Synthesis of trisaccharides containing internal galactofuranose O-linked in Trypanosoma cruzi mucins

Article abstract of DOI:10.1016/j.carres.2009.12.005

The trisaccharides β-d-Galf-(1→2)-β-d-Galf-(1→4)-d-GlcNAc (5) and β-d-Galp-(1→2)-β-d-Galf-(1→4)-d-GlcNAc (6) constitute novel structures isolated as alditols when released by reductive β-elimination from mucins of Trypanosoma cruzi (Tulahuen strain). Trisaccharides 5 and 6 were synthesized employing the aldonolactone approach. Thus, a convenient d-galactono-1,4-lactone derivative was used for the introduction of the internal galactofuranose and the trichloroacetimidate method was employed for glycosylation reactions. Due to the lack of anchimeric assistance on O-2 of the galactofuranosyl precursor, glycosylation studies were performed under different conditions. The nature of the solvent strongly determined the stereochemical course of the glycosylation reactions when the galactofuranosyl donor was substituted either by 2-O-Galp or 2-O-Galf.

Full text of DOI:10.1016/j.carres.2009.12.005

Carbohydrate Research 345 (2010) 385–396
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Synthesis of trisaccharides containing internal galactofuranose O-linked
in Trypanosoma cruzi mucins
*
Verónica M. Mendoza, Gustavo A. Kashiwagi, Rosa M. de Lederkremer, Carola Gallo-Rodriguez
CIHIDECAR, Departamento de Química Orgánica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Pabellón II, 1428 Buenos Aires, Argentina
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 21 October 2009
Received in revised form 24 November 2009
Accepted 5 December 2009
Available online 14 December 2009
The trisaccharides b-D-Galf-(1?2)-b-D-Galf-(1?4)-D-GlcNAc (5) and b-D-Galp-(1?2)-b-D-Galf-(1?4)-D-
GlcNAc (6) constitute novel structures isolated as alditols when released by reductive b-elimination from
mucins of Trypanosoma cruzi (Tulahuen strain). Trisaccharides 5 and 6 were synthesized employing the
aldonolactone approach. Thus, a convenient D-galactono-1,4-lactone derivative was used for the intro-
duction of the internal galactofuranose and the trichloroacetimidate method was employed for glycosyl-
ation reactions. Due to the lack of anchimeric assistance on O-2 of the galactofuranosyl precursor,
glycosylation studies were performed under different conditions. The nature of the solvent strongly
determined the stereochemical course of the glycosylation reactions when the galactofuranosyl donor
was substituted either by 2-O-Galp or 2-O-Galf.
Keywords:
Trypanosoma cruzi
Mucin
Trisaccharide
Ó 2009 Elsevier Ltd. All rights reserved.
Trichloroacetimidate glycosylation
Galactofuranose
Galactonolactone
1. Introduction
(Fig. 1).13–16 Pentasaccharide 4, the major oligosaccharide in the
G-strain, has two terminal Galp for possible sialylation. By prepara-
Trypanosoma cruzi, the agent of Chagas’ disease, the American
trypanosomiasis, presents surface dominated by carbohy-
tive trans-sialylation of 4 and analysis of the product, we have
demonstrated that selective monosialylation occurs on the
(1?3)-linked Galp.16 Recently, the synthesis of a mucin oligosac-
charide from the Y strain (group II), which lacks Galf, has been also
reported.17
a
drates.1,2 The mucins of T. cruzi stand out, not only because of their
crucial function but also from the structural point of view. They are
acceptors of sialic acid in the well-studied trans-sialidase reac-
tion,3–5 closely related to the infectivity of the parasite. A puzzling
fact is that the presence of galactofuranose is confined to strains of
a sylvatic origin grouped as T. cruzi I6 (Fig. 1). These are less infec-
tive than strains belonging to group II, from the domestic cycle,7
that only contain galactopyranose. The oligosaccharides 1–4 and
8 (Fig. 1) were first described in mucins of the G-strain.8,9 They
In this work, we describe the synthesis of b-
Galf-(1?4)- -GlcNAc (5) and b- -Galp-(1?2)-b-
-GlcNAc (6) and the corresponding alditols, employing the
D-Galf-(1?2)-b-
D-
D
D
D-Galf-(1?4)-
D
aldonolactone approach. Trisaccharide 5 is part of the higher oligo-
saccharides 7 and 9, whereas trisaccharide 6 is included in 8. Thus,
intermediates of these syntheses are useful for the construction of
the higher oligosaccharides. The two linear trisaccharides present
an internal galactofuranose unit substituted at position 2 by b-Galf
or b-Galp. Thus, stereoselective glycosidation was hampered by the
lack of neighboring group assistance.
The biosynthetic pathways involved in the construction of these
O-chains in the mucins have not been elucidated. Taking into ac-
count the various structures found in T. cruzi, the synthesis of these
oligosaccharides would be useful for studies on their biosynthesis.
present a unique core of b-D-Galf-(1?4)-GlcNAc which is a-O-gly-
cosidically linked to serine or threonine in the mucins. Later, the
same structures were found in the Dm28c clon together with the
novel oligosaccharide 7.10 More recently, in the Tulahuen strain,
trisaccharides 5 and 6 as well as pentasaccharide 9 were also
found.6 In 2009, from drug-resistant Colombiana strain, isolated
from a chronic human case in Colombia, oligosaccharides 1–4
and 8, as in the G-strain, were found.11 Among these oligosaccha-
rides, 5–9, present an internal Galf, 2-O-substituted by Galp or Galf.
Our laboratory has been involved in the synthesis of this family of
compounds with the aim to use them for trans-sialidase studies.12
To date, we have reported the synthesis of compounds 1–4
2. Results and discussion
The synthesis of Galf containing oligosaccharides from other
glycoconjugates of T. cruzi has been performed; however, in these
cases, Galf appeared as terminal non-reducing end.18,19 The syn-
thesis of oligosaccharides containing internal galactofuranose is
* Corresponding author. Tel./fax: +54 11 4576 3352.
E-mail address: cgallo@qo.fcen.uba.ar (C. Gallo-Rodriguez).
0008-6215/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2009.12.005

386
V. M. Mendoza et al. / Carbohydrate Research 345 (2010) 385–396
OH
HO
O
OH
O
HO
O
O
O
OH
HO
HO
OH
O
O
AcNH
O
OH
HO
OH
OH
AcNH
OH
OH
OH
OH
OH
1
2
HO
OH
HO OH
HO
OH
HO OH
O
O
O
O
O
O
HO
O
O
O
HO
OH
O
OH
OH
O
HO
OH
O
O
O
O
O
HO
HO
OH
OH
OH
OH
AcNH
AcNH
HO
OH
OH
OH
OH
OH
OH
OH
3
4
OH
OH
O
O
O
O
OH
O
HO
O
OH
AcNH
HO
OH
OH
AcNH
OH
HO
HO
O
O
O
O
OH
OH
OH
OH
OH
OH
OH
5
6
OH
OH
HO
OH
HO
OH
HO OH
HO OH
O
O
O
O
O
O
O
O
HO
HO
OH
HO
OH
HO
O
O
OH
O
OH
O
AcNH
O
O
O
O
O
HO
O
OH
HO
OH
OH
HO
HO
OH
AcNH
OH
OH
O
O
HO
HO
OH
O
O
OH
OH
OH
OH
OH
OH
OH
OH
OH
7
8
HO
OH
HO OH
O
O
O
O
HO
OH
OH
O
OH
O
O
HO
OH
AcNH
O
O
OH
OH
OH
9
OH
OH
OH
Figure 1. Oligosaccharides in mucins of Trypanosoma cruzi (group I).
more challenging, and involves the choice of convenient galacto-
furanosyl precursors as well as glycosylation methods. These as-
pects have been recently reviewed.20,21 Few internal b-Galf
containing oligosaccharides have been synthesized to date.22–29
The following methods were employed for the internal linkage
construction: Koenings–Knörr,22a cyanoorthoester,22b–d anomeric
acetate,23 trichloroacetimidate,24–26 and more recently, thioglyco-
side,27 20-carboxybenzyl-glycosides28,29 and glycosyl fluoride.29
Our strategy relied on the use of a convenient derivative of
present in glycoinositolphospholipids from Leishmania24 and oligo-
saccharide constituents of the arabinogalactan from Mycobacterium
tuberculosis.25 The stable lactone acts as a virtual-protected Galf
and may be also selectively substituted,30,31 avoiding the tedious
synthesis of the Galf unit precursor from galactose, thus, providing
a straightforward method for the oligosaccharide synthesis. On
the other hand, in our previous work related to mucin oligosaccha-
rides,15,16 we have succeeded in glycosylating the 4-OH of
GlcNAc derivative, benzyl 2-acetamido-3-O-benzoyl-6-O-t-butyldi
D-galactono-1,4-lactone as a common precursor of the internal Galf
phenylsilyl-2-deoxy-a-D-glucopyranoside (18) with a galactofur-
of both trisaccharides, 5 and 6. The glycosyl aldonolactone ap-
anosyl imidate donor with very good yield. For that reason, we
proach has been employed for the syntheses of a trisaccharide
decided to use the same derivative 18 considering, in addition, that

V. M. Mendoza et al. / Carbohydrate Research 345 (2010) 385–396
387
the orthogonal silyl group would allow further elongation to access
to hexasaccharides 7 and 8, and pentasaccharide 9, our future tar-
gets. In that case, both oligosaccharides 5 and 6 have to be assembled
from the non-reducing end.
(J = 4.3 Hz) at 5.04 ppm, 0.23 ppm downfield shifted compared to
10 due to O-2 glycosylation. To obtain the furanose-reducing end
for further activation, 15 was reduced with disiamylborane (DSB)
to give disaccharide 16 in 82% yield as an anomeric mixture, as
indicated by the 13C NMR spectrum (104.7 and 97.7 ppm for the
The fact that selective acylation of 5,6-O-isopropylidene-D-
galactono-1,4-lactone occurs on OH-2 due to stereoelectronic
factors,24,30 led us to evaluate the regioselectivity of trichloroace-
timidate glycosylation employing 1.1 equiv of O-(2,3,5,6-tetra-O-
a anomer and 105.1 and 101.9 ppm for the b anomer). In spite of
the superposition of signals in the 1H NMR spectrum, HSQC and
COSY experiments allowed full assignments. The integration of
the H-20 signals of both anomers (d 5.45 and 5.37 ppm) showed a
benzoyl-b-D-galactofuranosyl) trichloroacetimidate. However,
preliminary studies showed moderate regioselectivity toward the
7:3 b/a anomeric ratio of the mixture.
1?2 linkage formation.
The next step was the activation of the anomeric center as the
trichloroacetimidate derivative. This method has been widely used
in the construction of b-Galf linkages by anchimeric assis-
tance.15,24–26,27b,37 To our knowledge, there are no reports on the
use of a Galf donor with a 2-O-sugar substituent. Thus, treatment
of 16 with trichloroacetonitrile and DBU at rt gave a mixture of
imidates as shown by TLC, which was easily purified by column
chromatography. The less polar b-imidate 17b was obtained in
73% yield, and its stereochemistry was assigned on the basis of
the H-1 signal that appeared as a downfield shifted singlet (d
6.55 ppm) in the 1H NMR spectrum, and confirmed by the 13C
NMR spectrum where the anomeric carbons appeared at 105.0
and 104.8 ppm for both galactofuranosyl moieties in the b-config-
2.1. Synthesis of 3,4,5-tri-O-benzoyl-D-galactono-1,4-lactone
(10), the common precursor of trisaccharides 5 and 6
Compound 10 was prepared from 5,6-O-isopropylidene-D-
galactono-1,4-lactone (11)32 temporarily protected with a bulky
silyl group (Scheme 1). The fact that the isopropylidene group
requires careful reaction conditions and cautious purification to
avoid its partial hydrolysis along the synthesis15 led us to its early
replacement. Thus, tert-butyldiphenylsilyl was chosen as protect-
ing group, taking into account its resistance to acids. Treatment
of 11 with 1.2 equiv of tert-butyldiphenylchlorosilane gave the si-
lyl derivative 12, regioselectively in 83% yield; no 3-OH protection
was detected (Scheme 1). Deprotection of the isopropylidene by
HOAc–H2O at 80 °C followed by benzoylation with benzoyl chlo-
ride in pyridine gave 13 in 68% yield. The 1H NMR spectrum of
13 showed the H-5 and H-3 signals shifted downfield at 5.95 and
5.61 ppm, respectively, confirming the selective substitution at
OH-2 by TBDPS in 12. On the other hand, the TBDPS group resisted
the hydrolysis conditions as demonstrated by the absence of tetra-
uration. From the second fraction of the column, a sirupy
a-imidate
17 was obtained (10%) and, in this case, the H-1 resonance ap-
a
peared at 6.73 ppm as a doublet with a characteristic coupling con-
stant (J = 4.5 Hz), whereas in the 13C NMR spectrum the C-1
resonance appeared at 98.0 ppm confirming the
a stereochemistry.
It is interesting to point out that the sirupy -imidate transformed
a
partially in the b-imidate on standing in the freezer (ꢀ20 °C).
Considering the lack of anchimeric assistance in the imidate
17b, we evaluated the influence of the solvent in the glycosylation
reaction with GlcNAc acceptor 18,15 previously employed in the re-
lated synthesis of 3 and 4. The difficulties in glycosylating the 4-OH
group of GlcNAc encouraged many investigations in this area.38
However, Galf glycosylation of the 4-OH group of GlcNAc deriva-
tives has been performed with very good yields in our synthesis
of mucin oligosaccharides (3 and 4)15,16, and by others,39 employ-
O-benzoyl-D-galactono-1,4-lactone as byproduct. Upon aqueous
acetic acid deprotection of the isopropylidene group of the TBDMS
analog of 12, partial undesired removal of the silyl group oc-
curred.33 Removal of the TBDPS group of 13 with 5% HF (48 wt %
in water) in acetonitrile gave 10 in low yield with significant for-
mation of more polar byproducts. A mixture of tetrabutylammo-
nium fluoride buffered with acetic acid in THF–DMF solution34
was then employed to give 10 in 81% yield.
ing 2,3,5,6-tetra-O-benzoyl-D-galactofuranose trichloroacetimi-
date37b as the donor. This reactivity could be attributed to the
higher flexibility of the furanose ring. On reaction of glucosamine
acceptor 18 with imidate 17b in CH2Cl2 using TMSOTf as catalyst,
the best results were obtained by maintaining the temperature at
5 °C for three days to give the desired trisaccharide 19 in 41% yield
after column chromatography. The stereochemistry of the new gly-
cosidic linkage was established as b as indicated by the 13C NMR
spectrum. The anomeric signals appeared at 96.2 (GlcNAc), 105.5
(C-100) and 106.1 ppm (C-10), whereas the 1H NMR showed a dou-
blet at 4.92 ppm (J = 3.7 ppm) for H-1 of GlcNAc, and two singlets
(d 5.58 and 5.44 ppm) assigned to the internal (H-10) and terminal
(H-100) Galf anomeric hydrogen by HETCOR and COSY experiments.
2.2. Synthesis of trisaccharide 5
Having 10 in hand, the next step was the introduction of the ter-
minal Galf (Scheme 2). The absence of acid labile-protecting groups
in 10 allowed the use of the SnCl4-promoted glycosylation meth-
od13–15,35 with 1,2,3,5,6-penta-O-benzoyl-
D-galactofuranose (14)
as the donor, which was synthesized in one step from galact-
ose.14,36 Glycosyl lactone 15 crystallized from the crude mixture
in 78% yield. Further purification from the mother liquors in-
creased the yield to 92%. As expected, the new glycosidic linkage
has the b-configuration as determined by 13C NMR spectrum that
showed only one anomeric signal at 104.9 ppm. The 1H NMR spec-
trum showed the H-10 as a singlet at 5.57 ppm and H-20 as a dou-
blet with a small coupling constant (J = 1.4 Hz), a characteristic
pattern of the anomeric b stereochemistry for galactofuranosyl
The a-(1?4) glycosylation product 20 was also obtained in 18%
yield after column chromatography and crystallized from ethanol.
The 13C NMR spectrum showed the characteristic anomeric signals
of b-Galf and
third signal at 102.9 ppm, which suggested a a
a
-GlcNAc at 105.5 and 96.2 ppm, respectively, and a
-configuration for
linkages. The H-2 of the lactone appeared as
a
doublet
TBDPSCl
O
O
O
O
1) HOAc-H2O
(1.2 equiv.)
O
OH
O
O
O
OH
OBz
OBz
TBAF, AcOH
DMF, THF
81%
80 ºC
Imidazol
OH
OTBDPS
OH
OTBDPS
DMF (anh)
2) BzCl, C5H5N
0 ºC
O
O
O
O
OBz
OBz
OBz
OBz
83%
68%
11
12
13
10
Scheme 1. Synthesis of intermediate 10.

388
V. M. Mendoza et al. / Carbohydrate Research 345 (2010) 385–396
O
OBz
OBz
OTBDPS
O
O
O
OBz
O
OBz
O
OBz
CCl
3HO
BzO
O
OBz
OBz
OH
OBz
O
O
NH
AcNH
OBn
DSB, THF
82%
14
O
O
O
O
CCl3CN, DBU
CH2Cl2
10
OBz
18
OBz
OBz
OBz
OBz
SnCl4, Cl2CH2
92%
OBz
OBz
OBz
OBz
OBz
TMSOTf, 4A MS
CH2Cl2
OBz
OBz
OBz
OBz
15
OBz
OBz
OBz
OBz
17
β (73%)
16
+ 17α (10%)
OTBDPS
O
OR1
O
O
O
O
OBz
O
OR2
BzO
R2O
AcNH
AcNH
OBn HF 5 % (MeCN)
80%
OR3
O
O
OBz
O
O
OR2
OBz
OBz
OR2
OR2
OBz
OR2
OBz
OBz
OR2
OR2
OH
OH
19
O
(41%)
O
OH
OH
R = H, R2 = Bz, R3 = Bn
1
21
22
HO
+ 20
(α(1-4)) (18%)
NaOMe / MeOH
95%
AcNH
O
O
R , R = H, R3 = Bn
1
2
OH
H2 / Pd (C), 3 atm
90%
OH
OH
NaBH4
99%
23
5 R1, R2, R3 = H
OH
OH
OH
Scheme 2. Synthesis of b-D-Galf-(1?2)-b-D-Galf-(1?4)-D-GlcNAc (5) and alditol 23.
the new Galf linkage. In a HETCOR experiment, this signal corre-
lated to the corresponding H-10, which appeared at 5.40 ppm as a
doublet with a coupling constant of 3.2 Hz. This value was slightly
free trisaccharide 5 (90% yield), as a mixture of anomers as indi-
cated by the 13C NMR spectrum. The anomeric carbon of the
-Glc-
NAc unit appeared at 91.3 ppm whereas the anomeric carbon of
the b anomer appeared less intense at 95.6 ppm. The /b anomeric
ratio was established as 3:2 approximately based on the integra-
tion of the H-20 signals of the internal Galf units, which appeared
at 4.17 and 4.14 ppm as doublet of doublets. Further reduction of
5 with NaBH4 gave the corresponding alditol 23. The chemical
shifts in the 1H NMR spectrum were in agreement with those re-
ported for the novel alditol released by reductive b-elimination
from mucins of T. cruzi (Tulahen strain).6
a
smaller compared to other related
synthetized,27a,28,40–42 particularly, compared to the pentasaccha-
ride constituent of varianose,27a where the internal
-Galf is 2-O-
substituted by a glucopyranose. This small value was assigned to
the
-Galf stereochemistry considering that the H-20 appeared
a-Galf compounds previously
a
a
a-D
as a doublet of doublets (J = 3.2, 2.3 Hz), whereas the H-50appeared
downfield shifted (d 5.85 ppm) as expected for a benzoylated
exocyclic-OH of the Galf moiety. Moreover, C-20 resonated at
81.5 ppm, as expected, due to glycosylation.
The global yield of the glycosylation step was 59% (b/
a
ratio of
2.3. Synthesis of trisaccharide 6
2.3:1), with a moderate diastereoselectivity in favor of the b-(1?4)
linked product 19. This result prompted us to evaluate participat-
ing solvents, taking into account the strong influence of ether or
acetonitrile in diastereoselective control of glycosylations with
pyranose donors 2-O-substituted with non-participating protect-
ing groups.43 When acetonitrile was employed, the global yield
of glycosylation was lower (37%), with a 19:20 diastereomeric ratio
of 1:1 (after column chromatography). The use of ethyl ether in-
creased the global glycosylation yield (57%), however slightly
With the aim to construct the b-Galf linkage in 6, we chose
2,3,4,6-tetra-O-acetyl-D
-galactopyranosyl trichloroacetimidate44
(24) as the donor, taking into account that the acetyl group in O-
2 would provide the anchimeric assistance to afford the b linkage
diastereoselectively. However, reaction of lactone acceptor 10 with
imidate 24 afforded the b-(1?2) disaccharide lactone 25 in 54%
yield together with the unexpected
(Scheme 3).
a(1?2)linked 26 in 18% yield
favoring the
a
anomer (19:20 ratio 1:1.4).
The 13C NMR spectrum of 25 showed the anomeric carbon at
99.8 ppm indicating the b-configuration for the new glycosidic
linkage, confirmed by the doublet at 4.63 ppm for the H-10
(J = 7.9 Hz) in the 1H NMR spectrum. On the other hand, the 13C
NMR spectrum of the minor product 26 showed a signal at
97.1 ppm in the anomeric region. This signal correlated, in a HSQC
experiment, with a doublet at d 5.58 ppm with J = 3.1 Hz, and
With trisaccharide 19 in hand, the next step of the synthesis
was the removal of the TBDPS group at O-6 with 5% HF (48 wt %
in water) in acetonitrile to give 21 in 80% yield. No hydrolysis of
the labile Galf linkage was observed in the overnight acidic reac-
tion conditions. Debenzoylation of 21 with sodium methoxide gave
the benzyl glycoside 22 in 95% yield. Hydrogenolysis of 22 gave the

V. M. Mendoza et al. / Carbohydrate Research 345 (2010) 385–396
389
AcO
AcO
OAc
O
OTBDPS
O
AcO
O
NH
CCl3
O
CCl3
O
HO
O
OBz
O
OBz
O
BzO
OH
CCl3CN, DBU
AcO
OBz
OAc
O
TMSOTf
OAc
O
OAc
O
AcO
AcO
AcO
AcO
AcNH
OBn
NH
24
DSB, THF
86%
O
O
18
10
O
AcO
TMSOTf, 4A MS
CH2Cl2
CH2Cl2
AcO
OBz
OBz
25 (54%)
AcO
OBz
OBz
OBz
OBz
AcO
TMSOTf, 4A MS
CH3CN
27
28β (88%)
+ 26 (α(1-2)) (18%)
+ 28α
(9%)
OR1
OTBDPS
O
O
O
O
O
TBAF/AcOH
OR2
O
OR3
O
R2O
BzO
R3O
R3O
OBz
OAc
THF, DMF
AcO
AcO
AcNH
OR4
AcNH
O
O
OBn
O
82%
OR3 OR2
AcO
OBz
OBz
OR2
29
(40%)
32 R1 = H, R2 = Bz, R3 = Ac, R4 = Bn
OH
NaOMe / MeOH
97%
OH
O
+ 30
(α(1-4)) (12%)
O
33 R1, R2, R3 = H, R4 = Bn
OH
OH
OH
HO
HO
HO
H2 / Pd (C), 3 atm
92%
+ 31
(40% from 28β)
AcNH
NaBH4
1
2
3
4
O
6
R , R , R , R = H
O
93%
OH
OH
OH
34
Scheme 3. Synthesis of b-D-Galp-(1?2)-b-D-Galf-(1?4)-D-GlcNAc (6) and alditol 34.
confirmed the H-10/H-20 cis disposition, meaning a
for the anomeric center.
The moderate selectivity obtained toward the b linkage in the
latter glycosylation suggests that other factors are involved, and
this could be related to the reciprocal donor acceptor selectivity
(RDAS) concept introduced by Fraser-Reid.45 In this sense, an
a
-configuration
signals at 96.3 (C-1 GlcNAc), 99.5 (C-100, Galp unit), and 106.2 ppm,
the latter indicating a b-Galf linkage. The C-20, C-40, and C-30 reso-
nances of the internal Galf unit appeared at 86.7, 80.4, and
76.9 ppm, the first downfield shifted by glycosylation. On the other
hand, the broad singlet assigned to H-10 (d = 5.56 ppm) together
with the doublet (d = 4.21 ppm) with a coupling constant <1.5 Hz
due to the H-20 resonance, confirmed the b stereochemistry for
the new linkage. A second fraction from the column afforded the
unusual
a-glycosylation was reported with imidate 24, and other
2-O-acyl galactopyranose donors.46
Reduction of lactone 25 with diisoamylborane afforded the
a
-(1?4) product 30 in 27% yield. In this case, the 13C NMR spec-
furanose 27 in 86% yield as a
by the integration of b anomer H-1 (d = 5.61 ppm) and
a
/b mixture in 1:4 ratio as indicated
anomer H-
trum showed three anomeric signals at 95.9 (C-1, GlcNAc), 99.8
(C-100, Galp), and 102.7 ppm, the latter is shifted upfield compared
to the analogous signal in the b(1?4) product 29 (106.2 ppm), sug-
a
3 (d = 5.69 ppm). Reaction of 27 with acetonitrile and DBU gave a
mixture of imidates. After column chromatography, the less polar
b-imidate 28b was obtained in 88% yield. The 1H NMR spectrum
showed a downfield broad singlet (d 6.66 ppm) for H-1 whereas
H-2 resonated at 4.23 ppm as a singlet indicating a b stereochem-
istry for the imidate. On the other hand, the anomeric zone in the
13C NMR showed two signals, 101.1 ppm for C-10 and 104.3 ppm
gesting the
a
stereochemistry for the new Galf linkage. The 1H
NMR spectrum showed a doublet (d = 5.42 ppm) with a coupling
constant of 4.8 Hz assigned to H-10, and correlated with the signal
at 102 ppm in the HSQC experiment, confirming the
istry. No diastereomeric preference was obtained by employing the
low polar and non-participating solvent CH2Cl2 as the and b gly-
a stereochem-
a
for C-1, in agreement with the b stereochemistry. Also, the
date 28 was recovered (9%), as indicated by the H-1 signal
(d = 6.58 ppm; J = 4.7 Hz) in the 1H NMR spectrum.
a
-imi-
cosylation products 30 and 29 were obtained in 1:1 ratio and a to-
tal yield of 53%. Glycosidic substitution at O-2 of the Galf donor
plays a major influence in the stereochemistry of the glycosylation
reaction. In fact, Galp replacement by a Galf unit in O-2 of the
galactofuranosyl donor, under the same reaction conditions, affor-
ded the b product 19 as major component in the synthesis of trisac-
charide 5.
To increase the diastereoselectivity to obtain the desired b
linked product 29, other solvents were assayed for the glycosyla-
tion reaction. When participating ethyl ether was used, the total
glycosylation yield diminished to 35% and the diastereoselectivity
a
The next step was the construction of the Galf-(1?4)GlcNAc
glycosidic linkage considering that furanosyl donor 28b, with a b-
Galp substituent in O-2, lacks anchimeric assistance. On the other
hand, in the synthesis of trisaccharide 5, we used donor 17b with a
O-2 Galf substituent. In that case, CH2Cl2 was the best solvent in
terms of diastereoselectivity and obtained yields for the b linkage.
Thus, we performed our first glycosylation attempt of acceptor 18
with donor 28b in CH2Cl2 as solvent. The best results were obtained
employing the same conditions as described before for glycosyla-
tion with 17b, the galactofuranosyl equivalent of 28b. Column
chromatography purification gave the desired b(1?4) product 29
in 26% yield. The 13C NMR spectrum of 29 showed three anomeric
slightly favored the undesired
a product 30, giving rise to a b/a ra-
tio of 1:1.3 after purification. Similar diastereoselectivity was ob-
served for the analogous glycosylation in the synthesis of
trisaccharide 5.

390
V. M. Mendoza et al. / Carbohydrate Research 345 (2010) 385–396
At this point, we decided to evaluate the glycosidic reaction in
effected by exposure to UV light or by spraying with 10% (v/v)
H2SO4 in EtOH and charring. Column chromatography was per-
formed on Silica Gel 60 (230–400 mesh). NMR spectra were re-
corded at 500 MHz (1H) and 125.8 MHz (13C). Chemical shifts are
given relative to the signal of internal acetone standard at 2.22
and 30.89 ppm for 1H NMR and 13C NMR spectra, respectively,
when recorded in D2O. Assignments were supported by 2D 1H-
COSY and HSQC experiments. High resolution mass spectra (HRMS)
were recorded on an Agilent LCTOF with Windows XP based OS
and APCI/ESI ionization high resolution mass spectrometer ana-
lyzer or in a BRUKER micrOTOF-Q II spectrometer. Melting points
were determined with a Fisher–Johns apparatus and are uncor-
rected. Optical rotations were measured with a path length of
1 dm at 25 °C.
acetonitrile with low expectations because no diastereoselectivity
preferences were obtained in the synthesis of trisaccharide 5. How-
ever, to our surprise, the diastereoselectivity favored the desired b
product 29 (3:1 b/a ratio) with a total glycosidation yield of 52%.
This result confirmed that 2-O glycosyl substitution of the Galf do-
nor as well as the solvent play an important influence in the diaste-
reoselectivity of the reaction. Glycosyl substitution would
influence the conformation of the furanosyl oxocarbenium ion,
considering the flexibility of the furanose ring.
It is interesting to note that all glycosylations required long reac-
tion times, and in the case of acetonitrile as solvent, an imidate rear-
rangement byproduct with similar mobility of imidate 28b was
isolated.41 In fact, an inseparable mixture of 2,3,4,6-tetra-O-acetyl-
b-
-b-
pyranosyl-(1?2)-3,5,6-tri-O-benzoyl-N-trichloroacetyl-
tofuranosylamine (31b and ) was isolated in 40% yield from imidate
28b, and in 1:1 ratio as indicated by 1H NMR analysis. The H-1 of the
D
-galactopyranosyl-(1?2)-3,5,6-tri-O-benzoyl-N-trichloroacetyl
-galactofuranosylamine and 2,3,4,6-tetra-O-acetyl-b- -galacto-
-galac-
D
D
3.2. 2-O-tert-Butyldiphenylsilyl-5,6-O-isopropylidene-D-
galactono-1,4-lactone (12)
a
-
D
a
To a cooled solution (0 °C) of 5,6-O-isopropylidene-D-galactono-
a
and b anomers appeared as a doublet of doublets at 5.95 ppm
1,4-lactone32 (11, 1.2 g, 5.61 mmol) and imidazole (0.65 g,
9.54 mmol, 1.7 equiv) in anhyd DMF (8.5 mL), was added t-butyldi-
phenylsilyl chloride (1.67 g, 1.56 mL, 1.2 equiv) with stirring. After
stirring for 16 h at 5 °C, TLC revealed disappearance of starting
material and the formation of only one product (Rf 0.4, hexane–
EtOAc 2:1), the mixture was diluted with CH2Cl2 (200 mL), washed
with H2O (5 ꢁ 100 mL), dried (Na2SO4), and filtered. The filtrate
was concentrated under vacuum to give a sirup that crystallized
(J = 9.0, 4.7 Hz) and 5.85 ppm (J = 9.0, 2.3 Hz), respectively. The 13C
NMR spectrum showed the diagnostic signals at 87.4 and
81.9 ppm assigned to C-1 of b and
a anomers respectively. COSY
and HSQC experiments allowed complete assignment of the
mixture.
The next step was the removal of the TPBPS group. When 5% HF
(48 wt % in water) in acetonitrile was used, as in deprotection of
19, after 22 h at rt TLC still showed the presence of starting mate-
rial together with more polar compounds. Alternatively, treatment
of 29 with TBAF buffered with AcOH in anhydrous THF–DMF34
afforded crystalline desilylated product 32 in 82% yield. Deacyla-
tion with sodium methoxide in methanol gave the benzyl glycoside
33 in 98%, which crystallized from methanol. Debenzylation of 33
was accomplished by catalytic hydrogenation to give the free tri-
from n-hexane yielding 2.13 g of 12 (83%): mp 60–62 °C, [a]
D
ꢀ11.2 (c 0.7, CHCl3). 1H NMR (CDCl3, 500 MHz): d 7.81–7.41 (m,
10H, arom.), 4.40 (d, 1H, J = 8.2 Hz, H-2), 4.31 (td, 1H, J = 8.1,
3.9 Hz, H-3), 4.25 (td, 1H, J = 6.8, 3.9 Hz, H-5), 4.04 (dd, 1H,
J = 6.8, 8.6 Hz, H-6a), 3.92 (dd, 1H, J = 8.6, 6.9 Hz, H-6b), 3.87 (dd,
1H, J = 8.1, 3.9 Hz, H-4), 1.86 (d, 1H, J = 3.9 Hz, –OH), 1.42, 1.38
(2s, 6H, (CH3)2C), 1.13 (s, 9H, (CH3)3CSi). 13C NMR (CDCl3,
saccharide 6 with 92% yield. The
a/b ratio of the anomeric mixture
125.8 MHz,):
d
171.7 (CO), 136.0–128.0 (C-arom.), 110.2
was established as 3:2 by integration of the
a
and b anomeric H-1
((CH3)2C), 78.0 (C-4), 76.2 (C-2), 75.3 (C-3), 73.9 (C-5), 64.9 (C-6),
26.7 ((CH3)3CSi), 26.1, 25.5 ((CH3)2C), 19.2 ((CH3)3CSi). Anal. Calcd
for C25H32O6Siꢂ1/2H2O: C, 64.49; H, 7.14. Found: C, 64.25; H, 7.12.
that appeared at 5.19 ppm (J = 3.4 Hz) and 4.70 ppm (J = 8.3 Hz),
respectively. Upon reduction of 6 with sodium borohydride, alditol
34 was obtained and the chemical shifts in the 1H NMR spectrum
were in agreement with those reported for the novel alditol re-
leased from the T. cruzi mucins (Tulahen strain).6
3.3. 3,5,6-Tri-O-benzoyl-2-O-tert-butyldiphenylsilyl-D-
galactono-1,4-lactone (13)
In conclusion, two novel trisaccharide structures that contain an
internal Galf, b-
D
-Galf-(1?2)-b-
D
-Galf-(1?4)-
D
-GlcNAc (5) and b-
A suspension of 12 (2.0 g, 4.38 mmol) in a mixture of 2:1 HOAc–
H2O (13 mL) was warmed at 80 °C with stirring. After 30 min, TLC
revealed disappearance of starting material, and the solution was
cooled to rt, concentrated under vacuum and the residue was co-
evaporated with toluene (5 ꢁ 20 mL) to give crude 2-O-t-butyldi-
D-Galp-(1?2)-b-
D
-Galf-(1?4)- -GlcNAc (6), were synthesized
D
and their alditols confirmed the structure of the natural oligosac-
charides released from mucins of T. cruzi (Tulahuen strain). Inter-
mediates 21 and 32 would allow the construction of the
hexasaccharides 7 and 8 and the pentasaccharide 9. The glycosyl
aldonolactone approach was envisioned for the synthesis, a conve-
phenylsilyl-D-galactono-1,4-lactone as an colorless sirup (1.91 g,
4.24 mmol). The crude was dissolved in dry pyridine (8 mL), cooled
at 0 °C, and benzoyl chloride (1.92 mL, 16.5 mmol, 3.6 equiv) was
slowly added with stirring. After stirring 30 min at 0 °C and 1 h at
rt, the mixture was poured into ice-water (200 g). After 20 min,
the mixture was extracted with CH2Cl2 (200 mL) and the organic
phase was washed with H2O (100 mL), HCl 10% (80 mL), H2O
(100 mL), NaHCO3 10% (80 mL), H2O (2 ꢁ 150 mL) until pH 7, dried
(Na2SO4) and filtered. The solution was concentrated and the sirupy
residue was purified by column chromatography (6:1 hexane–
nient D-galactono-1,4-lactone derivative was used for the introduc-
tion of the internal galactofuranose. The trisaccharides were
assembled from the non-reducing to the reducing end, and the
trichloroacetimidate method was employed for glycosylation
reactions. The nature of the solvent strongly determined the ste-
reochemical course of glycosylation reactions when the Galf donor
lacks anchimeric assistance by substitution either by 2-O-Galp or
2-O-Galf. Dichloromethane favored the b stereochemistry employ-
ing the Galf-(1?2)-Galf donor, whereas acetonitrile favored the
b linkage when Galp-(1?2)-Galf was the donor.
EtOAc) to yield 13 (2.17 g, 68%). Rf 0.47 (3:1 hexane–EtOAc), [a]
D
ꢀ18.2 (c 1, CHCl3). 1H NMR (CDCl3, 500 MHz): d 7.95–7.05 (m,
25H, arom.), 5.95 (td, 1H, J = 6.2, 2.5 Hz, H-5), 5.61 (t, 1H,
J = 5.7 Hz, H-3), 4.77 (d, 1H, J = 6.0 Hz, H-2), 4.67 (m, 2H, H-6a, H-
6b), 4.64 (dd, 1H, J = 5.5, 2.5 Hz, H-4), 0.95 (s, 9H, (CH3)3CSi); 13C
NMR (CDCl3, 125.8 MHz): d 171.4 (C-1), 165.9, 165.3, 165.2 (PhCO),
136.1–127.7 (C-arom.), 78.7 (C-4), 76.7 (C-3), 73.3 (C-2), 70.1 (C-5),
62.4 (C-6), 26.5 ((CH3)3CSi), 19.1 (CH3)3CSi). Anal. Calcd for
C43H40O9Si: C, 70.86; H, 5.53. Found: C, 70.94; H, 5.39.
3. Experimental
3.1. General methods
Thin layer chromatography (TLC) was performed on 0.2 mm
Silica Gel 60 F254 aluminum-supported plates. Detection was

V. M. Mendoza et al. / Carbohydrate Research 345 (2010) 385–396
391
3.4. 3,5,6-Tri-O-benzoyl-
D
-galactono-1,4-lactone (10)
for the b anomer, only the diagnostic signals are listed for the
anomer: 8.11–7.23 (m, 35H), 6.06 (ddd, 0.3H, J = 6.6, 4.9, 3.7 Hz,
H-5 or H-50 anomer), 6.02 (ddd, 0.7H, J = 6.4, 4.8, 3.2 Hz, H-50),
5.99 (ddd, 0.3H, J = 6.7, 6.2, 3.4 Hz, H-5 or H-50a anomer), 5.93
(ddd, 0.7H, J = 6.6, 5.4, 4.1 Hz, H-5), 5.74 (br s, 0.3H, H-10
ano-
mer), 5.70 (br s, 0.7H, H-10), 5.62 (m, 1.4H, H-1 and H-3), 5.56
a
To a stirred cold solution (0 °C) of 13 (1.9 g, 2.61 mmol) in an-
hyd DMF (19 mL), was added glacial HOAc (0.31 mL, 2.11 equiv)
and a solution of 1 M TBAF in anhyd THF (5.2 mL, 2 equiv), and
the stirring continued at rt. After 2 h, TLC examination showed dis-
appearance of the starting material and the mixture was diluted
with CH2Cl2 (200 mL), washed with H2O (5 ꢁ 100 mL), dried
(Na2SO4), filtered, and concentrated under reduced pressure. The
resulting sirup was purified by silica gel column chromatography
(4:1 hexane–EtOAc) to afford 10 (1.04 g, 81%), which crystallized
a
a
(dd, 0.3H, J = 10.0, 4.0 Hz, H-1
a anomer), 5.54 (dd, 0.7H, J = 4.1,
1.3 Hz, H-3), 5.37 (d, 0.7H, J = 1.5 Hz, H-20), 4.86 (dd, 0.7H, J = 5.4,
4.1 Hz, H-4), 4.70 (dd, 0.7H, J = 5.2, 3.2 Hz, H-40), 4.47 (d, 0.7H,
J = 1.3 Hz, H-2), 4.10 (d, 0.3H, J = 10.0 Hz, –OH
a anomer), 3.36 (d,
0.7H, J = 4.0 Hz, –OH); 13C NMR (CDCl3, 125.8 MHz): d for b anomer
166.4–165.2 (COPh), 133.6–128.2 (arom.), 105.1 (C-10), 104.7 (C-10
from toluene: Rf 0.35 (3:2 hexane–EtOAc), mp 100–102 °C, [a]
D
ꢀ54.8 (c 1, CHCl3); 1H NMR (CDCl3, 500 MHz): d 8.09 – 7.96 (m,
6H, arom.), 7.63–7.37 (m, 9H, arom.), 5.90 (ddd, 1H, J = 6.5, 5.2,
3.0 Hz, H-5), 5.54 (t, 1H, J = 6.6 Hz, H-3), 4.94 (dd, 1H, J = 6.6,
3.0 Hz, H-4), 4.81 (d, 1H, J = 6.5 Hz, H-2), 4.77 (dd, 1H, J = 11.8,
5.2 Hz, H-6a), 4.67 (dd, 1H, J = 11.8, 6.5 Hz, H-6b), 3.76 (br s, 1H,
–OH); 13C NMR (CDCl3, 125.8 MHz): d 171.9 (C-1), 166.4, 165.9,
165.4 (PhCO), 134.1–128.0 (C-arom.), 77.8 (C-4), 76.9 (C-3), 72.9
(C-2), 69.4 (C-5), 62.4 (C-6). Anal. Calcd for C27H22O9 : C, 66.12;
H, 4.52. Found: C, 66.18; H, 4.63.
a anomer), 101.9 (C-1), 97.7 (C-1 a anomer), 84.8 (C-2), 82.3, 82.2
(C-20, C-40), 81.8 (C-4), 78.0 (C-3), 77.4 (C-30), 71.0 (C-5), 70.2 (C-50),
63.4, 63.2 (C-6, C-60). Anal. Calcd for C61H50O18: C, 68.40; H, 4.71.
Found: C, 68.33; H, 4.62.
3.7. Preparation of O-(2,3,5,6-tetra-O-benzoyl-b-
D
-galactofur-
anosyl-(1?2)-3,5,6-tri-O-benzoyl-b-D-galactofuranosyl) trich-
loroacetimidate (17b)
To a stirred solution of 16 (0.87 g, 0.81 mmol) and trichloroaceto-
nitrile (0.4 mL, 4.03 mmol) in CH2Cl2 (8 mL), cooled to 0 °C, DBU
3.5. 2,3,5,6-Tetra-O-benzoyl-b-
D-galactofuranosyl-(1?2)-3,5,6-
tri-O-benzoyl- -galactono-1,4-lactone (15)
D
(48 lL, 0.32 mmol, 0.4 equiv) was slowly added. After 1 h at rt the
solution was concentrated at rt under reduced pressure, and the res-
idue was purified by column chromatography (95:5:0.3 toluene–
EtOAc–TEA). The first fraction from the column (Rf 0.6, toluene–
EtOAc 9:1) gave a sirupy 17b (0.72 g, 73%) and was stable for one
To an externally cooled (0 °C) solution of 1,2,3,5,6-penta-O-ben-
zoyl- ,b-
-galactofuranose36 (14, 1.55 g, 2.21 mmol) in dry CH2Cl2
(45 mL), tin(IV) chloride (0.26 mL, 2.23 mmol) was slowly added
with stirring. After 15 min at 0 °C, 3,5,6-tri-O-benzoyl- -galacton-
a
D
D
day at –20 °C. [
a
]
D ꢀ22.6 (c 1, CHCl3); 1H NMR (CDCl3, 500 MHz) d
o-1,4-lactone (10, 0.98 g, 1.99 mmol) was added and the stirring
continued for 2 h until consumption of 14. The mixture was diluted
with CH2Cl2 (350 mL), washed with 10% NaHCO3 (2 ꢁ 100 mL) and
H2O (3 ꢁ 200 mL) until pH 6, and was dried (Na2SO4), filtered, and
concentrated. The resulting sirup was washed with small amounts
of cold ethanol and then CH3OH to afford a solid. Recrystallization
from isopropanol gave 1.66 g of 15 (78%). The combined mother li-
quors and the alcoholic washes were concentrated. Purification by
column chromatography of the residue (95:5 toluene–EtOAc) gave
additional 0.29 g of 15 (total yield 92%). Mp 82–85 °C (isopropanol),
8.57 (s, 1H, NH), 8.15–7.84 (m, 14H, arom.), 7.59–7.15 (m, 21H,
arom.), 6.55 (br s, 1H, H-1), 6.06 (m, 1H, H-5 or H-50), 6.03 (ddd,
1H, J = 7.0, 4.0, 5.0 Hz, H-5 or H-50), 5.79 (br s, 1H, H-10), 5.67 (dd,
1H, J = 5.1, 1.3 Hz, H-30), 5.63 (d, 1H, J = 3.5 Hz, H-3), 5.29 (d, 1H,
J = 1.3 Hz, H-20), 4.98 (dd, 1H, J = 5.0, 3.5 Hz, H-4 or H-40), 4.84 (dd,
1H, J = 12.0, 4.0 Hz), 4.82 (dd, 1H, J = 5.1, 3.5 Hz, H-4 or H-40), 4.81–
4.76 (m, 2H), 4.73 (br s, 1H, H-2), 4.68 (dd, 1H, J = 12.0, 7.0 Hz); 13C
NMR (CDCl3, 125.8 MHz) d 166.1, 166.0, 165.7, 165.6, 165.2 (PhCO),
160.3 (CNH), 133.6–128.2 (C-arom.), 105.0, 104.8 (C-1, C-10), 90.8
(CCl3), 84.6, 82.5, 82.3, 82.2 (C-2, C-20, C-4, C-40), 77.1, 76.9 (C-3, C-
30), 70.5, 70.2 (C-5, C-50), 63.7, 63.4 (C-6, C-60).
[
a]
D ꢀ11.9 (c 1, CHCl3), Rf 0.6 (9:1 toluene–EtOAc). 1H NMR (CDCl3,
500 MHz): d 8.07–7.15 (m, 35H, arom.), 6.01–5.98 (m, 2H, H-5, H-
50), 5.71 (t, 1H, J = 4.2 Hz, H-3), 5.60 (dd, 1H, J = 5.3, 1.4 Hz, H-30),
5.57 (br s, 1H, H-10), 5.04 (d, 1H, J = 1.4 Hz, H-20), 5.01 (dd, 1H,
J = 5.3, 3.6 Hz, H-40), 4.98 (dd, 1H, J = 4.1, 2.8 Hz, H-4), 4.93 (d, 1H,
J = 4.3 Hz, H-2), 4.79–4.68 (m, 4H, H-6a, H-6b, H-6a0, H-6b0), 13C
NMR (CDCl3, 125.8 MHz): d 170.6 (C-1), 166.0, 165.9, 165.6, 165.5,
165.4, 165.3, 164.9 (PhCO), 133.9–128.2 (C-arom.), 104.9 (C-10),
82.2 (C-40), 81.6 (C-20), 80.1 (C-4), 76.9 (C-30), 75.2 (C-3), 74.1 (C-
2), 71.1, 70.0 (C-5, C-50), 63.3, 62.5 (C-6, C-60). Anal. Calcd
C61H48O18 : C, 68.53; H, 4.53. Found: C, 68.52; H, 4.38.
A second fraction from the column (Rf 0.5, 9:1 toluene–EtOAc),
gave
(1?2)-3,5,6-tri-O-benzoyl-
date (17 , 100 mg, 10%): [a]
sirupy
O-(2,3,5,6-tetra-O-benzoyl-b-
-galactofuranosyl) trichloroacetimi-
D +11.7 (c 0.7, CHCl3), 1H NMR (CDCl3,
D-galactofuranosyl-
a-D
a
500 MHz): d 8.55 (s, 1H, NH), 8.13–7.14 (m, arom.), 6.73 (d, 1H,
J = 4.5 Hz H-1), 6.16 (t, 1H, J = 7.1 Hz, H-3), 6.02 (m, 1H, H-50),
5.78 (m, 1H, H-5), 5.59 (dd, 1H, J = 5.8, 1.4 Hz, H-30), 5.50 (s, 1H,
H-10), 5.47 (d, 1H, J = 1.4 Hz, H-20), 4.87 (dd, 1H, J = 12.3, 3.6 Hz,
H-6a or H-6a0), 4.82 (dd, 1H, J = 7.5, 4.5 Hz, H-2), 4.80–4.65 (m,
5H). 13C NMR (CDCl3, 125.8 MHz): d 166.1, 165.7, 165.6, 165.5,
165.3 ꢁ 2 (PhCO), 161.2 (CNH), 133.6–128.2 (C-arom.), 106.8 (C-
10), 98.0 (C-1), 82.6, 81.9, 80.5, 79.3 (C-2, C-20, C-4, C-40), 77.5,
74.2, 71.6, 70.1, 63.5, 62.8 (C-6, C-60).
3.6. 2,3,5,6-Tetra-O-benzoyl-b-
D-galactofuranosyl-(1?2)-3,5,6-
tri-O-benzoyl- ,b- -galactofuranose (16)
a
D
Unreacted 16 was recovered from the last fraction (118 mg,
13%).
A solution of bis(2-butyl-3-methyl)borane (6.5 mmol, 6 equiv)
in anhyd THF (2.2 mL) cooled to 0 °C under an argon atmosphere
was added to a flask containing dried 15 (1.16 g, 1.09 mmol). The
resulting solution was stirred for 18 h at 5 °C and 3 h at rt and then
processed as previously described.47 The organic layer was washed
with H2O, dried (Na2SO4), and concentrated. Boric acid was elimi-
nated by coevaporation with CH3OH (6 ꢁ 10 mL) at rt. The residue
was purified by column chromatography (95:5 toluene–EtOAc).
Unreacted 15 (0.10 g, 8.8%) was recovered from the first fraction.
The second fraction from the column gave 0.957 g (82%) of an
3.8. Benzyl 2,3,5,6-tetra-O-benzoyl-b-
-3,5,6-tri-O-benzoyl-b- -galactofuranosyl-(1?4)-2-acetamido-
3-O-benzoyl-6-O-tert-butyldiphenylsilyl-2-deoxy- -glucopy
ranoside (19) and benzyl 2,3,5,6-tetra-O-benzoyl-b- -galacto
furanosyl-(1?2)-3,5,6-tri-O-benzoyl- -galactofuranosyl-
(1?4)-2-acetamido-3-O-benzoyl-6-O-tert-butyldiphenylsilyl-2-
D-galactofuranosyl-(1?2)
D
a
-D
D
a
-D
deoxy-
a
-D
-glucopyranoside (20)
Benzyl
2-deoxy-a-D
2-acetamido-3-O-benzoyl-6-O-t-butyldiphenylsilyl-
amorphous solid 16 as a 3:7 a/b anomeric mixture: Rf 0.35 (9:1 tol-
-glucopyranoside 1815 (252 mg, 0.385 mmol) and
uene–EtOAc), [a]
D ꢀ11.1 (c 1, CHCl3); 1H NMR (CDCl3, 500 MHz): d

392
V. M. Mendoza et al. / Carbohydrate Research 345 (2010) 385–396
activated 4 Å powdered molecular sieves (300 mg) were dried in
vacuo and solution of trichloroacetimidate 17b (560 mg,
0.46 mmol, 1.2 equiv) in anhyd CH2Cl2 (20 mL) was added under
J = 12.3, 2.9 Hz, H-6a0), 4.42 (ddd, 1H, J = 10.9, 9.7, 3.7 Hz, H-2),
4.38 (dd, 1H, J = 11.9, 4.0 Hz, H-6b00), 4.36 (dd, 1H, J = 12.3, 8.0 Hz,
H-6b0), 4.23 (dd, 1H, J = 5.7, 3.4 Hz, H-40), 4.21 (br s, 1H, H-20),
4.17 (t, 1H, J = 9.7 Hz, H-4), 3.93 (dd, 1H, J = 11.7, 4.1 Hz, H-6a),
3.86 (dd, 1H, J = 11.7, 1.3 Hz, H-6b), 3.84 (ddd, 1H, J = 9.8, 4.1,
1.3 Hz, H-5), 1.78 (s, 3H, CH3CO), 0.99 (s, 9H, (CH3)3CSi); 13C NMR
(CDCl3, 125.8 MHz): d 166.9 (CH3CO), 165.9, 165.7, 165.6, 165.5,
165.3 ꢁ 2 (PhCO), 136.8–127.5 (C-arom.), 106.1 (C-10), 105.4 (C-
100), 96.2 (C-1), 85.0 (C-20), 82.6 (C-200), 82.3 (C-40), 81.9 (C-400),
77.7 (C-30), 77.3 (C-300), 72.3 (C-3, C-4), 72.1 (C-5), 70.6 (C-50),
70.2 (C-500), 69.5 (CH2Ph), 63.9 (C-60), 63.5 (C-600), 62.6 (C-6), 52.5
(C-2), 26.8 ((CH3)3CSi), 23.2 (CH3CO), 19.4 ((CH3)3CSi). HRMS
(ESI/APCI) m/z calcd for C99H91NNaO24Si (M+Na+) 1728.5598,
found 1729.5585.
a
argon and the mixture was cooled to ꢀ10 °C with vigorous stirring.
After 15 min, TMSOTf (0.116 mmol, 30 lL, 0.43 equiv) was added
and the stirring continued for 1 h at ꢀ10 °C and 48 h at 5 °C. TLC
monitoring showed the presence of 16 due to remaining trichloro-
acetimidate 17b. The reaction was stirred for additional 24 h, then
neutralized with TEA (23 lL, 0.43 equiv), filtered and concentrated
under reduced pressure. The sirupy residue was purified by col-
umn chromatography (2:1 hexane–EtOAc).
The first fraction of the column (Rf 0.64 2:1 hexane–EtOAc,
twice developed) was concentrated and after additional purifica-
tion by column chromatography (4:1 hexane–EtOAc) gave an
amorphous solid identified as 2,3,5,6-tetra-O-benzoyl-b-D-galacto-
furanosyl-(1?2)-3,5,6-tri-O-benzoyl-N-trichloroacetyl-b-
D
-galac-
3.9. Benzyl 2,3,5,6-tetra-O-benzoyl-b-
(1?2)-3,5,6-tri-O-benzoyl-b- -galactofuranosyl-(1?4)-2-
acetamido-3-O-benzoyl-2-deoxy- -glucopyranoside (21)
D-galactofuranosyl-
tofuranosylamine (75 mg, 13%): [
a
]
D
ꢀ3.4 (c 0.5, CHCl3); 1H NMR
D
(CDCl3, 500 MHz): d 8.16–7.78 (m, 14H, arom.), 7.75 (d, 1H,
J = 9.3 Hz, NH), 7.63–7.23 (m, 21H, arom.), 6.18 (dd, 1H, J = 8.7,
5.9, 3.0 Hz, H-5), 6.13 (m, 1H, H-50), 6.07 (dd, 1H, J = 9.3, 4.1 Hz,
H-1), 5.86 (br s, 1H, H-10), 5.72 (m, 2H, H-3, H-30), 5.53 (d, 1H,
J = 1.0 Hz, H-20), 4.96 (dd, 1H, J = 12.5, 3.0 Hz, H-6a), 4.83 (dd, 1H,
J = 12.5, 5.9 Hz, H-6b), 4.78–4.74 (m, 2H, H-40, H-6a0), 4.69 (dd,
1H, J = 11.9, 4.1 Hz, H-6b0), 4.58 (dd, 1H, J = 8.7, 1.0 Hz, H-4), 4.56
(d, 1H, J = 4.1 Hz, H-2); 13C NMR (CDCl3, 125.8 MHz): d 166.2,
166.1, 165.9, 165.8, 165.6, 165.5 (PhCO), 161.5 (CCl3CONH),
133.9–128.3 (C-arom), 104.9 (C-10), 92.0 (CCl3), 83.2 (C-20), 83.0
(C-40), 82.6 (C-1), 80.9 (C-4), 78.0 (C-2), 77.5, 76.8 (C-3, C-30),
70.8 (C-5), 70.2 (C-50), 63.6 (C-6), 63.5 (C-60). HRMS (ESI/APCI) m/
z calcd for C63H50Cl3NNaO18 (M+Na) 1236.1991, found 1236.1988.
The second fraction from the column (Rf 0.24 2:1 hexane–
EtOAc, twice developed) afforded 118 mg of trisaccharide 20
a-D
A solution of 5% HF (48 wt % in H2O) in acetonitrile (2.7 mL) was
added to 19 (88 mg, 0.052 mmol), and the resulting solution was
stirred at 28 °C for 16 h. The reaction mixture was diluted with
CH2Cl2 (30 mL), washed with H2O (5 ꢁ 8 mL) until pH 7, dried
(Na2SO4), filtered, and concentrated under reduced pressure. Col-
umn chromatography of the residue (3:2 hexane–EtOAc) gave sir-
upy 21 (61 mg, 80%): Rf 0.5 (5:7 hexane–EtOAc), [
a]
ꢀ1.7 (c 1,
D
CHCl3); 1H NMR (CDCl3, 500 MHz): d 8.04–7.21 (m, 45H, arom.),
6.05 (m, 1H, H-500), 5.78 (d, 1H, J = 9.6 Hz, NH), 5.68 (d, 1H,
J = 4.8 Hz, H-300), 5.62 (br s, 1H, H-100), 5.56 (dd, 1H, J = 10.8,
9.5 Hz, H-3), 5.54 (br s, 1H, H-10), 5.53 (m, 1H, H-50), 5.40 (d, 1H,
J = 3.6 Hz, H-30), 5.25 (br s, 1H, H-200), 4.96 (d, 1H, J = 3.7 Hz, H-1),
4.79 (dd, 1H, J = 12.0, 3.6 Hz, H-6a00), 4.74 (dd, 1H, J = 12.0, 7.3 Hz,
H-6b00), 4.72, 4.49 (2d, 2H, J = 11.9 Hz, CH2Ph), 4.71 (t, 1H,
J = 4.4 Hz, H-400), 4.45 (ddd, 1H, J = 10.8, 9.6, 3.7 Hz, H-2), 4.37 (br
s, 1H, H-20), 4.35 (dd, 1H, J = 12.2, 7.5 Hz, H-6a0), 4.29 (dd, 1H,
J = 12.2, 3.1 Hz, H-6b0), 4.22 (dd, 1H, J = 4.8, 3.6 Hz, H-40), 4.15 (t,
1H, J = 9.5 Hz, H-4), 3.90–3.83 (m, 3H, H-5, H-6a, H-6b), 2.37 (br
s, 1H, –OH), 1.77 (s, 3H, CH3CO), 13C NMR (CDCl3, 125.8 MHz): d
169.9 (CH3CONH), 166.9, 166.4, 165.8 ꢁ 2, 165.7, 165.5, 165.4,
165.1 (PhCO), 136.7–128.1 (C-arom.), 107.5 (C-10), 105.8 (C-100),
96.7 (C-1), 84.5 (C-20), 82.2 (C-40, C-400), 81.9 (C-200), 77.9 (C-30),
76.9 (C-300), 73.5 (C-4), 72.0 (C-3), 71.5 (C-5), 70.3 (C-50, C-500),
69.9 (CH2Ph), 63.6 (C-600), 63.4 (C-60), 60.7 (C-6), 52.3 (C-2), 23.0
(CH3CONH). Anal. Calcd for C83H73NO24Siꢂ1/2H2O: C, 67.47; H,
5.05; N, 0.95. Found: C, 67.45; H, 5.40; N, 0.98.
(18%), upon recrystallization from CH3OH gave: 100–104 °C, [a]
D
+42.2 (c 1, CHCl3). 1H NMR (CDCl3, 500 MHz): d 8.10–7.01 (m,
55H, arom.), 6.91 (d, 1H, J = 9.9 Hz, NH), 6.04 (ddd, 1H, J = 9.8,
3.0, 1.7 Hz, H-500), 5.85 (ddd, 1H, J = 7.5, 5.4, 3.0 Hz, H-50), 5.59
(dd, 1H, J = 10.9, 8.5 Hz, H-3), 5.58 (br s, 1H, H-100), 5.56 (d, 1H,
J = 4.5 Hz, H-300), 5.51 (dd, 1H, J = 3.5, 2.3 Hz, H-30), 5.43 (br s, 1H,
H-200), 5.40 (d, 1H, J = 3.2 Hz, H-10), 4.90 (d, 1H, J = 3.5 Hz, H-1),
4.88 (dd, 1H, J = 12.0, 9.8 Hz, H-6a00), 4.82 (dd, 1H, J = 12.0, 3.0 Hz,
H-6b00), 4.70 (dd, 1H, J = 12.4, 3.0 Hz, H-6a0), 4.63 (dd, 1H, J = 12.4,
7.5 Hz, H-6b0), 4.60 (dd, 1H, J = 4.5, 1.7 Hz, H-400), 4.51, 4.25 (2d,
2H, J = 11.3 Hz, CH2Ph), 4.50 (ddd, 1H, J = 10.9, 9.9, 3.5 Hz, H-2),
4.43 (t, 1H, J = 9.0 Hz, H-4), 4.32 (dd, 1H, J = 5.4, 3.5 Hz, H-40),
4.01 (dd, 1H, J = 3.2, 2.3 Hz, H-20), 3.98 (dd, 1H, J = 11.2, 3.5 Hz,
H-6a), 3.66 (ddd, 1H, J = 9.6, 3.5, 2.0 Hz, H-5), 3.56 (dd, 1H,
J = 11.2, 2.0 Hz, H-6b), 1.92 (s, 3H, CH3CO), 1.08 (s, 9H, (CH3)3CSi);
13C NMR (CDCl3, 125.8 MHz): d 170.6 (CH3CO), 166.2, 166.1, 166.0,
165.8, 165.7, 165.5, 165.2, 165.1 (PhCO), 136.2–127.5 (C-arom.),
105.5 (C-100), 102.9 (C-10), 96.2 (C-1), 83.3 (C-400), 81.5 (C-20), 78.8
(C-200), 78.3 (C-40), 78.0 (C-300), 76.1 (C-30), 75.5, 75.4 (C-3, C-4),
71.0 (C-50), 70.8 (C-5), 70.2 (C-500), 69.6 (CH2Ph), 65.6 (C-600), 63.5
(C-60), 61.9 (C-6), 51.5 (C-2), 26.8 ((CH3)3CSi), 22.9 (CH3CO), 19.4
((CH3)3CSi). HRMS (ESI/APCI) m/z calcd for C99H91NNaO24Si
(M+Na) 1728.5598, found 1729.5593.
3.10. Benzyl b-
D
-galactofuranosyl-(1?2)-b-
D-galactofuranosyl-
(1?4)-2-acetamido-2-deoxy-
a
-D
-glucopyranoside (22)
To a flask containing benzyl derivative 21 (39 mg, 0.027 mmol)
was added 1.5 mL of cooled 0.5 M NaOCH3 in CH3OH with stirring
and the resulting solution was stirred at rt. After 3 h, TLC examina-
tion showed only one compound (Rf 0.45, 14:1:1 n-propanol–
EtOH–H2O). The solution was passed through a column containing
Amberlite IR-120 plus resin 200 mesh, H+ form, and washed with
CH3OH. The solvent was evaporated and the methyl benzoate elim-
inated by successive coevaporation with H2O (3 ꢁ 1 mL). Further
purification through a C8 cartridge eluting with H2O followed by
concentration at 35 °C gave 16.3 mg of a glassy compound identi-
The last fraction of the column (Rf 0.17, 2:1 hexane–EtOAc,
twice developed) gave 19 as an amorphous solid (271 mg, 41%),
that crystallized from 4:1 hexane–toluene: mp 87–89 °C, [a]
D
+14 (c 1, CHCl3); 1H NMR (CDCl3, 500 MHz): d 8.05–7.25 (m, 55H,
arom.), 5.91 (ddd, 1H, J = 7.1, 4.0, 3.3 Hz, H-500), 5.79 (d, 1H,
J = 9.7 Hz, NH), 5.64 (ddd, 1H, J = 8.0, 5.7, 2.9 Hz, H-50), 5.58 (br s,
1H, H-10), 5.58 (dd, 1H, J = 5.6, 1.7 Hz, H-300), 5.55 (dd, 1H,
J = 10.9, 9.5 Hz, H-3), 5.44 (br s, 1H, H-100), 5.38 (d, 1H, J = 3.4 Hz,
H-30), 5.32 (d, 1H, J = 1.7 Hz, H-200), 4.92 (d, 1H, J = 3.7 Hz, H-1),
4.68, 4.44 (2d, 2H, J = 11.9 Hz, CH2Ph), 4.54 (dd, 1H, J = 11.9,
7.1 Hz, H-6a00), 4.49 (dd, 1H, J = 5.6, 3.3 Hz, H-400), 4.45 (dd, 1H,
fied as 22 (95%): [
a
]
ꢀ7.5 (c 0.8, H2O); 1H NMR (D2O, 500 MHz):
D
d 7.46–7.38 (m, 5H, arom.), 5.25 (br s, 1H, H-10), 5.19 (br s, 1H,
H-100), 4.94 (d, 1H, J = 3.0 Hz, H-1), 4.75, 4.55 (2d, 2H, J = 11.9 Hz,
CH2Ph), 4.23 (dd, 1H, J = 3.4, 6.6 Hz, H-30), 4.16 (d, 1H, J = 3.4 Hz,
H-20), 4.12–4.07 (m, 3H, H-200, H-300, H-40), 3.99 (dd, 1H, J = 5.9,
4.4 Hz, H-400), 3.88 (dd, 1H, J = 3.0, 10.9 Hz, H-2), 3.86–3.82 (m,
6H), 3.72 (dd, 1H), 3.69–3.60 (m, 4H), 1.96 (s, 3H, CH3CONH); 13C

V. M. Mendoza et al. / Carbohydrate Research 345 (2010) 385–396
393
NMR (D2O, 125.8 MHz): d 174.9 (CH3CONH), 137.6, 129.4, 129.2,
O-(2,3,4,6-tetra-O-acetyl-
a
-
D
-galactopyranoside) trichloroacetimi-
129.0 (C-arom.), 108.0 (C-100), 107.1 (C-10), 96.4 (C-1), 88.0 (C-20),
84.0 (C-400), 83.3, 81.9 (C-40, C-200), 77.5 (C-4), 77.3 (C-300), 76.1 (C-
30), 71.7, 71.5, 71.0, 70.0 (C-5, C-50, C-500, C-3), 70.5 (CH2Ph), 63.5,
63.3 (C-60, C-600), 60.6 (C-6), 54.3 (C-2), 22.4 (CH3CONH). HRMS
(ESI/APCI) m/z calcd. for C27H41NNaO16 (M+Na+) 658.2323, found
658.2308.
date44 (24, 2 g, 4.01 mmol, 1.3 equiv) and activated 4 Å powdered
molecular sieves, and the suspension was vigorously stirred at rt
for 5 min. After cooling to ꢀ12°
C during 15 min, TMSOTf
(220 L, 1.22 mmol, 0.4 equiv) was slowly added under an argon
l
atmosphere, and the stirring continued for 45 min until consump-
tion of imidate 24 showed by TLC. The reaction mixture was rap-
idly filtered, the molecular sieves were washed with CH2Cl2
(2 ꢁ 10 mL), and the filtrate was diluted with CH2Cl2 (250 mL).
The resulting solution was washed with 5% NaHCO3 (90 mL), H2O
(2 ꢁ 150 mL) until pH 7, dried (Na2SO4), filtered, and concentrated
under reduced pressure at rt. Column chromatography of the resi-
due (19:1 toluene–EtOAc, then 1:1) gave a first fraction of 26
(0.44 g, 18%, Rf 0.4, 3:2 hexane–EtOAc, twice developed) that crys-
3.11. b-
D
-Galactofuranosyl-(1?2)-b-
D-galactofuranosyl-(1?4)-
2-acetamido-2-deoxy-
a
,b- -glucopyranose (5)
D
A suspension of 22 (19.0 mg, 0.033 mmol) dissolved in 9:1
CH3OH–H2O (2.5 mL) and 10% Pd/C (7 mg), was hydrogenated for
20 h at 45 psi (3 atm) at rt. The catalyst was filtered and the filtrate
was concentrated. Evaporation of the solvent at rt afforded 5
tallized from 1:2 Et2O–hexane: mp 88–92 °C, [a]D +67 (c 1, CHCl3);
1H NMR (CDCl3, 500 MHz): d 8.07–7.39 (m, 15H, arom.), 5.92 (ddd,
1H, J = 3.4, 5.2, 6.3 Hz, H-5), 5.79 (t, 1H, J = 6.1 Hz, H-3), 5.58 (d, 1H,
J = 3.1 Hz, H-10), 5.30 (d, 1H, J = 2.5, 1.4 Hz, H-40), 5.18–5.13 (m, 2H,
H-20, H-30), 4.95 (dd, 1H, J = 5.9, 3.4 Hz, H-4), 4.77 (d, 1H, J = 6.2 Hz,
H-2), 4.74 (dd, 1H, J = 11.8, 5.2 Hz, H-6a), 4.69 (dd, 1H, J = 11.8,
6.3 Hz, H-6b), 4.09 (td, 1H, J = 6.7, 1.4 Hz, H-50), 3.87 (dd, 1H,
J = 11.2, 6.7 Hz, H-6a0), 3.79 (dd, 1H, J = 11.2, 6.7 Hz, H-6b0), 2.12,
2.09, 1.98, 1.77 (4s, 12H, CH3CO); 13C NMR (CDCl3, 125.8 MHz): d
170.7, 170.2, 170.0, 169.8, 169.6 (C-1, CH3CO), 165.8, 165.2, 165.1
(PhCO), 134.1–128.0 (C-arom.), 97.1 (C-10), 78.5 (C-4), 76.8 (C-2),
74.0 (C-3), 69.9 (C-5), 67.5 (C-40), 67.2, 67.1, 66.9 (C-20, C-30, C-
50), 62.2 (C-6), 61.4 (C-60), 20.7, 20.6, 20.5, 20.3 (CH3CO). Anal. Calcd
for C41H40O18: C, 60.00; H, 4.91. Found: C, 60.17; H, 4.94.
(14.7 mg, 90%) as a glassy hygroscopic solid: [
a
]
ꢀ77.5 (c 1,
D
H2O), Rf 0.35 and 0.30 (7:1:1 n-propanol–EtOH–H2O), 1H NMR
(D2O, 500 MHz): d anomeric zone and diagnostic signals: 5.24,
5.25 (2 br s, 1H,
1.6H, anomer
0.4H, J = 8.2 Hz, b anomer H-1), 4.17 (dd, 0.6H, J = 1.4, 3.4 Hz,
anomer H-20), 4.15 (dd, 0.4H, J = 1.3, 3.2 Hz, b anomer H-20), 2.04
(2s, 3H,
and b anomer CH3CONH); 13C NMR (D2O, 125.8 MHz):
d 175.4, 175.1 ( and b anomers CH3CONH), 108.0 ( and b ano-
mers C-100), 107.2 ( and b anomers C-10), 95.6 (b anomer C-1),
91.3 (
anomer C-1), 88.0, 84.0, 83.4, 83.3, 81.9 ꢁ 2, 77.6, 77.3,
77.2, 76.1, 75.7, 73.1, 71.5, 71.2, 71.0 ꢁ 2, 69.9, 63.6, 63.5, 63.3,
60.9, 60.8, 57.4 (b anomer C-2), 54.7 ( anomer C-2), 22.8, 22.5
and anomers CH3CONH). HRMS (ESI) m/z calcd for
C20H35NNaO16 (M+Na+) 568.1854, found 568.1851.
a
anomer H-10, b anomer H-10), 5.20–5.18 (m,
a
H-100, b anomer H-100,
a anomer H-1), 4.70 (d,
a
a
a
a
a
a
a
The next fraction from the column (Rf 0.3, 3:2 hexane–EtOAc,
twice developed) afforded 25 (1.33 g, 54%) and after crystalliza-
tion from isopropanol gave: mp 85–88 °C, [
(a
b
a
]
D
ꢀ29.4 (c 1, CHCl3);
1H NMR (CDCl3, 500 MHz): d 8.07–7.38 (m, 15H, arom.), 5.94
(ddd, 1H, J = 6.4, 5.5, 2.8 Hz, H-5), 5.82 (dd, 1H, J = 6.4, 5.5 Hz,
H-3), 5.19 (dd, 1H, J = 10.4, 7.9 Hz, H-20), 5.17 (dd, 1H, J = 3.3,
1.1 Hz, H-40), 5.02 (d, 1H, J = 6.4 Hz, H-2), 4.86 (dd, 1H, J = 5.5,
2.8 Hz, H-4), 4.82 (dd, 1H, J = 10.4, 3.3 Hz, H-30), 4.73 (dd, 1H,
J = 11.8, 5.5 Hz, H-6a), 4.67 (dd, 1H, J = 11.8, 6.4 Hz, H-6b), 4.63
(d, 1H, J = 7.9 Hz, H-10), 3.92 (dd, 1H, J = 11.4, 6.5 Hz, H-60a),
3.89 (dd, 1H, J = 11.4, 6.7 Hz, H-6b0), 3.43 (td, 1H, J = 6.6, 1.1 Hz,
H-50), 2.06, 2.01, 1.97, 1.94 (4s, 12H, CH3CO); 13C NMR (CDCl3,
125.8 MHz): d 170.4, 170.1, 169.9, 169.6, 168.7 (C-1, CH3CO),
165.8, 165.1, 164.9 (PhCO), 134.1–128.4 (C-arom.), 99.8 (C-10),
78.9 (C-4), 76.5 (C-2), 73.8 (C-3), 71.1 (C-50), 70.6 (C-30), 70.0
(C-5), 67.5 (C-20), 66.7 (C-40), 62.3 (C-6), 61.1 (C-60), 20.5 (ꢁ4)
(CH3CO). Anal. Calcd for C41H40O18: C, 60.00; H, 4.91. Found: C,
59.73; H, 5.15.
3.12. Preparation of b-
galactofuranosyl-(1?4)-2-acetamido-2-deoxy-
D
-Galactofuranosyl-(1?2)-b-
D-
D
-glucitol6 (23)
To a cooled solution (0 °C) of 5 (10 mg, 0.018 mmol) in 9:1
CH3OH–H2O (2 mL), sodium borohydride (21 mg, 0.53 mmol)
was added, and the mixture was stirred at rt for 16 h. The solution
was passed through a column of Amberlite IR-120 plus, and after
washing the resin with 4:1 CH3OH–H2O, the combined solutions
were concentrated to dryness. The residue was co-evaporated
with CH3OH (4 ꢁ 1 mL), dissolved in de-ionized H2O and the solu-
tion filtrated through C8 cartridge. Evaporation of the filtrate un-
der reduced pressure gave 10 mg of 23 (99%) as a glassy solid: Rf
0.14 (7:1:1 n-propanol–EtOH–H2O), [
a]
ꢀ9.2 (c 0.6, H2O); 1H
D
NMR (D2O, 500 MHz): d 5.29 (br s, 1H, H-10), 5.20 (d, 1H,
J = 1.5 Hz, H-100), 4.24–4.22 (m, 1H, H-30), 4.23 (br s, 1H, H-20),
4.16–4.11 (m, 2H, H-2, H-40), 4.10 (dd, 1H, J = 3.5, 1.5 Hz, H-200),
4.07 (dd, 1H, J = 6.1, 3.5 Hz, H-300), 4.00 (dd, 1H, J = 6.1, 4.3 Hz,
H-400), 3.93–3.90 (m, 2H), 3.86–3.80 (m, 3H), 3.77–3.61 (m, 8H),
2.05 (s, 3H, CH3CONH), d values matched lit.6; 13C NMR (D2O,
125.8 MHz): d 175.1 (CH3CONH), 107.6 (C-10, C-100), 87.7 (C-20),
84.0 (C-400), 83.9 (C-40), 81.9 (C-200), 78.3 (C-4), 77.3 (C-300), 76.2
(C-30), 71.7, 71.5, 71.2, 69.0 (C-3, C-5, C-50, C-500), 63.5, 63.3,
62.8, 61.4 (C-1, C-6, C-60, C-600), 53.4 (C-2), 22.8 (CH3CONH). HRMS
(ESI/APCI) m/z calcd. for C20H37NNaO16 (M+Na+) 570.2010, found
570.1997.
3.14. 2,3,4,6-Tetra-O-acetyl-b-
D-galactopyranosyl-(1?2)-3,5,6-
tri-O-benzoyl- ,b- -galactofuranose (27)
a
D
A freshly prepared solution of bis(2-butyl-3-methyl)borane
(5.48 mmol, 6 equiv) in anhyd THF (1.6 mL) cooled to 0 °C under
an argon atmosphere, was added to a flask containing dried 25
(758 mg, 0.914 mmol) with positive argon pressure and stirring.
The resulting solution was allow to reach rt and stirred for 16 h
when TLC showed small amount of lactone 25 (Rf 0.5, 2:1 tolu-
ene–EtOAc) and a new product (Rf 0.35, 2:1 toluene–EtOAc). The
mixture was cooled to 0 °C, H2O (0.5 mL) was slowly added and
then processed as previously described.47 The organic layer was
washed with H2O, dried (Na2SO4), and concentrated. Boric acid
was eliminated by coevaporation with CH3OH (4 ꢁ 25 mL) at rt.
The residue was purified by column chromatography (7:1 tolu-
ene–EtOAc). Unreacted 25 (76 mg, 10%) was recovered from the
first fraction (Rf 0.5, 2:1 toluene–EtOAc). Next fraction gave
646 mg of 27 (86%) as an amorphous solid: Rf 0.35 (2:1 toluene–
3.13. 2,3,4,6-Tetra-O-acetyl-b-
tri-O-benzoyl- -galactono-1,4-lactone (25) and 2,3,4,6-tetra-O-
acetyl- -galactopyranosyl-(1?2)-3,5,6-tri-O-benzoyl-
galactono-1,4-lactone (26)
D-galactopyranosyl-(1?2)-3,5,6-
D
a-
D
D-
A solution of 10 (1.5 g, 3.06 mmol) in anhyd CH2Cl2 (40 mL) was
transferred under positive argon pressure over a mixture of dried

394
V. M. Mendoza et al. / Carbohydrate Research 345 (2010) 385–396
EtOAc), [
a
]
D ꢀ10.6 (c 1, CHCl3); 1H NMR (CDCl3, 500 MHz): d for the
(CCl3), 82.6 (C-2), 78.5 (C-4), 73.9, 71.7, 70.9, 70.5, 68.2, 66.9,
62.5 (C-6), 61.6 (C-60), 20.7, 20.6, 20.5, 20.3 (CH3CO).
b anomer, only the diagnostic signals are listed for the
a anomer:
8.07–7.33 (m, 15H, arom.), 5.73 (ddd, 0.8H, J = 6.5, 5.8, 3.9 Hz, H-
5), 5.69 (dd, 0.2H, J = 5.1, 4.0 Hz, H-3 anomer), 5.61 (d, 0.8H,
a
3.16. Benzyl 2,3,4,6-tetra-O-acetyl-b-
3,5,6-tri-O-benzoyl-b- -galactofuranosyl-(1?4)-2-acetamido-3-
O-benzoyl-6-O-t-butyldiphenylsilyl-2-deoxy- -glucopy
ranoside (29) and benzyl 2,3,4,6-tetra-O-acetyl-b- -galacto
pyranosyl-(1?2)-3,5,6-tri-O-benzoyl- -galactofuranosyl-
(1?4)-2-acetamido-3-O-benzoyl-6-O-t-butyldiphenylsilyl-2-
deoxy- -glucopyranoside (30)
D-galactopyranosyl-(1?2)-
J = 1.5 Hz, H-1), 5.42 (dd, 0.8H, J = 5.9, 2.1 Hz, H-3), 5.39 (dd,
0.8H, J = 3.5, 1.1 Hz, H-40), 5.25 (dd, 0.8H, J = 10.5, 7.9 Hz, H-20),
5.03 (dd, 0.8H, J = 10.5, 3.5 Hz, H-30), 4.84 (t, 0.8H, J = 5.8 Hz, H-
D
a-D
4), 4.83 (d, 0.8H, J = 7.9 Hz, H-10), 4.78 (d, 0.2H, J = 8.0 Hz, H-10
a
D
a-D
anomer), 4.73 (dd, 0.8H, J = 12.2, 3.9 Hz, H-6a), 4.63 (dd, 0.8H,
J = 12.2, 6.5 Hz, H-6b), 4.45 (dd, 0.2 H, J = 6.5, 5.1 Hz, H-4 ano-
anomer), 4.34 (dd, 0.8H,
a
a
-D
mer), 4.36 (t, 0.2H, J = 4.0 Hz, H-2
a
J = 2.1, 1.1 Hz, H-2), 4.15–4.09 (m, 1.6H, H-60a, H-60b), 3.93 (m,
0.8H, H-50), 3.15 (d, 0.8H, J = 3.3 Hz, –OH), 2.15 (s, 2.4H, CH3CO),
1.98 (3 s, 7.2H, CH3CO); 13C NMR (CDCl3, 125.8 MHz): d for the b
To a flask containing dry benzyl 2-acetamido-3-O-benzoyl-6-O-
t-butyldiphenylsilyl-2-deoxy-
-glucopyranoside15 (18, 162 mg,
a-D
0.25 mmol, 1 equiv) and activated 4 Å powdered molecular sieves
was added a solution of imidate 28b 270 mg, 0.28 mmol, 1.2 equiv)
in anhyd CH3CN (10 mL) under argon pressure and the mixture
was cooled to 0 °C with vigorous stirring. After 15 min, TMSOTf
anomer, only the diagnostic signals are listed for the
170.4, 170.2, 170.0, 169.5 (CH3CO), 166.0, 165.6, 165.5 (PhCO),
a anomer:
133.5–128.3 (C-arom.), 101.7 (C-1), 100.6 (C-10
a anomer), 100.5
(C-10), 96.7 (C-1
(C-4), 77.9 (C-4
a
anomer), 87.5 (C-2), 82.2 (C-2
a
anomer), 79.7
(20 lL, 0.011 mmol, 0.44 equiv) was added and the stirring contin-
a
anomer), 77.6 (C-3), 75.8 (C-3
a anomer), 71.1
(C-50), 70.9, 70.8 (C-30, C-5), 68.4 (C-20), 67.1 (C-40), 63.0 (C-6),
61.4 (C-60), 20.6, 20.5 ꢁ 3 (CH3CO). Anal. Calcd for C41H42O18: C,
59.85; H, 5.15. Found: C, 59.96; H, 5.42.
ued for 1 h at 0 °C and for additional 48 h at 5 °C until disappear-
ance of imidate 28b shown by TLC. The mixture was filtered, the
molecular sieves were washed with CH2Cl2 (2 ꢁ 8 mL), and the
combined filtrate was diluted with CH2Cl2 (100 mL), washed with
5% NaHCO3 (50 mL) and H2O (2 ꢁ 50 mL) until pH 6, dried
(Na2SO4), filtered, and concentrated in vacuo at rt. The residue
was purified by column chromatography (5:1 toluene–EtOAc).
The first fraction from the column (Rf 0.5, 2:0.6 toluene–EtOAc,
twice developed) afforded 109 mg of an inseparable mixture of
3.15. Preparation of O-(2,3,4,6-tetra-O-acetyl-b-
D-galacto
pyranosyl-(1?2)-3,5,6-tri-O-benzoyl-b-
trichloroacetimidate (28b)
D-galactofuranoside)
To a solution of dry 27 (0.27 g, 0.324 mmol), trichloroacetoni-
trile (0.2 mL, 1.99 mmol, 6 equiv) in anhyd CH2Cl2 (3.5 mL) cooled
imidate transposition byproducts: 2,3,4,6-tetra-O-acetyl-b-
galactopyranosyl-(1?2)-3,5,6-tri-O-benzoyl-N-trichloroacetyl-b-
-galactofuranosylamine and 2,3,4,6-tetra-O-acetyl-b- -galacto-
pyranosyl-(1?2)-3,5,6-tri-O-benzoyl-N-trichloroacetyl- -galac-
tofuranosylamine (31b and
) in 1:1 ratio as indicated by 1H NMR
analysis (40% yield from imidate 28b): [
+2.5 (c 1, CHCl3); 1H
NMR (CDCl3, 500 MHz): d 8.09–7.94 (m, 12H, arom.), 7.90 (d, 1H,
J = 9.0 Hz, NH anomer), 7.72 6–7.30 (m, 19H, H-arom. and NH b
anomer), 5.95 (dd, 1H, J = 9.0, 4.7 Hz, H-1 anomer), 5.85 (dd, 1H,
J = 7.0, 2.3 Hz, H-1 b anomer), 5.76 (m, 1H, H-5 b anomer), 5.73
(ddd, 1H, J = 7.5, 6.3, 3.3 Hz, H-5 anomer), 5.60 (dd, 1H, J = 4.3,
2.7 Hz, H-3 b anomer), 5.57 (dd, 1H, J = 3.7, 1.6 Hz, H-3 anomer),
5.39–5.37 (m, 2H, H-40
and b anomers), 5.25 (dd, 1H, J = 7.9,
10.5 Hz, H-20b anomer), 5.23 (dd, 1H, J = 10.5, 8.0 Hz, H-20
ano-
mer), 5.05 (dd, 1H, J = 10.5, 3.5 Hz, H-30 b anomer), 4.98 (dd, 1H,
J = 10.5, 3.5 Hz, H-30 anomer), 4.94 (d, 1H, J = 7.9 Hz, H-10 b ano-
mer), 4.84 (dd, 1H, J = 5.7, 4.3 Hz, H-4 b anomer), 4.79 (dd, 1H,
J = 12.4, 3.3 Hz, H-6a anomer), 4.77 (dd, 1H, J = 12.3, 3.8 Hz, H-
6a b anomer), 4.71 (d, 1H, J = 8.0 Hz, H-10 anomer
), 4.70 (t, 1H,
J = 2.5 Hz, H-2 anomer b), 4.68 (dd, 1H, J = 12.3, 6.3 Hz, H-6b b ano-
mer), 4.63 (dd, 1H, J = 12.4, 6.3 Hz, H-6b anomer), 4.56 (dd, 1H,
J = 4.7, 1.6 Hz, H-2 anomer), 4.47 (dd, 1H, J = 7.5, 3.7 Hz, H-4
D-
to 0 °C, DBU (20
l
L, 0.133 mmol, 0.4 equiv) was added with stir-
D
D
ring. After 1 h at rt, TLC showed consumption of 19 and the mixture
was concentrated under reduced pressure at rt. Purification of the
sirupy residue by column chromatography (5:1 toluene–EtOAc,
0.3% TEA) gave a first fraction of 28b (0.274 g, 88%) as a sirup: Rf
a-D
a
a
]
D
0.55 (2:1 toluene–EtOAc), [
a
]
D
ꢀ12.8 (c 1, CHCl3), 1H NMR (CDCl3,
a
500 MHz): d 8.60 (s, 1H, NH), 8.06–7.15 (m, 15H, arom.), 6.66 (br s,
1H, H-1), 5.81 (ddd, 1H, J = 6.7, 5.2, 4.0 Hz, H-5), 5.51 (d, 1H,
J = 4.8 Hz, H-3), 5.40 (d, 1H, J = 3.5 Hz, H-40), 5.23 (dd. 1H.
J = 10.4, 8.1 Hz, H-20), 5.03 (dd, 1H, J = 10.4, 3.5 Hz, H-30), 4.92 (t,
1H, J = 5.0 Hz, H-4), 4.88 (d, 1H, J = 8.1 Hz, H-1), 4.76 (dd, 1H,
J = 12.2, 4.0 Hz, H-6a), 4.68 (dd, 1H, J = 12.2, 6.7 Hz, H-6b), 4.53
(br, 1H, H-2), 4.15 (dd, 1H, J = 11.3, 6.8 Hz, H-6a0), 4.13 (dd, 1H,
J = 11.3, 6.6 Hz, H-6b0), 3.97 (t, 1H, J = 6.7 Hz, H-50), 2.18, 2.05,
1.98, 1.95 (4s, 12H, CH3CO); 13C NMR (CDCl3, 125.8 MHz): d
170.3, 170.2, 170.0, 169.4 (CH3CO), 165.9, 165.6, 165.4 (PhCO),
160.4 (CNH), 133.7–128.4 (C-arom), 104.3 (C-1), 101.0 (C-10),
90.9 (CCl3), 86.8 (C-2), 82.8 (C-4), 77.0 (C-3), 71.1 (C-50), 70.8 (C-
30), 70.4 (C-5), 68.4 (C-20), 67.0 (C-40), 62.9 (C-6), 61.1 (C-60), 20.6,
20.5 (CH3CO).
a
a
a
a
a
a
a
a
a
a
a
The next fraction from the column gave 29 mg of a sirup iden-
anomer), 4.24 (dd, 1H, J = 11.4, 6.5 Hz, H-6a0a or b anomer),
4.13–4.06 (m, 3H, H-6b0a or b anomer, H-6b0a anomer , H-6b0b
anomer), 3.98–3.89(m, 2H, H-50a and b anomers), 2.14 ꢁ 2, 2.02,
1.98, 1.97, 1.96, 1.95, 1.93 (8s, 24H, CH3CO); 13C NMR (CDCl3,
125.8 MHz): d 170.5, 170.3, 170.2, 170.0 ꢁ 3, 169.9 ꢁ 2, 169.5
(CH3CO), 166.0, 165.8, 165.5, 165.1 (PhCO), 161.6, 161.2
tified as O-(2,3,4,6-tetra-O-acetyl-b-
3,5,6-tri-O-benzoyl- -galactofuranoside)
(28 , 9%): Rf 0.50 (2:1 toluene–EtOAc), [
D-galactopyranosyl-(1?2)-
a-
D
trichloroacetimidate
a
a
]
D
+5.8 (c 0.7, CHCl3);
1H NMR (CDCl3, 500 MHz): d 8.51 ppm (s, 1H, NH), 7.98–7.30 (m,
15H, arom.), 6.57 (d, 1H, J = 4.7 Hz, H-1), 6.18 (t, 1H, J = 7.6 Hz, H-
3), 5.71 (ddd, 1H, J = 6.5, 5.5, 3.4 Hz, H-5), 5.31 (dd, 1H, J = 3.4,
1.0 Hz, H-40), 5.18 (dd, 1H, J = 10.6, 7.9 Hz, H-20), 4.93 (dd, 1H,
J = 10.6, 3.4 Hz, H-30), 4.82 (dd, 1H, J = 12.3, 3.4 Hz, H-6a), 4.69
(dd, 1H, J = 7.2, 6.5 Hz, H-4), 4.66 (dd, 1H, J = 12.4, 5.5 Hz, H-6b),
4.61 (d, 1H, J = 7.9 Hz, H-10), 4.57 (dd, 1H, J = 8.0, 4.7 Hz, H-2),
4.02 (dd, 1H, J = 11.3, 7.0 Hz, H-6a0), 3.94 (dd, 1H, J = 11.3, 5.8 Hz,
H-6b0), 3.86 (ddd, 1H, J = 7.0, 5.8, 1.0 Hz, H-50), 2.09, 2.06, 1.94,
1.81 (4s, 12H, CH3CO); 13C NMR (CDCl3, 125.8 MHz): d 170.5,
170.1, 170.0, 169.0 (CH3CO), 165.8, 165.5, 164.7 (PhCO), 161.2
(CNH), 133.5–128.4 (C-arom.), 101.2 (C-10), 97.8 (C-1), 91.0
(CH3CONH), 135.6–127.8 (C-arom.), 100.2, 99.7 (C-10
mers), 87.4 (C-1 b anomer), 85.1 (C-2 b anomer), 81.9 (C-1
a
and b ano-
ano-
anomer), 77.5
and b ano-
and b
anomer), 67.0, 66.0
a and b anomers), 61.2,
a
mer, C-4 b anomer), 79.9 (C-2
(C-3 b anomer), 76.5 (C-3
a anomer), 79.2 (C-4 a
a
anomer), 71.2 ꢁ 2 (C-50
a
mers), 71.0 (C-5 b anomer), 70.6 ꢁ 3 (C-5
a a
anomer, C-30
anomers), 68.5 (C-20 b anomer), 68.1 (C-20
(C-40
a
a
and b anomers), 63.1 ꢁ 2 (C-6
61.0 (C-60a and b anomers), 20.6, 20.5 ꢁ 3 (CH3CO). HRMS (ESI/
APCI) m/z calcd. for C43H42Cl3NNaO18 (M+Na+) 988.1365, found
988.1389.

V. M. Mendoza et al. / Carbohydrate Research 345 (2010) 385–396
395
Next fraction from the column (Rf 0.35, 2:0.6 toluene–EtOAc,
J = 9.6 Hz, -NH), 5.59 (dd, 1H, J = 10.7, 9.5 Hz, H-3), 5.44 (s, 1H, H-
10), 5.39 (ddd, 1H, J = 7.3, 5.2, 3.6 Hz, H-50), 5.36 (d, 1H, J = 3.5 Hz,
H-400), 5.27 (dd, 1H, J = 5.2, 1.7 Hz, H-30), 5.16 (dd, 1H, J = 10.5,
8.0 Hz, H-200), 4.98 (d, 1H, J = 3.5 Hz, H-1), 4.97 (dd, 1H, J = 10.5,
3.5 Hz, H-300), 4.76 (d, 1H, J = 8.0 Hz, H-100); 4.76, 4.52 (2d, 2H,
J = 12.0 Hz, CH2Ph), 4.47 (ddd, 1H, J = 10.7, 9.6, 3.5 Hz, H-2), 4.32
(t, 1H, J = 5.2 Hz, H-40), 4.26 (d, 1H, J = 1.7 Hz, H-20), 4.26–4.10 (m,
3H, H-6a0, H-6b0, H-6a00), 4.14 (t, 1H, J = 9.5 Hz, H-4), 4.01 (dd, 1H,
J = 11.2, 7.5 Hz, H-6b00), 3.92–3.86 (m, 4H, H-5, H-500, H-6a, H-6b),
2.16, 1.98, 1.96, 1.93 (4s, 12H, CH3CO), 1.78 (s, 3H, CH3CONH);
13C NMR (CDCl3, 125.8 MHz): d 170.5, 170.2 ꢁ 2, 170.0 (CH3CO),
167.0 (CH3CONH), 165.8, 165.4, 165.3 (PhCO), 136.7–128.1
(C-arom.), 106.9 (C-10), 100.7 (C-100), 96.7 (C-1), 87.0 (C-20), 80.4
(C-40), 77.5 (C-30), 73.9 (C-4), 72.1 (C-3), 71.3 (C-5 or C-500), 70.8
(C-300, C-50 or C-500), 70.3 (C-50), 70.0 (CH2Ph), 68.3 (C-200), 66.8
(C-400), 62.9 (C-60), 61.0 (C-6), 60.8 (C-600), 52.3 (C-2), 23.0
(CH3CONH), 20.6, 20.5, 20.4 (CH3CO).
twice developed) gave 144 mg of an amorphous solid identified
as 29 (40%): [a]
+3.5 (c 1, CHCl3); 1H NMR (CDCl3, 500 MHz): d
D
8.07–7.25 (m, 35H, arom.), 5.87 (d, 1H, J = 9.7 Hz, NH), 5.60 (dd,
1H, J = 10.8, 9.3 Hz, H-3), 5.56 (s, 1H, H-10), 5.39 (ddd, 1H, J = 7.4,
6.2, 3.3 Hz, H-50), 5.33 (dd, 1H, J = 5.1, 1.2 Hz, H-30), 5.27 (dd, 1H,
J = 3.5, 1.2 Hz, H-400), 5.17 (dd, 1H, J = 10.4, 8.0 Hz, H-200), 4.97 (d,
1H, J = 3.8 Hz, H-1), 4.87 (dd, 1H, J = 10.4, 3.5 Hz, H-300), 4.70 (d,
1H, J = 8.0 Hz, H-100), 4.68, 4.47 (2d, 2H, J = 11.5 Hz, CH2Ph), 4.50
(ddd, 1H, J = 10.8, 9.7, 3.8 Hz, H-2), 4.31 (dd, 1H, J = 6.2, 5.1 Hz,
H-40), 4.30 (dd, 1H, J = 9.3, 8.6 Hz, H-4), 4.27 (dd, 1H, J = 12.1,
3.3 Hz, H-6a0), 4.21 (d, 1H, J = 1.2 Hz, H-20), 4.13 (dd, 1H, J = 12.1,
7.4 Hz, H-6b0), 3.97 (dd, 1H, J = 11.3, 6.7 Hz, H-6a00), 3.92 (dd, 1H,
J = 11.3, 6.5 Hz, H-6b00), 3.92–3.88 (m, 2H, H-5, H-6a), 3.81 (d, 1H,
J = 10.5 Hz, H-6b), 3.69 (td, 1H, J = 6.6, 1.2 Hz, H-500), 2.11, 1.96,
1.95, 1.91 (4s, 12H, CH3CO), 1.81 (s, 3H, CH3CONH), 1.01 (s, 9H,
(CH3)3CSi); 13C NMR (CDCl3, 125.8 MHz): d 170.2, 170.1, 169.9,
169.3 (CH3CO), 167.0 (CH3CONH), 165.7, 165.5, 165.2 (PhCO),
136.7–127.7 (C-arom.), 106.2 (C-10), 99.5 (C-100), 96.3 (C-1), 86.7
(C-20), 80.4 (C-40), 76.9 (C-30), 72.6 (C-4), 72.4 (C-3), 71.7 (C-5),
70.9 (C-300), 70.8 (C-500), 70.5 (C-50), 69.6 (CH2Ph), 68.1 (C-200),
67.2 (C-400), 62.9 (C-60), 62.5 (C-6), 61.1 (C-600), 52.4 (C-2), 26.6
((CH3)3CSi), 23.1 (CH3CONH), 20.6 ꢁ 2, 20.5, 20.4 (CH3CO), 19.2
((CH3)3CSi). Anal. Calcd for C79H83NO24Si: C, 65.05; H, 5.74. Found:
C, 64.82; H, 5.63.
Anal. Calcd for C63H65NO24ꢂH2O: C, 61.11; H, 5.45; N, 1.13.
Found: C, 61.50; H, 5.16; N, 1.39.
3.18. Benzyl b-
D
-galactopyranosyl-(1?2)-b-
D-galactofuranosyl-
(1?4)-2-acetamido-2-deoxy-
a-D
-glucopyranoside (33)
To a solution of 32 (38 mg, 0.031 mmol) in anhyd CH3OH (1 mL)
cooled to 0 °C, was added a solution of 1.15 M NaOCH3 in CH3OH
(0.72 mL) and the mixture was stirred at rt. After 3 h, TLC examina-
tion showed only one compound (Rf 0.5, 7:1:1 n-propanol–
EtOH–H2O) and the solution was processed as described for 22.
Evaporation of the solvent gave a white solid that after crystalliza-
The last fraction from the column (Rf 0.1, 2:0.6 toluene–EtOAc,
twice developed) gave 44 mg of 30 as an amorphous solid (12%):
[
a]
+15.3 (c 1, CHCl3). 1H NMR (CDCl3, 500 MHz): d 8.03–7.22
D
(m, 35H, arom.), 6.21 (d, 1H, J = 9.4 Hz, NH), 5.69 (dd, 1H, J = 10.8,
8.5 Hz, H-3), 5.62 (t, 1H, J = 6.4 Hz, H-30), 5.50 (dt, 1H, J = 6.0,
4.7 Hz, H-50), 5.42 (d, 1H, J = 4.8 Hz, H-10), 5.18 (dd, 1H, J = 3.6,
1.3 Hz, H-400), 5.13 (dd, 1H, J = 10.4, 8.0 Hz, H-200), 4.94 (d, 1H,
J = 3.7 Hz, H-1); 4.80, 4.49 (2d, 2H, J = 12.1 Hz, CH2Ph), 4.67 (dd,
1H, J = 10.4, 3.6 Hz, H-300), 4.50 (dd, 1H, J = 12.0, 4.8 Hz, H-6a0),
4.38 (ddd, 1H, J = 10.8, 9.4, 3.7 Hz, H-2), 4.36 (dd, 1H, J = 12.0,
6.0 Hz, H-6b0), 4.35 (dd, 1H, J = 6.5, 4.8 Hz, H-20), 4.26 (dd, 1H,
J = 6.3, 4.6 Hz, H-40), 4.22 (d, 1H, J = 8.0 Hz, H-100), 4.17–4.02 (m,
3H, H-4, H-5, H-6a), 3.77 (d, 1H, J = 11.0 Hz, H-6b), 3.74 (dd, 1H,
J = 11.1, 6.2 Hz, H-6a00), 3.66 (dd, 1H, J = 11.1, 7.8 Hz, H-6b00), 3.52
(ddd, 1H, J = 7.8, 6.2, 1.3 Hz, H-500); 2.13, 2.04, 1.95, 1.92 (4s, 12H,
CH3CO), 1.84 (s, 3H, CH3CONH), 1.01 (s, 9H, (CH3)3CSi); 13C NMR
(CDCl3, 125.8 MHz): d 170.2 ꢁ 2, 170.1, 170.0, 169.0 (CH3CO),
166.6 (CH3CONH), 165.7, 165.5, 164.8 (PhCO), 137.1 – 127.5 (C-
arom.), 102.7 (C-10), 99.8 (C-100), 95.9 (C-1), 79.1 (C-20), 78.7 (C-
40), 77.3 (C-4), 75.6 (C-30), 74.2 (C-3), 71.2 (C-5), 71.0 (C-300), 70.9
(C-50), 70.7 (C-500), 69.1 (CH2Ph), 67.9 (C-200), 66.6 (C-400), 63.0 (C-
6), 62.6 (C-60), 60.4 (C-600), 52.7 (C-2), 26.9 ((CH3)3CSi), 23.1
(CH3CONH), 20.6, 20.5 ꢁ 2, 20.4 (CH3CO), 19.4 ((CH3)3CSi).
HRMS (ESI/APCI) m/z calcd for C79H83NNaO24Si (M+Na+)
1480.4972, found 1480.4931.
tion from CH3OH yielded 33 (19 mg, 97%): mp 233–235 °C; [a]
D
+31 (c 0.4, H2O); 1H NMR (D2O, 500 MHz): d 7.46–7.38 (m, 5H,
arom.), 5.30 (br s, 1H, H-10), 4.94 (d, 1H, J = 3.5 Hz, H-1); 4.73;
4.55 (2d, 2H, J = 11.9 Hz, CH2Ph), 4.57 (d, 1H, J = 7.8 Hz, H-100),
4.29 (d, 1H, J = 3.0 Hz, H-20), 4.26 (dd, 1H, J = 6.4, 3.0 Hz, H-30),
4.12 (dd, 1H, J = 6.4, 3.7 Hz, H-40), 3.94 (dd, 1H, J = 3.4, 1.0 Hz, H-
400), 3.89 (dd, 1H, J = 10.7, 3.5 Hz, H-2), 3.84 (dd, 1H, J = 10.7,
8.5 Hz, H-3), 3.85–3.80 (m, 4H, H-50, H-5, H-6a or H-6a00, H-6b or
H-6b00), 3.79 (dd, 1H, J = 11.6, 7.4 Hz, H-6a or H-6a00), 3.76 (dd, 1H,
J = 11.6, 4.8 Hz, H-6b or H-6b00), 3.71 (ddd, 1H, J = 7.4, 4.8, 1.0 Hz, H-
500), 3.69 (dd, 1H, J = 9.6, 8.5 Hz, H-4), 3.68 (dd, 1H, J = 11.8, 4.3 Hz,
H-6a0), 3.66 (dd, 1H, J = 10.0, 3.4 Hz, H-300), 3.63 (dd, 1H, J = 11.8,
7.4 Hz, H-6b0), 3.53 (dd, 1H, J = 10.0, 7.8 Hz, H-200), 1.96 (s, 3H,
CH3CONH); 13C NMR (D2O, 125.8 MHz): d 174.9 (CH3CONH),
137.6, 129.4, 129.2, 129.0 (C-arom.); 107.2 (C-10), 103.2 (C-100),
96.5 (C-1), 89.2 (C-20), 83.6 (C-40), 77.1 (C-4), 76.3 (C-30), 75.9 (C-
500), 73.1 (C-300), 71.7 (C-5 or C-50), 71.4 (C-200), 71.1 (C-5 or C-50),
70.5 (CH2Ph), 70.0 (C-3), 69.2 (C-400), 63.6 (C-60); 61.6, 60.6 (C-6,
C-600), 54.4 (C-2), 22.4 (CH3CONH). Anal. Calcd for C27H41NO16
2H2O: C, 48.28; H, 6.75; N, 2.09. Found: C, 48.10; H, 6.52; N, 2.58.
.
3.19. b-
D-Galactopyranosyl-(1?2)-b-D-galactofuranosyl-(1?4)-
3.17. Benzyl 2,3,4,6-tetra-O-acetyl-b-
3,5,6-tri-O-benzoyl-b- -galactofuranosyl-(1?4)-2-acetamido-3-
O-benzoyl-2-deoxy- -glucopyranoside (32)
D
-galactopyranosyl-(1?2)-
2-acetamido-2-deoxy-
a
,b- -glucopyranose (6)
D
D
a
-
D
Compound 33 (12.8 mg, 0.020 mmol) dissolved in 8:1 CH3OH–
H2O (1.2 mL) was hydrogenated and purified as described for 5
to yield 6 (10 mg, 92%) as an amorphous solid: Rf 0.25–0.11
To a solution of 29 (163 mg, 0.134 mmol) in anhyd DMF
(1.7 mL) cooled at 0 °C, was added a freshly prepared solution of
(7:1:1 n-propanol–EtOH–H2O); [
a
]
D
ꢀ22.5 (c 0.8, H2O); 1H NMR
1 M TBAF in anhyd THF (223
l
L, 2 equiv) with vigorous stirring,
(D2O, 500 MHz) d anomeric zone and other diagnostic signals:
followed by glacial HOAc (14.8
l
L, 2.1 equiv). The solution was
5.31 (m, 1H, H-10a anomer and H-10 b anomer), 5.19 (d, 0.6H,
stirred at 25 °C for 2 h and left at ꢀ18 °C overnight. The solution
was diluted with CH2Cl2 (50 mL), washed with H2O (5 ꢁ 10 mL),
dried (Na2SO4), and concentrated under reduced pressure. Column
chromatography of the crude (1.1 to 1:2 n-hexane–EtOAc) gave 32,
(134 mg, 82%) that crystallized from THF–hexane (1:2): Rf 0.21 (1:2
J = 3.4 Hz, H-1
4.57 (d, 0.6H, J = 7.8 Hz, H-100
H-100 b anomer), 4.31 (d, 0.6H, J = 3 Hz, H-20
0.4H, J = 2.8 Hz, H-20 b anomer), 2.04 (2s, 3H, CH3CONH
anomers); 13C NMR (D2O, 125.8 MHz): d 175.4, 175.2 (CH3CONH
and b anomers), 107.3, 107.2 (C-10
and b anomers), 103.2
(C-100 a and b anomers), 95.6 (C-1 b anomer), 91.3 (C-1
anomer),
a
anomer), 4.70 (d, 0.4H, J = 8.3 Hz, H-1 b anomer),
anomer), 4.56 (d, 0.4H, J = 7.8 Hz,
anomer), 4.29 (d,
and b
a
a
a
hexane–EtOAc), mp 103–105 °C, [
a
]
D
ꢀ4.4 (c 1, CHCl3); 1H NMR
a
a
(CDCl3, 500 MHz): d 8.03–7.28 (m, 25H, arom.), 5.90 (d, 1H,
a

396
V. M. Mendoza et al. / Carbohydrate Research 345 (2010) 385–396
89.1 ꢁ 2 (C-20 a and b anomers); 83.8, 83.7 (C-40 a and b anomers);
77.2, 76.9, 76.4, 75.9, 75.7, 73.1, 73.0, 71.4, 71.2 ꢁ 2, 69.9, 69.2,
13. Gallo-Rodriguez, C.; Varela, O.; de Lederkremer, R. M. J. Org. Chem. 1996, 61,
1886–1889.
14. Gallo-Rodriguez, C.; Varela, O.; de Lederkremer, R. M. Carbohydr. Res. 1998, 305,
163–170.
63.6, 61.6, 60.9, 60.8; 57.4 (C-2 b anomer), 54.8 (C-2
a anomer);
15. Gallo-Rodriguez, C.; Gil-Libarona, M. A.; Mendoza, V. M.; de Lederkremer, R. M.
Tetrahedon 2002, 58, 9373–9380.
16. Mendoza, V. M.; Agusti, R.; Gallo-Rodriguez, C.; de Lederkremer, R. M.
Carbohydr. Res. 2006, 341, 1488–1497.
22.8, 22.5 (CH3CONH anomers
a and b). HRMS (ESI/APCI) m/z calcd
for C20H35NNaO16 (M+Na+) 568.1854, found 568.1840.
17. van Well, R. M.; Collet, B. Y. M.; Field, R. A. Synlett 2008, 2175–2177.
18. Hederos, M.; Konradsson, P. J. Am. Chem. Soc. 2006, 128, 3414–3419.
19. Randell, K. D.; Johnston, B. D.; Brown, P. N.; Pinto, B. M. Carbohydr. Res. 2000,
325, 253–264.
20. Peltier, P.; Euzen, R.; Daniellou, R.; Nugier-Chauvin, C.; Ferrières, V. Carbohydr.
Res. 2008, 343, 1897–1923.
21. Richards, M. R.; Lowary, T. L. ChemBioChem 2009, 10, 1920–1938.
22. (a) Veeneman, G. H.; Notermans, S.; Liskamp, R. M. J.; van der Marel, G. A.; van
Boom, J. H. Tetrahedron Lett. 1987, 28, 6695–6698; (b) Backinowsky, L. V.;
Nepogod’ev, S. A.; Kochetkov, N. K. Carbohydr. Res. 1989, 185, C1–C3; (c)
Kochetkov, N. K.; Nepogod’ev, S. A.; Backinowsky, L. V. Tetrahedron 1990, 46,
139–150; (d) Nepogod’ev, S. A.; Backinowsky, L. V.; Kochetkov, N. K. Mendeleev
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24. Gandolfi-Donadio, L.; Gallo-Rodriguez, C.; de Lederkremer, R. M. J. Org. Chem.
2002, 67, 4430–4435.
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Tetrahedron: Asymmetry 2005, 16, 733–738.
3.20. Preparation of b-
galactofuranosyl-(1?4)-2-acetamido-2-deoxy-
D
-galactopyranosyl-(1?2)-b-
D-
D
-glucitol6 (34)
Compound 6 (8.0 mg; 0.015 mmol) was reduced with NaBH4
(14 mg, 0.37 mmol) in CH3OH (0.5 mL) and then processed and
purified as described for 23 to yield 7.5 mg of 34 as a very hygro-
scopic solid (93%): Rf 0.27 (7:1:1.5 n-propanol–EtOH–H2O), [a]
D
ꢀ46 (c 0.5, H2O); 1H NMR (D2O, 500 MHz) d anomeric zone and
diagnostic signals: 5.36 (br s, 1H, H-10), 4.59 (d, 1H, J = 7.9 Hz, H-
100), 4.37 (d, 1H, J = 2.9 Hz, H-20), 4.26 (dd, 1H, J = 6.2, 2.9 Hz, H-
30), 4.14 (dd, 1H, J = 6.2, 3.8 Hz, H-40), 4.13 (m, 1H, H-2), 3.53 (dd,
1H, J = 10.0, 7.9 Hz, H-200), 2.05 (s, 3H, CH3CONH). d values matched
those described for the natural compound;6 13C NMR (D2O,
125.8 MHz): d 175.1 (CH3CONH), 107.8 (C-10), 102.9 (C-100), 88.9
(C-20), 84.2 (C-40), 78.5 (C-4), 76.5 (C-30), 76.0 (C-500), 73.2 (C-300),
71.7 (C-400), 71.4, 71.3 (C-50, C-200); 69.2, 69.0 (C-3, C-5), 63.6 (C-
60), 62.7 (C-600); 61.6, 61.4 (C-1, C-6), 53.3 (C-2), 22.8 (CH3CONH).
HRMS (ESI/APCI) m/z calcd for C20H37NNaO16 (M+Na+) 570.2010,
found 570.1997.
27. (a) Bai, Y.; Lowary, T. L. J. Org. Chem. 2006, 71, 9672–9680; (b) Completo, G. C.;
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29. (a) Lee, Y. J.; Fulse, D. B.; Kim, K. S. Carbohydr. Res. 2008, 343, 1574–1584; (b)
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2008, 10, 2373–2376.
30. de Lederkremer, R. M.; Varela, O. Adv. Carbohydr. Chem. Biochem. 1994, 50, 125–
209.
Acknowledgments
This work was supported by grants from Agencia Nacional de
Promoción Científica y Tecnológica, Universidad de Buenos Aires
and CONICET. R.M.L. and C.G.-R. are research members of CONICET.
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.carres.2009.12.005.
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