Synthesis of the hexasaccharide from Trypanosoma cruzi mucins with the Galp(1?→?2)Galf unit constructed with a superarmed thiogalactopyranosyl donor

Article abstract of DOI:10.1016/j.carres.2019.06.013

Hexasaccharide β-D-Galp-(1→ 2)-[β-D-Galp-(1 → 3)]-β-D-Galp-(1 → 6)-[β-D-Galp-(1 → 2)-β-D-Galf-(1 → 4)]-D-GlcNAc (1) was found O-linked in mucins of Trypanosoma cruzi epimastigotes and metacyclic trypomatigotes. Studies on the biological pathways and functionalities of the mucin oligosaccharides are prompted in order to understand the interactions of these molecules with the insect host. Trisaccharide constituent β-D-Galp-(1 → 2)-β-D-Galf-(1 → 4)-D-GlcNAc was constructed from the reducing to the non-reducing end. We discuss the difficulties to introduce a Galp unit at the O-2 position of a partially protected galactofuranosyl unit which were overcome using an anchimerically superarmed donor. By this route and employing a [3 + 3] nitrilium convergent approach hexasaccharide 1 was synthesized in moderate yield.

Full text of DOI:10.1016/j.carres.2019.06.013

Accepted Manuscript
Synthesis of the hexasaccharide from Trypanosoma cruzi mucins with the
Galp(1ꢀ→ꢀ2)fGuanlit constructed with a superarmed thiogalactopyranosyl donor
Gustavo A. Kashiwagi, Carmen R. Cori, Rosa M. de Lederkremer, Carola Gallo-
Rodriguez
PII:
S0008-6215(19)30339-8
https://doi.org/10.1016/j.carres.2019.06.013
CAR 7734
DOI:
Reference:
To appear in: Carbohydrate Research
Received Date: 31 May 2019
Revised Date: 19 June 2019
Accepted Date: 21 June 2019
Please cite this article as: G.A. Kashiwagi, C.R. Cori, R.M. de Lederkremer, C. Gallo-Rodriguez,
Synthesis of the hexasaccharide from Trypanosoma cruzi mucins with the Galp(1ꢀ→ꢀ2)fGuanlit
constructed with a superarmed thiogalactopyranosyl donor, Carbohydrate Research (2019), doi: https://
doi.org/10.1016/j.carres.2019.06.013.
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ACCEPTED MANUSCRIPT
TBDPSO
HO
OH
O
HO OH
O
O
O
O
RO
O
O
HO
OBz
BzO
O
OH
HO
BzO
BzO
O
AcNH
O
O
O
OBn
OH
O
OBz
BzO
O
HO
OH
low reactive acceptor
OH
AcNH
BzO
BzO
AcNH
NIS
OBn
HO
O
OH
O
OH
HO
TfOH
CH2Cl2
BnO
OBn
BnO
OBn
[3+3]
nitrile assisted
O
O
O
HO
OH
glycosylation
STol
BnO
BnO
HO
HO
OBz
OBz
O
only glycosylation with
anchimerically
superarmed donor
/
1.1:1
1
OH

ACCEPTED MANUSCRIPT
Synthesis of the hexasaccharide from Trypanosoma cruzi mucins with the
Galp(1→2)Galf unit constructed with a superarmed thiogalactopyranosyl donor
Gustavo A. Kashiwagi,1,2 Carmen R. Cori,1,2 Rosa M. de Lederkremer1,2 and
Carola Gallo-Rodriguez1,2*
1Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química
2
Orgánica. Buenos Aires, Argentina; CONICET- Universidad de Buenos Aires. Centro de
Investigaciones en Hidratos de Carbono (CIHIDECAR). Buenos Aires, Argentina.
*Corresponding author
E-mail address: cgallo@qo.fcen.uba.ar
1

Abstract
Hexasaccharide β-
Galf(1→4)]- -GlcNAc (1) was found O-linked in mucins of Trypanosoma cruzi epimastigotes and
ACCEPTED MANUSCRIPT
D-Galp-(1→2)-[β-D-Galp-(1→3)]-β-D-Galp-(1→6)-[β-D-Galp(1→2)-β-D-
D
metacyclic trypomatigotes. Studies on the biological pathways and functionalities of the mucin
oligosaccharides are prompted in order to understand the interactions of these molecules with the
insect host. Trisaccharide constituent β-D-Galp(1→2)-β-D-Galf(1→4)-D-GlcNAc was constructed
from the reducing to the non-reducing end. We discuss the difficulties to introduce a Galp unit at the
O-2 position of a partially protected galactofuranosyl unit which were overcome using an
anchimerically superarmed donor. By this route and employing a [3+3] nitrilium convergent
approach hexasaccharide 1 was synthesized in moderate yield.
Keywords: superarmed donor; galactofuranoside; galactofuranose; oligosaccharide; mucins;
Trypanosoma cruzi.
2

1. Introduction
ACCEPTED MANUSCRIPT
Around eight million people living mainly in rural areas of Latin America, and 300 thousand
people in the United States are infected with Trypanosoma cruzi, the protozoan parasite responsible
of Chagas disease [1]. Triatomine bugs act as transmission vectors in endemic areas. In recent years,
migration to non-endemic regions and/or countries increased the risk of transmission by blood
transfusion and organ transplants [2-3]. T. cruzi epimastigotes replicate in the midgut of a triatomine
insect, and down the intestine, they transform into non-proliferative metacyclic trypomastigotes that
pass from the hindgut to the feces. The infective metacyclic trypomastigotes enter the vertebrate host
through a skin wound or through mucosal membranes, invade cells and differentiate into amastigotes
which, after several cycles of binary division, differentiate back into trypomastigotes. The non-
replicative trypomastigotes are released into circulation upon host-cell rupture [4].
T. cruzi presents a surface covered by a dense glycocalyx, whose composition depends on the
differentiation stage of the living cycle [5-6]. The most abundant surface glycoproteins are GPI
anchored, highly glycosylated, mucin-like proteins (GP 35-50kDa). Mucins, composed by 60% in
mass of O-linked oligosaccharides, become negatively charged by accepting sialic acid residues (SA)
from host glycoconjugates [7]. A trans-sialidase from the parasite (TcTS), catalyzes the transfer of
sialic acid, a crucial process in pathogenesis [8-10]. The enzyme has been cloned [11] and used for in
vitro sialylation of oligosaccharides [12].
The structure of the mucin oligosaccharides depends not only on the differentiation stage of
the parasite, but also on the strains which were grouped into six evolutionary lineages classified as
TcI to TcVI on the basis of genetic and phenotypic characteristics [13]. A particular aspect of the O-
type oligosaccharide chains from Gp35/50 kDa mucins is that they are α-linked to threonine residues
via N-acetylglucosamine (GlcNAc) instead of N-acetylgalactosamine (GalNAc) as found in
mammalian mucins. Depending on the parasite strain, up to 20% of these α-D-GlcNAc residues may
remain unsubstituted or they may be elongated and branched, with various units of β-galactose in
different types of linkages and forms. The O-linked oligosaccharides may be derived from two cores:
3

ACCEPTED MANUSCRIPT
β-D-Galp(1→4)-GlcNAc or β-D-Galf(1→4)-GlcNAc [5,14]. Whereas the oligosaccharides carring
the first core contain galactose in the pyranose form [15-18], those with the latter core contain
galactose in both forms [19-23]. The biological reason of the diversity of this family of
oligosaccharides is still not known.
In addition to β-galactopyranose (βGalp) residues, TcI parasite strains display antigenic β-
galactofuranose (βGalf) units in the oligosaccharides. Several of these oligosaccharides have been
characterized [19-23] and most of them were chemically synthesized by our group [24-30]. It was
recently shown that the trisaccharide (Galfβ1-4[Galpβ1-6]GlcNAcα) and higher oligosaccharides that
contain this motif, in the mucins of O-linked glycans, inhibit the binding of T. cruzi epimastigotes to
the insect hindgut tissues [31].
Very recently, the 13C labeled tetrasaccharide alditol β-
D-Galp(1-3)-β-D-Galp-(1→6)[D-Galf-
(1→4)]- -GlcNAc-ol, was synthesized and offered to the scientific community as a standard to
D
facilitate analysis of native mucin O-glycans by mass spectroscopy [32]. The synthesis of a
tetrasaccharide from the Y strain mucins which contains only Galp has been described [33].
Hexasaccharides 1 and 2 (Figure 1) are the largest oligosaccharides present in mucins of T.
cruzi epimastigotes. These oligosaccharides were found not only in TcI strains but also in Tulahuen
strains classified as TcVI [23]. Accordingly, several Tulahuen-derived clones displayed TcI-like
features suggesting that the original ‘strain’ may have contained a mixture of parasite genotypes.
[34].
Isomers 1 and 2 differ in the form of the galactose residue β(1-2)linked to the internal
galactofuranose, which is pyranose in 1 and furanose in 2. We have already synthesized 1 and 2, and
their alditols [29,30], confirming the chemical structure found in the mucins. The synthesis of 2, the
Galf(1→2)Galf analogue, was accomplished with complete diastereoselectivity in each glycosylation
step[29]. The benzyl glycoside 4 was used as substrate of sialylation with recombinant TcTS and a
preparative synthesis allowed the characterization of the sialylated product establishing
unequivocally the site of sialylation at the less hindered (1-3)linked galactopyranose [30].
4

ACCEPTED MANUSCRIPT
Figure 1. Galf-containing hexasaccharides found in mucins of T. cruzi epimastigotes.
In the case of the the Galp(1→2)Galf analogue, HPAEC analysis of the trans-sialylation reaction on
benzyl glycoside 3 showed two monosialylated products whose relative abundance changed with
time. The low yield of the synthesis of 3 inhibited our ability to perform a preparative sialylation
reaction for characterization of the products [30].
The previous synthesis of 1 [30] involved the synthesis of linear trisaccharide β-
Galp(1→2)-β- -Galf(1→4)- -GlcNAc [28] using -galactono-1,4-lactone (5) as the internal
galactofuranosyl precursor. The glycosylation steps showed low stereoselectivity.
D-
D
D
D
In order to afford enough material for biological studies, we looked for other strategies for the
synthesis of hexasaccharide 1. In the present work we describe a new synthesis of hexasaccharide 1
which was planned with the aim of improving the stereoselectivity of each glycosylation step.
2. Results and discussion
Hexasaccharide 1 could be obtained by a [3+3] nitrilium based glycosylation of branched
trisaccharide imidate 6 [27] and a trisaccharide acceptor 7 (Scheme 1). Trisaccharide donor 6 has a
2-O-galactopyranosyl substituent, thus, lacking anchimeric assistance and was previous successfully
used for the synthesis of 1 [30] and other mucin oligosaccharides [27,29].
5

Compound 7 (Scheme 1) would be synthesized by glycosylation of known acceptor 9 [29]
ACCEPTED MANUSCRIPT
with galactopyranosyl donor 8 carrying an acyl group at O-2. Acceptor 9 was previously used for
synthesis of the analogous trisaccharide with a terminal β-D-Galf unit. Now 9 was synthesized using
benzyl α- -galactofuranoside 10, obtained in one step from free galactose. This strategy would
D
ensure the β-diastereoselective construction of the β-D-Galp(1→2)-D-Galf glycosidic bond
considering the 2-O-acyl substituent in compound 8.
Scheme 1. Retrosynthesis of 1 using linear trisaccharide 7 constructed in the upstream direction.
2.1 Synthesis of trisaccharide 7 following the upstream elongation
For glycosylation of 9 we first employed the trichloroacetimidate method [35]. However,
with the acyl trichloroacetimdate donors 11 [36] and 12 [37] (Table 1), no glycosylation product was
obtained (entries 1 and 2, Table 1) and the acceptor 9 was recovered together with the by-product of
hydrolysis of the donor (13 and 14). These unfavorable results suggested that a case of armed -
disarmed donor or acceptor selective reciprocity RDAS should be considered [38]. For this reason, a
glycosylation reaction was tested with a more reactive armed donor, the tetra benzyl
trichloroacetimidate 15 [39], using acetonitrile as solvent to favor the formation of the β product by
the nitrile effect [40-45] (entry 3, Table 1). However, the expected trisaccharide product was not
detected and acceptor 9 was recovered unaltered together with trichloroacetamide 16 [46]. The
formation of transposition products in the trichloroacetimidate method is associated with
6

glycosylation reactions in the presence of poorly nucleophilic or sterically hindered acceptors [28,
ACCEPTED MANUSCRIPT
35,47,48].
Table 1. Attempts of glycosylation of acceptor 9 with Galp donors 11, 12, 15, 17, 19 and 24.
Donor
Catalyst or Solvent
promoter
T/ °C
(duration)
By-Product
(yield)
Entry
17
(%
recovered)
AcO OAc
O
-15 → 0
(72h)
AcO
1
TMSOTf
CH2Cl2
72%
AcO
O
CCl3
NH
11
-20 → 0
(48h)
2
3
TMSOTf
TMSOTf
CH2Cl2
CH3CN
81%
77%
BnO OBn
O
-15 → 0
(72h)
BnO
BnO
O
CCl3
NH
15
4
5
NIS/TfOH
NIS/TfOH
CH2Cl2
-20
-18
72%
75%
CH2Cl2
CH2Cl2
6
DTBMP/
Tf2O
-78 → rt
49%
*
(20h)
a in all cases, concentration of acceptor 9 was 1 to 10 x 10-2 M. *: undetermined
7

We then tried the thioglycoside method of glycosylation without success. Reaction of 9 with
ACCEPTED MANUSCRIPT
donor 17 [49] using NIS/TfOH [50] as the promoting system gave 2,3,4,6-tetra-O-acetyl-β-
galactopyranosyl-(1→2)-1,3,4,6-tetra-O-acetyl-β- -galactopyranose (18) in 46% yield and 9 was
recovered unaltered (entry 4). Disaccharide 18 was previously obtained by condensation of 2,3,4,6-
D-
D
tetra-O-acetyl-α-D-galactopyranosyl bromide with 1,3,4,6-tetra-O-acetyl-α-D-galactopyranose as
acceptor [51].
When the benzoyl analogue of 17, the thiogalactopyranoside 19 was employed, a similar by-
product 20 was obtained (entry 5). Disaccharides 18 and 20 could be formed by an internal migration
of the acyl group at O-2 to the anomeric carbon, through a nucleophilic attack of triflic acid to the
carbon of the acyloxonium. A proton rearrangement in the trioxolane ion would leave the O-2
position free to react as an acceptor and yield the self-condensed product disaccharide 18 or 20,
respectively (Scheme 2).
AcO AcO
O
AcO
AcO
AcO
OAc
O
O
NIS
TfOH
AcO
AcO
OAc
O
O
AcO
AcO
OAc
O
AcO
AcO
OAc
AcO
AcO
OAc
O
AcO
AcO
OAc
O
O
STol
O
HO
O
AcO
AcO
OAc
O
OAc
O
AcO
HO
O
O
O
O
O
H
O
CF3S
O
17
O
O
Tf
O
Tf
AcO
18
H
Tf
Scheme 2. Suggested mechanism of formation of disaccharide 18 (and its benzoylated analogue 20).
The spectroscopic signals for the anomeric H and C of compound 18, matched with those
1
13
previously described [51]. In the case of 20, anomeric H- and C NMR displacements and JH1-H2
couplings were unambiguosly assigned by comparison to those of 18. Furthermore, HRMS analysis
showed that the molecular formula matched the proposed structure.
Due to the unusual result obtained using thioglycoside 19, we carried out the glycosylation of
21 [29], a galactofuranosyl derivative having the 2-OH free as a model reaction (Scheme 3). In this
case only glycosylation products were obtained and neither acyl transfer nor self-condensation by-
8

product were detected, indicating that the sugar linked to Galf in 9was responsible for the failure in
ACCEPTED MANUSCRIPT
glycosylate 17 or 19. Despite the anchimeric assistance provided by the benzoyl group, a mixture of
disaccharides 22 (β1-2) and 23 (α1−2) were produced in 9.5/1 β/α ratio (Scheme 3).
Scheme 3. Glycosylation of 29 with donor 19.
We also used the pivaloylated thiosulfoxide donor 24, taking into account that the pivaloyl group
has a lesser migratory aptitude, compared to benzoyl and acetyl groups [52] and the higher reactivity
of the thiosulfoxide. Although after 20 h of reaction [54,55] thin layer chromathography showed total
consumption of 24, several products of higher polarity, difficult to separate, were formed (entry 6,
Table 1) and no glycosylation product was detected.
To overcome the poor reactivity of acceptor 9, the anchimerically superarmed [56-58] donor p-
tolyl 3,4,6-tri-O-benzyl-2-O-benzoyl-1-thio-β-D-galactopyranoside (25) [59] was synthesized as
previously described, with slight modifications. Compound 25 was previously used as donor with
several pyranosyl acceptors [59-62].
Coupling of donor 25 with acceptor 9 (Table 2), in the presence of NIS/TfOH system [50] in
CH2Cl2 at 0 °C gave a new spot of higher polarity by TLC monitoring. As condensation products
could not be isolated by column chromatography, the fraction obtained was analyzed by High
Resolution Mass Spectroscopy (HRMS) and NMR, proving the presence of a mixture of
trisaccharides with terminal β-
D
-Galp- (1→2) and α-
D
-Galp-(1→2) units (26αβ). The mixture could
1
be separated after removal of the silyl ether group to afford the β diastereomer (27, JH-H = 8.0 Hz)
and α diastereomer (28, 1JH-H = 4.0 Hz) (total yield 62%, β/α 1.0:1.2) (Table 2, entry 1).
In order to improve the stereoselectivity in favor of the desired β diastereomer, different
temperatures, solvent systems and activators (conditions) were assayed (Table 2, entry 2-5). At -20
9

°C during 24 h (entry 2) the same yield of 62% was reached, but the diastereoselectivity was in a
ACCEPTED MANUSCRIPT
small degree reversed (β/α 1.1:1.0). At -78 ° C (entry 3), we expected to obtain an improvement of
the selectivity, but to our surprise, we did not obtain any glycosylation product. In an attempt to
improve the diastereoselectivity by the nitrile effect, a mixture of CH2Cl2 with CH3CN was used as
solvent in relation 1/6 v/v at -18 °C (entry 4). Although the diastereoselectivity was substantially
improved (β/α 2.5:1.0), the total yield of glycosylation dropped to 25%. Also, the use of p-
nitrophenyl sulfenyl chloride (p-NO2PhSCl)/silver triflate as activator system [63] (entry 5) of
glycosylation afforded a mixture of diastereomers with low yield (20%), favoring the unwanted
diastereomer (β/α 1.0:1.4).
In summary, the best result was obtained using NIS/TfOH as the promoting system in CH2Cl2
as solvent at -20 °C. Under these conditions, glycosylation reached 62 % of total yield with β/α ratio
1.1:1.0, after two steps (Table 2, entry 4).
Table 2. Glycosylation of superarmed donor 25 with disaccharide acceptor 9.
Entry
1
Promoter
Solvent
Temp.
Time
Method
Total Yield
27 + 28
(β/α ratio )
NIS/TfOH
NIS/TfOH
CH2Cl2
CH2Cl2
0 °C
5 h
B
A
62% (1.0:1.2)
62% (1.1:1.0)
-20 °C
24 h
2
3
NIS/TfOH
CH2Cl2
-78 °C
45 h
C
0%
10

4
5
NIS/TfOH
CH2Cl2/ CH3CN -18 °C
94 h
D
E
25% (2.5:1)
ACCEPTED MANUSCRIPT
1:6 v/v
p-NO2PhSCl/ AgTfO
CH2Cl2
-78 °C
80 h
20% (1.0:1.4)
Although coupling of donor 25 with acceptor 9 gave a diastereomeric mixture, this result was
encouraging, since, after many efforts, it was the first time that a galactopyranosyl unit could be
introduced. This would indicate that previously assayed donors (Table 1) lacked the appropriate
reactivity, and the “O-2/O-5 cooperative effect” [56-58] in 25 would be a requirement for
glycosylation.
The unexpected formation of the α-D-galactopyranoside diastereomer from 25 carrying an O-2
participating group was reported [61] as well as from other 2-O-acyl substituted galactopyranosyl
donors [28,64,65].
Beyond having obtained a mixture of diastereomers, the conclusion that emerges is that the
increase in reactivity provided by the mixed profile of protecting groups in 25 was the main
responsible for the introduction of the Galp unit to the unreactive acceptor 9 in the O-2 glycosylation
reaction. The present route of synthesis of the trisaccharide Galp(β1→2)Galf(β1→4)GlcNAc
involving a non-reducing end elongation is a better option than the previously described following
the opposite direction of elongation [28].
2.4 Synthesis of hexasaccharide 1
Despite the absence of anchimeric participation for glycosyation in trisaccharide donor 6, our
previous experience using this donor [27,29,30] moved us to follow a [3+3] convergent
glycosylation with 27 using acetonitrile as participating solvent. The coupling reaction afforded the
desired β-isomer 29 (Scheme 4) and no α-isomer was detected. Compound 29 co-eluted with the
hydrolyzed product of trisaccharide imidate 6. To isolate 29, acetylation was carried out, affording
hexasaccharide 29 with 43% yield after two steps. The by-product 1,4,6-tri-O-acetyl-2,3-di-O-
(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)-D-galactopyranose (31) [66] was obtained in 16 %
11

yield (Scheme 4). On the other hand, unreacted acceptor 27 was also recovered (34%). In our
ACCEPTED MANUSCRIPT
previous synthesis of hexasaccharide 1 [30], the [3+3] glycosylation step gave 33% yield but
acetylation of the column chromatography fraction was not required. Signals in the 1H and 13C-NMR
spectra of hexasaccharide 29 were assigned unequivocally by COSY, HSQC and NOESY
experiments. The β configuration of the new Galp(1→6)GlcNAc glycosidic linkage was evident by
the fact that C-1D signal of the internal β-
D
-Galp moiety appeared at 102.0 ppm in the HSQC, and
correlated with H-1D (δ 4.68) with a coupling constant of JH-H = 7.5 Hz. H-1D signal was partially
1
overlapped with a benzyl methylene hydrogen. This assignment was in agreement with the NOESY
H-H correlation signal between H-1D of the internal β- -Galp moiety and H-6aA and H-6bA of
D D-
GlcNAc moiety (See supporting information). Hexasaccharide 29 was further deprotected by
treatment with MeONa in MeOH to give 32 (78%) and in this case, H-1D appeared at δ 4.48 (7.9
Hz). Hydrogenolysis of 32 gave 1 with excellent yields. The spectra of hexasaccharide 1 matched
those previously described [30].
Scheme 4. Convergent synthesis of hexasaccharide 1.
3.
Conclusion
Hexasaccharide 1 was synthesized by a new strategy using benzyl α-D-galactofuranoside, as a
precursor of internal Galf. The overall yield led to a 65% improvement respect to the previous
12

synthetic route [30]. Construction of the linear trisaccharide from the reducing end to the non-
ACCEPTED MANUSCRIPT
reducing end ensured the complete diastereoselectivity of the galactofuranosyl linkage. However,
disaccharide acceptor 9 turned out to have limited reactivity. The use of anchimerically superarmed
donor p-tolyl 3,4,6-tri-O-benzyl-2-O-benzoyl-1-thio-β-D-galactopyranoside (19) enabled to
overcome this issue, although, diastereoselectivity could not be controlled with a O-2 participating
group.
4. Experimental
4.1 General Methods
TLC was performed on 0.2 mm Silica Gel 60 F254 aluminium supported plates. Detection was
effected by exposure to UV light or by spraying with 10 % (v/v) H2SO4 in EtOH and charring.
Column chromatography was performed on silica gel 60 (230-400 mesh). NMR spectra were
recorded with a Bruker AVANCE II 500 spectrometer at 500 MHz (1H) and 125.8 MHz (13C) or
with a Bruker AC 200 at 200 MHz (1H) and 50.3 MHz (13C). Chemical shifts are given relative to
1
13
the signal of internal acetone standard at 2.22 and 30.89 ppm for H NMR and C NMR spectra,
1
respectively when recorded in D2O. H and 13C assignments were supported by 2D COSY, and
HSQC experiments. High resolution mass spectra (HRMS) were recorded on a BRUKER
micrOTOF-Q II electrospray ionization mass spectrometer. Optical rotations were measured with a
Perkin-Elmer 343 polarimeter with a path length of 1 dm at 25 ºC.
4.2 General procedure of glycosylation reaction using the trichloroacetimidate method.
A dry solution of acceptor benzyl 3,5,6-tri-O-benzoyl-β-D-galactofuranosyl-(1→4)-2-
acetamido-3-O-benzoyl-6-O-ter-butyldiphenylsilyl-2-deoxy-α- -glucopyranoside (17, 1 equiv.) and
D
the corresponding trichloroacetimidate (11, 12 or 15, 1.1 equiv., Table 1) in freshly distilled anhyd
solvent (1 mL/10mg of 9), activated powdered 4 Å molecular sieves (30 mg/mL of solvent) was
13

added and the mixture was vigorously stirred at rt under argon. After 10 min., the mixture was
ACCEPTED MANUSCRIPT
cooled to the corresponding temperature, TMSOTf (0.25 equiv.) was added. The reaction was
quenched by addition of Et3N after TLC monitoring showed starting material disappeard. Then
mixture was filtered over celite and washed, the filtrates concentrated under vacuum and the residue
was purified by silica gel chromatography.
4.3.
galactopyranose (18)
To a solution of p-tolyl tetra-O-acetyl-1-thio-β-
mmol, 1.4 equiv.)[49], N-iodosuccinimide (16.0 mg, 0.071 mmol, 1.3 equiv.) and benzyl 3,5,6-tri-O-
benzoyl-β- -galactofuranosyl-(1→4)-2-acetamido-3-O-benzoyl-6-O-tert-butyl-diphenylsilyl-2-
deoxy-α- -glucopyranoside [29] (9, 60 mg, 0.053 mmol, 1.0 equiv.) in anhyd CH2Cl2 (2.5 mL),
2,3,4,6-Tetra-O-acetyl-β-D-galactopyranosyl-(1→2)-1,3,4,6-tetra-O-acetyl-α-D-
D-galactopyranoside (17, 33.5 mg, 0.074
D
D
activated powdered 4 Å molecular sieves was added under argon atmosphere. After 5 min. of
vigorous stirring at -20 °C, TfOH (3.5 µL, 0.040 mmol, 0.7 equiv.) was added. The stirring
continued for 22 h when a TLC analysis showed unchanged acceptor 9 (Rf 0.31, 3: 2 hexane-EtOAc),
a new polar compound (Rf 0.08, 1:1 hexane-EtOAc) and consumption of donor 7. The reaction was
quenched by addition of Et3N (6 ꢀL, 0.043 mmol), filtered through Celite washing with CH2Cl2. The
filtrate was washed with Na2S2O3 (2 x 30 mL), NaHCO3 (2 x 30 mL), H2O (1 x 30 mL), dried
(Na2SO4), filtered and concentrated to give a residue which was purified by silica column
chromatography (2:1 hexane-EtOAc).
The first fraction from the column gave unreacted acceptor 9 (49 mg, 72%). The next
fraction gave an amorphous solid which was identified as 2,3,4,6-tetra-O-acetyl-β-D-
galactopyranosyl-(1→2)-1,3,4,6-tetra-O-acetyl-α-D-galactopyranose (18, 11.4 mg, 46% from donor
1
1
17) with diagnostic signals similar to H NMR spectrum already described [51]. H NMR (CDCl3,
200 MHz): δ 6.32 (d, 1H, J = 3.9 Hz, H-1), 5.42 (dd, 1H, J = 3.4, 1.4 Hz, H-4 or H-4´), 5.36 – 5.31
(m, 1H, H-4 or H-4´), 5.31 (dd, J = 10.7, 3.3 Hz, H-3), 5.10 (dd, J = 10.6, 7.6 Hz, H-2´), 4.95 (dd, J
= 10.5, 3.4 Hz, H-3´), 4.57 (d, J = 7.7 Hz, H-1´), 4.27 – 3.87 (m, 7H, H-2, H-5, H-6ª, H-6b, H-5´, H-
14

6a´, H-6b), 2.17, 2.16, 2.15, 2.06, 2.04, 2.02, 1.99, 1.97 (8s, 24 H, CH CO). 13C NMR (CDCl , 50.3
ACCEPTED MANUSCRIPT
3
3
MHz): δ 167.5 (CH3CO), 101.5 (C-1´), 90.7 (C-1), 72.4, 70.7 (x 2), 69.1, 68.8, 68.1, 67.6, 66.7,
61.01 (C-6 or C-6´), 60.96 (C-6 or C-6´), 20.8, 20.6, 20.5 (CH3CO).
4.4.
galactopyranose (20)
To a solution of p-tolyl tetra-O-benzoyl-1-thio-β-
2,3,4,6-Tetra-O-benzoyl-β-D-galactopyranosyl-(1→3)-1,3,4,6-tetra-O-benzoyl-α-D-
D-galactopyranoside (19, 55 mg, 0.079
mmol, 1.5 equiv.), N-iodosuccinimide (17 mg, 0.077 mmol, 1.5 equiv.) and 9 (58 mg, 0.052 mmol,
1.0 equiv.) in anhyd CH2Cl2 (3 mL), activated 4Å powdered molecular sieves was added under argon
atmosphere. The mixture was cooled to -18 ºC and after 20 min of vigorous stirring, TfOH (3 µL,
0.034 mmol, 0.7 equiv.) was added and stirring continued for 2 h. Then the reaction was allowed to
warm to 0 °C and the stirring continued for 16 h. TLC analysis showed the presence of unreacted
acceptor 9 (Rf 0.25 3:2 hexane – EtOAc) and a new product (Rf 0,38 3:2 hexane – EtOAc). The
reaction was quenched by addition of Et3N (7 µL, 0.050 mmol) at 0 ºC, filtered over Celite washing
with CH2Cl2. The filtrate was washed with saturated aq. Na2S2O3 (2 x 30 mL), saturated aq.
NaHCO3 (2 x 30 mL) and H2O (1 x 30 mL), dried (Na2SO4) and concentrated under vacuum. The
residue was purified by silica gel column chromatography (30 : 1 toluene – EtOAc) to give, in the
first fraction, a colorless syrup identified as 2,3,4,6-tetra-O-benzoyl-β-D-galactopyranosyl-(1→2)-
1
1,3,4,6-tetra-O-acetyl-α-
D
-galactopyranose (20, 25 mg, 53% from 19). [α]D +85.3 (c 1, CHCl3), H
NMR (CDCl3, 500 MHz,): δ 8.24 – 7.14 (38H, H-arom.), 7.02 (d, 1H, J = 3.8 Hz, H-1), 6.94 – 6.91
(m, 2H, arom.), 6.01 (dd, 1H, J = 3.4, 1.0 Hz, H-4), 5.90 (dd, 1H, J = 3.4, 0.8 Hz, H-4´), 5.85 (dd,
1H, J = 10.4, 3.4 Hz, H-3), 5.63 (dd, 1H, J = 10.5, 7.8 Hz, H-2´), 5.44 (dd, 1H, J = 10.5, 3.4 Hz, H-
3´), 5.05 (d, 1H, J = 7.8 Hz, H-1´), 4.70 (dd, 1H, J = 11.3, 6.4 Hz, H-6a´), 4.68 – 4.62 (m, 1H, H-5),
4.57 (dd, 1H, J = 11.3, 6.2 Hz H-6a), 4.55 (dd, 1H, J = 10.4, 3.8 Hz, H-2), 4.42 (dd, 1H, J = 11.3, 6.8
13
Hz, H-6b´), 4.37 – 4.33 (m, 1H, H-5´), 4.32 (dd, 1H, J = 11.3, 7.3 Hz, H-6b). C NMR (CDCl3,
125.8 MHz): δ 166.1, 165.9, 165.3 (x 2), 165.2, 165.07, 165.01, 164.7 (PhCO), 136.6 – 128.0
(arom.), 102.1 (C-1´), 92.2 (C-1), 74.0 (C-2), 71.4 (C-3´), 71.2 (C-5´), 69.7 (C-3), 69.5 (C-2´), 68.6
15

(C-5), 68.3 (C-4), 67.6 (C-4´), 61.7 (C-6), 61.6 (C-6´). HRMS (ESI/APCI) m/z calcd for
ACCEPTED MANUSCRIPT
C68H54O19Na (M+Na+): 1197.3152. Found 1197.3183.
Unreacted 9 (49 mg, 75%) was recovered.
4.5.
galactofuranoside (22) and benzyl 2,3,4,6-tetra-O-benzoyl-α-
tri-O-benzoyl-α- -galactofuranoside (23)
A solution of p-tolyl 2,3,4,6-tetra-O-benzoyl-1-thio-β-
mmol, 1.5 equiv.) and dry benzyl 3,5,6-tri-O-benzoyl-α- -galactofuranoside [29] (21, 361 mg, 0.62
Benzyl
2,3,4,6-tetra-O-benzoyl-β-
D
-galactopyranosyl-(1→2)-3,5,6-tri-O-benzoyl-α-
D-
D-galactopyranosyl-(1→2)-3,5,6-
D
D-galactopyranoside (19, 655 mg, 0.93
D
mmol, 1.0 equiv.) in freshly distilled anhyd CH2Cl2, N-iodosuccinimide (209 mg, 0.93 mmol, 1.5
equiv.) and activated 4Å powdered molecular sieves (300 mg) was added under argon atmosphere.
The mixture was cooled to – 15 ºC, the stirring continued for 20 min and TfOH (38 µL, 0.43 mmol,
0.7 equiv.) was added. After 2.5 h, a TLC showed no acceptor 21 left (Rf 0.60, 3:2 hexane – EtOA)
and the reaction was quenched by addition of Et3N (65 µL, 0.47 mmol), filtered washing with
CH2Cl2. The organic phase was washed with Na2S2O3 (2 x 60 mL), NaHCO3 (2 x 70 mL), H2O (1 x
60 mL), dried (Na2SO4), filtered, and concentrated. The residue was purified by silica gel column
chromatography (2.5:1 hexane-EtOAc) to give in the first fraction 56 mg of 23 (8%) as a glassy
solid. [α]D +128.4 (c 0.7, CHCl3); 1H NMR (CDCl3, 500 MHz): δ 8.03 – 7.12 (40H, H-arom.), 6.09
(dd, 1H, J = 7.3, 6.5 Hz, H-3), 6.01 (dd, 1H, J = 10.6, 3.4 Hz, H-3´), 5.98 (dd, 1H, J = 3.4, 1.1 Hz,
H-4´), 5.76 (m, 1H, H-5), 5.72 (dd, 1H, J = 10.6, 3.7 Hz, H-2´), 5.54 (d, 1H, J = 3.7 Hz, H-1´), 5.15
(d, 1H, J = 4.4 Hz, H-1), 4.78 (d, 1H, J = 11.9, 4.4 Hz, H-6a), 4.70, 4.48 (2d, 2H, J = 12.3 Hz,
CH2Ph), 4.67 (dd, 1H, J = 11.9, 6.2 Hz, H-6b), 4.67 (dd, 1H, J = 7.3, 4.4 Hz, H-2), 4.67 – 4.63 (m,
1H, H-5´), 4.46 (dd, 1H, J = 6.5, 5.0 Hz, H-4), 4.39 (dd, 1H, J = 11.2, 6.4 Hz, H-6a´), 4.26 (dd, 1H, J
= 11.2, 6.9 Hz, H-6b´);13C NMR (CDCl3, 125.8 MHz): δ 166.2, 165.9, 165.68, 165.67, 165.5, 165.4,
165.2 (PhCO), 136.6, 133.5, 133.4, 133.11, 133.07, 133.0 (arom.), 130.0 – 127.7 (arom.), 98.1 (C-1),
95.9 (C-1´), 79.3 (C-2´), 79.0 (C-4), 75.3 (C-3), 71.5 (C-5), 69.5 (CH2Ph), 68.82, 68.80 (C-2´, C-4´),
16

68.1 (C-3´), 67.4 (C-5´), 62.9 (C-6), 61.9 (C-6´). HRMS (ESI/APCI) m/z calcd for C H NaO
ACCEPTED MANUSCRIPT
68 56
18
(M+Na+): 1183.3359. Found 1183.3382.
The next fraction gave 22 (546 mg, 76%) as a glassy solid: Rf 0.4 (11: 1 toluene-EtOAc), [α]D +96,7
(c 1, CHCl3); 1H NMR (CDCl3, 500 MHz): δ 8.10 – 6.97 (40H, H-arom.), 6.02 (dd, 1H, J = 8.1, 6.8
Hz, H-3), 5.95 (dd, 1H, J = 3.4, 0.8 Hz, H-4´), 5.89 (dd, 1H, J = 10.4, 8.0 Hz, H-2´), 5.70 (m, 1H, H-
5), 5.51 (dd, 1H, J = 10.4, 3.4 Hz, H-3´), 5.24 (d, 1H, J = 4.4 Hz, H-1), 5.03 (d, 1H, J = 8.0 Hz, H-
1´), 4.95, 4.65 (2d, 2H, J = 12.3 Hz, CH2Ph), 4.73 (dd, 1H, J = 11.9, 4.2 Hz, H-6a), 4.59 (dd, 1H, J =
11.9, 5.2 Hz, H-6b), 4.58 (dd, 1H, J = 8.1, 4.4 Hz, H-2), 4.46 (dd, 1H, J = 6.8, 5.3 Hz, H-4), 4.36
13
(dd, 1H, J = 11.4, 6.8 Hz, H-6a´), 4.34 (dd, 1H, J = 11.4, 4.6 Hz, H-6b), 4.24 (m, 1H, H-5´); C
NMR (CDCl3, 125.8 MHz): δ 165.90, 165.89, 165.7, 165.54, 165.47, 165.0, 164.9 (PhCO), 137.4,
133.7, 133.4, 133.3, 133.1, 132.92, 132.89, 132.8 (arom.), 130.0 – 127.6 (arom.), 101.9 (C-1´), 100.4
(C-1), 82.9 (C-2), 78.2 (C-4), 74.6 (C-3), 71.69, 71.66, 71.6 (C-3´, C-5, C-5´), 70.0 (CH2Ph), 69.3
(C-2´), 68.0 (C-4´), 62.9 (C-6), 62.0 (C-6´). HRMS (ESI/APCI) m/z calcd for C68H56NaO18
(M+Na+): 1183.33589. Found: 1183.34158.
4.6. (S) and (R)-p-Tolyl 2,3,4,6-tetra-O-pivaloyl-1-thio-β-
The procedure described for the phenyl analogue [53]was followed.
To a stirred solution of p-tolyl 1-thio-β- -galactopyranoside (185 mg, 0.64 mmol) and
D-galactopyranoside S-oxide (24)
D
DMAP (39 mg, 0.32 mmol) in anhyd pyridine (1.9 mL) at rt, freshly distilled pivaloyl chloride (0.80
mL, 0.64 mmol) was added and warmed to 110 °C. After 18 h of stirring at 110ºC, the reaction was
cooled to rt, and quenched by slow addition of methanol (2.5 mL) and diluted with CH2Cl2 (15 mL).
The mixture was washed with 1N HCl (3 x 15 mL), saturated aq. NaHCO3 (2 x 15 mL) and H2O (1 x
15 mL), dried (Na2SO4) and concentrated. The residue was purified by column chromatography
(25:1 hexane – EtOAc) to give p-tolyl 2,3,4,6-tetra-O-pivaloyl-1-thio-β-D-galactopyranoside (159
mg, 40%). Rf 0.45 (10:1 hexane – EtOAc); [α]D – 9.0º (c 1.2, CHCl3); 1H NMR (500 MHz, CDCl3):
δ 8.09 (d, 2H, J = 8.0 Hz, arom.), 7.89 (d, 2H, J = 8.0 Hz, arom.), 5.39 (d, 1H, J = 3.0 Hz, H-4), 5.17
(t, 1H, J = 9.8 Hz, H-2), 5.10 (dd, 1H, J = 10.0, 3.1 Hz, H-3), 4.65 (d, 1H, J = 9.7 Hz, H-1), 4.18 (m,
17

1H, H-6a), 4.02 – 3.98 (m, 2H, H-5, H-6b), 2.34 (s, 3H, CH Ph), 1.22, 1.18, 1.14, 1.09 (4s, 36H,
ACCEPTED MANUSCR3IPT
(CH3)3CCO). 13C NMR (CDCl3, 125.8 MHz): δ 177.9, 177.2, 176.8, 176.4 ((CH3)3CCO), 138.7,
134.3, 129.6, 127.3 (arom.), 85.9 (C-1), 74.6 (C-5), 72.2 (C-3), 66.9 (C-4), 66.6 (C-2), 61.4 (C-6),
38.9, 38.7, 38.7, 38.7 ((CH3)3CCO), 27.2, 27.1, 27.04, 26.95 ((CH3)3CCO), 21.2 (CH3Ph). HRMS
(ESI/APCI) m/z calcd for C33H50O9SNa (M+Na+): 645.30677. Found: 645.30775.
To a solution of p-tolyl 2,3,4,6-tetra-O-pivaloyl-1-thio-β-D-galactopyranoside (43 mg; 0.069
mmol) in CH2Cl2 (0.8 mL) cooled at -75 ºC, was added m-CPBA (77%) (20.4 mg, 0.083 mmol) in
CH2Cl2 (0.4 mL). After 3 h of vigorous stirring, TLC showed no starting material left and two more-
polar products (Rf 0.32 and 0.17, 4: 1 hexane - AcOEt). The mixture was quenched by addition of
saturated aq. NaHCO3 (3 mL) stirring for 5 min at rt. The mixture was diluted with CH2Cl2 (20 mL)
and organic phase was extracted with saturated aq. NaHCO3 (2 x 10 mL), H2O (1 x 10 mL), dried
(Na2SO4) and concentrated. Purification of the residue by silica gel column chromatography (4:1
hexane – EtOAc) gave a first fraction of the column afforded less polar tentatively assigned (R)-
1
diastereomer (Rf 0.32, 1:1 hexane – EtOAc) of 24 (12.6 mg, 28%) as a colorless syrup. H NMR
(CDCl3, 200 MHz): δ 7.54 (d, 2H, J = 8.3 Hz, arom.), 7.33 (d, 2H, J = 8.4 Hz, H arom.), 5.53 (t, 1H,
J = 9.8 Hz, H-2), 5.36 (d, 1H, J = 3.0 Hz, H-4), 5.16 (dd, 1H, J = 9.9, 3.0 Hz, H-3), 4.42 (d, 1H, J =
9.7 Hz, H-1), 4.14 – 3.88 (m, 3H, H-5, H-6a, H-6b), 2.42 (s, 3H, CH3Ph), 1.23, 1.13, 1.11, 1.10 (4s,
36H, (CH3)3CCO). HRMS (ESI/APCI) m/z calcd for C33H50NaO10S (M+Na+): 661.30169. Found:
661.30210.
The second fraction from the column gave 24.3 mg of a syrup tentatively identified as (S)-
1
diasteromer of 24 (55 %). H NMR (CDCl3, 200 MHz): δ 7.66 (d, 2H, J = 8.1 Hz, arom.), 7.33 (d,
2H, J = 8.1 Hz, arom.), 5.30 (d, 1H, J = 2.8 Hz, H-4), 5.14 (dd, 1H, J = 9.6, 2.8 Hz, H-3), 5.02 (t,
1H, J = 9.8 Hz, H-2), 4.64 (dd, 1H, J = 9.9 Hz, H-1), 4.15 – 4.00 (m, 2H), 3.82 – 3.70 (m, 1H), 2.42
(s, 3H, CH3Ph), 1.24, 1.15, 1.07, 0.93 (4s, 36H, (CH3)3CCO). HRMS (ESI/APCI) m/z calcd for
C33H50NaO10S (M+Na+): 661.30169. Found 661.30354.
13C NMR of (R) and (S) mixture: (CDCl3, 50.3 MHz): δ 129.6, 129.3, 127.3, 126.0, 92.0 (C-1
maj.), 89.2 (C-1 min.), 75.5 (min.), 74.9 (maj.), 72.2 (min.), 71.8 (maj.), 66.5 (min.), 66.1 (maj.),
18

64.9 (maj.), 64.3 (min.), 61.1 (min.), 60.2 (maj.), 38.9, 38.8, 38,7, 27.1, 27.0, 26.9, 26.7, 21.4
ACCEPTED MANUSCRIPT
(CH3Ph). The absolute sulfoxide configuration of each diastereomer was tentatively assigned based
on differences in the chemical shifts C-1 in 13C-NMR spectra comparing to the acetyl analogues
[67].
4.7. Reaction of benzyl 3,5,6-tri-O-benzoyl-β-
benzoyl-6-O-ter-butyldiphenylsilyl-2-deoxy-α- -glucopyranoside (9) with (S)/(R)-p-tolyl
2,3,4,6-tetra-O-pivaloyl-1-thio-β- -galactopyranoside S-oxide (24)
D-galactofuranosyl-(1→4)-2-acetamido-3-O-
D
D
To a stirred mixture of (R/S)-24 (33 mg, 0.051 mmol), 2,6-di-tert-butyl-4-methylpyridine
(DTBMP) (21 mg, 0.10 mmol, 2 equiv.) and activated 4 Å powdered molecular sieves, in anhyd
CH2Cl2 (3.5 mL) cooled to – 78 °C, was added Tf2O (10 ꢀL, 0.059 mmol, 1.2 equiv.) under argon
atmosphere. After 30 min. of vigorous stirring, a solution of acceptor 9 (59 mg, 0.052 mmol, 1
equiv.) in CH2Cl2 (1.8 mL) was added. The mixture was kept at -78 °C for 19 h until a TLC
examination showed disappearance of thiosulphoxide derivatives and several dispersed spots which
are presumed as decomposition products, together with a spot that comigrated with the acceptor 9.
The reaction was quenched by addition of saturated aq. NaHCO3 (5 mL) and the organic phase was
succesively washed with saturated aq. NaHCO3 (2 x 15 mL) and H2O (2 x 15 mL), dried (Na2SO4),
filtered, and concentrated under reduced pressure. The residue was purified by column
chromatography (6:1 hexane – EtOAc) giving several fractions lacking glycosylation product. 26 mg
of unreacted acceptor 9 was recovered (49%).
4.8. Benzyl 3,5,6-tri-O-benzyl-2-O-benzoyl-β-
-galactofuranosyl-(1→4)-2-acetamido-3-O-benzoyl-2-deoxy-α-
benzyl 3,5,6-tri-O-benzyl-2-O-benzoyl-α- -galactopyranosyl-(1→2)-3,5,6-tri-O-benzoyl-β-D-
D-galactopyranosyl-(1→2)-3,5,6-tri-O-benzoyl-β-
D
D-glucopyranoside (27) and
D
galactofuranosyl-(1→4)-2-acetamido-3-O-benzoyl-2-deoxy-α-D-glucopyranoside (28)
4.8.1.Method A
19

To a vigorously stirred mixture of 9 (0.90 g, 0.80 mmol) and activated powdered 4 Å
ACCEPTED MANUSCRIPT
molecular sieves (800 mg) in 20 mL of freshly distilled anhyd CH2Cl2 under argon atmosphere, was
added a solution of p-tolyl 3,4,6-tri-O-benzyl-2-O-benzoyl-1-thio-β-D-galactopyranoside [59] (25,
744 mg, 1.24 mmol, 1.6 equiv.) and N-iodosuccinimide (305 mg, 1.36 mmol, 1.7 equiv.) in 30 mL of
freshly distilled CH2Cl2 via cannula. The mixture was vigorously stirred for 10 min at rt and then
cooled to -20 °C under argon. After 5 min, TfOH (50 ꢀL, 0.57 mmol, 0.7 equiv.) was added. After
24 h of stirring at -20 °C, a TLC showed no donor left and a new spot (Rf 0.28, 7: 4 hexane-EtOAc,
twice developed). The mixture was diluted in CH2Cl2 (100 mL), washed with saturated aq. Na2S2O3
(3 x 70 mL), saturated aq. NaHCO3 (2 x 70 mL) and H2O (2 x 70 mL), dried (Na2SO4), filtered and
concentrated. Purification by column chromatography (7: 4 hexane-EtOAc) gave a fraction that was
1
13
analyzed by H and C NMR spectroscopy and showed the presence of two diastereomers benzyl
3,5,6-tri-O-benzyl-2-O-benzoyl-β- -galactopyranosyl-(1→2)-3,5,6-tri-O-benzoyl-β-
galactofuranosyl-(1→4)-2-acetamido-3-O-benzoyl-6-O-tert-butyldiphenylsilyl-2-deoxy-α-
glucopyranoside (26β) and benzyl 3,5,6-tri-O-benzyl-2-O-benzoyl-α- -galactopyranosyl-(1→2)-
D
D-
D-
D
3,5,6-tri-O-benzoyl-β-D-galactofuranosyl-(1→4)-2-acetamido-3-O-benzoyl-6-O-tert-
butyldiphenylsilyl-2-deoxy-α-
D
-glucopyranoside (26α). 13C NMR (CDCl3, 50.3 MHz) δ: anomeric
signals 107.0 (C-1', β anomer), 105.5 (C-1', α anomer), 100.5 (C-1'', β anomer), 96.1 (C-1'', α
anomer), 95.9 (C-1, α and β anomers). HMRS (ESI/APCI) analysis of this fraction gave two peaks
m/z C99H98NO21Si (MH+): 1664.6395 and C99H97NNaO21Si (M+Na+): 1686.6215, found 1664.6429
and 1686.6271, respectively.
To a stirred solution of a mixture of the isolated diastereomers 26αβ (1.44 g) in DMF (15
mL) cooled to 0 ° C, a solution of 1M TBAF in anhyd THF (1.7 mL, 1.7 mmol) was added, followed
by glacial acetic acid (104 ꢀL, 1.8 mmol). After 24 h of stirring, a TLC showed no material left (Rf
0.65, 2:3 hexane-EtOAc, twice developed) and two new spots of higher polarity: Rf 0.27 and 0.25 (2:
3 hexane-EtOAc, twice developed). The mixture was diluted in CH2Cl2 (150 mL), washed with H2O
(4 x 110 mL), dried (Na2SO4), filtered, and concentrated under reduced pressure. Column
chromatography of the residue (5: 4 hexane-EtOAc, then 1:1 hexane-EtOAc) gave a first fraction of
20

ACCEPTED MANUSCRIPT
28 (341 mg, 30% two steps) as a glassy solid: Rf 0.27 (1:1 hexane – EtOAc, twice developed), [α]D
1
+82.9 (c 0.6, CHCl3), H NMR (CDCl3, 500 MHz): δ 8.02 (dd, 2H, J = 8.3, 1.2 Hz, arom.), 7.95 –
7.92 (m, 4H, arom.), 7.87 (dd, 2H, J = 8.4; 1.3 Hz, arom.), 7.77 (dd, 2H, J = 8.4; 1.2 Hz, arom.), 7.61
– 7.14 (m, 35H, arom.), 5.69 (d, 1H, J = 9.6 Hz, NH), 5.55 (dd, 1H, J = 4.3, 1.2 Hz, H-3´), 5.47 (t,
1H, J = 10.2 Hz, H-3), 5.46 (d, 1H, J = 4.0 Hz, H-1´´), 5.34 (dd, 1H, J = 10.3, 4.0 Hz, H-2´´), 5.30
(ddd, 1H, J = 7.3, 4.7, 3.3 Hz, H-5´), 5.11 (s, 1H, H-1´), 4.94 (d, 1H, J = 3.7 Hz, H-1), 4.88 (d, 1H, J
= 11.3 Hz, CH2Ph), 4.67 (d, 1H, J = 11.9 Hz, CH2Ph), 4.63 (d, 1H, J = 11.8 Hz, CH2Ph), 4.60 (d,
1H, J = 11.8 Hz, CH2Ph), 4.54 (d, 1H, J = 11.3 Hz, CH2Ph), 4.50 (d, 1H, J = 11.9 Hz, CH2Ph), 4.44
(d, 1H, J = 11.9 Hz, CH2Ph), 4.41 (ddd, 1H, J = 10.7, 9.6, 3.7 Hz, H-2), 4.39 (d, 1H, J = 11.9 Hz,
CH2Ph), 4.30 (dd, 1H, J = 12.3, 7.3 Hz, H-6a´), 4.30 (d, 1H, J = 1.2 Hz, H-2´), 4.25 (dd, 1H, J =
12.3, 3.3 Hz, H-6b´), 4.20 (t, 1H, J = 6.8 Hz, H-5´´), 4.07 (t, 1H, J = 9.6 Hz, H-4), 4.02 (t, 1H, J =
4.7 Hz, H-4´), 3.98 (dd, 1H, J = 10.3, 2.8 Hz, H-3´´), 3.95 (d, 1H, J = 2.8 Hz, H-4´´), 3.75 – 3.65 (m,
3H, H-5; H-6a; H-6b), 3.55 – 3.47 (m, 2H, H-6a´; H-6b´), 2.48 (dd, 1H, J = 5.9, 2.8 Hz, -OH), 1.74
(s, 3H, CH3CONH); 13C NMR (CDCl3, 125.8 MHz): δ 169.8 (CH3CONH), 167.4 (PhCO), 166.8
(PhCO), 165.9 (PhCO), 165.4 (PhCO), 164.9 (PhCO), 106.5 (C-1´), 96.9 (C-1), 96.0 (C-1´´), 84.4
(C-2´), 81.2 (C-4´), 77.4 (C-3´), 76.8 (C-3´´), 74.9 (CH2Ph), 74.3 (C-4´´), 73.3 (CH2Ph), 73.1 (C-4),
72.9 (CH2Ph), 72.6 (C-2´´), 72.0 (C-3), 71.3 (C-5), 70.1 (CH2Ph), 69.8 (C-5´), 69.6 (C-5´´), 68.1 (C-
6´´), 63.0 (C-6´), 60.4 (C-6), 52.2 (C-2), 23.1 (CH3CONH). HRMS (ESI/APCI) m/z calcd for
C83H79NNaO21 (M+Na+): 1448.5037. Found: 1448.5063.
Second fraction from the column afforded 27 (365 mg, 32%, two steps) as a glassy solid. (Rf
1
0.25, 1:1 hexane – EtOAc, twice developed); [α]D +13,3 (c 0.6, CHCl3); H NMR (CDCl3, 500
MHz): δ 7.95 – 7.73 (m, 10H, arom.), 7.56 – 7.12 (m, 35H, arom.), 5.77 (d, 1H, J = 9.4 Hz, NH),
5.58 (dd, 1H, J = 10.0, 8.0 Hz, H-2´´), 5.55 (dd, 1H, J = 10.8, 9.3 Hz, H-3), 5.37 (s, 1H, H-1´), 5.29
(m, 1H, H-5´), 5.23 (dd, 1H, J = 5.1, 1.9 Hz, H-3´), 4.98 (d, 1H, J = 3.6 Hz, H-1), 4.92 (d, 1H, J =
11.6 Hz, CH2Ph), 4.73 (d, 1H, J = 11.9 Hz, CH2Ph), 4.67 (d, 1H, J = 8.0 Hz, H-1´´), 4.58 (d, 1H, J =
12.3 Hz, CH2Ph), 4.55 (d, 1H, J = 11.6 Hz, CH2Ph), 4.50 (d, 1H, J = 11.9 Hz, CH2Ph), 4.46 (d, 1H, J
= 12.2 Hz, CH2Ph), 4.42 (ddd, 1H, J = 10.8, 9.4, 3.6 Hz, H-2), 4.40 (d, 1H, J = 11.7 Hz, CH2Ph),
21

4.38 (s, 1H, H-2´), 4.33 (d, 1H, J = 11.7 Hz, CH Ph), 4.19 (t, 1H, H-4´), 4.15 (t, 1H, H-4), 4.06 (dd,
ACCEPTED 2MANUSCRIPT
1H, J = 12.2, 3.3 Hz, H-6a´), 3.98 (dd, 1H, J = 12.2, 7.7 Hz, H-6b´), 3.92 (d, 1H, J = 2.7 Hz, H-4´´),
3.91 – 3.87 (m, 1H, H-6a), 3.84 – 3.77 (m, 2H, H-5, H-6b), 3.61 (dd, 1H, J = 10.0, 2.7 Hz, H-3´´),
13
3.54 – 3.50 (m, 3H, H-5´´, H-6a´´, H-6b´´), 2.68 (t, 1H, J = 5.6 Hz, OH), 1.75 (s, 3H, CH3CO); C
NMR (CDCl3, 125.8 MHz): δ 169.8 (CH3CONH), 167.1 (PhCO), 165.6 (PhCO), 165.2 (PhCO),
138.3 – 127.6 (C arom.); 107.9 (C-1´), 100.7 (C-1´´), 96.7 (C-1), 85.7 (C-2´), 80.9 (C-4´), 79.6 (C-
3´´), 77.7 (C-3´), 74.5 (CH2Ph), 74.4 (C-4), 73.8 (C-5´´), 73.4 (CH2Ph), 72.6 (C-4´´), 72.5 (C-3),
71.9 (CH2Ph), 71.5 (C-5), 71.3 (C-2´´), 70.1 (C-5´), 69.8 (CH2Ph), 68.3 (C-6´´), 62.9 (C-6´), 60.8 (C-
6), 52.4 (C-2), 23.1 (CH3CONH). HRMS (ESI/APCI) m/z calcd for C83H79NNaO21 (M+Na+):
1448.5037. Found: 1448.5099.
4.8.2.Method B
Same procedure described for Method A was followed at 0 °C for 5 h. Compound 9 (33 mg,
0.03 mmol) and 25 (25 mg, 0.04 mmol), gave 16 mg of 28 (33%) and 14 mg of 27 (29%).
4.8.3. Method C
Same procedure described for Method A was followed except that the reaction was carried
out at -18 °C for 94 h using a mixture of 1:6 CH2Cl2 - CH3CN as solvent. Compound 9 (66 mg, 0.06
mmol) and 25 (47 mg, 0.07 mmol), gave 7 mg of 28 (7%) and 17 mg of 27 (18%).
4.8.4. Method D
Same procedure described for Method A was followed at -78 °C for 45 h. From 51 mg of 9
(0.05 mmol) and 36 mg of 25 (0.05 mmol), no products of glycosylation were obtained.
4.8.5. Method E
A suspension of donor 9 (40 mg, 0.04 mmol), 25 (51 mg, 0.05 mmol), silver triflate (30 mg,
0.11 mmol) and 4 Å molecular sieves in anhydrous CH2Cl2 (4 mL) was stirred, with the exclusion of
22

light, at room temperature under argon. After 10 min, the mixture was cooled to −78 °C and a
ACCEPTED MANUSCRIPT
solution of p-nitrobenzenesulfenyl chloride (9.2 mg, 0.05 mmol) in anhydrous CH2Cl2 (0.5 mL) was
added. The reaction was stirred for 80 h and was quenched by addition of saturated aq. NaHCO3
solution (0.5 mL). The mixture was diluted with CH2Cl2 (5 mL) and filtered. Then the mixture was
treated as described in Method A to give 7 mg of 28 (12%) and 5 mg of 27 (8%).
4.9. Benzyl 2,3,4,6-tetra-O-acetyl-β-
galactopyranosyl-(1→3)]-4,6-di-O-acetyl-β-
benzoyl-β- -galactopyranosyl-(1→2)-3,5,6-tri-O-benzoyl-β-
acetamido-3-O-benzoyl-2-deoxy-α- -glucopyranoside (29)
To a flask containing dried O-(2,3,4,6-tetra-O-acetyl-β-
tetra-O-acetyl-β- -galactopyranosyl-(1→3)]-4,6-di-O-acetyl-β-
D
-galactopyranosyl-(1→2)-[2,3,4,6-tetra-O-acetyl-β-
-galactopyranosyl-(1→6)- [3,5,6-tri-O-benzyl-2-O-
-galactofuranosyl-(1→4)]-2-
D-
D
D
D
D
D-galactopyranosyl)-(1→ 2)-[2,3,4,6-
D
D-galactopyranosyl
trichloroacetimidate [27] (6, 358 mg, 0.33 mmol, 1.2 equiv.) and activated powdered 4 Å molecular
sieves, a solution of acceptor 27 (389 mg, 0.27 mmol, 1.0 equiv.) in freshly distilled anhydr CH3CN
(16 mL) was added via cannula. The suspension was cooled to -20 ° C and after vigorous stirring for
10 min, TMSOTf (11 ꢀL, 0.061 mmol, 0.2 equiv.) was added. After 40 h of stirring, TLC analysis
showwed no trisaccharide donor 6 left (Rf 0.48, 2: 3 toluene-EtOAc, TEA 0.2%) and a new product
of higher polarity (Rf 0.33, 2: 3 toluene-EtOAc, TEA 0.2%). The reaction was quenched by addition
Et3N (10 ꢀL, 0.072 mmol), filtered whashing with CH3CN, and concentrated under reduced pressure
to give a residue which was purified by column chromatography (1: 1 toluene-EtOAc, followed by
5:6 toluene-EtOAc). Unreacted acceptor 27 (133 mg, 34%, Rf 0.55, 2: 3 toluene-EtOAc) was
recovered from the first fraction from the column. The next fraction afforded a mixture (405 mg) of
the desired hexasaccharide 29 (Rf 0.23, 2:3 toluene-EtOAc) which co-eluted with hydrolyzed
trisaccharide donor 2,3-di-O-(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)-4,6-di-O-acetyl-α,β-D-
galactopyranose (30) as suggested by NMR spectroscopy [66]. This fraction was acetylated with
acetic anhydride (4 mL) in anhydrous pyridine (4 mL) at room temperature for 40 min, followed by
addition of MeOH at 0 ºC. Evaporation of the mixture gave a residue which was purified by column
23

chromatography (2:3 hexane-EtOAc, followed by 2:5 hexane-EtOAc). The first fraction afforded
ACCEPTED MANUSCRIPT
1,4,6-tri-O-acetyl-2,3-di-O-(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)-D-galactopyranose
(31)
[66] (52 mg, 16 %, Rf 0.35, 1:2 hexane–EtOAc). The next fraction afforded 29 (270 mg, 43% two
steps, Rf 0.25 (1:2 hexane – EtOAc)) as a glassy solid: [α]D +11.3 (c 1, CHCl3), 1H NMR (CDCl3,
500 MHz): δ 8.01 – 7.64 (m, 10H, arom.), 7.55 – 7.04 (m, 35H, arom.), 5.83 (d, 1H, J = 9.3 Hz,
NH), 5.56 (dd, 1H, J = 10.7, 8.60 Hz, H-3A), 5.53 (dd, 1H, J = 9.7, 8.3 Hz, H-2C), 5.41 (m, 2H, H-3B
, H-4E), 5.38 (d, 1H, J = 2.9 Hz, H-4F), 5.33 (s, 1H, H-1B ), 5.31 (d, 1H, J = 3.6 Hz, H-4D), 5.26 (dd,
1H, J = 10.3, 8.2 Hz, H-2F), 5.18 (dd, 1H, J = 10.5, 3.3 Hz, H-3E), 5.12 (dd, 1H, J = 10.4, 7.5 Hz, H-
2E), 5.03 (dd, 1H, J = 10.5, 3.5 Hz, H-3F), 4.99 (m, 1H, H-5B), 4.99 (d, 1H, J = 4.0 Hz, H-1A), 4.99
(d, 1H, J = 7.5 Hz, H-1F), 4.96 (d, 1H, J = 7.6 Hz, H-1E), 4.90 (d, 1H, J = 11.6 Hz, CH2Ph), 4.88 (d,
1H, J = 7.7 Hz, H-1C), 4.79 (d, 1H, J = 11.9 Hz, CH2Ph), 4.69 (d, 1H, J = 7.5 Hz, H-1D), 4.68 (d, 1H,
J = 12.1 Hz, CH2Ph), 4.53 (d, 1H, J = 12.2 Hz, CH2Ph), 4.50 (d, 1H, J = 11.7 Hz, CH2Ph), 4.45 (m,
1H, H-2B), 4.37 (d, 1H, J = 11.6 Hz, CH2Ph), 4.36 (d, 2H, J = 12.6 Hz, CH2Ph), 4.35 (bs, 2H, H-2A,
H-6aE), 4.28 – 4.24 (m, 1H, H-4B), 4.23 – 4.08 (m, 9H, H-6aA, H-3D, H-4A, H-5E, H-5F, H-6aB, H-
6bE, H-6aF, H-6bF), 4.01 – 3.86 (m, 8H, H-2D, H-4C, H-5A, H-5D, H-6bA, H-6bB, H-6aD, H-6bD), 3.61
– 3.56 (m, 3H, H-3C, H-5C, H-6aC), 3.53 (m, 1H, H-6C), 2.17, 2.05, 2.03, 2.00, 2.00, 1.99, 1.96, 1.94,
1.94, 1.90 (10s, 30H, CH3CO), 1.74 (s, 3H, CH3CONH). 13C NMR (CDCl3, 125.8 MHz): δ 170.4 (x
3), 170.3, 170.2, 170.1, 169.9, 169.7 (x 2), 169.3, 169.1 (11C, CH3CO ), 167.2, 165.4, 165.3, 164.8
(4C, PhCO), 138.0, 137.7, 137.4, 136.9 (4C, C arom.), 133.3, 133.2, 133.0, 132.7 (4C, C arom.),
130.0 – 127.5 (C arom.), 107.7 (C-1B), 102.0 (C-1D), 100.2 (C-1F), 99.6 (C-1E), 99.3 (C-1C), 96.0 (C-
1A), 85.3 (C-2B), 80.2 (C-3C), 79.8(C-4B), 78.4 (C-2D), 76.4 (C-3B), 75.8 (C-4A or C-3D), 75.7 (C-4A
or C-3D), 74.4 (CH2Ph), 74.0 (C-5C), 73.3 (CH2Ph), 72.4 (C-3A), 72.2 (C-4C), 72.0 (CH2Ph), 71.26
(C-2C), 71.1 (C-3F), 70.8 (C-5E or C-5F), 70.7 (C-5A), 70.7 (C-5E or C-5F), 70.6 (C-3E), 70.4 (C-5D),
70.0 (C-2E), 69.8 (C-5B), 69.7 (C-6F), 69.6 (CH2Ph), 69.1 (C-4D), 68.7 (C-6A), 68.4 (C-6C), 66.9 (C-
4E), 66.9 (C-4F), 62.2 (C-6B), 61.8 (C-6D), 60.7 (C-6E or C-6F), 60.7 (C-6E or C-6F), 52.5 (C-2A), 23.0,
21.0, 20.8, 20.7, 20.7, 20.6 (x 2), 20.6, 20.6, 20.5, 20.3 (11C, CH3CO). HRMS (ESI/APCI) m/z calcd
for C121H130NO46 (M+): 2332.7859. Found: 2332.7842.
24

ACCEPTED MANUSCRIPT
4.10. Benzyl β-
(1→6)-[3,5,6-tri-O-benzyl-β-
acetamido-2-deoxy-α-
D
-galactopyranosyl-(1→2)-[β-
D
-galactopyranosyl-(1→3)]-β-
D
-galactopyranosyl-
D-galactopyranosyl-(1→2)-β-
D-galactofuranosyl-(1→4)]-2-
D-glucopyranoside (32)
To a solution of 29 (250 mg. 0.107 mmol) in CH3OH (2.0 mL) cooled to 0 °C, a solution of
NaOCH3 in CH3OH (2.6 M, 1.6 mL) was added under nitrogen atmosphere. After 4 h of stirring at
rt, a TLC analysis of an aliquot previously neutralized by the addition of an Amberlite resin particle
showed one new product (Rf 0.27; 7:1:1 n-propanol – EtOH - H2O). The mixture was passed through
an ion exchange column (Amberlite resin IR-120 plus, H+ form) eluting with 9: 1 CH3OH-H2O. The
eluate was concentrated under reduced pressure and coevaporated with H2O (5 x 1 mL) under high
vacuum to remove the methyl benzoate. The residue was dissolved in deionized H2O (2 mL) and
purified by passing the solution through a reverse phase C18 cartridge eluting with deionized water.
The concentration of the eluate afforded 32 (119 mg, 80%) as a glassy solid: [α]D +15.5 (c 1, H2O);
1H NMR (CD3OD-d4, 500 MHz): δ 7.37 – 7.35 (m, 2H, arom.), 7.29 – 7.10 (m, 18H, arom.), 5.60 (d,
1H, J = 2.1 Hz, H-1B), 4.76 (d, 1H, J = 3.50 Hz, H-1A), 4.71 (d, 1H, J = 12.1 Hz, CH2Ph), 4.70 (d,
1H, J = 7.9 Hz, H-1E), 4.68 (d, 1H, J = 10.8 Hz, CH2Ph), 4.65 (d, 1H, J = 11.6 Hz, CH2Ph), 4.60 (d,
1H, J = 12.1 Hz, CH2Ph), 4.52 (d, 1H, J = 12.1 Hz, CH2Ph), 4.48 (d, 1H, J = 7.9 Hz, H-1D), 4.43 (d,
1H, J = 7.7 Hz, H-1C), 4.40 (d, 1H, J = 7.8 Hz, H-1F), 4.37 (d, 1H, J = 11.9 Hz, CH2Ph), 4.33 (d, 1H,
J = 12.0 Hz, CH2Ph), 4.31 (d, 1H, J = 11.8 Hz, CH2Ph), 4.21 (dd, 1H, J = 4.8, 2.1 Hz; H-2B), 4.17
(dd, 1H, J = 7.9, 4.8 Hz; H-3B), 4.01-3.98 (m, 1H, H-6aA), 3.98 (dd, 1H, J = 7.9, 3.1 Hz, H-4B), 3.92
– 3.78 (m, 8H, H-4A, H-2A, H-2C, H-2D, H-6bA, H-6a*), 3.71 – 3.46 (m, 18H, H-6aB, H-6bB, H-6a*,
H-6b*, H-6a*, H-6b*, H-6b*, H-6aC, H-5B, H-3D, H-3A,H-5*, H-2E,H-2F, H-4E, H-4F), 3.44 – 3.41 (m,
1H, H-3C), 3.41 – 3.32 (m, 3H, H-3E, H-5*, H-6bC), 3,30 – 3,26 (m; 2H; H-5*; H-3F), 1,97 (s, 3H,
13
CH3CONH). C NMR (CD3OD-d4, 125.8 MHz): δ 173.8 (CH3CONH), 140.2, 140.2, 139.7; 139.0
(C-arom.), 130.0 – 128.9 (C-arom.), 109.3 (C-1B), 106.2 (C-1F), 104.8 (C-1E), 103.6 (C-1C), 103.4
(C-1D), 97.9 (C-1A), 89.1 (C-2B), 85.9 (C-3D), 83.8 (C-4B), 83.7 (C-3C), 80.6 (C-4A), 77.2 (2C, C-3B,
C-5*), 76.8 (C-2D), 76.5, 76.0 (C-5*), 75.7 (2C, CH2Ph, C-3E), 75.3, 75.1, 75.0, 74.53 (CH2Ph),
25

74.45 (CH Ph), 73.4, 73.1, 72.6, 71.8 (C-5B), 71.6, 71.4, 71.3, 71.1 (C-6C), 70.6 (2C, CH Ph), 70.5,
ACCEPTED MANUSCRIPT
2
2
69.0 (C-6A), 64.5 (C-6B), 62.8 (2C, C-6*, C-6*), 62.6 (C-6*), 55.5 (C-2A), 22.7 (CH3CONH). *could
be D, E or F. HRMS (ESI/APCI) m/z calcd for C66H89NNaO31 (M+Na+): 1414.5311. Found:
1414.5329.
4.11.
β-D-Galactopyranosyl-(1→2)-[β-D-galactopyranosyl-(1→3)]-β-D-galactopyranosyl-
(1→6)-[β-D-galactopyranosyl-(1→2)-β-D-galactofuranosyl-(1→4)]-2-acetamido-2-deoxy-α,β-D-
glucopyranose (1)
A mixture of hexasaccharide 32 (39 mg, 28 ꢀmol) and Pd/C 10% Deguzza type E101 NE/W
(40 mg) in 9: 1 CH3OH-H2O (2.8 mL) was hydrogenated at 3 atm at rt. After 10 h, a TLC showed no
39 left and a new product (Rf 0.22, 7: 2:2.5 n-propanol - EtOH - H2O, twice developed). The catalyst
was filtered through a bed of Celite washing with H2O (2 mL). The filtrate was concentrated at rt and
the residue was purified by passing through C-18 reverse phase cartridge, to give 1 (29 mg, 99%) as
a hygroscopic glassy solid, in an anomeric mixture of α/β 7:3: [α]D +0.5 (c 1, H2O), 1H NMR (D2O,
500 MHz): δ anomeric zone and diagnostic signals: 5.54 (m, 1H, H-1B α and β anomers), 5.21 (d,
0.7H, J = 3.4 Hz, H-1A α anomer), 4.83 (d, J = 7.7 Hz, Galp H-1), 4.70 (d, 0.3H, J = 8.4 Hz, H-1A β
anomer), 4.67 – 4.58 (m, 3H, Galp H-1, α and β anomers), 4.40 (dd, 0.7H, J = 4.1; 1.9 Hz, H-2B α
anomer), 4.37 (dd, 0.3H, J = 3.9; 1.7 Hz, H-2B β anomer), 2.05 (s, 3H, CH3CONH, α and β
anomers). 13C NMR (D2O, 125.8 MHz): δ 175.4, 175.10 (CH3CONH, α and β anomers), 133.1,
129.4, 128.9 (C arom.), 107.6, 107.5 (C-1B, α y β anomers), 104.7 (2C; C-1F, α and β anomers),
103.5, 103.4 (2C, C-1E, α and β anomers), 102.4, (2C, C-1D, α and β anómers), 102.2 (2C, C-1C, α
and β anomers), 95.8 (C-1A, β anomer), 91.4 (C-1A, α anomer), 87.7 (C-2B, β anomer), 87.6 (C-2B, α
anomer), 83.8, 83.2, 83.0, 78.7, 76.1, 76.1, 75.9, 75.8, 75.2, 73.7, 73.5, 73.2, 73.2, 72.1, 71.8, 71.6,
70.9, 70.0, 69.9, 69.7, 69.5, 69.4, 69.2, 63.4, 62.1, 61.8, 61.7, 61.5, 57.3 (C-2A, β anomer), 54.8 (C-
2A, α anomer), 22.8 (CH3CONH, β anomer), 22.5 (CH3CONH, α anomer). The NMR spectra
matched with already described [30].
26

ACCEPTED MANUSCRIPT
Acknowledgment. This work was supported by grants from Universidad de Buenos Aires
(20020150100070BA) and CONICET (PIP 11220150100366CO). R. M. de Lederkremer and C.
Gallo-Rodriguez are research members of CONICET.
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28