Novel glucopyranoside C2-derived 1,2,3-triazoles displaying selective inhibition of O-GlcNAcase (OGA)

Article abstract of DOI:10.1016/j.carres.2018.10.007

O-GlcNAcylation or O-GlcNAc modification is a post-translational modification of several proteins responsible for fundamental cellular processes. Dysregulation of the O-GlcNAc pathway has been linked to the etiology of several diseases such as neurodegenerative and cardiovascular diseases, type 2 diabetes and cancer. O-GlcNAcase (OGA) catalyzes the removal of O-GlcNAc from the modified proteins and several carbohydrate-based OGA inhibitors have been synthesized to understand the role of O-GlcNAc-modified proteins in physiological and pathological conditions. However, many of the inhibitors lack selectivity for OGA over lysosomal hexosaminidases A and B. Aiming the selectively inhibition of OGA, we propose herein the synthesis of twelve novel glucopyranoside derivatives exploring the bioisosteric replacement of the GlcNAc 2-acetamide group by 1,4-disubstituted 1,2,3-triazole ring, bearing a variety of central chains with different shapes. Compounds were readily prepared through “Copper(I) Catalyzed Azide/Alkyne Cycloaddition” (CuAAC) reaction between a sugar azide and different terminal alkynes. Initial Western Blot analyses and further inhibitory assays proved that compounds 6a (IC₅₀ = 0.50 ± 0.02 μM, OGA), 6k (IC₅₀ = 0.52 ± 0.01 μM, OGA) and 6l (IC₅₀ = 0.72 ± 0.02 μM, OGA) were the most potent and selective compounds of the series. Structure-activity relationship analyses and molecular docking simulations demonstrated that the bridge of two-carbon atoms between the C-4 position of the triazole and the phenyl ring (6a), which may be replaced by heteroatoms such as N (6k) or O (6l), is fundamental for accommodation and inhibition within OGA catalytic pocket.

Full text of DOI:10.1016/j.carres.2018.10.007

Accepted Manuscript
Novel glucopyranoside C2-derived 1,2,3-triazoles displaying selective inhibition of O-
GlcNAcase (OGA)
Michelle O. Igual, Paulo S.G. Nunes, Rafael M. da Costa, Susimaire P. Mantoani,
Rita C. Tostes, Ivone Carvalho
PII:
S0008-6215(18)30546-9
https://doi.org/10.1016/j.carres.2018.10.007
CAR 7617
DOI:
Reference:
To appear in: Carbohydrate Research
Received Date: 21 September 2018
Revised Date: 19 October 2018
Accepted Date: 22 October 2018
Please cite this article as: M.O. Igual, P.S.G. Nunes, R.M. da Costa, S.P. Mantoani, R.C. Tostes,
I. Carvalho, Novel glucopyranoside C2-derived 1,2,3-triazoles displaying selective inhibition of O-
GlcNAcase (OGA), Carbohydrate Research (2018), doi: https://doi.org/10.1016/j.carres.2018.10.007.
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ACCEPTED MANUSCRIPT
Graphical Abstract:

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Novel glucopyranoside C2-derived 1,2,3-triazoles displaying
selective inhibition of O-GlcNAcase (OGA)
Michelle O. Igual,a Paulo S. G. Nunes,a Rafael M. da Costa,b Susimaire P. Mantoani,a Rita C.
Tostesb and Ivone Carvalhoa*
a School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Av. do Café s/n,
Monte Alegre, 14040-930, Ribeirão Preto, Brazil.
b Ribeirão Preto Medical School, University of São Paulo, Av. Bandeirantes 3900, Monte Alegre,
14049-900, Ribeirão Preto, Brazil.
*Corresponding author e-mail: carronal@usp.br
Abstract
O-GlcNAcylation or O-GlcNAc modification is a post-translational modification of several
proteins responsible for fundamental cellular processes. Dysregulation of the O-GlcNAc
pathway has been linked to the etiology of several diseases such as neurodegenerative and
cardiovascular diseases, type 2 diabetes and cancer. O-GlcNAcase (OGA) catalyzes the
removal of O-GlcNAc from the modified proteins and several carbohydrate-based OGA
inhibitors have been synthesized to understand the role of O-GlcNAc-modified proteins in
physiological and pathological conditions. However, many of the inhibitors lack selectivity
for OGA over lysosomal hexosaminidases A and B. Aiming the selectively inhibition of
OGA, we propose herein the synthesis of twelve novel glucopyranoside derivatives exploring
the bioisosteric replacement of the GlcNAc 2-acetamide group by 1,4-disubstituted 1,2,3-
triazole ring, bearing a variety of central chains with different shapes. Compounds were
readily prepared through "Copper(I) Catalyzed Azide/Alkyne Cycloaddition" (CuAAC)
reaction between a sugar azide and different terminal alkynes. Initial Western Blot analyses
and further inhibitory assays proved that compounds 6a (IC50 = 0.50 ± 0.02 µM, OGA), 6k
(IC50 = 0.52 ± 0.01 µM, OGA) and 6l (IC50 = 0.72 ± 0.02 µM, OGA) were the most potent
and selective compounds of the series. Structure-activity relationship analyses and molecular
docking simulations demonstrated that the bridge of two-carbon atoms between the C-4
position of the triazole and the phenyl ring (6a), which may be replaced by heteroatoms such
as N (6k) or O (6l), is fundamental for accommodation and inhibition within OGA catalytic
pocket.

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Keywords: O-GlcNAcase, O-GlcNAcylation, selective inhibitors, click chemistry, 1,2,3-
triazole
1. Introduction
O-GlcNAcylation or O-GlcNAc modification of proteins has been recognized as an
important event in the post-translational modification of approximately 4,000 proteins that
are responsible for fundamental cellular processes, such as cellular signaling, transcription,
protein stability and degradation, among others [1,2]. O-GlcNAcylation is directly regulated
by two enzymes, a glycosyltransferase named O-GlcNAc transferase (OGT), which transfers
the sugar N-acetylglucosamine (GlcNAc) from uridine 5’-diphosphate N-acetylglucosamine
(UDP-GlcNAc) substrate to target proteins, and a glycoside hydrolase referred to as O-
GlcNAcase (OGA), which catalyzes the removal of O-GlcNAc from the modified proteins
[3].
Dysregulation of the O-GlcNAcylation pathway may affect innumerable cell signaling
processes [4], contributing to the etiology of several diseases such as neurodegenerative and
cardiovascular diseases, type 2 diabetes and cancer [5,2,6]. A range of carbohydrate-based
inhibitors of OGA has allowed identification of the role of O-GlcNAc-modified proteins in
physiological and pathological conditions [7,8]. These inhibitors, which have emerged as
novel therapeutic candidates, include PUGNAc, Streptozotocin, NAG-thiazoline, NButGT,
Thiamet-G, NAGstatin [8], NAM-thiazolines [9] and, more recently, the thioglycosyl-
naphthalimide hybrid molecules CAUS-A and CAUS-B [10] (Figure 1). However, many of
them lack selectivity for OGA over lysosomal hexosaminidases, β-N-hexosaminidase A
(HexA) and β-N-hexosaminidase (HexB).
Figure 1.
The cellular activities of OGA and HexA and B can be distinguished owing to the
environmental pH and substrate specificity, as observed for OGA activity at neutral pH and
GlcNAc substrate, and for HexA and B at acidic (lysosome) pH with affinity for GalNAc
units [11]. The role played by the latter on cell metabolism implies that any nonspecific
inhibition can give rise to lysosomal ganglioside accumulation that leads to the
neurodegenerative Tay-Sachs and Sandhoff diseases [8].
Based on the evidence that the catalytic site of OGA accommodates more flexible and
bulky substituents near the 2-acetamide group of the corresponding substrate, compared to
HexA and B enzymes, the methyl substituent of the NAG-thiazoline compound (KI 80 nM)
was extended to a propyl group, leading to NButGT (Figure 1) with higher selectivity

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(approximately 600-folds for OGA), although with lower potency (KI 600 nM) [7,12].
Further modifications of the alkyl chain of NButGT through the insertion of an amine group
provided Thiamet-G with remarkable potency (KI 21 nM) and selectivity (37,000-folds) [12].
The achievements on thiazoline-based GlcNAc derivatives encouraged us to pursue an
alternative approach to selectively target OGA by exploring the bioisosteric replacement of
the GlcNAc 2-acetamide by 1,4-disubstituted 1,2,3-triazole bearing a variety of side chains
with different shapes, length and polarity. In fact, triazole-based ring systems have been
intensively used in biomedical research in the discovery of new drug candidates and chemical
optimization of prototypes [13,14] due to its rigidity, stability and readily preparation using
"Copper(I) Catalyzed Azide/Alkyne Cycloaddition" (CuAAC) reaction as a click chemistry
strategy [15, 16] to produce easily purified products in good yields.
2. Results and Discussion
2.1. Synthesis of glucopyranoside triazoles library
For the synthesis of glucopyranoside triazole derivatives we followed the 1,3-dipolar
cycloaddition (CuAAC) reaction that enables the coupling of the sugar azide with terminal
alkynes to afford the acetylated triazole derivatives, which were then deacetylated to give the
target compounds.
Based on previously described methods [17,18,19], glucosamine hydrochloride (1)
was treated with acetyl bromide to give the highly unstable glycosyl bromide, which was
immediately used in the next glycosidation step to give the methyl 3,4,6-tri-O-acetyl-2-
amine-2-deoxy-D-glucopyranoside (2), containing the free amine group at the C-2 position
(61% yield over two steps, Scheme 1). This approach afforded product 2 as a mixture of β:α
anomers in a 10:1 ratio, respectively, that were maintained during its conversion to azide 3
(37%) using diazotransfer reaction in the presence of a freshly prepared triflyl azide solution
in pyridine [20,21].
Scheme 1.
For the synthesis of the acetylenic precursors 4g, 4h, 4j-l, different previously
reported synthetic approaches were applied. Therefore, treatment of propargylic alcohol with
benzyl bromide in dry tetrahydrofuran (THF) [22] afforded alkyne 4g (66%), while reductive
amination between benzaldehyde and propargylamine [23] under the presence of glacial
acetic acid (1.0 eq.) [24] gave product 4h in low yield (24%), though product 4h was
obtained in adequate proportion to proceed with the following cycloaddition reaction. On the

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other hand, the amide group of alkyne 4j (63%) was obtained by the treatment of 4-fluoro-
benzoic acid with propargylamine in triethylamine and the coupling reagent O-(benzotriazol-
1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU) [25]. For the acetylenes
4k and 4l, two similar approaches were used, e.g. potassium carbonate in the presence of 3-
bromopropyne treated with aniline [26] to give alkyne 4k (71%) or with phenol [27] to afford
alkyne 4l (47%). For all the alkyne precursors it was possible to identify the characteristic
1
acetylenes H NMR signals, in the range of δ 2.39-2.21 ppm and δ 4.70-3.44 ppm, both
presenting propargylic coupling constants (J 2.5 – 2.1 Hz).
The CuAAC reactions were carried out with azide precursor 3 and a series of
acetylenic precursors (4a-f and 4i, commercially available and 4g, 4h, 4j-l previously
synthesized). In general, the acetylated triazole derivatives 5a-l (Figure 1) were
straightforward obtained in moderate to good yields (43-79%) under microwave irradiation at
70 ºC for 10 minutes (min) in dimethylformamide (DMF), in the presence of 0.04 eq. of
CuSO4 and 0.1 eq. sodium ascorbate [28]. However, the coupling of acetylenes 4h and 4k
with azide 3 under these established conditions did not give the corresponding triazoles (5h,
5k), even after two sequential irradiations of 10 min. Therefore, an alternative CuAAC
procedure [29] was attempted using the 1,10-phenanthroline monohydrate ligand, in EtOH-
H2O at room temperature in order to increase CuAAC reaction’s efficiency, as the 1,10-
phenanthroline ligand has been reported to complex with copper(I) allowing its stabilization
for the cycloaddition reaction [30,31]. After 18 hours (h), the reaction was completed and
products 5h and 5k were obtained in 63% and 93% yields, respectively, after
chromatographic purification. The 1H NMR spectra of compounds 5a-l showed characteristic
singlet signals in a range of δ 7.55-7.42 ppm for triazole proton, whereas the sugar signals
were also observed in all derivatives with typical multiplicity, coupling constant and
integrals, notably indicating the downfield shift of the hydrogen at C-2 (δ 4.55-4.28 ppm)
compared to their azide precursor 3 (δ 3.51-3.45 ppm).
Finally, deacetylation reaction under standard conditions (catalytic NaOMe/MeOH)
[28] gave the glucopyranoside triazoles 6a-l (Figure 1) in good to excellent yields (59-98%).
1
The absence of H NMR singlet signals corresponding to the three acetyl groups was
confirmed for all products. Furthermore, it was observed that the hydrogen signals of the free
carbohydrate were slightly shifted upfield compared to the acetylated derivatives. The
accurate mass of the final compounds 6a-l was also confirmed by high-resolution mass
spectrometry and the data were in accordance with the calculated mass for each compound.

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Because of the OGA specificity to β substrates, separation of the anomeric mixture
was mandatory. Thus, an expedient separation of the β anomer by reversed phase HPLC was
accomplished using polar deacetylated derivatives 6a-l, though it was laborious and afforded
very low quantity of the pure products. To circumvent this issue, we managed to crystallize
the precursor azide 3 in EtOH, obtaining the pure β anomer as white needles with 34% yield.
Hence, triazole products 6a-l were promptly obtained as a single β-anomer as depicted in
Scheme 1 and then, further submitted to biological assays.
2.2. Cytotoxicity Assay
Prior to testing the potential OGA inhibitory activity of the glucopyranoside triazole
library, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was
performed. The potential toxicity of the glucopyranoside triazole compounds was assessed in
cultured vascular smooth muscle cells (VSMC) isolated from aorta of male Wistar rats. The
results shown in figure 2 indicate that none of the compounds was cytotoxic against the
evaluated cells and, therefore, the compounds were considered suitable for the subsequent
studies of inhibitory activity.
Figure 2.
2.3.Inhibitory Activity
The glucopyranoside triazoles 6a-l were initially obtained as a set of ten compounds
6a-j and their inhibitory activity over OGA was assessed by Western Blot analysis of O-
GlcNAc-modified proteins in lysates of cultured VSMCs from aorta of Wistar rats using an
antibody anti-β-O-linked N-acetylglucosamine (Sigma-Aldrich Inc., Germany). As shown in
figure 3A, compound 6a, but not the other compounds from the library 6b-l, increased O-
GlcNAc-modified proteins. The Compound 6a and Thiamet-G produced qualitatively similar
increases in O-GlcNAc-modified proteins. This preliminary result led us to an interesting
structure-activity relationship (SAR) analysis. Among compounds 6a-j, 6a is the unique
derivative bearing a central chain extension of two-carbon atoms bridging the C-4 position of
the triazole core to the phenol ring, suggesting that this length might be critical for
accommodation of the inhibitors in the OGA catalytic pocket. Conversely, three-carbon chain
displayed by compounds 6f-j may not adopt the correct orientation within the active site
pocket based on the weak ability to enhance protein O-GlcNAcylation, as determined in the
Western Blot experiments. Further evidence revealed a positive contribution of the aromatic

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ring on the activity of compound 6a, as compound 6e was not efficient in increasing O-
GlcNAcylated protein levels. In fact, novel derivatives 6k and 6l, containing heteroatoms (N
or O, respectively) that preserve the two carbons bridge extension of 6a linked to the
aromatic ring, exhibited similar activities in the Western Blot assays. These results indicate
that compounds 6a, 6k and 6l may create strong interactions at the OGA catalytic pocket,
which would enable them to qualitatively augment O-GlcNAc levels through OGA inhibition
(Figure 3B).
Figure 3.
In order to confirm the ability of the compounds to increase O-GlcNAcylation of
VSMCs proteins, observed in the Western Blot experiments, OGA enzymatic activity was
determined using cell lysates from Wistar rats, as previously described [32]. The enzymatic
activity was detected by the fluorescence intensity resulting from the breakdown of 4-
methylumbelliferyl N-acetyl-β-D-glucosaminide (4-MUNAG) substrate. The percentage of
inhibition was calculated by comparing the enzymatic activity in the presence of vehicle or
0.1, 1.0, 10 and 100 µM of the inhibitors. Thiamet-G was used as the reference compound.
Results shown in figure 4 are consistent with the former data, since only compound 6a
decreased OGA activity, by 91% to 93%, at concentrations of 1.0, 10 and 100 µM.
Compound 6a did not display inhibitory activity at lower concentrations, i.e. at 0.1 µM.
Figure 4.
Potential OGA inhibitory activity for compounds 6k and 6l was assessed under the
same conditions, but using 0.1 and 1.0 µM of the compounds. For comparisons, compound
6a and Thiamet-G were used. All three compounds were efficient at inhibiting OGA activity
at 1.0 µM (Figure 5). Likewise, compounds 6k and 6l were equally inactive at the
concentration of 0.1 µM. Furthermore, compound 6l, bearing an additional oxygen atom,
showed a lower percentage of inhibition (76%), whereas compound 6k, bearing an amine
group, was very effective, producing inhibitory effects similar to compound 6a (92%).
Figure 5.
Determination of IC50 corroborated the inhibitory activity of compounds 6a, 6k and 6l
at micromolar concentrations, with compound 6a (IC50 = 0.50 ± 0.02 µM) and 6k (IC50 =
0.52 ± 0.01 µM) being the most potent followed by compound 6l (IC50 = 0.72 ± 0.02 µM)
(Figure 6).
Figure 6.
2.4. Selectivity Assays

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The three most active compounds 6a, 6k and 6l were tested against β-N-
hexosaminidases A and B (HexA and B – GH20 family from prokaryotes) in order to assess
their potential selectivity for OGA.
Based on the formerly results obtained in cell lysates, compounds 6a, 6k and 6l were
initially evaluated at the concentration of 1.0 µM, whereas Thiamet-G was assayed at 1.0 mM
to ensure complete inhibition of the β-N-hexosaminidases A and B [33]. Compounds 6a, 6k
and 6l did not inhibit HexA or B at 1.0 µM concentration (Figure 7A). These promising
results led us to subsequently evaluate the compounds at a higher concentration (1.0 mM).
Figure 7B demonstrates that compounds 6a, 6k and 6l inhibited approximately 87% of HexA
and B at this concentration.
Figure 7.
IC50 determination revealed that compounds 6a (IC50 = 550 ± 0,004 µM), 6k (IC50 =
569 ± 0,009 µM) and 6l (IC50 = 571 ± 0,015 µM) are weak inhibitors of HexA and B (Figure
8), which allowed us to infer a high selective inhibitory effect of these three compounds for
OGA over HexA and B.
Figure 8.
2.5.Docking Studies
Molecular docking simulations were performed in order to analyze the binding mode
of the most potent inhibitors into the OGA catalytic site. The notable crystal structure of
Bacteroides thetaiotaomicron OGA (BtOGA) in complex with Thiamet-G (PDB code
2VVN) [33] was chosen to conduct the docking studies, based on its significant sequence
identity with the human O-GlcNAcase (hOGA) [34]. It is noteworthy that the three-
dimensional structure of hOGA was recently determined by Roth and co-workers, revealing
addition knowledge of the hOGA active site [35,36].
According to the structural similarity between BtOGA and hOGA, catalytic residues
in BtOGA were proposed to be Asp242 and Asp243 [33, 37]. The docking of compounds 6a,
6k and 6l were performed at BtOGA active site in parallel with Thiamet-G [12,33] (Figure
9).
Figure 9.
The docking studies indicated that the two-carbon atoms central chain, bridging the
triazole to the phenol ring, from compounds 6a, 6k and 6l, perfectly fit BtOGA catalytic

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pocket, being in agreement with the biological assays. Moreover, three compounds display
similar conformation within the catalytic site, as can be observed by the superposition of the
three structures represented on Figure 9A. Figure 9B shows the conformation of Thiamet-G
within BtOGA active site. Figures 9C-E represent compounds 6a, 6k and 6l, respectively,
and their interactions with the critical residues in the catalytic pocket.
The phenol ring from compounds 6a, 6k and 6l preserved hydrophobic interactions
with Cys278, Tyr282 and Trp337 residues. In addition, the sugar moiety allowed important
hydrogen bond interaction with catalytic residue Asp243, and only compound 6k is capable
to perform an addition hydrogen bond interaction with Asp242 (Figure 9D), due to the
presence of NH group in the central chain bridge, similar to Thiamet-G. Despite the
additional interaction of compound 6k with Asp242, it is observed that it does not
significantly influence the inhibitory potency.
Superposition of Thiamet-G and compound 6a into BtOGA active site, represented on
Figure 9F, showed that both compounds occupy the hydrophobic pocket displayed as an
extension of the catalytic site of the OGA enzyme. However, the sugar moiety from the
triazole derivatives does not occupy the same sugar recognition region of Thiamet-G, as well
as of the natural substrate O-GlcNAc [38], being displaced outside the catalytic pocket
(Figure 9F). On the other hand, the triazole core takes place performing additional
interactions that contributes to the activity, and, although the carbohydrate portion is shifted
outside the binding region, it is still able to make strong interactions with the key catalytic
residues. This observation allows us to the suggestion that the length of the central chain is
indeed critical for accommodation of the inhibitors in the OGA catalytic pocket and tightly
influence the inhibitory activity of the triazole derivatives over OGA enzyme.
3. Conclusions
We have synthesized twelve novel glucopyranoside triazole derivatives 6a-l, readily
prepared through CuAAC reaction between a sugar azide and different terminal alkynes.
MTT assay indicated that none of the compounds was cytotoxic. Screening with Western
Blot experiments revealed that compound 6a, 6k and 6l were qualitatively similar to
Thiamet-G in augment O-GlcNAc-modified proteins levels. Further inhibitory assays showed
that compounds 6a, 6k and 6l presented micromolar activity towards OGA with 6a (IC50 =
0.50 ± 0.02 µM) and 6k (IC50 = 0.52 ± 0.01 µM) being the most potent followed by
compounds and 6l (IC50 = 0.72 ± 0.02 µM). Moreover, compounds 6a, 6k and 6l

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demonstrated highly selectivity for OGA according to the weak inhibition obtained for HexA
and B. Additionally, molecular docking studies displayed the interaction mode of the
derivatives 6a, 6k and 6l within the OGA catalytic pocket, allowing us to conclude that these
compounds occupy all the extension of the catalytic site, being critical for the activity.
Furthermore, the sugar moiety from the triazole derivatives occupies a different region in the
active site, whilst performing strong interactions, which represents a great factor for the
inhibitory activity obtained for the triazole glucopyranoside compounds. These findings
provided promising clues for developing more potent OGA inhibitors.
4. Experimental section
4.1. General Information:
All chemicals were acquired as reagent grade (Sigma-Aldrich) and employed without
further purification. Solvents were treated according to standard procedures [39]. Reactions
were monitored by thin layer chromatography (TLC) on aluminum plates pre-coated with
silica gel (60 GF254, Sigma-Aldrich) using the indicated eluents. TLC spots were visualized
under ultraviolet (UV) light of 254 nm and/or by staining with ethanol-sulfuric acid (95:5,
v/v), ninhydrin or p-anisaldehyde solution followed by heating. Flash chromatography was
performed with a column with silica gel 60 (35 – 70 ꢀm) with the indicated eluents. The
microwave-assisted reactions were carried out on CEM® Discover System using sealed tubes.
1H and 13C Nuclear Magnetic Resonance spectra were obtained on Bruker Avance DRX 300,
DPX 400, DPX 500 spectrometers or Bruker UltraShield 300 FT-NMR spectrometer.
Chemical shifts (δ) were reported in parts per million (ppm) downfield from
tetramethylsilane (TMS) or relative to the deuterated solvent residual signal (Chloroform-d: δ
= 7.26 (1H), 77.16 (13C); Methanol-d4: δ = 3.31 (1H), 49.00 (13C); Deuterium oxide: 4.79
(1H)). Assignments were made with the aid of COSY, HSQC and HMBC experiments.
Optical rotations were measured on a Jasco P-2000 polarimeter at 20 ºC, using sodium lamp
and wavelength of 589ꢁnm. High Performance Liquid Chromatograph (HPLC) was performed
on a Shimadzu SCL-10AVP, equipped with software CLASS-VP 5.0 and SDP-M10A diode
array detector (DAD), using C-18 column (250 x 10 mm, 10 ꢀm, Macherey-Nagel
Nucleodur®). High-resolution mass spectra (HRMS) were obtained on a Bruker Daltonics
MicrOTOF-Q II ESI-TOF mass spectrometer.
4.2. Synthesis:

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4.2.1. Preparation of azide precursor 3
Methyl 3,4,6-tri-O-acetyl-2-amino-2-deoxy-
D
-glucopyranoside (2): [17,18,19] Acetyl
-glucose hydrochloride (1.0
bromide (3.20 mL; 42.9 mmol) was added to 2-amino-2-deoxy-
D
g; 4.6 mmol) and the mixture was stirred for 3 days at room temperature. The crude product
was dissolved in hot chloroform (distilled from P2O5) and filtered while still hot. Brown
crystals were formed as the solution cooled off. A small quantity of diethyl ether was added
until the solid precipitated from the solution. The product was filtered and washed with
diethyl ether. The resulting product was subsequently dissolved in methanol (44 mL) and dry
pyridine (0.44 mL) and the mixture was allowed to stir for 1 h at room temperature. The
mixture was concentrated by co-evaporation with toluene, diluted in chloroform (60 mL),
washed with Na2CO3 (aq) (5%, 2 x 100 mL) and water (100 mL), dried over Na2SO4, and
concentrated. The crude product was recrystallized in chloroform/hexane affording product 2
as a pale yellow solid as a mixture of β/α anomers in the ratio of 10/1, respectively (903.3
1
mg; 61% yield after two steps). H NMR (500 MHz, CDCl3) δH: 5.03 – 4.95 (2H, m, H-3, H-
4), 4.29 (1H, dd, J5,6a 4.7 Hz, J6a,6b 12.2 Hz, H-6a), 4.16 (1H, d, J1,2 8.0 Hz, H-1), 4.11 (1H,
dd, J5,6b 2.2 Hz, J6a,6b 12.2 Hz, H-6b), 3.68 (1H, ddd, J5,4 9.4 Hz, J5,6a 4.7 Hz, J5,6b 2.2 Hz, H-
5), 3.55 (3H, s, OCH3), 2.91 (1H, dd, J1,2 8.0 Hz, J2,3 9.4 Hz, H-2), 2.07, 2.07, 2.01 (9H, 3s, 3
CH3CO).
Methyl 3,4,6-tri-O-acetyl-2-azide-2-deoxy-D-glucopyranoside (3): [20,21] A solution of
triflyl azide was prepared dissolving sodium azide (170.5 mg; 2.6 mmol) in pyridine (2.9
mL), slowly adding trifluoromethanesulfonic anhydride (0.35 mL; 2.1 mmol) to the mixture
at 0 ºC. The mixture was stirred for 2 h at 0 ºC and the precipitated salts were filtered off
through celite. The freshly prepared triflyl azide solution was added dropwise at 0 ºC to a
solution of compound 2 (578 mg; 1.8 mmol), trimethylamine (0.5 mL; 3.6 mmol) and CuSO4
x 5 H2O (4.6 mg; 0.018 mmol) in pyridine (2.9 mL). The reaction was carried out for 16 h at
room temperature. The mixture was concentrated under reduced pressure followed by
purification by flash chromatography [Hexane:EtOAc (1:1)] affording product 3 as a yellow
oil as a mixture of β/α (10/1) (233.8 mg; 37% yield). Further crystallization of the β:α
mixture with ethanol afforded the β anomer (3β) as white needles with 34% yield. 1H NMR
(300 MHz, CDCl3) δH: 5.05 – 4.96 (2H, m, H-3, H-4), 4.32 – 4.26 (2H, m, H-1, H-6a), 4.12
(1H, dd, J5,6b 2.3 Hz, J6a,6b 12.3 Hz, H-6b), 3.66 (1H, ddd, J5,4 9.6 Hz, J5,6a 4.6 Hz, J5,6b 2.3
Hz, H-5), 3.60 (3H, s, OCH3), 3.51 – 3.45 (1H, m, H-2), 2.07 (6H, s, 2 CH3CO), 2.02 (3H, s,

ACCEPTED MANUSCRIPT
CH3CO). 13C NMR (75 MHz, CDCl3) δC: 170.8 (COCH3), 170.1 (COCH3), 169.7 (COCH3),
103.0 (C-1), 72.6 (C-5), 71.8 (C-2), 68.4 (C-3), 63.8 (C-4), 62.9 (C-6), 57.6 (OCH3), 20.8
(COCH3), 20.8 (COCH3), 20.7 (COCH3). IR (KBr) υmax: 3330, 3231, 2122, 1750, 1392,
1236, 1152, 1048, 607, 496 cm-1.
4.2.2. Synthesis of 1,2,3-triazole-modified glucopyranosides by CuAAC click
reaction
General procedure for the synthesis of compounds 5a-g, 5i-j and 5l: [28] Compound 3
containing the azide group (1.0 eq.) was dissolved in DMF (0.3 mL), followed by the
addition of sodium ascorbate (0.1 eq.), CuSO4 (10% aqueous solution; 0.04 eq.) and one of
the acetylene derivatives (1.0 eq.). The mixture was stirred for 10 min at 70 °C under
microwave irradiation (sealed microwave tube) (150 W). The reaction mixture was then
concentrated under reduced pressure by co-evaporation with toluene, diluted in water (20
mL) and extracted with EtOAc (3 x 25 mL). The combined organic layers were dried over
MgSO4, concentrated under reduced pressure and purified by flash chromatography
[Hexane:EtOAc (1:1)] to afford the desired products (5a-g, 5i-j and 5l).
General procedure for the synthesis of compounds 5h and 5k: [29] A solution of
CuSO4 1.0 M (5 mol%), 1,10-phenanthroline monohydrate (5 mol%), and sodium ascorbate
(1.02 eq.) in EtOH–H2O (2:1 v/v, 6 mL) was allowed to stir for 5 min at room temperature.
Subsequently, compound 3 containing the azide group (1.0 eq.) and one of the acetylene
derivatives 4h or 4k (1.0 eq.) were dissolved in EtOH–H2O (2:1 v/v, 1 mL) and added to the
mixture and the reaction was carried out for 18 h at room temperature. The reaction mixture
was then concentrated under reduced pressure and purified by flash chromatography [EtOAc
100%] to afford products (5h and 5k).
Methyl
3,4,6-tri-O-acetyl-2-[(4-(phenethyl)-1H-1,2,3-triazole-1-yl)]-2-deoxy-β-D-
1
glucopyranoside (5a): White solid (108 mg; 73% yield). H NMR (300 MHz, CDCl3) δH:
7.29 – 7.10 (6H, m, H-triazole, H-Ar.), 5.81 (1H, dd, J2,3 10.7 Hz, J3,4 9.3 Hz, H-3), 5.14 (1H,
t, J3,4 9.3 Hz, H-4), 4.91 (1H, d, J1,2 8.2 Hz, H-1), 4.38 (1H, dd, J5,6a 4.4 Hz, J6a,6b 12.3 Hz, H-
6a), 4.29 (2H, dd, J1,2 8.2 Hz, J2,3 10.7 Hz, H-2), 4.19 (1H, dd, J5,6b 2.1 Hz, J6a,6b 12.3 Hz, H-
6b), 3.91 (1H, ddd, J5,4 9.9 Hz, J5,6a 4.4 Hz, J5,6b 2.1 Hz, H-5), 3.41 (3H, s, OCH3), 3.08 –
2.97 (4H, m, CH2CH2), 2.11, 2.03, 1.80 (9H, 3s, 3 CH3CO). 13C NMR (75 MHz, CDCl3) δC:
170.8 (COCH3), 169.9 (COCH3), 169.1 (COCH3), 146.8 (C-Ar.), 141.2 (C-triazole), 128.6

ACCEPTED MANUSCRIPT
(CH-Ar.), 128.5 (CH-Ar.), 126.1 (CH-Ar.), 122.7 (CH-triazole), 101.8 (C-1), 72.3 (C-3), 71.9
(C-5), 68.8 (C-4), 63.8 (C-2), 61.9 (C-6), 57.7 (OCH3), 35.4 (CH2), 27.4 (CH2), 20.8
(COCH3), 20.7 (COCH3), 20.4 (COCH3).
Methyl
3,4,6-tri-O-acetyl-2-[(4-(benzyl)-1H-1,2,3-triazole-1-yl)]-2-deoxy-β-D-
glucopyranoside (5b): Colorless oil (115.7 mg; 84% yield). 1H NMR (300 MHz, CDCl3) δH:
7.33 – 7.20 (5H, m, H-Ar.), 7.15 (1H, s, H-triazole), 5.75 (1H, dd, J2,3 10.6 Hz, J3,4 9.3 Hz, H-
3), 5.14 (1H, t, J3,4 9.3 Hz, H-4), 5.00 (1H, d, J1,2 8.2 Hz, H-1), 4.38 (1H, dd, J5,6a 4.6 Hz,
J6a,6b 12.4 Hz, H-6a), 4.29 (2H, dd, J1,2 8.2 Hz, J2,3 10.6 Hz, H-2), 4.19 (1H, dd, J5,6b 2.2 Hz,
J6a,6b 12.4 Hz, H-6b), 4.13 (1H, d, JAB = 15.9 Hz, CHa), 4.05 (1H, d, JAB = 15.9 Hz, CHb),
3.91 (1H, ddd, J5,4 10.1 Hz, J5,6a 4.6 Hz, J5,6b 2.2 Hz, H-5), 3.44 (3H, s, OCH3), 2.11, 2.03,
1.77 (9H, 3s, 3 CH3CO). 13C NMR (75 MHz, CDCl3) δC: 170.7 (COCH3), 169.8 (COCH3),
169.0 (COCH3), 147.3 (C-triazole), 139.1 (C-Ar.), 128.7 (CH-Ar.), 126.6 (CH-Ar.), 123.2
(CH-triazole), 101.5 (C-1), 72.5 (C-3), 71.9 (C-5), 68.7 (C-4), 63.9 (C-2), 61.9 (C-6), 57.7
(OCH3), 32.1 (CH2), 20.8 (COCH3), 20.7 (COCH3), 20.2 (COCH3).
Methyl
3,4,6-tri-O-acetyl-2-[(4-(phenyl)-1H-1,2,3-triazole-1-yl)]-2-deoxy-D-
1
glucopyranoside (5c): White solid (128.9 mg; 43% yield). H NMR (300 MHz, CDCl3) δH:
7.84 – 7.81 (2H, m, H-Ar.), 7.75 (1H, s, H-triazole), 7.45 – 7.40 (2H, m, H-Ar.), 7.37 – 7.31
(1H, m, H-Ar.), 5.97 (1H, dd, J2,3 10.4 Hz, J3,4 9.0 Hz, H-3), 5.26 (1H, dd, J3,4 9.0 Hz, J5,4 9.8
Hz, H-4), 5.04 (1H, d, J1,2 7.9 Hz, H-1), 4.55 – 4.47 (2H, m, H-2, H-6a), 4.31 (1H, dd, J5,6b
2.2 Hz, J6a,6b 11.9 Hz, H-6b), 4.06 (1H, ddd, J5,4 9.8 Hz, J5,6a 4.4 Hz, J5,6b 2.2 Hz, H-5), 3.56
13
(3H, s, OCH3), 2.28, 2.21, 2.02 (9H, 3s, 3 CH3CO). C NMR (101 MHz, CDCl3) δC: 170.7
(COCH3), 169.9 (COCH3), 169.2 (COCH3), 147.4 (C-triazole), 130.3 (C-Ar.), 129.0 (CH-
Ar.), 128.5 (CH-Ar.), 125.8 (CH-Ar.), 121.3 (CH-triazole), 101.8 (C-1), 72.2 (C-5), 72.1 (C-
3), 69.0 (C-4), 64.2 (C-2), 61.9 (C-6), 57.8 (OCH3), 20.8 (COCH3), 20.7 (COCH3), 20.4
(COCH3).
Methyl
3,4,6-tri-O-acetyl-2-[(4-(p-tolyl)-1H-1,2,3-triazole-1-yl)]-2-deoxy-D-
glucopyranoside (5d): White solid (143.9 mg; 55% yield). 1H NMR (400 MHz, CDCl3) δH:
7.74 – 7.72 (3H, m, H-triazole, H-Ar.), 7.24 (2H, d, J 7.9 Hz, H-Ar.), 5.92 (1H, dd, J2,3 10.7
Hz, J3,4 9.3 Hz, H-3), 5.19 (1H, t, J3,4 9.3 Hz, H-4), 4.97 (1H, d, J1,2 8.2 Hz, H-1), 4.45 – 4.39
(2H, m, H-2, H-6a), 4.22 (1H, dd, J5,6b 2.3 Hz, J6a,6b 12.3 Hz, H-6b), 3.96 (1H, ddd, J5,4 10.1
Hz, J5,6a 4.5 Hz, J5,6b 2.3 Hz, H-5), 3.44 (3H, s, OCH3), 2.12, 2.05, 1.85 (9H, 3s, 3 CH3CO).

ACCEPTED MANUSCRIPT
13C NMR (101 MHz, CDCl3) δC: 170.7 (COCH3), 169.9 (COCH3), 169.2 (COCH3), 147.4
(C-triazole), 138.3 (C-Ar.), 129.6 (CH-Ar.), 127.5 (C-Ar.), 125.7 (CH-Ar.), 121.0 (CH-
triazole), 101.8 (C-1), 72.2 (C-5), 72.0 (C-3), 69.0 (C-4), 64.1 (C-2), 61.9 (C-6), 57.8
(OCH3), 21.4 (ArCH3), 20.8 (COCH3), 20.7 (COCH3), 20.4 (COCH3).
Methyl
3,4,6-tri-O-acetyl-2-[(4-(propyl)-1H-1,2,3-triazole-1-yl)]-2-deoxy-D-
glucopyranoside (5e): White solid (124.6 mg; 57% yield). 1H NMR (400 MHz, CDCl3) δH:
7.27 (1H, s, H-triazole), 5.81 (1H, dd, J2,3 10.6 Hz, J3,4 9.4 Hz, H-3), 5.16 (1H, t, J3,4 9.4 Hz,
H-4), 4.96 (1H, d, J1,2 8.2 Hz, H-1), 4.41 – 4.32 (2H, m, H-2, H-6a), 4.20 (1H, dd, J5,6b 2.2
Hz, J6a,6b 12.3 Hz, H-6b), 3.92 (1H, ddd, J5,4 10.1 Hz, J5,6a 4.5 Hz, J5,6b 2.2 Hz, H-5), 3.43
(3H, s, OCH3), 2.69 (2H, t, J 7.4 Hz, CH2), 2.11, 2.04, 1.84 (9H, 3s, 3 CH3CO), 1.69 (2H, m,
CH2), 0.94 (3H, t, J 7.4 Hz, CH3). 13C NMR (101 MHz, CDCl3) δC: 170.7 (COCH3), 169.9
(COCH3), 169.1 (COCH3), 147.8 (C-triazole), 122.3 (CH-triazole), 101.8 (C-1), 72.5 (C-5),
72.0 (C-3), 68.9 (C-4), 63.9 (C-2), 62.0 (C-6), 57.7 (OCH3), 27.6 (CH2), 22.6 (CH2), 20.8
(COCH3), 20.7 (COCH3), 20.3 (COCH3), 13.7 (CH3).
Methyl
3,4,6-tri-O-acetyl-2-[(4-(3-phenylpropyl)-1H-1,2,3-triazole-1-yl)]-2-deoxy-D-
glucopyranoside (5f): White solid (157.6 mg; 54% yield). 1H NMR (300 MHz, CDCl3) δH:
7.08 – 6.94 (6H, m, H-triazole, H-Ar.), 5.64 (1H, dd, J2,3 10.3 Hz, J3,4 9.0 Hz, H-3), 5.00 (1H,
t, J3,4 9.0 Hz, H-4), 4.79 (1H, d, J1,2 8.0 Hz, H-1), 4.28 – 4.18 (2H, m, H-2, H-6a), 4.07 (1H,
dd, J5,6b 2.2 Hz, J6a,6b 12.0 Hz, H-6b), 3.80 (1H, ddd, J5,4 9.8 Hz, J5,6a 4.3 Hz, J5,6b 2.2 Hz, H-
5), 3.32 (3H, s, OCH3), 2.66 (2H, t, J 7.4 Hz, CH2), 2.58 (2H, t, J 7.4 Hz, CH2), 2.05, 1.78
(6H, 2s, 2 CH3CO), 1.97 – 1.89 (5H, m, CH3CO; CH2). 13C NMR (75 MHz, CDCl3) δC: 170.7
(COCH3), 169.9 (COCH3), 169.1 (COCH3), 147.5 (C-triazole), 141.9 (C-Ar.), 128.6 (CH-
Ar.), 128.5 (CH-Ar.), 126.0 (CH-Ar.), 122.4 (CH-triazole), 101.7 (C-1), 72.4 (C-3), 72.0 (C-
5), 68.8 (C-4), 63.9 (C-2), 61.9 (C-6), 57.7 (OCH3), 35.4 (CH2), 31.0 (CH2), 25.1 (CH2), 20.9
(COCH3), 20.7 (COCH3), 20.4 (COCH3).
Methyl
3,4,6-tri-O-acetyl-2-[(4-(benzyloxymethyl)-1H-1,2,3-triazole-1-yl)]-2-deoxy-D-
1
glucopyranoside (5g): Yellow oil (203 mg; 79% yield). H NMR (300 MHz, CDCl3) δH:
7.55 (1H, s, H-triazole), 7.36 – 7.29 (5H, m, H-Ar.), 5.84 (1H, t, J3,4 9.6 Hz, H-3), 5.17 (1H, t,
J3,4 9.6 Hz, H-4), 4.94 (1H, d, J1,2 8.2 Hz, H-1), 4.67 (2H, s, CH2-triazole), 4.58 (2H, s, OBn),
4.38 (2H, dd, J5,6a 4.3 Hz, J6a,6b 12.3 Hz, H-2, H-6a), 4.20 (1H, dd, J5,6b 2.2 Hz, J6a,6b 12.3 Hz,
H-6b), 3.92 (1H, ddd, J5,4 10.0 Hz, J5,6a 4.3 Hz, J5,6b 2.2 Hz, H-5), 3.42 (3H, s, OCH3), 2.11,
2.03, 1.83 (9H, 3s, 3 CH3CO). 13C NMR (101 MHz, CDCl3) δC: 170.7 (COCH3), 169.8

ACCEPTED MANUSCRIPT
(COCH3), 169.2 (COCH3), 137.8 (C-Ar.), 128.6 (CH-Ar.), 128.0 (CH-Ar.), 128.0 (CH-Ar.),
124.2 (CH-triazole), 101.6 (C-1), 72.3 (C-3), 72.6 (OBn), 72.0 (C-5), 68.9 (C-4), 64.2 (C-2),
63.7 (CH2-triazole), 61.9 (C-6), 57.7 (OCH3), 20.8 (COCH3), 20.7 (COCH3), 20.4 (COCH3).
Methyl 3,4,6-tri-O-acetyl-2-[(4-((benzylamino)methyl)-1H-1,2,3-triazole-1-yl)]-2-deoxy-
β-D
-glucopyranoside (5h): Yellow oil (214 mg; 63% yield). 1H NMR (400 MHz, CDCl3) δH:
7.47 (1H, s, H-triazole), 7.33 – 7.28 (5H, m, H-Ar.), 5.83 (1H, dd, J3,2 10.7 Hz, J3,4 9.3 Hz, H-
3), 5.17 (1H, t, J3,4 9.3 Hz, H-4), 4.93 (1H, d, J1,2 8.2 Hz, H-1), 4.41 – 4.33 (2H, m, H-2, H-
6a), 4.19 (1H, dd, J5,6b 2.2 Hz, J6a,6b 12.3 Hz, H-6b), 3.94 – 3.89 (3H, m, H-5, CH2NH), 3.80
(2H, s, CH2-triazole), 3.42 (3H, s, OCH3), 2.11, 2.03, 1.84 (9H, 3s, 3 CH3CO). 13C NMR (101
MHz, CDCl3) δC: 170.7 (COCH3), 169.8 (COCH3), 169.2 (COCH3), 146.4 (C-triazole), 139.8
(C-Ar.), 128.6 (CH-Ar.), 128.4 (CH-Ar.), 127.2 (CH-Ar.), 123.4 (CH-triazole), 101.7 (C-1),
72.4 (C-3), 72.1 (C-5), 68.9 (C-4), 64.1 (C-2), 61.9 (C-6), 57.7 (OCH3), 53.4 (CH2NH), 44.1
(CH2-triazole), 20.8 (COCH3), 20.7 (COCH3), 20.4 (COCH3).
Methyl 3,4,6-tri-O-acetyl-2-[(4-((benzyl(methyl)amino)methyl)-1H-1,2,3-triazole-1-yl)]-
2-deoxy-D
-glucopyranoside (5i): Yellow oil (131.5 mg; 60% yield). 1H NMR (300 MHz,
CDCl3) δH: 7.51 (1H, s, H-triazole), 7.33 – 7.27 (5H, m, H-Ar.), 5.82 (1H, dd, J2,3 10.6 Hz,
J3,4 9.4 Hz, H-3), 5.18 (1H, t, J3,4 9.4 Hz, H-4), 4.98 (1H, d, J1,2 8.2 Hz, H-1), 4.42 – 4.35 (2H,
m, H-2, H-6a), 4.20 (1H, dd, J5,6b 2.2 Hz, J6a,6b 12.3 Hz, H-6b), 3.93 (1H, ddd, J5,4 10.1 Hz,
J5,6a 4.4 Hz, J5,6b 2.2 Hz, H-5), 3.73 (2H, s, CH2-triazole), 3.52 (2H, s, NCH2-benzyl), 3.43
(3H, s, OCH3), 2.22 (3H, s, NCH3), 2.12, 2.04, 1.79 (9H, 3s, 3 CH3CO). 13C NMR (75 MHz,
CDCl3) δC: 170.7 (COCH3), 169.8 (COCH3), 169.1 (COCH3), 129.1 (CH-Ar.), 128.4 (CH-
Ar.), 127.2 (CH-Ar.), 124.3 (CH-triazole), 101.7 (C-1), 72.4 (C-3), 72.0 (C-5), 68.7 (C-4),
64.0 (C-2), 61.9 (C-6), 61.3 (NCH2-benzyl), 57.7 (OCH3), 52.0 (CH2-triazole), 42.1 (NCH3),
20.8 (COCH3), 20.7 (COCH3), 20.3 (COCH3).
Methyl 3,4,6-tri-O-acetyl-2-[(4-((p-fluorobenzamido)methyl)-1H-1,2,3-triazole-1-yl)]-2-
1
deoxy-
D
-glucopyranoside (5j): White solid (121.2 mg; 71% yield). H NMR (400 MHz,
CDCl3) δH: 7.82 – 7.79 (2H, m, H-Ar.), 7.62 (1H, s, H-triazole), 7.12 – 7.08 (2H, m, H-Ar.),
7.01 (1H, t, JCH ,NH 5.6 Hz, NH), 5.76 (1H, dd, J2,3 10.6 Hz, J3,4 9.3 Hz, H-3), 5.16 (1H, t, J3,4
2
9.3 Hz, H-4), 4.99 (1H, d, J1,2 8.2 Hz, H-1), 4.72 (1H, dd, JAB = 15.2 Hz, JH,NH = 5.6 Hz,
CHa), 4.65 (1H, dd, JAB = 15.2 Hz, JH,NH = 5.6 Hz, CHb), 4.40 – 4.33 (2H, m, H-2, H-6a),
4.20 (1H, dd, J5,6b 2.3 Hz, J6a,6b 12.4 Hz, H-6b), 3.91 (1H, ddd, J5,4 10.1 Hz, J5,6a 4.5 Hz, J5,6b

ACCEPTED MANUSCRIPT
2.3 Hz, H-5), 3.43 (3H, s, OCH3), 2.11, 2.02, 1.81 (9H, 3s, 3 CH3CO). 13C NMR (101 MHz,
CDCl3) δC: 170.7 (COCH3), 169.7 (COCH3), 169.2 (COCH3), 166.4 (CONH), 144.1 (C-
triazole), 130.3 (C-Ar., d, JF,C(para) 3.2 Hz), 129.5 (2CH-Ar., d, JF,C(meta) 8.7 Hz), 124.1 (CH-
triazole), 115.8 (2CH-Ar., d, JF,C(ortho) 21.9 Hz), 101.5 (C-1), 72.5 (C-3), 72.1 (C-5), 68.8 (C-
4), 64.2 (C-2), 61.9 (C-6), 57.7 (OCH3), 35.4 (CH2), 20.8 (COCH3), 20.7 (COCH3), 20.3
(COCH3).
Methyl 3,4,6-tri-O-acetyl-2-[(4-((phenylamino)methyl)-1H-1,2,3-triazole-1-yl)]-2-deoxy-
1
β-D
-glucopyranoside (5k): Yellow oil (136.3 mg; 93% yield). H NMR (300 MHz, CDCl3)
δH: 7.42 (1H, s, H-triazole), 7.19 – 7.14 (2H, m, H-Ar.), 6.75 – 6.71 (1H, m, H-Ar.), 6.66 –
6.62 (2H, m, H-Ar.), 5.78 (1H, dd, J2,3 10.7 Hz, J3,4 9.3 Hz, H-3), 5.14 (1H, t, J3,4 9.3 Hz, H-
4), 4.96 (1H, d, J1,2 8.2 Hz, H-1), 4.46 (2H, s, CH2-triazole), 4.41 – 4.32 (2H, m, H-2, H-6a),
4.19 (1H, dd, J5,6b 2.2 Hz, J6a,6b 12.4 Hz, H-6b), 3.91 (1H, ddd, J5,4 10.1 Hz, J5,6a 4.5 Hz, J5,6b
2.2 Hz, H-5), 3.41 (3H, s, OCH3), 2.11, 2.03, 1.74 (9H, 3s, 3 CH3CO). 13C NMR (75 MHz,
CDCl3) δC: 170.7 (COCH3), 169.8 (COCH3), 169.2 (COCH3), 147.5 (C-Ar.), 145.9 (C-
triazole), 129.4 (CH-Ar.), 123.3 (CH-triazole), 118.2 (CH-Ar.), 113.3 (CH-Ar.), 101.6 (C-1),
72.3 (C-3), 72.0 (C-5), 68.7 (C-4), 64.0 (C-2), 61.8 (C-6), 57.7 (OCH3), 39.9 (CH2-triazole),
20.8 (COCH3), 20.7 (COCH3), 20.2 (COCH3).
Methyl 3,4,6-tri-O-acetyl-2-[(4-(phenoxymethyl)-1H-1,2,3-triazole-1-yl)]-2-deoxy-β-D-
1
glucopyranoside (5l): White solid (104.3 mg; 75% yield). H NMR (300 MHz, CDCl3) δH:
7.59 (1H, s, H-triazole), 7.32 – 7.26 (2H, m, H-Ar.), 6.99 – 6.95 (3H, m, H-Ar.), 5.82 (1H,
dd, J2,3 10.7 Hz, J3,4 9.3 Hz, H-3), 5.23 (2H, s, CH2-triazole), 5.16 (1H, t, J3,4 9.3 Hz, H-4),
4.97 (1H, d, J1,2 8.2 Hz, H-1), 4.42 – 4.34 (2H, m, H-2, H-6a), 4.20 (1H, dd, J5,6b 2.2 Hz, J6a,6b
12.4 Hz, H-6b), 3.92 (1H, ddd, J5,4 10.1 Hz, J5,6a 4.5 Hz, J5,6b 2.2 Hz, H-5), 3.43 (3H, s,
OCH3), 2.11, 2.03, 1.76 (9H, 3s, 3 CH3CO). 13C NMR (75 MHz, CDCl3) δC: 170.8 (COCH3),
169.9 (COCH3), 169.2 (COCH3), 158.1 (C-Ar.), 144.1 (C-triazole), 129.6 (CH-Ar.), 124.4
(CH-triazole), 121.4 (CH-Ar.), 114.9 (CH-Ar.), 101.6 (C-1), 72.3 (C-3), 72.0 (C-5), 68.8 (C-
4), 64.3 (C-2), 62.1 (CH2-triazole), 62.0 (C-6), 57.8 (OCH3), 20.8 (COCH3), 20.7 (COCH3),
20.3 (COCH3).
General procedure for deacetylation reactions: [28] Compounds 5a-l were dissolved in
MeOH and treated with sodium methoxide (1.0 M in MeOH) until pH 9–10 was achieved.

ACCEPTED MANUSCRIPT
The mixtures were stirred for 1 h at room temperature, neutralized with Dowex 50WX8-200
(H+), filtered and concentrated under reduced pressure affording products 6a-l.
Methyl 2-[(4-(phenethyl)-1H-1,2,3-triazole-1-yl)]-2-deoxy-β-D-glucopyranoside (6a):
Colorless oil (55.2 mg; 40% yield). [ꢀ]ꢂꢁꢂ.ꢃ -1.9 (c 1.29, H2O). 1H NMR (300 MHz, D2O) δH:
7.51 (1H, s, H-triazole), 7.21 – 7.00 (5H, m, H-Ar.), 4.75 (1H, d, J1,2 8.2 Hz, H-1), 4.12 (1H,
dd, J1,2 8.2 Hz, J2,3 10.5 Hz, H-2), 3.97 (1H, t, J3,4 8.5 Hz, H-3), 3.85 (1H, d, J6a,6b 12.3 Hz, H-
6b), 3.67 (1H, dd, J5,6a 5.4 Hz, J6a,6b 12.3 Hz, H-6a), 3.54 – 3.49 (1H, m, H-5), 3.43 (1H, t,
J3,4 8.5 Hz, H-4), 3.21 (3H, s, OCH3), 2.97 – 2.83 (4H, m, CH2CH2). 13C NMR (75 MHz,
D2O) δC: 147.3 (C-triazole), 140.9 (C-Ar.), 128.7 (CH-Ar.), 128.4 (CH-Ar.), 126.1 (CH-Ar.),
123.6 (CH-triazole), 101.1 (C-1), 76.0 (C-5), 73.3 (C-3), 69.8 (C-4), 66.0 (C-2), 60.4 (C-6),
57.4 (OCH3), 34.4 (CH2), 26.0 (CH2). HRMS-ESI: calcd for C17H23N3NaO5 [M + Na]+:
372.1530; found: 372.1532.
Methyl
2-[(4-(benzyl)-1H-1,2,3-triazole-1-yl)]-2-deoxy-β-
D
-glucopyranoside
(6b):
Colorless oil (51.9 mg; 62% yield). [ꢀ]ꢂꢁꢂ.ꢃ -1.2 (c 0.76, H2O). 1H NMR (300 MHz, D2O) δH:
7.86 (1H, s, H-triazole), 7.38 – 7.25 (5H, m, H-Ar.), 4.91 (1H, d, J1,2 8.3 Hz, H-1), 4.31 (1H,
t, J1,2 8.3 Hz, H-2), 4.12 (1H, t, J3,4 8.6 Hz, H-3), 4.07 (2H, s, CH2), 3.96 (1H, dd, J5,6b 1.3 Hz,
J6a,6b 12.3 Hz, H-6b), 3.79 (1H, dd, J5,6a 5.4 Hz, J6a,6b 12.3 Hz, H-6a), 3.67 – 3.61 (1H, m, H-
5), 3.56 (1H, t, J3,4 8.6 Hz, H-4), 3.34 (3H, s, OCH3). 13C NMR (75 MHz, D2O) δC: 147.6 (C-
triazole), 138.9 (C-Ar.), 128.8 (CH-Ar.), 128.5 (CH-Ar.), 126.7 (CH-Ar.), 123.7 (CH-
triazole), 101.0 (C-1), 76.0 (C-5), 73.4 (C-3), 69.7 (C-4), 66.1 (C-2), 60.4 (C-6), 57.2
(OCH3), 30.8 (CH2). HRMS-ESI: calcd for C16H22N3O5 [M + H]+: 336.1554; found:
336.1553.
Methyl 2-[(4-(phenyl)-1H-1,2,3-triazole-1-yl)]-2-deoxy-β-D-glucopyranoside (6c): White
solid (73 mg; 98% yield). [ꢀ]ꢁꢂꢂ.ꢃ +4.3 (c 0.41, H2O). 1H NMR (300 MHz, D2O) δH: 8.44 (1H,
s, H-triazole), 7.84 – 7.82 (2H, m, H-Ar.), 7.55 – 7.42 (3H, m, H-Ar.), 5.03 (1H, d, J1,2 8.2
Hz, H-1), 4.43 (1H, dd, J1,2 8.2 Hz, J2,3 10.2 Hz, H-2), 4.23 (1H, dd, J2,3 10.2 Hz, J3,4 8.8 Hz,
H-3), 4.00 (1H, dd, J5,6b 1.9 Hz, J6a,6b 12.2 Hz, H-6b), 3.82 (1H, dd, J5,6a 5.3 Hz, J6a,6b 12.2
Hz, H-6a), 3.70 (1H, ddd, J5,4 10.4 Hz, J5,6a 5.3 Hz, J5,6b 1.9 Hz, H-5), 3.61 (1H, t, J3,4 8.8 Hz,
13
H-4), 3.41 (3H, s, OCH3). C NMR (101 MHz, D2O) δC: 147.6 (C-triazole), 129.3 (C-Ar.),
129.2 (CH-Ar.), 129.0 (CH-Ar.), 125.8 (CH-Ar.), 122.5 (CH-triazole), 101.0 (C-1), 76.2 (C-

ACCEPTED MANUSCRIPT
5), 73.5 (C-3), 69.9 (C-4), 66.4 (C-2), 60.6 (C-6), 57.3 (OCH3). HRMS-ESI: calcd for
C15H20N3O5 [M + H]+: 322.1397; found: 322.1396.
Methyl 2-[(4-(p-tolyl)-1H-1,2,3-triazole-1-yl)]-2-deoxy-β-D-glucopyranoside (6d): White
solid (49.9 mg; 67% yield). [ꢀ]ꢁꢂꢂ.ꢃ +5.1 (c 0.55, CH3OH). 1H NMR (300 MHz, CD3OD) δH:
8.31 (1H, s, H-triazole), 7.71 (2H, d, J 7.9 Hz, H-Ar.), 7.26 (2H, d, J 7.9 Hz, H-Ar.), 4.24
(1H, dd, J1,2 8.0 Hz, J2,3 10.5 Hz, H-2), 4.15 (1H, dd, J2,3 10.5 Hz, J3,4 8.0 Hz, H-3), 3.97 (1H,
dd, J5,6b 1.9 Hz, J6a,6b 11.9 Hz, H-6b), 3.79 (1H, dd, J5,6a 5.1 Hz, J6a,6b 11.9 Hz, H-6a), 3.56 –
13
3.46 (2H, m, H-4, H-5), 3.42 (3H, s, OCH3), 2.37 (3H, s, ArCH3). C NMR (75 MHz,
CD3OD) δC: 148.3 (C-triazole), 139.3 (C-Ar.), 130.6 (CH-Ar.), 128.8 (CH-Ar.), 126.6 (CH-
Ar.), 123.1 (CH-triazole), 102.8 (C-1), 78.1 (C-5), 75.6 (C-3), 72.0 (C-4), 68.3 (C-2), 62.5
(C-6), 57.3 (OCH3), 21.2 (ArCH3). HRMS-ESI: calcd for C16H22N3O5 [M + H]+: 336.1554;
found: 336.1551.
Methyl
2-[(4-(propyl)-1H-1,2,3-triazole-1-yl)]-2-deoxy-β-
D
-glucopyranoside
(6e):
1
Colorless oil (65.5 mg; 94% yield). [ꢀ]ꢂꢁꢂ.ꢃ -3.5 (c 1.16, H2O). H NMR (300 MHz, D2O) δH:
7.85 (1H, s, H-triazole), 4.93 (1H, d, J1,2 8.3 Hz, H-1), 4.30 (1H, dd, J1,2 8.3 Hz, J2,3 10.5 Hz,
H-2), 4.12 (1H, dd, J2,3 10.5 Hz, J3,4 8.7 Hz, H-3), 3.96 (1H, dd, J5,6b 1.6 Hz, J6a,6b 12.3 Hz, H-
6b), 3.78 (1H, dd, J5,6a 5.4 Hz, J6a,6b 12.3 Hz, H-6a), 3.67 – 3.61 (1H, m, H-5), 3.55 (1H, t,
J3,4 8.7 Hz, H-4), 3.36 (3H, s, OCH3), 2.66 (2H, t, J 7.4 Hz, CH2), 1.69 – 1.57 (2H, m, CH2),
0.87 (3H, t, J 7.4 Hz, CH3). 13C NMR (75 MHz, D2O) δC: 148.6 (C-triazole), 123.2 (CH-
triazole), 101.0 (C-1), 76.0 (C-5), 73.4 (C-3), 69.7 (C-4), 66.0 (C-2), 60.4 (C-6), 57.2
(OCH3), 26.3 (CH2), 21.9 (CH2), 12.6 (CH3). HRMS-ESI: calcd for C12H22N3O5 [M + H]+:
288.1554; found: 288.1554.
Methyl
2-[(4-(3-phenylpropyl)-1H-1,2,3-triazole-1-yl)]-2-deoxy-β-D-glucopyranoside
(6f): Colorless oil (87.8 mg; 96% yield). [ꢀ]ꢂꢁꢄ.ꢅ +2.3 (c 1.33, CH3OH). 1H NMR (300 MHz,
D2O) δH: 7.82 (1H, s, H-triazole), 7.36 – 7.23 (5H, m, H-Ar.), 4.93 (1H, d, J1,2 8.3 Hz, H-1),
4.30 (1H, dd, J1,2 8.3 Hz, J2,3 10.5 Hz, H-2), 4.13 (1H, dd, J2,3 10.5 Hz, J3,4 8.6 Hz, H-3), 3.97
(1H, dd, J5,6b 1.9 Hz, J6a,6b 12.3 Hz, H-6b), 3.79 (1H, dd, J5,6a 5.5 Hz, J6a,6b 12.3 Hz, H-6a),
3.68 – 3.62 (1H, m, H-5), 3.56 (1H, t, J3,4 8.6 Hz, H-4), 3.37 (3H, s, OCH3), 2.70 (2H, t, J 7.5
Hz, CH2), 2.62 (2H, t, J 7.5 Hz, CH2), 1.96 (2H, quint, J 7.5 Hz, CH2). 13C NMR (75 MHz,
D2O) δC: 148.2 (C-triazole), 142.3 (C-Ar.), 128.5 (CH-Ar.), 125.9 (CH-Ar.), 123.4 (CH-
triazole), 101.1 (C-1), 76.0 (C-5), 73.4 (C-3), 69.8 (C-4), 66.0 (C-2), 60.4 (C-6), 57.2

ACCEPTED MANUSCRIPT
(OCH3), 34.0 (CH2), 30.0 (CH2), 23.7 (CH2). HRMS-ESI: calcd for C18H26N3O5 [M + H]+:
364.1867; found: 364.1865.
Methyl 2-[(4-(benzyloxymethyl)-1H-1,2,3-triazole-1-yl)]-2-deoxy-β-D-glucopyranoside
(6g): Yellow oil (80 mg; 75% yield). [ꢀ]ꢂꢁꢂ.ꢃ -2.7 (c 0.91, H2O). 1H NMR (300 MHz, D2O)
δH: 8.09 (1H, s, H-triazole), 7.43 – 7.34 (5H, m, H-Ar.), 4.96 (1H, d, J1,2 8.3 Hz, H-1), 4.71
(2H, s, CH2-triazole), 4.61 (2H, s, OBn), 4.36 (1H, dd, J1,2 8.3 Hz, J2,3 10.1 Hz, H-2), 4.17
(1H, t, J3,4 8.8 Hz, H-3), 3.97 (1H, dd, J5,6b 1.2 Hz, J6a,6b 12.4 Hz, H-6b), 3.79 (1H, dd, J5,6a
5.4 Hz, J6a,6b 12.4 Hz, H-6a), 3.69 – 3.63 (1H, m, H-5), 3.57 (1H, t, J3,4 8.8 Hz, H-4), 3.37
13
(3H, s, OCH3). C NMR (75 MHz, D2O) δC: 144.0 (C-triazole), 136.8 (C-Ar.), 128.7 (CH-
Ar.), 128.5 (CH-Ar.), 128.3 (CH-Ar.), 125.5 (CH-triazole), 101.0 (C-1), 76.0 (C-5), 73.3 (C-
3), 72.1 (OBn), 69.7 (C-4), 66.1 (C-2), 62.1 (CH2-triazole), 60.4 (C-6), 57.2 (OCH3). HRMS-
ESI: calcd for C17H23N3NaO6 [M + Na]+: 388.1479; found: 388.1480.
Methyl
2-[(4-((benzylamino)methyl)-1H-1,2,3-triazole-1-yl)]-2-deoxy-β-D-
glucopyranoside (6h): Yellow oil (35 mg; 59% yield). [ꢀ]ꢂꢁꢂ.ꢃ -1.9 (c 1.07, H2O). 1H NMR
(300 MHz, D2O) δH: 7.96 (1H, s, H-triazole), 7.41 – 7.29 (5H, m, H-Ar.), 4.96 (1H, d, J1,2 8.3
Hz, H-1), 4.34 (1H, dd, J1,2 8.3 Hz, J2,3 10.5 Hz, H-2), 4.15 (1H, dd, J2,3 10.5 Hz, J3,4 8.6 Hz,
H-3), 3.96 (1H, dd, J5,6b 2.0 Hz, J6a,6b 12.3 Hz, H-6b), 3.88 (2H, s, CH2-triazole), 3.81 – 3.75
(3H, m, H-6a, CH2NH), 3.68 – 3.62 (1H, m, H-5), 3.56 (1H, t, J3,4 8.6 Hz, H-4), 3.37 (3H, s,
13
OCH3). C NMR (75 MHz, D2O) δC: 145.2 (C-triazole), 138.2 (C-Ar.), 128.6 (CH-Ar.),
127.5 (CH-Ar.), 124.6 (CH-triazole), 101.0 (C-1), 76.0 (C-5), 73.3 (C-3), 69.7 (C-4), 66.1 (C-
2), 60.4 (C-6), 57.2 (OCH3), 51.4 (CH2NH), 41.8 (CH2-triazole). HRMS-ESI: calcd for
C17H25N4O5 [M + H]+: 365.1819; found: 365.1819
.
Methyl 2-[(4-((benzyl(methyl)amino)methyl)-1H-1,2,3-triazole-1-yl)]-2-deoxy-β-
D
-
glucopyranoside (6i): Yellow oil (66 mg; 98% yield). [ꢀ]ꢂꢁꢂ.ꢃ -1.5 (c 1.53, H2O). 1H NMR
(500 MHz, D2O) δH: 8.04 (1H, s, H-triazole), 7.44 – 7.34 (5H, m, H-Ar.), 4.99 (1H, d, J1,2 8.4
Hz, H-1), 4.39 (1H, dd, J1,2 8.4 Hz, J2,3 10.2 Hz, H-2), 4.20 (1H, dd, J2,3 10.2 Hz, J3,4 9.3 Hz,
H-3), 4.00 (1H, d, J6a,6b 12.4 Hz, H-6b), 3.82 (1H, dd, J5,6a 5.7 Hz, J6a,6b 12.4 Hz, H-6a), 3.78
(2H, s, NCH2-triazole), 3.70 – 3.67 (1H, m, H-5), 3.62 – 3.59 (3H, m, H-4; NCH2-benzil),
3.41 (3H, s, OCH3), 2.22 (3H, s NCH3). 13C NMR (126 MHz, D2O) δC: 130.1 (CH-Ar.), 128.7
(CH-Ar.), 127.9 (CH-Ar.), 125.8 (CH-triazole), 101.3 (C-1), 76.3 (C-5), 73.5 (C-3), 70.0 (C-

ACCEPTED MANUSCRIPT
4), 66.3 (C-2), 60.7 (C-6), 60.1 (NCH2-benzil), 57.5 (OCH3), 50.0 (NCH2-triazole), 40.8
(NCH3). HRMS-ESI: calcd for C18H27N4O5 [M + H]+: 379.1976; found: 379.1975.
Methyl
2-[(4-((p-fluorobenzamido)methyl)-1H-1,2,3-triazole-1-yl)]-2-deoxy-β-D-
1
glucopyranoside (6j): Colorless oil (52.5 mg; 98% yield). [ꢀ]ꢂꢁꢂ.ꢃ -2.3 (c 0.57, H2O). H
NMR (300 MHz, D2O) δH: 8.04 (1H, s, H-triazole), 7.80 – 7.76 (2H, m, H-Ar.), 7.23 – 7.17
(2H, m, H-Ar.), 4.96 (1H, d, J1,2 8.3 Hz, H-1), 4.65 (2H, s, CH2), 4.34 (1H, dd, J1,2 8.3 Hz, J2,3
10.0 Hz, H-2), 4.15 (1H, t, J3,4 8.7 Hz, H-3), 3.96 (1H, dd, J5,6b 1.1 Hz, J6a,6b 12.4 Hz, H-6b),
3.78 (1H, dd, J5,6a 5.1 Hz, J6a,6b 12.4 Hz, H-6a), 3.68 – 3.63 (1H, m, H-5), 3.56 (1H, t, J3,4 8.7
Hz, H-4), 3.36 (3H, s, OCH3). 13C NMR (75 MHz, D2O) δC: 169.8 (CONH), 164.8 (C-Ar., d,
JF,C(ipso) 250.3 Hz), 144.7 (C-triazole), 129.6 (2 CH-Ar., d, JF,C(meta) 9.4 Hz), 129.4 (C-Ar., d,
JF,C(para) 3.2 Hz), 124.1 (CH-triazole), 115.6 (2 CH-Ar., d, JF,C(ortho) 22.3 Hz), 100.9 (C-1),
76.0 (C-5), 73.3 (C-3), 69.7 (C-4), 66.1 (C-2), 60.4 (C-6), 57.2 (OCH3), 34.9 (CH2). HRMS-
ESI: calcd for C17H21FN4NaO6 [M + Na]+: 419.1337; found: 419.1338.
Methyl
2-[(4-((phenylamino)methyl)-1H-1,2,3-triazole-1-yl)]-2-deoxy-β-D-
glucopyranoside (6k): Yellow oil (81.4 mg; 81% yield). [ꢀ]ꢂꢁꢂ.ꢃ +1.4 (c 1.22, H2O). 1H NMR
(300 MHz, D2O) δH: 7.92 (1H, s, H-triazole), 7.25 – 7.20 (2H, m, H-Ar.), 6.85 – 6.80 (3H, m,
H-Ar.), 4.84 (1H, d, J1,2 8.3 Hz, H-1), 4.43 (2H, s, CH2-triazole), 4.28 (1H, dd, J1,2 8.3 Hz, J2,3
10.5 Hz, H-2), 4.12 (1H, dd, J2,3 10.5 Hz, J3,4 8.4 Hz, H-3), 3.95 (1H, dd, J5,6b 1.9 Hz, J6a,6b
12.4 Hz, H-6b), 3.78 (1H, dd, J5,6a 5.4 Hz, J6a,6b 12.4 Hz, H-6a), 3.64 – 3.59 (1H, m, H-5),
3.54 (1H, t, J3,4 8.4 Hz, H-4), 3.25 (3H, s, OCH3). 13C NMR (75 MHz, D2O) δC: 146.9 (C-
triazole), 146.0 (C-Ar.), 129.4 (CH-Ar.), 124.0 (CH-triazole), 119.4 (CH-Ar.), 115.1 (CH-
Ar.), 101.0 (C-1), 76.0 (C-5), 73.2 (C-3), 69.7 (C-4), 66.1 (C-2), 60.4 (C-6), 57.3 (OCH3),
38.8 (CH2-triazole). HRMS-ESI: calcd for C16H23N4O5 [M + H]+: 351.1663; found:
351.1663.
Methyl
2-[(4-(phenoxymethyl)-1H-1,2,3-triazole-1-yl)]-2-deoxy-β-D-glucopyranoside
(6l): White solid (86.3 mg; 98% yield). [ꢀ]ꢂꢁꢂ.ꢃ -0.13 (c 0.85, H2O). 1H NMR (300 MHz,
D2O) δH: 8.12 (1H, s, H-triazole), 7.36 – 7.31 (2H, m, H-Ar.), 7.06 – 7.01 (3H, m, H-Ar.),
5.23 (2H, s, CH2-triazole), 4.89 (1H, d, J1,2 8.3 Hz, H-1), 4.33 (1H, dd, J1,2 8.3 Hz, J2,3 10.6
Hz, H-2), 4.15 (1H, dd, J2,3 10.6 Hz, J3,4 8.5 Hz, H-3), 3.96 (1H, dd, J5,6b 2.0 Hz, J6a,6b 12.4
Hz, H-6b), 3.78 (1H, dd, J5,6a 5.4 Hz, J6a,6b 12.4 Hz, H-6a), 3.65 – 3.60 (1H, m, H-5), 3.55
(1H, t, J3,4 8.5 Hz, H-4), 3.29 (3H, s, OCH3). 13C NMR (75 MHz, D2O) δC: 168.2 (C-triazole),

ACCEPTED MANUSCRIPT
157.0 (C-Ar.), 143.2 (CH-triazole), 129.8 (CH-Ar.), 122.0 (CH-Ar.), 115.5 (CH-Ar.), 101.0
(C-1), 76.1 (C-5), 73.3 (C-3), 69.9 (C-4), 66.9 (C-2), 61.1 (CH2-triazole), 60.5 (C-6), 57.3
(OCH3). HRMS-ESI: calcd for C16H21N3NaO6 [M + Na]+: 374.1323; found: 374.1320.
4.3. Biological Assays
4.3.1. Cytotoxicity assay
To determine potential cytotoxic effects of the compounds, the colorimetric assay
with 4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was performed [40].
Cultured vascular smooth muscle cells (VSMCs) isolated from aorta of Wistar rats were
seeded (100 ꢀl/well) at seeding densities of 1x106 cells/ml into 96-well plates and allowed to
adhere for 24 h. VSMCs were incubated with 0.10, 0.25, 0.50, 0.75, 1.0, 10.0 µM of
compounds 6a-l for 6 h in a CO2 (5%) chamber, at 37 ºC. Following the incubation period, 20
µL of MTT (5 mg/mL) were added to the wells and the plates were incubated at 37 ºC for 4
h. Then, 50 µL isopropanol/HCl were added to the wells and the plates were kept at room
temperature for 1 h. The absorbance at 570 nm of the plates was read with the Epoch
Microplate Spectrophotometer (BioTek®, Winooski, VT, USA). Absorbance values were
blanked against dimethyl sulfoxide (DMSO) and the absorbance of cells exposed to medium
only (i.e. no compounds or vehicle added) was taken as 100 % cell viability (i.e. the control).
4.3.2. Western Blot analysis
Western Blot analysis was assessed in lysates of cultured VSMCs from aorta from
Wistar rats using antibodies anti-β-O-linked N-acetylglucosamine, anti-OGA and anti-
GAPDH (Sigma-Aldrich Inc., Germany), according to the previously reported method [32].
4.3.3. Enzymatic Activity Assays
O-GlcNAcase OGA) and β-N-Acetylhexosaminidase A and B activity were
performed as previously described [32]. Proteins were extracted (3 ꢀg), quantified using the
Bradford method [41] and eluted in citrate buffer following the procedure reported [32]. IC50
measurements of OGA activity against the compounds 6a-l were performed using the
fluorogenic 4-methylumbelliferyl N-acetyl-β-D-glucosaminide (4-MUNAG, 300 µg/ml,
Sigma-Aldrich Inc., Germany) substrate. After elution, the OGA substrate 4-MUNAG,

ACCEPTED MANUSCRIPT
compounds 6a-l or the OGA inhibitor Thiamet-G (positive control), at concentrations of 0.10,
0.25, 0.50, 0.75, 1.0, 10.0 µM were added. Proteins were kept in a chamber at 37 °C for 30
min and the reaction was stopped by addition of glycine buffer (0.1 M, pH 12). The
fluorescence of the released 4-MU was quantified after excitation of 362 nm and emission of
448 nm in a FlexStation 3 Multi-Mode Microplate Reader (Molecular Devices®, San Jose,
CA, USA). Percent of inhibition was calculated comparing the enzyme activity in the absence
of the inhibitors with the activity in the presence of the inhibitors. Assays were performed in
triplicate under identical conditions.
β-N-Acetylhexosaminidase A and B from prokaryote (EC 3.2.1.52, Megazyme,
Ireland, UK) assays toward compounds 6a, 6k and 6l were measured in duplicate under the
same conditions as described above
.
4.4.Molecular Docking
Ligands were docked in silico into the proposed active site of BtOGA (PDB code
2VVN) [33]. All docking runs were performed with the GOLD 5.2 software [42,43]. For the
calculations, the orientation of highest score (top-ranked) was selected for each of
compounds here investigated. Top-ranked orientations were selected by GOLD via an
empirical energy function (the ChemPLP) [44]. Based on this function, the software classifies
the orientations of the molecules by a decreasing ordering of affinity (the fitness) with the
binding site of the O-GlcNAcase. The simulations were performed inside a sphere of 10 Å
radius in a point centered among Asp242 and Asp243 catalytic residues and Cys278, Tyr282,
TRp337, Asn339 and Asp334 residues, with 100 orientations (docking runs) for each
molecule.
5. Acknowledgments
This research was supported by grants from São Paulo Research Foundation
(FAPESP Proc. 2016/04003-2), Coordenação de Aperfeiçoamento de Pessoal de Nível
Superior (CAPES) and Conselho Nacional de Desenvolvimento Científico e Tecnológico
(CNPq).
6. References

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Figures:
Figure 1. O-GlcNAcase (OGA) inhibitors previously reported.

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Figure 2. Cell viability assay. Cultured vascular smooth muscle cells were incubated for 6 h with
1.0 µM of the glucopyranoside triazole compounds 6a-l. The potential cytotoxicity of the
compounds was determined with the MTT assay. Results are expressed as mean ± SEM for n=5
in each experimental group. One-way ANOVA.

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(B)