Glycosyl triazoles as novel insect β-N-acetylhexosaminidase OfHex1 inhibitors: Design, synthesis, molecular docking and MD simulations

Article abstract of DOI:10.1016/j.bmc.2018.11.032

The insect enzyme GH20 β-N-acetyl-D-hexosaminidase OfHex1 represents an important chitinolytic enzyme found in the agricultural pest Ostrinia furnacalis (Guenée) and inhibition of this enzyme has been considered a promising strategy for the development of eco-friendly pesticides. In this article, based on the structure of the catalytic domains of OfHex1, a series of novel glycosyl triazoles were designed and synthesized via Cu-catalyzed azide-alkyne [3+2] cycloaddition reaction. To investigate the potency and selectivity of these glycosyl triazoles, the inhibition activities towards OfHex1 and HsHexB (human β-N-acetylhexosaminidase B) were studied. Particularly compound 17c (OfHex1, K_i = 28.68 μM; HsHexB, K_i > 100 μM) exhibited a suitable activity and selectivity against OfHex1. Furthermore, the possible inhibitory mechanisms of 17c with OfHex1 were studied using molecular docking and MD simulations. The structure-activity relationship results as well as the formed binding patterns may provide promising insights into the further development of novel OfHex1 inhibitors.

Full text of DOI:10.1016/j.bmc.2018.11.032

Accepted Manuscript
Glycosyl triazoles as novel insect β-N-acetylhexosaminidase OfHex1 inhibi-
tors: Design, synthesis, molecular docking and MD simulations
Lili Dong, Shengqiang Shen, Wei Chen, Huizhe Lu, Dongdong Xu, Shuhui Jin,
Qing Yang, Jianjun Zhang
PII:
S0968-0896(18)31783-8
https://doi.org/10.1016/j.bmc.2018.11.032
BMC 14636
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
17 October 2018
16 November 2018
21 November 2018
Please cite this article as: Dong, L., Shen, S., Chen, W., Lu, H., Xu, D., Jin, S., Yang, Q., Zhang, J., Glycosyl triazoles
as novel insect β-N-acetylhexosaminidase OfHex1 inhibitors: Design, synthesis, molecular docking and MD
simulations, Bioorganic & Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.2018.11.032
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Graphical Abstract
Glycosyl triazoles as novel insect β-N-acetylhexosaminidase OfHex
inhibitors: Design, synthesis, molecular docking and MD simulations
Lili Donga, Shengqiang Shena, Wei Chenb, Huizhe Lua, Dongdong Xua, Shuhui Jina, Qing Yangb,c,*, Jianjun
Zhanga,*
aDepartment of Applied Chemistry, College of Science, China Agricultural University, Beijing, China
bSchool of Life Science and Biotechnology, Dalian University of Technology, Dalian, China
cInstitute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China.
The insect enzyme GH20 β-N-acetyl-D-hexosaminidase OfHex1 represents an important chitinolytic
enzyme found in the agricultural pest Ostrinia furnacalis (Guenée) and inhibition of this enzyme has
been considered a promising strategy for the development of eco-friendly pesticides. In this article,
based on the structure of the catalytic domains of OfHex1, a series of novel glycosyl triazoles were
designed and synthesized via Cu-catalyzed azide-alkyne [3+2] cycloaddition reaction. To investigate
the potency and selectivity of these glycosyl triazoles, the inhibition activities towards OfHex1 and
HsHexB (human β-N-acetylhexosaminidase B) were studied. Particularly compound 17c (OfHex1, Ki
= 28.68 μM; HsHexB, Ki>100 μM) exhibited a suitable activity and selectivity against OfHex1.
Furthermore, the possible inhibitory mechanisms of 17c with OfHex1 were studied using molecular
docking and MD simulations. The structure–activity relationship results as well as the formed binding
patterns may provide promising insights into the further development of novel OfHex1 inhibitors.
O
4 N
O
HO
HO
HO
O
O
N
N
S
N
H
To acquire additional
affinity at +1 subsite
and byond the pocket
N
NHAc
To interact with -1 subsite
17c
OfHex1: K =28.68

M
i
HsHexB: K >100

M
i
OH
R
O
HO
HO
X
linker
N
N
N
AcHN
To prevent hydrolysis by OfHex1
X= N, S
linker=
NHCOCH2, CH2CONHCH2
O
O
n N
N
R=
R1
H
O
MD simulations of OfHex1 in complex with 17c

Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com
Glycosyl triazoles as novel insect β-N-acetylhexosaminidase OfHex1 inhibitors:
Design, synthesis, molecular docking and MD simulations
Lili Donga, Shengqiang Shena, Wei Chenb, Huizhe Lua, Dongdong Xua, Shuhui Jina, Qing Yangb,c,*, Jianjun
Zhanga,*
a
Department of Applied Chemistry, College of Science, China Agricultural University, Beijing, China
bSchool of Life Science and Biotechnology, Dalian University of Technology, Dalian, China
cInstitute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China.
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
The insect enzyme GH20 β-N-acetyl-D-hexosaminidase OfHex1 represents an important
chitinolytic enzyme found in the agricultural pest Ostrinia furnacalis (Guenée) and inhibition of
this enzyme has been considered a promising strategy for the development of eco-friendly
pesticides. In this article, based on the structure of the catalytic domains of OfHex1, a series of
novel glycosyl triazoles were designed and synthesized via Cu-catalyzed azide-alkyne [3+2]
cycloaddition reaction. To investigate the potency and selectivity of these glycosyl triazoles, the
inhibition activities towards OfHex1 and HsHexB (human β-N-acetylhexosaminidase B) were
studied. Particularly compound 17c (OfHex1, Ki = 28.68 μM; HsHexB, Ki>100 μM) exhibited a
suitable activity and selectivity against OfHex1. Furthermore, the possible inhibitory
mechanisms of 17c with OfHex1 were studied using molecular docking and MD simulations.
The structure–activity relationship results as well as the formed binding patterns may provide
promising insights into the further development of novel OfHex1 inhibitors.
Available online
Keywords:
β-N-acetyl-D-hexosaminidase
OfHex1
glycosyl triazoles
enzyme inhibitors
molecular docking
MD simulations
2017 Elsevier Ltd. All rights reserved.
1. Introduction
Therefore, OfHex1 has been considered to be a promising target
for developing eco-friendly pesticides.6-11
Family 20 (GH20) β-N-acetylhexosaminidases (EC 3.2.1.52)
are responsible for catalyzing the hydrolysis of terminal non-
reducing β-linked N-acetyl-D-glucosamine (GlcNAc) or N-
As reported previously,12 the crystal structure of OfHex1
(PDB: 3NSM) shows that the enzyme features a long substrate-
binding pocket and is composed of two subsites (subsite -1 and
subsite +1). The -1 subsite is responsible for catalysis the
substrates via residues such as His303, Asp367, and Glu368.2 The
+1 subsite binding domain mainly contains two conserved
residues, namely Val327 and Trp490, able to form sandwich
structures with the related substrate moieties and resulting in high
affinity and specificity.2 These crystallographic investigations on
OfHex1 laid a solid foundation for the design of novel inhibitors.
acetyl-D-galactosamine
(GalNAc)
residues
from
oligosaccharides, glycoproteins and glycolipids.1-3 These
enzymes are widely distributed in organisms and involved in
diverse physiological functions, such as pathogen defense, virus
infections, lysosomal storage, N-glycan modification, chitin
degradation, and cell proliferation.1-5
Remarkably, GH20 β-N-acetyl-D-hexosaminidase OfHex1
has attracted much attention due to its important target enzyme
roles in the field of pest control.6-9 OfHex1 represents a
chitinolytic enzyme from the agricultural pest Ostrinia furnacalis
(Guenée).10 The function of this enzyme has been shown to
involve insect chitin (old chitinous cuticles) degradation during
molting, pupation and eclosion processes.10 Moreover, OfHex1
exhibits high substrate specificity, which can hydrolyse β-1,4-
linked chitooligo-saccharides efficiently but is unable to
hydrolyse β-1,2-linked GlcNAc from N-glycans.11 It is well
known that chitin is absent in higher animals and plants.2
A number of small molecule inhibitors against OfHex1 have
been reported to date, including NAG-thiazoline,13 PUGNAc,11
TMG-chitotriomycin,8,12 and naphthalimides14-15. Among these
compound species, PUGNAc is one of the potent inhibitors
against OfHex1 but lacks selectivity. The complex crystal
structure of OfHex1-PUGNAc (PDB: 3OZP) exhibited that the
glycosyl moiety bound the -1 subsite of the active pocket as well
as the phenyl group is sandwiched by residues Val327 and
Trp490 (+1 subsite) at the entrance of the pocket.11 TMG-

To acquire additional
affinity at +1 subsite
and byond the pocket
-1 subsite
+1 subsite
To interact with -1 subsite
HO
O
H
O
N
HO
HO
N
O
OH
NHAc
O
outside of pocket
R
O
HO
X
linker
N
HO
N
N
HO
HO
HO
HO
HO
AcHN
design
O
N
O
O
O
HO
O
O
HO
HO
OH
HO
To prevent hydrolysis by OfHex1
X= N, S
NHAc
NHAc
NHAc
O
N
O
N
N
H
N
N
linker=
NHCOCH2, CH2CONHCH2
S
O
O
N
O
n
N
R=
R1
H
Reported OfHex1 inhibitors PUGNAc, TMG-chitotriomycin, Q2
Figure 1. Design of glycosyl triazole derivatives for OfHex1.
chitotriomycin has been shown to be able to only inhibit β-N-
acetylhexosaminidases from chitin-containing organisms,
however, applicability is limtited due to the complicated
synthetic process.8 The crystal structure of TMG-chitotriomycin
complexed with OfHex1 (PDB: 3NSN) revealed that the N,N,N-
triMe-GlcNH2 and GlcNAcI moieties were localized at the -1 and
+1 subsites in the active pocket of OfHex1, respectively.12
Meanwhile, GlcNAcII and GlcNAcIII of TMG-chitotriomycin
have been demonstrated to be extended out from the active
pocket and stabilized via formation of hydrogen bonds with
corresponding residues.12 Moreover, one of the naphthalimide
inhibitors, Q2, was found to possess Ki values of 0.31 μM
against OfHex1 and more than 100 μM against HsHex (human β-
N-acetylhexosaminidase).15 The Q2-complexed OfHex1 (PDB:
3WMC) showed that the 2-methylthiadiazole group formed
interactions with the -1 subsite of OfHex1 and the larger
hydrophobic naphthalimide moiety tightly bound to the +1
subsite via π-π stacking interactions within the indolyl group of
Trp490.15 (Figure 1)
material and reacted with acetyl chloride to obtain glycosyl
chloride, and then stirred with NaN3 in anhydrous DMF to afford
glycosyl azide 2.21 Subsequently, glycosyl amine 3 was
synthesized by catalytic hydrogenation from 2 with 97% yield.22
And after treatment of 3 with 2-azidoacetyl chloride in dry
CH2Cl2, the key intermediate 4 was produced. Meanwhile, the
benzamide derivatives 6a-6z were obtained according to
published procedures from 5a-5z.23-24 The coupling of azide 4
with 6a-6z in THF/water (1:1, v:v) catalyzed by sodium
ascorbate and copper sulfate yielded the acetyl-protected
precursors 7a-7z. Finally, deacetylation of 7a-7z via methanol-
ammonia catalysis resulted in the target compounds 8a-8z with
78-94% yield.
To further investigate the structure-activity relationship
between compounds 8a-8z and OfHex1, we replaced the
substituted phenyl group in 8a-8z with a 2-pyridinyl or allyloxy
group, respectively. The synthetic routes to obtain target
compounds 9 and 10 are outlined in Scheme 2. Briefly,
intermediate 4 was reacted with N-(prop-2-yn-1-yl)picolinamide
and allyl prop-2-yn-1-yl carbamate to furnish the corresponding
acetyl-protected precursors. These were then deprotected by
addition of methanol-ammonia to afford 9 and 10.
The Cu-catalyzed azide-alkyne [3+2] cycloaddition reaction
has evolved as a widely used reaction for the efficient
construction of novel bioactive molecules.16-20 The triazole
moiety possesses favorable structural characteristics for the
formation of hydrogen bonds and π-π stacking interactions with
the biological targets.17 Since the +1 subsite of OfHex1 features a
hydrophobic domain (Val327, Trp490), the triazole moiety with
an appropriate linker synthesized via ‘click chemistry’ may act as
an important active fragment to bind to OfHex1. (Figure 1)
We then introduced naphthalimide moieties with different
linker lengths to glycosyl triazoles via ‘click chemistry’. The
corresponding synthetic routes are shown in Scheme 3. First,
thiol 11 was obtained according to literature procedures in three
steps.25 Subsequently, thiol 11 was treated with 2-chloro-N-
(prop-2-yn-1-yl) acetamide in acetone/water (2:1, v:v) to provide
intermediate 12. Meanwhile, azides 15a-15d were prepared as
published in the literature.26 Cu-catalyzed cycloaddition reactions
of glycosyl alkyne 12 and 15a-15d in the ratio of 1:1.1 were
completed upon vigorous stirring at room temperature to form
triazoles 16a-16d. Finally, the target compounds 17a-17d were
obtained by removal of the protecting group from 16a-16d with
89-92% yield.
These observations prompted us to design glycosyl triazole
derivatives as novel OfHex1 inhibitors. Thus, in this study we
selected a GlcNAc moiety as the structural fragment to obtain
strong interactions towards the -1 subsite of OfHex1.
Furthermore, the glycosidic bond was converted to an
aminoglycosidic or thioglycosidic bond to prevent hydrolysis by
OfHex1. In additon, triazole-linked diverse hydrophobic groups
(substituted phenyl or naphthalimides) were introduced via ‘click
chemistry’ in an effort to add additional affinity with residues at
the +1 subsite and beyond the pocket of OfHex1. Finally, the
glycosyl triazole inhibitors were synthesized and the inhibitory
activity against GH20 OfHex1 and HsHexB was evaluated.
(Figure 1)
2.2. Bioevaluation of inhibitory potency
There the synthesized compounds 8a-8z, 9, 10, and 17a-17d
at a concentration of 100 μM were evaluated for their in vitro
inhibitory potency against OfHex1 and HsHexB by using 4-MU-
GlcNAc as substrate. As shown in Table 1, the phenyl triazoles
8a-8z exhibited a lower inhibitory potency against OfHex1 and
HsHexB. After changing the phenyl group of 8a-8z into pyridinyl
(heterocyclic group) and allyloxy (linear chain) to obtain 9 and
10, similarly poor inhibition activities were observed. These
results suggested that the structural fragments located at the
phenyl group of 8a-8z exhibited little effects on the activity
towards these two enzymes. Comparatively, glycosyl triazoles
2. Results and discussion
2.1. Chemical synthesis
Substituted phenyl group-bearing glycosyl triazoles (8a-8z)
were synthesized following a multi-step strategy as shown in
Scheme 1. N-acetyl-D-glucosamine 1 was selected as the starting

HO
HO
HO
AcO
AcO
AcO
AcO
AcO
AcO
AcO
AcO
AcO
O
iii
iv
O
i, ii
O
H
O
N
NH2
NHAc
N3
NHAc
N3
OH
NHAc
NHAc
O
1
3
2
4
O
COOH
v
vi
N
R
R
H
5a-5z
vii
6a-6z
OH
OAc
O
O
H
H
O
O
HO
HO
AcO
AcO
N
N
N
H
N
N
N
8a-8z
N
N
N
R
R
H
N
NHAc
NHAc
O
O
7a-7z
8a
8j
8b
8c
8d
8e
8f
8g
8h
8i
: R=H; : R=2-OCH3; : R=3-OCH3; : R=4-OCH3;
R=2-Cl; : R=3-Cl; : R=4-Cl; : R=2-NO2; : R=3-NO2;
8k 8l 8m 8n
8o 8p 8q 8r
: R=4-NO2; : R=2-Ph; : R=3-Ph; : R=4-Ph; : R=2-NH2; : R=3-NH2; : R=4-NH2; : R=2-F; : R=3-F;
8s 8t 8u 8v 8w 8x 8y 8z
: R=4-F; : R=2-OH; : R=3-OH; : R=2-CF3; : R=2-iPr-4-Cl; : R=2,4-di-Cl; : R=2,6-di-Cl; : R=2-NH2-3-CH3
Scheme 1. Synthetic route for glycosyl triazoles 8a-8z. (i) AcCl; (ii) NaN3, DMF; (iii) Pd/C, H2; (iv) 2-azidoacetyl chloride, Et3N, DCM; (v) prop-2-yn-1-amine,
EDCI, DCM; (vi) CuSO4, sodium ascorbate, THF, H2O; (vii) NH3, MeOH.
OH
AcO
AcO
AcO
O
H
O
H
O
N
i, ii
HO
HO
N
N
N
N
N
N3
H
NHAc
N
NHAc
O
O
4
9
O
OH
H
O
iii, iv
HO
HO
N
O
N
N
H
N
NHAc
N
O
10
Scheme 2. Synthetic route for glycosyl triazoles 9 and 10. (i) N-(prop-2-yn-1-yl) picolinamide, CuSO4, sodium ascorbate, THF, H2O; (ii) NH3, MeOH; (iii) allyl
prop-2-yn-1-yl carbamate, CuSO4, sodium ascorbate, THF, H2O; (iv) NH3, MeOH.
HO
AcO
AcO
AcO
AcO
AcO
AcO
O
O
i, ii, iii
iv
O
O
HO
HO
S
SH
OH
N
NHAc
12
NHAc
H
NHAc
11
1
O
O
N
O
O
N
v
vi
vii
NH
O
Br
N3
n
n
O
13
14a-14d
15a-15d
O
O
N
O
HO
HO
HO
AcO
AcO
AcO
O
O
viii
O
O
n
n
N
O
N
N
N
N
N
16a-16d
S
S
N
N
H
H
N
NHAc
NHAc
17a-17d
17a
17b
17c
17d
: n=4; : n=5
: n=2;
: n=3;
Scheme 3. Synthetic route for glycosyl triazoles 17a-17d. (i) AcCl; (ii) thiourea, acetone; (iii) Na2S2O5, DCM, H2O; (iv) 2-chloro-N-(prop-2-yn-1-yl)acetamide,
K2CO3, acetone, H2O; (v) α,ω-dibromoalkane, K2CO3, CH3CN; (vi) NaN3, DMF; (vii) CuSO4, sodium ascorbate, THF, H2O; (viii) NH3, MeOH.
bearing a naphthalimide group (compounds 17a-17d) featured an
OfHex1, with an inhibition rate of 54.5 % at a concentration of
improved inhibitory potency against OfHex1.
100 μM.
Although 8a-8z exhibited a relatively weak activity towards
OfHex1, these compounds could also provide some valuable
clues regarding the structure-activity relationship. Specifically,
compounds 8q, 8r, 8s, and 8v bearing the electron-withdrawing
groups NO2 and CF3 on the phenyl ring could weaken the
inhibitory activity against OfHex1 compared to 8a (phenyl ring
not bearing any substituents). Moreover, the position of the
substituent on the phenyl ring showed a significant influence on
the activity towards OfHex1, but the activity order of the effect
of ortho, meta, and para substitution on benzene was not fixed.
For example, compound 8l bearing a meta-phenyl on benzene
The inhibitory activity assays of the naphthalimide triazoles
17a-17d against OfHex1 and HsHexB are shown in Table 1 and
Table 2. Analysis of the structure-activity relationship between
17a-17d and OfHex1 revealed that the length of the linker
between triazole and naphthalimide had a direct influence on the
inhibitory efficiency. In detail, the optimal length of the linker
was determined to be about four carbon atoms (i.e. 17c, n=4).
Otherwise, the potency was observed to be lower when the linker
length was either shortened or extended (compared to 17b and
17d). Moreover, it is worth noting that 17c exhibited the lowest
inhibition rate agaisnt HshexB among 17a-17d. Subsequently,
the Ki value showed that 17c (Ki= 28.68 ± 0.18 μM against
OfHex1, Ki>100 μM against HsHexB) exhibited a relatively
good activity and selectivity for OfHex1.
ring exhibited
a higher inhibitory potency compared to
compound 8k (bearing a ortho-phenyl group) as well as 8m
(bearing a para-phenyl group). However, the activity order of
compounds substituted with a methoxy group, i.e. compounds
8b, 8c, 8d, was ortho> meta> para, and the order of compounds
bearing chlorine, i.e. compounds 8e, 8f, 8g, was para> meta>
ortho. Among the phenyl triazoles 8a-8z, 8x bearing a 2,4-
dichlorophenyl group exhibited the highest potency against
Table 1. Inhibition rate of compounds 8a-8z, 9, 10, and 17a-
17d against OfHex1 and HsHexB at a concentration of 100
μM.

Inhibition rate at 100 μM a (%)
Inhibition rate at 20 μM
Ki values (μM)
Compound
(%)
Compound
OfHex1
HsHexB
OfHex1
23.6 ± 2.0
52.4 ± 1.4
55.5 ± 2.6
67.1 ± 0.2
57.0 ± 2.5
HsHexB
2.1 ± 0.4
OfHex1
HsHexB
>100
8a
8b
29.2
13.1
8x
>100
32.0
17.4
0.0
19.5
18.2
6.9
17a
17b
17c
17d
13.8 ± 1.2
16.9 ± 1.9
11.6 ± 1.5
16.8 ± 0.1
54.47 ± 1.41
42.86 ± 0.15
28.68 ± 0.18
39.39 ± 0.52
>100
8c
>100
8d
>100
8e
9.8
3.8
>100
8f
15.7
23.4
21.0
24.5
17.8
0.0
13.8
23.0
13.9
10.2
16.2
28.5
6.6
The dixon plot of compound 17c against OfHex1 was shown
in Figure 2. The results demonstrated that 17c was a competitive
inhibitor of OfHex1 due to the trendlines drawn for each
concentration of substrate that intersected in quadrant two.
Accordingly, 17c was able to compete with the substrate for
binding directly to the active pocket of OfHex1. Then, density
functional theory (DFT) calculations at the B3LYP/6-31G (d)
basis set were carried out to optimize 17c using the Gaussian 16
program (Figure S2). Subsequently, molecular docking was
performed using the reported complex crystal structure of
OfHex1-PUGNAc (PDB ID: 3OZP)11 as templates. The docked
conformation of 17c was found to tightly bind to the entire active
pocket of OfHex1 (Figure 3c). Specifically, the glycosyl moiety
of inhibitor 17c was shown to be inserted into the -1 subsite of
OfHex1 via H-bond interactions with residues Arg220, Glu328,
Asp367, Glu368, Asp477. These interactions were coherent with
those found in the complex structure of OfHex1-PUGNAc11,
confirming the accuracy of the docking result. The triazole ring
was located at the +1 subsite of the active pocket entrance
guarded by Val327 and Trp490 of OfHex1. Furthermore, the
naphthalimide group was extended out from the active pocket
(Figure 3c).
8g
8h
8i
8j
8k
8l
44.1
17.6
0.0
8m
8n
26.2
5.1
8o
16.6
0.0
10.9
13.5
2.4
8p
8q
19.9
19.8
24.3
19.3
17.6
22.6
31.1
54.5
23.5
13.6
22.6
25.7
81.4
83.6
86.2
82.8
98.9
8r
3.3
8s
8.0
8t
0.9
8u
3.1
8v
11.1
9.2
8w
8x
7.0
8y
2.7
8z
17.8
6.6
9
10
16.4
46.6
48.7
42.9
49.1
97.3
17a
17b
17c
17d
PUGNAc
a Values were assessed as the averages of at least three experiments. Errors
were in the range of ± 0.0-3.1% of the reported value.
Figure 2. Dixon plot for compound 17c against OfHex1.
2.3. Inhibition mechanism studies of inhibitor 17c against
OfHex1
In order to shed further light on the appropriate binding
pattern of compound 17c when bound to OfHex1, we performed
MD simulations of 30 ns. As shown in Figure 3a, the root-mean-
square deviations (RMSD) of OfHex1-17c were ultimately
maintained around 3.2 Å, which revealed that an equilibrated and
converged state was achieved after 24 ns simulation. Besides,
OfHex1 receptors were found to exhibit some larger fluctuations
located at the loop areas of the proteins (Figure 3b), a finding that
To further explore the inhibition mechanism of these glycosyl
triazoles towards OfHex1, the most potent inhibitor 17c was
selected for investigating the binding mode using molecular
docking and MD simulation.
Table 2. Inhibitory activity tests of compounds 8x and 17a-
17d against OfHex1 and HsHexB.

(b)
(a)
(d)
(c)
Figure 3. Predicted binding mechanism of compound 17c in OfHex1 calculated by molecular docking and MD simulations. (a) RMSD changes. (b) RMSF
changes. (c) Molecular docking and (d) 30 ns MD simulations of OfHex1 in complex with 17c. The protein is shown in cartoon style and 17c is shown as a stick
structure.
The
essential
residues
are
shown
as
lines
and
hydrogen
bonds
are
highlighted
as
red
dashed
lines.
was in agreement with the high conformational flexibility of the
glycoprotein.27 The conformation of 17c combined in the active
pocket of OfHex1 at 30 ns of MD simulations was extracted as
shown in Figure 3d. Compound 17c was found to exhibit a great
deal of folding and insert more deeply into the OfHex1 pocket
relative to the conformation before MD simulation (Figure 3c).
Therefore, the triazole ring could bind to the catalytic residue
Asp367 via H-bond interactions at the -1 subsite of OfHex1.
Moreover, the naphthalimide moiety entered into the +1 subsite
of OfHex1 resulting in π-π stacking interactions with Trp490. It
is worth noting that the glycosyl moiety of 17c could only form
two hydrogen bonds with residues Asp477 and Asn489, with
reduced interactions in its docking mode (Figure 3c and Figure
3d). This result was in agreement with the inhibitory potency of
17c against OfHex1 (Ki = 28.68 μM, a moderately active
inhibitor), and further confirmed the MD simulation method was
more accurate than docking (the docking results showed that 17c
demonstrated a strong interaction with OfHex1). Furthermore,
the MD simulation studies revealed that the large hydrophobic
group (triazole-linked naphthalimide) of 17c could bind well in
the +1 subsite of OfHex1, however, it pushed the glycosyl moiety
of 17c out of the -1 subsite. Therefore, the affinities between
these glycosyl triazole derivatives with OfHex1 were determined
to be relatively weak, resulting in a moderate inhibitory activity
against OfHex1.
and allyloxy (10) against OfHex1. Notably, compound 17c (Ki =
28.68 μM for OfHex1 and Ki>100 μM for HsHexB) exhibited a
relatively good potency and selectivity against OfHex1.
Moreover, molecular docking and MD simulation studies
allowed for the rationalization of possible binding mechanisms of
these glycosyl triazoles towards OfHex1. The triazole-linked
hydrophobic groups, e.g. substituted phenyl or naphthalimide
groups, in these glycosyl triazole derivatives might acquire
strong affinity at the +1 subsite or beyond the pocket of OfHex1.
However, this would render a weak binding of the glycosyl
moiety the -1 subsite of OfHex1. Taken in concert, the novel
glycosyl triazole species reported herein may provide useful
insights into the rational molecular design of OfHex1 inhibitors.
4. Experimental
4.1. General materials and methods
All reagents and solvents were purchased from commercial
1
sources and used without further purification. H NMR and 13C
NMR spectra were recorded on a Bruker AVANCE600
1
spectrometer (300 MHz, H; 75 MHz, 13C) in CDCl3 or DMSO-
d6 at 25°C and referenced to TMS. Chemical shifts were reported
in ppm (δ). High-resolution mass spectra (ESI-HRMS) was
performed on
a Bruker Daltonics Bio-TOF-Q III mass
spectrometer. Reaction progress was monitored using thin layer
chromatography (TLC) on silica gel GF254 plates and the spots
were detected by UV light (254 nm) or by charring with 15 %
(v/v) H2SO4 in methanol.
3. Conclusions
In summary, we showed the molecular design, synthesis, and
inhibitory potencies of various glycosyl triazole derivatives
against OfHex1 and HsHexB. The activity evaluation by
enzymatic assay revealed that glycosyl triazoles bearing
naphthalimide group (17a-17d) proved be more effective than
glycosyl triazoles bearing substituted phenyl (8a-8z), pyridyl (9),
4.2 Chemical synthesis
4.2.1. 2-(acetylamino)-3,4,6-tri-O-acetyl-2-deoxy-β-D-gluco-
pyranosylamine (3)22

To a solution of compound 2 (3.7 g, 10 mmol) in THF (50
mL), 10% Pd/C (0.4 g) was added. The reaction was then stirred
vigorously under hydrogen atmosphere for 5 h at room
temperature, the completion of the reaction was confirmed by
TLC (DCM /MeOH, 20:1 v/v) analysis. The reaction mixture
was filtered through celite. The filtrate was concentrated under
reduced pressure to afford compound 3 (3.4 g, 97.1%) as a
4.2.3.2. Data for compounds 7b-7z can be found in
Supporting Information.
4.2.4. Synthesis of compounds 8a-8z
A solution of 7a-7z (1 mmol) were dissolved in MeOH (20
mL), saturated solution of NH3 in MeOH (4 mL) was added to
the solution. The mixture was stirred for 20 h at room
temperature, TLC (EtOAc: MeOH: H2O = 8:1:1) indicated the
completion of the reaction. Then the mixture was concentrated in
vacuo and recrystallised from MeOH /ether, and as a result, the
final compounds 8a-8z were obtained.
1
colorless syrup. [α]D25 -2.1 (c=1.0, MeOH); H NMR (300 MHz,
CDCl3) δ 5.60 (d, J = 8.7 Hz, 1H, NH), 5.15 – 4.97 (m, 2H, H-3,
H-4), 4.22 (dd, J = 12.3, 4.8 Hz, 1H, H-6b), 4.16 – 4.08 (m, 2H,
H-1, H-6a), 4.02 (dd, J = 19.2, 9.2 Hz, 1H, H-2), 3.64 (m, 1H, H-
5), 2.13 (br s, 2H, NH2), 2.11, 2.05, 2.04 (3 s, 9H, 3 OAc), 1.98
(s, 3H, NAc).
4.2.4.1. N-((1-(2-((2-acetamido-2-deoxy-β-D-glucopyranosyl)
amino)-2-oxoethyl)-1H-1,2,3-triazol-4-yl)methyl)benzamide
(8a). light yellow solid; (0.41 g, 89.1 %) yield; [α]D25 67.3 (c=1,
4.2.2. N-(3,4,6-tri-O-acetyl-2-acetamido-2-deoxy-β-D-gluco-
pyranosyl) azidoacetamide (4)
1
DMSO); H NMR (300 MHz, DMSO-d6) δ 9.07 (t, J = 5.8 Hz,
1H, CH2NH), 8.65 (d, J = 9.1 Hz, 1H, CHNH), 8.00 – 7.77 (m,
4H, NHAc, ArH), 7.61 – 7.41 (m, 3H, ArH), 5.19 – 4.96 (m, 4H,
H-3, H-4, COCH2Ar), 4.82 (t, J = 9.4 Hz, 1H, H-1), 4.60 (t, J =
5.4 Hz, 1H, OH), 4.53 (d, J = 5.7 Hz, 2H, ArCH2NH), 3.67 (dd, J
= 11.7, 4.4 Hz, 1H, H-6b), 3.58 (dd, J = 19.2, 9.8 Hz, 1H, H-2),
3.50 – 3.43 (m, 1H, H-6a), 3.38 – 3.26 (m, 1H, H-5), 3.11 (m, 2H,
2 OH), 1.80 (s, 3H, NAc); 13C NMR (75 MHz, DMSO-d6) δ
170.14, 166.29, 166.02, 145.03, 134.24, 131.41, 128.41, 127.37,
124.57, 79.11, 79.04, 74.57, 70.54, 60.97, 54.70, 51.65, 34.98,
22.96; HRMS (ESI) calcd for C20H27N6O7(M+H+) 463.1941,
found 463.1936.
A solution of compound 3 (2.8 g, 8 mmol) in anhydrous
CH2Cl2 (30 mL) was mixed with Et3N (1.7 mL, 12 mmol) at 0°C,
then 2-azidoacetyl chloride (1.1 g, 9.6 mmol) in anhydrous
CH2Cl2 (15 ml) was added dropwise. The solution was stirred for
2.5 h at room temperature and the completion of the reaction was
confirmed by TLC (petroleum ether/EtOAc, 1:1 v/v) analysis.
The reaction was then quenched with chilled H2O (150 mL), and
the mixture was extracted with CH2Cl2 (3×100 ml). The organic
phase was washed with water (2×40 mL), dried over Na2SO4,
filtered, concentrated in vacuo and purified by flash column
chromatography to yield compound 4 (2.8 g, 82.3%) as a white
solid. [α]D -2.0 (c=1.0, CHCl3), [Lit. [α]D -1.7 (c=1.0,
CHCl3)]28; 1H NMR (300 MHz, DMSO-d6) δ 8.87 (d, J = 9.1 Hz,
1H, NHCO), 8.05 (d, J = 9.2 Hz, 1H, NHAc), 5.26 (t, J = 9.5 Hz,
1H, H-3), 5.19 (t, J = 9.9 Hz, 1H, H-4), 4.90 (t, J = 9.8 Hz, 1H,
H-1), 4.24 (dd, J = 12.4, 4.3 Hz, 1H, H-6b), 4.08 – 4.01 (m, 1H,
H-6a), 4.01 – 3.92 (m, 2H, H-2, H-5), 3.91 (s, 2H, CH2N3), 2.07,
2.04, 1.98 (3 s, 9H, 3 OAc), 1.82(s, 3H, NAc); 13C NMR (75
MHz, DMSO-d6) δ 170.14, 169.72, 169.66, 169.42, 168.31,
78.17, 73.33, 72.50, 68.46, 61.91, 52.18, 50.69, 22.67, 20.59,
20.48, 20.44.
25
25
4.2.4.2. Data for compounds 8b-8z can be found in
Supporting Information.
4.2.5. Synthesis of compounds 9, 10
Compounds 9 and 10 were synthesized from compound 4
using the procedures described for the synthesis of compound 8a-
8z.
4.2.5.1.N-((1-(2-((2-acetamido-2-deoxy-β-D-glucopyranosyl)
amino)-2-oxoethyl)-1H-1,2,3-triazol-4-yl)methyl)picolinamide
25
(9). yellow solid; (0.40 g, 86.9 %) yield; [α]D -78.3 (c=1,
4.2.3. Synthesis of compounds 7a-7z
1
DMSO); H NMR (300 MHz, DMSO-d6) δ 9.23 (t, J = 6.0 Hz,
Compound 4 (0.86 g, 2 mmol) and alkynes 6a-6z were
dissolved in THF/H2O (1:1, 30mL) , then sodium ascorbate (60
mg, 0.3 mmol) and copper(II) sulfate (50 mg, 0.2 mmol) were
added. The resulting suspension was stirred at room temperature
for 10 h, and TLC analysis (DCM/MeOH, 20:1 v/v) was
performed to identify the completion of the reaction. The mixture
was extracted with DCM (3×80 ml). The organic extracts were
combined, dried over Na2SO4, filtered, and concentrated. Finally,
the residue was purified by column chromatography to afford
compounds 7a-7z.
1H, CH2NH), 8.74 – 8.57 (m, 2H, CHNH, ArH), 8.09 – 7.95 (m,
2H, ArH), 7.87 (s, 1H, ArH), 7.80 (d, J = 8.8 Hz, 1H, NHAc),
7.65 – 7.53 (m, 1H, ArH), 5.17 – 4.94 (m, 4H, H-3, H-4,
COCH2Ar), 4.80 (t, J = 9.4 Hz, 1H, H-1), 4.64 – 4.48 (m, 3H,
OH, ArCH2NH), 3.64 (dd, J = 11.6, 5.7 Hz, 1H, H-6b), 3.56 (dd,
J = 19.1, 9.7 Hz, 1H, H-2), 3.41 (dd, J = 11.8, 5.9 Hz, 1H, H-6a),
3.32 – 3.22 (m, 1H, H-5), 3.13 – 3.03 (m, 2H, 2 OH), 1.78 (s, 3H,
NAc); 13C NMR (75 MHz, DMSO-d6) δ 170.09, 166.02, 163.98,
149.90, 148.59, 144.72, 137.92, 126.73, 124.53, 122.04, 79.11,
79.05, 74.59, 70.54, 60.97, 54.70, 51.64, 34.71, 22.95; HRMS
(ESI) calcd for C19H26N7O7(M+H+) 464.1894, found 464.1884.
4.2.3.1.N-((1-(2-((3,4,6-tri-O-acetyl-2-acetamido-2-deoxy-β-
D-glucopyranosyl)amino)-2-oxo-ethyl)-1H-1,2,3-triazol-4-yl)
methyl)benzamide (7a). light yellow syrup; (0.91 g, 77.1 %)
4.2.5.2 Allyl ((1-(2-((2-acetamido-2-deoxy-β-D-glucopyrano-
syl)amino)-2-oxoethyl)-1H-1,2,3- triazol-4-yl)methyl)carbamate
(10). light yellow solid; (0.38 g, 88.4 %) yield; [α]D25 -55.8 (c=1,
1
yield; [α]D25 35.1 (c=0.5, CHCl3); H NMR (300 MHz, DMSO-
d6) δ 9.13 – 9.01 (m, 2H, 2 NHCO), 7.98 (d, J = 9.2 Hz, 1H,
NHAc), 7.93 – 7.84 (m, 3H, ArH), 7.58 – 7.41 (m, 3H, ArH),
5.27 – 5.02 (m, 4H, H-3, H-4, COCH2Ar), 4.86 (t, J = 9.7 Hz, 1H,
H-1), 4.53 (d, J = 5.7 Hz, 2H, ArCH2NH), 4.18 (dd, J = 12.5, 4.2
Hz, 1H, H-6b), 4.04 – 3.91 (m, 2H, H-6a, H-2), 3.91 – 3.82 (m,
1H, H-5), 1.99 (s, 3H, OAc), 1.97 (s, 3H, OAc), 1.92 (s, 3H,
OAc), 1.76 (s, 3H, NAc); 13C NMR (75 MHz, DMSO-d6) δ
170.15, 169.78, 169.68, 169.43, 166.37, 166.28, 145.07, 134.25,
131.39, 128.38, 127.37, 124.63, 78.25, 73.34, 72.55, 68.44,
61.88, 52.28, 51.53, 34.96, 22.70, 20.60, 20.48, 20.44; HRMS
(ESI) calcd for C26H33N6O10(M+H+) 589.2258, found 589.2267.
1
DMSO); H NMR (300 MHz, DMSO-d6) δ 8.63 (d, J = 9.1 Hz,
1H, CHNH), 7.94 – 7.80 (m, 2H, ArH, NHAc), 7.76 (t, J = 5.7
Hz, 1H, CH2NH), 6.04 – 5.77 (m, 1H, CH=CH2), 5.37 – 4.92 (m,
6H, H-3, H-4, CH=CH2, COCH2Ar), 4.81 (t, J = 9.4 Hz, 1H, H-
1), 4.60 (t, J = 5.7 Hz, 1H, OH), 4.47 (d, J = 5.2 Hz, 2H), 4.23 (d,
J = 5.8 Hz, 2H, CH2CH=CH2), 3.73 – 3.51 (m, 2H, H-6b, H-2),
3.50 – 3.42 (m, 1H, H-6a), 3.37 – 3.23 (m, 1H, H-5), 3.15 – 3.02
(m, 2H, 2 OH), 1.80 (s, 3H, NAc); 13C NMR (75 MHz, DMSO-
d6) δ 170.17, 166.00, 156.14, 145.15, 133.76, 124.26, 117.16,
79.13, 79.03, 74.56, 70.55, 64.55, 60.98, 54.70, 51.67, 36.06,
22.94; HRMS (ESI) calcd for C17H27N6O8(M+H+) 443.1890,
found 443.1885.

temperature, TLC (EtOAc: MeOH: H2O = 10:1:1) indicated that
the reaction was completed. The solution was concentrated in
vacuo and recrystallised from MeOH /ether, resulting in
compounds 17a-17d.
4.2.6. 2-(2-acetamido-3,4,6-tri-O-acetyl-β-D-glucopyranosyl-
thio)-N-(prop-2-yn-1-yl) acetamide (12)
The thiol 11 (1.0 g, 2.8 mmol) and 2-chloro-N-(prop-2-yn-1-
yl)acetamide (0.36g, 2.8 mmol) were dissolved in acetone/H2O
(2:1, 45mL), and potassium carbonate (0.46 g, 3.3 mmol) were
then added. The mixture was stirred at room temperature for 12 h,
TLC (EtOAc) showed that the reaction was completed, then the
solution was diluted with CH2Cl2 (50 mL). The layers were
separated, with the aqueous layer being re-extracted with further
CH2Cl2 (50 mL). The combined organic layers were dried over
Na2SO4 and concentrated in vacuo. Chromatography of the
residue gave the compounds 12 (1.05 g, 82.1%) as a white solid;
[α]D25 50.1 (c=0.5,CHCl3); 1H NMR (300 MHz, DMSO-d6) δ 8.36
(t, J = 5.3 Hz, 1H, NH), 8.01 (d, J = 9.5 Hz, 1H, NH), 5.06 (t, J =
9.7 Hz, 1H, H-3), 4.87 (t, J = 9.7 Hz, 1H, H-4), 4.74 (d, J = 10.4
Hz, 1H, H-1), 4.17 (dd, J = 12.2, 4.7 Hz, 1H, H-6b), 4.10–3.89
(m, 2H, H-6a, H-2), 3.85 (dd, J = 5.3, 2.4 Hz, 2H, ≡CCH2), 3.83–
3.75 (m, 1H, H-5), 3.41–3.18 (m, 2H, SCH2), 3.11 (t, J = 2.5 Hz,
1H, ≡CH), 2.02, 1.96, 1.91, 1.77 (3 s, 9H, 3 OAc), 1.77 (s, 3H,
NAc); 13C NMR (75 MHz, DMSO-d6) δ 170.19, 169.71, 169.39,
168.31, 82.99, 80.92, 74.85, 73.66, 73.26, 68.64, 62.08, 52.10,
32.51, 28.36, 22.74, 20.64, 20.52, 20.43; HRMS (ESI) calcd for
C19H27N2O9S(M+H+) 459.1437, found 459.1439.
4.2.9.1. N-[[1-[2-(1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-
yl)ethyl]-1H-1,2,3-triazol-4-yl] meth yl]-2-(2-acetamido-β-D-
glucopyranosylthio)acetamide (17a). light yellow solid; (0.55 g,
25
1
91.8%) yield; [α]D -44.5 (c=0.2, DMF); H NMR (300 MHz,
DMSO-d6) δ 8.47–8.31 (m, 5H, 4 ArH, CONHCH2), 8.00 (s, 1H,
ArH), 7.89–7.76 (m, 3H, 2 ArH, NHAc), 4.66 (t, J = 5.6 Hz, 2H,
CH2), 4.54–4.38 (m, 3H, H-1, H-3, H-4), 4.26 (d, J = 5.0 Hz, 2H,
CH2), 3.68 (d, J = 11.4 Hz, 1H, H-6b), 3.56 (dd, J = 18.9, 9.3 Hz,
1H, H-2), 3.44 (d, J = 9.1 Hz, 1H, H-6a), 3.38–3.20 (m, 3H, H-5,
CH2), 3.14–3.07 (m, 2H, CH2), 1.80 (s, 3H, NAc); 13C NMR (75
MHz, DMSO-d6) δ 169.40, 169.10, 163.36, 144.68, 134.55,
131.34, 130.92, 127.45, 127.28, 123.54, 121.79, 83.97, 81.23,
75.42, 70.66, 61.38, 54.58, 48.74, 47.11, 34.58, 33.00, 23.11;
HRMS (ESI) calcd for C27H31N6O8S(M+H+) 599.1924, found
599.1921.
4.2.9.2. Data for compounds 17b-17d can be found in
Supporting Information.
4.3. Inhibition activity assays
4.2.7. Synthesis of compounds 15a-15d
Compounds 15a-15d were prepared according to published
methods26. Data for 15a-15d can be found in Supporting
Information.
OfHex1 was overexpressed in Pichia pastoris and then
12
purified as described previously.
Human β-N-
acetylhexosaminidase B (HsHexB) was obtained according to
previous methods.25
The inhibitory activities of OfHex1 and HsHexB were assayed
4.2.8. Synthesis of compounds 16a-16d
by end-point experiment,
and 4-MU-GlcNAc (4-
To a solution of compound 12 (1 mmol, 1eq) and compounds
15a-15d (1mmol, 1eq) in THF/H2O (1:1, 20mL), sodium
ascorbate (0.38mmol, 0.25eq) and copper (II) sulfate (0.3mmol,
0.2eq) were then added. The mixture was stirred at room
temperature for 12 h, and TLC (DCM/MeOH, 20:1 v/v) showed
that the reaction was completed. The mixture was extracted with
DCM (3×80 ml), dried over Na2SO4, and concentrated in vacuo.
Finally, the residue was subjected to chromatography, from
which the acetylated intermediates 16a-16d were obtained.
methylumbelliferyl N-acetyl-β-D-glucosaminide, Sigma) was
used as the substrate. The assay components were incubated in a
o
final volume of 100 μL at 30 C for 30 min in the presence of
Britton-Robinson buffer (pH 6.5), dimethylsulfoxide (at the final
concentration of 2%), enzyme, compound and 40 μM 4-MU-
GlcNAc. Subsequently, the enzymatic reaction was terminated by
the addition of 100 μl of 0.5 M sodium carbonate solution. The
fluorescence of the released 4-methylumbelliferone was
quantified (excitation at 366 nm, emission at 445 nm) with a
Varioskan Flash microplate reader (Thermo Fisher Scientific,
Waltham, MA, USA). The inhibition constant (Ki) was obtained
by changing the concentration of the 4-MU-GlcNAc at a constant
concentration (50, 25 and 12.5 μM) and then the reciprocal plots
of 1/velocity versus inhibitor were constructed.
4.2.8.1. N-[[1-[2-(1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-
yl)ethyl]-1H-1,2,3-triazol-4-yl] methyl]-2-(2-acetamido-3,4,6-tri-
O-acetyl-β-D-glucopyranosylthio)acetamide (16a). light yellow
solid; (0.54 g, 74.5 %) yield; [α]D25 32.1(c=0.2, DMF); H NMR
1
(300 MHz, DMSO-d6) δ 8.51–8.29 (m, 5H, ArH), 8.03 (d, J = 8.7
Hz, 2H, CONHCH2, NHAc), 7.89–7.73 (m, 2H, ArH), 5.08 (t, J
= 9.7 Hz, 1H, H-3), 4.88 (t, J = 9.7 Hz, 1H, H-4), 4.78 (d, J =
10.4 Hz, 1H, H-1), 4.67 (t, J = 5.9 Hz, 2H, CH2), 4.45 (t, J = 5.9
Hz, 2H, CH2), 4.37–4.21 (m, 2H, CH2), 4.15 (dd, J = 12.5, 4.6
Hz, 1H, H-6b), 4.01–3.84 (m, 2H, H-6a, H-2), 3.84–3.74 (m, 1H,
H-5), 3.35–3.20 (m, 2H, CH2), 1.99, 1.96, 1.91 (3 s, 9H, 3 OAc),
1.77(s, 3H, NAc); 13C NMR (75 MHz, DMSO-d6) δ 170.18,
169.72, 169.42, 169.39, 168.37, 163.40, 134.57, 131.40, 130.93,
127.52, 127.30, 123.58, 121.88, 82.98, 74.84, 73.67, 68.66,
62.05, 52.22, 47.15, 34.58, 32.66, 28.38, 22.74, 20.60, 20.51,
20.43; HRMS (ESI) calcd for C33H37N6O11S(M+H+) 725.2241,
found 725.2240.
4.4. Molecular docking
The complex crystal structure of OfHex1-PUGNAc (PDB
entry code: 3OZP)11 was used as the starting model for molecular
docking employing the Sybyl Software (Version 7.3).29 Before
docking calculations, the compound 17c was first optimized at
the B3LYP/6-31G(d) basis set using density functional theory
(DFT) in Gaussian 16. For the protein, all water molecules were
removed and missing hydrogen atoms were then added. After
that, the protomol, appropriate putative ligand pose, was created
by ligand mode based on the Hammerhead scoring function with
the molecular similarity algorithm in the active domain of the
receptor.30-31 The Surflex-Dock program was used for docking
studies and the ring flexibility is considered. All other parameters
were set as default.
4.2.8.2. Data for compounds 16b-16d can be found in
Supporting Information.
4.2.9. Synthesis of compounds 17a-17d
4.5. MD simulation
A solution of compounds 16a-16d (1 mmol) were dissolved in
MeOH (10 mL), then saturated solution of NH3 in MeOH (5 mL)
was added. The reaction mixture was stirred for 40 h at room
Molecular dynamics (MD) simulations of OfHex1-17c system
was performed using the Amber14 package32. AMBER 03 force

field33 was selected for the protein, and GAFF34 was used for the
inhibitor 17c. The complex was immersed in a truncated
octahedral box with TIP3P water molecules and electrostatic
neutralized by adding counterions (Cl- or Na+). Initially, Sander
module was used to realize the energy minimizations of the
system. First, the hydrogen atoms and water molecules were
minimized with the 2500 cycles of steepest descent followed by
the conjugated-gradient methods. Second, another 2500 steps of
the steepest-descent and the conjugated-gradient methods were
carried out to minimize all atoms of the system without
restriction. Next, the complex was heated gradually from 0 to 300
K in the NVT ensemble and equilibrated to 300 K. The SHAKE
method35 was applied to constrain hydrogen atoms and the
particle mesh Ewald (PME) method36 was used to treat the long-
range electrostatic interactions under periodic boundary
conditions. During the sampling process, coordinate trajectories
were saved every 10 ps. Finally, 30 ns MD simulation was
performed at a constant temperature of 300 K and a constant
pressure of 105 Pa using the PMEMD module.
12. Liu T, Zhang H-T, Liu F-Y, Wu Q-Y, Shen X, Yang Q. Structural
determinants of an insect β-N-acetyl-D- hexosaminidase
specialized as a chitinolytic enzyme. J Biol Chem. 2011;286:
4049-4058.
13. Liu T, Xia M, Zhang H, Zhou H, Wang J, Shen X, Yang Q.
Exploring NAG-thiazoline and its derivatives as inhibitors of
chitinolytic β-acetylglucosaminidases. FEBS Lett. 2015;589: 110-
116.
14. Chen W, Shen S, Dong L, Zhang J, Yang Q. Selective inhibition
of β-N-acetylhexosaminidases by thioglycosyl-naphthalimide
hybrid molecules. Bioorg Med Chem. 2018;26: 394-400.
15. Liu T, Guo P, Zhou Y, et al. A crystal structure-guided rational
design switching non-carbohydrate inhibitors' specificity between
two β-GlcNAcase homologs. Sci Rep. 2014;4: 6188-6193.
16. Slamova K, Marhol P, Bezouska K, Lindkvist L, Hansen S, Kren
V, Jensen H. Synthesis and biological activity of glycosyl-1H-
1,2,3-triazoles. Bioorg Med Chem Lett. 2010;20: 4263-4265.
17. Li T, Guo L, Zhang Y, Wang J, Li Z, Lin L, Zhang Z, Li L, Lin J,
Zhao W, Li J, Wang P. Design and synthesis of O-GlcNAcase
inhibitors via 'click chemistry' and biological evaluations.
Carbohydr Res. 2011;346: 1083- 1092.
18. Kong H, Chen W, Lu H, Yang Q, Dong Y, Wang D, Zhang J.
Synthesis of NAG-thiazoline-derived inhibitors for β-N-acetyl-D-
hexosaminidases. Carbohydr Res. 2015;413: 135-144.
19. Hottin A, Carrion-Jimenez S, Moreno-Clavijo E, Moreno-Clavijo
A, Carmona A, Robina I, Behr J. Expanding the library of divalent
fucosidase inhibitors with polyamino and triazole-benzyl bridged
bispyrrolidines. Org Biomol Chem. 2016;14: 3212-3220.
20. Sugawara A, Maita N, Gouda H, Yamamoto T, Hirose T, Kimura
S, Saito Y, Nakano H, Kasai T, Nakano H, Shiomi K, Hirono S,
Conflicts of interest
There are no conflicts to declare.
Acknowledgments
Watanabe T, Taniguchi H, O̅mura S, Sunazuka T. Creation of
customized bioactivity within a 14-membered macrolide scaffold:
Design, synthesis, and biological evaluation using a family-18
chitinase. J Med Chem. 2015;58: 4984-4997.
We acknowledge the financial support by the National Natural
Science Foundation (21772230) of China, and the National Key
R&D Program (2018YFD0200100) of China.
21. Paul KJV, Sahoo L, Sorna V, Loganathan D. An improved
procedure for the synthesis of fully acetylated N-(β-glycosyl)
azidoacetamides and N-(β-glycosyl)iodoacetamides useful as
chemical ligating agents. Trends Carbohydr Res. 2010;2: 21-28.
22. Chai H, Le Mai Hoang K, Vu MD, Pasunooti K, Liu C-F, Liu X-
W. N-linked glycosyl auxiliary-mediated native chemical ligation
on aspartic acid: application towards N-glycopeptide synthesis.
Angew Chem Int Ed. 2016;55: 10363-10367.
References
1. Kongbacterial chitinolytic H, Chen W, Liu T, Lu H, Yang Q,
Dong Y, Liang X, Jin S, Zhang J. Synthesis of NAM-thiazoline
derivatives as novel O-GlcNAcase inhibitors. Carbohydr Res.
2016;429: 54-61.
2. Liu T, Chen L, Ma Q, Shen X, Yang Q. Structural insights into
chitinolytic enzymes and inhibition mechanisms of selective
inhibitors. Curr Pharm Design. 2014;20: 754-770.
23. Sun G, Zhang J, Jin S, Zhang J. Synthesis and insecticidal activity
of 4 ''-Avermectin triazole derivatives containing amide unit.
Chinese J Org Chem. 2018;38: 1214-1222.
24. Mai S, Rao C, Chen M, Su J, Du J, Song Q. Merging gold
catalysis, organocatalytic oxidation, and Lewis acid catalysis for
chemodivergent synthesis of functionalized oxazoles from N-
propargylamides. Chem Commun. 2017;53: 10366-10369.
25. Shen S, Chen W, Dong L, Yang Q, Lu H, Zhang J. Design and
synthesis of naphthalimide group-bearing thioglycosides as novel
β-N-acetylhexosaminidases inhibitors. J Enzyme Inhib Med Chem.
2018;33: 445-452.
26. Ramchander J, Rameshvvar N, Reddy TS, Raju G, Reddy AR.
Synthesis and photophysical properties of 1, 4-disubstituted
naphthyloxymethyl-N-alkyl naphthimido-1,2,3-triazole. J Chem
Sci. 2014;126: 1063-1074.
3. Liu T, Duan Y, Yang Q. Revisiting glycoside hydrolase family 20
beta-N-acetyl-D-hexosaminidases:
Crystal
structures,
physiological substrates and specific inhibitors. Biotechnol Adv.
2018;36: 1127-1138.
4. Shikhman AR, Brinson DC, Lotz M. Profile of
glycosaminoglycan-degrading glycosidases and glycoside
sulfatases secreted by human articular chondrocytes in
homeostasis and inflammation. Arthritis Rheum. 2000;43: 1307-
1314.
5. Li DC. Review of fungal chitinases. Mycopathologia. 2006;161:
345-360.
6. Usuki H, Yamamoto Y, Kumagai Y, Nitoda T, Kanzaki H,
Hatanaka T. MS/MS fragmentation-guided search of TMG-
chitooligomycins and their structure-activity relationship in
specific beta-N-acetylglucosaminidase inhibition. Org Biomol
Chem. 2011;9: 2943-2951.
27. Elsen NL, Patel SB, Ford RE, et al. Insights into activity and
inhibition from the crystal structure of human O-GlcNAcase. Nat
Chem Biol. 2017;13: 613-U205.
28. Aich
U,
Loganathan
D.
Synthesis
of
N-(β-
Glycopyranosyl)azidoacetamides. J Carbohydr Chem. 2005;24: 1-
12.
29. SYBYL Molecular Modeling Software, 7.3, 2006, Tripos, St.
Louis, MO.
7.
Liu J, Liu M, Yao Y, Wang J, Li Y, Li G, Wang Y. Identification
of novel potential β-N-acetyl-D-hexosaminidase inhibitors by
virtual screening, molecular dynamics simulation and MM-PBSA
calculations. Int J Mol Sci. 2012;13: 4545-4563.
30. Jain, A N. Scoring noncovalent protein–ligand interactions: a
continuous differentiable function tuned to compute binding
affinities. J Comput Aided Mol Des. 1996;10: 427–440.
31. Clark RD, Strizhev A, Leonard J M, Blake JF, Matthew J B.
Consensus scoring for ligand/protein interactions. J Mol Graph
Model. 2002;20: 281–295.
32. Case DA, Babin V, Berryman JT, Betz RM, Cai Q, Cerutti DS,
Cheatham III TE, Darden TA, Duke RE, Gohlke H, Goetz AW,
Gusarov S, Homeyer N, Janowski P, Kaus J, Kolossváry I,
Kovalenko A, Lee TS, LeGrand S, Luchko T, Luo R, Madej B,
Merz KM, Paesani F, Roe DR, Roitberg A, Sagui C, Salomon-
Ferrer R, Seabra G, Simmerling CL, Smith W, Swails J, Walker
RC, Wang J, Wolf RM, Wu X, Kollman PA. 2014, AMBER 14.
University of California, San rancisco
8.
Yang Y, Liu T, Yang Y, Wu Q, Yang Q, Yu B. Synthesis,
evaluation, and mechanism of N,N,N-Trimethyl-D-glucosamine-
(1→4)-chitooligosaccharides as selective inhibitors of glycosyl
hydrolase
Chembiochem. 2011;12: 457-467.
Chen L, Liu T, Duan Y, Lu X, Yang Q. Microbial secondary
metabolite, Phlegmacin B-1, as novel inhibitor of insect
chitinolytic enzymes. J Agr Food Chem. 2017;65: 3851-3857.
family
20
β-N-acetyl-D-hexosaminidases.
9.
a
10. Yang Q, Liu T, Liu F, Qu M, Qian X. A novel beta-N-acetyl-D-
hexosaminidase from the insect Ostrinia furnacalis (Guenee).
FEBS J. 2008;275: 5690-5702.
11. Liu T, Zhang H, Liu F, Chen L, Shen X, Yang Q. Active-pocket
size differentiating insectile from bacterial chitinolytic β-N-acetyl-
D-hexosaminidases. Biochem J. 2011;438: 467-474.

33. Duan Y, Wu C, Chowdhury S, Lee M C, Xiong G, Zhang W,
Yang R, Cieplak P, Luo R, Lee T, Caldwell J, Wang J, Kollman P.
A point-charge force field for molecular mechanics simulations of
proteins based on condensed-phase quantum mechanical
calculations. J Comput Chem. 2003;24: 1999-2012.
34. Wang J, Wolf R M, Caldwell, J W, Kollman, P A, Case, D A.
Development and testing of a general amber force field. J Comput
Chem. 2004;25: 1157-1174.
35. Darden T, York D, Pedersen L. Particle mesh Ewald: an N·log(N)
method for Ewald sums in large systems. J Chem Phys. 1993;98:
10089-92.
36. Ryckaert J P, Ciccotti G, Berendsen H J C. Numerical integration
of the Cartesian equations of motion of a system with constraints:
molecular dynamics of n-alkanes. J Comput Phys. 1977;23: 327-
341.
O
HO
O
O
HO
HO
4 N
O
N
N
S
N
H
To acquire additional
affinity at +1 subsite
and byond the pocket
N
NHAc
To interact with -1 subsite
17c
OfHex1: K =28.68

M
i
HsHexB: K >100

M
i
OH
R
O
HO
HO
X
linker
N
N
N
AcHN
To prevent hydrolysis by OfHex1
X= N, S
linker=
NHCOCH2, CH2CONHCH2
O
O
n N
N
R=
R1
H
O
MD simulations of OfHex1 in complex with 17c