Trypanosoma cruzi trans-sialidase alternative substrates: Study of the effect of substitution in C-6 in benzyl β-lactoside

Article abstract of DOI:10.1016/j.carres.2019.04.003

Trypanosoma cruzi trans-sialidase (TcTS) is a cell surface protein that participates in the adhesion and invasion mechanisms of the parasite into the host cells, making it an attractive target for inhibitors design. In order to contribute to the knowledge of the interaction between TcTS and their acceptor substrates, we designed and synthesized a library of 20 benzyl lactosides substituted in C-6 of the glucose residue with a series of 1,2,3-triazole derivatives containing different aromatic substituents in the C-4 position. The library was prepared by alkyne-azide cycloaddition reaction catalyzed by Cu(I) (“click chemistry”) between a benzyl β-lactoside functionalized with an azide group in the C-6 position and a series of 2-propargyl phenyl ethers. Herein we analyzed the chromatographic behavior on high performance anion exchange chromatography (HPAEC) of the triazoyl-lactose derivatives and their activity as acceptors of TcTS and inhibitors of the sialylation of N-acetyllactosamine. The triazoyl derivatives were obtained with excellent yields and all of them behaved as moderate alternative substrates. The presence of bulky hydrophobic substituents dramatically increased the retention times in HPAEC but did not affect significantly their acceptor properties toward TcTS.

Full text of DOI:10.1016/j.carres.2019.04.003

Accepted Manuscript
Trypanosoma cruzi trans-sialidase alternative substrates: Study of the effect of
substitution in C-6 in benzyl β-lactoside
Pablo Morrone-Pozzuto, Maria Laura Uhrig, Rosalia Agusti
PII:
S0008-6215(19)30090-4
https://doi.org/10.1016/j.carres.2019.04.003
CAR 7699
DOI:
Reference:
To appear in: Carbohydrate Research
Received Date: 18 February 2019
Revised Date: 9 April 2019
Accepted Date: 16 April 2019
Please cite this article as: P. Morrone-Pozzuto, M.L. Uhrig, R. Agusti, Trypanosoma cruzi trans-sialidase
alternative substrates: Study of the effect of substitution in C-6 in benzyl β-lactoside, Carbohydrate
Research (2019), doi: https://doi.org/10.1016/j.carres.2019.04.003.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Trypanosoma cruzi trans-sialidase alternative substrates: Study of the
effect of substitution in C-6 in benzyl β-lactoside.
1,2
1,2*
1,2*
Pablo Morrone-Pozzuto , Maria Laura Uhrig ,Rosalia Agusti
1
Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales.
2
Departamento de Química Orgánica. Buenos Aires, Argentina; CONICET-
Universidad de Buenos Aires. Centro de Investigaciones en Hidratos de
Carbono (CIHIDECAR). Buenos Aires, Argentina.
*
Corresponding authors:
E-mail address: ragusti@qo.fcen.uba.ar (R Agusti)
mluhrig@qo.fcen.uba.ar (ML Uhrig)
1

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Abstract
Trypanosoma cruzi trans-sialidase (TcTS) is a cell surface protein that
participates in the adhesion and invasion mechanisms of the parasite into the
host cells, making it an attractive target for inhibitors design. In order to
contribute to the knowledge of the interaction between TcTS and their acceptor
substrates, we design and synthesized a library of 20 benzyl lactosides
substituted in C-6 of the glucose residue with a series of 1,2,3-triazole
derivatives containing different aromatic substituents in the C-4 position. The
library was prepared by alkyne-azide cycloaddition reaction catalyzed by Cu(I)
(“click chemistry”) between a benzyl β-lactoside functionalized with an azide
group in the C-6 position and a series of 2-propargyl phenyl ethers. Herein we
analyzed the chromatographic behavior on high performance anion exchange
chromatography (HPAEC) of the triazoyl-lactose derivatives and their activity as
acceptors of TcTS and inhibitors of the sialylation of N-acetyllactosamine. The
triazoyl derivatives were obtained with excellent yields and all of them behaved
as moderate alternative substrates. The presence of bulky hydrophobic
substituents dramatically increased the retention times in HPAEC but did not
affect significantly their acceptor properties toward TcTS.
Keywords: Trypanosoma cruzi trans-sialidase, click chemistry, HPAEC,
acceptor substrates, triazole.
2

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1
. Introduction
Chagas disease, or American trypanosomiasis, is one of the most prevalent
neglected tropical diseases [1]. It is occasioned by the protozoan parasite
Trypanosoma cruzi and disseminated by the triatomine insect vector. The
parasite can also be transmitted by blood or organ donation; or from infected
mothers onto their babies during pregnancy [2]. There are currently only two
drugs available to treat this disease, nifurtimox and benznidazole, both
displaying low efficacy in the chronic phase and several toxic side effects [3]. In
order to address this issue, specific enzymes of the parasite biochemical
pathways, such as cysteine proteases, trypanothione reductase, and trans-
sialidase, are considered to be potential drug targets [4].
Trypanosoma cruzi trans-sialidase (TcTS) is a retaining glycosyltransferase
anchored to the parasite membrane by glycosylphosphatidylinositol (GPI) [5,6],
which primary function consists in the transfer of sialic acid residues from
mammalian host glycoconjugates to terminal β-Galp residues on the mucins
that cover the parasite cell surface, generating (α2→3)-sialylated β-
galactopyranose units [7–9]. By altering the membrane profile, TcTS provides
direct protection from recognition by the host’s immune system [10], resistance
to the complement [11] and recognition of and attachment to host cells [12–15].
The enzyme follows a ping-pong bi-bi kinetic mechanism with formation of a
sialyl-enzyme intermediate. The active site shows two distinct sites, one
responsible for the interaction with the sialic acid residue (donor site) and the
other with the β-galactose acceptor molecule (acceptor site). The donor site
contains an arginine triad (Arg35, Arg245 and Arg314) that contributes to the
3

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stabilization of the transition state, two acidic amino acids (Asp59 and Glu230)
that function as acid-base catalysts and Tyr342 that binds the sialic acid to form
the sialyl-enzyme intermediate. The acceptor site is comprised mainly by two
aromatic amino acids (Tyr119 and Trp312) which forms an aromatic sandwich
that binds the β-galactose-containing acceptor by π-CH and π-OH interactions
[
16,17]. Asp59 (common to both sites) and Glu362 cooperate in the catalysis of
the reverse reaction in which the acceptor substrate attacks the sialyl-enzyme
intermediate [18].
In order to exploit TcTS as a drug target for chemotherapy use, it is
essential to understand the characteristics of the enzyme/substrates interaction.
Numerous approaches had been taken to address this point, including
spectroscopic techniques [19–22], X-ray crystallography [18,23,24], virtual
screening [4,25–28], natural product library screening [29] and chemical
synthesis of transition state or substrate analogues [7,30–38].
TcTS inhibitors are usually classified in two main groups according to their
binding site target: sialic acid mimetics or acceptor substrate analogues. 2-
Deoxy-2,3-didehydro-D-N-acetylneuraminic acid (DANA), a transition state
analogue, is a potent inhibitor of the Influenza neuraminidase but a poor
inhibitor of TcTS [39]. Regarding the inhibitors that target the acceptor binding
site, lactitol and other lactose derivatives prevent sialylation of N-
acetyllactosamine by behaving as a better acceptor substrate than the
conventional acceptor [30,32,40,41]. Acceptor substrate studies performed on a
series of systematically modified octyl galactosides and octyl N-
acetyllactosamines determined that TcTS activity largely diminished if galactose
is substituted at 2 and 4 positions but substitution at 6 position is well tolerated
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[
37]. Giorgi et al covalently bound oseltamivir, a well-known sialidase inhibitor,
with lactose and lactobionolactone with the aim to obtain a bi-substrate potential
inhibitor. The synthesized compounds were shown to be moderate acceptors
and inhibitors of TcTS [31].
In the last years a growing interest to the synthesis of 1,2,3-triazole
derivatives of carbohydrates has been developed due to the improved stability
and physical properties of the triazole ring when compared to an amide, ester or
ether linkage [42]. Besides, Cu(I)-assisted 1,3-dipolar azide–alkyne
cycloaddition (CuAAC) is a fast, selective and easily executed reaction that
allows the introduction of a variety of substituents from the proper azide and
alkyne precursors. In this respect, Carvalho et al [36] synthesized a library of
substrate analogues based on 1,4-disubstituted 1,2,3-triazole derivatives of
galactose modified at either the C-1 or C-6 positions. They behaved as poor
inhibitors of TcTS even though they were able to act as acceptor substrates and
even had in vitro trypanocidal activity. More recently, mixed multivalent cyclic
and linear triazole–linked oligomers were synthesized that showed to be
acceptor substrates and blocked macrophage invasion by T. cruzi.[43].
Multivalent β-thio-galactopyranosides and β-thio-lactosides were used as
acceptor substrates using 3’-sialyllactose as the sialic acid donor and allowing
the chemoenzymatic synthesis of polysialoside glycoclusters [44,45].
With the aim to explore the influence of the presence of aromatic
substituents in the acceptor substrates, taking into account the key function of
the aromatic sandwich in the binding site of TcTS, herein we report the
synthesis of a library of lactose analogues modified at C-6 of the glucose ring
with 1,2,3-triazole derivatives substituted at C-4 with different aromatic groups.
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The ability of the synthesized compounds to act as substrates of TcTS and
inhibitors of the sialylation of N-acetyllactosamine, as well as their
chromatographic behavior on high performance anion exchange
chromatography was studied.
2
. Results and discussion
For the synthesis of lactose analogues modified at C-6 with different
substituents, it was important to develop a method to selectively modify the C-6
of the glucose residue with high yield. Two different pathways were taken for
the synthesis of the key 6-azido lactose derivative: the first one involved the
utilization of a lactosan intermediate while the other proceeds via the 4’,6’-
benzyliden lactose derivative.
2
.1. Synthesis of benzyl peracetyl-6-azido-6-deoxy-β-lactoside
2
.1.1 Lactosan pathway: per-acetyllactosan, 2,3,4,6-tetra-O-acetyl-β-D-
galactopyranosyl-(1→4)-2,3-di-O-acetyl-1,6-anhydro-β-D-glucopyranose (3) was
synthesized from lactose (1) according to the procedure described by Tejima
with minor modifications (Scheme 1) [46]. Lactose was peracetylated and
glycosylated with phenol in the presence of p-TsOH/Ac O to obtain phenyl
2
hepta-O-acetyl-lactoside (2) as pure β anomer according to its NMR spectra (δ
5
.06, J1,2 7.4 Hz H-1; δ 4.51. J1’,2’ 7.8 Hz, H-1’; δ 98.7, C-1; δ 101.1, C-1’). For
the synthesis of the hexa-O-acetyl-lactosan 3, alkaline treatment was performed
on the β-phenyl lactoside since the anhydrosugar formation occurred
6

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exclusively on the β anomer. After acetylation, the attainment of 3 was
confirmed by the observation of two broad singlets at 5.47 and 5.17 ppm in the
1
H-NMR spectrum, corresponding respectively to H-1 and H-2 of the anhydro
residue. The opening of the anhydro bridge was achieved by treatment with
TiCl . Further glycosylation with benzyl alcohol required catalytic amounts of
4
silver triflate added to the silver carbonate used as promoter, affording 4 as a
single anomer. Compound 4 was a convenient intermediate for the introduction
1
of the azide group at the 6-position of the Glc residue. The NMR- H spectra of 4
showed two signals at 4.63 and 4.59 ppm corresponding to the anomeric
protons of the galactose and glucose residues, respectively. Additionally, in the
13
C-NMR spectrum only two signals appeared in the anomeric region at 100.9
and 99.6 ppm for the C-1 of galactose and glucose, respectively. The azide
substituent in the C-6 position was introduced converting the free hydroxyl into
an appropriate leaving group by standard mesylation (5) followed by
1
displacement of the mesylate with sodium azide. The H-NMR spectrum of 6
showed the signals of H-6 at higher fields (3.4 and 3.5 ppm) in comparison with
the analogous signals in the mesylate 5. The same behavior was observed for
C-6, which appeared upfield at 50.5 ppm.
Scheme 1- Lactosan pathway for the synthesis of the azido lactoside 6
7

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2
.1.2. Benzyliden pathway: The alternative pathway showed in Scheme 2 was
explored in the search of a more readily synthetic procedure to obtain the key
precursor 6. In the first place, benzyl β- -lactoside (8) was synthesized from
D
octa-O-acetyl-lactose (7) by glycosylation with benzyl alcohol in the presence of
BF .Et O followed by deacetylation. The 4’:6’-benzyliden acetal (9) was
3
2
obtained in very good yield (81%) from 8 using dimethoxytoluene and p-TsOH
as acid catalyst and allowing the reaction to proceed in a rotavap, following the
procedure described for phenyl β-D-lactoside [47]. The in situ evaporation of the
solvent led to the precipitation of the product and the concomitant displacement
of the equilibrium reaction, accounting for the good yield obtained. As direct
sulphonylation of the 6-OH was shown to give poor yields for the glucose
configuration [48], 9 was regioselectively protected with a tert-butyldimethylsilyl
group, and then acetylated in all free positions to obtain 10. The silyl protecting
group was then removed with tetrabutylammonium fluoride to give 11. After
mesylation and displacement with sodium azide, 13 was obtained. Benzylidene
acetal cleavage and in situ acetylation of the diol was performed using
perchloric acid immobilized on silica as catalyst [49]. The reaction progressed
fast and with excellent yield and the solid catalyst could be removed by filtration,
avoiding tedious work up, to give 6 in an overall yield from 10 of 86% without in
between purifications. The spectroscopic data of 6 obtained through this
sequence matched exactly to the observed for the same compound obtained by
the lactosan pathway.
For the synthesis of the triazole derivatives library, we selected this last
pathway since the total yield for the synthesis of the azide derivative 6 was 5-
fold superior than the one summarize in Scheme 1. The lactosan pathway, on
8

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the other hand, allowed us to obtain compounds 2 and 3 to be analyzed as
substrates of TcTS after deacetylation.
Mesylated derivative 12 was also used for the synthesis of the 6-deoxy-β-
lactoside derivative. Treatment of 12 with sodium borohydride in DMF afforded
1
4, which NMR spectra showed the characteristic signals for the 6-deoxy sugar;
1
a doublet at 1.37 ppm in the H-NMR spectrum, and a peak at 17.3 ppm in the
13
C-NMR spectrum corresponding to the 6-CH . C-5 was also shifted 4 ppm
3
upfield.
Scheme 2- Benzyliden pathway for the synthesis of the azido lactoside 6
9

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2
1
.2. Synthesis of benzyl β-lactosides modified at C-6 with 1,4-substituted-
,2,3-triazoles
CuAAC reactions on the 6-azido modified benzyl lactoside 6 were carried
out with a set of terminal alkynes (a-r, Figure S1), either commercially available
or synthesized according to the procedure described by Wang et al [50].
Cycloadditions were performed under microwave irradiation and proceeded with
excellent yields (85-96 %) and complete regioselectivity to obtained 1,4-
disubstituted triazoles (15a-15r, Figure 1), as certified by NMR spectroscopy.
Cycloaddition with propiolic acid (a) required 2 microwave cycles at 120 °C and
afforded the monosubstituted triazole derivative 15a with a moderate yield (51
%
). To our knowledge, this is the first report of a successful alkyne-azide
cycloaddition using propiolic acid in a carbohydrate derivative, followed by
decarboxylation. Finally, compounds 15a-15r were deacetylated with catalytic
NaOMe in methanol, desalted with a cation exchange resin and the deprotected
products recovered with high yields and characterized by NMR and HR-MS.
Carboxymethyl ester-containing compounds 16q and 16r were further
saponified with aqueous NaOH to afford the carboxylic acid derivatives 16s and
1
6t, respectively. Thus, a library of 20 triazole lactosides (16a-16t) was
obtained, to be analyzed as potential TcTS substrates.
1
0

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Figure 1- Synthesis of benzyl β-lactosides modified at C-6 with 1,4-substituted-
1
,2,3-triazoles
2
.3. Analysis of lactose derivatives by HPAEC
Compounds 2,3,5,6 were deacetylated with catalytic sodium methoxide in
methanol, and desalted with a cation exchange resin to afford compounds 17-
0, respectively (Figure 2). Compound 14 was deprotected by one-pot removal
2
of the benzylidene acetal and the acetate groups yielding compound 21. All
products were recovered with high yields and characterized by NMR and HR-
MS. These free compounds, together with the library of 20 triazole lactosides
(compounds 16a-16t) provided a suitable set of lactose-related products to be
1
1

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studied by HPAEC, prior to the investigation of their activity towards the TcTS
enzyme.
Figure 2- Lactose derivatives analyzed by HPAEC
The chromatographic resolution of lactose derivatives was studied using a
Carbopac PA-10 analytical column under Condition A, as described in the
Experimental Section. Retention times of the triazole derivatives highly
increased with the incorporation of an aromatic ring (Table 1, see below).
Moreover, compounds 16o and 16p, with naphtyl substituents were highly
retained and eluted after 70 min under these conditions. Among the phenyl-
substituted derivatives, compounds containing bulky hydrophobic substituents,
such as tert-butyl and bromine (compounds 16i and 16j) were more retained in
the chromatographic column than those with polar substituents -OMe, -NO , -
2
NHAc (compounds 16h, 16m, 16n). Benzoic acid derivatives, compounds 16s
and 16t, were highly retained due to the presence of the acid and this effect
was more pronounced for para isomer. The chromatographic behavior of their
ester precursors, compounds 16q and 16r, could not be assessed since they
hydrolyzed under the alkaline condition of the chromatography.
2
.4. Lactose derivatives as acceptors of TcTS. Analysis by HPAEC
1
2

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The 6-modified lactose derivatives 17-21 and the 6-triazolyl lactose
derivatives 16a-16t were analyzed as acceptor substrates of TcTS. The
reaction is shown in Scheme 3. Incubations were performed as previously
described [32] using 1 mM 3’-SL as donor, 1 mM of the potential substrate and
recombinant TcTS. The incubation mixtures were analyzed by HPAEC (PA10,
various conditions, Table 1). The CarboPac PA-10 column was chosen for this
experiment since sialylated derivatives of the most hydrophobic compounds
(16o and 16p) were highly retained in a PA100 column. The percentage of sialic
acid transference in each case was calculated from the amount of sialylated
product with respect to the sum of all sialylated species present in the reaction
mixture [51]. The results are included in Table 1 (fourth column). As an
example, the chromatograms obtained for lactosan (18), and the click
derivatives with propargylic alcohol (16c) and p-tert-butylphenyl propargyl ether
(16i) are depicted in Figure 3. Control experiments without the addition of trans-
sialidase were performed for all the samples to discard the occurrence of other
peaks, besides the sialylated species. Moreover, the presence of the sialylated
derivative of compound 21 was verified by MALDI-TOF mass spectrometry of
the incubation mixture (Figure Sx). Peaks at m/z 752.326 and m/z 730.348
+
+
corresponding to the [M-H+2Na] and [M+Na] confirmed the presence of the
sialylated product in the incubation mixture.
Scheme 3- Sialylation of lactose derivatives by T. cruzi trans-sialidase
1
3

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Figure 3- HPAEC of the incubations of compounds 18 (panel a), 16c (panel b)
and 16i (panel c) with TcTS (300 ng) in 20 mM Tris–HCl, pH 7.6 buffer, 30 mM
NaCl, containing 1 mM 3’-sialyllactose as a sialic acid donor. The mixtures were
analyzed using a Carbopac PA-10 column under conditions C (panels a and b)
and A (panel c). HPAEC conditions A-C are detailed in Experimental. L, lactose;
SA, sialic acid; SL, 3’-sialyllactose; S-18, sialylated compound 18; S-16c,
sialylated compound 16c; S-16i, sialylated compound 16i.
1
4

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All compounds were good acceptors of sialic acid showing that the
presence of the triazoyl substituent did not impair their acceptor capacity,
unless substituted with hydrophobic bulky groups such as tert-butylphenyl (16i)
or β-naphtyl (16p). When comparing compounds with similar characteristics,
more polar derivatives were better as sialic acid acceptors. Compound 16c,
containing only an –CH OH substituent in the triazoyl moiety, was among the
2
best substrates and inhibitors tested (see below). Additionally, the p-
methylphenyl derivative 16g is a worse acceptor than the unsubstituted phenyl
(
16d) but a better one than the tert-butyl derivative 16i. The three positional
isomers of the nitrophenyl derivatives behaved similarly being the o-nitrophenyl
6k a better substrate than the m- and p-isomers 16l and 16m. Interestingly,
1
even though the glucose residue in compound 18 is conformationally locked in
1
a C conformation, 18 still showed a moderate activity as acceptor of sialic
4
acid. Percentages of transference were similar than those obtained for other
lactose derivatives previously synthesized [31,32,52,53].
2
.5. Lactose derivatives as inhibitors of sialylation of N-acetyllactosamine by
TcTS.
All synthesized compounds were analyzed as inhibitors of sialylation of N-
acetyllactosamine by the enzyme, a model reaction that can be considered as
representative of the natural process that occurs during infection with the
parasite. Incubations were performed as mentioned above with the addition of 1
mM N-acetyllactosamine. Inhibition percentages were calculated as the
1
5

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decrease in the percentage of sialyl-N-acetyllactosamine in comparison with the
value obtained in a control experiment performed in absence of the proposed
inhibitor. Reaction mixtures were also analyzed by HPAEC and the results are
shown in Table 1, fifth column.
The analysis of the values obtained in the inhibition experiments suggests
that the presence of the triazole ring generally improves inhibition; taking into
account the values obtained for the non-triazoyl containing derivatives (cf. 17,
1
9-21 with the 16 series). On the other hand, interestingly, compound 21, which
lacks the oxygenated functionality at C-6, resulted as active as the benzyl
glycoside 8, which can be considered as reference compound to evaluate the
activity behavior of our library.
Compound 16c, containing only an –CH OH substituent in the triazoyl
2
moiety, was among the best substrates and inhibitors tested. On the other
hand, 16d, which poses a –CH OPh in the same position, shows a similar
2
activity to that of 16c, a fact that suggests that steric effects are tolerable to a
certain extent. On the other hand, the incorporation of a o-methyl group in the
aromatic ring (compound 16e), causes a substantial drop in the inhibition (cf.
entries 10 and 11, Table 1), also observed for the meta and para isomers
(compounds 16f and 16g). This effect is more pronounced in the case of 16i,
having the bulky and hydrophobic p-tert-butylphenyl methyl ether, which
rendered the worst acceptor and inhibition results.
In summary, all compounds resulted good to moderate inhibitors, with the
exception of the p-tert-butylphenyl derivative 16i, that showed only 4.9% of
inhibition. It is noticeable that the β-naphtyl derivative 16p was among the worst
1
6

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substrates tested but showed the highest inhibitory value, suggesting that an
alternative mode of action is taking place.
Table 1- Chromatographic behavior and activity towards TcTs of the 6-
substituted lactose derivatives.
R
t
(min)
HPAEC
Condition
PA-10
a
b
Entry
Compound
(condition A)
PA-10
% T
% I
1
2
3
4
5
6
7
8
9
8
17
18
19
20
21
5.5
5.7
2.2
6.3
7.2
2.9
5.1
25.7
5.4
22.1
24.7
25.5
25.4
21.6
35.9
37.2
24.5
25.7
23.4
20.6
88.4
74.1
- (i)
46.0
48.7
33.5
38.0
51.0
48.4
42.3
42.0
58.8
53.3
44.8
45.5
29.2
48.5
22.5
39.3
48.3
39.0
37.7
32.3
44.5
30.1
35.8
30.7
35.1
33.2
43.2
32.9
42.5
34.5
39.8
44.8
50.4
45.3
59.6
60.2
35.4
53.6
49.5
56.5
4.9
55.2
42.7
60.2
44.0
35.2
50.9
68.7
40.7
42.5
45.8
32.8
C
C
C
C
C
C
C
A
C
B
A
A
A
B
A
A
B
A
A
B
A
A
A
A
A
A
16a
16b
16c
16d
16e
16f
16g
16h
16i
16j
16k
16l
16m
16n
16o
16p
16q
16r
16s
16t
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
- (i)
62.5
41.2
(
i) Not determined because the ester hydrolyzed under the alkaline chromatographic
a
conditions. %T, Percentage of transference: lactose derivatives were incubated with TcTS
300 ng) in 20 mM Tris–HCl, pH 7.6 buffer, 30 mM NaCl, containing 1 mM 3’-sialyllactose
bas a sialic acid donor and the mixture analyzed by HPAEC in the indicated conditions A-C.
I, Percentage of inhibition: each compound (1 mM) was incubated with TcTS in 20 mM
(
%
Tris–HCl, 30 mM NaCl, pH 7 buffer (20 µL), containing 1 mM 3’-SL as donor and 1 mM N-
acetyllactosamine for 15 min at room temperature. % Transfer and inhibition were
calculated by integration of the chromatographic peaks. HPAEC conditions A-C are detailed
in Experimental.
3
. Conclusions
A library of 20 benzyl lactosides substituted in C-6 of the glucose residue
with a series of 1,2,3-triazole derivatives containing mostly aromatic
substituents was synthesized and each component was analyzed as substrate
1
7

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of TcTs and inhibitor of the sialylation of N-acetyllactosamine. A nine step
synthesis starting from readily available benzyl β-lactoside (8) was carried out
allowing the obtaining of highly pure lactose derivatives with yields ranging from
5
0 to 55 %. Additionally, the synthetic routes explored for the synthesis of 6
provided some interesting derivatives (compounds 17-21) that were also
included in this study.
Regarding the chromatographic conditions used for the analysis of the
enzymatic determinations, the PA-100 column generally used for sialylated
species was not appropriate for this set of compounds. As the hydrophobicity of
the substituents increased, the retention times for the sialylated derivatives
became too high to be analyzed. The Carbopac PA-10 column, with smaller
particle size, proved to be more effective. Even so, four different
chromatographic conditions had to be used in order to allow the resolution of
the different sialylated species, required for the inhibition determinations.
In general, the 6-triazoyl lactose derivatives showed better inhibitory
properties than the previously described 6-substituted β-galactosides, although
a different inhibitory assay was employed in this case (based on MuNANA
hydrolysis) [36]. The inhibition properties of compounds 16a-16t were in the
same range than the S- and N-lactosides previously studied by us, having a
triazoyl ring in the anomeric substituent [44] These results provided additional
information on the selectivity of the enzyme and the structural features that
modulate the effective transfer of sialic acid residues. , the development of
active small structures is the first necessary approach for the development of
more complex conjugates with greater bioavailability or activity
1
8

ACCEPTED MANUSCRIPT
These results open the possibility for the incorporation of a variety of other
groups (carbohydrate or even peptide residues) through the position 6- of the
glucose residue, towards the development of new acceptors of sialic acid.
Moreover, by combination with the CuAAC click reaction, even multivalent
species can be obtained, an approach that has proved to be effective in this
respect [45].
4
. ExperimentalGeneral Information
Reagents were purchased from commercial suppliers and were used
without further purification. Solvents were treated according to standard
procedures [54]. TLC was performed on Silica Gel 60 F254 plates (Merck) and
visualization was accomplished using UV light or by charring at ∼150°C with 5%
(v/v) sulfuric acid in EtOH, containing 0.5% p-anisaldehyde. Column
chromatography was carried out with Silica Gel 60 (230-400 mesh). Optical
rotations were measured at 25 °C in a 1 dm cell with a Perkin-Elmer 343
polarimeter. Microwave irradiation was carried out in a CEM Discover MW
instrument with a System Internal IR probe type at 70 ºC (power max 300 W).
High resolution mass spectra (HRMS) were obtained by Electrospray Ionization
(ESI) and Q-TOF detection with a BRUKER microTOF-Q II ESI-Qq-TOF
1
13
spectrometer. H and C nuclear magnetic resonance (NMR) spectra were
recorded at 25 °C at 500 and 126 MHz, respectively, using a Bruker Avance II
1
13
5
00 spectrometer. For H, C nuclear magnetic resonance (NMR) spectra,
chemical shifts are reported in parts per million relative to tetramethylsilane or a
1
13
residual solvent peak (CDCl : H: δ = 7.26 ppm, C: δ = 77.2 ppm, DMSO-d :
3
6
1
9

ACCEPTED MANUSCRIPT
1
1
13
1
H: δ = 3.33 ppm, C: δ = 39.5 ppm , D O: H: δ = 4.79 ppm). Assignments of
2
13
1
1
13
H and C were assisted by 2D H–COSY and 2D H– C experiments. For the
sialylation experiments a recombinant TcTS expressed in Escherichia coli was
kindly provided by the group of O. Campetella from Universidad Nacional
General San Martin (Buenos Aires, Argentina).
Analysis by HPAEC was performed using a Dionex ICS 5000 HPLC system
equipped with a pulse amperometric detector. A CarboPac PA-10 ion exchange
analytical column (4 × 250 mm) equipped with a guard column PA-10 (4 × 50
mm) was used eluted with one of the following solvent systems. Condition A:
1
00 mM NaOH 20 mM AcONa for 10 minutes followed by a gradient system
from 100 mM NaOH-20 mM AcONa to 100 mM NaOH-500 mM AcONa in 50
min at a flow rate of 0.9 ml/min at 25 ºC. Condition B: 50 mM NaOH 20 mM
AcONa for 10 minutes followed by a gradient system from 50 mM NaOH-20 mM
AcONa to 50 mM NaOH-500 mM AcONa in 50 min at a flow rate of 0.9 ml/min
at 25 ºC. Condition C: gradient system from 150 mM NaOH to 150 mM NaOH-
2
00 mM AcONa in 60 min at a flow rate of 0.9 ml/min at 25 ºC.
4
.2. General procedure for the click reaction
Sugar derivative 6, containing an azide group at C-6 (0.21 mmol) was
dissolved in dioxane (2.6 mL) in a microwave flask equipped with a stirring bar.
After dissolution, one of the acetylene derivatives b-t (0.23 mmol, Figure S1)
was added. Then, sodium ascorbate (0.23 mmol), CuSO (0.11 mmol) and
4
water (0.48 mL) were added and the tube was sealed. The mixture was stirred
at 85 °C (5 W) for 1 h in the microwave, unless stated otherwise. The reaction
2
0

ACCEPTED MANUSCRIPT
was followed by TLC and after completion, the solvent was evaporated and the
residue chromatographed in a silica gel column (toluene/ethyl acetate as mobile
phase). Yields ranged between 85 and 96% (For a complete characterization of
products 15b-15r see Supporting information).
4
.3. Click reaction with propiolic acid
The azido lactose derivative 6 (0.17 g, 0.24 mmol) was dissolved in
anhydrous DMF (3 mL) in a microwave flask equipped with a stirring bar. After
dissolution, the propiolic acid (a, 0.024 mL, 0.4 mmol) was added together with
sodium ascorbate (0.1 mmol), CuI (0.03 g, 0.16 mmol) and DBU (0.02 mL, 0.13
mmol) and the tube was sealed. The mixture was stirred at 120 °C (7 W) for 1 h.
Then, another addition of propiolic acid, sodium ascorbate, CuI and DBU in the
same amounts as before was made and the reaction stirred at the microwave
for another 2 h. After TLC showed no further advance in the reaction, the
mixture was purified as described above affording 0.09 g of the
monosubstituted triazole derivative 15a (51 % yield, Supporting information).
4
.4. General procedure for deacetylation
Deacetylation was performed with sodium methoxide, unless otherwise
indicated. The acetylated compound was dissolved in CH OH (10 mL) and 0.1
3
M NaOCH -CH OH (1.5 mL) and stirred for 3 h. The mixture was desalted with
3
3
+
an Amberlite IR-120 (H ) column and concentrated.
2
1

ACCEPTED MANUSCRIPT
4
1
.5. 1-[Benzyl β-
D-galactopyranosyl-(1→4)-6-deoxy-β-D-glucopyranosid-6-yl]-
H-1,2,3-triazole (16a).
25
From 71 mg of 15a, 45 mg of 16a were obtained (97 % yield); [α]D -17.4 (c
1
1
, H O); H NMR (D O, 500 MHz) δ 7.85, 7.76 (2H, 2s, Trz-H), 7.31 (3H, m,
2
2
aromatics), 7.14 (2H, m, aromatics), 4.93 (1H, m, J5,6a 2.5 Hz, J6a,6b 14.5, H-6a),
.59 (1H, dd, J5,6b 8.6 Hz, H-6b), 4.59, 4.48 (2H, 2d, J 11.7 Hz, CH Ph), 4.50
4
2
(1H, d, J1’,2’ 7.5 Hz, H-1’), 4.29 (1H, d, J1,2 8.0 Hz, H-1), 3.86 (1H, m, J3’,4’ 3.4 Hz,
H-4’), 3.82 (1H, ddd, J4,5 = J5,6b 9.0 Hz, H-5), 3.75-3.63 (3H, m, H-5’, H-6’), 3.62
(1H, dd, J2’,3’ 9.6, H-2’), 3.57–3.48 (3H, m, H-3, H-4, H-3’), 33.26 (1H, dd, J2,3
1
3
8
1
7
.6 Hz, H-2). C NMR (D O, 126 MHz) δ 133.9, 126.8 (Trz C-H x 2), 136.1,
2
28.7 x 2, 128.7 x 2, 128.5 (aromatics), 103.0 (C-1’), 100.4 (C-1), 80.1 (C-4),
5.5 (C-5’), 74.2 (C-3), 72.8 (C-3’), 72.7 (C-2), 72.5 (C-5), 71.4 (C-2’), 70.9
(
PhCH ), 68.5 (C-4’), 61.0 (C-6’), 50.4 (C-6). HRMS (ESI) m/z calcd. for
2
+
C H N O [M + H] : 484.1926; found: 484.1920.
21
30
3
10
4
.6. {1-[Benzyl β-
D-galactopyranosyl-(1→4)-6-deoxy-β-D-glucopyranosid-6-yl]-
1
H-1,2,3-triazol-4-yl}-benzene (16b).
25
Starting from 98 mg of 15b, 61 mg of 16b were obtained (91 % yield); [α]D
1
-
12.9 (c 1, DMSO); mp 239-240 °C (ethanol); H NMR (DMSO-d /D O, 500
6
2
MHz): δ 8.56 (1H, s, Trz-H), 7.84 (2H, m, aromatics), 7.45 (2H, m, aromatics),
.34 (1H, m, aromatic), 7.25 (3H, m, aromatics), 7.15 (2H, m, aromatics), 5.07
1H, m, J5,6a 2.6 Hz, J6a,6b 14.3, H-6a), 4.56 (1H, dd, J5,6b 9.0 Hz, H-6b), 4.56,
.39 (2H, 2d, J 12.3 Hz, CH Ph), 4.40 (1H, d, J1’,2’ 7.3 Hz, H-1’), 4.25 (1H, d, J1,2
7
(
4
2
2
2

ACCEPTED MANUSCRIPT
7
4
.9 Hz, H-1), 3.77 (1H, ddd, J4,5 = J5,6b 9.0 Hz, H-5), 3.66 (1H, m, J3’,4’ 3.7 Hz, H-
’), 3.59-3.47 (3H, m, H-5’, H-6’), 3.45 (1H, dd, J2’,3’ 9.6, H-2’), 3.42–3.39 (2H,
13
m, H-3, H-4), 3.37 (1H, dd, H-3’), 3.13 (1H, dd, J2,3 8.9 Hz, H-2); C NMR
(
DMSO-d /D O, 126 MHz): δ 146.6 (Trz C-C-R), 137.8, 131.2, 129.4 x 2, 128.6
6
2
x 2, 128.3 x 2, 128.2 x 2, 128.0, 122.9 (aromatics), 125.5 (Trz C-C-H), 104.0 (C-
’), 101.6 (C-1), 81.7 (C-4), 76.1 (C-5’), 75.0 (C-3), 73.5 (C-3’), 73.3 (C-2), 72.8
1
(
C-5), 70.7 (C-2’), 69.9 (PhCH ), 68.5 (C-4’), 60.9 (C-6’), 51.0 (C-6); HRMS
2
+
(
ESI) m/z calcd. for C H N O [M + H] : 560.2239; found: 560.2244.
27
34
3
10
4
.7. {1-[Benzyl β-
D-galactopyranosyl-(1→4)-6-deoxy-β-D-glucopyranosid-6-yl]-
1
H-1,2,3-triazol-4-yl}-methanol (15c).
Starting from 100 mg of 15c, 64 mg of 16c were obtained (96 % yield);
2
5
1
[
α]D -13.7 (c 1, H O); H NMR (D O, 500 MHz) δ 7.83 (1H, s, Trz-H), 7.29 (3H,
2 2
m, aromatics), 7.13 (2H, dd, J 4.8, 1.9 Hz, aromatics), 4.88 (1H, m, H-6a), 4.64
(
2H, m, Trz-CH ), 4.58, 4.48 (2H, 2d, J 12.0 Hz, CH Ph), 4.57 (1H, m, H-6b),
2
2
4
3
.49 (1H, d, J1’,2’ 8.1 Hz, H-1’), 4.28 (1H, d, J1,2 8.1 Hz, H-1), 3.85 (1H, m, H-4’),
.80 (1H, m, H-5), 3.68 (3H, m, H-5’, H-6’), 3.61 (1H, dd, J2’,3’ 6.5 Hz, J3’,4’ 2.5
Hz, H-3’), 3.50 (2H, m, H-3, H-2’), 3.47 (1H, m, H-4), 3.25 (1H, dd, J1,2 8.0 Hz,
13
J2,3 9.0 Hz, H-2); C NMR (D O, 126 MHz) δ 146.7 (Trz C-C-R), 136.1, 128.6 x
2
4
7
, 128.5 (aromatics), 125.2 (Trz C-C-H), 103.0 (C-1’), 100.4 (C-1), 80.1 (C-4),
5.5 (C-5’), 74.1 (C-3), 72.7 (C-5), 72.6 (C-2), 72.5 (C-3’), 71.3 (PhCH ), 70.9
2
(
C-2’), 68.5 (C-4’), 61.0 (C-6’), 54.5 (Trz-CH ), 50.6 (C-6); HRMS (ESI) m/z
2
+
calcd. for C H N O [M + H] : 514.2031; found: 514.2029; calcd. for
22
32
3
11
+
C H N NaO [M + Na] : 536.1851; found: 536.1858.
22
31
3
11
2
3

ACCEPTED MANUSCRIPT
4
.8. {1-[Benzyl β-
D-galactopyranosyl-(1→4)-6-deoxy-β-D-glucopyranosid-6-yl]-
1
H-1,2,3-triazol-4-yl}-methanol O-phenyl ether (16d).
Starting from 100 mg of 15d, 62 mg of 16d were obtained (89 % yield);
2
5
1
[
α]D -13.2 (c 1, DMSO); mp 208-209 °C (ethanol); H NMR (DMSO-d /D O,
6 2
5
00 MHz) δ 8.16 (1H, s, Trz-H), 7.29-7.23 (5H, m, aromatics), 7.20 (2H, m,
aromatics), 6.98 (2H, m, aromatics), 6.91 (1H, m, aromatic), 5.13 (2H, m, Trz-
CH OH), 5.03 (1H, dd, J5,6a 2.5 Hz, J6a,6b 14.3, H-6a), 4.53, 4.34 (2H, 2d, J 12.3
2
Hz, CH Ph), 4.51 (1H, dd, J
8.6 Hz, H-6b), 4.34 (1H, d, J1’,2’ 7.7 Hz, H-1’),
5,6b
2
4
3
3
.22 (1H, d, J1,2 7.9 Hz, H-1), 3.71 (1H, ddd, J4,5 8.8 Hz, H-5), 3.63 (1H, d, J3’,4’
.2 Hz, H-4’), 3.55-3.48 (3H, m, H-5’, H-6’), 3.41 (1H, dd, J2’,3’ 9.7 Hz, H-2’), 3,
8 (1H, t, J =J 8.5 Hz, H-3), 3.35 (1H, dd, H-3’), 3.33 (1H, t, H-4), 3.10 (1H,
2,3
3,4
13
dd, H-2); C NMR (DMSO-d /D O, 126 MHz) δ 143.0 (Trz C-C-R), 158.5,
6
2
1
1
7
37.9, 130.0 x 2, 128.6 x 2, 128.2 x 2, 128.0, 121.3, 115.1 x 2 (aromatics),
26.0 (Trz C-C-H), 104.1 (C-1’), 101.7 (C-1), 81.8 (C-4), 76.1 (C-5’), 74.9 (C-3),
3.5 (C-3’), 73.3 (C-2), 72.8 (C-5), 70.7 (C-2’), 70.0 (PhCH ), 68.5 (C-4’), 61.4
2
(
Trz-CH OH), 60.8 (C-6’), 50.8 (C-6); HRMS (ESI) m/z calcd. for C H N O
2 28 36 3
11
+
+
[
M + H] : 590.2344; found: 590.2367; calcd. for C H N NaO [M + Na] :
28 35 3 11
6
12.2164; found: 612.2194.
4
.9. {1-[Benzyl β-
D-galactopyranosyl-(1→4)-6-deoxy-β-D-glucopyranosid-6-yl]-
1
H-1,2,3-triazol-4-yl}-methanol O-(o-methylphenyl) ether (16e)
2
4

ACCEPTED MANUSCRIPT
Starting from 100 mg of 15e, 68 mg of 15e were obtained (96 % yield);
2
5
1
[
α]D -12.8 (c 1, DMSO); mp 109-110 °C (ethanol); H NMR (DMSO-d /D O,
6 2
5
00 MHz): δ 8.11 (1H, s, Trz-H), 7.30-7.20 (3H, m, aromatics), 7.15-7.02 (5H,
m, aromatics), 6.80 (1H, m, aromatic), 5.12 (2H, m, Trz-CH OH), 5.00 (1H, dd,
2
J5,6a 2.5 Hz, J6a,6b 14.3, H-6a), 4.51 (1H, dd, J5,6b 8.6 Hz, H-6b), 4.46, 4.29 (2H,
2
d, J 12.0 Hz, CH Ph), 4.32 (1H, d, J1’,2’ 7.6 Hz, H-1’), 4.18 (1H, d, J1,2 7.9 Hz,
2
H-1), 3.70 (1H, td, J4,5 9.2 Hz H-5), 3.63 (1H, d, J3’,4’ 3.5 Hz, H-4’), 3.55-3.46
3H, m, H-5’, H-6’), 3.39 (1H, dd, J2’,3’ 9.6 Hz, H-2’), 3.33 (1H, dd, J = J 8.5
(
2,3
3,4
Hz, H-3), 3.34 (1H, dd, H-3’), 3.31 (1H, dd, H-4), 3.09 (1H, dd, H-2), 1.99 (3H, s,
13
CH ); C NMR (DMSO-d /D O, 126 MHz): δ 143.5 (Trz C-C-R), 156.5, 137.7,
3
6
2
1
1
7
31.1, 128.7 x 2, 128.4 x 2, 128.2, 127.5, 126.5, 121.2, 112.3 (aromatics),
26.5 (Trz C-C-H), 104.1 (C-1’), 101.6 (C-1), 81.8 (C-4), 76.1 (C-5’), 74.9 (C-3),
3.4 (C-3’), 73.2 (C-2), 72.8 (C-5), 70.8 (C-2’), 70.1 (PhCH ), 68.6 (C-4’), 61.7
2
(
Trz-CH OH), 60.9 (C-6’), 50.9 (C-6), 16.4 (CH ); HRMS (ESI) m/z calcd. for
2
3
+
C H N O [M + H] : 604.2501; found: 604.2509.
29
38
3
11
4
.10.{1-[Benzyl β-D-galactopyranosyl-(1→4)-6-deoxy-β-D-glucopyranosid-6-yl]-
1
H-1,2,3-triazol-4-yl}-methanol O-(m-methylphenyl) ether (16f)
25
Starting from 101 mg of 15f, 67 mg of 16f were obtained (96 % yield); [α]D
1
-
13.1 (c 1, DMSO); mp 184-185 °C (ethanol); H NMR (DMSO-d /D O, 500
6
2
MHz): δ 8.15 (1H, s, Trz-H), 7.32-7.24 (3H, m, aromatics), 7.23-7.17 (2H, m,
aromatics), 7.21 (1H, m, aromatic), 6.79-6.69 (3H, m, aromatics), 5.10 (2H, m,
Trz-CH OH), 5.03 (1H, dd, J
2.5 Hz, J6a,6b 14.3, H-6a), 4.53, 4.33 (2H, 2d, J
5,6a
2
1
1.9 Hz, CH Ph), 4.51 (1H, dd, J
8.9 Hz, H-6b), 4.34 (1H, d, J1’,2’ 7.6 Hz, H-
2
5,6b
2
5

ACCEPTED MANUSCRIPT
1
3
’), 4.22 (1H, d, J1,2 7.9 Hz, H-1), 3.71 (1H, td, J4,5 9.2 Hz H-5), 3.63 (1H, d, J3’,4’
.3 Hz, H-4’), 3.54-3.46 (3H, m, H-5’, H-6’), 3.41 (1H, dd, J2’,3’ 9.6 Hz, H-2’), 3.38
(
1H, dd, J = J 8.8 Hz, H-3), 3.34 (1H, dd, H-3’), 3.33 (1H, dd, H-4), 3.10 (1H,
2,3 3,4
13
dd, H-2), 2.21 (3H, s, CH ); C NMR (DMSO-d /D O, 126 MHz): δ 142.9 (Trz
3
6
2
C-C-R), 158.2, 139.2, 137.6, 129.4, 128.4 x 2, 128.0 x 2, 127.7, 121.8, 115.5,
11.7 (aromatics), 125.7 (Trz C-C-H), 103.8 (C-1’), 101.4 (C-1), 81.5 (C-4), 75.8
C-5’), 74.6 (C-3), 73.2 (C-3’), 73.0 (C-2), 72.5 (C-5), 70.5 (C-2’), 69.7 (PhCH2),
1
(
6
8.2 (C-4’), 61.1 (Trz-CH OH), 60.6 (C-6’), 50.6 (C-6), 21.3 (CH ); HRMS (ESI)
2
3
+
m/z calcd. for C H N O [M + H] : 604.2501; found: 604.2494.
29
38
3
11
4
.11.{1-[Benzyl β-
D-galactopyranosyl-(1
→4)-6-deoxy-β-D-glucopyranosid-6-yl]-
1
H-1,2,3-triazol-4-yl}-methanol O-(4-methylphenyl) ether (16g)
Starting from 100 mg of 15g, 62 mg of 16g were obtained (88 % yield);
2
5
1
[
α]D -8.4 (c 1, DMSO); mp 125 °C (dec., ethanol); H NMR (DMSO-d /D O,
6 2
5
00 MHz): δ 8.16 (1H, s, Trz-H), 7.30-7.23 (3H, m, aromatics), 7.20 (2H, m,
aromatics), 7.01 (2H, m, aromatics), 6.84 (2H, m, aromatic), 5.08 (2H, m, Trz-
CH OH), 5.00 (1H, dd, J5,6a 2.5 Hz, J6a,6b 14.3, H-6a), 4.49, 4.31 (2H, 2d, J 12.3
2
Hz, CH Ph), 4.51 (1H, dd, J
8.6 Hz, H-6b), 4.33 (1H, d, J1’,2’ 7.9 Hz, H-1’),
5,6b
2
4
3
.19 (1H, d, J1,2 7.9 Hz, H-1), 3.69 (1H, m, H-5), 3.63 (1H, d, J3’,4’ 3.3 Hz, H-4’),
.55-3.46 (3H, m, H-5’, H-6’), 3.40 (1H, dd, J2’,3’ 9.2 Hz, H-2’), 3, 38-3.33 (2H, m,
H-3, 3’, 3.31 (1H, dd, J3,4 9.4 Hz, J4,5 8.7 Hz, H-4), 3.09 (1H, dd, J2,3 8.3 Hz, H-
1
3
2
1
), 2.16 (3H, s, CH ); C NMR (DMSO-d /D O, 126 MHz): δ 143.2 (Trz C-C-R),
3 6 2
58.2, 137.7, 130.2 x 2, 130.0, 128.6 x 2, 128.2 x 2, 128.0, 114.9 x 2
(aromatics), 125.9 (Trz C-C-H), 104.0 (C-1’), 101.5 (C-1), 81.7 (C-4), 76.0 (C-
2
6

ACCEPTED MANUSCRIPT
5
6
’), 74.8 (C-3), 73.3 (C-3’), 73.2 (C-2), 72.7 (C-5), 70.7 (C-2’), 70.0 (PhCH2),
8.5 (C-4’), 61.3 (Trz-CH OH), 60.8 (C-6’), 50.8 (C-6), 20.4 (CH ); HRMS (ESI)
2
3
+
m/z calcd. for C H N O [M + H] : 604.2501; found: 604.2493; calcd. for
29
38
3
11
+
C H N NaO [M + Na] : 626.2320; found: 626.2316.
29
37
3
11
4
.12.{1-[Benzyl β-D-galactopyranosyl-(1→4)-6-deoxy-β-D-glucopyranosid-6-yl]-
1
H-1,2,3-triazol-4-yl}-methanol O-(4-methoxyphenyl) ether (16h)
Starting from 102 mg of 15h, 67 mg of 16h were obtained (93 % yield);
2
5
1
[
α]D -8.0 (c 1, DMSO); mp 209-210 °C (ethanol); H NMR (DMSO-d /D O, 500
6 2
MHz): δ 8.13 (1H, s, Trz-H), 7.31-7.25 (3H, m, aromatics), 7.23-7.16 (2H, m,
aromatics), 6.91 (2H, d, J 9.1 Hz, aromatics), 6.80 (2H, d, J 9.1 Hz, aromatics),
5
4
.07 (2H, m, Trz-CH OH), 5.02 (1H, dd, J5,6a 2.5 Hz, J6a,6b 14.3, H-6a), 4.53,
2
.34 (2H, 2d, J 12.1 Hz, CH Ph), 4.51 (1H, dd, J5,6b 8.8 Hz, H-6b), 4.34 (1H, d,
2
J1’,2’ 7.9 Hz, H-1’), 4.22 (1H, d, J1,2 7.9 Hz, H-1), 3.71 (1H, m, H-5), 3.65 (3H, s,
OCH ),.3.63 (1H, d, J 3.3 Hz, H-4’), 3.55-3.46 (3H, m, H-5’, H-6’), 3.41 (1H,
3
3’,4’
dd, J2’,3’ 9.6 Hz, H-2’), 3.38 (1H, t, J =J 8.6 Hz, H-3), 3.34 (1H, dd, H-3’), 3.32
2,3
3,4
1
3
(
1H, dd, J4,5 9.0 Hz, H-4), 3.10 (1H, dd, H-2); C NMR (DMSO-d /D O, 126
6 2
MHz): δ 142.9 (Trz C-C-R), 153.6, 152.2, 137.6, 128.3 x 2, 127.9 x 2, 127.7,
1
8
2
15.8 x 2, 114.7 x 2 (aromatics), 125.6 (Trz C-C-H), 103.8 (C-1’), 101.4 (C-1),
1.5 (C-4), 75.8 (C-5’), 74.6 (C-3), 73.2 (C-3’), 72.9 (C-2), 72.4 (C-5), 70.4 (C-
’), 69.7 (PhCH ), 68.2 (C-4’), 61.7 (Trz-CH OH), 60.5 (C-6’), 55.4 (OCH ), 50.5
2
2
3
+
:
(
C-6); HRMS (ESI) m/z calcd. for C H N O [M + H]
620.2450; found:
29
38
3
12
+
:
6
20.2473; calcd. for C H N NaO [M + Na]
642.2269; found: 642.2290.
29
37
3
12
2
7

ACCEPTED MANUSCRIPT
4
.13.{1-[Benzyl β-D-galactopyranosyl-(1→4)-6-deoxy-β-D-glucopyranosid-6-yl]-
1
H-1,2,3-triazol-4-yl}-methanol O-(4-terbutylphenyl) ether (16i)
25
Starting from 99 mg of 15i, 61 mg of 16i were obtained (85 % yield); [α]D
-
1
1
3.1 (c 1, DMSO); mp 220-221 °C (ethanol); H NMR (DMSO-d /D O, 500
6
2
MHz): δ 8.15 (1H, s, Trz-H), 7.31-7.24 (3H, m, aromatics), 7.24 (2H, m,
aromatics), 7.22-7.16 (2H, m, aromatics), 6.90 (2H, m, aromatic), 5.11 (2H, m,
Trz-CH OH), 5.03 (1H, dd, J
2.5 Hz, J6a,6b 14.3, H-6a), 4.53, 4.33 (2H, 2d, J
5,6a
2
1
1
4
2.3 Hz, CH Ph), 4.51 (1H, dd, J
8.1 Hz, H-6b), 4.34 (1H, d, J1’,2’ 7.7 Hz, H-
2
5,6b
’), 4.21 (1H, d, J1,2 7.9 Hz, H-1), 3.71 (1H, m, H-5), 3.63 (1H, d, J3’,4’ 3.3 Hz, H-
’), 3.54-3.46 (3H, m, H-5’, H-6’), 3.41 (1H, dd, J2’,3’ 9.5 Hz, H-2’), 3.39 (1H, dd,
J2,3 7.7 Hz, J3,4 8.8 Hz, H-3), 3.34 (1H, dd, H-3’), 3.33 (1H, dd, J4,5 8.7 Hz, H-4),
1
3
3
1
1
7
.10 (1H, dd, H-2), 1.20 (9H, s, CH x 3); C NMR (DMSO-d /D O, 126 MHz): δ
3 6 2
43.2 (Trz C-C-R), 156.0, 143.0, 137.5, 128.3 x 2, 128.0 x 2, 127.7, 126.2 x 2,
14.3 x 2 (aromatics), 125.6 (Trz C-C-H), 103.8 (C-1’), 101.3 (C-1), 81.5 (C-4),
5.8 (C-5’), 74.6 (C-3), 73.2 (C-3’), 73.0 (C-2), 72.5 (C-5), 70.4 (C-2’), 69.7
(
PhCH ), 68.2 (C-4’), 61.2 (Trz-CH OH), 60.6 (C-6’), 50.6 (C-6), 33.9 (C(CH ) ),
2 2 3 3
+
3
1.5 (CH X 3); HRMS (ESI) m/z calcd. for C H N O [M + H] : 646.2970;
3 32 44 3 11
+
found: 646.2969; calcd. for C H N NaO [M + Na] : 668.2790; found:
32
43
3
11
6
68.2783.
4
.14.{1-[Benzyl β-
D-galactopyranosyl-(1
→4)-6-deoxy-β-D-glucopyranosid-6-yl]-
1
H-1,2,3-triazol-4-yl}-methanol O-(4-bromo-phenyl) ether (16j)
2
8