Computer-aided rational design of novel EBF analogues with an aromatic ring

Article abstract of DOI:10.1007/s00894-016-3011-3

Odorant binding proteins (OBPs) are important in insect olfactory recognition. These proteins bind specifically to insect semiochemicals and induce their seeking, mating, and alarm behaviors. Molecular docking and molecular dynamics simulations were performed to provide computational insight into the interaction mode between AgamOBP7 and novel (E)-β-farnesene (EBF) analogues with an aromatic ring. The ligand-binding cavity in OBP7 was found to be mostly hydrophobic due to the presence of several nonpolar residues. The interactions between the EBF analogues and the hydrophobic residues in the binding cavity increased in strength as the distance between them decreased. The EBF analogues with an N-methyl formamide or ester linkage had higher docking scores than those with an amide linkage. Moreover, delocalized π–π and electrostatic interactions were found to contribute significantly to the binding between the ligand benzene ring and nearby protein residues. To design new compounds with higher activity, four EBF analogues D1–D4 with a benzene ring were synthesized and evaluated based on their docking scores and binding affinities. D2, which had an N-methyl formamide group linkage, exhibited stronger binding than D1, which had an amide linkage. D4 exhibited particularly strong binding due to multiple hydrophobic interactions with the protein. This study provides crucial foundations for designing novel EBF analogues based on the OBP structure. [Figure not available: see fulltext.]

Full text of DOI:10.1007/s00894-016-3011-3

J Mol Model (2016) 22: 144
DOI 10.1007/s00894-016-3011-3
ORIGINAL PAPER
Computer-aided rational design of novel EBF analogues
with an aromatic ring
Shanshan Wang1 & Yufeng Sun2 & Shaoqing Du1 & Yaoguo Qin1 & Hongxia Duan1
&
Xinling Yang1
Received: 29 March 2016 /Accepted: 19 May 2016 /Published online: 1 June 2016
#
Springer-Verlag Berlin Heidelberg 2016
Abstract Odorant binding proteins (OBPs) are important in
insect olfactory recognition. These proteins bind specifically
to insect semiochemicals and induce their seeking, mating,
and alarm behaviors. Molecular docking and molecular dy-
namics simulations were performed to provide computational
insight into the interaction mode between AgamOBP7 and
novel (E)-β-farnesene (EBF) analogues with an aromatic ring.
The ligand-binding cavity in OBP7 was found to be mostly
hydrophobic due to the presence of several nonpolar residues.
The interactions between the EBF analogues and the hydro-
phobic residues in the binding cavity increased in strength as
the distance between them decreased. The EBF analogues
with an N-methyl formamide or ester linkage had higher
docking scores than those with an amide linkage. Moreover,
delocalized π–π and electrostatic interactions were found to
contribute significantly to the binding between the ligand ben-
zene ring and nearby protein residues. To design new com-
pounds with higher activity, four EBF analogues D1–D4 with
a benzene ring were synthesized and evaluated based on their
docking scores and binding affinities. D2, which had an N-
methyl formamide group linkage, exhibited stronger binding
than D1, which had an amide linkage. D4 exhibited particu-
larly strong binding due to multiple hydrophobic interactions
with the protein. This study provides crucial foundations for
designing novel EBF analogues based on the OBP structure.
.
.
Keywords EBF analogues Odorant binding protein
.
.
Rational design Semiochemicals Insect behavior regulators
Introduction
Aphids are a major agricultural pest because they not only
directly damage the leaves of many cultivated plants via a
sap-feeding mode but also indirectly impact plant growth
through a virus-transmitting mode [1]. Most aphids release
the aphid alarm pheromone when they are attacked by other
species, so this semiochemical could be used to keep aphids
away from crops to protect them. The active ingredient in the
aphid alarm pheromone was identified as (E)-β-farnesene
(EBF) [2]. It is well known that EBF is unstable because of
its multiple double bonds, which are easily oxidized in the
environment. Therefore, EBF has been modified continually
in recent years to develop novel EBF analogues. In particular,
the EBF conjugated double bond has been modified in several
studies (Fig. 1). An early study by Dawson et al. reported that
some EBF analogues in which a few double bonds in the
middle or terminal position were removed had better repellent
responses [3]. EBF analogues without a terminal conjugated
double bond were also found to exhibit good repellent activity
by Li et al. [4]. At the same time, dimers of EBF analogues
exhibited good alarm responses to peach aphids when the
terminal conjugated double bonds in both EBF analogues
were hydrogenated [5]. Recently, the EBF conjugated double
bond was replaced with heteroatoms or heterocyclics, and
Yang et al. synthesized approximately 200 new EBF
Electronic supplementary material The online version of this article
(doi:10.1007/s00894-016-3011-3) contains supplementary material,
which is available to authorized users.
* Hongxia Duan
hxduan@cau.edu.cn
* Xinling Yang
yangxl@cau.edu.cn
1
Department of Applied Chemistry, College of Science, China
Agricultural University, Beijing 100193, China
2
Laboratory of Agro-products Quality Safety Risk Assessment,
Institute of Agro-products Processing Science and Technology,
Chinese Academy of Agricultural Sciences, Beijing 100193, China

144 Page 2 of 9
J Mol Model (2016) 22: 144
Fig. 1 The aphid alarm
pheromone EBF and some of its
analogues. EBF analogues
synthesized by other groups and
by our group are shown in the left
and right boxes, respectively
analogues [6–10]. Many EBF analogues with an oxadiazine or
pyrazole ring were found to have excellent repellent responses
to aphids [7, 8].
Moreover, new EBF analogues were designed and synthe-
sized based on the determined ligand–protein interaction
mode. This study provides significant guidance for the design
of novel EBF analogues based on OBPs.
It is well known that odorant binding proteins (OBPs) can
bind specifically to insect semiochemicals to activate their
seeking, mating, and alarm behaviors. Therefore, OBPs have
become an interesting potential target in attempts create new
insect behavior regulators in recent years, and the binding
between some OBPs in aphids and EBF has received particu-
lar attention [11–13]. Pelosi et al. reported that ApisOBP3 is
involved in the detection and recognition of EBF, which ex-
hibits a high binding affinity to it [13]. SaveOBP7 in
Hemiptera species was also found to play an important role
in EBF recognition [14]. Recently, ApisOBP7 was proposed
by Yang et al. to bind specifically not only to EBF but also to
its analogues with a pyrazole ring [15]. More importantly,
some EBF analogues with high binding affinities surprisingly
exhibited good repellent responses to aphids [15]. Moreover,
OBP, which is an important protein in insect olfactory recog-
nition, could transfer the bound ligand to downstream olfac-
tory receptors (ORs) to further stimulate insect behavioral re-
sponses. If OBP and OR constitute a two-step filter, OR would
be another potential target in attempts to control insect behav-
ior by olfactory perception because OBP and OR share a com-
mon ligand [16]. However, studies of the binding between OR
and the ligand are currently still limited.
Increasing attention has recently focused on ligand–protein
interactions. Recently, the crystal structures of insect OBP1,
OBP7, OBP4, and OBP14 were successfully solved, and they
could stimulate new research on novel insect behavioral con-
trols based on OBPs [16–21]. Surprisingly, in our previous
work, some EBF analogues with a pyrazole or benzene ring
were found to exhibit not only good binding affinity to
ApisOBP7 but also good repellent responses to aphids [15].
However, the interaction mechanism between these EBF an-
alogues and OBP7 is unknown, and it is unclear if new ana-
logues can be designed based on the OBP7 binding cavity.
Therefore, the interaction mode between OBP7 and EBF an-
alogues with an aromatic ring was investigated by molecular
docking and molecular dynamics simulations in this study.
Materials and methods
In our previous work, twelve EBF analogues with a pyrazole
or benzene ring were synthesized [8, 15], and their binding
affinities to ApisOBP7 were measured by recording the fluo-
rescence peak values of different EBF analogues and N-phe-
nyl-1-naphthylamine (1-NPN) during their competitive bind-
ing to the protein [13, 22]. However, the interaction mode
between these EBF analogues and ApisOBP7 was not clear.
To our knowledge, no ApisOBP7 crystal structure has been
reported to date. AgamOBP7 complexed with a palmitic acid
(PA) (PDB code: 3R1P) shares a certain sequence identity
with ApisOBP7 (Fig. S1 in the BElectronic supplementary
material,^ ESM) and co-crystallizes with a long linear ligand
(PA) similar to EBF [19]. More importantly, AgamOBP7 has a
hydrophobic, elongated, and open binding cavity similar to
that of the ApisOBP7 model (Fig. S2 in the ESM) surrounded
by nearly the same nonpolar residues (Table S1 in the ESM)
[23]. This binding cavity can accommodate elongated linear
ligands similar to EBF analogues (Fig. S3 in the ESM) [19].
Therefore, twelve EBF analogues were docked into
AgamOBP7 as a model for ApisOBP7 using the Surflex-
Dock software to explore the binding mode. The docking
results for PA, EBF, and its I-5 and D-4 analogues were ver-
ified by 20-ns molecular dynamics simulations, which were
performed using the GROMACS 4.0.5 package [24, 25]. The
G43a1 force field was used to calculate the protein energy, and
the topology files and force-field parameters of the ligand
were generated using the PRODRG program [26]. Each com-
plex was solvated with explicit solvent (SPC) water and neu-
tralized by adding 6 Na+ ions. The steepest-descent and
conjugated-gradient methods were used to minimize the ener-
gy of each system. The reference temperature was fixed at
300 K, and all bonds were constrained with LINCS [27].

J Mol Model (2016) 22: 144
Page 3 of 9 144
Long-range electrostatics was handled using PME during
each simulation [28]. Unless otherwise specified, OBP7 actu-
ally refers to AgamOBP7 in this study. The initial conforma-
tions of all the EBF analogues were optimized at the B3LYP/
6-31G level in Gaussian 03 [29]. The Protomol based on the
ligand mode was generated as the ligand binding pocket. The
docking score represents the ligand binding affinity to the
protein, which is usually correlated to the ligand pIC50 value
[30, 31]. It is well known that Surflex-Dock is an accurate,
rapid, and efficient tool for protein-ligand binding simula-
tions, and is especially successful at eliminating false-
positive results [32]. This software has become a commercial
module in the SYBYL software package [33]. A prototype
molecule algorithm was employed in Surflex-Dock, and li-
gand flexibility was considered in the docking process [30].
Four new EBF analogues were designed based on the interac-
tion mode between the known EBF analogues and OBP7, and
were then synthesized according to Scheme 1. The binding
affinities (IC50 values) were measured by recording the fluo-
rescence peak values of the four EBF analogues and 1-NPN
during competitive binding to ApisOBP7. The protein was
prepared and purified according to previously reported proce-
dures [13].
PA, stabilizing the OBP7 protein during the whole docking
process, as confirmed by the 20-ns MD results (Fig. S4 in the
ESM). The results clearly showed that the OBP7 binding cav-
ity was suitable for binding the linear EBF analogues. Twelve
EBF analogues with an aromatic ring [8] were docked into
OBP7 to determine their binding modes. The obtained
docking scores of all the EBF analogues are given in
Table 1. The scores of compounds I-1, II-1, I-2, and II-2 in
group A correlated well with the binding affinities (pIC50
values), with a high coefficient of 0.96 (Fig. S5 in the ESM).
As shown in Fig. 2a, the ligand binding cavity in OBP7 was
found to be a long, narrow hydrophobic tunnel with several
nonpolar residues (Phe120, Leu124, Val125, and Val117)
along the top and some hydrophobic residues (Leu72, Ile57,
Phe54, Leu53, Ile50, and Val107) along the bottom [19]. It is
similar to the ApisOBP7 model binding domain with the hy-
drophobic residues Phe52, Ile109, Leu57, and Val88 [23],
which also has the same residues as the ApisOBP3 model
except for Tyr84 [13]. A similar inner hydrophobic cavity
was also found in the SlitOBP1 model [34]. Figure 2b shows
that I-1 and II-1, which have ester and amide linkages, respec-
tively, both fit into the long hydrophobic OBP7 cavity well.
However, the I-1 molecule was better accommodated in the
hydrophobic pocket—it had a higher clogP value (6.34) than
that of II-1 (5.34). This explained why the score and binding
affinity of I-1 were better than those of II-1. As indicated in
Table 1, when the methyl group on the pyrazole ring in I-1 and
II-1 was replaced with a bulky propyl group to give I-2 and
II-2, respectively, the docking scores decreased. Steric hin-
drance caused I-2 and II-2 to move outside the binding do-
main, where they could not interact with the hydrophobic
residues in the binding cavity. These results are consistent
with previous reports suggesting that the presence of a short
chain on the pyrazole ring enhances the binding affinity of
EBF analogues [23]. In short, the size of the hydrophobic
group could be an important consideration when modifying
EBF to optimize its contacts with protein residues in the OBP7
binding domain.
Results and discussion
Difference between the binding modes
of pyrazole-substituted EBF analogues with ester
and amide linkages
Although the OBP7 binding cavity was reported to be flexible
and adaptable [19], EBF acted as a good regulator, like native
Binding modes of the pyrazole-substituted EBF analogues
with an N-methyl formamide or amide linkage
As shown in Table 1, the docking scores of the pyrazole-
substituted EBF analogues III-1, which has an N-methyl
formamide linkage, and II-3, which has an amide linkage,
were similar. As shown in Fig. 3a, both III-1 and II-3 of group
B could be accommodated in the hydrophobic OBP7 binding
region. As shown in Fig. 3b, part of II-4 was located outside
the narrow entrance of the protein binding pocket because of
the bulky isopropyl group. In addition, the amide group in II-4
formed a weak H-bond with the carbonyl of the hydrophilic
Leu53 residue, preventing its entry into the binding cavity.
Therefore, the II-4 docking score was lower than the other
Scheme 1 Synthesis route for the newly designed EBF analogues D1–
D4

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Table 1 Structures, docking scores, and binding affinities of the EBF analogues with an aromatic ring
No.
I-1
Structure
Score a
IC50 (μM) b
pIC50
7.06
2.8
5.55
6.69
6.8
5.17
I-2
7.95
6.64
8.22
3.0
4.8
1.3
5.52
5.32
5.89
I-3
I-4
I-5
7.51
6.38
6.05
3.6
9.3
5.44
5.03
4.85
I-6
II-1
II-2
14.1
7.82
7.69
7.91
7.86
8.2
11
5.09
4.96
5.29
5.28
II-3
II-4
5.1
5.3
III-1
III-2
a
docking score for binding to AgamOBP7
binding affinity to ApisOBP7
b

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A
B
Fig. 2a–b OBP7 binding cavity and the interaction modes between
different ligands and OBP7. a Long, narrow hydrophobic binding
cavity in OBP7. The inner helix surface, which has some hydrophobic
residues, is shown in brown, and the external helix surface, which has
some hydrophilic residues, is shown in blue. The key residues in the
OBP7 binding cavity are labeled with a three-letter ID and depicted
using a line model. b Alignment of compounds I-1 (magenta) and II-1
(tan) in the OBP7 binding cavity. The long chain of each EBF analogue is
well matched with the long hydrophobic OBP7 cavity
docking scores. However, in a previous study, the bind-
ing affinities of III-1 and III-2, which have an N-meth-
yl formamide group, were found to be higher than those
of II-3 and II-4, respectively, which have an amide
group, based on the binding assay results [15]. The results
of this study can be explained by the hydrophobicity of
III-1 and III-2, which enabled them to penetrate into
the cell membrane more easily. In short, hydrophobic
interactions are very important for the binding between
these pyrazole-substituted EBF analogues and OBP7.
Some hydrophobic side chains could be included on the
EBF long chain to improve its hydrophobic interactions
with OBP.
Binding affinity analysis for EBF analogues with different
aromatic rings
Because an ester group was previously used as a linkage be-
tween EBF and a pyrazole ring, analogues with an ester group
linkage between EBF and a different aromatic benzene ring
were studied [23]. Molecular docking simulations were used
to provide insight into the binding mode between OBP7 and
these EBF analogues, which constitute group C.
As shown in Table 1, the binding affinities and docking
scores of compounds I-3 and I-5 with benzene rings were
significantly higher than those of I-4 and I-6 with pyrazole
rings. As shown in Fig. 4a, I-3 with a benzene ring could be
A
B
Fig. 3a–b Hydrophobic interactions between different EBF analogues
and OBP7. a Compounds III-1 (green) and II-3 (purple). b Compounds
II-3 (purple) and II-4 (orange). The inner helix surface, which has some
hydrophobic residues, is shown in brown, and the external helix surface,
which has some hydrophilic residues, is shown in blue. The key residues
in the OBP7 binding cavity are labeled with a three-letter ID and depicted
using a line model

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Fig. 4a–b Electrostatic interactions between different EBF analogues
and OBP7. a Compounds I-3 (light blue) and I-4 (pink) in the OBP7
binding domain. The potentials on the I-3 benzene ring and I-4
pyrazole ring are positive (blue) and negative (red), respectively. (b)
Electrostatic interactions between compound I-5 (crimson) and OBP7.
The positive potential (blue) on the I-5 benzene ring interacts with the
negative potential (red) on OBP7 residues Leu72 and Leu73
accommodated in the binding domain and formed strong con-
tacts with the surrounding hydrophobic OBP7 residues.
Moreover, delocalized electrons on the I-3 benzene ring re-
sulted in a positive electrostatic potential surface, whereas the
electrostatic potential of the I-4 pyrazole ring was negative
(Fig. 4a). The I-3 benzene ring formed a π–π stacking inter-
action with residue Phe120 of OBP7 at a distance of only
3.52 Å. These results could explain those of a previous work
which noted that replacing the EBF conjugated double bond
with a benzene ring led to desirable binding properties [23]. In
that study, only a few of the EBF analogues were tested for
repellent activity, and aphids were found to have a repellent
response to I-3, which exhibited a good binding affinity IC50
value of 3.0 μM [23]. It was clear that not only did hydropho-
bic interactions contribute to the binding affinity of EBF ana-
logues to OBP7, but an additional π-electron effect was also
beneficial for the binding [35, 36].
In addition to the hydrophobic t-butyl substituent on the
benzene ring of the EBF analogue I-3, electron-withdrawing
F substituents were placed on the benzene ring of the EBF
analogue I-5. The binding assays indicated that I-5 with
two F atoms had a much higher binding affinity with an IC50
value of 1.3 μM, as shown in Table 1 [23]. It also had a higher
docking score of 8.22, as confirmed by a 20-ns MD simulation
(Fig. S6 in the ESM). These results can be explained by the fact
that the electron-withdrawing F atoms greatly increased the
electropositive potential of the I-5 benzene ring, which could
induce electrostatic interactions with several nearby OBP7 res-
idues, especially Leu72 and Leu73, as shown in Fig. 4b. Thus,
substituting the EBF conjugated double bond with an aromatic
benzene ring was clearly beneficial for improving the binding.
Moreover, including some electron-withdrawing groups on the
benzene ring of each EBF analogue also enhanced the binding
by increasing the electrostatic interactions.
Newly designed EBF analogues based on the OBP7
binding cavity
Using an N-methyl formamide group as the linkage was
shown to increase the hydrophobic contacts between EBF
analogues and OBP7, thereby improving their binding.
Moreover, electron delocalization over the EBF analogue ben-
zene ring was also found to enhance the protein–ligand bind-
ing. Therefore, four new EBF analogues were designed and
synthesized with a benzene ring and an N-methyl formamide
linkage or amide linkage as a control in this study (Scheme 1).
They were denoted D1–D4 of group D, and their binding
affinities and docking scores were calculated to provide in-
sight into their abilities to bind to OBP7. The results are listed
in Table 2.
As shown in Table 2 and Fig. S5 of the ESM, the docking
scores of newly designed EBF analogues D1–D4 were well
correlated with their binding affinities (pIC50). The binding
affinities and docking scores of D1 and D2 (Table 2) were
found to be much lower than those of I-5 (Table 1). Clearly,
when the EBF conjugated double bond was replaced with a
benzene ring with two F atoms, the binding abilities of ana-
logues with different linkages decreased as follows: ester>N-
methyl formamide>amide. Increasing the hydrophobicity of
the linkage, by using an ester group for instance, enabled
stronger hydrophobic interactions between the EBF analogues
and the OBP7 binding domain, as discussed previously. To
further evaluate the effect of the substituent position on the
benzene ring, a Br atom was placed in the position para to the
EBF long chain to synthesize D3. Its binding affinity
(IC50 = 3.3 μM) was slightly higher than that of D2
(IC50 =6.0 μM) (Table 2). The para substituent on the benzene
ring might have enabled the EBF analogues to fit into the long,
narrow OBP7 binding cavity better.

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Table 2 Structures, docking scores, and binding affinities of the newly synthesized EBF analogues
Score a
No.
D1
Structure
IC50 (μM) b
pIC50
7.20
8.5
5.07
7.63
8.09
8.41
6.0
3.3
1.7
5.22
5.48
5.77
D2
D3
D4
a
docking score for binding to AgamOBP7
binding affinity to ApisOBP7
b
As discussed previously, the EBF analogues with an ester
OBP7 binding cavity. The hydrophobic t-butyl group in D4
interacted with the hydrophobic Leu72 and Ile57 residues at
the binding pocket entrance. In contrast, as shown in Fig. 5b,
the electron-withdrawing F atoms in I-5 greatly increased the
electropositive potential of the benzene ring, inducing electro-
static interactions with nearby polar protein residues such as
Leu53. Moreover, the N-methyl formamide linkage in D4 led
to a higher binding affinity to the protein than the ester linkage
in I-3. The methyl group on the D4 N-methyl formamide
fragment provided an additional contact point in the hydro-
phobic binding cavity. Similarly to I-3, D4 also formed a π–π
linkage were found to exhibit stronger binding than those with
an N-methyl formamide linkage. Because the protein binding
cavity had a long, narrow hydrophobic channel, a new EBF
analogue with a t-butyl substituent on the benzene ring and an
N-methyl formamide linkage was designed, synthesized, and
denoted D4. Surprisingly, the binding affinity of D4 was high,
with an IC50 value of 1.7 μM (Table 2), which was close to the
IC50 value of the known I-5 analogue (Table 1). As shown in
Fig. 5a and confirmed by a 20-ns MD simulation (Fig. S6), D4
was closely surrounded by key hydrophobic residues in the
A
B
Fig. 5a–b Binding modes between the newly synthesized EBF
analogues and OBP7. a Hydrophobic interactions between OBP7 and
D4, which had a high binding affinity. The D4 docking conformation
fitted well into the OBP7 hydrophobic binding cavity. b Alignment of
the newly synthesized D4 (yellow) and the reported I-5 (crimson) in the
OBP7 binding cavity. The conformation of D4 in the OBP7 binding
cavity was narrower, whereas one of the I-5 F atoms interacted with the
polar Leu53 residue of OBP7

144 Page 8 of 9
J Mol Model (2016) 22: 144
interaction with the nonpolar Phe120 residue in the protein
binding domain. Clearly, the hydrophobic interactions were
dominant in the binding between the EBF analogues and
OBP7 because its binding domain is a long, narrow hydro-
phobic tunnel. In addition, electronic effects were also pro-
posed to strengthen the binding between the EBF conjugated
double bond or aromatic ring and nearby OBP7 residues.
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Molecular docking and molecular dynamics simulations of
EBF analogues in the OBP7 binding cavity were performed
to provide insight into the intermolecular binding mode and
thus facilitate the design of novel EBF analogues with higher
stability than EBF. Newly designed and synthesized com-
pound D4, which had a t-butyl-substituted benzene ring and
N-methyl formamide linkage, exhibited good binding affinity
and had a high docking score because of multiple hydropho-
bic interactions with OBP7. The docking scores and binding
affinities of the known pyrazole-substituted EBF analogues
with an N-methyl formamide or ester linkage were higher than
those of the analogues with an amide linkage. When the un-
stable conjugated double bond of EBF was replaced with a
benzene ring, the binding affinity and docking score increased
due to an additional π–π interaction between the delocalized
electrons on the benzene and nearby protein residues.
Substituting two electron-withdrawing F atoms on the ben-
zene ring further delocalized the electrons, leading to electro-
static interactions and thus a significant increase in the binding
affinity to the protein. Therefore, because the OBP7 binding
cavity is hydrophobic, EBF analogues should first be
modified with hydrophobic groups to increase the hy-
drophobic contact area between the ligand and protein
binding tunnel. In addition, electron delocalization over
the conjugated double bond or aromatic ring in each
EBF analogue should strengthen the binding by increas-
ing its electrostatic interactions with the protein. This
study provides an insight that should aid the future de-
velopment of new stable EBF analogues with good
binding affinities based on the OBP structure.
Acknowledgments We thank Dr. Pelosi for fruitful discussions and
writing assistance. This work was supported by the National Scientific
and Technology Supporting Program of China (2011BAE06B05-5) and
the National Natural Science Foundation of China (21132003,
31371946).
Author contributions S.S. Wang, S.Q. Du, and H.X. Duan conceived
and designed the experiments. Y.F. Sun, Y.G. Qin, and X.L. Yang pre-
pared the EBF analogues. S.S. Wang, Y.F. Sun, and H.X. Duan analyzed
the data. S.S. Wang and H.X. Duan wrote the first draft of the manuscript.
H.X. Duan and X.L. Yang made critical revisions and approved the final
manuscript. All the authors reviewed and approved the final manuscript.
The authors declare no conflict of interest.
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