fluorescence spectra of BSA + o-MAC-Calix, BSA + m-MAC-Calix and BSA + p-MAC-Calix solutions with o-MAC-Calix, m-MAC-Calix and p-MAC-Calix concentrations
ꢀ
5
ꢀ5
ꢀ5
ꢀ5
(
from 0.00 ꢁ 10 mol/L to 2.50 ꢁ 10 mol/L at 0.50 ꢁ 10 mol/L intervals) ([BSA] = 1.00 ꢁ 10 mol/L, [Tris–HCl] = [NaCl] = 50 mmol/L, pH = 7.40, Tsolu = 37.00 ± 0.02 °C
and Vtotal = 25.00 mL).
In controlled experiment, four 12.5 mL BSA stock solutions were
added to four 25.0 mL volumetric flasks marked as a–d. And then,
attributed to tryptophan (Trp), tyrosine (Tyr) and phenylalanine
(Phe) residues. As could be seen in Fig. 1, the intrinsic fluorescence
of BSA molecules at 345 nm could be greatly quenched by
azocalix[4]arene derivatives (o-MAC-Calix, m-MAC-Calix and
p-MAC-Calix). It is confirmed that this is owing to the effect of the
interaction of azocalix[4]arene derivatives as quencher with BSA.
5
.0 mL three azocalix[4]arene derivatives stock solutions were
added into above volumetric flasks b, c and d, respectively. Finally,
all of the solutions were diluted to the mark with Tris–HCl–NaCl
buffer solution. The final BSA and azocalix[4]arene derivatives con-
ꢀ5
centration were both 1.00 ꢁ 10 mol/L. Each of the sample solu-
tions was also measured by Circular dichroism (CD) spectrometer.
3.2. Binding constant and binding site number
3
. Results and discussion
The interaction between the azocalix[4]arene derivatives and
BSA molecules in Tris–HCl–NaCl buffer solution could be stud-
ied by measuring the change of intrinsic fluorescence of BSA
molecules around 345 nm in the presence of azocalix[4]arene
derivatives with different concentrations. The data of fluores-
cence intensities could be analyzed by using the Stern–Volmer
Eq. (1):
3
.1. Fluorescence Spectra of BSA solution with azocalix[4]arene
derivatives (o-MAC-Calix, m-MAC-Calix and p-MAC-Calix)
As a protein molecule, bovine serum albumin (BSA) naturally
fluoresces around 345 nm (at 278 nm excitation), which is mainly

P. Fan et al. / Bioorganic Chemistry 58 (2015) 88–95
91
Table 1
q
Quenching constants (Ksv and K ), binding constants, stable constants and binding site numbers calculated according to Stern–Volmer plots, Lineweaver–Burk plots and double
logarithm plots of BSA + o-MAC-Calix, BSA + m-MAC-Calix and BSA + p-MAC-Calix solutions with o-MAC-Calix, m-MAC-Calix and p-MAC-Calix concentrations. (from
ꢀ
5
ꢀ5
mol/L at 0.50 ꢁ 10 mol/L intervals) ([BSA] = 1.00 ꢁ 10ꢀ5 mol/L, [Tris–HCl] = [NaCl] = 50 mmol/L, pH = 7.40,
ꢀ5
0
V
.00 ꢁ 10 mol/L to 2.50 ꢁ 10
Tsolu = 37.00 ± 0.02 °C and
total = 25.00 mL).
System
Stern–Volmer plot
R2
K
sv (L/mol)
K
q
(L/molꢃs)
5
4
4
13
12
12
o-MAC-Calix
m-MAC-Calix
p-MAC-Calix
F
0
F
0
F
0
/F = 1.0178[Q] + 1
/F = 0.8409[Q] + 1
/F = 0.4571[Q] + 1
0.9826
0.9885
0.9980
1.0178 ꢁ 10
8.4090 ꢁ 10
4.5710 ꢁ 10
1.0178 ꢁ 10
8.4090 ꢁ 10
4.5710 ꢁ 10
System
Lineweaver–Burk plot
R2
f
K
LB (L/mol)
D
K (mol/L)
4
4
4
ꢀ5
ꢀ5
ꢀ5
o-MAC-Calix
m-MAC-Calix
p-MAC-Calix
1/[(F
1/[(F
1/[(F
0
0
0
ꢀ F)/F
ꢀ F)/F
ꢀ F)/F
0
0
0
] = 1.1598/[Q] + 0.925
] = 1.4878/[Q] + 0.8615
] = 2.2052/[Q] + 1.0210
0.9994
0.9997
0.9976
1.0811
1.1608
0.9794
7.9755 ꢁ 10
5.7904 ꢁ 10
4.6300 ꢁ 10
1.2538 ꢁ 10
1.7270 ꢁ 10
2.1598 ꢁ 10
0
System
Double logarithm plot
R2
K
A
(L/mol)
n
DG (kJ/mol)
5
5
4
o-MAC-Calix
m-MAC-Calix
p-MAC-Calix
log[(F
log[(F
log[(F
0
0
0
ꢀ F)/F] = 1.1048log[Q] + 5.4939
ꢀ F)/F] = 1.1414log[Q] + 5.5858
ꢀ F)/F] = 1.0194log[Q] + 4.7480
0.9949
0.9977
0.9973
3.118 ꢁ 10
3.853 ꢁ 10
5.598 ꢁ 10
1.1048
1.1414
1.0194
ꢀ32.604
ꢀ33.149
ꢀ28.177
F
0
=F ¼ 1 þ KSV½Q ꢂ ¼ 1 þ K
q
s
0
½QFꢂ
ð1Þ
mechanism may be static and the azocalix[4]arene derivatives
have interaction with BSA molecules [36].
F
To further confirm that the fluorescence quenching mecha-
nisms of azocalix[4]arene derivatives to BSA static quenching are
process, the fluorescence quenching data was analyzed again
according to the Lineweaver–Burk double reciprocal equation
(modified Stern–Volmer equation or double reciprocal) (2).
where the F
0
and F are the fluorescence intensities of BSA solutions
at 345 nm in the absence and presence of azocalix[4]arene deriva-
tives with various concentrations, respectively. K
quenching constant of bimolecular fluorescence,
q
is the apparent
0
is the life time
s
of the fluorophore, Ksv is the Stern–Volmer fluorescence quenching
constant and [Q ] is the concentration of azocalix[4]arene deriva-
tives as a quencher.
F
1=½ðF
0
ꢀ FÞ=F
0
ꢂ ¼ 1=ðfKLB½Q ꢂÞ þ 1=f
ð2Þ
F
Fig. 2a displayed the Stern–Volmer plots of BSA solutions in the
presence of azocalix[4]arene derivatives with various concentra-
tions. All three plots exhibited a comparatively good linear rela-
0 F
In the present case, F , F and Q are same as Eq. (1). The f is the frac-
tion of accessible fluorescence, and the KLB is the static fluorescence
quenching association constant. From the corresponding plots in
Fig. 2b and dates in Table 1, the f and KLB could be obtained
2
2
tionship (R = 0.9826 for BSA + o-MAC-Calix system, R = 0.9885
2
4
for BSA + m-MAC-Calix system and R = 0.998 for BSA + p-MAC-
(f = 1.0811 and KLB = 7.9755 ꢁ 10 L/mol for BSA + o-MAC-Calix sys-
4
Calix system). From Stern–Volmer Eq. (1), the Ksv of these three
tem, f = 1.1608 and KLB = 5.7904 ꢁ 10 L/mol for BSA + m-MAC-Calix
5
4
systems could be obtained (Ksv = 1.0178 ꢁ 10 L/mol for BSA +
system and f = 0.9794 and
K
LB = 4.6300 ꢁ 10 L/mol for BSA +
4
o-MAC-Calix system, Ksv = 8.4090 ꢁ 10 L/mol for BSA + m-MAC-
p-MAC-Calix system). Apparently, the KLB are relatively large.
4
Calix system and Ksv = 4.5710 ꢁ 10 L/mol for BSA + p-MAC-Calix
Whereas the corresponding dissociation constant K
D
(K
D
= 1.2538
ꢀ5
ꢀ5
system). In general, for the most of protein molecules, the
s
0
is
could be cal-
= 1.0178 ꢁ 10 L/molꢃs for BSA + o-MAC-Calix system,
ꢁ 10 mol/L for BSA + o-MAC-Calix system, K
D
= 1.7270 ꢁ 10
ꢀ
8
ꢀ5
known to be approximately 10 s. Therefore, the K
q
mol/L for BSA + m-MAC-Calix system and K
D
= 2.1598 ꢁ 10 mol/L
13
culated (K
q
for BSA + p-MAC-Calix system) were quite small. This result esti-
mates that the quenching is static process [36]. In addition, from
Table 1, it could be found that all of these Lineweaver–Burk plots
1
2
K
K
q
= 8.4090 ꢁ 10 L/molꢃs for BSA + m-MAC-Calix system and
12
q
= 4.5710 ꢁ 10 L/molꢃs for BSA + p-MAC-Calix system) and all
2
corresponding results were showed in Table 1. It can be seen
that the array order is
had better linear relationship (R = 0.9994 for BSA + o-MAC-Calix sys-
2
2
K
q(BSA+o-MAC-Calix) > Kq(BSA+m-MAC-Calix)
>
tem, R = 0.9997 for BSA + m-MAC-Calix system and R = 0.9976 for
BSA + p-MAC-Calix system) than the corresponding Stern–Volmer
plots. Thus, it can be confirmed again that the fluorescence quenching
mechanism of azocalix[4]arene derivatives to BSA is mainly static
quenching procedure indeed.
K
q(BSA+p-MAC-Calix). All of the above K values are greater than the
q
10
maximum value (2.0 ꢁ 10 L/mol s) of the diffusion controlled
quenching process of biological macromolecules. The results
provide preliminary evidences that the dominating quenching
Fig. 2. Stern–Volmer plot (a), Lineweaver–Burk plot (b) and double logarithm plot (c) of BSA + o-MAC-Calix, BSA + m-MAC-Calix and BSA + p-MAC-Calix solutions with
ꢀ
5
ꢀ5
ꢀ5
ꢀ5
o-MAC-Calix, m-MAC-Calix and p-MAC-Calix concentrations (from 0.00 ꢁ 10 mol/L to 2.50 ꢁ 10 mol/L at 0.50 ꢁ 10 mol/L intervals) ([BSA] = 1.00 ꢁ 10 mol/L,
[
Tris–HCl] = [NaCl] = 50 mmol/L, pH = 7.40, Tsolu = 37.00 ± 0.02 °C and Vtotal = 25.00 mL).

9
2
P. Fan et al. / Bioorganic Chemistry 58 (2015) 88–95
According to Fig. 2c, the equilibrium constants (K
binding site numbers (n) could be calculated by using the double
logarithm Eq. (3).
A
) and the
where r is the binding distance between the donor and acceptor,
and R is the critical binding distance when the efficiency (E) of
energy transfer is 50%, which could be calculated by the Eq. (6):
0
log½ðF
0
ꢀ FÞ=FÞꢂ ¼ log K
A
þ n log½QFꢂ
ð3Þ
6
ꢀ25
2
ꢀ4
R ¼ 8:8 ꢁ 10 ðK n uD JÞ
ð6Þ
0
The results were shown in Table 1, K
A
and n could be measured
where the K2 is the spatial orientation factor of the dipole, n is the
refractive index of medium, is the quantum yield of the donor in
the absence of acceptor and J is the overlap integral of the emission
spectrum of the donor and the absorption spectrum of the acceptor.
In the present case, K , n and
respectively. And then, the J could be calculated by the Eq. (7).
from the intercept and slope by plotting log[(F
log[Q ]. Obviously, being similar to the KLB, the array order was also
It illus-
0
ꢀ F)/F)] against
u
D
F
KA(BSA+o-MAC-Calix) > KA(BSA+m-MAC-Calix) > KA(BSA+p-MAC-Calix).
trates that o-MAC-Calix has more efficient binding to BSA molecules
than m-MAC-Calix and p-MAC-Calix. The reason may be that the
bonding site of the methyl on azocalix[4]arene derivatives affect
the interaction with BSA. The methyl of azo group are in the
ortho-position so that the o-MAC-Calix gets hydrophobic interac-
tion with BSA easier. The three n values are equal to about 1, indicat-
ing that there is the same type of binding site for azocalix[4]arene
derivatives to BSA.
2
D
u are 2/3, 1.336 and 0.15 for BSA,
X
X
ðkÞk4Dk=
J ¼
FðkÞe
FðkÞ
D
k
ð7Þ
where the k is the wavelength of fluorescence spectrum of BSA and
maximum absorption wavelength of azocalix[4]arene derivative.
The F(k) is the fluorescence intensity of BSA and the
e (k) is the
Furthermore, the titration curve of BSA with the addition of
azocalix[4]arene derivatives were used to prove the binding site
number (n). The initial fluorescence intensity of BSA started to
decrease as a function of increasing concentration of azoca-
lix[4]arne and reached an approximately saturation point after
the addition of 1 equiv azocalix[4]arene derivatives (Fig. 3). It indi-
cates the binding site number (n) is in line with the data calculated
according to the double logarithm equation, which strongly sug-
absorbance of azocalix[4]arene derivative at k. Fig. 4 showed the
spectral overlap of fluorescence emission of BSA solution and UV–
vis absorption of azocalix[4]arene derivatives solutions at 200–
5
00 nm, respectively. The efficiency (E) of energy transfer could
be determined by the Eq. (8):
E ¼ 1 ꢀ F=F
0
ð8Þ
where F and F are the fluorescence intensities of BSA solutions with
0
gests the existence of a single binding site in BSA protein.
ꢀ5
1
.0 ꢁ 10 mol/L at 345 nm in the absence and presence of azoca-
0
Utilizing K
A
, the free energy change (
D
G ) could be calculated
ꢀ5
lix[4]arene derivatives with 1.0 ꢁ 10 mol/L, respectively. Accord-
from the relationship (4):
ing to the above equations from (4)–(8), E, R and J could be
0
0
D
G ¼ ꢀRT ln K
A
ð4Þ
calculated and the corresponding results were given in Table 2.
The binding distances (r) between BSA and azocalix[4]arene
derivatives were calculated and the corresponding results were
Here, R = 8.314 J/molꢃK and T = 310 K, and the
D
G0 were
ꢀ
32.604 kJ/mol for BSA + o-MAC-Calix system, ꢀ33.149 kJ/mol for
2
.2902 nm for BSA + o-MAC-Calix system, 2.6508 nm for BSA + m-
BSA + m-MAC-Calix system and ꢀ28.177 kJ/mol for BSA + p-MAC-
MAC-Calix system and 2.7194 nm for BSA + p-MAC-Calix system.
The connection of azocalix[4]arene derivatives with BSA may be
formed due to the hydrogen bond, benzene ring stacking, the
hydrophobic interaction of the methyl was showed in Fig. 5. The
benzene ring stacking and hydrogen bond are almost same
between azocalix[4]arene and BSA molecular. However, their
hydrophobic interactions display some differences, because hydro-
phobic interactions depend on the distance and angle between
methyl group of azobenzene and BSA molecules. The o-MAC-Calix
may be easier forming hydrophobic interaction with BSA than the
other two azocalix[4]arene derivatives. Therefore, the order of
binding intensity is o-MAC-Calix-BSA > m-MAC-Calix-BSA > p-
0
Calix system. The negative sign for
D
G
indicates that the
binding of azocalix[4]arene derivatives to BSA molecule is
spontaneous.
3.3. Binding distances between BSA with azocalix[4]arene derivatives
(
o-MAC-Calix, m-MAC-Calix and p-MAC-Calix)
In the preceding paragraph, the interactions of three azoca-
lix[4]arene derivatives with BSA molecules could be speculated
through the fluorescence quenching of BSA solutions along with
the increase of azocalix[4]arene derivatives concentration. The
binding distance (r) between BSA and azocalix[4]arene derivatives
could be determined according to Foster’s non-radioactive energy
transfer theory (FRET) [37,38]. Based on this theory, the efficiency
A
MAC-Calix-BSA. This result is consistent with the K previously
obtained. Apparently, the binding distances of three azoca-
lix[4]arene derivatives with BSA are all less than 7.0 nm, which
indicates that the energy transfer from BSA to azocalix[4]arene
derivatives occur with high possibility [39]. It also suggests that
the fluorescence quenching of BSA molecule is through energy
transfer from BSA to corresponding azocalix[4]arene.
(E) of energy transfer between the donor (BSA) and acceptor could
be calculated by using Eq. (5):
h
i
6
E ¼ 1= 1 þ ðr=R
0
Þ
ð5Þ
–5
Fig. 3. The titration curve of BSA with the addition of azocalix[4]arene derivatives(o-MAC-Calix, m-MAC-Calix and p-MAC-Calix) ([BSA] = 1.00 ꢁ 10 mol/L, [Tris–
HCl] = [NaCl] = 50 mmol/L, pH = 7.40, Tsolu = 37.00 ± 0.02 °C and Vtotal = 25.00 mL).

P. Fan et al. / Bioorganic Chemistry 58 (2015) 88–95
93
Fig. 4. Spectral overlap of fluorescence (kex = 278 nm) of BSA solution and absorption of BSA + o-MAC-Calix, BSA + m-MAC-Calix and BSA + p-MAC-Calix solutions ([BSA] =
ꢀ
5
[
o-MAC-Calix] = [m-MAC-Calix] = [p-MAC-Calix] = 1.00 ꢁ 10 mol/L, [Tris–HCl] = [NaCl] = 50 mmol/L, pH = 7.40, Tsolu = 37.00 ± 0.02 °C and Vtotal = 25.00 mL).
Table 2
3.4. Synchronous fluorescence spectra of BSA + o-MAC-Calix, BSA + m-
MAC-Calix and BSA + p-MAC-Calix solutions
Energy transfer efficiency (E), critical binding distance (R), overlap integral (J) and
binding distance (r) calculated according to Foster’s non-radioactive energy transfer
ꢀ
5
theory ([BSA] = [o-MAC-Calix] = [m-MAC-Calix] = [p-MAC-Calix] = 1.00 ꢁ 10 mol/L,
Synchronous fluorescence spectroscopy technique was intro-
duced by Lloyd [40]. Its spectra could provide much valuable infor-
mation about the microenvironment around fluorogens in
[
Tris–HCl] = [NaCl] = 50 mmol/L,
pH = 7.40,
T
solu = 37.00 ± 0.02 °C
and
V
total = 25.00 mL).
System
3
E (%)
R (nm)
J (cm ꢃL/mol)
r (nm)
biomolecules [41]. For protein molecules, when the
fixed, the spectrum characteristic of Tyr residues can be observed,
and when k = 60 nm is done, the spectrum characteristic of Trp
residues can be obtained in general [42,43]. In this research, the
Dk = 15 nm is
ꢀ14
ꢀ15
ꢀ15
o-MAC-Calix
m-MAC-Calix
p-MAC-Calix
48.01
42.15
29.90
2.5560
2.5147
2.3594
1.00949 ꢁ 10
9.15440 ꢁ 10
6.24610 ꢁ 10
2.2902
2.6508
2.7194
D
Fig. 5. Difference of hydrophobic interaction of BSA + azocalix[4]arene derivatives (o-MAC-Calix, m-MAC-Calix or p-MAC-Calix).
Fig. 6. Synchronous fluorescence spectra of BSA + o-MAC-Calix, BSA + m-MAC-Calix and BSA + p-MAC-Calix solutions with o-MAC-Calix, m-MAC-Calix and p-MAC-Calix
ꢀ
5
ꢀ5
ꢀ5
ꢀ5
concentrations (from 0.00 ꢁ 10 mol/L (o) to 2.50 ꢁ 10 mol/L (e) at 0.50 ꢁ 10 mol/L intervals) ([BSA] = 1.00 ꢁ 10 mol/L, [Tris–HCl] = [NaCl] = 50 mmol/L, pH = 7.40,
T
solu = 37.00 ± 0.02 °C and Vtotal = 25.00 mL).

9
4
P. Fan et al. / Bioorganic Chemistry 58 (2015) 88–95
Fig. 7. (a) Docking model of o-MAC-Calix at the active sites of BSA. (b) Minimum energy docking conformation obtained from docking simulation. The BSA is presented by
ribbon structure whereas o-MAC-Calix by stick model.
state may affect their secondary structure [44]. As we know, the
CD spectra of BSA exhibits two negative bands at 208 and
2
22 nm, which are characteristics of
a
p
-helix structure of proteins.
⁄
The band at 208 nm corresponds to
helix, whereas the band at 222 nm corresponds to n ?
?
p
transition of the
a-
⁄
p
transi-
tion of the -helix [45,46]. In general situation, the
a
a-helix content
of BSA could decrease along with the increase of micromolecule
compound concentration. From Fig. 8, it could be seen that the
negative peak decreases (curves b, c and d) comparing with that
of BSA original solution (curve a) at 208 nm when the BSA mole-
cules were mixed with azocalix[4]arene derivatives in Tris–HCl–
NaCl buffer solution. It is proves that the helicity of BSA decreased
significantly with adding azocalix[4]arene derivatives [45,47]. And
the decreasing degree is o-MAC-Calix > m-MAC-Calix > p-MAC-
Calix. However, the negative peaks of BSA (curves b, c and d)
slightly increase when m-MAC-Calix and p-MAC-Calix added to
BSA at 222 nm. It may be due to the presence of potential chirality
Fig. 8. CD spectras of BSA + azocalix[4]arene derivatives (o-MAC-Calix, m-MAC-
Calix or p-MAC-Calix) solutions. ([BSA] = [o-MAC-Calix] = [m-MAC-Calix] = [p-MAC-
ꢀ
5
ꢀ1
ꢀ1
Calix] = 1.0 ꢁ 10 mol L , pH = 7.40, [NaCl] = 50 mmol L , Tsolu = 37.0 ± 0.2 °C and
V
total = 25.00 mL).
of m-MAC-Calix and p-MAC-Calix, which maintains or increases a-
helix content of BSA molecules [48].
synchronous fluorescence spectroscopy was used to estimate the
binding site of azocalix[4]arene derivatives to BSA molecules.
From Fig. 6, it could be seen that three courses the fluorescence
4
. Conclusion
intensities at
Dk = 15 nm (upper) and Dk = 60 nm (nether) both
decreased gradually with the increase of azocalix[4]arene deriva-
tives. Three azocalix[4]arene derivatives have stronger fluores-
In this paper, three water-soluble azocalix[4]arene derivatives
o-MAC-Calix, m-MAC-Calix and p-MAC-Calix) were synthesized.
(
cence intensity for
Dk = 60 nm than for Dk = 15 nm. It proves
Their interactions with BSA molecules in Tris–HCl–NaCl buffer
solution were studied by fluorescence spectroscopy. The experi-
mental results shows that three azocalix[4]arene derivatives could
all bind to BSA molecules obviously, and then quenched their
intrinsic fluorescence efficiently. According to the fluorescence
that three azocalix[4]arene derivatives can bind the Trp residues
much stronger than Tyr residues.
In order to illustrate the o-MAC-Calix has better hydrophobic
interaction. Molecular docking experiment was been used. Based
on the Molegro Virtual Docker docking, the o-MAC-Calix is located
at the active sites, which is on the surface of the BSA. It could be
seen that the Trp residue of BSA molecular had strong interaction
with o-MAC-Calix, as seen in Fig. 7a. This result is consistent with
the experimental data above-mentioned. From Fig. 7b, the mini-
mum energy docking conformation was obtained in docking simu-
lation. It shows that the o-MAC-Calix is embedded in the
hydrophobic cavity of helical structure of BSA molecular, which
may be due to the strong hydrophobic interaction between methyl
group of azobenzene and BSA molecule.
quenching calculation, the bimolecular quenching constant (K
apparent quenching constant (Ksv), equilibrium constants (K ), cor-
responding dissociation constant (K ) and binding site number (n)
q
),
A
D
were measured. The quenching mechanisms are all static process.
In addition, the synchronous fluorescence spectroscopy indicates
that three azocalix[4]arene derivatives were more vicinal to Trp
residues than Tyr residues. The CD spectroscopy shows the content
of
a-helix changing under the effect of azocalix[4]arene deriva-
tives. It is expected that this work could offer some valuable refer-
ences for promoting the application of calixarene derivatives in the
biological field.
3.5. Circular dichroism (CD) spectra of BSA, BSA + o-MAC-Calix,
BSA + m-MAC-Calix and BSA+p-MAC-Calix solutions
Acknowledgments
CD spectroscopy is one of the most powerful techniques for
examining the conformational changes of proteins whose bound
The authors greatly acknowledge the National Natural Science
Foundation of China (No. 21171081), the Science Foundation of

P. Fan et al. / Bioorganic Chemistry 58 (2015) 88–95
95
the Education Department of Liaoning Province (No. L2011007)
and the Science and Technology Planning Project of Shenyang
Technology Bureau (No. F13-318-1-51) for financial supports. The
authors thank Molegro ApS for kindly providing a free evaluation
copy of their software package and the laboratory of Prof. Chun
Hu of Shenyang Pharmaceutical University. The authors also would
like to thank our colleagues and other students for their participat-
ing in this work.
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