Accepted Manuscript Exploring host-guest interactions of sulfobutylether-β- cyclodextrin and phenolic acids by chemiluminescence and site-directed molecular docking Xunyu Xiong, Xinfeng Zhao, Zhenghua Song PII: DOI: Reference:
S0003-2697(14)00227-9 http://dx.doi.org/10.1016/j.ab.2014.05.016 YABIO 11749
To appear in:
Analytical Biochemistry
Received Date: Revised Date: Accepted Date:
2 December 2013 19 May 2014 20 May 2014
Please cite this article as: X. Xiong, X. Zhao, Z. Song, Exploring host-guest interactions of sulfobutylether-βcyclodextrin and phenolic acids by chemiluminescence and site-directed molecular docking, Analytical Biochemistry (2014), doi: http://dx.doi.org/10.1016/j.ab.2014.05.016
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1
Exploring
host-guest
interactions
of
sulfobutylether-β-
2
cyclodextrin and phenolic acids by chemiluminescence and
3
site-directed molecular docking
4
Xunyu Xionga, b, Xinfeng Zhaoc, Zhenghua Songa*
5 6
a. Key Laboratory of Synthetic and Natural Functional Molecule Chemistry of
7
Ministry of Education, College of Chemistry and Materials Science, Northwest
8
University, Xi’an, 710069, China
9 10
b. College of Chemistry & Chemical Engineering, Xi’an Shiyou University, Xi’an 710065, China
11
c. Key Laboratory of Resource Biology and Biotechnology in Western China,
12
Ministry of Education, College of Life Sciences, Northwest University, Xi’an
13
710069, China
14 15
Subject category: Carbohydrates
16
Short title: Interactions of β- cyclodextrin and phenolic acids
17 18 19 20 21 22
*Corresponding author: Zhenghua Song
23
Fax: (+86) 029 88302604;
24
Tel: (+86) 029 88303798;
25
E-mail: [email protected]; [email protected]
26 27 1
28
Abstract:
29
We have developed a rapid method that allows us to characterize the binding
30
interaction of sulfobutylether-β-cyclodextrin (SBE-β-CD) with five therapeutically
31
important phenolic acids: ferulic acid, caffeic acid, gallic acid, protocatechuic acid
32
and vanillic acid. The method utilizes a flow-injection chemiluminescence (FI-CL)
33
technique that relies on the inhibition of a cyclodextrin-luminol chemiluminescence
34
(CL) by increasing amounts of the phenolic acids (PAs). This loss of CL with
35
increasing amounts of PAs fits the equation lg[(I0 -Is)/Is] = lgKPAs + nlg[PAs],
36
allowing calculation of the binding constant (KPAs) and stoichiometry (n). The five
37
phenolic acids and SBE-β-CD/PAs formed complexes with a stoichiometric ratio of
38
1:1. The binding constants were on the order of 107 M-1. These results showed a good
39
correlation with the scores calculated by molecular docking. Further investigation by
40
site-directed molecular docking and linear correlation analysis revealed that PAs
41
entered the larger cavity of SBE-β-CD and the formation constants mainly depended
42
upon the number of hydrogen bond acceptors in the PA structures. All these results
43
indicate that the CL based affinity method can be used for direct determination of the
44
host-guest inclusion interactions and has great potential to become a reliable
45
alternative for quantitatively studying host-guest binding and drug-protein
46
interactions.
47 48
Keywords: Chemiluminescence; Molecular docking; Luminol; Phenolic acids
49 2
50
Introduction
51
Phenolic acids (PAs) are natural antioxidants abundantly distributed in grains [1],
52
vegetables [2, 3], fruits [4] and medicinal plants [5]. Recent studies have shown
53
increasing
54
anti-inflammatory activities [6-9]. Among these acids, ferulic acid [9], caffeic acid
55
[10], gallic acid [11], protocatechuic acid [12] and vanillic acid [13, 14] have the
56
potential to become remedies for fighting diseases of the cardiovascular and
57
cerebrovascular systems and attract growing attention from pharmacologists and
58
chemists. However, the therapeutic application of these compounds is limited because
59
of their unfavorable physicochemical properties, especially their very poor water
60
solubility and the low stability of the molecules [15-17]. This issue may be addressed
61
by complexation with cyclodextrins (CDs).
62
In the pharmaceutical field, CDs prove to be capable of forming stable inclusion
63
complexes with various small lipophilic molecules through non-covalent interactions
64
[18-20]. Several reports have performed analytical characterization of inclusion
65
complexes between ferulic acid and α-CD, hydroxypropyl-β-CD and γ-CD by
66
UV-visible spectroscopy, Fourier transform infrared spectroscopy, differential
67
scanning calorimetry, X-ray diffractometry, scanning electron microscopy and nuclear
68
magnetic resonance spectroscopy [21-23]. The thermodynamic properties of inclusion
69
complexation between caffeic acid and β-CD have also been investigated by
70
fluorescence spectroscopy [24]. Additionally, another report has confirmed the
71
microencapsulation of gallic acid in β-CD [25]. To the best of our knowledge, there
interest
in
these
compounds due
3
to
their
antioxidative
and
72
have been no reports of CD interactions with protocatechuic and vanillic acids in
73
previous publications.
74
Sulfobutylether-β-cyclodextrin (SBE-β-CD), an important derivative of β-CD, can
75
enhance luminol chemiluminescence intensity by forming a 1:1 complex, while rutin
76
and risperidone are capable of quenching this enhancement [26-27]. However, to
77
validate the application of the method would require additional studies describing the
78
binding of other typical compounds and other binding analysis techniques to make
79
valid comparisons and reveal the mechanism of the CL system. In the present work, a
80
flow injection (FI)-chemiluminescence (CL) method is designed to determine the
81
inclusion behaviors of SBE-β-CD and PAs. The presentation also aims to correlate the
82
calculated constants from the CL assay with the data from site-directed docking
83
technology and to reveal the relationship between the photochemical parameters of
84
the compounds and the formation constants.
85 86
Experimental
87
Apparatus
88
The FI-CL system includes a peristaltic pump of the IFFL-DD type flow injection
89
chemiluminescence analyzer (Xi’an Remax Electronic Science-Tech. Co. Ltd., Xi’an,
90
China) for delivering solution streams, PTFE tubing (1.0 mm i.d.) throughout the
91
manifold for carrying the CL reagents, a six-way valve with a loop of 100.0 µL for
92
sampling and an IFFL-DD client system for data acquisition and processing.
93 4
94
Reagents
95
Reference standards of the phenolic acids (Fig. 1): ferulic acid, caffeic acid, gallic
96
acid, protocatechuic acid and vanillic acid were all purchased from the Institute of
97
Drug and Biological Product Control of China (Beijing, China). Luminol (Fluka,
98
Biochemika) was obtained from Xi’an Medicine Purchasing and Supply Station
99
(Xi’an, China). SBE-β-CD (in which the degree of substitution of the sulfobutyl ether
100
group is 7) was acquired from Zibo Qianhui Fine Chemical Co., LTD (Zibo, China).
101
Doubly deionized water was produced by a Milli-Q system (Millipore, Bedford, MA,
102
USA). All other reagents were of analytical grade unless otherwise noted.
103 104
Methods
105
Preparation of solutions
106
Standard stock solutions of 1.0 M ferulic acid, caffeic acid, gallic acid, protocatechuic
107
acid and vanillic acid were prepared in ethanol/water mixture (1:9, V:V). A series of
108
working standards solutions were freshly prepared by diluting the stock solutions as
109
required. Doubly deionized water was used to prepare 10.0 M SBE-β-CD stock
110
standard solution. The stock solution of 25 mM luminol was prepared by dissolving
111
0.44 g luminol in 100 mL of 10 nM NaOH solution in a brown calibrated flask. The
112
resulting solution was stored in a dark place to protect it from light. The stock solution
113
of 25 mM NaOH was prepared by dissolving 0.25 g NaOH powder and diluting it to
114
250 mL in a calibrated flask using doubly deionized water. Working solutions of
115
luminol/NaOH were prepared daily using the above stock solutions. All the solutions 5
116
were kept at 4℃ for future use.
117
2.3.2 FI-CL assay
118
The FI-CL method used in this work was similar to our previous reports [26, 27]
119
except that the drugs were changed to ferulic acid, caffeic acid, gallic acid,
120
protocatechuic acid and vanillic acid, respectively (Fig. 2.). Briefly, flow lines
121
consisted of luminol/NaOH, carrier (doubly deionized water) and SBE-β-CD
122
solutions. At initialization, the whole FI-CL instrument system was equilibrated using
123
doubly deionized water at a flow rate of 2.0 mL/min. Subsequently, 100.0 µL of
124
luminol solution was pumped into the system and merged with SBE-β-CD through the
125
six-way valve. When the baseline became stable, drug solutions were sucked into the
126
line and mixed with the luminol/SBE-β-CD solution. The total mixture was delivered
127
to the luminescence analyzer for detection. The PMT negative voltage was -750 V.
128 129
Calculation of inclusion constants and inclusion sites by the FI-CL method
130
According to our previous work [26, 27], the inclusion constant of a drug binding to
131
SBE-β-CD can be calculated by equation (1):
132
lg
I0 − I s = lg K PAs + n lg[ PAs] Is
(1)
133
where lg represents the base ten logarithm, and I0 and Is are the CL intensity of the
134
luminol/SBE-β-CD system in the absence and presence of the drug, respectively. [PAs]
135
denotes the concentration of the drug when an equilibrium has been reached. KPAs
136
means the inclusion constant of a drug to SBE-β-CD. The constant n describes the
137
stoichiometric ratio of the inclusion reaction. In this case, the plot of equation (1) 6
138
predicts a linear relationship between lg[(I0-Is)/Is] and lg[PAs]. The inclusion constant
139
and stoichiometric ratio can be calculated from the intercept and slope of the curve.
140 141
Molecular docking
142
Molecular docking was performed to obtain the host-guest binding energies and to
143
identify the potential ligand binding sites. The docking experiments were performed
144
with the help of the LibDock program, which was implemented in the software
145
platform Discovery Studio 2.5 (DS 2.5, Accelrys Software Inc., San Diego, CA). The
146
energy-based DS scoring function included terms accounting for short range van der
147
Waals and electrostatic interactions, loss of entropy upon ligand binding, hydrogen
148
bonding and solvation. A PDB file (3M3R) containing the molecular structure
149
information for β-CD was downloaded from the Protein Data Bank (PDB,
150
www.rcsb.org/pdb/). SBE-β-CD was constructed with DS by replacing seven
151
hydroxyls in β-CD with sulfobutyls. Docking preference was defined as ‘high quality’
152
and the number of maximum hits to save was 10. The radius of the ligand-binding site
153
was set to 6 Å with a docking tolerance of 0.25 Å after considering the length of
154
SBE-β-CD molecule. The number of hotspots and the maximum number of
155
conformation hits were set to 100 and 30, respectively. These values were selected
156
regarding the results of preliminary calculations and a best conformation model
157
generation method with a charms force field.
158
Statistical analysis
159
The relationship between the calculated KPAs obtained using the CL method and the 7
160
corresponding score values from molecular docking was explored by comparing the
161
rank order of the two parameters. The correlation between KPAs and physico-chemical
162
data for the phenolic acids calculated by molecular docking with a Charms force field
163
was analyzed. Correlation constants (r2) were obtained from linear regression analysis
164
using SPSS 12.0. A level of p < 0.05 was considered statistically significant.
165 166
Results and discussion
167
The relative CL intensity-time profiles
168
The relative CL intensity-time profile of luminol/SBE-β-CD reaction system was
169
investigated showed in Fig. 3. The maximum CL intensity time (Tmax) and CL
170
intensity (Imax) of luminol/O2 were 8.4 s and 182, respectively. The two parameters
171
changed to 7.8 s and 565 when the CL system of luminol/SBE-β-CD was used. When
172
varied concentrations of caffeic acid were pumped into luminol/SBE-β-CD CL system,
173
Tmax remained same but Imax decreased. These results showed that the CL intensity of
174
luminol was enhanced by SBE-β-CD; caffeic acid substantially blocked the enhanced
175
CL signal in a concentration-dependent manner. These discoveries indicated that
176
caffeic acid could be determined by the concentration-dependent order of
177
luminol/SBE-β-CD/PAs CL reaction system.
178 179
Optimum conditions for the luminol/SBE-β-CD CL system
180
The CL intensity of luminol in basic medium was investigated at the concentrations of
181
0.5, 5, 10, 25, 50, 100, 250 and 500 µM without any addition of SBE-β-CD and PAs 8
182
to the CL system. It was found that the CL intensity was enhanced with increasing
183
luminol concentration up to 25 µM, above which, CL intensity decreased slightly.
184
Thus, 25 µM was the optimum concentration of luminol. Keeping the concentration of
185
luminol as 25 µM, we tested the influence of SBE-β-CD on luminol CL intensity at
186
the concentrations 1.0, 5.0, 10, 50, 100, 200, 500 and 1000 µM. The CL intensity
187
increased rapidly with growing SBE-β-CD concentrations lower than 100 µM, and
188
then decreased slowly when the concentrations continued increasing. Regarding the
189
stable and strong CL intensity, we used 100.0 µM as the best SBE-β-CD
190
concentration for the subsequent experiments.
191
It is widely known that the luminol CL reaction has medium-dependent properties. In
192
this work, NaOH was employed to generate a basic environment for the
193
luminol/SBE-β-CD CL system. The effect of NaOH concentrations on the intensity of
194
the CL system was tested over the range of 5.0 to 250 mM. The concentration of 25
195
mM was used as the basic medium in subsequent experiments, because it produced
196
the strongest and most stable CL signals.
197
The length of the mixing tube and the flow rate have important impacts on the CL
198
intensity. In this work, the effects of these two factors were also explored to determine
199
the best conditions for the luminol/SBE-β-CD CL system. Ten centimeters proved to
200
be the optimum length, resulting in the best sensitivity and repeatability and a fast rate
201
of the CL reaction. The inhibition of CL intensity in the presence of the PAs
202
positively correlated with increasing flow rates, and 2.0 mL/min was determined to be
203
a good choice considering reagent consumption and sensitivity. 9
204
Determination of KPAs and the stoichiometric ratio n
205
Standard solutions of ferulic acid, caffeic acid, gallic acid, protocatechuic acid and
206
vanillic acid were measured by the proposed FI-CL method under the
207
luminol/SBE-β-CD optimized conditions. For each PA, the decrement of CL intensity
208
was proportional to the logarithm of the drug concentration (presented in Table 1).
209
The linear ranges for the PAs were from 3.0 to 1000.0 pM. When setting the ratio of
210
signal to noise as 3, the detection limits of the PAs were determined at the level of 1.0
211
pM. The whole run, including sampling, washing and analyzing procedures, was
212
accomplished in 36 s at a flow rate of 2.0 mL/min, providing an analytical throughput
213
of 100 samples/h with less than 3.0% relative standard deviation.
214
In accordance with Eq. (1), the relationships between the concentrations of the PAs
215
and the decreased CL intensity were further analyzed. The results for KPAs and the
216
stoichiometric ratio for the inclusion of the PAs in SBE-β-CD were listed in Table 2.
217
The rank order of the affinity of the PAs for SBE-β-CD was gallic acid > ferulic
218
acid > vanillic acid > caffeic acid > protocatechuic acid, with stoichiometric ratios of
219
approximate 0.69, 0.77, 0.74, 0.73 and 0.70, respectively.
220 221
Molecular docking analysis
222
Previous publications by Maeztu et al [28] and Yuan et al [29] have reported that the
223
primary hydroxyls on the narrow rim of β-CD were not ionized while the wide rim
224
would be negatively charged in alkaline media. In luminol/SBE-β-CD CL reaction
225
system,
the
luminescent
intermediate 10
of the
reaction (luminol*),
excited
226
3-aminophthalatewith negatively charged entered into the narrow neutral rim of
227
SBE-β-CD and formed 1:1 complex (SBE-β-CD…luminol*), which accelerated the
228
electrons transferring rate of excited 3-aminophthalate, giving the enhanced CL
229
intensity of luminol and producing the effect of complexation enhancement of CL.
230
CD serves as a host of inclusion complex and can include more than one molecule [30,
231
31]. Especially, SBE-β-CD which could provide an extended hydrophobic cavity by
232
the SBE groups on the wide rim of CD. When the drugs with hydrophobicity entered
233
this wide rim and formed 1:1 complex, it resulted in the quenching CL intensity of
234
SBE-β-CD…luminol* and causing the effect of complexation enhancement of
235
quenching.
236
In this work, ten conformers were chosen for the docking of the PAs and SBE-β-CD
237
based on the free energy of binding and score ranking. The minimum binding energy
238
conformer was shown in Fig. 4, and all the data related to these complexations and
239
binding processes were reported in Table 3.
240
In SBE-β-CD, there is a principal binding site that is located in the small cavity with
241
XYZ values of 52.416 Å, 33.14 Å and 28.842 Å and a radius of 6 Å. The minimum
242
energy conformer showed that all the PAs bind to SBE-β-CD within this small cavity
243
and are surrounded by the hydrophobic side chains. This result indicates that the
244
interaction between the PAs and SBE-β-CD is mainly driven by hydrophobic forces.
245
Moreover, there are many hydrogen bonding interactions, due to the presence of
246
several polar groups, such as O-41, O-45, O-47, O-63 and O-65, in SBE-β-CD near
247
the ligands, as shown in Table 3. Considering the distance between donor and 11
248
acceptor atoms, we found one hydrogen bond between H-19 of ferulic acid and O-41
249
of SBE-β-CD. Three hydrogen bonds were found between H-17 and O-47, H-18 and
250
O-45, and H-21 and O-63 during the binding of caffeic acid and SBE-β-CD. Gallic
251
acid formed two hydrogen bonds between H-15 and O-65 and between H-17 and
252
O-41 by its inclusion in SBE-β-CD. For protocatechuic acid and vanillic acid, only
253
one hydrogen bond was formed by complexation with SBE-β-CD. Accordingly, it is
254
believed that the migration of the ligand to the pore of SBE-β-CD is due to a
255
hydrophobic effect, and the final positioning is directed by hydrogen bonds.
256 257
The relationship between KPAs determined by the CL assay and docking scores
258
In molecular docking, the fit score positively corresponds to the affinity of the binding
259
of a ligand to a receptor or a guest to a host. The KPAs values determined by an
260
experimental method should, in theory, be related to the docking scores. The
261
relationship between the KPAs values measured by the proposed CL method and the
262
scores determined by molecular docking was further investigated to assess the
263
reliability of the CL assay for determining guest-host interactions. As shown in Table
264
3, the fit scores of the PAs binding to SBE-β-CD were 86.62 for ferulic acid, 83.16 for
265
caffeic acid, 89.74 for gallic acid, 76.48 for protocatechuic acid and 84.97 for vanillic
266
acid. The rank order was gallic acid > ferulic acid > vanillic acid > caffeic acid >
267
protocatechuic acid. In addition, it is known that the docking scores correlate with the
268
values of free energy of the binding interaction in molecular docking. A higher score
269
means a more negative free energy of the binding by this method. In this case, the 12
270
pattern of fit scores for the five phenolic acids correlated with the pattern of free
271
energy change during binding. Consequently, it was expected that the fit scores’ order
272
highly agreed with that of the KPAs values determined by the CL assay in this work,
273
and this agreement indicated that the proposed CL method will most likely be an
274
alternative for exploring guest-host interaction.
275
Currently, florescence spectrometry and nuclear magnetic resonance spectroscopy are
276
the classic approaches used in studying the guest-host interactions. Using ferulic acid
277
as a probe, the reproducibility and stability of the luminol/SBE-β-CD system was
278
investigated. The experiment lasted for 3 days and the flow system was regularly used
279
over 8 h per day. The relative standard deviation (RSD) for every seven separate
280
determinations was 1.0% and the RSD for the stability investigation during the three
281
days were below 1.2%. These results suggested that luminol/SBE-β-CD CL system
282
exerted good reproducibility and stability. Accordingly, it is concluded: in contrast to
283
the two methods, the proposed CL assay has several advantages such as good stability,
284
high sensitivity and a potential application in complicated matrix through coupling to
285
other approaches with separating capacity [32].
286 287
Relationship between KPAs values and the physico-chemical parameters of the
288
phenolic compounds
289
Different physico-chemical parameters, such as the molecular weight (MW),
290
calculated the logarithm of n-octanol/water partition coefficient (AlogP), number of
291
hydrogen bond donors (NHD), number of hydrogen bond acceptors (NHA) and dipole 13
292
moment (µ), were chosen as molecular descriptors to explore the effect of these
293
parameters on KPAs and reveal the binding mechanism of the five guests targeting
294
SBE-β-CD. All the values of these descriptors were calculated by molecular docking
295
and listed in Table 4. A linear analysis method in SPSS 12.0 was used to correlate the
296
KPAs values and the structural parameters (Table. 5.). NHD was found to negatively
297
correspond to the KPAs values, which indicated that the hydrogen bond contributed to
298
the interaction much more than electrostatic interactions and van der Waals' forces.
299
The reason may be that the hydrogen bond is flexible and easy to bend and can
300
become longer, increasing the targeting area of a guest to a host. On the second
301
section, hydrogen bonds often take place in highly competitive aqueous media under
302
basic conditions. In this work, 25 mM of NaOH was used to provide a basic
303
environment for luminol/SBE-β-CD/PAs CL system. This special environment
304
contributed greatly to the form of hydrogen bonds during the binding of phenolic
305
acids to SBE-β-CD.
306 307
Relationship between KPAs and the antioxidant activity of the PAs
308
A survey of the literature showed that the antioxidant activity, as hydrogen donating
309
free radical scavengers, correlates with the sutures of phenolic acids. In this case, we
310
hypothesized that inclusion in SBE-β-CD should alter the antioxidant activity and
311
performed further work to reveal the relationship between the association constants
312
and these activities. The antioxidant activity data for gallic acid, ferulic acid, caffeic
313
acid, protocatechuic acid and vanillic acid were retrieved from the report by 14
314
Rice-Evans et al [33], where these values were defined as Trolox equivalent
315
antioxidant activity (TEAC).
316
solution with equivalent antioxidant potential to a 1 mM concentration of the
317
compound under investigation. Gallic acid, the 3,4,5-trihydroxy benzoic acid, had an
318
antioxidant capacity of 3.0 mM, corresponding to the three available hydroxyl groups.
319
Incorporation of a hydroxyl group into p-coumaric acid adjacent to that in the
320
para-position, as in caffeic acid, generated a TEAC of 1.26 mM. The antioxidant
321
activity of protocatechuic acid is almost the same as that of caffeic acid. Ferulic acid,
322
which substitutes the 3-hydroxyl group of caffeic acid with a methoxy group,
323
presented a considerably enhanced antioxidant effectiveness of 1.9 mM. The benzoic
324
acid derivative, vanillic acid, yielded an antioxidant activity of 1.43 mM, which is
325
influenced by the adjacency to the carboxylate groups on the phenyl ring.
326
The TEACs of gallic acid, ferulic acid, vanillic acid, caffeic acid and protocatechuic
327
acid (Table 6) showed a good linear relationship with the reciprocals of the
328
corresponding KPAs values calculated in this work. The regression equation was y =
329
-0.0694x + 0.92 with a correlation coefficient of 0.9394. This linear relationship
330
indicated that the KPAs values of PAs depended on the number of hydroxyl groups in
331
the
332
electron-withdrawing property of the carboxylate group in benzoic acids had a
333
negative influence on the H-donating abilities of the hydroxy benzoates. Accordingly,
334
hydrogen bonds should be the main driving force during the binding of phenolic
335
compounds to SBE-β-CD. This result confirmed the conclusion of section 3.5, where
molecule
that
would
The TEAC is defined as the concentration of Trolox
be
strengthened
15
by
steric
hindrance.
The
336
hydrogen interaction proved to be the main force for the inclusion of PAs in
337
SBE-β-CD.
338 339
Conclusion
340
A sensitive FI-CL method was developed and successfully applied in determining the
341
inclusion constants for PAs binding to SBE-β-CD. The KPAs values are substantially
342
different from the different acids, indicating that the proposed affinity method can be
343
rapidly used to determine guest-host interactions. In addition, the data set of the
344
inclusion constants measured in this work exhibits a positive correlation with the
345
scores obtained by molecular docking. The number of hydrogen bond donors
346
contributes much more than other descriptors to KPAs values. This CL-based affinity
347
method proves to be a rapid and highly sensitive, as well as feasible for quantitatively
348
predicting the SBE-β-CD/PAs inclusion interactions. It will most likely be an
349
alternative for revealing the interaction between a guest and a host.
350
351
Acknowledgments
352
The authors gratefully acknowledge Professor Jiang Ru in Fourth Military University
353
for providing DS software. This work received financial support from the National
354
Nature Science Foundation of China (No. 21275118, 21005060), the NWU Graduate
355
Innovation and Creativity Fund (No. 10YZZ29), and the Open Funds from the Key
356
Laboratory of Synthetic and Natural Functional Molecule Chemistry of the Ministry
357
of Education, China.
358
16
359
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445
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peroxyoxalate chemiluminescence. J. Lumin. 39 (1987) 19-27.
447 448
[31].
S.
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Peroxyoxalate
449
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450
Chem. 38 (1992) 788.
451
[32]. P. Khan , D. Idrees, M.A. Moxley, J.A. Corbett, F. Ahmad, G. Figura, W.S. Sly, A.
452
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453
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454
333–355
455
[33]. C.A. Rice-Evans, N.J. Miller, G. Paganga, Structure-antioxidant activity
456
relationships of flavonoids and phenolic acids, Free. Radical. Bio. Med. 20
457
(1996) 933–956.
458 459
21
460
Fig. 1. Chemical structures of the phenolic acids. A, Ferulic acid; B, caffeic acid; C,
461
gallic acid; D, protocatechuic acid; E, vanillic acid
462 463 OH
O
464
O
OH O
OH
465 466 467
HO
468
OH
OH
OH
C
B
A
469
O
470 O
OH
OH
471 472 473
H3CO
HO OH
474
OH
HO
H3CO
OH
E
D
475 476 477 478 479 480 481 22
482
Fig.2. Schematic diagram of FI-CL. Luminol: 25 µM; NaOH 25 mM; SBE-β-CD:
483
100.0 µM; Flow rate: 2.0 mL/min; Mixing tube length: 10.0 cm; High voltage: –750
484
V
485
Pump
486 487 488 489
Luminol/NaOH
Black box Flow cell
Carrier SBE-β-CD
Valve Mixing tube
Sample 490
Detector
Waste
491 492 493 494 495 496 497 498 499 500 501 502 503 23
PC
504
Fig. 3. Relative CL intensity–time profiles of luminol/SBE-β-CD system. Curve 1:
505
luminol CL system; curve 2: luminol CL system with 4.0 pM caffeic acid; curves 3–6:
506
luminol/SBE-β-CD CL system with 4.0, 40.0, 400.0 and 800 pM caffeic acid,
507
respectively; curve 7: luminol/SBE-β-CD CL system
508 509
512 513 514 515 516
500 Relative CL intensity
511
7
600
510
1
400
Tmax = 7.8 s 300 200
Tmax = 8.4 s
100 0 0
9
18
517
Time/s
518 519 520 521 522 523 524 525 24
27
36
526
Fig. 4. An overview of the docking complexes of phenolic acids and SBE-β-CD. A
527
surface was added to SBE-β-CD, and the acids were described as sticks. A, Ferulic
528
acid; B, caffeic acid; C, gallic acid; D, protocatechuic acid; E, vanillic acid.
529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565
A
B
C
D
E
25
566 567
Table 1. Calibration curves and detection limit of phenolic acids Drugs
Linear equation/R2
Linear range/LOD pM
Protocatechuic acid
∆I = 82.6 lg[PAs] + 223.7 / 0.9992
3.0-1000.0/1.0
Caffeic acid
∆I = 137.1 lg[PAs] + 330.7 / 0.9922
5.0-1000.0/2.0
Vanillic acid
∆I = 139.5 lg[PAs] + 392.6 / 0.9986
3.0-700.0/1.0
Ferulic acid
∆I = 169.3 lg[PAs] + 415.1 / 0.9964
5.0-1000.0/2.0
Gallic acid
∆I = 173.1 lg[PAs] + 213.3 / 0.9944
3.0-1000.0/1.0
568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 26
586
Table 2. Inclusion constants and stoichiometric ratio of phenolic acids targeting
587
SBE-β-CD determined by the FI-CL method Drugs
Linear equation
KPAs×107
n
R2
Protocatechuic acid
lg[(I0 -Is)/Is]= 0.70 lg[PAs] + 7.01
1.02
0.70
0.9966
Caffeic acid
lg[(I0 -Is)/Is]= 0.73 lg[PAs] + 7.27
1.86
0.73
0.9978
Vanillic acid
lg[(I0 -Is)/Is]= 0.74 lg[PAs] + 7.64
4.37
0.74
0.9971
Ferulic acid
lg[(I0 -Is)/Is]= 0.77 lg[PAs] + 7.68
4.79
0.77
0.9958
Gallic acid
lg[(I0 -Is)/Is]= 0.69 lg[PAs] + 7.93
8.51
0.69
0.9912
588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 27
616
Table 3. Simulation results of SBE-β-CD/PAs complexations calculated by
617
site-directed molecular docking Drugs
Binding Energy (kcal/mol)
Hydrogen bonds (Numbers)
Hydrogen bonds (Atoms)
Score
Ferulic acid
-8.31
1
H19-O41
86.62
H17-O47 Caffeic acid
-6.24
3
H18-O45
83.16
H21-O63 H15-O65 Gallic acid
-3.49
3
H17-O41
89.74
H18-O45 Protocatechuic acid
-4.78
1
H18-O47
76.48
Vanillic acid
-7.15
1
H17-O47
84.97
618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 28
643
Table 4. Physico-chemical parameters of phenolic acids calculated by site-directed
644
molecular docking with a Charms force. Drugs
MW
AlogP
NHA
NHD
µ
Ferulic acid
194.19
1.315
5
3
3.55
Caffeic acid
180.16
1.443
4
3
2.83
Gallic acid
170.12
0.733
5
4
1.83
Protocatechuic acid
154.12
0.401
4
4
0.83
Vanillic acid
168.15
0.627
4
3
2.60
645 646 647 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 29
676
Table. 5. Linear regression of the relationship between KPAs and structural descriptors.
MW
PC Sig. N
MW
AlogP
NHA
NHD
µ
KPAs
1
0.554 0.254
0.655 0.158
-0.219 0.677
0.816* 0.048
0.092 0.863
6
6
6
6
6
6
0.554 0.254
1
0.113 0.832
-0.692 0.128
0.763 0.078
0.387 0.449
6
6
6
6
6
6
0.655 0.158
0.113 0.832
1
0.333 .519
0.339 0.511
-0.145 0.784
6
6
6
6
6
6
-0.219 0.677
-0.692 0.128
0.333 0.519
1
-0.722 0.105
-0.844* 0.034
6
6
6
6
6
6
0.816 0.048
0.763 0.078
0.339 0.511
-0.722 0.105
1
0.587 0.221
6
6
6
6
6
6
0.587 0.221
1
6 6 6 6 6 PC: Pearson correlation; * Correlation is significant at the level of 0.05 (2-tailed).
6
AlogP
PC Sig. N
NHA
PC Sig. N
NHD
PC Sig. N
µ
PC Sig. N
KPAs
PC Sig.
*
0.092 0.863
0.387 0.449
-0.145 0.784
N
677 678 679 680 681 682 683 684 685 686 687 688 689 690 691 692
30
-0.844 0.034
*
693
Table. 6. Trolox equivalent antioxidant activities (TEAC) of the five phenolic acids Position of -OH
TEAC a(mM)
KPAs×107
Protocatechuic acid
3, 4
1.19 ± 0.03
1.02
Caffeic acid
3, 4
1.26 ± 0.01
1.86
Vanillic acid
4-hydroxy, 3-methoxy
1.43 ± 0.05
4.37
Ferulic acid
4-hydroxy, 3-methoxy
1.90 ± 0.02
4.79
Gallic acid
3, 4, 5
3.01 ± 0.05
8.51
Drugs
694
a. Data collected from the work by Rice-Evans et al. [33]
695
b.
31
S0003-2697(14)00227-9 http://dx.doi.org/10.1016/j.ab.2014.05.016 YABIO 11749
To appear in:
Analytical Biochemistry
Received Date: Revised Date: Accepted Date:
2 December 2013 19 May 2014 20 May 2014
Please cite this article as: X. Xiong, X. Zhao, Z. Song, Exploring host-guest interactions of sulfobutylether-βcyclodextrin and phenolic acids by chemiluminescence and site-directed molecular docking, Analytical Biochemistry (2014), doi: http://dx.doi.org/10.1016/j.ab.2014.05.016
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1
Exploring
host-guest
interactions
of
sulfobutylether-β-
2
cyclodextrin and phenolic acids by chemiluminescence and
3
site-directed molecular docking
4
Xunyu Xionga, b, Xinfeng Zhaoc, Zhenghua Songa*
5 6
a. Key Laboratory of Synthetic and Natural Functional Molecule Chemistry of
7
Ministry of Education, College of Chemistry and Materials Science, Northwest
8
University, Xi’an, 710069, China
9 10
b. College of Chemistry & Chemical Engineering, Xi’an Shiyou University, Xi’an 710065, China
11
c. Key Laboratory of Resource Biology and Biotechnology in Western China,
12
Ministry of Education, College of Life Sciences, Northwest University, Xi’an
13
710069, China
14 15
Subject category: Carbohydrates
16
Short title: Interactions of β- cyclodextrin and phenolic acids
17 18 19 20 21 22
*Corresponding author: Zhenghua Song
23
Fax: (+86) 029 88302604;
24
Tel: (+86) 029 88303798;
25
E-mail: [email protected]; [email protected]
26 27 1
28
Abstract:
29
We have developed a rapid method that allows us to characterize the binding
30
interaction of sulfobutylether-β-cyclodextrin (SBE-β-CD) with five therapeutically
31
important phenolic acids: ferulic acid, caffeic acid, gallic acid, protocatechuic acid
32
and vanillic acid. The method utilizes a flow-injection chemiluminescence (FI-CL)
33
technique that relies on the inhibition of a cyclodextrin-luminol chemiluminescence
34
(CL) by increasing amounts of the phenolic acids (PAs). This loss of CL with
35
increasing amounts of PAs fits the equation lg[(I0 -Is)/Is] = lgKPAs + nlg[PAs],
36
allowing calculation of the binding constant (KPAs) and stoichiometry (n). The five
37
phenolic acids and SBE-β-CD/PAs formed complexes with a stoichiometric ratio of
38
1:1. The binding constants were on the order of 107 M-1. These results showed a good
39
correlation with the scores calculated by molecular docking. Further investigation by
40
site-directed molecular docking and linear correlation analysis revealed that PAs
41
entered the larger cavity of SBE-β-CD and the formation constants mainly depended
42
upon the number of hydrogen bond acceptors in the PA structures. All these results
43
indicate that the CL based affinity method can be used for direct determination of the
44
host-guest inclusion interactions and has great potential to become a reliable
45
alternative for quantitatively studying host-guest binding and drug-protein
46
interactions.
47 48
Keywords: Chemiluminescence; Molecular docking; Luminol; Phenolic acids
49 2
50
Introduction
51
Phenolic acids (PAs) are natural antioxidants abundantly distributed in grains [1],
52
vegetables [2, 3], fruits [4] and medicinal plants [5]. Recent studies have shown
53
increasing
54
anti-inflammatory activities [6-9]. Among these acids, ferulic acid [9], caffeic acid
55
[10], gallic acid [11], protocatechuic acid [12] and vanillic acid [13, 14] have the
56
potential to become remedies for fighting diseases of the cardiovascular and
57
cerebrovascular systems and attract growing attention from pharmacologists and
58
chemists. However, the therapeutic application of these compounds is limited because
59
of their unfavorable physicochemical properties, especially their very poor water
60
solubility and the low stability of the molecules [15-17]. This issue may be addressed
61
by complexation with cyclodextrins (CDs).
62
In the pharmaceutical field, CDs prove to be capable of forming stable inclusion
63
complexes with various small lipophilic molecules through non-covalent interactions
64
[18-20]. Several reports have performed analytical characterization of inclusion
65
complexes between ferulic acid and α-CD, hydroxypropyl-β-CD and γ-CD by
66
UV-visible spectroscopy, Fourier transform infrared spectroscopy, differential
67
scanning calorimetry, X-ray diffractometry, scanning electron microscopy and nuclear
68
magnetic resonance spectroscopy [21-23]. The thermodynamic properties of inclusion
69
complexation between caffeic acid and β-CD have also been investigated by
70
fluorescence spectroscopy [24]. Additionally, another report has confirmed the
71
microencapsulation of gallic acid in β-CD [25]. To the best of our knowledge, there
interest
in
these
compounds due
3
to
their
antioxidative
and
72
have been no reports of CD interactions with protocatechuic and vanillic acids in
73
previous publications.
74
Sulfobutylether-β-cyclodextrin (SBE-β-CD), an important derivative of β-CD, can
75
enhance luminol chemiluminescence intensity by forming a 1:1 complex, while rutin
76
and risperidone are capable of quenching this enhancement [26-27]. However, to
77
validate the application of the method would require additional studies describing the
78
binding of other typical compounds and other binding analysis techniques to make
79
valid comparisons and reveal the mechanism of the CL system. In the present work, a
80
flow injection (FI)-chemiluminescence (CL) method is designed to determine the
81
inclusion behaviors of SBE-β-CD and PAs. The presentation also aims to correlate the
82
calculated constants from the CL assay with the data from site-directed docking
83
technology and to reveal the relationship between the photochemical parameters of
84
the compounds and the formation constants.
85 86
Experimental
87
Apparatus
88
The FI-CL system includes a peristaltic pump of the IFFL-DD type flow injection
89
chemiluminescence analyzer (Xi’an Remax Electronic Science-Tech. Co. Ltd., Xi’an,
90
China) for delivering solution streams, PTFE tubing (1.0 mm i.d.) throughout the
91
manifold for carrying the CL reagents, a six-way valve with a loop of 100.0 µL for
92
sampling and an IFFL-DD client system for data acquisition and processing.
93 4
94
Reagents
95
Reference standards of the phenolic acids (Fig. 1): ferulic acid, caffeic acid, gallic
96
acid, protocatechuic acid and vanillic acid were all purchased from the Institute of
97
Drug and Biological Product Control of China (Beijing, China). Luminol (Fluka,
98
Biochemika) was obtained from Xi’an Medicine Purchasing and Supply Station
99
(Xi’an, China). SBE-β-CD (in which the degree of substitution of the sulfobutyl ether
100
group is 7) was acquired from Zibo Qianhui Fine Chemical Co., LTD (Zibo, China).
101
Doubly deionized water was produced by a Milli-Q system (Millipore, Bedford, MA,
102
USA). All other reagents were of analytical grade unless otherwise noted.
103 104
Methods
105
Preparation of solutions
106
Standard stock solutions of 1.0 M ferulic acid, caffeic acid, gallic acid, protocatechuic
107
acid and vanillic acid were prepared in ethanol/water mixture (1:9, V:V). A series of
108
working standards solutions were freshly prepared by diluting the stock solutions as
109
required. Doubly deionized water was used to prepare 10.0 M SBE-β-CD stock
110
standard solution. The stock solution of 25 mM luminol was prepared by dissolving
111
0.44 g luminol in 100 mL of 10 nM NaOH solution in a brown calibrated flask. The
112
resulting solution was stored in a dark place to protect it from light. The stock solution
113
of 25 mM NaOH was prepared by dissolving 0.25 g NaOH powder and diluting it to
114
250 mL in a calibrated flask using doubly deionized water. Working solutions of
115
luminol/NaOH were prepared daily using the above stock solutions. All the solutions 5
116
were kept at 4℃ for future use.
117
2.3.2 FI-CL assay
118
The FI-CL method used in this work was similar to our previous reports [26, 27]
119
except that the drugs were changed to ferulic acid, caffeic acid, gallic acid,
120
protocatechuic acid and vanillic acid, respectively (Fig. 2.). Briefly, flow lines
121
consisted of luminol/NaOH, carrier (doubly deionized water) and SBE-β-CD
122
solutions. At initialization, the whole FI-CL instrument system was equilibrated using
123
doubly deionized water at a flow rate of 2.0 mL/min. Subsequently, 100.0 µL of
124
luminol solution was pumped into the system and merged with SBE-β-CD through the
125
six-way valve. When the baseline became stable, drug solutions were sucked into the
126
line and mixed with the luminol/SBE-β-CD solution. The total mixture was delivered
127
to the luminescence analyzer for detection. The PMT negative voltage was -750 V.
128 129
Calculation of inclusion constants and inclusion sites by the FI-CL method
130
According to our previous work [26, 27], the inclusion constant of a drug binding to
131
SBE-β-CD can be calculated by equation (1):
132
lg
I0 − I s = lg K PAs + n lg[ PAs] Is
(1)
133
where lg represents the base ten logarithm, and I0 and Is are the CL intensity of the
134
luminol/SBE-β-CD system in the absence and presence of the drug, respectively. [PAs]
135
denotes the concentration of the drug when an equilibrium has been reached. KPAs
136
means the inclusion constant of a drug to SBE-β-CD. The constant n describes the
137
stoichiometric ratio of the inclusion reaction. In this case, the plot of equation (1) 6
138
predicts a linear relationship between lg[(I0-Is)/Is] and lg[PAs]. The inclusion constant
139
and stoichiometric ratio can be calculated from the intercept and slope of the curve.
140 141
Molecular docking
142
Molecular docking was performed to obtain the host-guest binding energies and to
143
identify the potential ligand binding sites. The docking experiments were performed
144
with the help of the LibDock program, which was implemented in the software
145
platform Discovery Studio 2.5 (DS 2.5, Accelrys Software Inc., San Diego, CA). The
146
energy-based DS scoring function included terms accounting for short range van der
147
Waals and electrostatic interactions, loss of entropy upon ligand binding, hydrogen
148
bonding and solvation. A PDB file (3M3R) containing the molecular structure
149
information for β-CD was downloaded from the Protein Data Bank (PDB,
150
www.rcsb.org/pdb/). SBE-β-CD was constructed with DS by replacing seven
151
hydroxyls in β-CD with sulfobutyls. Docking preference was defined as ‘high quality’
152
and the number of maximum hits to save was 10. The radius of the ligand-binding site
153
was set to 6 Å with a docking tolerance of 0.25 Å after considering the length of
154
SBE-β-CD molecule. The number of hotspots and the maximum number of
155
conformation hits were set to 100 and 30, respectively. These values were selected
156
regarding the results of preliminary calculations and a best conformation model
157
generation method with a charms force field.
158
Statistical analysis
159
The relationship between the calculated KPAs obtained using the CL method and the 7
160
corresponding score values from molecular docking was explored by comparing the
161
rank order of the two parameters. The correlation between KPAs and physico-chemical
162
data for the phenolic acids calculated by molecular docking with a Charms force field
163
was analyzed. Correlation constants (r2) were obtained from linear regression analysis
164
using SPSS 12.0. A level of p < 0.05 was considered statistically significant.
165 166
Results and discussion
167
The relative CL intensity-time profiles
168
The relative CL intensity-time profile of luminol/SBE-β-CD reaction system was
169
investigated showed in Fig. 3. The maximum CL intensity time (Tmax) and CL
170
intensity (Imax) of luminol/O2 were 8.4 s and 182, respectively. The two parameters
171
changed to 7.8 s and 565 when the CL system of luminol/SBE-β-CD was used. When
172
varied concentrations of caffeic acid were pumped into luminol/SBE-β-CD CL system,
173
Tmax remained same but Imax decreased. These results showed that the CL intensity of
174
luminol was enhanced by SBE-β-CD; caffeic acid substantially blocked the enhanced
175
CL signal in a concentration-dependent manner. These discoveries indicated that
176
caffeic acid could be determined by the concentration-dependent order of
177
luminol/SBE-β-CD/PAs CL reaction system.
178 179
Optimum conditions for the luminol/SBE-β-CD CL system
180
The CL intensity of luminol in basic medium was investigated at the concentrations of
181
0.5, 5, 10, 25, 50, 100, 250 and 500 µM without any addition of SBE-β-CD and PAs 8
182
to the CL system. It was found that the CL intensity was enhanced with increasing
183
luminol concentration up to 25 µM, above which, CL intensity decreased slightly.
184
Thus, 25 µM was the optimum concentration of luminol. Keeping the concentration of
185
luminol as 25 µM, we tested the influence of SBE-β-CD on luminol CL intensity at
186
the concentrations 1.0, 5.0, 10, 50, 100, 200, 500 and 1000 µM. The CL intensity
187
increased rapidly with growing SBE-β-CD concentrations lower than 100 µM, and
188
then decreased slowly when the concentrations continued increasing. Regarding the
189
stable and strong CL intensity, we used 100.0 µM as the best SBE-β-CD
190
concentration for the subsequent experiments.
191
It is widely known that the luminol CL reaction has medium-dependent properties. In
192
this work, NaOH was employed to generate a basic environment for the
193
luminol/SBE-β-CD CL system. The effect of NaOH concentrations on the intensity of
194
the CL system was tested over the range of 5.0 to 250 mM. The concentration of 25
195
mM was used as the basic medium in subsequent experiments, because it produced
196
the strongest and most stable CL signals.
197
The length of the mixing tube and the flow rate have important impacts on the CL
198
intensity. In this work, the effects of these two factors were also explored to determine
199
the best conditions for the luminol/SBE-β-CD CL system. Ten centimeters proved to
200
be the optimum length, resulting in the best sensitivity and repeatability and a fast rate
201
of the CL reaction. The inhibition of CL intensity in the presence of the PAs
202
positively correlated with increasing flow rates, and 2.0 mL/min was determined to be
203
a good choice considering reagent consumption and sensitivity. 9
204
Determination of KPAs and the stoichiometric ratio n
205
Standard solutions of ferulic acid, caffeic acid, gallic acid, protocatechuic acid and
206
vanillic acid were measured by the proposed FI-CL method under the
207
luminol/SBE-β-CD optimized conditions. For each PA, the decrement of CL intensity
208
was proportional to the logarithm of the drug concentration (presented in Table 1).
209
The linear ranges for the PAs were from 3.0 to 1000.0 pM. When setting the ratio of
210
signal to noise as 3, the detection limits of the PAs were determined at the level of 1.0
211
pM. The whole run, including sampling, washing and analyzing procedures, was
212
accomplished in 36 s at a flow rate of 2.0 mL/min, providing an analytical throughput
213
of 100 samples/h with less than 3.0% relative standard deviation.
214
In accordance with Eq. (1), the relationships between the concentrations of the PAs
215
and the decreased CL intensity were further analyzed. The results for KPAs and the
216
stoichiometric ratio for the inclusion of the PAs in SBE-β-CD were listed in Table 2.
217
The rank order of the affinity of the PAs for SBE-β-CD was gallic acid > ferulic
218
acid > vanillic acid > caffeic acid > protocatechuic acid, with stoichiometric ratios of
219
approximate 0.69, 0.77, 0.74, 0.73 and 0.70, respectively.
220 221
Molecular docking analysis
222
Previous publications by Maeztu et al [28] and Yuan et al [29] have reported that the
223
primary hydroxyls on the narrow rim of β-CD were not ionized while the wide rim
224
would be negatively charged in alkaline media. In luminol/SBE-β-CD CL reaction
225
system,
the
luminescent
intermediate 10
of the
reaction (luminol*),
excited
226
3-aminophthalatewith negatively charged entered into the narrow neutral rim of
227
SBE-β-CD and formed 1:1 complex (SBE-β-CD…luminol*), which accelerated the
228
electrons transferring rate of excited 3-aminophthalate, giving the enhanced CL
229
intensity of luminol and producing the effect of complexation enhancement of CL.
230
CD serves as a host of inclusion complex and can include more than one molecule [30,
231
31]. Especially, SBE-β-CD which could provide an extended hydrophobic cavity by
232
the SBE groups on the wide rim of CD. When the drugs with hydrophobicity entered
233
this wide rim and formed 1:1 complex, it resulted in the quenching CL intensity of
234
SBE-β-CD…luminol* and causing the effect of complexation enhancement of
235
quenching.
236
In this work, ten conformers were chosen for the docking of the PAs and SBE-β-CD
237
based on the free energy of binding and score ranking. The minimum binding energy
238
conformer was shown in Fig. 4, and all the data related to these complexations and
239
binding processes were reported in Table 3.
240
In SBE-β-CD, there is a principal binding site that is located in the small cavity with
241
XYZ values of 52.416 Å, 33.14 Å and 28.842 Å and a radius of 6 Å. The minimum
242
energy conformer showed that all the PAs bind to SBE-β-CD within this small cavity
243
and are surrounded by the hydrophobic side chains. This result indicates that the
244
interaction between the PAs and SBE-β-CD is mainly driven by hydrophobic forces.
245
Moreover, there are many hydrogen bonding interactions, due to the presence of
246
several polar groups, such as O-41, O-45, O-47, O-63 and O-65, in SBE-β-CD near
247
the ligands, as shown in Table 3. Considering the distance between donor and 11
248
acceptor atoms, we found one hydrogen bond between H-19 of ferulic acid and O-41
249
of SBE-β-CD. Three hydrogen bonds were found between H-17 and O-47, H-18 and
250
O-45, and H-21 and O-63 during the binding of caffeic acid and SBE-β-CD. Gallic
251
acid formed two hydrogen bonds between H-15 and O-65 and between H-17 and
252
O-41 by its inclusion in SBE-β-CD. For protocatechuic acid and vanillic acid, only
253
one hydrogen bond was formed by complexation with SBE-β-CD. Accordingly, it is
254
believed that the migration of the ligand to the pore of SBE-β-CD is due to a
255
hydrophobic effect, and the final positioning is directed by hydrogen bonds.
256 257
The relationship between KPAs determined by the CL assay and docking scores
258
In molecular docking, the fit score positively corresponds to the affinity of the binding
259
of a ligand to a receptor or a guest to a host. The KPAs values determined by an
260
experimental method should, in theory, be related to the docking scores. The
261
relationship between the KPAs values measured by the proposed CL method and the
262
scores determined by molecular docking was further investigated to assess the
263
reliability of the CL assay for determining guest-host interactions. As shown in Table
264
3, the fit scores of the PAs binding to SBE-β-CD were 86.62 for ferulic acid, 83.16 for
265
caffeic acid, 89.74 for gallic acid, 76.48 for protocatechuic acid and 84.97 for vanillic
266
acid. The rank order was gallic acid > ferulic acid > vanillic acid > caffeic acid >
267
protocatechuic acid. In addition, it is known that the docking scores correlate with the
268
values of free energy of the binding interaction in molecular docking. A higher score
269
means a more negative free energy of the binding by this method. In this case, the 12
270
pattern of fit scores for the five phenolic acids correlated with the pattern of free
271
energy change during binding. Consequently, it was expected that the fit scores’ order
272
highly agreed with that of the KPAs values determined by the CL assay in this work,
273
and this agreement indicated that the proposed CL method will most likely be an
274
alternative for exploring guest-host interaction.
275
Currently, florescence spectrometry and nuclear magnetic resonance spectroscopy are
276
the classic approaches used in studying the guest-host interactions. Using ferulic acid
277
as a probe, the reproducibility and stability of the luminol/SBE-β-CD system was
278
investigated. The experiment lasted for 3 days and the flow system was regularly used
279
over 8 h per day. The relative standard deviation (RSD) for every seven separate
280
determinations was 1.0% and the RSD for the stability investigation during the three
281
days were below 1.2%. These results suggested that luminol/SBE-β-CD CL system
282
exerted good reproducibility and stability. Accordingly, it is concluded: in contrast to
283
the two methods, the proposed CL assay has several advantages such as good stability,
284
high sensitivity and a potential application in complicated matrix through coupling to
285
other approaches with separating capacity [32].
286 287
Relationship between KPAs values and the physico-chemical parameters of the
288
phenolic compounds
289
Different physico-chemical parameters, such as the molecular weight (MW),
290
calculated the logarithm of n-octanol/water partition coefficient (AlogP), number of
291
hydrogen bond donors (NHD), number of hydrogen bond acceptors (NHA) and dipole 13
292
moment (µ), were chosen as molecular descriptors to explore the effect of these
293
parameters on KPAs and reveal the binding mechanism of the five guests targeting
294
SBE-β-CD. All the values of these descriptors were calculated by molecular docking
295
and listed in Table 4. A linear analysis method in SPSS 12.0 was used to correlate the
296
KPAs values and the structural parameters (Table. 5.). NHD was found to negatively
297
correspond to the KPAs values, which indicated that the hydrogen bond contributed to
298
the interaction much more than electrostatic interactions and van der Waals' forces.
299
The reason may be that the hydrogen bond is flexible and easy to bend and can
300
become longer, increasing the targeting area of a guest to a host. On the second
301
section, hydrogen bonds often take place in highly competitive aqueous media under
302
basic conditions. In this work, 25 mM of NaOH was used to provide a basic
303
environment for luminol/SBE-β-CD/PAs CL system. This special environment
304
contributed greatly to the form of hydrogen bonds during the binding of phenolic
305
acids to SBE-β-CD.
306 307
Relationship between KPAs and the antioxidant activity of the PAs
308
A survey of the literature showed that the antioxidant activity, as hydrogen donating
309
free radical scavengers, correlates with the sutures of phenolic acids. In this case, we
310
hypothesized that inclusion in SBE-β-CD should alter the antioxidant activity and
311
performed further work to reveal the relationship between the association constants
312
and these activities. The antioxidant activity data for gallic acid, ferulic acid, caffeic
313
acid, protocatechuic acid and vanillic acid were retrieved from the report by 14
314
Rice-Evans et al [33], where these values were defined as Trolox equivalent
315
antioxidant activity (TEAC).
316
solution with equivalent antioxidant potential to a 1 mM concentration of the
317
compound under investigation. Gallic acid, the 3,4,5-trihydroxy benzoic acid, had an
318
antioxidant capacity of 3.0 mM, corresponding to the three available hydroxyl groups.
319
Incorporation of a hydroxyl group into p-coumaric acid adjacent to that in the
320
para-position, as in caffeic acid, generated a TEAC of 1.26 mM. The antioxidant
321
activity of protocatechuic acid is almost the same as that of caffeic acid. Ferulic acid,
322
which substitutes the 3-hydroxyl group of caffeic acid with a methoxy group,
323
presented a considerably enhanced antioxidant effectiveness of 1.9 mM. The benzoic
324
acid derivative, vanillic acid, yielded an antioxidant activity of 1.43 mM, which is
325
influenced by the adjacency to the carboxylate groups on the phenyl ring.
326
The TEACs of gallic acid, ferulic acid, vanillic acid, caffeic acid and protocatechuic
327
acid (Table 6) showed a good linear relationship with the reciprocals of the
328
corresponding KPAs values calculated in this work. The regression equation was y =
329
-0.0694x + 0.92 with a correlation coefficient of 0.9394. This linear relationship
330
indicated that the KPAs values of PAs depended on the number of hydroxyl groups in
331
the
332
electron-withdrawing property of the carboxylate group in benzoic acids had a
333
negative influence on the H-donating abilities of the hydroxy benzoates. Accordingly,
334
hydrogen bonds should be the main driving force during the binding of phenolic
335
compounds to SBE-β-CD. This result confirmed the conclusion of section 3.5, where
molecule
that
would
The TEAC is defined as the concentration of Trolox
be
strengthened
15
by
steric
hindrance.
The
336
hydrogen interaction proved to be the main force for the inclusion of PAs in
337
SBE-β-CD.
338 339
Conclusion
340
A sensitive FI-CL method was developed and successfully applied in determining the
341
inclusion constants for PAs binding to SBE-β-CD. The KPAs values are substantially
342
different from the different acids, indicating that the proposed affinity method can be
343
rapidly used to determine guest-host interactions. In addition, the data set of the
344
inclusion constants measured in this work exhibits a positive correlation with the
345
scores obtained by molecular docking. The number of hydrogen bond donors
346
contributes much more than other descriptors to KPAs values. This CL-based affinity
347
method proves to be a rapid and highly sensitive, as well as feasible for quantitatively
348
predicting the SBE-β-CD/PAs inclusion interactions. It will most likely be an
349
alternative for revealing the interaction between a guest and a host.
350
351
Acknowledgments
352
The authors gratefully acknowledge Professor Jiang Ru in Fourth Military University
353
for providing DS software. This work received financial support from the National
354
Nature Science Foundation of China (No. 21275118, 21005060), the NWU Graduate
355
Innovation and Creativity Fund (No. 10YZZ29), and the Open Funds from the Key
356
Laboratory of Synthetic and Natural Functional Molecule Chemistry of the Ministry
357
of Education, China.
358
16
359
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456
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458 459
21
460
Fig. 1. Chemical structures of the phenolic acids. A, Ferulic acid; B, caffeic acid; C,
461
gallic acid; D, protocatechuic acid; E, vanillic acid
462 463 OH
O
464
O
OH O
OH
465 466 467
HO
468
OH
OH
OH
C
B
A
469
O
470 O
OH
OH
471 472 473
H3CO
HO OH
474
OH
HO
H3CO
OH
E
D
475 476 477 478 479 480 481 22
482
Fig.2. Schematic diagram of FI-CL. Luminol: 25 µM; NaOH 25 mM; SBE-β-CD:
483
100.0 µM; Flow rate: 2.0 mL/min; Mixing tube length: 10.0 cm; High voltage: –750
484
V
485
Pump
486 487 488 489
Luminol/NaOH
Black box Flow cell
Carrier SBE-β-CD
Valve Mixing tube
Sample 490
Detector
Waste
491 492 493 494 495 496 497 498 499 500 501 502 503 23
PC
504
Fig. 3. Relative CL intensity–time profiles of luminol/SBE-β-CD system. Curve 1:
505
luminol CL system; curve 2: luminol CL system with 4.0 pM caffeic acid; curves 3–6:
506
luminol/SBE-β-CD CL system with 4.0, 40.0, 400.0 and 800 pM caffeic acid,
507
respectively; curve 7: luminol/SBE-β-CD CL system
508 509
512 513 514 515 516
500 Relative CL intensity
511
7
600
510
1
400
Tmax = 7.8 s 300 200
Tmax = 8.4 s
100 0 0
9
18
517
Time/s
518 519 520 521 522 523 524 525 24
27
36
526
Fig. 4. An overview of the docking complexes of phenolic acids and SBE-β-CD. A
527
surface was added to SBE-β-CD, and the acids were described as sticks. A, Ferulic
528
acid; B, caffeic acid; C, gallic acid; D, protocatechuic acid; E, vanillic acid.
529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565
A
B
C
D
E
25
566 567
Table 1. Calibration curves and detection limit of phenolic acids Drugs
Linear equation/R2
Linear range/LOD pM
Protocatechuic acid
∆I = 82.6 lg[PAs] + 223.7 / 0.9992
3.0-1000.0/1.0
Caffeic acid
∆I = 137.1 lg[PAs] + 330.7 / 0.9922
5.0-1000.0/2.0
Vanillic acid
∆I = 139.5 lg[PAs] + 392.6 / 0.9986
3.0-700.0/1.0
Ferulic acid
∆I = 169.3 lg[PAs] + 415.1 / 0.9964
5.0-1000.0/2.0
Gallic acid
∆I = 173.1 lg[PAs] + 213.3 / 0.9944
3.0-1000.0/1.0
568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 26
586
Table 2. Inclusion constants and stoichiometric ratio of phenolic acids targeting
587
SBE-β-CD determined by the FI-CL method Drugs
Linear equation
KPAs×107
n
R2
Protocatechuic acid
lg[(I0 -Is)/Is]= 0.70 lg[PAs] + 7.01
1.02
0.70
0.9966
Caffeic acid
lg[(I0 -Is)/Is]= 0.73 lg[PAs] + 7.27
1.86
0.73
0.9978
Vanillic acid
lg[(I0 -Is)/Is]= 0.74 lg[PAs] + 7.64
4.37
0.74
0.9971
Ferulic acid
lg[(I0 -Is)/Is]= 0.77 lg[PAs] + 7.68
4.79
0.77
0.9958
Gallic acid
lg[(I0 -Is)/Is]= 0.69 lg[PAs] + 7.93
8.51
0.69
0.9912
588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 27
616
Table 3. Simulation results of SBE-β-CD/PAs complexations calculated by
617
site-directed molecular docking Drugs
Binding Energy (kcal/mol)
Hydrogen bonds (Numbers)
Hydrogen bonds (Atoms)
Score
Ferulic acid
-8.31
1
H19-O41
86.62
H17-O47 Caffeic acid
-6.24
3
H18-O45
83.16
H21-O63 H15-O65 Gallic acid
-3.49
3
H17-O41
89.74
H18-O45 Protocatechuic acid
-4.78
1
H18-O47
76.48
Vanillic acid
-7.15
1
H17-O47
84.97
618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 28
643
Table 4. Physico-chemical parameters of phenolic acids calculated by site-directed
644
molecular docking with a Charms force. Drugs
MW
AlogP
NHA
NHD
µ
Ferulic acid
194.19
1.315
5
3
3.55
Caffeic acid
180.16
1.443
4
3
2.83
Gallic acid
170.12
0.733
5
4
1.83
Protocatechuic acid
154.12
0.401
4
4
0.83
Vanillic acid
168.15
0.627
4
3
2.60
645 646 647 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 29
676
Table. 5. Linear regression of the relationship between KPAs and structural descriptors.
MW
PC Sig. N
MW
AlogP
NHA
NHD
µ
KPAs
1
0.554 0.254
0.655 0.158
-0.219 0.677
0.816* 0.048
0.092 0.863
6
6
6
6
6
6
0.554 0.254
1
0.113 0.832
-0.692 0.128
0.763 0.078
0.387 0.449
6
6
6
6
6
6
0.655 0.158
0.113 0.832
1
0.333 .519
0.339 0.511
-0.145 0.784
6
6
6
6
6
6
-0.219 0.677
-0.692 0.128
0.333 0.519
1
-0.722 0.105
-0.844* 0.034
6
6
6
6
6
6
0.816 0.048
0.763 0.078
0.339 0.511
-0.722 0.105
1
0.587 0.221
6
6
6
6
6
6
0.587 0.221
1
6 6 6 6 6 PC: Pearson correlation; * Correlation is significant at the level of 0.05 (2-tailed).
6
AlogP
PC Sig. N
NHA
PC Sig. N
NHD
PC Sig. N
µ
PC Sig. N
KPAs
PC Sig.
*
0.092 0.863
0.387 0.449
-0.145 0.784
N
677 678 679 680 681 682 683 684 685 686 687 688 689 690 691 692
30
-0.844 0.034
*
693
Table. 6. Trolox equivalent antioxidant activities (TEAC) of the five phenolic acids Position of -OH
TEAC a(mM)
KPAs×107
Protocatechuic acid
3, 4
1.19 ± 0.03
1.02
Caffeic acid
3, 4
1.26 ± 0.01
1.86
Vanillic acid
4-hydroxy, 3-methoxy
1.43 ± 0.05
4.37
Ferulic acid
4-hydroxy, 3-methoxy
1.90 ± 0.02
4.79
Gallic acid
3, 4, 5
3.01 ± 0.05
8.51
Drugs
694
a. Data collected from the work by Rice-Evans et al. [33]
695
b.
31
