Binding of triazole-linked galactosyl arylsulfonamides to galectin-3 affects Trypanosoma cruzi cell invasion

Article abstract of DOI:10.1016/j.bmc.2017.09.042

The synthesis of the O-3 triazole-linked galactosyl arylsulfonamides 1–7 as potential inhibitors of Trypanosoma cruzi cell invasion is described. These target compounds were synthesized by Cu(I)-catalysed azide-alkyne cycloaddition reaction (‘click chemis

Full text of DOI:10.1016/j.bmc.2017.09.042

Accepted Manuscript
Binding of triazole-linked galactosyl arylsulfonamides to galectin-3 affects
Trypanosoma cruzi cell invasion
Marcelo Fiori Marchiori, Thalita B. Riul, Leandro Oliveira Bortot, Peterson
Andrade, Getúlio G. Junqueira, Giuseppina Foca, Nunzianna Doti, Menotti
Ruvo, Marcelo Dias-Baruffi, Ivone Carvalho, Vanessa Leiria Campo
PII:
S0968-0896(17)31366-4
https://doi.org/10.1016/j.bmc.2017.09.042
BMC 14002
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
4 July 2017
22 September 2017
29 September 2017
Please cite this article as: Marchiori, M.F., Riul, T.B., Oliveira Bortot, L., Andrade, P., Junqueira, G.G., Foca, G.,
Doti, N., Ruvo, M., Dias-Baruffi, M., Carvalho, I., Campo, V.L., Binding of triazole-linked galactosyl
arylsulfonamides to galectin-3 affects Trypanosoma cruzi cell invasion, Bioorganic & Medicinal Chemistry (2017),
doi: https://doi.org/10.1016/j.bmc.2017.09.042
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

1
Binding of triazole-linked galactosyl arylsulfonamides to galectin-3 affects Trypanosoma cruzi
cell invasion
Marcelo Fiori Marchiori1, Thalita B. Riul1, Leandro Oliveira Bortot1, Peterson Andrade1, Getúlio G.
Junqueira1, Giuseppina Foca2, Nunzianna Doti2, Menotti Ruvo2, Marcelo Dias-Baruffi1, Ivone
Carvalho1, Vanessa Leiria Campo1
1. Faculty of Pharmaceutical Sciences of Ribeirão Preto - USP. Av. do Café S/N, CEP 14040-903,
Ribeirão Preto - SP, Brazil.
2. Istituto di Biostrutture e Bioimmagini, CNR, via Mezzocannone, 16, I-80134, Napoli.
vlcampo@fcfrp.usp.br
Abstract
The synthesis of the O-3 triazole-linked galactosyl arylsulfonamides 1-7 as potential inhibitors of
Trypanosoma cruzi cell invasion is described. These target compounds were synthesized by Cu(I)-
catalysed azide-alkyne cycloaddition reaction (‘click chemistry’) between different azide
arylsulfonamides and the alkyne-based sugar 3-O-propynyl-βGalOMe. Inhibition assays of T. cruzi
cell invasion with compounds 1-7 showed reduced values of infection index (~20) for compounds 3
and 5, bearing the corresponding 5-methylisoxazole and 2,4-dimethoxypyrimidine groups, which
also presented higher binding affinities to galectin-3 (EC50 17-18 µM) in Corning Epic label-free
assays. In agreement with experimental results, the assessment of the theoretical binding of
compounds 1-7 to galectin-3 by MM/PBSA method displayed higher affinities for compounds 3 (-
9.7 Kcal/ mol) and 5 (-11.1 Kcal/ mol). Overall, these achievements highlight compounds 3 and 5
as potential T. cruzi cell invasion blockers by means of a galectin-3 binding-related mechanism,
revealing galectin-3 as an important host target for design of novel anti-trypanosomal agents.
Keywords
Triazole, Galactosyl arylsulfonamides, “Click Chemistry”, Trypanosoma cruzi, Cell invasion,
Galectin-3, trans-Sialidase
1. Introduction
1 Corresponding author. Tel.: +55 16 33154285; fax: +55 16 33154879; e-mail: vlcampo@fcfrp.usp.br

2
Chagas disease is among the most prevalent neglected tropical diseases, affecting an
estimated 6 to 7 million people mostly with low socioeconomic status. Although this disease is
endemic in Latin America, it is spreading as a worldwide threat due to globalization and population
mobility to North America, Europe and others areas where it is non-endemic.1,2 Chagas disease is
mainly transmitted by triatomine bugs that carry the protozoan parasite Trypanosoma cruzi, and
make its eradication exceptionally difficult; furthermore, due to parasite's efficient ways of evading
the host immune system, vaccination remains as a great challenge.2,3 Thus, the use of anti-
trypanosomatid drugs is the unique choice for treatment and still relies only on benznidazole and
nifurtimox, drugs with poor efficacy in chronic infection phase and recognized toxicity.3 In this
regard, development of multi-target drug candidates able to block parasite invasion in the early
stages of infection may represent an effective strategy against this neglected disease.
T. cruzi adhesion and invasion into host cells are regulated by complex interactions among
parasite cell surface components, such as trans-sialidase (TcTS),4 mucins5 and cruzipain,6 as well as
galectins-3 and -1,7,8 and Toll-like receptors present in host cells.9,3 T. cruzi trans-sialidase is
anchored to the parasite membrane by glycosylphosphatidylinositol (GPI) and has a fundamental
role on T. cruzi invasion since it catalyses the transfer of sialic acid from host glycoconjugates to β-
galactopyranosyl acceptor groups on T. cruzi mucins.4,10-12 This transglycosylation reaction enables
the parasite to be recognized by host cells and protects it against complement-independent lyses
induced by human anti-α-galactosyl antibodies due to the generated negatively charged sialylated
barrier.13,14 In addition, TcTS may induce the early escape of T. cruzi from lysosome-enriched
parasitophorous vacuole (PV) formed in the host cell by removing sialic acid from lysosome-
associated membrane glycoproteins (LAMP), followed by disruption of PV membranes with the aid
of a parasite trypsin sensitive enzyme (TcTox).15 In turn, T. cruzi mucins (TcMUC) are also GPI
anchored plasma membrane glycoproteins (about 60% carbohydrate by weight) highly expressed on
T. cruzi surface, with about 2 x 106 copies per parasite. TcMUC play a significant role in the

3
parasite recognition process and invasion of host cells based on its acceptor substrate preferences
for sialic acid, transferred by TcTS.16,17
On the other hand, galectin-3 is a carbohydrate-binding lectin found in different host cell
compartments that is also importantly involved in T. cruzi infection. It contains a conserved
carbohydrate recognition domain (CRD) with affinity for β-galactosides containing glycoconjugates
and acts as one of the receptors that mediates the adhesion of the parasite to the host cells, besides
being involved in cell invasion and intracellular trafficking of the parasite.3,7,18 Galectin-3 is able to
interact effectively with both T. cruzi surface mucins and laminin present in the extracellular matrix,
establishing thus a bridge between these two components, which increases the recruitment of
trypomastigotes to the extracellular matrix and their adhesion to cells.18 Galectin-3 can also act as
marker of the cell biology events in T. cruzi infection, since it is able to interact with lysosome-
associated membrane glycoproteins (LAMP) of macrophage cell phagosomes, accumulating around
T. cruzi parasites that lysed the parasitophorous vacuole during the host cell infection.19
Novel compounds from distinct chemical classes have been recently reported as
antitrypanosomal agents.20-28 For instance, potent trypanocidal activities have been described for
tetrahydroquinoline-sulfonamide derivatives against epimastigote forms (Y strain) (I,25 IC50 11.4
µM and II,26 IC50 31.7 µM) (Fig. 1A), as well as strong inhibitory activity towards TcTS by
chalcone-derived sulfonamides (III,27 IC50 0.6 µM) (Fig. 1B). In addition, a series of seven
galactosyl-triazolo-benzenesulfonamides comprising β-galactosyl unit anomeric-linked to distinct
arylsulfonamides via triazole ring was reported by Carvalho et al.,28 being verified significant
trypanocidal activity against trypomastigote forms (Tulahuen strain) for the 2,4-
dimethoxypyrimidine-derivative IV (IC50 44.0 µM), and strong inhibition against TcTS for the
pyridine-derivative V (81% at 1 mM) (Fig. 1C). Differently from this series containing anomeric
triazole-substituted galactosyl sugar, we had described the synthesis of 1,2,3-triazole amino acids-
derived-3-O-galactosides as galectin-3 inhibitors, based on the known potential of O-3 triazole-
galactose analogs to interact with galectin-3 CRD. High binding affinities for galectin-3 were

4
verified for Phe (VI)/ Lys (VII)-derived-3-O-galactosides (Fig. 1D), with corresponding KD values
of 7.8 µM and 4.6 µM.29
Figure 1. Representative compounds with significant trypanocidal effect (I, II and IV), and
inhibitory activities against TcTS (III and V) and galectin-3 (VI and VII).
All these findings led us to postulate that novel hybrid glycoconjugates represented by 1,2,3-
triazole arylsulfonamides-derived-3-O-galactosides, such as 1-7 (Fig. 2), may hamper T. cruzi cell
invasion by possible dual inhibition of TcTS and galectin-3. Therefore, here we describe the
synthesis of O-3 triazole-linked galactosyl arylsulfonamides 1-7, via microwave-assisted Cu(I) 1,3-
dipolar azide-alkyne cycloaddition (CuAAC), along with their evaluation on T. cruzi cell invasion
process and inhibition of both TcTS and galectin-3.

5
Figure 2. Chemical structures of the target triazole-linked galactosyl arylsulfonamides 1-7.
2. RESULTS AND DISCUSSION
2.1. Synthesis
The syntheses of the O-3 triazole-linked galactosyl arylsulfonamides 1-7 by CuAAC required
the previous preparation of the azide-arylsulfonamides 8-1428 and the alkyne-based sugar 3-O-
propynyl-βGalOMe 1529 (Scheme 1) as their direct precursors.
Starting from the commercially available arylsulfonamides, named sulfanilamide 16,
sulfacetamide 17, sulfamethoxazole 18, sulfamethazine 19, sulfadimethoxine 20, sulfapyridine 21
and sulfamerazine 22, the preparation of their corresponding azide arylsulfonamides 9-15 was
firstly attempted by the treatment of 16-22 with tert-butyl nitrite and sodium azide under microwave
irradiation.28 However, the desired products 8-14 were obtained in very low yields (10-30%),
possibly due to poor solubility of most precursors in the tested solvents (DMF, acetonitrile, dioxane
and THF).31 To circumvent this limiting factor, we applied an alternative method based on the
previous protonation of free 4-aminoaryl groups of 16-22, turning them water-soluble and thus
allowing the use of common NaNO2 reagent for diazotation reaction (Scheme 1A).32 Therefore,
treatment of 16-22 with aqueous sulfuric acid solution 17% (v/v), followed by dropwise addition of
NaNO2 solution for few minutes, led to diazo intermediates, which were subsequently treated with
solid urea and NaN3 (Scheme 1A). The ongoing formation of the azide derivatives 8-14 was
indicated by their decreasing solubility until complete precipitation in the reaction mixture. After
filtration, the products 8-14 were obtained in yields varying from 38% to 91%, being their

6
1
structures confirmed by H NMR and IR [2100 cm-1 (N3)].28 Then, the alkyne-based sugar 3-O-
propynyl-βGalOMe 15 was synthesized from commercial βGalOMe, under our previously
1
described method applied to αGalOMe, giving 15 in 70% yield.30 The H NMR of 15 showed a
characteristic doublet of H-1 at δ 4.15 (J 7.8 Hz), confirming its β configuration.
Once the azide-arylsulfonamides 8-14 and alkyne-functionalyzed 15 precursors were
obtained, the syntheses of O-3 triazole-linked galactosyl arylsulfonamides 1-7 were carried out by
Cu(I)-assisted 1,3-dipolar azide-alkyne cycloaddition reactions (CuAAC), in a microwave reactor
utilizing the catalytic system CuSO4/sodium ascorbate and DMF as solvent.29,30,33 Thus, the
peracetylated triazole-derivatives 23-29 were obtained after 30 min. of microwave irradiation at
100°C (150W), with regioselective formation of 1,4-disubstituted triazoles (Scheme 1B), in
satisfactory yields varying from 60 to 70% after purification by column chromatography, except for
compound 27 (47%), possibly due to the influence of the bulkier 4,6-dimethoxypyrimidine group
on the CuAAC reaction. The structures of compounds 23-29 were confirmed by 1H NMR analysis,
which showed a characteristic singlet of CH-triazole around δ 8.0, signals of sugar at δ 4.9 (H-1), δ
3.3 (OCH3) and δ 2.11-2.06 (OCOCH3), as well as CH aromatic signals of arylsulfonamides at δ
8.4-7.8. Finally, compounds 23-29 were submitted to deacetylation reactions using 1 M NaOMe in
MeOH,29 affording the final triazole-linked galactosyl arylsulfonamides 1-7 (63-100%) after
purification by column chromatography. (Scheme 1B).

7
Scheme 1. (A) Syntheses of azide-arylsulfonamides 8-14 precursors from the commercial
arylsulfonamides 16-22. Reagents and conditions: i. H2SO4/H2O, 0°C; ii. NaNO2/H2O, rt.; iii.
(NH2)2CO, NaN3, H2O, rt. (B) Syntheses of 1,2,3-triazole-linked galactosyl arylsulfonamides 1-7.
Reagents and conditions: i. Na ascorbate, CuSO4, DMF, microwave heating (100°C, 15 min); ii.
NaOMe, MeOH, rt.
2.2. Biological assays
2.2.1. Inhibition of T. cruzi cell invasion
The capacity of compounds 1-7 to block invasion of fibroblasts cells (LLCMK2- monkey
kidney epithelial cells) by T. cruzi trypomastigotes (Y strain) was evaluated by treatment of pre-
formed adhered cells in circular coverslips with compounds 1-7 (250 µM), followed by addition of
trypomastigotes (1 x 105) after 30 min of contacting of the compounds with cells. After the
incubation of the cultures for 18 h, the coverslips were fixed and stained for measuring the level of
cell infection.34,35

8
Firstly, it was observed a reduction in the percentage of infected fibroblasts treated with
compounds 3 and 5, bearing the corresponding 5-methylisoxazole and 2,4-dimethoxypyrimidine
groups, with decreased values of 41% and 38%, respectively, if compared to 63% observed for non-
treated cells, while the other tested compounds varied from 47% to 54% (Fig. 3A). In addition,
compounds 3 and 5 also showed a significant reduction in the mean number of amastigotes within
each cell, presenting 0.49 parasite/cell versus 1.0 for non-treated cells and a range of 0.81 to 0.61
for the remaining compounds (Fig. 3B). Lastly, the infection index values, obtained by multiplying
the percentage of infected cells by the mean number of amastigotes per cell, were also determined
for compounds 1-7. It was verified a reduced infection index of about 20 for both compounds 3 and
5 versus 68 for the control without treatment. The infection index for the other tested compounds
ranged from 44 to 28 (Fig. 3C).
150
100
50
Figure 3. Inhibition of T. cruzi cell invasion by compounds 1-7. (A) Percentage of infected
fibroblasts; (B) Mean number of parasites (amastigotes) per cell; (C) Infection index assessed by
optical microscopy after 18-hours treatment. * represents p<005 of treated cells when compared to
control (only medium) of a triplicate representative experiment; Cl-control.
0
Subsequently, we also investigated the cytotoxicity of compounds 1-7 in LLCMK2 fibroblasts
as well as their direct trypanocidal effect, using the corresponding colorimetric MTT and resazurin-
based methods as indicators of cell metabolic activity.36 At the tested concentrations, none of the
compounds were toxic for fibroblasts (67.5-500 µM) or T. cruzi tripomastigote forms (31.25-250
150
100
50
0

9
µM), as shown in Fig. 4. Thus, since no trypanocidal effect was observed for compounds 1-7, we
may say that it has no correlation with the observed blocking of T. cruzi cell invasion.
Therefore, the satisfactory capacities of the tested compounds, remarkably 3 and 5, to block T.
cruzi invasion towards fibroblasts cells, prompted us to investigate their inhibitory activities against
T. cruzi trans-sialidase (TcTS) and galectin-3 found in host cells, since they are essential targets
involved in T. cruzi cell invasion.
Figure 4. Evaluation of citotoxicity and trypanocidal effect of compounds 1-7. (A) Citotoxicity of
compounds 1-7 in LLCMK2 fibroblasts assessed by MTT; (B) Trypanocidal effect of compounds
1-7 by rezasurin method, both after 48-hours treatment. BZD, benznidazole as a reference
trypanocidal drug.

10
2.2.2. Inhibition of T. cruzi trans-sialidase (TcTS)
The inhibitory activities of compounds 1-7 against T. cruzi trans-sialidase were evaluated by
the continuous fluorimetric method, which is based on TcTS-catalyzed hydrolysis of 2’-(4-
methylumbelliferyl)-α-D-N-acetyl-neuraminic acid (MuNANA), with release of the
methylumbelliferone (MU) fluorophore.37 Compounds 1-7 were tested at 1.0 mM concentration in
the presence of MuNANA (0.1 mM), and in parallel to the measured activities of pyridoxal
phosphate (PLP), used as positive control at the same concentration.38
According to the Fig. 5, highest inhibition of TcTS was verified for compound 2 (88%), the
N-acetamide derivative, whereas almost no inhibitory activity was detected for the other
compounds (4.5-16%). Since these results did not correlate with the low parasite infection index
values verified for compounds 3 and 5, we may say that TcTS inhibition was not a possible
mechanism by which these compounds blocked T. cruzi cell invasion.
Figure 5. TcTS inhibition by compounds 1-7 performed by fluorimetric assay; PLP-pyridoxal
phosphate, used as positive control.
2.2.3. Galectin-3 binding assays
Compounds 1-7 were applied to human recombinant galectin-3 (hr galectin-3)39 binding
assays using the Corning Epic label-free technology, which measures changes in the refraction
index upon a binding event.40 Given the partial insolubility of compounds 1-7, they were firstly
dissolved in pure DMSO and then diluted to the final working concentrations with a 1% or 2%

11
residual solvent. Compounds 1-7 tested in the presence of only 1% DMSO did not provide binding
curves of sufficient quality for the determination of binding values, which led us to test them in the
presence of 2% DMSO. In this case, signals obtained with compounds 1 through 5 were stronger
and showed the expected dose-response trend (Fig. 6). By fitting of data with a non-linear
algorithm, we determined the EC50 binding values for compounds 1-5 (Table 1), which ranged from
17 µM, for the highest affinities for compounds 3 and 5, up to 200 µM for the remaining
compounds. Regarding compounds 6 and 7, upon fitting, the obtained data did not converge to a
meaningful value.
Figure 6. Dose-response binding curves of compounds 1-5 to hr galectin-3. Compounds 1-5
dissolved in PBS 1X, DMSO 2% at pH 7.4 were tested at concentrations ranging between 3 µM and
400 µM, on µ-plate surfaces coated with galectin-3. Results were analyzed using the EnSpire label-
free user interface software. Graphs were generated using GraphPad Prism V-5.01 (GraphPad
Software, La Jolla, CA). Results are means of triplicates.
Table 1. EC50 values of compounds 1-5 towards hr galectin-3.
Compound
EC50 (µM)
40.7
205
1
2
3
4
5
17.2
67.7
18.3
The higher binding affinities to galectin-3 observed for compounds 3 (17.2 µM) and 5 (18.3
µM) were in agreement with their increased capacities to block T. cruzi cell invasion. Thus, we

12
believe that galectin-3 inhibition by compounds 3 and 5 may have blocked the host cell infection
process, contributing to its destruction still in the vacuole, considering the essential role of galectin-
3 in the escape of T. cruzi from the parasitophorous vacuole to the cell cytoplasm, which is an
important phenomenon for the permanence and multiplication of the parasite.19 To corroborate this
hypothesis, we performed a Western Blotting (WB) assay to investigate the galectin-3 expression
by LLCMK2 fibroblasts. In fact, galectin-3 was detected in LLCMK2 cell extracts, being verified a
band with the same molecular weight of hr galectin-3 (data not shown).
2.2.3.1. Molecular modeling studies with hr galectin-3
As label-free assays showed effective binding to hr galectin-3 for most tested compounds, we
found valuable to assess their theoretical binding to hr galectin-3 by molecular modeling studies.
Hence, relative affinities of compounds 1-7 towards hr galectin-3 were estimated by the
MM/PBSA method in combination with molecular dynamics simulations,41,42 which showed
compounds 3 and 5 as the ones with higher affinities for hr galectin-3 followed by compounds 1, 2,
4, 6 and 7 (Table 2). These data agreed with experimental results for the EC50 measured with the
immobilized protein. Actually, for the condition with 1% DMSO, the compounds that showed
relative affinities higher than -5 kcal/mol did not converge in the experiments, as a consequence of
their low affinities towards the protein. On the other hand, the linear correlation between the data
obtained with 2% DMSO and the MM/PBSA method was R2=0.31 for all compounds with
converged EC50 values and R2=0.96, if we consider compound 2 as an outlier (Fig. S15).

13
Table 2. Relative affinities of compounds 1-7 to hr galectin-3 as calculated using the MM/PBSA
method.
Relative affinity
[kcal/mol]
Compound
-6.5 0.8
1
2
3
4
5
6
7
-5
1
-9.7 0.9
-3.3 0.8
-11.1 0.9
0
1
0 1
From the structural point of view, the galactose portion of compounds 1-7 kept the same
interactions at the conserved carbohydrate binding site of hr galectin-3 that LacNAc displays in the
crystallographic structure.29 The main interactions between compounds 1-7 and residues outside the
carbohydrate binding site are stacking with residues Pro-117 and Arg-144 and hydrogen bonding
with Ser-237 (Fig. 7). However, residue-wise decomposition of the MM/PBSA energies showed
that Pro-117 is the only residue outside the carbohydrate binding site that contributes favorably to
the interaction. The interactions with Arg-144 and Ser-237 are unfavorable to the binding because
of the high energetic cost associated with breaking their interaction with water from the solvent.
Figure 7. Representative snapshots from the molecular dynamics trajectories showing the main
interactions between hr galectin-3 and representative compounds 3 (A) and 5 (B). All hydrogen,
nitrogen, oxygen and sulfur atoms are displayed in white, blue, red and yellow, respectively.

14
Carbon atoms of the ligand are displayed in green and from residues of the carbohydrate binding
site are displayed in cyan. Pro-117, Arg-144 and Ser-237 are displayed with gray carbons.
Additionally, stacking interactions are highlighted by surface representation of the ligand, Pro-117
and Arg-144.
3. Conclusions
In summary, we have synthesized the O-3 triazole-linked galactosyl arylsulfonamides 1-7
from different azide arylsulfonamides and alkyne-functionalized galactose as precursors. In this
regard, the use of Cu(I)-catalyzed azide-alkyne cycloaddition reaction proved to be effective for
generating modified O-3 1,2,3-triazole derivatives of galactose as potential anti-trypanosomal
agents.
The inhibition assays of T. cruzi cell invasion showed compounds 3 and 5 as the most potent
in blocking parasite invasion, with reduced values of infection index (~20). Subsequently,
fluorimetric inhibitory assays of compounds 1-7 against T. cruzi trans-sialidase (TcTS) showed
highest TcTS inhibition for compound 2 (88%), whilst evaluation of their binding to hr galectin-3
using the Corning Epic label-free technology displayed higher binding affinities for compounds 3
(EC50 17.2 µM) and 5 (EC50 18.3 µM). In agreement with these later experimental results, the
assessment of the theoretical binding of compounds 1-7 to hr galectin-3 by MM/PBSA method also
showed higher affinities for compounds 3 (-9.7 Kcal/ mol) and 5 (-11.1 Kcal/ mol), which, in
addition, established effective interactions with galectin-3 active site. Overall, compounds 3 and 5
that showed higher capacity of inhibiting fibroblast cells invasion by T. cruzi are the ones with
higher experimental and theoretical binding affinities for hr galectin-3. Thus, these results highlight
compounds 3 and 5 as potential T. cruzi cell invasion blockers by means of a galectin-3 binding-
related mechanism, revealing galectin-3 as an important host target for design of novel anti-
trypanosomal agents.

15
4. Experimental
4.1. General
All chemicals were purchased as reagent grade and used without further purification.
Reactions were monitored by thin layer chromatography (TLC) on 0.25 nm precoated silica gel
plates (Whatman, AL SIL G/UV, aluminium backing) with the indicated eluents. Compounds were
visualized under UV light (254 nm) and/or dipping in ethanol-sulfuric acid (95:5, v/v), followed by
heating the plate for a few minutes. Column chromatography was performed on silica gel 60
(Fluorochem, 35-70 mesh). Nuclear magnetic resonance spectra were recorded on Bruker Advance
DRX 300 (300 MHz), DPX 400 (400 MHz) or DPX 500 (500 MHz) spectrometers. Chemical shifts
(δ) are given in parts per million downfield from tetramethylsilane. Assignments were made with
the aid of HMQC and COSY experiments. Accurate mass electrospray ionization mass spectra
(ESI-HRMS) were obtained using positive or negative ionization modes on a Bruker Daltonics
MicroOTOF II ESI-TOF mass spectrometer.
4.2. Synthesis
4.2.1. Synthesis of azide-benzenesulfonamides 8-14
Each 4-aminobenzenesulfonamide (3.0 mmol) was added in a solution of concentrated
sulfuric acid (0.5 mL) and H2O (3.0 mL) and then cooled to 0°C in an ice bath. A solution of
NaNO2 (3.0 mmol) in water (2.1 mL) was added dropwise to the reaction and left stirring for 10
minutes. After a color change to a yellowish tone and appearance of foam in the medium, solid urea
(150 mg) was added followed by NaN3 solution (9.0 mmol, 1.5 eq) in H2O (3.2 mL) dropwise.
Finally, after stirring for few minutes each reaction was filtered through Buchner funnel and washed
subsequently with 5% NaHCO3 and H2O. Each obtained product was dried under reduced pressure
and analysed by TLC, being verified at increased retention factors (Rf) if compared to the
corresponding starting materials. Compounds 8-14 were obtained in corresponding yields of 38%

16
(8), 45% (9), 91% (10), 52% (11), 42,5% (12), 40% (13) and 76% (14). 1H NMR and IR [2100 cm_1
(N3)] (2100 cm-1) analysis of 8-14 were in accordance with the literature.28
4.2.2. Synthesis of 1,2,3-triazole-linked galactosyl arylsulfonamides 1-7
4.2.2.1. General procedure for CuAAC reactions29,30
A solution of methyl-2,4,6-tri-O-acetyl-3-prop-2-ynyl-β-D-galactopyranose 15 (1 equiv), 4-
azidobenzenesulfonamides 8-14) (1 equiv), sodium ascorbate (0.5 equiv) and CuSO4 (0.1 equiv) in
DMF (0.5 mL) was placed into a microwave tube. Then, the tube was sealed and submitted to
microwave irradiation for 30 min (100 ºC, 150 W). The reaction was followed by TLC (EtOAc) and
after completion the reaction mixture was concentrated under reduced pressure and purified by
column chromatography (EtOAc/Hexane 7:3 v/v) to afford the desired 1,2,3-triazole-linked
galactosyl arylsulfonamides 23-29.
4.2.2.2. Methyl 2,4,6-tri-O-acetyl-3-O-methyl[(4-benzenesulfonamide)-1H-1,2,3-triazol-4-yl]-β-
D-galactopyranoside 23
Following procedure described in Section 4.2.2.1 the reaction of 4-azidobenzenesulfonamide
8 (30.0 mg, 0.15 mmol), methyl-2,4,6-tri-O-acetyl-3-prop-2-ynyl-β-D-galactopyranose 15 (50.0 mg,
0.15 mmol), sodium ascorbate (12.0 mg, 0.075 mmol) and CuSO4 (0.015 mmol, 0.1 equiv) in DMF
(1.0 mL) gave the product 23 as a pale yellow oil (57.8 mg, 0.10 mmol, 69%) after purification by
column chromatography (EtOAc/Hexane 7:3 v/v). 1H NMR (300 MHz, CDCl3): δ 8.06 (s, 1H, CH-
triazole), 7.99 (d, 2H, Jortho 8.4 Hz, Ar-H), 7.81 (d, 2H, Jortho 8.4 Hz, Ar-H), 5.72 (s, 2H, NH2), 5.52
(d, 1H, J 2.7 Hz, H-4), 5.13 (dd, 1H, J1,2 7.8 Hz, J2,3 9.7 Hz, H-2), 4.92 (d, 1H, Jgem 11.9 Hz, O-CH2
triazole), 4.78 (d, 1H, Jgem 11.9 Hz, O-CH2 triazole), 4.45 (d, 1H, J1,2 7.8 Hz, H-1), 4.26 (d, 2H, J5,6
6.8 Hz, H-6, H-6'), 3.98 (t, 1H, J5,6 6.8 Hz, H-5), 3.88 (dd, 1H, J3,4 3.1 Hz, J2,3 9.7 Hz, H-3), 3.60 (s,
3H, OCH3), 2.21-2.17 (s, 9H, 3x CH3CO).13C NMR (75 MHz, CDCl3): δ 170.8-169.7 (CH3CO),
142.6 (C-triazole), 139.5 (Ar, C-SO2 and C-N-triazole), 128.3 (Ar, o-CSO2), 121.3 (CH-triazole),

17
120.4 (Ar, m-CSO2), 102.0 (C-1), 77.9 (C-3), 70.8 (C-2), 70.6 (C-5), 66.6 (C-4), 63.6 (OCH2), 61.9
(C-6), 56.8 (OCH3), 21.0-20.7 (CH3CO). ESI-HRMS: calcd. for C16H22N4O8S [M +H]+: 557.1548,
found: 557.1537.
4.2.2.3.
Methyl
2,4,6-tri-O-acetyl-3-O-methyl-N-{[(phenyl)sulfonyl]acetamide]-1H-1,2,3-
triazol-4-yl}-β-D-galactopyranoside 24
Following procedure described
in Section 4.2.2.1, the
reaction of
N-[(4-
azidophenyl)sulphonyl]acetamide 9 (33.5 mg, 0.14 mmol), methyl-2,4,6-tri-O-acetyl-3-prop-2-
ynyl-β-D-galactopyranose 15 (50 mg, 0.14 mmol), sodium ascorbate (13.8 mg, 0.07 mmol) and
CuSO4 (0.014 mmol, 0.1 equiv) in DMF (1.0 mL) gave the product 24 as a pale yellow oil (54.0
mg, 0.09 mmol, 64%) after purification by column chromatography (EtOAc/Hexane 1:1 v/v).1H
NMR (300 MHz, CDCl3): δ 7.44 (d, 2H, Jortho 8.5 Hz, Ar-H), 7.34 (s, 1H, CH-triazole), 7.17 (d, 2H,
Jortho 8.5 Hz, Ar-H), 4.77 (d, 1H, J 2.7 Hz, H-4), 4.41 (dd, 1H, J1,2 7.8 Hz, J2,3 9.6 Hz, H-2), 4.18 (d,
1H, Jgem 12.3 Hz, O-CH2 triazole), 4.07 (d, 1H, Jgem 12.3 Hz, O-CH2 triazole), 3.70 (d, 1H, J1,2 7.8
Hz, H-1), 3.54-3.51 (m, 2H, H-6, H-6'), 3.22 (t, 1H, J5,6 6.5 Hz, H-5), 3.12 (dd, 1H, J3,4 3.3 Hz, J2,3
9.7 Hz, H-3), 2.87 (s, 3H, OCH3), 1.57 (s, 3H, CH3CO-sulfonamide), 1.49-1.47 (s, 9H, 3x CH3CO).
13C NMR (75 MHz, CDCl3): δ 170.6-169.7 (CH3CO, CO-sulfonamide), 146.2 (C-triazole), 142.3
(Ar, C-SO2), 139.7 (Ar, C-N-triazole), 128.4 (Ar, o-CSO2), 121.0 (CH-triazole), 120.5 (Ar, m-
CSO2), 101,9 (C-1), 78.0 (C-3), 70.8 (C-2), 70.6 (C-5), 66.6 (C-4), 63.8 (OCH2), 61.8 (C-6), 56.8
(OCH3), 20.9-20.7 (CH3CO, CH3-sulfonamide). ESI-HRMS: calcd. for C18H24N4O9S [M+H]+:
599.1654, found: 599.1677.
4.2.2.4. Methyl 2,4,6-tri-O-acetyl-3-O-methyl-N-{[(5-methylisoxazol-3-yl)benzesulfonamide]-
1H-1,2,3-triazol-4-yl}-β-D-galactopyranoside 25
Following procedure described in Section 4.2.2.1, the reaction of 4-azido-N-(5-
methylisoxazol-3-yl) benzenesulfonamide 10 (25.2 mg, 0.09 mmol), methyl-2,4,6-tri-O-acetyl-3-

18
prop-2-ynyl-β-D-galactopyranose 15 (34.0 mg, 0.09 mmol), sodium ascorbate (9.0 mg, 0.045
mmol) and CuSO4 (0.009 mmol, 0.1 equiv) in DMF (0,8 mL) gave the product 25 as a pale yellow
oil (38.1 mg, 0.06 mmol, 66%) after purification by column chromatography (EtOAc/Hexane 6:4
1
v/v). H NMR (300 MHz, CDCl3): δ 8.03 (s, 1H, CH-triazole), 7.99 (d, 2H, Jortho 8.4 Hz, Ar-H),
7.85 (d, 2H, Jortho 8.4 Hz, Ar-H), 6.25 (s, 1H, H-isoxazole), 5.48 (d, 1H, J 3.2 Hz, H-4), 5.15 (dd,
1H, J1,2 7.8 Hz, J2,3 9.6 Hz, H-2), 4.91 (d, 1H, Jgem 12.3 Hz, O-CH2 triazole), 4.79 (d, 1H, Jgem 12.3
Hz, O-CH2 triazole), 4.43 (d, 1H, J1,2 7.8 Hz, H-1), 4.24-4.27 (m, 2H, H-6, H-6'), 3.95 (t, 1H, J5,6
6.5 Hz, H-5), 3.85 (dd, 1H, J3,4 3.3 Hz, J2,3 9.8 Hz, H-3), 3.60 (s, 3H, OCH3), 2.52 (s, 3H, CH3-
isoxazole), 2.29-2.17 (s, 9H, 3x CH3CO). 13C NMR (75 MHz, CDCl3): δ 171.3 (C-O-isoxazole),
170.6-169.7 (CH3CO), 157.3 (C-N-isoxazole), 146.2 (C-triazole), 142.3 (Ar, C-SO2), 139.1 (Ar, C-
N-triazole), 129.1 (Ar, o-CSO2), 121.1 (CH-triazole), 120.5 (Ar, m-CSO2), 101.9 (C-1), 95.6 (CH-
isoxazole), 77.9 (C-3), 70.8 (C-2), 70.6 (C-5), 66.7 (C-4), 63.7 (OCH2), 61.8 (C-6), 56.8 (OCH3),
21.0-20.6 (CH3CO), 12.7 (CH3-isoxazole). ESI-HRMS: calcd. for C20H25N5O9S [M + H]+:
638.1763, found: 638.1785.
4.2.2.5.
Methyl
2,4,6-tri-O-acetyl-3-O-methyl-N-{[(4,6-dimethylpyrimidin-2-yl)
benzesulfonamide]-1H-1,2,3-triazol-4-yl}-β-D-galactopyranoside 26
Following procedure described in Section 4.2.2.1, the reaction of 4-azido-N-(4,6-
dimethypyrimidin-2-yl) benzenesulfonamide 11 (34.0 mg, 0.11 mmol), methyl-2,4,6-tri-O-acetyl-3-
prop-2-ynyl-β-D-galactopyranose 15 (40.0 mg, 0.11 mmol), sodium ascorbate (11.0 mg, 0.055
mmol) and CuSO4 (0.011 mmol, 0.1 equiv) in DMF (1.0 mL) gave the product 26 as a pale yellow
oil (42.2 mg, 0.064 mmol, 58%) after purification by column chromatography (EtOAc/Hexane 6:4
v/v). 1H NMR (300 MHz, CDCl3): δ 8.29 (d, 2H, Jortho 8.6 Hz, Ar-H), 8.02 (s, 1H, CH-triazole),
7.87 (d, 2H, Jortho 8.6 Hz, Ar-H), 6.67 (s, 1H, H-pyrimidine), 5.49 (d, 1H, J 2.6 Hz, H-4), 5.15 (dd,
1H, J1,2 7.8 Hz, J2,3 9.6 Hz, H-2), 4.92 (d, 1H, Jgem 12.2 Hz, O-CH2 triazole), 4.79 (d, 1H, Jgem 12.2
Hz, O-CH2 triazole), 4.43 (d, 1H, J1,2 7.8 Hz, H-1), 4.28-4.25 (m, 2H, H-6, H-6'), 3.95 (t, 1H, J5,6

19
6.3 Hz, H-5), 3.84 (dd, 1H, J3,4 3.3 Hz, J2,3 9.6 Hz, H-3), 3.60 (s, 3H, OCH3), 2.53 (s, 6H, 2x CH3-
pyrimidine), 2.30-2.17 (s, 9H, 3x CH3CO). 13C NMR (75 MHz, CDCl3): δ 170.5-169.6 (CH3CO),
168.3 (C-2'), 155.9 (C-4' and C-6'), 146.1 (C-triazole), 140.0 (Ar, C-SO2), 139.9 (Ar, C-N-triazole),
130.8 (Ar, o-CSO2), 121.0 (CH-triazole), 119.7 (Ar, m-CSO2), 115.1 (C-5'), 102.0 (C-1), 77.8 (C-
3), 70.8 (C-2), 70.5 (C-5), 66.6 (C-4), 63.8 (OCH2), 61.7 (C-6), 56.8 (OCH3), 23.5 (CH3-
pyrimidine), 20.9-20.6 (CH3CO). ESI-HRMS: calcd. for C28H34N6O11S [M + H]+: 663.2079, found:
663.2066.
4.2.2.6.
Methyl
2,4,6-tri-O-acetyl-3-O-methyl-N-{[(4,6-dimethoxylpyrimidin-4-yl)
benzesulfonamide] -1H-1,2,3-triazol-4-yl}-β-D-galactopyranoside 27
Following procedure described in Section 4.2.2.1, the reaction of 4-azido-N-(2,6-
dimethoxypyrimidin-4-yl) benzenesulfonamide 12 (36.0 mg, 0.11 mmol), methyl-2,4,6-tri-O-
acetyl-3-prop-2-ynyl-β-D-galactopyranose 15 (38.0 mg, 0.11 mmol), sodium ascorbate (11.0 mg,
0.055 mmol) and CuSO4 (0.011 mmol, 0.1 equiv) in DMF (1.0 mL) gave the product 26 as a pale
yellow oil (36 mg, 0.051 mmol, 47%) after purification by column chromatography (EtOAc/Hexane
7:3 v/v). 1H NMR (300 MHz, CDCl3): δ 8.10 (d, 2H, Jortho 7.7 Hz, Ar-H), 8.04 (s, 1H, CH-triazole),
7.89 (d, 2H, Jortho 7.8 Hz, Ar-H), 6.23 (s, 1H, H-pyrimidine), 5.48 (d, 1H, J 2.8 Hz, H-4), 5.14 (dd,
1H, J1,2 7.8 Hz, J2,3 9.6 Hz, H-2), 4.91 (d, 1H, Jgem 12.0 Hz, O-CH2 triazole), 4.79 (d, 1H, Jgem 12.1
Hz, O-CH2 triazole), 4.43 (d, 1H, J1,2 7.8 Hz, H-1), 4.27-4.24 (m, 2H, H-6, H-6'), 3.94 (t, 1H, J5,6
6.4 Hz, H-5), 3.84 (dd, 1H, J3,4 3.1 Hz, J2,3 9.7 Hz, H-3), 3.60 (s, 3H, OCH3), 3.58 (s, 6H, 2x OCH3-
pyrimidine), 2.29-2.17 (s, 9H, 3x CH3CO). 13C NMR (75 MHz, CDCl3): δ 172.7 (C-6'), 170.6-169.7
(CH3CO), 164.4 (C-2'), 158.6 (C-4'), 146.2 (C-triazole), 140.2 (Ar, C-SO2), 139.5 (Ar, C-N-
triazole), 129.4 (Ar, o-CSO2), 121.0 (CH-triazole), 120.5 (Ar, m-CSO2), 101.9 (C-1), 85.9 (C-5'),
77.9 (C-3), 70.8 (C-2), 70.6 (C-5), 66.6 (C-4), 63.8 (OCH2), 61.7 (C-6), 56.8 (OCH3), 54,9 (OCH3-
methoxazole), 54.3 (OCH3-methoxazole), 20.9-20.6 (CH3CO). ESI-HRMS: calcd. for
C28H34N6O13S [M + H]+: 695.1977, found: 695.1980.

20
4.2.2.7. Methyl 2,4,6-tri-O-acetyl-3-O-methyl-N-[(benzesulfa-ortho-pyridine)-1H-1,2,3-triazol-
4-yl]-β-D-galactopyranoside 28
Following procedure described in Section 4.2.2.1, the reaction of 4-azido-N-pyridin-2-yl
benzenesulfonamide 13 (40.0 mg, 0.16 mmol), methyl-2,4,6-tri-O-acetyl-3-prop-2-ynyl-β-D-
galactopyranose 15 (50.0 mg, 0.16 mmol), sodium ascorbate (16.0 mg, 0.08 mmol) and CuSO4
(0.016 mmol, 0.1 equiv) in DMF (1.0 mL) gave the product 28 as a pale yellow oil (63 mg, 0.10
mmol, 62%) after purification by column chromatography (EtOAc/Hexane 7:3 v/v). 1H NMR (300
MHz, CDCl3): δ 8.28 (broad s, 1H, H-pyrimidine), 8.04 (d, 2H, Jortho 7.6 Hz, Ar-H), 8.00 (s, 1H,
CH-triazole), 7.82 (d, 2H, Jortho 7.6 Hz, Ar-H), 7.70 (m, 1H, H-pyrimidine), 7.41 (broad s, 1H, H-
pyrimidine), 6.83 (broad s, 1H, H-pyrimidine), 5.46 (d, 1H, J3,4 3.0 Hz, H-4), 5.10 (t, 1H, J1,2 = J2,3
8.54 Hz, H-2), 4.87 (d, 1H, Jgem 12.2 Hz, O-CH2 triazole), 4.76 (d, 1H, Jgem 12.2 Hz, O-CH2
triazole), 4.40 (d, 1H, J1,2 7.7 Hz, H-1), 4.22 (d, 2H, J5,6 6.4 Hz, H-6, H-6'), 3.92 (t, 1H, J5,6 6.3 Hz,
H-5), 3.82 (dd, 1H, J3,4 3.0 Hz, J2,3 9.7 Hz, H-3), 3.57 (s, 3H, OCH3), 2.26-2.13 (s, 9H, 3x CH3CO).
13C NMR (75 MHz, CDCl3): δ 170.4-169.5(CH3CO), 155.5 (C-2'), 146.0 (C-triazole), 143.1 (C-6'),
139.5 (Ar, C-SO2), 139.2 (C-3'), 139.5 (Ar, C-N-triazole), 128.6 (Ar, o-CSO2), 121.1 (C-H
triazole), 120.5 (Ar, m-CSO2), 115.8 (C-4'), 113.9 (C-5'), 101.9 (C-1), 70.8 (C-2), 70.5 (C-5), 66.6
(C-4), 63.7 (OCH2), 61.7 (C-6), 56.8 (OCH3), 20.8-20.6 (CH3CO). ESI-HRMS: calcd. for
C27H31N5O11S [M + H]+: 634.1814, found: 634.1836.
4.2.2.8.
Methyl
2,4,6-tri-O-acetyl-3-O-methyl-N-{[(4-methylpyrimidin-2-
yl)benzesulfonamide]-1H-1,2,3-triazol-4-yl}-β-D-galactopyranoside 29
Following procedure described in Section 4.2.2.1, the reaction of 4-azido-N-(4-
methylpyrimidin-2-yl) benzenesulfonamide 14 (26.2 mg, 0.09 mmol), methyl-2,4,6-tri-O-acetyl-3-
prop-2-ynyl-β-D-galactopyranose 15 (34.0 mg, 0.09 mmol), sodium ascorbate (9.0 mg, 0.045
mmol) and CuSO4 (0.009 mmol, 0.1 equiv) in DMF (0.8 mL) gave the product 29 as a pale yellow
oil (57.8 mg, 0.10 mmol, 69%) after purification by column chromatography (EtOAc/Hexane 7:3

21
1
v/v). H NMR (300 MHz, CDCl3): δ 8.42 (broad s, 1H, H-pyrimidine), 8.29 (d, 2H, Jortho 8.3 Hz,
Ar-H), 8.01 (s, 1H, CH-triazole), 7.88 (d, 2H, Jortho 8.6 Hz, Ar-H), 6.83 (m, 1H, H-pyrimidine), 5.48
(d, 1H, J3,4 2.9 Hz, H-4), 5.48 (dd, 1H, J1,2 7.9 Hz, J2,3 9.5 Hz, H-2), 5.15 (d, 1H, Jgem 11.9 Hz, O-
CH2 triazole), 4.92 (d, 1H, Jgem 11.9 Hz, O-CH2 triazole), 4.43 (d, 1H, J1,2 7.8 Hz, H-1), 4.28-4.25
(m, 2H, H-6, H-6'), 3.96 (t, 1H, J5,6 6.3 Hz, H-5), 3.84 (dd, 1H, J3,4 2.9 Hz, J2,3 9.7 Hz, H-3), 3.61 (s,
3H, OCH3), 2.58 (s, 3H, CH3-pyrimidine), 2.53 (s, 6H, 2x CH3-pyrimidine), 2.30-2.17 (s, 9H, 3x
CH3CO). 13C NMR (75 MHz, CDCl3): δ 170.5-170.4 (CH3CO), 169.6 (C-2'), 156.8 (C-4'), 146.1
(C-triazole), 140.0 (Ar, C-SO2), 139.9 (Ar, C-N-triazole), 130.8 (Ar, o-CSO2), 121.0 (CH-triazole),
119.8 (Ar, m-CSO2), 115.2 (C-5'), 102.0 (C-1), 77.9 (C-3), 70.8 (C-2), 70.6 (C-5), 66.6 (C-4), 63.8
(OCH2), 61.7 (C-6), 56.8 (OCH3), 24.1 (CH3-pyrimidine), 20.9-20.6 (CH3CO). ESI-HRMS: calcd.
for C27H32N6O11S [M + H]+: 649.1923, found: 649.1930.
4.2.3. General procedure for deacetylation reactions29
To the solutions of compounds 23-29 in methanol (2.0 mL) was added 1 M NaOMe until pH
9–10 was achieved. The mixtures were stirred for 2-4 h at room temperature, neutralized with ion
exchange resin (Dowex 50WX8-200 H+), filtered and concentrated under reduced pressure.
4.2.3.1.
Methyl-3-O-methyl[(4-benzenesulfonamide)-1H-1,2,3-triazol-4-yl]-β-D-
galactopyranoside 1
Following the procedure described in Section 4.2.3. deacetylation reaction of compound 24
(55 mg, 0.01 mmol) under conditions described gave the final product 1 as a white amorphous solid
(29.9 mg, 0.007 mmol, 70.2%). 1H NMR (400 MHz, D2O): δ 8.41 (s, 1H, CH-triazole), 7.92 (d, 2H,
Jortho 8.8 Hz, Ar-H), 7.86 (d, 2H, Jortho 8.8 Hz, Ar-H), 5.02 (dd, 1H, J1,2 7.8 Hz, J2,3 9.7 Hz, H-2),
4.79 (d, 1H, Jgem 12.2 Hz, O-CH2 triazole), 4.64 (d, 1H, Jgem 12.2 Hz, O-CH2 triazole), 4.29 (d, 1H,
J1,2 7.8 Hz, H-1), 4.14 (d, 1H, J3,4 3.4 Hz, H-4), 3.77-3.73 (m, 2H, H-6, H-6'), 3.66 (dd, 1H, J3,4 3.4
13
Hz, J2,3 9.7 Hz, H-3), 3.55 (t, 1H, J5,6 5.6 Hz, H-5), 3.42 (s, 3H, OCH3). C NMR (75 MHz, D2O):

22
δ 146.0 (C-triazole), 143.9 (Ar, C-SO2), 139.2 (Ar, C-N-triazole), 127.7 (Ar, m-CSO2), 121.9 (CH-
triazole), 120.2 (Ar, m-CSO2), 102.1 (C-1), 79.5 (C-3), 75.2 (C-5), 70.9 (C-2), 65.6 (C-4), 62.0
(OCH2), 60.9 (C-6), 55.5 (OCH3). ESI-HRMS: calcd. for C18H23N4O9S [M ￚ H]ￚ: 471.1191, found:
471.1181.
4.2.3.2.
Methyl
3-O-methyl-N-{[(phenyl)sulfonyl]acetamide]-1H-1,2,3-triazol-4-yl}-β-D-
galactopyranoside 2
Following the procedure described in Section 4.2.3. deacetylation reaction of compound 25
(18 mg, 0.03 mmol) under conditions described gave the final product 2 (13.8 mg, 0.029 mmol,
1
97%) as an amorphous solid. H NMR (400 MHz, D2O): δ 8.50 (s, 1H, CH-triazole), 8.07 (d, 2H,
Jortho 8.3 Hz, Ar-H), 7.93 (d, 2H, Jortho 8.3 Hz, Ar-H), 5.00 (dd, 1H, J1,2 7.8 Hz, J2,3 9.7 Hz, H-2),
4.72 (broad s, 2H, O-CH2 triazole), 4.33 (d, 1H, J1,2 6.7 Hz, H-1), 4.80-4.90 (m, 1H, H-2), 4.22 (d,
1H, J3,4 2.5 Hz, H-4), 3.79 (d, 1H, J5,6 4.0 Hz, H-5), 3.58 (broad s, 2H, H-6, H-6'), 3.51 (d, 1H, J3,4
13
2.5 Hz, H-3), 3.37 (s, 3H, OCH3), 2.14 (s, 3H, CH3CO-sulfonamide). C NMR (75 MHz, D2O): δ
170.5 (CO-sulfacetamide), 146.0 (C-triazole), 143.9 (Ar, C-SO2), 139.2 (Ar, C-N-triazole), 127.7
(Ar, m-CSO2), 121.9 (CH-triazole), 120.2 (Ar, m-CSO2), 102.1 (C-1), 79.5 (C-3), 75.2 (C-5), 70.9
(C-2), 65.6 (C-4), 62.0 (OCH2), 60.9 (C-6), 55.5 (OCH3), 23.6 (CH3-sulfacetamide). ESI-HRMS:
calcd. for C18H24N4O9S [M + Na]+: 495.1156, found: 495.1156.
4.2.3.3. Methyl 3-O-methyl-N-{[(5-methylisoxazol-3-yl)benzenesulfonamide]-1H-1,2,3-triazol-
4-yl}-β-D-galactopyranoside 3
Following the procedure described in Section 4.2.3. deacetylation reaction of compound 25
(35 mg, 0.055 mmol) under conditions described gave the final product 3 (28 mg, 0.055 mmol,
100%) as an amorphous solid. 1H NMR (400 MHz, D2O): δ 8.40 (s, 1H, CH-triazole), 7.92 (d, 2H,
Jortho 8.5 Hz, Ar-H), 7.87 (d, 2H, Jortho 8.5 Hz, Ar-H), 6.05 (s, 1H, H-isoxazole), 5.02 (dd, 1H, J1,2
7.8 Hz, J2,3 9.7 Hz, H-2), 4.79 (d, 1H, Jgem 12.3 Hz, O-CH2 triazole), 4.64 (d, 1H, Jgem 12.3 Hz, O-

23
CH2 triazole), 4.29 (d, 1H, J1,2 7.8 Hz, H-1), 4.13 (d, 1H, J3,4 2.8 Hz, H-4), 3.77-3.74 (m, 2H, H-6,
H-6'), 3.65 (dd, 1H, J3,4 2.8 Hz, J2,3 9.7 Hz, H-3), 3.54 (t, 1H, J5,6 5.6 Hz, H-5), 3.42 (s, 3H, OCH3),
2.32 (s, 1H, CH3-isoxazole). 13C NMR (75 MHz, D2O): δ 171.0 (C-O-isoxazole), 157.6 (C=N-
isoxazole), 146.0 (C-triazole), 140.1 (Ar, C-SO2), 139.6 (Ar, C-N-triazole), 128.9 (Ar, m-CSO2),
122.0 (CH-triazole), 120.4 (Ar, p-CSO2), 102.1 (C-1), 95.1 (CH-isoxazole), 79.5 (C-3), 75.1 (C-5),
70.9 (C-2), 65.6 (C-4), 61.9 (OCH2), 60.9 (C-6), 55.5 (OCH3), 10.9 (CH3-isoxazole). ESI-HRMS:
calcd. for C22H27N5O10S [M ￚ H]ￚ: 552.1406, found: 552.1417.
4.2.3.4. Methyl 3-O-methyl-N-{[(4,6-dimethylpyrimidin-2-yl) benzenesulfonamide]-1H-1,2,3-
triazol-4-yl}-β-D-galactopyranoside 4
Following the procedure described in Section 4.2.3. deacetylation reaction of compound 26
(39 mg, 0.059 mmol) under conditions described gave the final product 4 (20 mg, 0.037 mmol,
63%) as an amorphous solid. 1H NMR (400 MHz. D2O): δ 8.39 (s, 1H, CH-triazole), 8.06 (d, 2H,
Jortho 8.5 Hz, Ar-H), 7.84 (d, 2H, Jortho 8.5 Hz, Ar-H), 6.55 (s, 1H, H-pyrimidine), 5.00 (dd, 1H, J1,2
7.8 Hz, J2,3 9.6 Hz, H-2), 4.79 (d, 1H, Jgem 12.2 Hz, O-CH2 triazole), 4.62 (d, 1H, Jgem 12.3 Hz, O-
CH2 triazole), 4.27 (d, 1H, J1,2 7.8 Hz, H-1), 4.10 (d, 1H, J3,4 2.9 Hz, H-4), 3.74-3.71 (m, 2H, H-6,
H-6'), 3.62 (dd, 1H, J3,4 2.9 Hz, J2,3 9.6 Hz, H-3), 3.51 (t, 1H, J5,6 6.1 Hz, H-5), 3.40 (s, 3H, OCH3),
2.30 (s, 6H, 2x CH3-pyrimidine). 13C NMR (75 MHz, D2O): δ 171.9 (C-2'), 157.6 (C-6', C-4'),
147.3 (C-triazole), 142.8 (Ar, C-SO2), 140.9 (Ar, C-N-triazole), 131.4 (Ar, m-CSO2), 123.4 (CH-
triazole), 120.9 (Ar, p-CSO2), 114.1 (C-5'), 103.5 (C-1), 80.9 (C-3), 76.6 (C-5), 72.3 (C-2), 67.0 (C-
4), 63.2 (OCH2), 62.3 (C-6), 56.9 (OCH3), 21.0 (CH3-pyrimidine). ESI-HRMS: calcd. for
C24H30N6O9S [M + Na]+: 601.1687, found: 601.1708.
4.2.3.5. Methyl 3-O-methyl-N-{[(4,6-dimethoxylpyrimidin-4-yl) benzenesulfonamide] -1H-
1,2,3-triazol-4-yl}-β-D-galactopyranoside 5
Following the procedure described in Section 4.2.3. deacetylation reaction of compound 27

24
(32 mg, 0.046 mmol) under conditions described gave the final product 5 (26.4 mg, 0.046 mmol,
100%) as an amorphous solid. 1H NMR (400 MHz, D2O): δ 8.39 (s, 1H, CH-triazole), 8.0 (d, 2H,
Jortho 8.5 Hz, Ar-H), 7.88 (d, 2H, Jortho 8.5 Hz, Ar-H), 5.95 (s, 1H, H-pyrimidine), 5.01 (dd, 1H, J1,2
7.8 Hz, J2,3 9.6 Hz, H-2), 4.79 (d, 1H, Jgem 12.2 Hz, O-CH2 triazole), 4.63 (d, 1H, Jgem 12.3 Hz, O-
CH2 triazole), 4.28 (d, 1H, J1,2 7.8 Hz, H-1), 4.11 (d, 1H, J3,4 3.0 Hz, H-4), 3.80 (s, 3H, OCH3
pyrimidine), 3.79 (s, 3H, OCH3 pyrimidine), 3.75-3.72 (m, 2H, H-6, H-6'), 3.64 (dd, 1H, J3,4 3.0 Hz,
13
J2,3 9.6 Hz, H-3), 3.53 (t, 1H, J5,6 6.1 Hz, H-5), 3.41 (s, 3H, OCH3). C NMR (75 MHz, D2O): δ
174.1 (C-6'), 161.2 (C-4', C-2'), 147.4 (C-triazole), 141.8 (Ar, C-SO2), 141.4 (Ar, C-N-triazole),
130.6 (Ar, m-CSO2), 123.4 (CH-triazole), 121.7 (Ar, p-CSO2), 103.5 (C-1), 86.20 (C-5'), 80.9 (C-
3), 76.6 (C-5), 72.3 (C-2), 67.0 (C-4), 63.3 (OCH2), 62.4 (C-6), 56.9 (OCH3), 55.6 (OCH3-
pyrimidine), 54.8 (OCH3-pyrimidine). ESI-HRMS: calcd. for C24H30N6O11S [M + H]+: 611.1766,
found: 611.1798.
4.2.3.6.
Methyl
3-O-methyl-N-[(benzenesulfa-ortho-pyridine)-1H-1,2,3-triazol-4-yl]-β-D-
galactopyranoside 6
Following the procedure described in Section 4.2.3. deacetylation reaction of compound 28
(60 mg, 0.095 mmol) under conditions described gave the final product 6 as an amorphous solid
(46,1 mg, 0,091 mmol, 95.7%). 1H NMR (400 MHz, D2O): δ 8.35 (s, 1H, CH-triazole), 7.91 (d, 2H,
Jortho 8,5 Hz, Ar-H), 7.78 (m, 3H, Ar-H, H-pyridine), 7.58 (ddd, 1H, J 1.8 Hz, J 6.7 Hz, J 8.7 Hz, H-
pyridine), 7.14 (d, 1H, J 8.6 Hz, H- pyridine), 6.72 (ddd, 1H, J 0.9 Hz, J 5.7 Hz, J 6.8 Hz, H-
pyridine), 4.99 (dd, 1H, J1,2 7.8 Hz, J2,3 9.7 Hz, H-2), 4.79 (d, 1H, Jgem 12.2 Hz, O-CH2 triazole),
4.61 (d, 1H, Jgem 12.3 Hz, O-CH2 triazole), 4.27 (d, 1H, J1,2 7.8 Hz, H-1), 4.11 (d, 1H, J3,4 3.0 Hz,
H-4), 3.74-3.71 (m, 2H, H-6, H-6'), 3.63 (dd, 1H, J3,4 3.0 Hz, J2,3 9.7 Hz, H-3), 3.53 (t, 1H, J5,6 6.0
Hz, H-5), 3.39 (s, 3H, OCH3). 13C NMR (75 MHz, D2O): δ 146.0 (C-triazole), 142.5 (Ar, C-SO2),
141.9 (Ar, C-N-triazole), 139.9-139.2 (C-5' and C-2'), 128.4 (Ar, m-CSO2), 121.7 (CH-triazole),
120.2 (Ar, m-CSO2), 115.9-114.6 (C-3' and C-4'), 102.1 (C-1), 79.7 (C-3), 75.0 (C-5), 71.0 (C-2),

25
65.7 (C-4), 62.2 (OCH2), 61.0 (C-6), 56.0 (OCH3). ESI-HRMS: calcd. for C23H27N5O9S [M + H]+:
550.1602, found: 550.1621.
4.2.3.7.
Methyl
3-O-methyl-N-{[(4-methylpyrimidin-2-yl)benzenesulfonamide]-1H-1,2,3-
triazol-4-yl}-β-D-galactopyranoside 7
Following the procedure described in Section 3.2.3. deacetylation reaction of compound 29
(33 mg, 0.051 mmol) under conditions described gave the final product 7 as an amorphous solid
(19.9 mg, 0.038 mmol, 74.7%). 1H NMR (400 MHz, D2O): δ 8.41 (s, 1H, CH-triazole), 8.16-8.11
(m, 3H, Ar-H, H-pyrimidine), 7.90 (d, 2H, Jortho 8.3 Hz, Ar-H), 6.74 (s, 1H, H-pyrimidine), 5.11-
5.05 (m, 1H, H-2), 4.79 (d, 1H, Jgem 12.2 Hz, O-CH2 triazole), 4.69 (d, 1H, Jgem 12.2 Hz, O-CH2
triazole), 4.33 (d, 1H, J1,2 7.8 Hz, H-1), 4.16 (broad s, 1H, H-4), 3.82-3.78 (m, 2H, H-6, H-6'), 3.67
(dd, 1H, J3,4 2.0 Hz, J2,3 9.5 Hz, H-3), 3.55 (broad s, 1H, H-5), 3.48 (s, 3H, OCH3), 3.34 (s, 3H,
CH3-pyrimidine). 13C NMR (75 MHz, D2O): δ 169.4 (C-2'), 156.7 (C-6'), 156.5 (C-4'), 146.0 (C-
triazole), 140.7 (Ar, C-SO2), 139.7 (Ar, C-N-triazole), 130.0 (Ar, m-CSO2), 121.9 (CH-triazole),
119.7 (Ar, m-CSO2), 114.4 (C-5'), 102.1 (C-1), 79.6 (C-3), 75.1 (C-5), 71.0 (C-2), 65.6 (C-4), 62.0
(OCH2), 61.0 (C-6), 55.8 (OCH3), 22.6 (CH3-pyrimidine). ESI-HRMS: calcd. for C23H28N6O9S [M
+ H]+: 565.1711, found: 565.1726.
4.3. Biological assays
4.3.1. Inhibition of T. cruzi cell invasion
Inhibition of fibroblast cells invasion by T. cruzi was carried out by treating with 250 µM of
compounds 1-7 previously adhered LLCMK2 fibroblasts in circular coverslips (2 x 104 cells per
well in 500 µL of RPMI 1640 supplemented with penicillin, streptomycin, L-glutamine, and 10%
FBS) and, after 30 minutes, adding 1 x 105 trypomastigote forms of T. cruzi (in a proportion of 5
o
parasites for each fibroblast). The cultures were incubated for 18 hours at 37 C and 5% CO2, then
the coverslips were fixed and stained with fast panoptic dye (Laborclin, Brazil). The coverslips

26
were mounted to glass slides with Entellan (Merck Millipore, USA) and examined under an optical
microscope. The level of parasitic cell invasion was determined using three parameters: i)
percentage of infected cells; ii) mean number of T. cruzi amastigotes by cell; iii) infection index that
was determined by multiplying the percentage of infected cells times by the mean number of
parasites per cell, as described previously.34,35
4.3.2. Citotoxicity assay
LLCMK2 fibroblasts were seeded in 96-wells plates (1 x 104 cells in 200 µL per well) in
RPMI 1640 supplemented with penicillin, streptomycin, L-glutamine, and 10% FBS. After adhering
overnight at 37oC and 5% CO2, the medium was replaced by fresh RPMI medium with different
o
concentrations of compounds 1-7 (500 to 67,5 µM) in triplicate and cultured at 37 C and 5% CO2
for 48 hours. Then, 20 µL of a 5 mg/mL of (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium
Bromide, MTT, Sigma-Aldrich, USA) were added to each well and incubated for 4 h at 37oC and
5% CO2. The medium was discarded and 200 µL of dimethylsulfoxide was added to each well to
dissolve formazan crystals, and then the plate was read at 570 nm (UV mini 1240, Shimadzu).
4.3.3. Trypanocidal assay
Culture derived tripomastigote forms of T. cruzi (Y strain) were seeded in 96-wells plates (1 x
106 cells in 200 µL per well) in RPMI 1640 supplemented with penicillin, streptomycin, L-
glutamine, and 10% FBS containing different concentrations of the compounds (ranging from 250
to 31,25 µM) and incubated at 37oC and 5% CO2 for 48 hours. Then, 5 µL of a 1 mg/mL solution of
resazurin (Sigma-Aldrich, USA) were added to each well and, 6 hours after 37oC and 5% CO2
incubation the plates were read at 570 and 600 nm (UV mini 1240, Shimadzu). The parasites
viability were assessed subtracting the OD in 600 nm from the OD in 570 nm and comparing with
untreated cells.

27
4.3.4. Inhibition of T. cruzi trans-sialidase (TcTS)
trans-Sialidase used in this study was a His-tagged 70 kDa recombinant material truncated to
remove C-terminal repeats but retaining the catalytic N-terminal domain of the enzyme.43 Inhibition
was assessed using the continuous fluorimetric assay described by Douglas and co-workers.37
Briefly, the assay was performed in triplicate in 96-well plates containing phosphate buffer solution
at pH 7 (25 µL), recombinant enzyme solution (25 µL) and inhibitor solution (25 µL of 4.0 mM
solution). This mixture was incubated for 10 min. at 26 ºC followed by addition of MuNANA (25
µL of a 0.4 mM solution affording an assay concentration of 0.1 mM). The fluorescent product
released was measured over 5 min., with excitation and emission wavelengths of 360 and 460 nm,
respectively, and the data were analyzed with GraphPad Prism software version 4.0 (San Diego,
CA, USA). Inhibition percentages were calculated according to the equation: % I = 100 ₓ [1-
(Vi/V0)], where Vi is the velocity in the presence of inhibitor and V0 is the velocity in absence of
inhibitor.
4.3.5. Galectin-3 binding assays
4.3.5.1. Isolation and purification of human recombinant galectin-3
The purified preparation of hr galectin-3 was prepared as described previously39 using a crude
extract of transformed BL21 E.coli strain contained cDNA of human galectin-3 and affinity
chromatography on lactosyl-Sepharose (Sigma).
4.3.5.2. Corning Epic label-free assays
Binding assays of small molecules to hr galectin-3 were performed with the Corning Epic
label-free technology using the EnSpire Multimode Plate Reader.40 Galectin-3 was immobilized on
the microplate optical biosensor surface preactivated with N-hydroxy-succinimide esters groups.
Protein solutions at 150 µg/mL in 20 mM sodium acetate at pH 4.0, were dispensed into the wells
(15 µL) and left in incubation for 16 h at 4 °C. The microplate was subsequently washed three times
in the binding buffer (PBS 1X, DMSO 1% or 2% at pH 7.4). A baseline reading was recorded

28
before and after stabilization in the binding buffer for 3 h. Given their partial insolubility in aqueous
buffers, compounds were first dissolved in pure DMSO and then diluted to the final working
concentrations dissolved in the binding buffer containing 1% or 2% DMSO. Compound solutions
were dispensed into the wells (total volume 30 µL) in triplicate at different concentrations, ranging
from 0.09 to 400 µM. Final readings were taken up to a period of 3 h at 25 °C. Label-free responses
were measured as shifts in reflected wavelength and were expressed in picometers. The difference
between the last baseline measurements and the maximum signal obtained after 3 h incubation was
used to obtain plots of compound concentration versus picometers. Readings from blank wells
treated with the solvent alone were further subtracted to obtain the net binding values. EC50 binding
values were obtained by non-linear fitting of values using a one-site ligand binding model. Results
were analyzed using the EnSpire label-free user interface software. Plots were generated using
GraphPad Prism V-5.01 (GraphPad Software, La Jolla, CA).
4.4. Molecular modeling studies with hr galectin-3
The 3D structure of compounds 1-7 were optimized with the RM1 semi-empirical
hamiltonian44 using the MOPAC 201645 software and then at the HF/6-31G* theory level using
Gaussian 03. The electronic density was calculated at the same theory level for obtaining RESP
charges with the antechamber package from AmberTools16.46
The initial structure for the human galectin-3 carbohydrate recognition domain was that
described by the crystallographic structure PDB ID 1KJL.47 The initial structures of the complexes
were obtained by structural alignment of the galactosyl portion of glycoconjugates 1-7 with the
galactose residue from LacNAc present in the crystallographic structure.
Molecular dynamics simulations were performed with the GROMACS 5.1.2 suite41 with the
AMBER99SB-ILDN forcefield48 for describing the protein, water and ions and GAFF49 for
describing ligands. TIP3P water model was used in a dodecahedric box containing about 32
thousand atoms with about 30 NaCl to account for charge neutralization and the ionic strength of

29
150 mM. The system was submitted to energy minimization with the steepest descent algorithm
until convergence to simple precision and then to 1 ns equilibration phase using the v-rescale
thermostat and berendsen barostat to keep temperature and pressure at 309K and 1bar, respectively.
Production runs were carried out for 20 ns repeated 3 times from different initial velocities using the
Nose-Hoover thermostat and Parrinello-Rahman barostat. The time step was 2 fs.
Estimation of the relative affinity between human galectin-3 and the ligands 1-7 was made
with the MM/PBSA method as implemented in AmberTools16.41 The value for the internal
dielectric constant was set to 1. A total of 100 frames of each system was used for the calculations
and errors were calculated by bootstrapping with 200 resamplings. Solute entropic contributions
were not calculated and, hence, the final results can only be interpreted in a comparative way to
rank the compounds.
The hardware was provided by a Brazilian startup called Mining Information for You (MI4U)
and was composed of machines hosted at the IBM SoftLayer infrastructure. Each of them had 24
physical cores at 2.60 GHz and two nVIDIA Tesla K80 GPUs.
1
Supporting Information Available: The Supporting Information encloses the H NMR and ESI-
MS analyses of the main compounds, as well as a complementary figure related to molecular
modeling studies.
Acknowledgments
We acknowledge the financial support and fellowships from FAPESP (Fundação de Amparo
à Pesquisa do Estado de São Paulo, Proc. 2012/19390-0) and CNPq (Conselho Nacional de
Pesquisa e Desenvolvimento Científico e Tecnológico, Grant: 31.1947/2015-8). We are grateful to
Prof. Gino Del Ponte for valuable suggestions in the synthesis of the target compounds, to Vinícius
Palaretti for NMR analyses, to José Carlos Tomaz for ESI-MS analyses, to Prof. Antonio Caliri for
discussions about the molecular dynamics simulations, and to Prof. Richard D. Cummings for
kindly providing E. coli BL21 (DE3) containing the plasmid expressing human galectin-3.