Synthesis of divalent ligands of β-thio- And β-N-galactopyranosides and related lactosides and their evaluation as substrates and inhibitors of Trypanosoma cruzi trans-sialidase

Article abstract of DOI:10.3762/bjoc.10.324

In this work we describe the synthesis of mono- and divalent β-N - and β-S-galactopyranosides and related lactosides built on sugar scaffolds and their evaluation as substrates and inhibitors of the Trypanosoma cruzi trans-sialidase (TcTS). This enzyme catalyzes the transfer of sialic acid from an oligosaccharidic donor in the host, to parasite βGalp terminal units and it has been demonstrated that it plays an important role in the infection. Herein, the enzyme was also tested as a tool for the chemoenzymatic synthesis of sialic acid containing glycoclusters. The transfer reaction of sialic acid was performed using a recombinant TcTS and 3'-sialyllactose as sialic acid donor, in the presence of the acceptor having βGalp non reducing ends. The products were analyzed by high performance anion exchange chromatography with pulse amperometric detection (HPAEC-PAD). The ability of the different S-linked and N-linked glycosides to inhibit the sialic acid transfer reaction from 3'-sialyllactose to the natural substrate N-acetyllactosamine, was also studied. Most of the substrates behaved as good acceptors and moderate competitive inhibitors. A di-N -lactoside showed to be the strongest competitive inhibitor among the compounds tested (70% inhibition at equimolar concentration). The usefulness of the enzymatic trans-sialylation for the preparation of sialylated ligands was assessed by performing a preparative sialylation of a divalent substrate, which afforded the monosialylated compound as main product, together with the disialylated glycocluster.

Full text of DOI:10.3762/bjoc.10.324

Synthesis of divalent ligands of β-thio- and β-N-galacto-
pyranosides and related lactosides and their
evaluation as substrates and inhibitors of
Trypanosoma cruzi trans-sialidase
María Emilia Cano‡1, Rosalía Agusti‡1, Alejandro J. Cagnoni1,
María Florencia Tesoriero1, José Kovensky2, María Laura Uhrig*1
and Rosa M. de Lederkremer*1
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2014, 10, 3073–3086.
1CIHIDECAR-CONICET, Departamento de Química Orgánica,
Facultad de Ciencias Exactas y Naturales, Universidad de Buenos
Aires, Pabellón 2, Ciudad Universitaria, 1428 Buenos Aires,
Argentina, Fax: (+) 541145763346 and 2Laboratoire de Glycochimie,
des Antimicrobiens et des Agroressources (LG2A)-CNRS FRE 3517,
Université de Picardie Jules Verne, 33 rue Saint Leu, 80039 Amiens
Cedex, France
doi:10.3762/bjoc.10.324
Received: 08 October 2014
Accepted: 03 December 2014
Published: 19 December 2014
Associate Editor: S. Flitsch
Email:
© 2014 Cano et al; licensee Beilstein-Institut.
License and terms: see end of document.
María Laura Uhrig* - mluhrig@qo.fcen.uba.ar;
Rosa M. de Lederkremer* - lederk@qo.fcen.uba.ar
* Corresponding author ‡ Equal contributors
Keywords:
β-galactopyranosides; multivalent ligands; sialic acid; sugar scaffolds;
T. cruzi trans-sialidase
Abstract
In this work we describe the synthesis of mono- and divalent β-N- and β-S-galactopyranosides and related lactosides built on sugar
scaffolds and their evaluation as substrates and inhibitors of the Trypanosoma cruzi trans-sialidase (TcTS). This enzyme catalyzes
the transfer of sialic acid from an oligosaccharidic donor in the host, to parasite βGalp terminal units and it has been demonstrated
that it plays an important role in the infection. Herein, the enzyme was also tested as a tool for the chemoenzymatic synthesis of
sialic acid containing glycoclusters. The transfer reaction of sialic acid was performed using a recombinant TcTS and 3’-sialyllac-
tose as sialic acid donor, in the presence of the acceptor having βGalp non reducing ends. The products were analyzed by high
performance anion exchange chromatography with pulse amperometric detection (HPAEC-PAD). The ability of the different
S-linked and N-linked glycosides to inhibit the sialic acid transfer reaction from 3’-sialyllactose to the natural substrate N-acetyllac-
tosamine, was also studied. Most of the substrates behaved as good acceptors and moderate competitive inhibitors. A di-N-lacto-
side showed to be the strongest competitive inhibitor among the compounds tested (70% inhibition at equimolar concentration). The
usefulness of the enzymatic trans-sialylation for the preparation of sialylated ligands was assessed by performing a preparative
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sialylation of a divalent substrate, which afforded the monosialylated compound as main product, together with the disialylated
glycocluster.
Introduction
Trypanosoma cruzi, the agent of American trypanosomiasis, of 1,6-linked cyclic pseudo-galacto oligosaccharides and their
affects millions of people in Latin America [1,2] and is trans- in vitro sialylation by recombinant TcTS [30]. Conjugation of
mitted to animals, including humans, by triatomine insects. The lactose analogs with multiarm poly(ethylene glycol) increases
parasite is passed from the mother to the fetus during preg- the bioavailability in vivo [31]. Triazole-substituted β-galacto-
nancy, and also by blood transfusions and organ transplants [3]. pyranosides and triazole-sialyl mimetics have been synthesized
North American and European countries are now at risk as a by click chemistry and their inhibitory activity on the hydrol-
consequence of globalization and immigration, as Chagas’ ysis of 2’-(4-methylumbelliferyl)-β-D-N-acetylneuraminic acid
disease is usually not tested in blood banks [4-6].
by TcTS and trypanocidal activity were evaluated [32,33].
Terminal β-galactopyranosides (βGalp) present in T. cruzi In the present work, we selected thioglycosidic and N-glyco-
mucins play an important role in the interaction between the sidic bonds to link the acceptor sugars to a platform by click
parasite and the host since they are the acceptors for sialic acid chemistry, taking into consideration that they are highly resis-
transferred by the unique trans-sialidase (TcTS) [7-9] from host tant to enzymatic hydrolysis [34,35]. We have previously
cells instead of using a sialyltransferase and the donor described the synthesis of multivalent β-thiogalactopyranosides
nucleotide CMP-sialic acid [10]. Although TcTS can be consid- and their inhibitory activity against the β-galactosidase from
ered as “promiscuous” with respect to the sialyl donor and the E. coli [36,37]. The study of β-galactopyranosides as acceptor
β-galactopyranoside acceptor, it should be noted that the reac- substrates for sialic acid is in general concomitant with the
tion is in fact specific in vivo. Only sialic acid-linked α(2→3) to study of their inhibitory properties, as they usually behave as
β-galactopyranosides in glycoconjugates is transferred to competitive acceptors. Thus, both aspects have been explored,
terminal β-galactopyranoside units in the acceptor substrate, to even though our main goal was the use of the TcTS enzyme as a
construct the same type of linkage [11,12]. TcTS also transfers, tool for the synthesis of sialylated biantennary β-N- and β-S-
efficiently, α(2→3)-linked N-glycolylneuraminic acid to galactopyranosides and related lactosides. On the other hand,
terminal βGalp groups [13,14].
we considered imperative the purification and characterization
of the sialylated products, an aspect that has not been often
The search of efficient inhibitors for TcTS is an attractive field exploited in previous reports. A preparative method based on
of research not only for their potential use for chemotherapy, anion exchange chromatography using AG1X2 resin, followed
since there is no equivalent enzymatic activity in the human by analytical HPAE-PAD chromatography was optimized. The
host, but also because it could provide a tool for probing the inhibitory behavior of the substrates for the transfer of sialic
biological functions of the enzyme. Given the 3D structure of acid to the natural acceptor N-acetyllactosamine was also evalu-
TcTS [15-18], inhibitors may be directed to the sialic acid ated.
binding site or to the galactose acceptor site. Inhibitors of TcTS
binding to the βGalp acceptor site would be highly selective, as Results and Discussion
other sialidases lack this interaction. In this direction, a group of As part of our project on the synthesis and biological evalua-
octyl β-galactopyranosides and octyl N-acetyllactosaminides tion of multivalent ligands, two families of mono- and divalent
were described as substrates as well as inhibitors of the enzyme structures were synthesized in order to study their ability as
[19]. Also, the synthesis of the mucin oligosaccharides allowed acceptors or inhibitors of the reaction catalyzed by the T. cruzi
the study of their acceptor and inhibitory properties [20,21]. trans-sialidase. Taking into consideration that mainly ester but
Lactose derivatives were shown to be good inhibitors of the also glycosidic linkages are labile in biological fluids, we
transfer of sialic acid to the natural acceptor, N-acetyllac- choose amide and thioglycosidic bonds to attach the sugar
tosamine (LN) [22]. In particular, lactitol efficiently controlled residues to the platforms.
the apoptosis triggered by TcTS [23]. The synthesis of multiva-
lent glycoclusters designed to be high affinity ligands for 2,3,4,6-Tetra-O-acetyl-β-D-galactosylamine (1) was obtained
specific proteins has been an active area of research during the by catalytic hydrogenation of the azide precursor and then
last years [24-28]. Among them, tetravalent glycoclusters treated with succinic anhydride to afford 2 in excellent yield,
bearing β-lactosyl residues showed to have trypanocidal activity using a methodology similar to that previously described [38].
[29]. On the other hand, a recent paper described the synthesis Reaction with propargylamine in the presence of DCC yielded
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precursor 3. A similar procedure applied to the lactosylamine the 13C spectrum and anomeric carbons of the αGlc (96.8 ppm)
derivative 4, led to compound 6 (Scheme 1). On the other hand, scaffold, and the N-linked residue (78.5 ppm for the βNGal of
thiolactose derivative 8 was prepared by reaction of the thio- 10 and 78.2 for the βNGlc moiety of 12) were clearly distin-
uronium salt 7 [37] and propargyl bromide in the presence of guishable. When precursors 3 and 6 reacted with α,α-trehalose
triethylamine.
diazide derivative 14, the two divalent acetylated products 15
and 17 were respectively obtained (Scheme 2B). As a conse-
Compounds 3, 6 and 8, functionalized with terminal alkynyl quence of the symmetry of these trehalose-based divalent prod-
residues, were convenient precursors for the synthesis of mono- ucts, the NMR spectra of 15 showed to be similar to those of
and divalent ligands based on the azide scaffolds 9 and 14, 10, with the exception of the signal of the anomeric CH3O
readily available in our laboratory [36,37]. Thus, by cycloaddi- group in the case of 10. The same observation applied for NMR
tion reaction of monoazide 9 and N-galactopyranoside 3 or spectra of compound 17, with respect to those of 12.
N-lactoside 6, two monovalent derivatives (10 and 12, respect-
ively) were obtained after purification by column chromatog- On the other hand, β-thiolactosides 19 and 21 were prepared
raphy (Scheme 2A). The 1H NMR spectra showed the diag- (Scheme 3). Again, they showed very similar NMR spectra. For
nostic signals corresponding to the aromatic protons of the tria- example, in the 13C spectra the anomeric signals appeared at
zole ring (≈7.65 ppm), as well as the anomeric signals. For ca. 101 ppm, 91–97 ppm and 82 ppm, corresponding respective-
compound 10, the H-1 of the βNGal appeared at 5.22 ppm (J ≈ ly to the terminal βGal, αGlc from the scaffold and the βSGlc
9.1 Hz) and the H-1 of the αGlc at 4.92 ppm (J = 3.6 Hz). In the residues.
case of 12, an additional anomeric signal corresponding to the
terminal βGal residue was observed at 4.46 ppm (J = 7.9 Hz). N-linked precursors 10, 12, 15 and 17 and S-glycosides 19 and
Triazole carbon signals appeared at ca. 145.0 and 124.0 ppm in 21 were treated with base in mild conditions (TEA/MeOH/
Scheme 1: Synthesis of the alkynyl precursors 3, 6 and 8.
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Scheme 2: Synthesis of the mono-(A)- and di-(B)-N-galactopyranosides and lactosides.
H2O), to give the final deacetylated products. The solutions Thiolactosides 20 and 22, together with the thiogalactosides 23
were desalted using a mixed bed exchange resin and purified by and 24 previously reported (Table 1 and Table 2) [36], consti-
passing through a reversed phase mini-column. The NMR tuted thio-linked analogs to the above mentioned N-linked
spectra of the monovalent ligands 11, 13 and 20, as well as derivatives, and thus, a set of 8 structurally related mono- and
those corresponding to the divalent 16, 18 and 22 confirmed divalent acceptors was available to study the trans-sialylation
their identity and purity.
reaction.
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Scheme 3: Synthesis of the mono- and di-S-lactosides.
prolonging the incubation times. It should be noted that,
remarkably, in the case of compound 18, almost one third of the
molecules incorporated two sialyl residues. In a pioneering
work using TcTS to sialylate radiolabeled alditols obtained
from parasite mucins, analysis by paper electrophoresis showed
Mono- and divalent β-N- and β-S-galacto-
pyranosides and related lactosides as sialic
acid acceptors and inhibitors in the trans-
sialidase reaction
The N-galactopyranoside 11, N-lactoside 13, S-galactopyra- that, after incorporation of the first sialyl residue, the incorpor-
noside 23 [36], S-lactoside 20 (Table 1) and the divalent analo- ation of sialic acid on a second galactosyl unit in the same
gous 16, 18, 24 [36] and 22 (Table 2) were first analyzed as molecule was highly reduced [39]. It was then inferred that
acceptor substrates for TcTS. The reaction is depicted for sub- sialylation of one residue in T. cruzi glycans modulates the
strate 18 (Scheme 4). Conditions for incubations were as previ- susceptibility of nearby sites, and thus, polysialylated complex
ously described [22] using 1 mM of 3’-sialyllactose (SL) as multiantennary glycans would not be reachable by using the
donor and 1 mM of the substrate if not otherwise indicated. The enzyme. Our results suggest that the amount of disialylated
reaction was analyzed by high pH anion exchange chromatog- glycoclusters obtained (mainly in the case of 18, and also for
raphy with pulse amperometric detection (HPAEC-PAD) as 16, although at a lesser extent) is related to the structure of the
shown in Figure 1 for the sialylation of 18. In all cases, the acceptor and the experimental conditions. When both arms of
reaction was fast and reached the equilibrium in about 15 min. the divalent precursors are sufficiently distant one from the
All the compounds were good acceptors of sialic acid (Table 1 other, they may be independently available for the enzyme.
and Table 2). As expected, the new sialylated compounds were Since determinations were performed under equilibrium condi-
more retained in the anion exchange column than the original tions, we cannot rule out the possibility that the percentages
substrates. In the case of the divalent compounds 16, 18, 24 and obtained also depend on the stability of the sialylated products
22 the disialylated compounds were also observed as minor that could act as donor substrates. However, incubations
products with the highest retention times. The extent of total performed at different times between 15 and 120 min gave very
sialylation reached about 60% for most of the divalent similar results (not shown). A steric effect operating on the
substrates (Table 2). To evaluate the disialylated species sialylation of multiple Galp residues has been previously
obtained from divalent substrates, experiments using 2 equiva- suggested for lactosyl lipids attached to membrane
lents of SL were performed (Table 2 and Figure 1C). Although microdomains [40]. The dependence of the amount of disialyla-
the disialylated compound was always the minor product an tion of the divalent glycans on the concentration of 3’-sialyllac-
increase in the ratio between di- and monosialylated derivatives tose in the incubation mixture was shown using equimolar or
was evident. No significant changes were observed by stoichiometric ratios of SL to acceptor (Table 2).
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Table 1: Evaluation of monovalent substrates in the TcTS reaction.
Entry
Compound
Transfer (%)a
Inhibition (%)
1
2
3
4
11
13
23
20
47
41
52
55
26
32
35
41
aCalculated by integration of the peaks of all sialylated compounds observed in the HPAEC.
Table 2: Evaluation of the divalent substrates in the TcTS reaction.
X
SL:X = 1:1
SL:X = 2:1
SL:X = 1:1
% Xa
% S-Xa
% S2-Xa
% Xa
% S-Xa
% S2-Xa
Inhibition (%)
16
18
24
22
35
40
53
46
57
40
41
46
8
20
12
8
29
33
41
29
49
39
47
58
22
28
12
13
16
70
48
53
aRelated to the total amount of X (X + SX + S2X) by integration of the peaks observed in the HPAEC. X, compound tested, S-X, monosialylated com-
pound X, S2-X, disialylated compound X, SL, sialyllactose, SL:X indicates de molar ratio of SL with respect to X.
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Figure 1: Analysis of 18 as acceptor substrate of TcTS. A: 18 (1 mM) and 3’-sialyllactose (SL, 1 mM), without enzyme; B: 18 (1 mM) was incubated
with SL (1 mM) and TcTS for 15 min at 25 °C; C: the same as B but using 2 equivalents of SL (2 mM). The incubation mixtures were analyzed by
HPAEC using a CarboPac PA-10 ion exchange analytical column eluted with a linear gradient over 30 min from 20 to 200 mM NaAcO in 100 mM
NaOH at a flow rate of 0.9 mL/min. Structures for compounds 18, 25 and 26 are shown in Scheme 4.
The inhibition of the sialylation of the natural substrate, per lactose residue than the monovalent 20, showing that not
N-acetyllactosamine (LN), by the synthetic derivatives was also even a statistical effect on the inhibition is operative. This result
studied (Table 1 and Table 2). Equimolar amounts of SL, the may be a consequence of the linker structure, a fact that can be
natural acceptor LN, and the potential inhibitors were incu- also playing a role in the proportion of sialylated species listed
bated with TcTS and the reaction mixtures analyzed by HPAEC above (Table 1 and Table 2). On the other hand, the multivalent
and compared with the sialylation of LN in absence of the effect for the inhibition of certain glycosidases was recently
inhibitor. An example is shown in Figure 2 for compounds 13 described [41,42]. To our knowledge, there is only one previous
and 18. The best competitive inhibitor was compound 18 which report on the inhibition properties of multivalent ligands on the
reached 70% of inhibition of transfer to LN (Table 2). In fact, TcTS [31].
18 is a divalent compound, and so, the concentration of lactosyl
groups can be considered as twice as that of 13, which showed a It should be noted that by using SL as donor substrate and quan-
32% inhibition (Table 1). Therefore, there is no multivalent tifying the new sialylated compounds we assess that only the
effect in the inhibition of the sialylation of LN. On the other trans-sialidase activity is measured, and not an alternative
hand, when comparing monovalent 20 (41% inhibition) and sialidase activity. Only traces of free sialic acid have been
divalent 22 (53% inhibition), the latter is actually less effective detected (Figure 2A).
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Scheme 4: Sialylation of 18. SL: sialyllactose.
In order to prove the usefulness of the trans-sialidase reaction anomeric region, a doublet corresponding to both anomeric
for the synthesis of sialylated derivatives, a preparative reaction protons of the two indistinguishable β-N-linked Glc residues
was performed with the divalent N-lactoside 18, as it was appeared at 4.89 ppm (J = 9.3 Hz), which correlated to a 13C
shown to be the most sensitive, among the compounds tested, to anomeric signal at 79.3 ppm in the HSQC spectrum. The two
the concentration of the SL used as donor (Scheme 4). The reac- unresolved signals of both anomeric protons of αGlc residues of
tion mixture, containing unreacted 18 and sialyl derivatives 25 trehalose (T) were observed at 4.49 ppm (J = 3.9 Hz) and the
and 26, was purified using an AG1X2 (acetate form) resin corresponding signal at 93.4 ppm in the 13C spectrum was
column. After elution of neutral compounds with water, acidic detected. Finally, two spots were observed at 4.44 and 4.36 ppm
derivatives were eluted with different concentrations of pyri- which correlated with signals at 102.6 and 103.1 ppm, respect-
dinium acetate buffer. The eluted fractions were monitored by ively. These signals can be ascribed to both βGal residues, one
HPAEC. The fractions containing the monosialylated product, of which is sialylated [(NeuNAc)βGal and terminal βGal
which appeared as a single peak at 18 min, were pooled, and 25 (Supporting Information File 2)]. The appearance of signals at
was characterized on the basis of the 1H NMR and two-dimen- 2.66 ppm (H-3eq) and 1.72 ppm (H-3ax), which correlated with
sional HSQC spectra, by comparison with the spectrum of 18 a signal at 34.4 ppm (C-3, NeuNAc) in the 13C spectrum, was
(Figure 3A and Supporting Information File 2). The 1H NMR also diagnostic of the sialic acid residue. Also, a singlet at
spectrum of 25 was complex, but diagnostic signals were 1.95 ppm, corresponding to the CH3CON group was observed.
detected (Figure 3B and Supporting Information File 2). In the The structure of 25 was also confirmed by HRMS (ESI) with
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Beilstein J. Org. Chem. 2014, 10, 3073–3086.
Figure 2: Inhibition of sialylation of LN by compounds 13 and 18. A: N-acetyllactosamine (LN, 1 mM), 3’-sialyllactose (SL, 1 mM) and TcTS were incu-
bated for 15 min at 25 °C. B: The same as A, in the presence of 13 (1 mM) as inhibitor. C: The same as A, in the presence of 18 (1 mM) as inhibitor.
The incubation mixtures were analyzed by HPAEC using a CarboPac PA-10 ion exchange analytical column eluted with a linear gradient over 30 min
from 20 to 200 mM NaAcO in 100 mM NaOH at a flow rate of 0.9 mL/min. L: lactose; SA: sialic acid; SLN: sialyl N-acetyllactosamine; S13: monosialyl
compound 13.
the presence of a peak at m/z 842.7806, corresponding to the a single peak at 26 min in the HPAEC. Although only
[M + 2Na]2+ cation.
2 mg of compound 26 were obtained, analysis by ESIMS was
possible, and a peak at m/z 988.3291, corresponding to the
Further elution of the anion exchange column with 500 mM [M + 2Na]2+ cation, was observed consistent with the proposed
AcOPy gave disialylated compound 26, which appeared as structure.
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Beilstein J. Org. Chem. 2014, 10, 3073–3086.
Figure 3: Comparison of the 1H NMR spectra of 18 (A) and the sialylated derivative 25 (B).
Conclusion
lated, which means that both arms are accessible to the enzyme.
Mono- and bivalent β-N and β-S-galactopyranosides and lacto- By increasing the proportion of SL used as donor, a higher yield
sides supported on sugar scaffolds were synthesized by a of disialylated products could be obtained and thus, this ap-
convergent approach using the CuAAC reaction. Monovalent as proach can be envisaged as a chemoenzymatic methodology for
well as divalent compounds were shown to be good acceptors of the synthesis of sialylated biantennary sugar derivatives. All the
sialic acid residues. Divalent substrates could also be disialy- compounds tested were shown to be competitive inhibitors for
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Beilstein J. Org. Chem. 2014, 10, 3073–3086.
the sialylation of the natural acceptor N-acetyllactosamine by Hz, 1H, H-5´), 3.78 (t, J3,4 = 8.8, J4,5 = 9.9 Hz, 1H, H-4), 3.74
TcTS. The best result was obtained for compound 18 which (ddd, J5,6a = 1.6, J5,6b = 4.1, J4,5 = 10.1, 1H, H-5), 2.59–2.45
showed a 70% inhibition when equimolar amounts of substrates (m, 4H, CH2-CH2), 2.15, 2.12, 2.06, 2.04 (3×), 2.03, 2.01 (2×),
and inhibitor were used. The divalent N- and S-β-galacto- 1.96 (10 s, 30H, CH3CO); 13C NMR (125 MHz, CDCl3) δ
pyranosides and their sialylated products are potentially useful 172.7, 171.8, 171.1, 170.5 (2×), 170.3 (2×), 170.2, 170.0, 169.9,
as inhibitors of other clinically relevant receptors of β-galacto- 169.6, 169.1 (COCH3), 145.2 (C-4 triazole), 124.1 (C-5 tria-
side or sialic acid binding proteins. On the other hand, due to zole), 101.1 (C-1´), 91.7 (C-1T), 78.1 (C-1), 76.0 (C-4), 74.5
the ability of the compounds synthesized to inhibit the sialic (C-5), 72.9 (C-3), 71.1 (C-3´), 71.0 (C-2), 70.8 (C-5´), 69.9
acid transfer reaction from 3’-sialyllactose to the natural sub- (C-4T) 69.5 (C-5T), 69.4 (C-3T), 69.1 (C-2´), 69.0 (C-2T), 66.7
strate N-acetyllactosamine, they are potential candidates for (C-4´), 62.0 (C-6), 60.9 (C-6´), 50.8 (C-6T), 35.2 (CH2NH),
chemotherapy of Chagas’ disease, since TcTS is a fundamental 31.4, 30.8 (CH2-CH2), 21.0 (2×), 20.8 (6×), 20.7 (2×)
enzyme in the infection process.
(CH3CO-); anal. calcd for C90H120N10O53·2H2O: C, 48.56; H,
5.61; N, 6.29; found: C, 48.20; H, 5.59; N, 6.01. HRMS–ESI
(m/z): [M + H]+ calcd for C90H121N10O53, 2189.7075; found,
Experimental
The synthetic general methods are described in the Supporting 2189.7081.
Information File 1.
General procedure for O-deacetylation
Synthesis of compounds 10, 12, 15, 17, 20 and 22. General Compounds 10, 12, 15, 17, 19 and 21 (0.10 mmol) were
procedure for the click reaction [43,44]. The corresponding deacetylated by treatment with a solution of Et3N/MeOH/H2O
azido-saccharides 9 or 14 [34] (0.20 mmol) and N-linked glyco- 1:4:5 as previously described [45]. Further purification by a
sides 3 or 6, or S-linked lactoside 8 (0.20 mmol per mol of mixed bed ion-exchange resin and an octadecyl (C18) mini
reacting azide) were dissolved in 2.5 mL of a dioxane/H2O mix- column was accomplished. Purity was checked by TLC
ture (8:2). Copper sulfate (0.05 mmol per mol of reacting azide) (n-BuOH/EtOH/H2O, 2.5:1:1 or 1:1:1) and the corresponding
and sodium ascorbate (0.10 mmol per mol of azide reacting Rf are indicated in each case.
group) were added, and the mixture was stirred at 70 °C under
microwave irradiation during 50 min. The mixture was then Compound 18: Yield: 126 mg, 93%; [α]D20 +40.6 (c 0.6,
poured into a 1:1 H2O/NH4Cl solution (20 mL) and extracted H2O); Rf 0.34 (BuOH/EtOH/H2O 1:1:1); 1H NMR (500 MHz,
with EtOAc (4 × 15 mL). The organic layer was dried CDCl3) δ 7.80 (s, 1H, H-triazole), 4.89 (d, J1,2 = 9.2 Hz, 1H,
(Na2SO4), filtered, and the solvent was removed under reduced H-1), 4.71 (dd, J5T,6aT = 2.0, J6aT,6bT = 14.5 Hz, 1H, H-6aT),
pressure. The residue was purified by flash chromatography, 4.49 (d, J1T,2T = 3.9 Hz, 1H, H-1T), 4.45 (dd, J5T,6aT = 8.0,
using the solvent system indicated in each case.
J6aT,6bT = 14.5 Hz, 1H, H-6bT), 4.36 (d, J1´,2´ ≈ 7.8 Hz, 1H,
H-1´), 4.35 (s, 2H, NH-CH2), 3.93 (ddd, J5T,6aT = 2.2, J5T,6bT =
Compound 17: Compound 17 was obtained by reaction of 7.9, J4T,5T = 10.2 Hz, 1H, H-5T), 3.72–3.55 (m, 9H, H-3´, H-3,
alkyne 6 and diazide 14. Yield: 193 mg, 44%; mp 151–152 °C; H-3T, H-4, H-5´, H-5, H-6a´, H-6b´, H-6b), 3.45 (dd, J1´,2´ =
[α]D20 +12.5 (c 1.0, CHCl3); Rf 0.18 (EtOAc/MeOH 9:1); 1H 7.8, J2´,3´ = 9.8 Hz, 1H, H-2´), 3.36 (dd, J1T,2T = 3.9, J2T,3T =
NMR (500 MHz, CDCl3) δ 7.61 (H-triazole), 7.19 (d, J1,NH = 9.9 Hz, 1H, H-2T), 3.34 (t, J1,2 = J2,3 = 8.8 Hz, 1H, H-2), 3.12
9.3 Hz, 1H, NH), 6.69 (t, JCH2,NH = 5.4 Hz, 1H, NH), 5.41 (t, (t, J3T,4T = J4T,5T = 9.5 Hz, 1H, H-4T), 2.59–2.47 (m, 4H, CH2-
J3T,4T = J2T,3T = 9.7 Hz, 1H, H-3T), 5.34 (dd, J4´,5´ = 0.7, J3´,4´ CH2); 13C NMR (125 MHz, D2O) δ 175.9, 174.6 (CO), 144.7
= 3.4 Hz, 1H, H-4´), 5.27 (t, J2,3 = J3,4 = 9.2 Hz, 1H, H-3), 5.24 (C-4 triazole), 124.7 (C-5 triazole), 102.9 (C-1´), 93.3 (C-1T),
(t, J1,2 = J1,NH = 9.3 Hz, 1H, H-1), 5.09 (dd, J1´,2´ = 7.9, J2´,3´ = 79.1 (C-1), 77.8, 76.3, 75.3, 75.0, 72.6, 72.5, 71.5, 70.9, 70.8
10.4 Hz, 1H, H-2´), 4.96 (dd, J1T,2T = 3.8, J2T,3T = 9.9 Hz, 1H, (C-2´, C-2, C-2T, C-3´, C-3, C-3T, C-4, C-5´, C-5), 70.7
H-2T), 4.94 (dd, J3´,4´ = 3.5, J2´,3´ = 10.4 Hz, 1H, H-3´), 4.92 (t, (C-4T), 70.4 (C-5T), 68.5 (C-4´), 61.0 (C-6´), 59.9 (C-6), 50.8
J3T,4T = J4T,5T = 9.7 Hz, 1H, H-4T), 4.87 (t, J1,2 = J2,3 = 9.5 (C-6T), 34.4 (NH-CH2), 30.8, 30.4 (CH2-CH2); Anal. calcd for
Hz, 1H, H-2), 4.74 (d, J1T,2T = 3.8 Hz, 1H, H-1T), 4.59 (dd, C50H80N10O33·2H2O: C, 43.35; H, 6.11; N, 10.11; found: C,
JCH2,NH = 5.0, Jgem = 15.0 Hz, 1H, CH2N), 4.54 (dd, J5T,6aT = 43.04; H, 5.95; N, 9.80; HRMS–ESI (m/z): [M + Na]+ calcd for
0.9, J6aT,6bT = 13.9 Hz, 1H, H-6aT), 4.46 (d, J1´,2´ = 7.9 Hz, C50H80N10O33Na, 1371.4781; found, 1371.4797.
1H, H-1´), 4.40 (dd, J5,6a = 1.4, J6a,6b = 12.1 Hz, 1H, H-6a),
4.33 (dd, JCH2,NH = 4.5, Jgem = 15.0 Hz, 1H, CH2N), 4.26 (dd, Enzyme catalysis
J5T,6bT = 9.4, J6aT,6bT = 14.5 Hz, 1H, H-6bT), 4.14 (dd, J5,6a´ = Compounds 11, 13, 16, 18, 20, and 22–24 were incubated with
6.2, J6a´,6b´ = 11.1 Hz, 1H, H-6a´), 4.09–4.02 (m, 3H, H-5T, TcTS in 20 mM Tris–HCl, pH 7 buffer, 30 mM NaCl,
H-6b, H-6b´), 3.86 (ddd, J4´,5´ = 0.7, J5´,6a´ = 6.7, J5´,6b´ = 7.2 containing 1 mM 3’-sialyllactose as donor, in a similar manner
3083

Beilstein J. Org. Chem. 2014, 10, 3073–3086.
as described before [46]. Analysis of the reaction mixture was 4.6, 12.3 Hz, 1H, H-3eq-NeuNAc), 2.58–2.47 (m, 8H, 4 ×
performed by HPAEC-PAD. For comparison of their capacity CH2), 1.93 (s, 3H, CH3CON), 1.72 (t, J = 12.3 Hz, 1H, H-3ax-
to act as acceptors, 1 mM of each, SL and mono- or divalent NeuNAc); 13C NMR (125 MHz, D2O), partial assignments
substrates were used. In the case of divalent substrates, an assisted by the HSQC spectrum: δ 124.8 (CH-triazole), 103.1
experiment using a 2-fold excess of SL (2 mM) was also carried (C-1-(NeuNAc)βGal*), 102.6 (C-1-βGal*), 93.4 (C-1T), 79.3
out. The percentage of sialylation was calculated by integration (C-1-βNGlc), 77.8, 77.7, 76.3, 75.4 (C-6-NeuNAc), 75.1, 72.6,
of all the sialylated species present (in the case of monovalent 72.5, 71.6, 71.4 (C-2T*), 70.9, 70.8 (C-4T), 70.6 (C-2-βNGlc*),
compounds) or by integration of mono-, di- and non-sialylated 70.4, 70.4 (C-5T), 69.3, 68.4, 68.1 (2×), 67.5, 62.6 (2×), 61.0,
remaining species (in the case of divalent compounds).
59.8 (2×), 51.6, 50.8 (C-6T), 39.4 (C-3-NeuNAc), 34.4 (CH2N),
30.7, 30.4 (CH2-CH2), 22.2 (CH3CON); ESIMS (m/z):
Inhibition of sialylation of N-acetyllactosamine [M + 2Na]2+ calcd for C61H97N11Na2O41, 842.7814; found,
The inhibition experiments were performed as described before 842.7806.
[22]. Briefly, monovalent compounds 11, 13, 20, and 23, or
divalent 16, 18, 22, and 24, (1 mM) were incubated in 20 mM Disialylated compound 26 (2 mg) was obtained by using the
Tris–HCl, pH 7 buffer (20 µL), 30 mM NaCl, containing 1 mM anion exchange column and elution with 500 mM AcOPy:
3’-sialyllactose as donor, 1 mM N-acetyllactosamine, and ESIMS (m/z): [M + 2Na]2+ calcd for C72H114N12Na2O49:
recombinant TcTS (300 ng) for 15 min at room temperature. 988.3291; found: 988.3291.
After dilution with deionized water, analysis by HPAEC-PAD
was performed. Inhibition was calculated considering the
Supporting Information
amount of 3’-sialyl-N-acetyllactosamine with respect to the
total amount of sialylated compounds, obtained with or without
Supporting Information File 1
inhibitor.
Experimental section and data of 1H and 13C NMR spectra
of compounds 2, 3, 5, 6, 8, 10–13, 15–22 and 25.
[http://www.beilstein-journals.org/bjoc/content/
supplementary/1860-5397-10-324-S1.pdf]
Preparative sialylation of compound 18
Compound 18 (10 mg, 6.5 μmol) and SL (9.0 mg, 14 μmol)
were incubated with 13 μg of recombinant TcTS in 0.2 mL of
20 mM Tris buffer pH 7.6 containing 30 mM NaCl for 14 h at
25 °C. The reaction mixture was analyzed by HPAEC. The
sialylated products were purified by passing through an anion
exchange resin (AG1X2, acetate form, BioRad, 1.2 × 15 cm).
Neutral compounds, namely 18 and lactose, were eluted with
H2O and sialylated compounds with a stepped gradient from
50 mM to 500 mM pyridinium acetate buffer pH 5.4. Fractions
Supporting Information File 2
Copies of 1H and 13C NMR spectra of compounds 2, 3, 5,
6, 8, 10–13, 15–22 and 25.
[http://www.beilstein-journals.org/bjoc/content/
supplementary/1860-5397-10-324-S2.pdf]
(1.5 mL) were collected and analyzed by HPAEC. Compound Acknowledgements
25, the product of sialylation of 18, was eluted with 100 mM We thank O. Campetella and his group from Universidad
pyridinium acetate while the remaining sialyllactose was eluted Nacional General San Martín (UNSAM) Argentina, for the kind
with 200 mM pyridinium acetate buffer. Further elution with gift of trans-sialidase from T. cruzi. Support for this work from
500 mM buffer afforded the disialylated compound 26 (2 mg). the National Agency for Promotion of Science and Technology,
The pooled fractions were concentrated by lyophilization. Com- ANPCyT, Project PICT Bicentenario 2010 – No. 1080, the
pound 25 was further purified by passing through a SepPack C8 National Research Council CONICET and the University of
cartridge (Alltech) eluting with H2O to obtain 5 mg of a colour- Buenos Aires is gratefully acknowledged. María E. Cano is a
less syrup: 1H NMR (500 MHz, D2O), partial assignments fellow from CONICET. Rosalia Agusti, María Laura Uhrig and
assisted by the HSQC spectrum: δ 7.81 (s, 2H, H-triazole), 4.89 Rosa M. de Lederkremer are research members of CONICET.
(d, J = 9.3 Hz, 2H, H-1-βNGlc), ≈4.69 (m, under the suppressed
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