Design, synthesis and the effect of 1,2,3-triazole sialylmimetic neoglycoconjugates on Trypanosoma cruzi and its cell surface trans-sialidase

Article abstract of DOI:10.1016/j.bmc.2011.11.022

This work describes the synthesis of a series of sialylmimetic neoglycoconjugates represented by 1,4-disubstituted 1,2,3-triazole-sialic acid derivatives containing galactose modified at either C-1 or C-6 positions, glucose or gulose at C-3 position, and by the amino acid derivative 1,2,3-triazole fused threonine-3-O-galactose as potential TcTS inhibitors and anti-trypanosomal agents. This series was obtained by Cu(I)-catalysed azide-alkyne cycloaddition reaction ('click chemistry') between the azido-functionalized sugars 1-N₃-Gal (commercial), 6-N ₃-Gal, 3-N₃-Glc and 3-N₃-Gul with the corresponding alkyne-based 2-propynyl-sialic acid, as well as by click chemistry reaction between the amino acid N₃-ThrOBn with 3-O-propynyl-GalOMe. The 1,2,3-triazole linked sialic acid-6-O-galactose and the sialic acid-galactopyranoside showed high Trypanosoma cruzi trans-sialidase (TcTS) inhibitory activity at 1.0 mM (approx. 90%), whilst only the former displayed relevant trypanocidal activity (IC₅₀ 260 μM). These results highlight the 1,2,3-triazole linked sialic acid-6-O-galactose as a prototype for further design of new neoglycoconjugates against Chagas' disease.

Full text of DOI:10.1016/j.bmc.2011.11.022

Bioorganic & Medicinal Chemistry 20 (2012) 145–156
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Bioorganic & Medicinal Chemistry
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Design, synthesis and the effect of 1,2,3-triazole sialylmimetic
neoglycoconjugates on Trypanosoma cruzi and its cell surface trans-sialidase
Vanessa L. Campo a, Renata Sesti-Costa b, Zumira A. Carneiro a, João S. Silva b, Sergio Schenkman c,
Ivone Carvalho a,
⇑
a Faculdade de Ciências Farmacêuticas de Ribeirão Preto, USP, Av. Café S/N, CEP 14040-903, Ribeirão Preto, SP, Brazil
b Faculdade de Medicina de Ribeirão Preto, USP, Av. Bandeirantes 3900, CEP 14049-900, Ribeirão Preto, SP, Brazil
c Departament of Microbiology, Immunology and Parasitology, Universidade Federal de São Paulo, Rua Botucatu 862, 8 Andar, 04023-062 São Paulo, SP, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
This work describes the synthesis of a series of sialylmimetic neoglycoconjugates represented by
1,4-disubstituted 1,2,3-triazole-sialic acid derivatives containing galactose modified at either C-1 or
C-6 positions, glucose or gulose at C-3 position, and by the amino acid derivative 1,2,3-triazole fused
threonine-3-O-galactose as potential TcTS inhibitors and anti-trypanosomal agents. This series was
obtained by Cu(I)-catalysed azide–alkyne cycloaddition reaction (‘click chemistry’) between the azido-
functionalized sugars 1-N3-Gal (commercial), 6-N3-Gal, 3-N3-Glc and 3-N3-Gul with the corresponding
alkyne-based 2-propynyl-sialic acid, as well as by click chemistry reaction between the amino acid
N3-ThrOBn with 3-O-propynyl-GalOMe. The 1,2,3-triazole linked sialic acid-6-O-galactose and the sialic
acid-galactopyranoside showed high Trypanosoma cruzi trans-sialidase (TcTS) inhibitory activity at
Received 19 September 2011
Revised 4 November 2011
Accepted 11 November 2011
Available online 22 November 2011
Keywords:
Triazole
Sialylmimetics
Click chemistry
Trypanosoma cruzi
trans-Sialidase
1.0 mM (approx. 90%), whilst only the former displayed relevant trypanocidal activity (IC50 260 lM).
These results highlight the 1,2,3-triazole linked sialic acid-6-O-galactose as a prototype for further design
of new neoglycoconjugates against Chagas’ disease.
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
acids of the donor site are represented by an arginine triad
(Arg35, Arg245 and Arg314) that interacts with the carboxylate
Trypanosoma cruzi trans-sialidase (TcTS) is a retaining glycosyl-
transferase that plays a key role in the pathogenesis of Chagas’
disease—a parasitic blood-borne infection which affects many peo-
ple in South and Central America.1–3 The primary function of TcTS
consists in the acquisition of sialic acid residues from mammalian
host glycoconjugates and transfer of these monosaccharides to
terminal b-Gal residues on the mucins that cover the parasite cell
group of sialic acid, besides other important residues essential
for stabilization of the transition state (Tyr342, Glu230) and catal-
ysis (Asp59). The acceptor site contains the amino acids Tyr119
and Trp312, which are crucial for the transglycosylation process,
in addition to Asp59 (common to both sites) and Glu362 that
directly interact with b-galactose acceptor.8
The most potent TcTS inhibitors described so far are
represented by compounds that are able to occupy both donor
(sialic acid) and acceptor (b-galactose) binding sites, so that the
sialic acid transfer reaction is hindered or even blocked. As out-
lined in Figure 1, GM3 ganglioside 1, when modified in its sialic
acid residue, for instance at C-4 (deoxy or methoxy) or C-8 (deoxy),
surface, generating
a
-2,3-sialylated-b-galactopyranose units.4–6
Scavenging sialic acid from the host glycoconjugates aids the
recognition and attachment of the parasite to host cells, and allows
its evasion from the host’s immune response, resulting in the host
cells invasion where the parasite can complete its life cycle. TcTS is
also shed from the parasite surface into the blood of the host and is
able to act far from the infection site by inducing apoptosis in the
spleen, thymus and peripheral ganglia.7
According to the determined crystal structure of TcTS by
Buschiazzo et al. (2002),8,9 the active site of this enzyme has sev-
eral common features with microbial sialidases, being, however,
composed by two distinct sites related to sialic acid (donor site)
and to b-galactose molecule (acceptor site). The principal amino
acted as TcTS inhibitor in a concentration range of 10–100
two sulfonamide chalcones 2 (IC50 2.5 M) and 3 (IC50 0.9
and the quinolinone 4 (IC50 0.6 M), obtained from chemical
l
l
M,10
M),
l
l
synthesis showed remarkable improvement against TcTS,11 and a
series of flavonoids and anthraquinones with also strong inhibitory
activity against TcTS were identified from biological screening,
being the lowest IC50 value observed for compound 5 (IC50
0.58 l
M).12 More recently, 6-modified octyl-galactosides such as
6 and 7 showed strong TcTS inhibition (75% and 84%) in a
radiochemical assay,13 and the C-sialoside 8 obtained by cross
metathesis displayed high affinity to TcTS (Kd 0.16 mM), as
measured by surface plasmon resonance (SPR).14
⇑
Corresponding author. Tel.: +55 16 36024709; fax: +55 16 36024879.
E-mail address: carronal@usp.br (I. Carvalho).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.11.022

146
V. L. Campo et al. / Bioorg. Med. Chem. 20 (2012) 145–156
Figure 1. Representative TcTS inhibitors able to occupy both donor and acceptor sites of this enzyme.
Figure 2. Structures of 1,2,3-triazole sialylmimetic neoglycoconjugates 9–13 as potential TcTS inhibitors.
Sialic acid derivatives and sialylmimetics have been largely uti-
ment of sialic acid moiety by other containing carboxylate mole-
cules like amino acids, as represented by the 1,2,3-triazole
sialylmimetic 13 (Fig. 2). Thus, in this work we report the synthesis
of the 1,2,3-triazole sialylmimetic neoglycoconjugates 9–13 and
their biological evaluation as TcTS inhibitors and anti-trypanoso-
mal agents.
lized as scaffolds for the development of new drugs against various
therapeutic targets.15–28 In this context, Cu(I)-assisted 1,3-dipolar
azide–alkyne cycloaddition (CuAAC) reactions have proved to be
attractive to synthesize new sialylmimetics, considering that
azides and alkynes precursors are relatively straightforward to
introduce into organic molecules, besides being, generally, easily
executed, fast and highly selective.29,30 Indeed, they represent a
valuable alternative strategy to overcome the main difficulties re-
lated to the synthesis of sialic acids glycosides, particularly the
presence of carboxylate group at C-2, which may favors 2,3-elimi-
nation in glycosidation reactions, and the absence of C-3 substitu-
ent that impair the neighboring group participation to direct the
stereochemical outcome of the formed glycoside.15 Moreover, the
physicochemical properties of the formed triazole group are partic-
ularly favourable, since it acts as a rigid link, displaying pharmaco-
phore groups in well defined spatial orientation, and cannot be
hydrolytically cleaved, oxidised or reduced.31
In fact, the potential of compounds 9–13 to interact with essen-
tials amino acids of both donor and acceptor sites was investigated
32
by performing docking calculations into the TcTS active site.
According to the results, all compounds were able to establish
hydrogen bonding interactions with the arginine triad and Tyr342
at the donor site. Concerning interactions with the acceptor site,
compounds 9–12 displayed hydrophobic ‘p stacking’ interactions be-
tween their triazole rings and Trp312, while galactosyl moiety of 13
was capable to interact, additionally, with both Trp312 and Tyr119
residues. The supra-cited interactions can be visualized in Figure 3,
comprising compound 9, representative of the designed 1,2,3-tria-
zole sialic acid-based derivatives 9–12 and the sialylmimetic 13.
Based only on docking results it is possible to assert that sialic acid
unit seems not to be a requisite for TcTS inhibition, considering that
carboxylate group from threonine amino acid (compound 13) was
also able to interact with the donor active site, likewise sialic acid
(compounds 9–12). Furthermore, the different galactose, glucose
and gulose sugar units of compounds 9–13 were capable to interact
quite similarly with TcTS acceptor site, which means that galactose
Therefore, we asked whether or not the mimicking of the termi-
nal sugars
a-D-Neu5Ac(2?3)-b-D-Gal of T. cruzi mucins by the
1,2,3-triazole sialic acid-based neoglycoconjugates 9–12 (Fig. 2),
obtained by using CuAAC reactions, may contribute for strong
and multivalent interactions at both donor and acceptor regions
of TcTS active site. For purposes of further comparative enzymatic
assays with TcTS the design of compounds 9–12 was envisaged
based on the utilization of different azido-derived sugars (galacto-
pyranose, glucopyranose or gulopyranose) along with an alkyne-
functionalized sialic acid as precursors. Nevertheless, the fact that
the negatively charged carboxylate group of sialic acid represent
the most important group able to interact with sialic acid donor
site through strong interactions with the arginine triad led us to
also envision the design of relative simpler structures by replace-
Previously to docking studies of compound 9–13 we performed docking between
DANA and the donor active site of TcTS in order to validate the method with this
enzyme. The good superposition between the DANA structure oriented with GOLD
and the same molecule in the crystallographic orientation (PDB code 1MS8)
suggested that the chosen method was appropriate.

V. L. Campo et al. / Bioorg. Med. Chem. 20 (2012) 145–156
147
Figure 3. Docking results of compounds 9 (A) and 13 (B) in the TcTS active site.
does not constitute a requirement for design of TcTS inhibitors.
Summing up, the number of significant interactions described for
compounds 9–13 corroborate their capacity to interact with donor
and acceptor sites, turning them, at least theoretically, potential TcTS
inhibitors.
relative to H-3 [a isomer (d 3.93, J 10.3 Hz); b isomer (d 3.66, J
10.3 Hz)], being the presence of azido group also confirmed by IR
analysis [2104 cmꢀ1 (N3)].
Regarding the preparation of 3-azido-Gul 17, different ap-
proaches were investigated, such as the azide substitution reaction
of the intermediate methyl-2-O-benzoyl-3-O-triflate-4,6-O-ben-
zylidene-a-D aGulOMe 23
-Gal 22,37,38 which afforded the 3-azido-
2. Results and discussion
2.1. Synthesis
with inverted C-3 configuration, albeit in low yield (33%) and still
requiring additional laborious deprotections and acetylation steps
(Scheme 1).39 Alternatively, a synthetic approach to obtain the cor-
responding 3-azido-aGalOMe from the supra-cited intermediate
22 by means of double inversions at C-3 was not effective40; even
though the subsequent reactions of 22 with NaNO2 and Tf2O fur-
The target sialylmimetic neoglycoconjugates are represented by
1,4-disubstituted 1,2,3-triazole-sialic acid derivatives containing
galactose modified at either C-1 (9) or C-6 positions (10), glucose
(11) or gulose (12) at C-3 positions, and by the amino acid deriva-
tive 1,2,3-triazole fused threonine-3-O-galactose 13 (Fig. 2). The
synthesis of compounds 9–13 by ‘click chemistry’ involved the
use of azido-functionalized precursors represented by the sugars
galactosyl azide 14 (commercial), 6-N3-Gal 15, 3-N3-Glc 16 and
3-N3-Gul 17, and the amino acid N3-ThrOBn 18, as well as the uti-
lization of the corresponding alkyne-based monomers 2-propynyl-
sialic acid 19 and 3-O-propynyl-GalOMe 20.
nished the corresponding 3-O-Tf-
the following azide substitution did not give the desired 3-azido-
GalOMe.38 To obtain the 3-azido-Gul 17 by a synthetic route
aGulOMe intermediate (76%),
a
involving fewer steps and in a better yield, Hindsgaul’s method
was followed as described for 3-azido-Glc 16.36a Thus, triflation
of galactofuranose diacetonide 24 followed by azide substitution
reaction furnished the 3-azido 25 in 55% yield (Scheme 1), which
was treated with aqueous trifluoroacetic acid and then per-O-acet-
ylated to afford the final product 17 as an inseparable mixture of
both anomers (a/b 0.5:1.0) in total yield of 80%. The structure of
compound 17 was confirmed by IR [2112 cmꢀ1 (N3)] and NMR
1H spectroscopy, which showed characteristic signals for H-1 [b
2.1.1. Synthesis of azido-functionalized sugars 15–17 and amino
acid 18
The synthesis of 6-azido-functionalized galactopyranose 15 was
isomer (d 5.1, J1,2 8.2 Hz,);
a isomer (d 6.40, J1,2 3.0 Hz)], H-3 [b iso-
performed in three steps, being the
a isomer 15 isolated in 48%
mer (d 5.08, J3,4 3.4 Hz, J2,3 10.4 Hz)], and COCH3 (d 2.19–1.99).
HRESI-MS analysis of 17 showed the characteristic adduct
[M+Na]+ 396.10.
yield.33–35 The synthesis of both 3-azido-functionalised glucopyra-
nose 16 and gulopyranose 17 were carried out following the
modified method described by Hindsgaul et al. (1994).36a Thus,
starting with the synthesis of 3-azido-Glc 16,36a,b the treatment
of the allofuranose diacetonide 21, obtained by sequential
triflation/azide substitution reactions, with aqueous trifluoroacetic
acid followed by per-acetylation gave compound 16 as a mixture of
Concerning the preparation of the azido-functionalized N3-
ThrOBn 18, the treatment of Fmoc- -threonine benzyl ester 26
with 20% piperidine in DMF, followed by diazo transfer reaction
of the resulting -Thr-OBn 27 (78%), utilizing in situ generated tri-
L-
L
L
flyl azide and CuSO4, gave the amino acid 18 (60%) (Scheme
1).41,42,19 Its structure was confirmed by 1H NMR [characteristic
signals: OBn aromatic hydrogens (d 7.39–7.33); bCHThr (d 4.26)
anomers (
sis of 16 showed characteristic doublets of H-1 [
J1,2 3.6 Hz); b isomer, d 5.63, J1,2 8.2 Hz)] and shielded triplets
a
/b 1:1) in total yield of 82% (Scheme 1). 1H NMR analy-
isomer (d 6.25,
a
and a
CHThr (d 3.83)] and IR [2109 cmꢀ1 (N3)] spectra.

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V. L. Campo et al. / Bioorg. Med. Chem. 20 (2012) 145–156
obtained intermediate 31 (81%) under standard conditions pro-
vided the final product 20 (quantitative) after purification by col-
umn chromatography (EtOAc–Hexane 1:1) (Scheme 2). 1H NMR
analysis of 20 showed a doublet of H-1 (d 4.96, J1,2 3.8 Hz), as well
as characteristic signals of propargyl functional group [d 2.44 ppm,
J 2.3 Hz, (CH); d 4.29 ppm, J 2.5 Hz, J 15.8 Hz, (CH2)] and COCH3
groups (d 2.18–2.07).
2.1.3. Synthesis of potential TcTS inhibitors 9–13 by ‘click
chemistry’ reactions
In general, the synthesis of compounds 9–13 by Cu(I)-assisted
1,3-dipolar azide–alkyne cycloaddition reactions were performed
in a microwave reactor utilizing the catalytic system CuSO4/so-
dium ascorbate and DMF as solvent.51,52 The progress of the reac-
tions was followed by TLC analysis, which revealed complete
consumption of starting materials and no further changes after
15 min irradiation bursts (3) at 100 °C. Thus, the condensation of
sugars 14, 15, 16 and 17 with 2-propynyl-sialic acid 19 afforded
the per-acetylated products 32 (32%), 33 (34%), 34 (74%) and 35
(43.4%) after purification by column chromatography (EtOAc),
being compounds 34 and 35 isolated as single
a and b anomers,
Scheme 1. Synthesis of the azido-functionalized sugars 3-N3-Glc 16 and 3-N3-Gul
17, and the amino acid N3-ThrOBn 18. Reagents and conditions: (i) TFA 80%, rt; (ii)
Ac2O, Py, rt; (iii) NaN3, 120–130 °C; (iv) Tf2O, DCM–Py (19:1); (v) NaOMe, MeOH;
(vi) AcOH 80%, microwave heating (110 °C, 15 min); (vii) Ph3CBF4, DCM, rt; (viii)
20% piperidine/DMF, rt; (ix) Tf2O, NaN3; CuSO4, H2O, MeOH, CH2Cl2, rt.
respectively (Scheme 3). The structures of 32–35 were confirmed
by 1H NMR analysis, which showed a characteristic singlet of CH-
triazole around d 8.0, singlets relative to COCH3 and NHCOCH3
groups between d 2.2 and 1.9, with integration value of 27, besides
other characteristic signals of sugars and sialic acid units. Other
relevant evidence was the absence of the propargyl-CH signal at
d 2.43 ppm. As described for compound 19, 3JC-1,H-3ax heteronuclear
coupling constant values in the range of 8.0–8.7 Hz were verified
2.1.2. Synthesis of 2-propynyl-sialic acid 19 and 3-O-propynyl-
GalOMe 20
The synthesis of 2-propynyl-sialic acid 19 was performed by
glycosylation reaction of the chloride 28 with propargyl alcohol,
in the presence of AgOTf as catalyst, being obtained in 66% yield
after purification by column chromatography (EtOAc–Hexane
7:3) (Scheme 2).43 Alternatively, BF3ꢁEt2O-catalysed glycosylation
of the per-acetylated derivative 29 with propargyl alcohol also
gave the desired product 19 in 70% yield.44 The quite similar yields
of 19 observed for both glycosylation procedures justify the
employment of BF3ꢁEt2O-catalyzed reaction considering that it
requires one less step. The presence of propargyl functional group
in compound 19 was evident from characteristic signals of its
1H NMR spectra [d 2.43 ppm (CH) and d 4.34, 4.02 ppm (CH2)].
for sialosides 32–35, confirming their
a-anomeric configura-
tion.45,46 HRESI-MS analysis showed the characteristic adducts of
[M+Na]+ 925.28 for compounds 32, 34 and 35, and [M+H]+
903.29 for compound 33.
Compounds 32–35 were then deacetylated by treatment with
1 M NaOMe, followed by de-methyl-esterification using 0.2 M
KOH (Scheme 3).27 The resulting materials were purified using re-
verse-phase HPLC, affording the corresponding deprotected prod-
ucts 9–12 in low to moderate yields (13–47%), considering that
their purification by HPLC was not straightforward, as it required
the use of several experimental conditions. The structures of com-
pounds 9–12 were confirmed by 1H NMR spectroscopy, which
showed absence of singlets relative to CO2CH3 and COCH3 groups,
being verified only one singlet of NHCOCH3 at d 1.9 ppm.
Regarding the synthesis of compound 13, click reaction be-
The
a-anomeric configuration of 19 was confirmed with the aid
of a 2D Heteronuclear Correlation Experiment (G-BIRDR,X-CPMG-
3
HSQMBC), being verified the value of 8.0 Hz for its JC-1,H-3ax
heteronuclear coupling constant.45,46
tween N3-L-ThrOBn 18 and 3-O-propynyl-GalOMe 20 gave the
The sugar 3-O-propynyl-GalOMe 20 was synthesized from com-
per-acetylated product 36 in 48% yield after purification by column
chromatography (EtOAc–Hexane 7:3) (Scheme 3). Its 1H NMR anal-
ysis showed characteristic signals of sugar (OCOCH3, d 2.11–2.06)
and amino acid (OBn, d 5.31–5.22), as well as CH-triazole (d 7.97)
and CH2-triazole (d 4.69–4.4; J 12.4 Hz) signals. Subsequently,
removal of the benzyl ester from 36 by means of standard hydro-
genation (10% Pd–C/H2) and deacetylation reaction in the presence
of 1 M NaOMe in MeOH afforded the final deprotected product 13
in 80% yield.
mercial
a-GalOMe 30, by means of dibutylstannylene acetal for-
mation with dibutyltin oxide, followed by in situ reaction with
propargyl bromide.47–50 Subsequently, per-O-acetylation of the
2.2. Biological assays
2.2.1. 1,2,3-Triazole sialylmimetic neoglycoconjugates 9–13 as
inhibitors of Trypanosoma cruzi trans-sialidase
The enzymatic inhibition assay involving compounds 9–13
was performed using the continuous fluorimetric method, which
is based on TcTS-catalyzed hydrolysis of MuNANA.53 Compounds
9–13 were tested at 1.0 and 0.5 mM concentrations, along with
DANA as control, which is reported to be a weak TcTS inhibitor.54
According to the obtained results (Fig. 4), compounds 9 (88%), 10
(91%), 11 (67%) and 12 (69%) showed high inhibition of TcTS at
Scheme 2. Synthesis of alkyne-based sugars 2-propynyl-sialic acid 19 and 3-O-
propynyl-GalOMe 20. Reagents and conditions: (i) propargyl alcohol, AgOTf, DCM,
rt; (ii) propargyl alcohol, BF3ꢁEt2O, DCM, rt; (iii) Bu2SnO, MeOH, rt; (iv) propargyl
bromide, TBAI, toluene, rt; (v) Ac2O, Py, rt.

V. L. Campo et al. / Bioorg. Med. Chem. 20 (2012) 145–156
149
Scheme 3. Synthesis of 1,2,3-triazole-linked sialic acid and amino acid glycosides 9–13 by ‘click chemistry’. Reagents and conditions: (i) CuSO4, Na ascorbate, DMF,
microwave heating (100 °C, 15 min); (ii) NaOMe, MeOH; (iii) KOH 0.2 M; (iv) H2/10% Pd/C.
inhibition of TcTS by compounds 9–12 at 1.0 mM are in accor-
dance with docking results (Section 1), despite the fact that com-
pounds 9 and 10, containing galactosyl units, exhibited higher
activity against TcTS than compounds 11 and 12, constituted by
corresponding glucose and gulose sugar units. On the other hand,
the very low inhibition displayed by compound 13 (1.0 mM), ob-
tained from threonine amino acid in replacement of sialic acid, is
not in agreement with its docking result, indicating that sialic
acid unit may have a relevant role in TcTS inhibition. Therefore,
the significant TcTS inhibition showed by compounds 9–12,
albeit only at 1.0 mM, may direct the development of new
sialylmimetics as TcTS inhibitors, pointing out the necessity to
maintain sialic acid and galactosyl units for efficient inhibition
of this enzyme. Lastly, the increasing repertoire of biological
Figure 4. TcTS inhibition of compounds 9–13.
activities described for sialylmimetics opens up the possibility
to test compounds 9–13 against other essential therapeutic
targets, such as neuraminidase.15,16
1.0 mM, whereas almost no inhibitory activity was detected for
compound 13 (5%) at this concentration. However, when tested
at 0.5 mM concentration, only compound 10 (30%) displayed a
moderate inhibition of TcTS, being weak inhibition values
(1.5–17%) verified for the remaining compounds. The effective
2.2.2. In vitro trypanocidal activities of compounds 9–13 and
cytotoxicity towards mammalian cells
The five 1,2,3-triazole sialylmimetic neoglycoconjugates 9–13
were evaluated against trypomastigote forms of T. cruzi Tulahuen

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V. L. Campo et al. / Bioorg. Med. Chem. 20 (2012) 145–156
Figure 5. Trypanocidal activities of compounds 9–13 and reference compound benznidazole (Bz).
strain using benznidazole (N-benzyl-2-nitro-1-imidazolaceta-
mide), the current frontline drug used to treat Chagas’ disease, as
control.55
cidal activity comparable to the reference benznidazole (Bz, IC50try
125 M) in the concentrations range from 0.5 to 0.06 mM. Regard-
ing the remaining compounds, overall relative low activity (9 and
11–13) was observed, despite the high trypanocidal activity pre-
sented by 9, 11 and 13 at 0.5 mM concentration.
Concerning the mammalian cell toxicity, compounds 9–13 were
screened against cultured mouse spleen cells. According to Figure 6,
high levels of cytotoxicity were verified only for compounds 9 and
13 (above 80%) at 0.5 mM, followed by compound 10 that showed
around 55% mouse cell kill at this increased concentration, being,
however, not cytotoxic in lower concentrations. For the remaining
compounds 11 and 12 no cytotoxicity was observed in the 500–
l
Results of parasite viability, measured based on a colorimetric
reaction with Chlorophenol red-b-D-galactoside (CPRG) as sub-
strate for T. cruzi b-galactosidase, are summarised in Figure 5.56
The concentrations of compounds corresponding to 50% trypanoci-
dal activity are expressed as IC50try (Table 1). As shown in Figure 5
and Table 1, amongst the tested compounds 10 displayed higher
trypanocidal activity, presenting an IC50try value of 260 lM against
T. cruzi Tulahuen strain. In fact, this compound presented trypano-
7.8
l
M range.
Table 1
Although TcTS is essential to enabling parasite evasion of the
Trypanocidal activities of compounds 9–13 and benzni-
dazole (Bz) expressed as IC50Try
human immune response, adhesion to and invasion of host cells,
it is not known to play a role in parasite survival in culture.51 In
this context, the obtained TcTS inhibition and anti-trypanosomal
activities were not directly correlated, suggesting that the tested
compounds may act against T. cruzi by a different mechanism of
action. Furthermore, considering that significant cytotoxicity
against mouse spleen cells was observed only at high concentra-
tions of the tested compounds, a specific mode of anti-parasite
action rather than a generic cytotoxic effect may be suggested.
Compound
IC50try (mM)
9
0.475 0.054
0.26 0.018
0.726 0.073
>2
0.369 0.076
0.125 0.034
10
11
12
13
Bz
Figure 6. Percentage cell death caused by compounds 9–13 and Bz, evaluated against cultured mouse spleen cells.

V. L. Campo et al. / Bioorg. Med. Chem. 20 (2012) 145–156
151
pressure and the residue was diluted with DCM. The solution
was washed with 1 M HCl, and satd aq NaHCO3 solution, dried over
MgSO4, concentrated under reduced pressure and purified by col-
umn chromatography (EtOAc–hexane 1:1) to give product 16
3. Conclusions
In summary, we have developed the synthesis of five new
1,2,3-triazole sialylmimetic neoglycoconjugates from different
azido-derived sugars (Gal, Glc, Gul)/amino acid (Thr) and alkyne-
functionalized sialic acid/galactose as precursors. The use of
Cu(I)-catalysed azide–alkyne cycloaddition reaction proved to be
effective for generating the more resistant 1,2,3-triazole rings as
mimics of natural O-glycosidic linkages, thus representing a
valuable strategy to get a repertoire of neoglycoconjugates.
Trypanosoma cruzi trans-sialidase (TcTS) inhibition assays with
compounds 9–13 provided evidence that sialic acid and galactosyl
units are relevant for TcTS inhibition, due to higher inhibitory
activities verified for 9 and 10 at 1.0 mM, whilst trypanocidal as-
says showed higher anti-parasite activity for compound 10 (IC50try
(126 mg, 0.34 mmol, 82%) as a mixture of anomers (
a:b 1:1). dH
(CDCl3, 300 MHz). anomer: 6.25 (1H, d, J1,2 3.6 Hz, H-1), 4.98
a
(1H, t, J 10.14 Hz, H-4), 4.90 (1H, dd, J1,2 3.5 Hz, J2,3 10.6 Hz, H-2),
4.17 (1H, dd, J5,6 4.6 Hz, J
12.0 Hz, H-6), 4.05 (1H, m, H-60),
6,60
0
3.93 (1H, t, J 10.29 Hz, H-3), 3.75 (1H, ddd, J5,6 2.3 Hz, J5,6 4.6 Hz,
J4,5 9.8 Hz, H-5), 2.15–2.03 (12H,
4 s, COCH3). dC (CDCl3,
100 MHz) 171.0, 169.7, 168.6 (COCH3), 87.8 (C-1), 73.2 (C-5),
69.9 (C-2), 69.8 (C-4), 61.5 (C-6), 60.5 (C-3), 21.1–20.8 (COCH3).
b anomer: 5.63 (1H, d, J1,2 8.2 Hz, H-1), 4.99 (1H, dd, J1,2 8.2 Hz,
0
J2,3 10.14 Hz, H-2), 4.18 (1H, dd, J5,6 4.6 Hz, J 6,6 12.0 Hz, H-6), 4.04–
0
0
3.96 (3H, m, H-6 , H-4, H-5), 3.66 (1H, t, J 10.14 Hz, H-3), 2.15–2.03
(12H, 4 s, COCH3). dC (CDCl3, 100 MHz) 171.0, 169.7, 168.6 (COCH3),
91.2 (C-1), 69.5 (C-5), 67.3 (C-2), 69.7 (C-4), 64.3 (C-3), 61.5 (C-6),
21.1–20.8 (COCH3).
260 lM). Taken together, these data highlights the 1,2,3-triazole
linked sialic acid-6-O-galactose 10 as a prototype for the develop-
ment of more effective candidates against Chagas’ disease.
IR (KBr) mmax 2961, 2104, 1759, 1372, 1234, 1040 cmꢀ1
.
ESI-HRMS: calcd for C14H19N3O9Na [M+Na]+: 396.1121, found:
396.1008.
4. Experimental
4.1. General
4.2.2. 3-Azido-3-deoxy-1:2,5:6-di-O-isopropylidene-a-D-
gulofuranose 25
All chemicals were purchased as reagent grade and used with-
out further purification. Solvents were dried according to standard
To a solution of 1:2,5:6-di-O-isopropylidene-a-D-galactofura-
nose 24 (280 mg, 1.07 mmol), in anhydrous DCM (10 mL) and pyr-
idine (0.5 mL) at 0 °C was added dropwise triflic anhydride
(0.7 mL) under N2. After stirring for 45 min the reaction mixture
was washed with satd aq NaHCO3 solution, brine, dried over
MgSO4 and evaporated to an orange liquid. The obtained product
methods.57 MuNANA (20-(4-methylumbelliferyl)-
a-D-N-acetylneu-
raminic acid sodium salt), used as a donor substrate for silylation
reactions, was acquired from Toronto Research Chemicals Inc.
The trans-sialidase used in this study was a His-tagged 70 kDa re-
combinant material truncated to remove C-terminal repeats but
retaining the catalytic N-terminal domain of the enzyme.58 Reac-
tions were monitored by thin layer chromatography (TLC) on
0.25 nm precoated silica gel plates (Whatman, AL SIL G/UV, alu-
minium 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) or on a Biotage Horizon High-
Performance FLASH Chromatography system using 12 or 25 mm
flash cartridges with the eluents indicated. The microwave-
assisted reactions were carried out in a laboratorial oven (CEM
DISCOVER) using sealed tubes. HPLC purifications were performed
in the Shimadzu HPLC system using a Shim-PaK CLC-ODS (M)
semipreparative reverse phase column (250 ꢂ 10.0 mm). 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 (d) are given in parts per million
downfield from tetramethylsilane. Assignments were made with
the aid of HMQC and COSY experiments. Optical rotations were
measured at ambient temperature on a Jasco DIP-370 digital
polarimeter using a sodium lamp. Accurate mass electrospray
ionization mass spectra (ESI-HRMS) were obtained using positive
ionization mode on a Bruker Daltonics UltrOTOF-Q-ESI-TOF mass
spectrometer.
3-O-triflate-1:2,5:6-di-O-isopropylidene-a-D-galactofuranose 37
(318 mg, 0.86 mmol, 80%) was directly dissolved in dry DMF
(2.5 mL) and cooled to 0 °C and sodium azide (244 mg, 3.75 mmol)
was added. After stirring for 18 h at room temperature the reaction
mixture was diluted with DCM, washed with water and brine,
dried over MgSO4 and concentrated under reduced pressure. Puri-
fication of the obtained residue by column chromatography
(EtOAc–hexane 1:1) afforded product 25 as a pale yellow solid
(125 mg, 0.48 mmol, 55.4%). dH (CDCl3, 300 MHz) 5.55 (1H, d, J1,2
4.9 Hz, H-1), 4.63 (1H, dd, J 2.3 Hz, J 7.8 Hz, H-3), 4.33 (1H, dd, J
2.3 Hz, J 4.9 Hz, H-2), 4.20 (1H, dd, J 1.8 Hz, J 7.7 Hz, H-4), 3.91
0
0
(1H, dd, J 1.5 Hz, J 7.3 Hz, H-5), 3.51 (1H, dd, J 8.0 Hz, J 12.7 Hz,
0
0
H-6), 3.36 (1H, dd, J 5.1 Hz, J 12.7 Hz, H-6 ), 1.55 (3H, s, CH3),
1.46 (3H, s, CH3), 1.34 (6H, s, (CH3)2CO2). dC (CDCl3, 100 MHz):
96.5 (C-1), 70.8 (C-3), 70.5 (C-2), 71.4 (C-4), 67.0 (C-5), 50.9 (C-
6), 26.8, 26.5, 26.2, 24.9 (CH3)2CO2). ESI-HRMS: calcd for
C
12H20N3O5 [M+H]+: 286.1325, found: 286.1340.
4.2.3. 1,2,4,6-Tetra-O-acetyl-3-azido-3-deoxy-a,b-D-
gulopyranose 17
A solution of azido 25 (125 mg, 0.48 mmol) in 80% aqueous TFA
(1 mL) was stirred at room temperature for 1 h. The mixture was
then evaporated in vacuo and to the resulting oil were added pyr-
idine (3 mL) and acetic anhydride (2 mL). After stirring for 20 h at
room temperature, the solvents were removed under reduced
pressure and the residue was diluted with DCM. The solution
was washed with 1 M HCl, and satd aq NaHCO3 solution, dried over
MgSO4, concentrated under reduced pressure and purified by col-
umn chromatography (EtOAc–hexane 1:1) to give product 17
4.2. Synthesis
4.2.1. 1,2,4,6-Tetra-O-acetyl-3-azido-3-deoxy-a,b-D-
glucopyranose 1636a,b
(142 mg, 0.38 mmol, 80%) as a mixture of anomers a:b (0.5:1). dH
(CDCl3, 300 MHz). b anomer: 5.71 (1H, d, J1,2 8.2 Hz, H-1), 5.38–
5.30 (1H, dd, J1,2 8.2 Hz, J2,3 10.4 Hz, H-2; 1H, m, H-4), 5.08 (1H,
dd, J3,4 3.4 Hz, J2,3 10.4 Hz, H-3), 3.95 (1H, m, H-6), 3.45 (1H, dd,
A solution of azido 21 (108 mg, 0.41 mmol) in 80% aqueous TFA
(1 mL) was stirred at room temperature for 1 h. The mixture was
then evaporated in vacuo and to the resulting oil were added pyr-
idine (3 mL) and acetic anhydride (2 mL). After stirring for 12 h at
room temperature, the solvents were removed under reduced
J5,6 7.4 Hz, J6,6 12.9 Hz, H-60), 3.20 (1H, dd, J4,5 1.2 Hz, J5,6 5.4 Hz,
H-5), 2.19–1.99 (12H, 4 s, COCH3). dC (CDCl3, 100 MHz) 170.8,
0
0

152
V. L. Campo et al. / Bioorg. Med. Chem. 20 (2012) 145–156
169.6, 168.5 (COCH3), 85.7 (C-1), 72.5 (C-5), 67.2 (C-2), 65.2 (C-4),
60.2 (C-6), 58.9 (C-3), 21.3–20.7 (COCH3).
product 19 as a pale yellow amorphous solid in 70% yield
(82 mg, 0.15 mmol, 70%). [ +69.4 (c 0.5, CH3CN). dH (CDCl3,
a]
D
a
anomer: 6.40 (1H, d, J1,2 3.0 Hz, H-1), 5.48 (1H, m, H-4), 5.42–
300 MHz) 5.60 (1H, d, J5,NH, 9.6 Hz, NH), 5.47 (1H, dd, J8,9’ 3.2 Hz,
J8,9 = J7,8 6.8 Hz, H-8), 5.31 (1H, dd, J6,7 2.9 Hz, J7,8 7.3 Hz, H-7),
4.62 (1H, dd, J3a,4 3.1 Hz, J3e,4 12.3 Hz, H-4), 4.41–4.34 (1H, dd, J
2.4 Hz, J 15.7 Hz, CH2aC„CH), 4.27 (1H, dd, J8,9’ 2.7 Hz, J9,9’
12,4 Hz, H-90), 4.21–4.02 (4H, m, H-5, H-6, H-9, CH2bC„CH), 3.77
(3H, s, CO2CH3), 2.60 (1H, dd, J3e,4 4,6 Hz, J3a,3e 13,7 Hz, H-3e),
0
5.32 (2H, m, H-2, H-3), 4.23 (1H, m, H-6), 3.53 (1H, dd, J5,6 7,4 Hz,
J6,6 12.9 Hz, H-60), 3.24 (1H, dd, J4,5 1.2 Hz, J5,6 5.4 Hz, H-5), 2.19–
0
1.99 (12H, 4s, COCH3). IR (KBr) max 2935, 2112, 1759, 1410,
1254 cmꢀ1
.
ESI-HRMS: calcd for C14H19N3O9Na [M+Na]+:
396.1121, found: 396.1114.
2.43 (1H, t,
J
2.4 Hz, CH2C„CH), 2.12–1.98 (4 ꢂ 3H, 4s,
4.2.4. Azido-
The amino acid N-(9-fluorenylmethoxycarbonyl)-
benzyl ester 26 (300 mg, 0.69 mmol, prepared from commercially
L
-threonine benzyl ester 18
4 ꢂ OCOCH3), 1.95 (3H, s, NHCOCH3), 1.91 (1H, dd, J3a,4 11.2, J3a,3e
13.7, H-3a). dC (100 MHz, CDCl3): 171.0, 170.7, 170.6, 170.3,
170.0 (4 ꢂ OCOCH3 and CONH), 166.8 (C-1), 98.7 (C-2), 79.0
(C„CH), 75.3 (C„CH), 72.4 (C-6), 68.7 (C-4), 68.3 (C-8), 67.3 (C-
7), 62.4 (C-9), 52.8 (OCH3), 52.2 (OCH2C„), 49.2 (C-5), 37.1 (C-3),
23.1 (NHCOCH3), 21.0, 20.8, 20.7, 20.6 (4 ꢂ OCOCH3).
ESI-HRMS: calcd for C23H31NO13Na [M+Na]+: 552.1795, found:
552.1706.
L-threonine
available amino acid Fmoc-L-threonine by treatment with cesium
carbonate and benzyl bromide in DMF)59 was treated with 20%
piperidine/DMF (1 mL) and stirred for 20 min at room tempera-
ture. After concentration in vacuo the residue was purified by col-
umn chromatography (EtOAc/hexane 7:3 v/v; MeOH/DCM 1:9 v/v).
The obtained product
L-threonine benzyl ester 27 (112.6 mg,
0.54 mmol, 78%) was submitted to diazo transfer reaction utilizing
the method described by Wong et al.41 Triflyl azide preparation:
Sodium azide (0.6 g, 9.23 mmol) was dissolved in distilled water
(2.0 mL) with DCM (2.5 mL) and cooled on an ice bath. Triflyl anhy-
dride (0.3 mL, 1.79 mmol) was added slowly over 5 min while stir-
ring continued for 2 h. The mixture was placed in a separatory
funnel and the DCM phase was removed. The aqueous portion
was extracted with DCM and the organic fractions, containing
the triflyl azide, were pooled and washed once with saturated
4.2.6. Methyl 3-prop-2-ynyl-
a-D-galactopyranoside 31
Methyl- -galactopyranose 30 (500 mg, 2.57 mmol) was dis-
a
-D
solved in MeOH (20 mL). Subsequently, bis-dibutyltin oxide (671.
7 mg, 2.69 mmol) was added and the reaction mixture was heated
under reflux for 2 h. Then, the solvent was evaporated and the
residual solid was dried in vacuo for several hours. The obtained
white solite was diluted with toluene (20 mL) and treated with tet-
rabutylammonium iodide (949.3 mg, 2.57 mmol) and propargyl
bromide (1.2 g, 10.28 mmol, 0.9 mL). The reaction was stirred at
room temperature for 12 h, then filtered through a silica path.
The resulting organic solution was concentrated under reduced
pressure and purified by column chromatography (EtOAc–Hexane
7:3 v:v) to afford 31 (482.5 mg, 2.07 mmol, 81%) as an yellow oil.
dH (CDCl3, 500 MHz), 4.72 (1H, d, J1,2 3.8 Hz, H-1), 4.29 (2H, dd, J
2.5 Hz, J 15.8 Hz, CH2C„CH), 4.10 (1H, d, J 2.8, H-4), 3.86 (1H, dd,
NaHCO3 and used without further purification. Subsequently, L-
threonine benzyl ester 27 (112.6 mg, 0.54 mmol) was dissolved
in water and treated with CuSO4 (8.6 mg, 0.05 mmol). Methanol
(4 mL) and previously prepared triflyl azide in DCM (6 mL) were
then added, and the solution was stirred at room temperature
overnight. Solvents were then removed under reduced pressure
to give a blue-green residue which was suspended in DCM (2 ml)
and filtered through a plug of silica gel (3 cm in a Pasteur pipette);
silica gel was washed with additional DCM (10 ml) and the com-
bined filtrates were concentrated to give the product 18 as a pale
oil in 60% yield (97.3 mg, 0.41 mmol). dH (CDCl3, 500 MHz) 7.39–
7.33 (5H, m, OCH2Ph), 5.25 (2H, AB, JAB = 12.2 Hz, OCH2Ph), 4.26
0
J1,2 3.8 Hz, J2,3 9.8 Hz, H-2), 3.76 (1H, dd, J5,6 7.7 Hz, J6,6 12.0 Hz,
H-6), 3.72–3.68 (2H, m, H-60, H-5), 3,65 (1H, dd, J3,4 31 Hz, J2,3
9.8 Hz, H-3), 3.35 (3H, s, OCH3), 2,44 (1H, t, J 2.3 Hz, CH2C„CH).
dC (100 MHz, CDCl3): 99.9 (C-1), 78.9 (C„CH), 78.0 (C-3), 74.4
(C„CH), 69.2 (C-5), 68.5 (C-4), 63.4 (C-6), 63.1 (C-2), 57.4
(OCH2C„), 55.18 (OCH3).
(1H, dd, J 6.4 Hz, J 3.8 Hz, bCHThr), 3.83 (1H, d, J 3.8 Hz,
1.27 (3H, d, 6.4 Hz, CH3Thr). dC (100 MHz, CDCl3): 169.5
(COCH2Ph), 135.23 (Cquat. OCH2Ph), 129.1–128.8 (CHPh), 68.13
(OCH2Ph), 68.9 (bCHThr), 67.6 ( CHThr), 20.3 (CH3 Thr). IR (KBr)
max 3450, 2109, 1600, 1100 cmꢀ1
ESI-HRMS: calcd for
14H13N3O3Na [M+Na]+: 258.0957, found: 258.0948.
aCHThr),
ESI-HRMS: calcd for C10H16O6Na [M+Na]+: 255.0947, found:
255.0958.
J
a
4.2.7. Methyl 2,4,6-tri-O-acetyl-3-prop-2-ynyl-a-D-
galactopyranoside 20
.
C
A solution of the sugar 31 (88 mg, 0.37 mmol), anhydrous pyri-
dine (2.7 mL) and acetic anhydride (2 mL) was stirred at room tem-
perature for 20 h. After removal of solvents, the residue was
dissolved in DCM and the solution was washed with 1 M HCl, and
satd aq NaHCO3 solution, dried over Mg2SO4, concentrated under
reduced pressure and purified by column chromatography
(EtOAc–Hexane 1:1) to give 20 as a pale oil (135.74 mg, 0.37 mmol,
100%). dH (CDCl3, 500 MHz) 5.47 (1H, d, J 3.3, H-4), 5.07 (1H, dd, J1,2
3.6 Hz, J2,3 10.3 Hz, H-2), 4.96 (1H, d, J1,2 3.8 Hz, H-1), 4.20 (2H, dd, J
4.2.5. 2-Propynyl 5-acetamido-3,5-dideoxi-4,7,8,9-tetra-O-
acetyl-D-glycerol-a-D-galacto-non-2-ulopyranoside methyl ester
19
Method A. b-Acetochloroneuraminic acid 28 (221 mg,
0.43 mmol), propargyl alcohol (0.86 mmol, 50 L) and 4 Å molecu-
l
lar sieves (200 mg) in dry DCM (5 mL) was stirred at ꢀ20 °C under
N2 during 1 h before adding AgOTf (132.6 mg, 0.52 mmol) in dry
toluene (1 mL). The reaction was allowed to warm up to room
temperature. After 2 h, the solution was neutralized with triethyl-
amine, filtered through Celite and concentrated in vacuo. Column
chromatography (EtOAc–Hexane 7:3 v:v) afforded the product
19 as a pale yellow amorphous solid in 66% yield (150.3 mg,
0.28 mmol).
0
2.3 Hz, J 16.0 Hz, CH2C„CH), 4.14 (1H, dd, J5,6 4.4 Hz, J6,6 12.19 Hz,
H-6), 4.13–4.08 (3H, m, H-60, H-5, H-3), 3.40 (3H, s, OCH3), 2.45 (1H,
t, J 2.3 Hz, CH2C„CH), 2.14–2.07 (9H, 3s, COCH3). dC (100 MHz,
CDCl3): 170.9, 170.8, 170.7 (COCH3), 98.0 (C-1), 79.5 (C„CH), 75.3
(C„CH), 72.6 (C-3), 69.8 (C-2), 67.5 (C-4), 66.8 (C-5), 62.8 (C-6),
57.3 (CH2C„CH), 55.8 (OCH3), 21.4, 21.1, 20.9 (COCH3).
ESI-HRMS: calcd for C16H22O9Na [M+Na]+: 381.1264, found:
381.1156.
Method B. To a solution of the per-acetate sialic acid 29 (117 mg,
0.22 mmol) in dry DCM (2.0 mL) was added propargyl alcohol
(0.26 mmol, 16 lL) and BF3. Et2O (0.33 mmol, 40 lL) at 0 °C, being
the reaction mixture stirred at room temperature for 5 h. The
resultant solution was washed successively with dilute HCl solu-
tion, satd NaHCO3 solution, dried over MgSO4 and concentrated.
Column chromatography (EtOAc–Hexane 1:1 v:v) afforded the
4.3. General procedure for ‘click chemistry’ reactions
A solution of 2-propynyl-sialic acid 18/3-O-propynyl-GalOMe
20 (1 equiv), azido-functionalized sugars 14–17/amino acid 18

V. L. Campo et al. / Bioorg. Med. Chem. 20 (2012) 145–156
153
(1 equiv), sodium ascorbate (0.5 equiv) and CuSO4 (0.1 equiv) in
4.3.3. Methyl {5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-acetyl-
2-oxymethyl-[1-(1,2,4,6-tetra-O-acetyl- -glucopyranosid-3-
-galacto-non-2-
ulopyranoside}onate 34
Following procedure described in Section 4.3, the reaction of
2-propynyl-sialic acid 19 (30 mg, 0.05 mmol), 1,2,4,6-tetra-O-acet-
DMF (0.5 mL) was placed into a microwave tube. Then, the tube
was sealed and submitted to microwave irradiation in 15 min
bursts (100 °C, 50 W). The reaction was followed by TLC (EtOAc)
taking samples at 15 min intervals. After completion the reaction
mixture was partitioned between H2O and EtOAc and the aqueous
phase was extracted with EtOAc. The organic phase was dried over
MgSO4, filtered, concentrated under reduced pressure and purified
by column chromatography (EtOAc/EtOAc–Hexane 7:3 v:v) to af-
ford the desired 1,2,3-triazole sialylmimetic neoglycoconjugates
32–36.
a-D
yl)-1H-1,2,3-triazol-4-yl]-D-glycerol-a-D
yl-3-azido-3-deoxy-
sodium ascorbate (5.54 mg, 0.03 mmol) and CuSO4 (0.9 mg,
5.6 mol, 6 L of 1 M solution) in DMF (0.5 mL) afforded the
a,b-D-glucopyranose 16 (35 mg, 0.09 mmol),
l
l
product 34 as a pale yellow solid (38 mg, 0.04 mmol, 74%). (CD3OD,
300 MHz) 8.3 (1H, s, CH triazole), 6.42 (1H, d, J1,2 3.5 Hz, H-1), 5.72
0
(1H, J1,2 3.5 Hz, J2,3 11.2 Hz, H-2), 5.43 (1H, dd, J8,9 2.0 Hz, J8,9
4.8 Hz, H-8), 5.37 (1H, dd, J6,7 2.3 Hz, J7,8 9.3 Hz, H-7), 5.25 (1H, t,
4.3.1. Methyl {5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-acetyl-
J 10.4 Hz, H-3), 5.15 (1H, dd, J3 a,4 3.9 Hz, J3 e,4 12.9 Hz, H-40),
4.82–4.66 (2H, m, CHaHb-triazole), 4.42–4.30 (2H, m, H-6a, H-9),
4.20 (1H, dd, J5,6b 2.1 Hz, J6a,6b 10.6 Hz, H-6b), 4.14–4.03 (4H, m,
H-5, H-50, H-60, H-90), 3.99 (1H, J3,4 3.1, H-4), 3.82 (3H, s, CO2CH3),
0
0
0
2-oxymethyl-[1-(2,3,4,6-tetra-O-acetyl-b-
D
-galactopyranosyl)-
1H-1,2,3-triazol-4-yl]-D-glycerol-a-D-galacto-non-2-ulopyrano-
side}onate 32
Following procedure described in Section 4.3, the reaction of
2-propynyl-sialic acid 19 (50 mg, 0.09 mmol), azido-2,3,4,6-tetra-
2.42 (1H, dd, J3 e,4 4.2 Hz, J3 a,3 e 12.17 Hz, H-30e), 2.17–1.91 (8 ꢂ 3H,
0
0
0
0
0
8s, 4 ꢂ OCOCH3), 1.89 (3H, s, NHCOCH3), 1.87 (1H, dd, J3 a,4 4.9 Hz,
O-acetyl-1-deoxy-
dium ascorbate (9.31 mg, 0.05 mmol) and CuSO4 (1.5 mg, 9.4
9.4 L of 1 M solution) in DMF (0.5 mL) afforded the product 32 as
a-D
-galactopyranose 14 (35 mg, 0.09 mmol), so-
J3 a,3 e 14.3 Hz, H-30a). dC (100 MHz, CDCl3): 172.1–167.3
(8 ꢂ OCOCH3 and CONH), 124.2 (CH triazole), 88.1 (C-1), 70.5 (C-
7), 68.1 (C-40), 67.9 (C-2), 67.5 (C-8), 61.3 (C-9), 61.6 (C-5), 61.2
(C-50), 60.9 (C-3), 60.7 (C-60), 60.3 (C-6), 56.8 (CHaHb-triazole),
51.4 (C-4), 51.9 (OCH3), 36.4 (C-30), 21.4 (NHCOCH3), 19.7–18.7
(8 ꢂ OCOCH3). ESI-HRMS: calcd for C37H50N4O22Na [M+Na]+:
925.2917, found: 925.2878.
0
0
lmol,
l
a pale yellow solid (27.5 mg, 0.03 mmol, 32.4%). dH (CD3OD,
300 MHz) 8.16 (1H, s, CH triazole), 6.11 (1H, d, J1,2 9.3 Hz, H-1),
5.69 (1H, dd, J1,2 9.2 Hz, J2,3 10.14 Hz, H-2), 5.56 (1H, d, J3,4
0
2.8 Hz, H-4), 5.48 (1H, m, H-8), 5.42 (1H, dd, J3 a,4 3.2 Hz, J2,3
10.14 Hz, H-3), 5.35 (1H, dd, J6,7 1.8 Hz, J7,8 9.2 Hz, H-7), 4.80–
0
0
4.63 (2H, m, CHaHb-triazole), 4.58 (1H, dd, J3 a,4 4.9 Hz, J3 e,4
4.3.4. Methyl {5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-acetyl-
12.3 Hz, H-40), 4.48 (1H, t, J 5.9 Hz, H-50), 4.32 (1H, dd, J8,9 2.6 Hz,
2-oxymethyl-[1-(1,2,4,6-tetra-O-acetyl-b-
D-gulopyranosid-3-yl)-
J9,9 12.4 Hz, H-9), 4.21 (1H, m, H-60), 4.16–4.05 (3H, m, H-6a, H-
0
1H-1,2,3-triazol-4-yl]-D-glycerol-a-D-galacto-non-2-ulopyrano-
side}onate 35
Following procedure described in Section 4.3, the reaction of
2-propynyl-sialic acid 19 (26.5 mg, 0.05 mmol), 1,2,4,6-tetra-O-acet-
6b, H-90), 4.0 (1H, dd, J4,5 7.3 Hz, J5,6 9.6, H-5), 3.77 (3H, s, CO2CH3),
2.66 (1H, dd, J3 e,4 4.6 Hz, J3 a,3 e 12.7 Hz, H-30e), 2.23–1.95 (8 ꢂ 3H,
0
0
0
0
8s, 8 ꢂ OCOCH3), 1.89 (3H, s, NHCOCH3), 1.87 (1H, dd, J3 a,4
5.1 Hz, J3 a,3 e 13.5 Hz, H-30a). dC (100 MHz, CDCl3): 172.1–168.1
(8 ꢂ OCOCH3 and CONH), 123.1 (CH triazole), 85.7 (C-1), 73.9
(C-50), 70.6 (C-8), 67.9 (C-2), 67.8 (C-3), 67.4 (C-4), 67.3 (C-7),
62.1 (C-9), 61.5 (C-60), 60.6 (C-6), 57.4 (CHaHb-triazole), 57.1
(C-40), 52.06 (OCH3), 48.5 (C-5), 37.1 (C-30), 21.3 (NHCOCH3),
19.9–18.8 (8 ꢂ OCOCH3). ESI-HRMS: calcd for C37H50N4O22 Na
[M+Na]+: 925.2917, found: 925.2867.
0
0
yl-3-azido-3-deoxy-
dium ascorbate (5.0 mg, 0.02 mmol) and CuSO4 (0.8 mg, 5.0
L of1 M solution)inDMF(0.5 mL)affordedtheproduct35 as a pale
a,b-D-gulopyranose17(18.7 mg, 0.05 mmol),so-
lmol,
5
l
yellow solid (19.6 mg, 0.02 mmol, 43.4%). (CD3OD, 300 MHz) 7.98
(1H, s, CH triazole), 5.78 (1H, d, J1,2 8.1 Hz, H-1), 5.52 (1H, d, J3,4 3.1,
H-4), 5.44 (1H, m, H-7), 5.39–5.30 (2H, m, H-3, H-8), 5.25 (1H, dd,
J1,2 7.8 Hz, J2,3 10.3 Hz, H-2), 5.17 (1H, m, H-40), 4.80–4.73 (2H, m,
CHaHb-triazole), 4.70–4.52 (2H, m, H-6a, H-9), 4.26–4.18 (2H, m, H-
6b, H-60), 4.11–3.96 (3H, m, H-5, H-90, H-50), 3.82 (3H, s, CO2CH3),
4.3.2. Methyl {5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-acetyl-
-galactopyranosid-
6-yl)-1H-1,2,3-triazol-4-yl]-D-glycerol-a-D-galacto-non-2-ulo-
2.46 (1H, dd, J3 e,4 5.14 Hz, J3 a,3 e 13.10 Hz, H-30e), 2.19–1.94
2-oxymethyl-[1-(1,2,3,4-tetra-O-acetyl-
a-
D
0
0
0
0
0
0
(8 ꢂ 3H, 8s, 4 ꢂ OCOCH3), 1.85 (3H, s, NHCOCH3), 1.84 (1H, dd, J3 a,4
4.8 Hz, J3 a,3 e 13,1 Hz, H-30a). dC (100 MHz, CDCl3): 172.1–167.3
(8 ꢂ OCOCH3 and CONH), 124.3 (CH triazole), 70.5 (C-40), 68.7 (C-3),
68.4 (C-8), 67.9 (C-7), 67.7 (C-1), 68.5 (C-4), 66.9 (C-2), 61.9 (C-9),
61.7 (C-6), 61.6 (C-60), 60.8 (C-50), 56.5 (CHaHb-triazole), 50.3
(OCH3), 47.9 (C-5), 37.2 (C-30), 21.7 (NHCOCH3), 19.6–18.8
(8 ꢂ OCOCH3). ESI-HRMS: calcd for C37H50N4O22Na [M+Na]+:
925.2917, found: 925.2871.
pyranoside}onate 33
0
0
Following procedure described in Section 4.3, the reaction of
2-propynyl-sialic acid 19 (50 mg, 0.09 mmol), 1,2,3,4-tetra-O-
acetyl-6-azido-6-deoxy-
mmol), sodium ascorbate (9.31 mg, 0.05 mmol) and CuSO4
(1.5 mg, 9.4 mol, 9.4 L of 1 M solution) in DMF (0.5 mL) affor-
a-D-galactopyranose 15 (35 mg, 0.09
l
l
ded the product 33 as a pale yellow solid (29 mg, 0.03 mmol,
34%). dH (CD3OD, 300 MHz) 7.97 (1H, s, CH triazole), 5.68–4.92
(8H, m, H-1, H-4, H-3, H-2, H-8, H-7, CHaHb-triazole), 4.58 (1H,
4.3.5. Methyl 2,4,6-tri-O-acetyl-3-oxymethyl-[1-(3-hydroxy)-
butanoic acid)-1H-1,2,3-triazol-4-yl]-a-D-galactopyranoside
benzyl ester 36
dd, J3 a,4 6.2 Hz, J3 e,4 11.7 Hz, H-40), 4.48 (1H, dd, J5,6a 2.0 Hz,
0
0
9,90
J6a,6b12.7 Hz, H-6a), 4.32 (1H, dd, J8,9 2.4 Hz, J
12.3 Hz, H-9),
4.25 (1H, m, H-5), 4.20 (1H, dd, J5,6b 2.2 Hz, J6a,6b 10.8 Hz, H-6b),
Following procedure described in Section 4.3, the reaction
of azido- -threonine benzyl ester 18 (37.2 mg, 0.10 mmol),
methyl-2,4,6-tri-O-acetyl-3-prop-2-ynyl- -galactopyranose 20
4.09–3.94 (3H, m, H-50, H-90, H-60), 3.82 (3H, s, CO2CH3), 2.65
L
(1H, dd, J3 e,4 4.8 Hz, J3 a,3 e 12.3 Hz, H-30e), 2.17–1.91 (8 ꢂ 3H, 8s,
0
0
0
a
-D
0
8 ꢂ OCOCH3), 1.89 (3H, s, NHCOCH3), 1.86 (1H, dd, J3 a,4 5.1 Hz,
(24.4 mg, 0.10 mmol), sodium ascorbate (10.20 mg, 0.05 mmol)
and CuSO4 (0.01 mmol, 0.1 equiv) in DMF (0.5 mL) gave the
product 36 as a pale yellow solid (29.3 mg, 0.05 mmol, 48.3%)
after purification by column chromatography (EtOAc–Hexane
7:3 v:v). dH (CDCl3, 500 MHz) 7.97 (1H, s, CH triazole), 7.38–
7.27 (5H, m, OCH2Ph), 5.49 (1H, d, J3,4 3.6 Hz, H-4), 5.37 (1H,
J3 a,3 e 13.5 Hz, H-30a). dC (100 MHz, CDCl3): 172.5–167.9
(8 ꢂ OCOCH3 and CONH), 125.3 (CH triazole), 92.05 (C-1), 70.3
(C-5), 70.6 (C-8), 68.2 (C-4), 68.1 (C-2), 67.6 (C-3), 67.0 (C-7),
62.1 (C-9), 61.7 (C-60), 57.4 (C-6), 57.7 (CHaHb-triazole), 51.6
(OCH3), 49.5 (C-40), 47.9 (C-50), 37.4 (C-30), 21.6 (NHCOCH3),
21.4–19.2 (8 ꢂ OCOCH3). ESI-HRMS: calcd for C37H52N4O22
[M+H]+: 903.2917, found: 903.2986.
0
0
d, J 3.3 Hz,
aCHThr), 5.31–5.22 (2H, AB, JAB = 12.2 Hz, OCH2Ph),
5.04 (1H, dd, J1,2 3.6 Hz, J2,3 10.3 Hz, H-2), 4.96 (1H, d, J1,2

154
V. L. Campo et al. / Bioorg. Med. Chem. 20 (2012) 145–156
3.6 Hz, H-1), 4.84–4.69 (2H, d, J 12.4 Hz, CHaHb-triazole), 4.68
(1H, m, bCHThr), 4.12–4.02 (3H, m, H-6, H-60, H-5), 3.98 (1H,
dd, J3,4 3.3 Hz, J2,3 10.3 Hz, H-3), 3.37 (3H, s, OCH3), 2.11–2.06
(9H, 3s, COCH3), 1.06 (3H, d, J 6.4 Hz, CH3Thr). dC (100 MHz,
CDCl3) 171.5, 170.9, 170.8 (COCH3), 169.5 (COCH2Ph), 135.23
(Cquat. OCH2Ph), 129.1–128.2 (CHPh), 124.6 (CH triazole), 97.1
(C-1), 73.5 (C-3), 69.6 (C-2), 67.6 (C-4), 67.4 (OCH2Ph), 66.8
ESI-HRMS: calcd for C20H33N4O14 [M+H]+: 553.1915, found:
553.1983.
4.3.9. 5-Acetamido-3,5-dideoxy-2-oxymethyl-[1-(
a-
D-glucopyr-
anosid-3-yl)-1H-1,2,3-triazol-4-yl]-D-glycerol-a-D-galacto-non-
2-ulopyranosidic acid 11
Compound 34 (37 mg, 0.04 mmol) was reacted as described in
the general procedure (Section 4.3.6). The deprotected product
11 was obtained as colourless amorphous solid in 15.4% yield
(aCHThr), 66.5 (C-5), 64.3 (CHaHb-triazole), 63.7 (bCHThr), 61.8
(C-6), 55.4 (OCH3), 21.4–21.0 (COCH3).
ESI-HRMS: calcd for C27H36N3O12 [M+ H]+: 594.2221, found:
594.2286.
(3.4 mg, 6.1 lmol) after HPLC purification. dH (CD3OD, 300 MHz)
8.19 (1H, s, CH triazole), 5.38 (1H, d, J 3.6 Hz, H-1), 4.73 (1H, d, J
11.67 Hz, CHa triazole), 4.50 (1H, d, J 11.67 Hz, CHb triazole), 4.23
(1H, dd, J1,2 3.6 Hz, J2,3 10.6 Hz, H-2), 4.04 (1H, m, H-40), 4.02–
3.74 (6H, m, H-8, H-7, H-5, H-9a, H-60, H-50), 3.68 (2H, dd,
4.3.6. General procedure for deprotection reactions of
compounds 32–35
0
0
To a solution of 32–35 in methanol (0.5 mL) was added 1 M
NaOMe until pH 9–10 was achieved. The mixture was stirred for
3 h at room temperature, neutralized with ion exchange resin
(Dowex 50WX8-200H+), filtered and concentrated under reduced
pressure. The obtained product was treated with 0.2 M KOH
(0.5 mL) and the solution was stirred for 12 h at room temperature.
After neutralization with ion exchange resin (Dowex 50WX8–
200H+) the solution was filtered and the solvent was removed un-
der reduced pressure. The obtained deprotected compounds 9–12
were purified by HPLC on a semi-preparative C18 column (Shim-
PaK CLC-ODS (M)) using gradient elution with 0.1% aq CF3CO2H
(A)–CH3CN (B), 0–100% B in 25 min, with detection of peaks at
252 nm with a UV detector.
J5,6 5.7 Hz, J6,6 11.9 Hz, H-6a, H-6b), 3.58 (1H, m, H-9b), 2.38 (1H,
dd, J3 e,4 4.6 Hz, J
13.2 Hz, H-30e), 2.05 (3H, s, NHCOCH3),
0
0
30a,30e
1.71 (1H, t, J3 a,3 e 11.6 Hz, H-30a). dC (100 MHz, CDCl3) 174.0
(CONH), 124 (CH triazole), 85.8 (C-1), 74.4 (C-60), 72.1 (C-2), 68.9
(C-9), 68.3 (C-4), 68.15 (C-5), 66.8 (C-40), 63.3 (C-6), 62.8 (C-3),
56.2 (CHaHb-triazole), 52.3 (C-50), 39.6 (C-30), 21.8 (NHCOCH3).
0
0
ESI-HRMS: calcd for
629.1115, found 629.1106.
C
20H32N4O14ꢁ3NH4Na [M+3NH4+Na]+
4.3.10. 5-Acetamido-3,5-dideoxy-2-oxymethyl-[1-(b-
pyranosid-3-yl)-1H-1,2,3-triazol-4-yl]-D-glycerol-a-D-galacto-
non-2-ulopyranosidic acid 12
D
-gulo-
Compound 35 (19 mg, 0.02 mmol) was reacted as described in
the general procedure (Section 4.3.6). The deprotected product 12
was obtained as colourless amorphous solid in 47% yield (5.6 mg,
0.01 mmol) after HPLC purification. dH (CD3OD, 300 MHz) 7.99 (1H,
s, CH triazole), 4.59 (1H, d, J 11.4 Hz, CHa triazole), 4.53 (1H, d, J1,2
8.3 Hz, H-1), 4.45 (1H, m, H-3), 4.39 (1H, d, J 10.9 Hz, CHb triazole),
4.22 (1H, m, H-2), 4.07–3.81 (4H, m, H-7, H-8, H-40, H-60, H-9a),
3.78–3.73 (3H, m, H-6a, H-9b, H-4), 3.65–3.53 (1H, m, H-5, 1H, dd,
4.3.7. 5-Acetamido-3,5-dideoxy-2-oxymethyl-[1-(b-
D-galacto-
pyranosyl)-1H-1,2,3-triazol-4-yl]-D-glycerol-a-D-galacto-non-
2-ulopyranosidic acid 9
Compound 32 (23 mg, 0.025 mmol) was reacted as described in
the general procedure (Section 4.3.6). The deprotected product 9
was obtained as colourless amorphous solid in 13% yield (1.8 mg,
3.2 lmol) after HPLC purification. dH (CD3OD, 300 MHz) 8.14 (1H,
J5,6b 5.7 Hz, J6a,6b11.9 Hz, H-6b), 3.46 (1H, d, J5 ,6 9.6 Hz, H-50), 2.28
0
0
s, CH triazole), 5.56 (1H, d, J1,2 9.3 Hz, H-1), 4.80 (1H, d, J 12.0 Hz,
CH2a triazole), 4.53 (1H, d, J 12.0 Hz, CH2b triazole), 4.08 (1H, dd,
J1,2 9.2 Hz, J2,3 10.14 Hz, H-2), 3.95 (1H, d, J 2.8 Hz, H-4), 3.90–
3.80 (2H, m, H-8, H-60), 3.78–3.70 (3H, m, H-3, H-5, H-9), 3.68–
3.61 (3H, m, H-7, H-50, H-90), 3.54–3.43 (3H, m, H-40, H-6a, H-6b),
(1H, dd, J3 e,4 4.1 Hz, J3 a,3 e 13.4 Hz, H-30e), 1.94 (3H, s, NHCOCH3),
0
0
0
0
1.59 (1H, t, J3 a,3 e 11.9 Hz, H-30a). dC (100 MHz, CDCl3) 174.0 (CONH),
126.1 (CH triazole), 75.3 (C-1), 73.2 (C-7), 70.3 (C-8), 69.6 (C-2), 68.3
(C-9), 67.8 (C-5), 66.5 (C-40), 63.9 (C-60), 63.4 (C-4), 63.2 (C-6), 56.0
(CHaHb-triazole), 53.6 (C-3), 39.8 (C-300), 21.7 (NHCOCH3).
0
0
2.61 (1H, dd, J3 e,4 4.6 Hz, J3 a,3 e 12.6 Hz, H-30e), 1.90 (3H, s,
0
0
0
0
ESI-HRMS: calcd for
611.3515, found 611.3528.
C
20H34N4O14ꢁKNH4 [M+K+NH4+2H]+
NHCOCH3), 1.55 (1H, t, J3 a,3 e 12.0 Hz, H-30a). dC (100 MHz, CDCl3)
173.2 (CONH), 124.0 (CH triazole), 88.0 (C-1), 78.0 (C-8), 72.8 (C-
9), 71.9 (C-7), 69.7 (C-60), 69.5 (C-2), 68.2 (C-6), 68.1 (C-4), 62.7
(C-3), 62.15 (C-40), 57.5 (CHaHb-triazole), 40.0 (C-30), 21.8
(NHCOCH3).
0
0
4.3.11. Methyl 3-oxymethyl-[1-(3-hydroxy)-butanoic acid)-1H-
1,2,3-triazole-4-yl]- -galactopyranoside 13
a
-D
ESI-HRMS: calcd for C20H33N4O14 [M+H]+: 553.1915, found:
553.1987.
A solution of compound 36 (29 mg, 0.05 mmol) in MeOH
(0.5 mL) was treated with glacial AcOH (0.05 mL) and 10% Pd/C
(10 mg) for removal of the O-Bn group. The reaction mixture was
stirred and kept under H2 (ꢃ1.5 atm) for 5 h. The reaction mixture
was then filtered through Celite, concentrated in vacuo and puri-
fied by column chromatography (DCM–MeOH 9:1 v/v). The ob-
tained product (19.4 mg, 0.04 mmol, 79%) was then dissolved in
MeOH (0.5 mL) and made basic with 1 M NaOMe in MeOH. The
reaction mixture was stirred for 3 h, and then neutralized with
Dowex 50WX8-200 resin. Filtration and concentration of the reac-
tion mixture gave the product 12 (11.32 mg, 0.03 mmol, 80%) as a
white amorphous solid. dH (CDCl3, 500 MHz) 8.17 (1H, s, CH tria-
4.3.8. 5-Acetamido-3,5-dideoxy-2-oxymethyl-[1-(
a
-D
-galacto-
pyranosid-6-yl)-1H-1,2,3-triazol-4-yl]-D-glycerol-a-D-galacto-
non-2-ulopyranosidic acid 10
Compound 33 (12 mg, 0.013 mmol) was reacted as described in
the general procedure (Section 4.3.6). The deprotected product 10
was obtained as colourless amorphous solid in 45% yield (3.3 mg,
5.9 lmol) after HPLC purification. dH (CD3OD, 300 MHz) 7.95 (1H,
s, CH triazole), 4.81 (1H, d, J 12.0 Hz, CHa triazole), 4.59 (1H, d, J
12.0 Hz, CHb triazole), 4.51 (1H, d, J1,2 3.9 Hz, H-1) 4.50–3.80 (6H,
m, H-4, H-8, H-7, H-50, H-3, H-2), 3.77–3.70 (2H, m, H-9, H-90),
zole), 5.15 (1H, d, J 3.6 Hz,
aCHThr), 4.77 (2H, dd, J 7.7 Hz, J
3.65 (1H, dd, J3 a,4 4.9 Hz, J3 e,4 12.3 Hz, H-40), 3.57–3.43 (4H, m,
0
0
0
12.2 Hz, CHaHb-triazole), 4.73 (1H, d, J1,2 3.6 Hz, H-1), 4.64 (1H,
m, bCHThr), 4.10 (1H, d, J3,4 3.0 Hz, H-4), 3.92 (1H, dd, J1,2 3.8 Hz,
J2,3 10.1 Hz, H-2), 3.78–3.70 (3H, m, H-6, H-60, H-5), 3.67 (1H, dd,
J3,4 2.8 Hz, J2,3 9.8 Hz, H-3), 3.41 (3H, s, OCH3), 1.06 (3H, d, J
6.4 Hz, CH3Thr). dC (100 MHz, CDCl3) 129.3 (CH triazole), 100.4
H-5, H-60, H-6a, H-6b), 2.60 (1H, dd, J3 e,4 4.9 Hz, J3 a,3 e 12.9 Hz,
H-30e), 1.91 (3H, s, NHCOCH3), 1.66 (1H, t, J 13.41 Hz, H-30a). dC
(100 MHz, CDCl3) 173.0 (CONH), 123.7 (CH triazole), 74.2 (C-1),
70.25 (C-8), 69.9 (C-2), 69.2 (C-7), 68.2 (C-60), 67.7 (C-40), 67.4
(C-5), 66.3 (C-3), 63.2 (C-9), 62.7 (C-6), 58.9 (C-50), 53.8 (CHaHb-tri-
azole), 52.0 (C-4), 38.7 (C-30), 21.5 (NHCOCH3).
0
0
0
0
(C-1), 79.3 (C-3), 71.4 (
(bCHThr), 66.6 (C-4), 63.1 (CHaHb-triazole), 61.5 (C-6), 54.8 (OCH3).
aCHThr), 71.2 (C-5), 68.6 (C-2), 67.7

V. L. Campo et al. / Bioorg. Med. Chem. 20 (2012) 145–156
155
ESI-HRMS: calcd for C13H21N3O9ꢁKNH4 [M+K+NH4]+: 434.2434,
Brazil). We are grateful to Dr. Rose Mary Zumstein Georgetto Naal
for the fluorimeter facilities, to José Carlos Tomaz for the ESI-MS
analysis, to the Vinícius Palaretti for the NMR analysis and to Clau-
dio Rogerio Oliveira for preparing recombinant TcTS.
found: 434.2325.
–
1H NMR and ESI-HRMS spectra of compounds 32–35 and 9–12
are presented in the Supplementary data (ESI) .
– A figure related to the 2D Heteronuclear Correlation Experiment
(G-BIRDR,X-CPMG-HSQMBC), confirming the -anomeric configu-
Supplementary data
a
ration of the obtained sialosides is showed in the ESI .
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.11.022.
4.4. Biological assays
References and notes
4.4.1. Fluorimetric TcTS inhibition assays
trans-Sialidaseused in this study was a His-tagged 70 kDa recom-
binant material truncated to remove C-terminal repeats but retain-
ing the catalytic N-terminal domain of the enzyme.58 Inhibition was
assessed using thecontinuousfluorimetric assay describedby Doug-
las and co-workers.53 Briefly, the assay was performedin triplicatein
96-well plates containing phosphate buffer solution at pH 7.4
1. Previato, J. O.; Andrade, A. F.; Pessolani, M. C. V.; Mendonca-Previato, L. Mol.
Biochem. Parasitol. 1985, 16, 85.
2. Zingales, B.; Carniol, C.; de Lederkremer, R. M.; Colli, W. Mol. Biochem. Parasitol.
1987, 26, 135.
3. Cross, G. A. M.; Takle, G. B. Annu. Rev. Microbiol. 1993, 47, 385.
4. Schenkman, S.; Jiang, M. S.; Hart, G. W.; Nussenzweig, V. Cell 1991, 65,
1117.
5. de Lederkremer, R. M.; Agusti, R. Adv. Carbohydr. Chem. Biochem. 2009, 62, 311.
6. Buscaglia, C. A.; Campo, V. A.; Frasch, A. C. C.; Di Noia, J. M. Nat. Rev. Microbiol.
2006, 4, 229.
7. Mucci, J.; Risso, M. G.; Leguizamon, M. S.; Frasch, A. C. C.; Campetella, O. Cell
Microbiol. 2006, 8, 1086.
8. Buschiazzo, A.; Amaya, M. F.; Cremona, M. L.; Frasch, A. C.; Alzari, P. M. Mol. Cell
2002, 10, 757.
9. Previato, L. M.; Previato, J. O.; Jones, C.; Xavier, M. T.; Parodi, A. J.; Wait, R.;
Travassos, L. R. J. Biol. Chem. 1995, 270, 7241.
10. Vandekerckhove, F.; Schenkman, S.; de Carvalho, L. P.; Tomlinson, S.; Kiso, M.;
Yoshida, M.; Hasegawa, A.; Nussenzweig, V. Glycobiology 1992, 2, 541.
11. Kim, J. H.; Won Ryu, H.; Shim, J. H.; Park, K. H.; Withers, S. G. ChemBioChem
2009, 10, 2475.
(25
(25
l
l
L), recombinant enzyme solution(25
lL) and inhibitorsolution
L of 4.0 mM/2.0 mM solution). This mixture was incubated for
10 min at 26 °C followed by addition of MuNANA (Km = 0.68 mM53
;
25 lL of a 0.4 mM solution giving an assay concentration of 0.1 mM).
The fluorescence of the released product (Mu) was measured after
10 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 percent-
ages were calculated by the equation: % I = 100 ꢂ [1 ꢀ (Vi/V0)],
where Vi is the velocity in the presence of inhibitor and V0 is the
velocity in absence of inhibitor.
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Acknowledgements
We acknowledge financial support from FAPESP (Fundação de
Amparo à Pesquisa do Estado de São Paulo-Brazil) and the CNPq
(Conselho Nacional de Desenvolvimento Científico e Tecnológico-

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