Synthesis of disaccharide congeners of the Trichinella spiralis glycan and binding site mapping of two monoclonal antibodies

Article abstract of DOI:10.1139/v02-117

The tetrasaccharide epitope, β-D-Tyvp(1→3)β-D-GalNAcp(1→4)[α-L-Fucp(1→3)] β-D-GlcNAcp (1) is the major constituent of the N-glycan expressed on the cell surface of the parasite Trichinella spiralis. Two monoclonal antibodies (Mabs 9D4 and 18H1) that prote

Full text of DOI:10.1139/v02-117

1141
Synthesis of disaccharide congeners of the
Trichinella spiralis glycan and binding site
mapping of two monoclonal antibodies
Ping Zhang, Judith Appleton, Chang-Chun Ling, and David R. Bundle
Abstract: The tetrasaccharide epitope, ꢀ-D-Tyvp(1J3)ꢀ-D-GalNAcp(1J4)[ꢁ-L-Fucp(1J3)] ꢀ-D-GlcNAcp (1) is the
major constituent of the N-glycan expressed on the cell surface of the parasite Trichinella spiralis. Two monoclonal an-
tibodies (Mabs 9D4 and 18H1) that protect rats against infection by T. spiralis bind the terminal disaccharide epitope
ꢀ-D-Tyvp(1J3)ꢀ-D-GalNAcp conjugated to BSA. The syntheses of disaccharide congeners containing mono-deoxy,
mono-methyl, as well as modifications to replace the acetamido group are reported. These target disaccharides were
assayed for binding to the protective MAbs. For each antibody different clusters of three hydroxyl groups, that include
C-2 and C-4 of tyvelose and for 18H1, the GalNAc acetamido group, provide the key polar interactions with the
antibody binding sites. Mapping of the sites by functional group replacement revealed a similar pattern of recognition
for the dideoxyhexose by the two MAbs while each recognizes distinct surfaces of the GalNAc residue. Consequently
although both antibodies bury the 4-OH of tyvelose, the principal contact surface occurs on opposite sides of the 3,6-
dideoxyhexose.
Key words: ꢀ-tyveloside, 3,6-dideoxy-D-arabino-hexose, Trichinella carbohydrate antigen, antibody mapping, Trichinella
spiralis, N-glycans, molecular recognition of carbohydrates, antigen topology, functional group replacement.
Résumé : Le tétrasaccharide épitope, ꢀ-D-Tyvp(1J3)ꢀ-D-GalNAcp(1J4)[ꢁ-L-Fucp(1J3)]ꢀ-D-GlcNAcp (1) est un
constituant majeur du N-glucane extrait de la surface de la cellule du parasite Trichinella spiralis. Deux anticorps
monoclonaux (Mabs 9D4 et 18H1) qui protègent les rats contre les infections par le T. spiralis se lient au disaccharide
terminal épitope ꢀ-D-Tyvp(1J3)ꢀ-D-GalNAcp conjugué au BSA. On rapporte les synthèses de congénères du
disaccharide comportant des modifications de type monodésoxy- ou monométhyle ainsi que le remplacement du groupe
acétamido. On a évalué les capacités de ces disaccharides à se lier aux MAbs protecteurs. Pour chacun des anticorps,
différents ensembles de trois groupes hydroxyles incluant les C-2 et C-4 du tyvelose ainsi que le groupe acétamido
GalNAc du 18H1 fournissent les interactions polaires clés avec les sites de liaison de l’anticorps. Une carte des sites
obtenue par le remplacement des groupes fonctionnels a permis de réaliser qu’un patron semblable existe pour la re-
connaissance du didésoxyhexose par les deux MAbs alors que chacun d’eux reconnaît des surfaces distinctes du résidu
GalNAc. En conséquence, même si les deux anticorps ensevelissent le 4-OH du tyvelose, le principal contact de sur-
face se produit sur les faces opposées du 3,6-didésoxyhexose.
Mots clés : ꢀ-tyveloside, 3,6-didésoxy-D-arabino-hexose, hydrate de carbone antigène du Trichinella, carte de
l’anticorps, Trichinella spiralis, N-glycanes, reconnaissance moléculaire des hydrates de carbone, topologie des
antigènes, remplacement d’un groupe fonctionnel.
[Traduit par la Rédaction] Zhang et al. 1161
Introduction
glycoproteins that are key to successful colonization of the
intestine by the parasite. Antibodies against the glycan por-
tion of the glycoproteins can prevent parasitic invasion and
cause expulsion of the T. spiralis larvae from the intestine
(2, 3). Here the syntheses of congeners of the native, termi-
nal disaccharide ꢀ-D-Tyvp(1J3)ꢀ-D-GalNAcp are reported.
The inhibitory activities of these ligands are used to define
the precise specificity and topology of the epitope’s contact
surface with the binding sites of two protective rat
The causative agent of trichinosis a parasitic nematode,
Trichinella spiralis, has an exceptionally large host range
and is endemic in many carnivorous animals. The parasite
establishes itself in the intestinal epithelia of the host ani-
mal, following consumption of infected tissue and causes
acute muscle pain, fatigue, fever, and in severe cases, death
(1). During invasion, T. spiralis secretes an array of
Received 4 December 2001. Published on the NRC Research Press Web site at http://canjchem.nrc.ca on 4 September 2002.
We dedicate this paper to the memory of Professor Raymond U. Lemieux.
P. Zhang, C.-C. Ling, and D.R. Bundle.1 Department of Chemistry, The University of Alberta, Edmonton, AB T6G 2G2, Canada.
J. Appleton. James A. Baker Institute, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, U.S.A.
1Corresponding author (e-mail: dave.bundle@ualberta.ca).
Can. J. Chem. 80: 1141–1161 (2002)
DOI: 10.1139/V02-117
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Can. J. Chem. Vol. 80, 2002
Fig. 1. The structure of the terminal tetrasaccharide 1 of the
N-glycan expressed by the L1 stage larvae of the T. spiralis parasite,
and its component terminal trisaccharide 2 and disaccharide 3.
monoclonal antibodies (MAbs) produced from lymphocytes
of rats infected with T. spiralis.
In common with many other nematodes (4), Trichinella
secretes and displays on its cuticle a large number of pro-
teins that display a rich repertoire of N-linked glycans (5, 6).
Complex-type N-glycans in eukaryotes are characterized by
the presence of variable numbers of antennae (usually two,
three, or four, called bi-, tri-, and tetraantennary, respec-
tively). In mammals the antennae stubs are usually elongated
by the addition of ꢀ-Gal to give ꢀ-Gal(1J4)GlcNAc or
“LacNAc”. In a restricted number of mammalian glyco-
proteins, ꢀ-GalNAc is added to the GlcNAc stubs in place of
ꢀ-Gal and thus they have ꢀ-GalNAc(1J4)GlcNAc or
“LacdiNAc” antennae. Both types of antennae are found in
vertebrates but LacdiNAc is the preferred antennae building
block in many helminths including Trichinella (5, 6). The L1
stage of T. spiralis modifies LacdiNAc with phosphoryl-
choline moieties or by substitution with 3,6-dideoxy-D-
arabino-hexose (D-tyvelose), a sugar normally confined to
the cell wall lipopolysaccharide (LPS) of gram-negative bac-
teria. Both modifications create highly immunogenic epitopes.
Trichinella spiralis faces an aggressive immune response
from its animal host, but avoids extinction by changing its
expression of antigenic molecules as it changes life stages
(7, 8) and by residing in intracellular habitats (9, 10). Anti-
body responses induced by L1 stage larvae in the intestine
and in the muscle are largely directed at N-glycans unique to
this stage (11–14). Glycans capped by tyvelose are the target
of protective immunity against T. spiralis and antibodies
specific for one of these glycans protect rats against reinfec-
tion with T. spiralis (3, 15, 16). The structure of the anten-
nae of the protective antigen is shown (1, Fig. 1). The most
striking and unusual feature is the presence of tyvelose as its
ꢀ-pyranoside form rather than the ꢁ-anomer, which previ-
ously was the only naturally occurring form to have been
observed in bacterial LPS antigens of pathogenic Salmonella (17).
Some monoclonal tyvelose-specific antibodies that bind
the T. spiralis glycan provide protection to passively immu-
nized rats (3, 5). Antibody 9D4 has high protective efficacy
in rats and exhibits high affinity for tetrasaccharide 1. An-
other tyvelose specific antibody 18H1, also shows poor pro-
tective efficacy (3, 15). It fails to cause expulsion of larvae
from the intestines of rats, even though it exhibits most of
the inhibitory effects shown by antibody 9D4.
No structural details of these antibody binding sites are
available, although preliminary crystallographic data has
been collected for MAb 9D4, and both antibody Fv regions
have been partially sequenced (18). The 3,6-dideoxy
arabinohexose was inferred to be linked to the N-glycans via
the rare ꢀ-linkage based on binding studies with di- (16, 19),
tri- (20, 21), and tetrasaccharides (22, 23). MAb 9D4
showed the highest affinity for tetrasaccharide 1, while
trisaccharide 2 had five times weaker affinity than the
tetrasaccharide, and three times higher affinity than the
disaccharide 3 (Table 1) (21, 24). This indicates the binding
site of this antibody has contacts to all four sugars of the
tetrasaccharide 1, while the majority of its binding energy is
focused around the dideoxyhexose containing disaccharide,
an observation that has been found for other antibodies rec-
ognizing glycans containing dideoxyhexoses (25). MAb
18H1 unexpectedly recognized the trisaccharide 2 with an
HO
OH
OH
O
HO
OH
O
OH
O
NHAc
OMe
O
O
HO
O
O
NHAc
OH
1
HO
O
OH
OH
O
NHAc
OMe
O
HO
O
HO
O
NHAc
OH
2
HO
O
OH
OH
O
O
HO
OMe
NHAc
3
affinity comparable to that of the tetrasaccharide 1 and rec-
ognized the disaccharide 3 with six times higher affinity.
This data is clearly inconsistent with earlier rationalization
of the poor protective efficacy of MAb 18H1 that hypothe-
sized it had been raised against a non-fucosylated linear arm
of the glycan (16), and that the linear trisaccharide 2 was the
preferred epitope. This would require that trisaccharide 2 be
bound more tightly than the branched tetrasaccharide 1.
Consistent with the mapping data from the three different
sized ligands (1–3), MAb 9D4 shows a higher affinity for its
complementary ligand 1 than 18H1 shows for its smaller and
complementary disaccharide epitope 3 (Table 1).
Chemical mapping by functional group replacement has
proven to be a very successful tool to acquire information
about carbohydrate-binding sites of antibodies (26–30), en-
zymes (31, 32), and lectins (26, 33). The strategy as devised
by Lemieux and co-workers (34) involves the use of mono-
deoxy and mono-O-methyl congeners to identify the most
important polar contacts between the carbohydrate epitope
and protein. When the binding data of the modified epitopes
with protein are combined with computer-assisted modeling,
an overall picture of the binding site emerges, and the carbo-
hydrate surface located in the site vs. that exposed to solvent
water as well as the complementary interacting surfaces can
be identified.
Results and discussion
The methyl glycoside of ꢀ-D-Tyvp(1J3)ꢀ-D-GalNAcp 3,
together with 11 other synthetic targets (Fig. 2) are used in
this chemical mapping study. Disaccharides 4 and 5 are used
to probe the importance of the 2-acetamido-2-deoxy group
for binding, while analogs 6–8 contain modifications to
probe the 4-position of GalNAc and disaccharides 9 and 10,
© 2002 NRC Canada

Zhang et al.
1143
Fig. 2. The structures of the synthetic disaccharide congeners 4–14 of the native disaccharide 3.
HO
OH
O
OH
O
OH
O
OH
O
HO
O
HO
HO
O
OMe
OMe
R
NHAc
6
4 R = NH2
5 R = OH
R
O
HO
OH
O
R
OH
O
OH
O
O
HO
HO
OMe
O
OMe
NHAc
NHAc
9
R = H
10 R = OMe
7 R = H
8 R = OMe
HO
HO
OH
OH
O
R
OH
O
O
O
HO
R
O
OMe
O
OMe
NHAc
NHAc
13 R = H
14 R = OMe
11 R = H
12 R = OMe
cially when an extended reaction sequence was involved.
This necessitated an alternative route for creation of the ꢀ-
tyveloside linkage.
Table 1. Epitope size of the Trichinella spiralis glycan recognized
by MAbs 9D4 and 18H1.
Relative
Use of a 3,6-dideoxy-D-ribohexopyranose (paratose) de-
rivative as a precursor of the target tyvelosides became the
strategy of choice, since the anomeric ꢀ-linkage is easily in-
troduced by exploiting a C-2 participating group. The C-2
hydroxyl group can be inverted to its C-2 epimer through an
oxidation–reduction sequence (36, 37). It was reported that
by carefully choosing the reducing agent, the axial OH-2
epimer could be obtained in high yield. Recently, we pub-
lished a new methodology to allow the preparation of 3,6-
dideoxy-D-hexopyranoses (abequose and paratose) on a large
scale and in high yields (38); furthermore, this methodology
permits the facile introduction of different protecting groups
at the C-2 and C-4 positions. For this purpose we employed
a previously prepared paratose thioglycoside 20 as starting
material (38).
Since benzoyl and pivaloyl protecting groups are superior
participating groups when compared to the corresponding
acetate ester, we converted 20 to the 2-OH derivative 21 and
then prepared the corresponding benzoylated and pivaloyl-
ated donors 22 and 23 (Scheme 2). In our preliminary inves-
tigation, it was found that 22 and 23 gave similar yields
when reacted with 2-azido galactosyl acceptor 24 (39). Due
to the relative ease with which the benzoate group can be re-
moved, the benzoylated donor 22 was used for all glycosyl-
ations. Using N-iodosuccinimide – triflic acid as the
activation conditions, the disaccharide 25 was obtained in
83% yield. After removal of benzoate (J26, 94%), the
2>-hydroxyl group was oxidized to the corresponding ketone
27 by DMSO–Ac2O. The intermediate ketone 27 was not
isolated but immediately reduced. When excess L-selectride
was used as the reducing reagent, the corresponding
ꢀ-tyveloside was obtained in excellent stereoselectivity
(J28, 92%), no paratose derivative was detected by NMR,
IC50 ( M)a
9D4
Oligosaccharide
inhibitor
inhibitory powerb
18H1
9D4
18H1
15
58
178
Tetrasaccharide 1
Trisaccharide 2
Disaccharide 3
>500
~480
93
100
26
8
< 9
~19
100
aCalculated from 50% inhibition data with an estimated accuracy of
~ ±5%.
bInhibition potency of 1 or 3 set as 100.
the 6-position of GalNAc. Modifications were made to the
tyvelose residue to investigate the contributions of the
hydroxyl groups at the C-2> and C-4> positions. Compounds
11 and 12 are 2>-monodeoxy and 2>-O-monomethyl conge-
ners while compounds 13 and 14 are 4>-monodeoxy and 4>-
O-monomethyl analogues.
Initially, disaccharides 5 and 6 were prepared from an eas-
ily accessible tyvelose donor 15 (22) (Scheme 1), using in-
soluble silver zeolite as promoter. When reacting with
galactosyl acceptor 16 (35) or glucosamine acceptor 17, the
disaccharides 18 and 19 were obtained in only poor yields
(J18 (15%), J19 (26%)). These are typical and representa-
tive yields in the preparation of ꢀ-mannopyranosides using
insoluble silver salt and halides as donors. Both the
disaccharides 18 and 19 were deprotected by hydrogenation
to give targeted compounds 5 (89%) and 6 (97%).
In our synthesis of several targets, differentiation between
the hydroxyl group OH-4> and OH-2> of the tyvelose residue
was essential. Obviously the donor 15 was not satisfactory
since both positions are protected by benzyl groups. Even
though the synthetic scheme seems short, the fact that the
glycosylations proceeded in low yields also prevented us
from obtaining final products in reasonable amounts, espe-
© 2002 NRC Canada

1144
Can. J. Chem. Vol. 80, 2002
Scheme 1. (a) Silver zeolite – MS 4 Å – CH2Cl2; (b) H2–Pd(OH)2/C–MeOH.
Ph
O
OBn
O
O
Ph
O
BnO
O
O
O
HO
HO
OMe
OMe
OBn
NHAc
Cl
16
15
17
a
a
Ph
O
O
BnO
Ph
OBn
O
O
O
O
OBn
O
O
BnO
BnO
O
OMe
OMe
O
NHAc
18
19
b
b
5
6
Scheme 2. (a) MeONa–MeOH; (b) BzCl–Py–CH2Cl2; (c) PivCl–Py–CH2Cl2; (d) NIS – TfOH – MS 4 Å – CH2Cl2; (e) DMSO–Ac2O;
( f ) L-selectride–THF; (g) Na–NH3 (liq.); (h) Ac2O–MeOH; (i) H2–Pd(OH)2–MeOH; (j) (i) Bu2SnO–MeOH, (ii) MeI–CsF–DMF.
Ph
Ph
O
O
O
O
O
O
O
d
BnO
O
BnO
+
SPh
O
OMe
HO
OMe
N3
OR
OR
20 R=Ac
21 R=H
22 R=Bz
23 R=Piv
N3
25 R=Bz
26 R=H
24
a
b
a
c
e
Ph
Ph
O
O
O
O
OH
O
f
g
O
O
O
O
BnO
BnO
4
O
OMe
O
OMe
NHR
N3
28 R=H
29 R=Ac
27
h
i
j
3
10
and the 2-azido group was simultaneously reduced to the
amine. The amino disaccharide 4 (90%) was obtained from
28 following removal of benzyl and benzylidene groups by
Birch reduction. The fully protected 2-amino disaccharide
28 was acetylated using acetic anhydride in methanol (J29,
93%), and the protecting groups were successfully removed
by hydrogenation to afford the native disaccharide 3 (88%).
When compound 3 was subjected to a dibutyltin-oxide-assisted
© 2002 NRC Canada

Zhang et al.
1145
Scheme 3. (a) MeI–NaH–DMF; (b) H2–Pd(OH)2/C–MeOH; (c) Im2CS–toluene, 90°C; (d) n-Bu3SnH–AIBN–toluene, reflux.
N
Ph
Ph
N
O
O
O
O
O
S
OH
O
O
O
O
c
BnO
BnO
O
OMe
O
OMe
NHAc
NHAc
29
31
a
d
Ph
Ph
O
O
O
O
OMe
O
O
O
O
BnO
BnO
O
OMe
O
OMe
30
32
NHAc
NHAc
b
b
11
12
Scheme 4. (a) BzCl–Py–CH2Cl2; (b) NH4HCO2–Pd/C–MeOH, reflux; (c) Im2CS–toluene, 90°C; (d) n-Bu3SnH–AIBN–toluene, reflux;
(e) (i) MeONa–MeOH, (ii) H2–Pd(OH)2/C–MeOH.
Ph
Ph
O
O
O
OR
O
O
OBz
O
O
O
b
BnO
HO
O
OMe
O
OMe
NHAc
NHAc
29 R=H
33 R=Bz
34
a
c
Ph
Ph
O
O
O
O
S
OBz
OBz
O
N
N
d
O
O
O
O
O
OMe
O
OMe
NHAc
NHAc
35
36
e
13
methylation, the corresponding 6-O-methyl ether derivative
10 was obtained in moderate yield (44%).
give the 2>-OMe disaccharide 12 (93% yield). To prepare the
corresponding 2>-deoxgenated disaccharide 11, 29 was trans-
formed to 31 (98%) by reaction with 1,1>-thiocarbonyldiimi-
dazole, and 2>-deoxygenation was realized after subjecting
31 to reaction with tributyltin hydride (J32, 95%) (40). Af-
ter hydrogenation, the desired 2>-deoxy compound 11 was
obtained in excellent yield (95%).
With compound 29 in hand, the 2>-hydroxyl group was
methylated using sodium hydride (Scheme 3) and a con-
trolled amount of methyl iodide as reagent, the methylated
derivative 30 was obtained (37%). The poor yield reflected
incomplete methylation which results from the conditions
employed which are designed to avoid N-methylation. The
benzyl and benzylidene were removed by hydrogenation to
The 4>-deoxygenated disaccharide 13 was prepared from
disaccharide 29 (Scheme 4). Benzoylation of 29 gave 33
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Can. J. Chem. Vol. 80, 2002
Scheme 5. (a) NH4HCO2–Pd/C–MeOH, reflux; (b) Bz2O–Py–DMAP; (c) H2–Pd(OH)2/C–MeOH; (d) TsCl–Py; (e) LiBr–KI–DMF,
100°C; ( f ) n-Bu3SnH–AIBN–toluene, reflux; (g) NaOMe–MeOH.
Ph
O
OH
O
OR1
O
HO
O
O
OBz
O
c
O
R2O
BzO
O
OMe
OMe
NHAc
NHAc
29 R1=H, R2=Bn
37 R1=H, R2=H
38 R1=R2=Bz
39
a
b
d
HO
O
OBz
O
R
g
O
9
BzO
OMe
NHAc
40 R=OTs
41 R=Br
42 R=H
e
f
Scheme 6. (a) MeI–NaH–DMF; (b) 10% H2SO4 – THF, 65°C; (c) BzCl–Py–CH2Cl2; (d) Me3SiSPh – TMSOTf – MS 4 Å – CH2Cl2;
(e) NIS – TfOH – MS 4 Å – CH2Cl2; ( f ) MeONa–MeOH; (g) DMSO–Ac2O; (h) L-selectride–THF; (i) H2–Pd(OH)2/C–MeOH.
O
O
O
O
RO
b
d
MeO
MeO
OR
SPh
OR
OBz
O
47
45 R=H
46 R=Bz
c
e
+ 24
43 R=H
44 R=Me
a
Ph
Ph
O
O
O
O
g
O
O
O
O
O
MeO
MeO
O
OMe
O
OMe
N3
N3
OR
48 R=Bz
49 R=H
f
50
h
Ph
O
O
OH
O
O
i
MeO
14
O
OMe
NHAc
51
(87%), and the 4>-benzyl group was selectively removed by
transfer hydrogenation in the presence of the benzylidene
acetal using ammonium formate – Pd-C conditions (J34,
78%). The 4>-OH was deoxygenated via a similar sequence
to that described for the synthesis of compound 32, (J35
(92%), J36 (83%)), and the final disaccharide 13 was ob-
tained after removing the protecting groups by Zemplen
transesterification and hydrogenation (J13, 88%).
© 2002 NRC Canada

Zhang et al.
1147
Scheme 7. (a) (Bu3Sn)2O–BzCl–toluene; (b) MeOTf–DTBMP–CH2Cl2, reflux; (c) NaOMe–MeOH; (d) t-BuMe2SiCl–Py; (e) NIS –
TfOH – MS 4 Å – CH2Cl2; ( f ) DMSO–Ac2O; (g) (i) L-selectride–THF, (ii) Ac2O–MeOH; (h) (i) H2–Pd(OH)2/C–MeOH, (ii) TBAF–THF.
OR
O
OR
O
OBz
O
HO
MeO
HO
MeO
BzO
b
c
RO
a
OMe
OMe
OMe
N3
N3
55 R=H
56 R=SiMe2Bu-t
N3
52 R=H
53 R=Bz
54
d
+ 22
e
OSiMe2Bu-t
OSiMe2Bu-t
MeO
O
MeO
O
O
O
O
O
f
BnO
BnO
OMe
OMe
N3
O
N3
OR
59
57 R=Bz
58 R=H
g
c
MeO
O
OSiMe2Bu-t
OH
O
O
BnO
h
OMe
8
NHAc
60
Scheme 8. (a) BzCl–Py–CH2Cl2; (b) NaBH3CN – HCl – MS 4 Å –THF; (c) Im2CS–toluene, 90°C; (d) n-Bu3SnH–AIBN–toluene, re-
flux; (e) MeONa–MeOH; ( f ) NIS – TfOH – MS 4 Å – CH2Cl2; (g) DMSO–Ac2O; (h) L-selectride–THF; (i) Na–NH3.
OBn
OBn
Ph
O
O
RO
b
d
O
O
O
RO
BzO
OMe
RO
OMe
NHAc
OMe
NHAc
NHAc
62 R=H
63
R=(imidazolyl)thiocarbonyl
17 R=H
61 R=Bz
64 R=Bz
65 R=H
e
f
c
a
+ 22
OBn
O
OBn
O
O
O
O
g
BnO
BnO
O
OMe
O
OMe
NHAc
NHAc
OR
e
66 R=Bz
67 R=H
68
h
OBn
O
OH
O
BnO
i
O
OMe
7
NHAc
69
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Can. J. Chem. Vol. 80, 2002
Fig. 3. Representative inhibition curves obtained with the
The synthesis of 6-deoxyGalNAc disaccharide 9 also
monoclonal antibody 9D4 and the following disaccharides:
disaccharide 10 (6-O-methyl GalNAc congener) (᭹), disaccharide
4 (2-amino GalNAc congener) (᭿), native disaccharide 3 (᭢),
disaccharide 5 (2-hydroxy GalNAc congener) (ꢀ). Microtiter
plates were coated with [ꢀ-D-Tyv(1J3)ꢀ-D-GalNAc]8-BSA glyco-
conjugate and diluted monoclonal antibody solutions containing
increasing amounts of inhibitor were added to the plate. Bound
antibody was detected by a species-specific antibody–horseradish
peroxidase conjugate and percentage inhibitions are expressed
relative to wells that contained no inhibitor. Each point is the av-
erage of three values and the standard deviation on each point
was ±5% or better.
started from compound 29 (Scheme 5). The benzyl ether
was removed selectively in the presence of the benzylidene
acetal in a manner similar to the conversion of 33 to 34 to
give 37 (79%), together with 3 (11%) resulting from loss of
the benzylidene acetal. The diol 37 was benzoylated (J38,
89%), and the acetal was removed under standard hydroge-
nation conditions using palladium hydroxide on charcoal as
catalyst (J39, 89%). The 6-OH was reduced through a se-
quence of tosylation (J40), displacement by bromide to
give 41, and reduction using tributyltin hydride (J42). The
intermediates 40–42 were not isolated, and after
transesterification of the 2>,4>-dibenzoate 42, the targeted
compound 9 was obtained in moderate yield (49% from 39).
Initially it was planned to obtain the 4>-OMe disaccharide
14 by methylation of intermediate 34. However, when 34
was methylated using methyl triflate and 2,6-di-tert-butyl-4-
methylpyridine, no methylated product was isolated. It was
then decided to use a 4-O-methylated paratose donor 47 as
starting material (Scheme 6). The paratose thioglycoside 47
was obtained in several transformation steps. Thus, starting
from compound 43 (38), the 4-OH was methylated (J44,
92%), and the 1,2-propylidene acetal was hydrolyzed and
the diol 45 benzoylated (J46, 96%). The anomeric benzoate
was converted to the mixture of anomeric thioglycosides 47
(89%) using PhSSiMe3–TMSOTf as reagents. Disaccharide
48 was obtained in good yield (78%) after coupling 47 with
acceptor 24 using N-iodosuccinimide – triflic acid as pro-
moter. After removing the 2>-benzoate (J49, 94%), the 4>-
O-methylated disaccharide 10 was obtained by oxidation to
the keto compound 50 followed by reduction and
deprotection (J51 (76%), J14 (94%)).
To prepare the 4-O-monomethylated disaccharide 8, we
decided to use compound 56 as an acceptor (Scheme 7).
Compound 56 has a methyl group preinstalled at the 4-
position, and was prepared starting from 2-azido galacto-
pyranosyl derivative 52 (39). Thus, by heating a solution of
triol 52 with bis(tributyltin)oxide, the 3,6-positions were
benzoylated to give 53 in very good yield (86%) and selec-
tivity. The 4-OH was methylated (J54, 94%) using methyl
triflate as reagent in the presence of the bulky base, 2,6-di-
tert-butyl-4-methylpyridine. The benzoates were removed
(J55, 97%) and the primary hydroxyl group was selectively
silylated using tert-butyldimethylsily chloride as reagent to
give the acceptor 56 (84%). Glycosylation of 56 with donor
22 afforded the disaccharide 57 in 71% yield. After remov-
ing the 2>-O-benzoate (J58, 98%), the 2>-equatorial
hydroxyl group was inverted to the corresponding 2>-axial
configuration by an oxidation–reduction sequence as de-
scribe above (J60, 76%), and the disaccharide 8 (88%) was
obtained following deprotection.
The synthesis of 4-mono-deoxygenated disaccharide 7
(Scheme 8) started with glucosamine alcohol 17. The 3-
hydroxyl group was protected as a benzoate (J61, 96%),
and the 4,6-O-benzylidene acetal was regioselectively opened
to afford the alcohol 62 in excellent yield (95%). The 4-OH
position was deoxygenated through a two-step Barton–
McCombie deoxygenation reaction (40) as describe above
(J63 (94%), J64 (88%)). After removing the 3-O-benzo-
ate (J65, 94%), the acceptor 65 was glycosylated by donor
22 to produce the disaccharide 66 in moderate yield (54%).
Compound 66 was debenzoylated to give the alcohol 67
(91%). Oxidation gave the keto derivative 68, which was re-
duced to give 69. The protecting groups were finally re-
moved by Birch reduction to afford the pure 4-deoxy-
disaccharide 7 (overall 23% yield from 67).
We have described the synthesis of a comprehensive set of
disaccharide congeners 4–14 (Fig. 2) related to the T. spiralis
epitope ꢀ-D-Tyvp(1J3)ꢀ-D-GalNAcp (3). The congeners are
modified at each position bearing a hydroxyl or acetamido
group. The ability of these disaccharides to inhibit binding
of the monoclonal antibodies 9D4 and 18H1 to a ꢀ-D-
Tyvp(1J3)ꢀ-D-GalNAcp BSA glyconconjugate (16) was as-
sayed in a solid-phase ELISA format. Briefly the dis-
accharide conjugate was coated on ELISA plates and incubated
with antibody in the presence of increasing amounts of in-
hibitor. Bound antibody was detected by a goat anti-rat IgG
antibody and percentage inhibition was calculated relative to
wells that contained no inhibitor. The structure activity data
was used to infer a topological map of the epitope surface
for each antibody binding site. The quality of the inhibition
data obtained in this work is illustrated in Fig. 3 and the in-
hibitory activities for compounds 1–14 with both mono-
clonal antibodies 9D4 and 18H1 are recorded in Tables 1–3.
In designing the set of congeners we followed the ap-
proach pioneered by Lemieux and co-workers (34) for
detecting intermolecular hydrogen bonds present in a protein–
oligosaccharide complex. The inhibitory power of mono-
deoxy and mono-O-methyl congeners identifies hydroxyl
© 2002 NRC Canada

Zhang et al.
1149
Table 2. Inhibitory activities of disaccharides 3–14 with the IgG
Table 3. Inhibitory activities of disaccharides 3–14 with the IgG
MAb 9D4.
MAb 18H1.
Compound
IC50 ( M)a
Relative inhibitory powerb
Compound
IC50 ( M)a
Relative inhibitory powerb
3
4
5
6
7
100
588
90
0
89
0
75
1780
94
890
0
3
4
5
6
7
93
–ve
–ve
60
178
30
100
0
0
220
–ve
199
–ve
239
10
190
20
–ve
–ve
150
137
75
80
200
25
500
0
68
8
9
8
9
127
112
45
370
18
10
11
12
13
14
10
11
12
13
14
–ve
–ve
0
0
aCalculated from 50% inhibition data with an estimated accuracy of ~ ±5%.
aCalculated from 50% inhibition data with an estimated accuracy of ~ ±5%.
bInhibition potency of 3 set as 100.
bInhibition potency of 3 set as 100.
groups that are buried and involved in key polar interactions
with the protein, as well as those hydroxyl groups exposed
to solvent water. Hydroxyl groups that are positioned around
the periphery of the complex may also be identified from the
relative inhibitory power of monodeoxy and mono-O-methyl
congeners thus providing a detailed topology of the interact-
ing surface. Inactive monodeoxy and mono-O-methyl conge-
ners pinpoint hydroxyl groups that are buried and involved
in key polar interactions. When these modifications are made
to solvent exposed hydroxyl groups only minor changes are
registered in their binding activity. However, when mono-O-
methylation or monodeoxygenation replaces hydroxyl groups
that lie at the periphery of the protein–oligosaccharide com-
plex large swings in activity for either one or both of the de-
rivatives can be observed. Frequently the changes may result
in enhanced binding energy. In general, binding at the pe-
riphery is indicated when the deoxy congener is active but
the mono-O-methyl compound is inactive (41). However, if
a hydroxyl at the periphery is an acceptor of a hydrogen
bond the O-methyl derivative may be even more active than
the deoxy congener (42, 43).
At first appearance the recognition of the dideoxyhexose
residue by both antibodies is similar. The C-4 hydroxyl
group is buried in the binding site and makes essential polar
contacts. The hydroxyl group at C-2 appears to make much
weaker polar contacts perhaps via a hydrogen bond either
from the protein or via a water molecule. Mono-O-methyl
disaccharide 12 is five to eight times more active than 3 with
each antibody, whereas, with the antibody 9D4, the deoxy
compound is almost as active as the native disaccharide 3,
while with 18H1 the same compound is fourfold less active.
In both cases this suggests that this hydroxyl group resides
close to the periphery of the binding site. When interacting
with 18H1 12 may either accept a hydrogen bond or the
methyl group can make favourable contacts with the protein,
and (or) displace a water molecule.
active than 3, and the 4-deoxy and mono-O-methyl
disaccharides 7 and 8 have activities close to that of 3. The
C-6 hydroxyl must reside close to the periphery of the bind-
ing site since the methyl ether 10 is twice as active as 3.
The recognition of GalNAc by MAb 9D4 is quite distinct.
The acetamido group is not important to binding, since Gal
may substitute for GalNAc without impact, and in fact the
free amino disaccharide 5 is almost six times more active
than 3. Steric changes at C-4 are prohibitive since the C-4
epimer 6 is inactive, as is the mono-O-methyl disaccharide
8. However, the 4-deoxy disaccharide 7 is almost unchanged
compared to 3. The 6-deoxy disaccharide 9 is only slightly
less active than 3, while the methyl ether 10 is 18 times
more active than 3. This suggests that both C-4 and C-6
hydroxyl group reside at the edge of the binding site and the
C-6 O-methyl group is able to make a productive interaction
at the periphery of the binding site via van der Waal contacts
or by replacing a water molecule.
These data (Table 3) may be integrated with the initial in-
hibition data for tetra, tri, and disaccharides 1–3 (Table 1)
and lead to the conclusion that the poorly protective MAb
18H1 only interacts with the terminal disaccharide of the T.
spiralis N-glycan chains. There are no antibody contacts with
the proximal branching fucose residue, since the underside
of the Tyv and GalNAc residues contact the binding site
leaving Tyv O-2 at the periphery and the major polar con-
tacts being O-4 (Tyv) and the 2-acetamido group of GalNAc.
This is consistent with the noninvolvement of GalNAc 4-
OH. The key polar groups and those at the periphery of the
binding site are shown in the stereo plots as CPK plots and
as a Connolly surface (44) (Fig. 4).
While the recognition of Tyv by MAb 9D4 initially ap-
peared to be similar to that by 18H1, the most deeply buried
surface of the Tyv likely extends around the opposite face in-
volving the C-6 methyl group and ring oxygen atoms. For
9D4 the involvement of its binding site with the GalNAc res-
idue is almost the exact opposite of the situation with 18H1,
and must involve the top face of the GalNAc residue.
Whereas, the acetamido group can be replaced by OH with-
out significant loss of binding, both O-4 and O-6 are close to
the periphery of the site, and since the fucose residue is
adjacent to these groups and protrudes from this upper face,
It is immediately obvious that the two monoclonal anti-
bodies recognize the GalNAc residue via completely differ-
ent surfaces. The 2-acetamido group is of major importance
for MAb 18H1, since disaccharides modified at C-2 (4 and
5) are inactive. The C-4 hydroxyl group is clearly not in-
volved in protein contacts since the C-4 epimer 6 is more
© 2002 NRC Canada

1150
Can. J. Chem. Vol. 80, 2002
Fig. 4. Stereo drawings of the epitopes recognized by antibodies 9D4 and 18H1. Functional groups shown in red make crucial hydro-
gen bonds with the binding sites. Groups shown in pink are involved in contacts at the periphery of the binding site. Groups shaded
blue are also located at the periphery. (a) Space-filling model showing the epitope residues Tyv O-4 and O-2 and GalNAc O-4 that
create the contact surface for the 9D4 epitope. (b) Connolly surface of the 9D4 epitope. (c) Space-filling model showing the epitope
residues Tyv O-4 and O-2 and GalNAc acetamido groups that create the contact surface for the 18H1 epitope. (d) Connolly surface of
the 18H1 epitope.
its involvement in binding together with a minor contribution
from the GlcNAc residue are readily appreciated (Fig. 4, Ta-
bles 1 and 2).
tional polar contacts. At this stage we would infer that this is
most likely to be O-2 or O-3 of fucose.
The superior protective ability of MAb 9D4 correlates
with the higher affinity for its larger tetrasaccharide target
epitope, which is about 20 times the affinity of the poorly
protective MAb 18H1 for its smaller disaccharide target
epitope (Table 1) (21, 24). Although the key recognition ele-
ment in both cases is the terminal disaccharide, the surface
of the oligosaccharide epitope that is buried determines the
degree to which the buried surface area can be extended. For
MAb 9D4 the inclusion of the fucose residue can be con-
trasted with the restriction of MAb 18H1 to contacts that do
not extend beyond the terminal disaccharide and involve po-
lar contacts with O-4 and O-2 (Tyv) and NHAc (GalNAc)
and possible non-polar interactions with this group. It
appears that the additional specificity element and larger sur-
face area of interaction lead to tighter binding and that this
is essential for full bioactivity.
Investigation of a number of antibody binding sites by
functional group replacement studies has shown that a small
number of hydroxyl groups, and where appropriate,
acetamido groups provide from two to four key polar con-
tacts. Without these complex formation does not occur. The
binding of T. spiralis N-glycans to antibody 18H1 conforms
to this pattern, where O-4 of tyvelose and the acetamido
group of GalNAc are involved in crucial polar interactions
and to a lesser extent O-2 of tyvelose. Monoclonal 9D4 is
quite different and its recognition of the disaccharide in-
volves only one key polar contact, which is again O-4 of
tyvelose. The tyvelose residue is recognized from the oppo-
site face, which would require its 6-deoxy group to be buried
and involved in hydrophobic contacts. Involvement of
GalNAc O-4 and O-6 at the periphery is consistent with this,
as is the ability to modify the acetamido function without
penalty. The complete recognition surface of the carbohy-
drate epitope for 9D4 must extend to include the GlcNAc
and Fuc residues since both trisaccharide 2 and tetra-
saccharide 1 are more active inhibitors than the disaccharide
3 (Table 1). One or both of these residues may provide addi-
Experimental
General methods
Optical rotations were measured with a PerkinElmer 241
polarimeter for samples in a 10-cm cell at 22 ± 2°C and [ꢁ]D
© 2002 NRC Canada

Zhang et al.
1151
values are given in units of 10–1 deg cm2 g–1. Analytical
TLC was performed on plates of Silica Gel 60-F254 (Merck,
Darmstadt) with detection by quenching of fluorescence and
(or) by charring with 5% sulfuric acid in water. All commer-
cial reagents were used as supplied and chromatography sol-
vents were distilled prior to use. Column chromatography
was performed on Silica Gel 60 (Silicycle, Quebec, Canada).
1H NMR spectra were recorded on Varian Unity 300, 500,
or 600 MHz. The first-order proton chemical shifts ꢂH are
referenced to either residual CHCl3 (ꢂH 7.24, CDCl3) or re-
sidual CD2HOD (ꢂH 3.30, CD3OD), or internal acetone (ꢂH
2.225, D2O). First order coupling constants are given in Hz.
HMQC-NMR spectra were recorded on Varian spectrome-
ters operating at 300, 500, or 600 MHz. The 13C chemical
shifts (ꢂC) are referenced to internal CDCl3 (ꢂC 77.00,
CDCl3). Organic solutions were dried prior to concentration
under vacuum at <40°C (bath). Final compounds were puri-
fied by reverse-phase chromatography performed on a
Waters 600 HPLC system, using a Beckman semipreparative
C-18 column (10 × 250 mm, 5 ), and the products were
detected with a Waters 2487 UV detector or a Waters 2410
refractive index monitor. Microanalyses and electrospray
mass spectra were performed by the analytical services of
this department. Reaction yields were not optimized.
anhyd CH2Cl2 (3 mL) was stirred for 30 min at room
temperature, and cooled to ꢃ40°C. A solution of tyvelosyl
chloride 15 (125 mg, 0.36 mmol, prepared by reacting the
corresponding hemi-acetal with oxalyl chloride (22)) in
anhyd CH2Cl2 (2.5 mL) was added dropwise and the mixture
was slowly warmed to room temperature and stirred for
24 h. The mixture was filtered off, and the filtrate was con-
centrated. The disaccharide 18 was obtained by chromatog-
raphy on silica gel using 15% EtOAc – toluene as eluent
(29 mg, 15%), [ꢁ]D + 27.3 (c 2.1, CHCl3). 1H NMR (CDCl3)
ꢂ: 7.51–7.58 (m, 2H, Ar), 7.10–7.36 (m, 18H, Ar), 5.56 (s,
1H, PhCH), 4.83 (d, 1H, J = 11.2 Hz, Bn), 4.77 (d, 1H, J =
13.2 Hz, Bn), 4.75 (“s”, 1H, H-1>), 4.68 (d, 1H, J = 13.2 Hz,
Bn), 4.55 (d, 1H, J = 11.5 Hz, Bn), 4.27–4.37 (m, 4H, H-
1, H-6a, H-4, Bn), 4.06 (dd, 1H, J = 1.6, 12.3 Hz, H-6b),
3.76–3.84 (m, 2H, H-2, H-3), 3.57 (s, 3H, OMe), 3.28–3.52
(m, 4H, H-5, H2>, H-4>, H-5>), 2.23 (d>t>, 1H, J = 4.0,
13.4 Hz, H-3e>), 1.31 (d, 3H, J = 6.1 Hz, H-6>), 1.18 (m,
1H, H-3a>). Anal. calcd. for C41H46O9: C 72.12, H 6.79;
found: C 72.19, H 6.49.
Methyl 3-O-(3,6-dideoxy-ꢀ-D-arabino-hexopyranosyl)-ꢀ-
D-galactopyranoside (5)
A mixture of disaccharide 18 (26 mg, 0.038 mmol) and
10% Pd(OH)2 on charcoal (20 mg) in MeOH (10 mL) was
hydrogenated overnight. The catalyst was filtered and the fil-
trate was concentrated, and the residue was purified by
reverse phase HPLC using H2O–MeOH (linear gradient 0 J
20%) as eluent to afford 5 (11 mg, 89%), [ꢁ]D –21.2 (c 0.6,
Enzyme immunoassay
Inhibition assays were performed in triplicate in 96 well
microtiter plates with the previously described rat mono-
clonal antibodies 18H1 (IgG1) and 9D4 (IgG2c) (3). Microtiter
plates were incubated for 18 h at 4°C with a solution of [ꢀ-
D-Tyv(1J3)ꢀ-D-GalNAc]8-BSA glycoconjugate (100 L/well)
dissolved in 0.01 M phosphate-buffered saline solution at a
concentration of 1 g mL–1 (16). The plate was washed with
PBST (5×) and blocked for 1 h at room temperature by incu-
bation with 2% BSA in PBS (100 L). After the plate had
been washed with PBST (3×), antibody solution with or
without inhibitor was added (100 L) to the plate incubated
for 18 h at room temperature. Monoclonal antibody 18H1
was used from ascites fluid at a final dilution of 5 × 10–5 and
9D4 at a dilution of 1.5 × 10–3. The plate was washed with
PBST (5×), and goat anti-rat IgG antibody conjugated to
horse radish peroxidase (Kirkegaard and Perry Lab, Mandel,
Guelph, ON) solution (100 L) diluted (1:5000) in PBS was
incubated for 1 h at room temperature. The plate was washed
with PBST (5×), 3,3>,5,5>-tetramethylbenzidine (TMB, 100 L)
was added, and after 2 min the colour reaction was stopped
by the addition of 1 M phosphoric acid (100 L). Absorb-
ance was read at 450 nm and percent inhibition was calcu-
lated using wells containing no inhibitor as the reference
point. Triplicate samples typically exhibited OD values with
a standard deviation of ±5% or better. Wells with OD values
outside this error range were discarded and eliminated from
the data analysis. Inhibition curves were drawn using the
software package Origin 5.0 (OriginLab, Northampton,
MA), and the IC50 value was computed by the software.
1
MeOH). H NMR (CD3OD) ꢂ: 4.76 (d, 1H, J = 1.1 Hz,
H-1>), 4.16 (m, 1H, H-1, high order), 4.03 (m, 1H, H-4),
3.96 (m, 1H, H-2>), 3.69–3.79 (m 2H, H-6a, H-6b), 3.59–
3.61 (m, 2H, H-2, H-3), 3.45–3.57 (m, 6H, H-5, H-4>, OMe,
H-4), 3.30 (dq, 1H, J = 6.1, 9.3 Hz, H-5>), 2.14 (ddd, 1H,
J = 3.3, 4.6, 13.6 Hz, H-3e>), 1.55 (ddd, 1H, J = 2.9, 11.4,
13.6 Hz, H-3a>), 1.27 (d, 3H, J = 6.1 Hz, H-6>). HR-ES-MS
m/e calcd. for C13H24O9 (MNa+): 347.13180; found: 347.131922.
Methyl 2-acetamido-3-O-(2,4-di-O-benzyl-3,6-dideoxy-ꢀ-
D-arabino-hexopyranosyl)-4,6-O-benzylidene-2-deoxy-ꢀ-
D-glucopyranoside (19)
A mixture of acceptor 17 (45) (88 mg, 0.27 mmol), silver
zeolite (300 mg), and 4 Å molecular sieves (400 mg) in
anhyd CH2Cl2 (3 mL) was stirred for 30 min at room tem-
perature, and cooled to ꢃ40°C. A solution of tyvelosyl chlo-
ride 15 (173 mg, 0.50 mmol, prepared by reacting the
corresponding hemi-acetal with oxalyl chloride (22)) in
anhyd CH2Cl2 (2.5 mL) was added dropwise and the mixture
was slowly warmed to room temperature and stirred for 24 h.
The mixture was filtered off, and the filtrate was concen-
trated. The disaccharide 19 was obtained by chromatography
on silica gel using 35% EtOAc – toluene as eluent (44 mg,
1
26%), [ꢁ]D –0.8 (c 1.7, CHCl3). H NMR (CDCl3) ꢂ: 7.47–
7.52 (m, 2H, Ar), 7.23–7.37 (m, 13H, Ar), 5.52 (s, 1H,
PhCH), 5.45 (d, 1H, J = 7.1Hz, NH), 4.86 (d, 1H, J = 8.2 Hz,
H-1), 4.70 (d, 1H, J = 1.2 Hz, H-1>), 4.64 (d, 1H, J =
12.5 Hz, Bn), 4.58 (d, 1H, J = 12.5 Hz, Bn), 4.38–4.50 (m,
3H, 2 × Bn, H-3), 4.32 (dd, 1H, J = 4.9, 10.4 Hz, H-6a),
3.76 (t, 1H, J = 10.2 Hz, H-4), 3.65 (t, 1H, J = 9.2 Hz,
H-6b), 3.59 (m, 1H, H-2>), 3.47 (s, 3H, OMe), 3.36–3.53
(m, 3H, H-5, H-4>, H-5>), 3.13 (m, 1H, H-2), 2.27 (ddd, 1H,
Methyl 2-O-benzyl-4,6-O-benzylidene-3-O-(2,4-di-O-
benzyl-3,6-dideoxy-ꢀ-D-arabino-hexopyranosyl)-ꢀ-D-
galactopyranoside (18)
A mixture of acceptor 16 (35) (105 mg, 0.28 mmol), sil-
ver zeolite (300 mg), and 4 Å molecular sieves (300 mg) in
© 2002 NRC Canada

1152
Can. J. Chem. Vol. 80, 2002
J = 4.5, 4.5, 13.5 Hz, H-3e>), 1.74 (s, 3H, Ac), 1.43 (br m,
1H, H-3a>), 1.20 (d, 3H, J = 5.7 Hz, H-6>). Anal. calcd. for
C36H43O9N: C 68.23, H 6.84, N 2.21; found: C 68.01, H 6.81, N 2.19.
10.9 Hz, H-4), 2.84 (d>t>, 1H, J = 4.6, 11.9 Hz, H-3e), 1.66
(d>t>, 1H, J = 11.1, 11.1 Hz, H-3a), 1.39 (d, 1H, J = 6.1 Hz,
H-6); ꢁ-anomer ꢂ: 8.08 (m, 1H, Bz), 7.57 (m, 1H, Bz), 7.42–
7.49 (m, 4H, Ar), 7.20–7.37 (m, 8H, Ar), 5.76 (d, 1H, J =
5.0 Hz, H-1), 5.29 (d>t>, 1H, J = 5.1, 10.1 Hz, H-2), 4.68 (d, 1H,
J = 11.6 Hz, Bn), 4.51 (d, 1H, J = 11.6 Hz, Bn), 4.28 (dq, 1H,
J = 6.1, 9.2 Hz, H-5), 3.30 (ddd, 1H, J = 4.6, 7.1, 11.0 Hz, H-4),
2.54 (dd>t>, 1H, J = 1.2, 4.6, 11.8 Hz, H-3e), 2.00 (d>t>, 1H, 12.0,
12.0 Hz, H-3a), 1.29 (d, 3H, J = 6.2 Hz, H-6). Anal. calcd. for
C26H26O4S: C 71.86, H 6.03; found: C 71.95, H 6.16.
Methyl 2-acetamido-3-O-(3,6-dideoxy-ꢀ-D-arabino-
hexopyranosyl)-2-deoxy-ꢀ-D-glucopyranoside (6)
Disaccharide 19 (40 mg, 0.063 mmol) was deprotected in
a similar fashion as compound 5 and compound 6 (22.4 mg,
97%) was purified by reverse-phase HPLC using H2O–
MeOH (linear gradient 0 J 20%) as eluent. [ꢁ]D –29.7 (c
1
0.7, MeOH). H NMR (D2O) ꢂ: 4.63 (d, 1H, J = 0.9 Hz,
Phenyl 4-O-benzyl-3,6-dideoxy-2-O-pivaloyl-1-thio-ꢁ,ꢀ-D-
ribo-hexopyranoside (23)
H-1>), 4.50 (d, 1H, J = 8.6 Hz, H-1), 3.94 (dd, 1H, J = 2.3,
13.0 Hz, H-6a), 3.90 (m, 1H, H-2>), 3.80 (dd, 1H, J = 6.8,
10.4 Hz, H-2), 3.77 (dd, 1H, J = 5.5, 13.0 Hz, H-6b), 3.70
(dd, 1H, J = 9.0, 10.8 Hz, H-3), 3.57 (m, 1H, H-4>), 3.53
(“t”, 1H, 10.8 Hz, H-4), 3.52 (s, 3H, OMe), 3.46–3.52 (m,
2H, H-5, H-5>), 2.19 (ddd, 1H, J = 3.7, 4.6, 13.9 Hz, H-3e>),
2.04 (s, 3H, Ac), 1.68 (ddd, 1H, J = 3.1, 11.5, 14.3 Hz,
H-3a>), 1.29 (d, 3H, J = 6.2 Hz, H-6>). HR-ES-MS m/e
calcd. for C15H27O9N (MNa+): 388.15835; found: 388.158837.
The alcohol 21 (80 mg, 0.24 mmol) was reacted with
pivaloyl chloride (89.4 L, 0.73 mmol) in a similar fashion
to afford the pivaloyl donor 23 (ꢁ/ꢀ: 37:73, 92 mg, 92%).
1H NMR (CDCl3) for ꢀ-anomer ꢂ: 7.21–7.48 (m, 10H, Ar),
4.64–4.73 (m, 2H, H-1, H-2), 4.61 (d, 1H, J = 11.4 Hz, Bn),
4.43 (d, 1H, J = 11.4 Hz, Bn), 3.44 (dq, 1H, J = 6.1, 9.0 Hz,
H-5), 3.20 (ddd, 1H, J = 4.5, 9.1, 10.9 Hz, H-4), 2.65 (d>t>,
1H, J = 4.5, 12.1 Hz, H-3e), 1.50 (m, 1H, H-3a), 1.34 (d,
1H, J = 6.1 Hz, H-6), 1.23 (s, 9H, Piv); ꢁ-anomer ꢂ: 7.21–
7.48 (m, 10H, Ar), 5.65 (d, 1H, J = 5.2 Hz, H-1), 4.96 (d>t>,
1H, J = 5.1, 10.1 Hz, H-2), 4.67 (d, 1H, J = 11.5 Hz, Bn),
4.48 (d, 1H, J = 11.5 Hz, Bn), 4.22 (dq, 1H, J = 6.1, 9.2 Hz,
H-5), 3.23 (m, 1H, m, H-4), 2.36 (dd>t>, 1H, J = 1.2, 4.6,
12.0 Hz, H-3e), 1.84 (d>t>, 1H, J = 12.1, 12.1 Hz, H-3a),
1.26 (d, 3H, J = 6.2 Hz, H-6), 1.25 (s, 9H, Piv). Anal. calcd.
for C24H30O4S: C 69.53, H 7.29; found: C 69.33, H 7.52.
Phenyl 4-O-benzyl-3,6-dideoxy-1-thio-ꢁ,ꢀ-D-ribo-hexopy-
ranoside (21)
Thioglycoside 20 (38) (ꢁ/ꢀ: 37:73, 3.50 g, 9.40 mmol)
was transesterified in anhyd MeOH with a catalytic amount
of NaOMe. After 3 h, the mixture was neutralized with
Amberlite IR-120 (H+), and evaporated to dryness to give al-
cohol 21 (ꢁ/ꢀ: 37:73, 3.10 g, quantitative). 1H NMR
(CDCl3) for ꢀ-anomer ꢂ: 7.25–7.53 (m, 10H, Ar), 4.61 (d,
1H, J = 11.7 Hz, Bn), 4.46 (d, 1H, J = 11.7 Hz, Bn), 4.45 (d,
1H, J = 9.5 Hz, H-1), 3.40–3.49 (m, 2H, H-2, H-5), 3.15
(ddd, 1H, J = 4.4, 8.8,10.8 Hz, H-4), 2.63 (d>t>, 1H, J = 4.6,
12.3 Hz, H-3e), 1.49 (d>t>, 1H, J = 11.4, 11.4 Hz, H-3a),
1.34 (d, 1H, J = 6.3 Hz, H-6); ꢁ-anomer ꢂ: 7.25–7.51 (m,
10H, Ar), 5.40 (d, 1H, J = 4.6 Hz, H-1), 4.64 (d, 1H, J =
11.5 Hz, Bn), 4.50 (d, 1H, J = 11.5 Hz, Bn), 4.12 (dq, 1H,
J = 6.1, 9.2 Hz, H-5), 3.93 (d>t>, 1H, J = 4.6, 9.0, 11.5 Hz,
H-2), 3.16 (m, 1H, H-4), 2.40 (dd>t>, 1H, J = 0.7, 4.2,
12.3 Hz, H-3e), 1.57 (d>t>, 1H, 11.7, 11.7 Hz, H-3a), 1.30 (d,
3H, J = 6.1 Hz, H-6). Anal. calcd. for C19H22O3S: C 69.06,
H 6.71; found: C 68.73, H 6.77.
Methyl 2-azido-3-O-(2-O-benzoyl-4-O-benzyl-3,6-
dideoxy-ꢀ-D-ribo-hexopyranosyl)-4,6-O-benzylidene-2-
deoxy-ꢀ-D-galactopyranoside (25)
A mixture of donor 22 (ꢁ/ꢀ: 37:73, 807 mg, 1.86 mmol),
acceptor 24 (39) (519 mg, 1.70 mmol), and 4 Å molecular
sieves (1.5 g) in anhyd CH2Cl2 (16 mL) was cooled to –60°C,
and NIS (796 mg, 3.70 mmol) was added. After stirring for
1 h, TfOH (31 L) was added dropwise, the reaction was con-
tinued for 3 h and warmed to –30°C. Et3N (1.0 mL) was added
to quench the reaction. The insoluble material was filtered off
and washed with CH2Cl2. The organic solution was washed
with a 1:1 mixture of aq Na2S2O3 (10%) and NaHCO3 (sat.)
(1 × 50 mL), dried and concentrated. Disaccharide 25 was
obtained by chromatography on silica gel using 5% EtOAc –
toluene as eluent (891 mg, 83% yield), [ꢁ]D –14.9 (c 0.8,
Phenyl 2-O-benzoyl-4-O-benzyl-3,6-dideoxy-1-thio-ꢁ,ꢀ-D-
ribo-hexopyranoside (22)
Benzoyl chloride (2.4 mL, 20.5 mmol) was added
dropwise to an ice-cold solution of alcohol 21 (2.80 g,
8.47 mmol) in a mixture of anhyd CH2Cl2 (60 mL) and an-
hydrous pyridine (30 mL), and the mixture was stirred for
3 h. Methanol (2 mL) was added to quench the reaction, and
the mixture was concentrated to dryness. The residue was
dissolved in EtOAc (150 mL), and the organic solution was
washed with 2 M HCl (1 × 50 mL), sat. NaHCO3 (1 ×
50 mL), and saturated brine (1 × 50mL), dried and evapo-
rated. Chromatography on silica gel using 5% EtOAc – hex-
ane as eluent gave the donor 22 as an inseparable mixture of
1
CHCl3). H NMR (CDCl3) ꢂ: 8.08 (m, 2H, Bz), 7.56 (m, 1H,
Bz), 7.20–7.51 (m, 12H, Ar), 5.52 (s, 1H, PhCH), 4.87–4.95
(m, 2H, H-1>, H-2>), 4.63 (d, 1H, J = 11.4 Hz, Bn), 4.45 (d,
1H, J = 11.4 Hz, Bn), 4.31 (dd, 1H, J = 1.5, 12.4 Hz, H-6a),
4.21–4.25 (m, 2H, H-1, H-4), 3.78 (dd, 1H, J = 8.0, 10.6 Hz,
H-2), 3.50–3.59 (m, 5H, OMe, H-3, H-5>), 3.40 (m, 1H, H-5),
3.30 (ddd, 1H, J = 4.5, 9.1, 10.9 Hz, H-4>), 2.85 (d>t>, 1H, J =
4.5, 12.1 Hz, H-3e>), 1.59 (d>t>, 1H, J = 11.1, 11.1 Hz, H-3a>),
1.34 (d, 3H, J = 6.1 Hz, H-6>). Anal. calcd. for C34H37O9N3:
C 64.65, H 5.90, N 6.65; found: C 64.47, H 5.61, N 6.29.
1
anomers (ꢁ/ꢀ: 37:73, 3.39 g, 92% yield). H NMR (CDCl3)
Methyl 2-azido-3-O-(4-O-benzyl-3,6-dideoxy-ꢀ-D-ribo-
hexopyranosyl)-4,6-O-benzylidene-2-deoxy-ꢀ-D-
galactopyranoside (26)
Disaccharide 25 (550 mg, 0.811 mmol) was dissolved in
anhyd MeOH (5 mL), and a solution of NaOMe in MeOH
for ꢀ-anomer ꢂ: 8.05 (m, 2 H, Bz), 7.57 (m, 1H, Bz), 7.42–
7.49 (m, 4H, Ar), 7.20–7.37 (m, 8H, Ar), 4.91 (ddd, 1H, J =
4.9, 9.9, 10.9 Hz, H-2), 4.82 (d, 1H, J = 9.9 Hz, H-1), 4.62
(d, 1H, J = 11.5 Hz, Bn), 4.46 (d, 1H, J = 11.5 Hz, Bn), 3.52
(dq, 1H, J = 6.1, 9.0 Hz, H-5), 3.27 (ddd, 1H, J = 4.5, 9.0,
© 2002 NRC Canada

Zhang et al.
1153
(100 L) was added. After 2 h, the solution was neutralized
with Amberlite IR-120 (H+) resin and filtered. The filtrate
was concentrated and the disaccharide 26 was obtained by
chromatography on silica gel using 20% EtOAc–toluene as
eluent (432 mg, 94% yield), [ꢁ]D +39.0 (c 0.5, CHCl3).
1H NMR (CDCl3) ꢂ: 7.53 (m, 2H, Ph), 7.26–7.38 (m, 8H,
Ar), 5.53 (s, 1H, PhCH), 4.62 (d, 1H, J = 11.6 Hz, Bn), 4.45
(d, 1H, J = 11.6 Hz, Bn), 4.44 (d, 1H, J = 7.3 Hz, H-1>), 4.32
(dd, 1H, J = 1.6, 12.4 Hz, H-6a), 4.25 (dd, 1H, J = 0.5,
3.6 Hz, H-4), 4.22 (d, 1H, J = 8.0 Hz, H-1), 4.05 (dd, 1H,
J = 1.7, 12.4 Hz, H-6b), 3.86 (dd, 1H, J = 8.0, 10.6 Hz, H-2),
3.58 (s, 2H, OMe), 3.42–2.56 (m, 3H, H-3, H-2>, H-5>), 3.40
(m, 1H, H-5), 3.30 (ddd, 1H, J = 4.4, 8.8, 10.9 Hz, H-4>), 2.50
(d>t>, 1H, J = 4.7, 12.3 Hz, H-3e>), 1.44 (d>t>, 1H, J = 11.7,
11.7 Hz, H-3a>), 1.29 (d, 3H, J = 6.2 Hz, H-6>). Anal. calcd.
for C27H33O8N3: C 61.64, H 6.30, N 7.96; found: C 61.88,
H 6.34, N 7.88.
was allowed to evaporate. After the evaporation of NH3, the
residue was purified by reverse-phase chromatography using
a gradient of H2O–MeOH (0 J 30%) as eluent to yield com-
pound 4 (20.5 mg, 90% yield), [ ] –7.1 (c 0.2, MeOH).
ꢁ
1H NMR (D2O) ꢂ: 4.84 (d, 1H, J =D0.7 Hz, H-1>), 4.60 (d,
1H, J = 8.6 Hz, H-1), 4.28 (“d”, 1H, J = 2.9 Hz, H-4), 4.10
(m, 1H, H-2>), 4.03 (dd, 1H, J = 2.9, 10.8 Hz, H-3), 3.84
(dd, 1H, J = 7.9, 11.7 Hz, H-6a), 3.78 (dd, 1H, J = 4.2,
11.7 Hz, H-6b), 3.76 (m, 1H, H-5), 3.61 (s, 3H, OMe), 3.58
(ddd, 1H, J = 4.8, 9.34, 11.5 Hz, H-4>), 3.48 (dq, 1H, J =
6.0, 9.3 Hz, H-5>), 3.32 (dd, 1H, J = 8.6, 10.6 Hz, H-2), 2.20
(ddd, 1H, J = 3.5, 4.6, 13.9 Hz, H-3e>), 1.69 (ddd, 1H, J =
2.9, 11.5, 14.1 Hz, H-3e>), 1.28 (d, 3H, J = 6.1 Hz, H-6>).
HR-ES-MS m/e calcd. for C13H25O8N (MNa+): 346.14779;
found: 346.148284.
Methyl 2-acetamido-3-O-(4-O-benzyl-3,6-dideoxy-ꢀ-D-
arabino-hexopyranosyl)-4,6-O-benzylidene-2-deoxy-ꢀ-D-
galactopyranoside (29)
Methyl 2-amino-3-O-(4-O-benzyl-3,6-dideoxy-ꢀ-D-
arabino-hexopyranosyl)-4,6-O-benzylidene-2-deoxy-ꢀ-D-
galactopyranoside (28)
The intermediate ketone 27 (106 mg, 0.2 mmol) was dis-
solved in anhyd THF (3 mL) at 0°C, a solution of L-
selectride in THF (1.0 M, 1.5 mL, 1.5 mmol) was added
dropwise, and the reaction was continued at 0°C for 1 h.
Water (0.3 mL) was added, after 10 min, Ac2O (2 mL) was
added dropwise, the reaction was continued for 30 min. Af-
ter concentration, the mixture was dissolved in CH2Cl2
(100 mL), the organic solution was washed with a 10%
aqueous solution of Na2S2O3 (1 × 50 mL) and saturated
brine (50 mL), dried, and evaporated. The mixture was puri-
fied by chromatography on silica gel using 2.5% MeOH–
CH2Cl2 as eluent to afford compound 29 (102 mg, 93%
yield), [ꢁ]D +56.5 (c 0.6, CHCl3). 1H NMR (CDCl3) ꢂ: 7.46–
7.52 (m, 2H, Ar), 7.22–7.36 (m, 8H, Ar), 5.95 (d, 1H, J =
7.0 Hz, NH), 5.52 (s, 1H, PhCH), 5.02 (d, 1H, J = 8.2 Hz,
H-1), 4.73 (dd, 1H, J = 3.4, 11.1 Hz, H-3), 4.57 (d, 1H, J ~
1 Hz, H-1>), 4.56 (d, 1H, J = 11.5 Hz, Bn), 4.42 (d, 1H, J =
11.5 Hz, Bn), 4.26–4.36 (m, 2H, H-6a, H-4), 4.04 (dd, 1H,
J = 1.6, 12.4 Hz, H-6b), 3.83 (br m, 1H, H-2>), 3.51 (br, 4H,
OMe, H-5), 3.38–3.50 (m, 3H, H-2, H-4>, H-5>), 2.33–2.39
(m, 2H, H-3e>, OH-2>), 1.85 (s, 3H, Ac), 1.38 (m, 1H, H-
3a>), 1.30 (d, 3H, J = 5.6 Hz, H-6>). HR-ES-MS m/e calcd.
for C29H37O9N (MNa+): 566.23660; found: 566.236763.
The alcohol 26 (289 mg, 0.548 mmol) was dissolved in a
mixture of anhyd DMSO (4 mL) and Ac2O (2 mL) at 0°C.
The reaction was left at ambient temperature overnight. Af-
ter removing the solvents under reduced pressure, the mix-
ture was filtered through a thin bed of silica gel using 40%
EtOAc–hexane as eluent to give the intermediate ketone 27
(235 mg, 82% yield) which was divided in two parts for the
preparation of both compound 28 and 29.
A portion of 27 (40 mg, 0.076 mmol) was dissolved in
anhyd THF (3 mL) at 0°C, a solution of L-selectride in THF
(1.0 M, 300 L, 0.3 mmol) was added dropwise and the re-
action was continued for 1 h. Water (1 mL) was added and
the reaction mixture was concentrated. The syrupy mixture
was dissolved in CH2Cl2 (50 mL), washed with a 10% aque-
ous solution of Na2S2O3 (20 mL) and saturated brine
(20 mL), dried and concentrated. The mixture was purified
by chromatography on silica gel using 5% MeOH–CH2Cl2
as eluent to afford the amine 28 (35 mg, 92% yield), [ꢁ]D
+40.2 (c 1.5, CHCl3). 1H NMR (CDCl3) ꢂ: 7.49 (m, 2H, Ar),
7.22–7.36 (m, 8H, Ar), 5.53 (s, 1H, PhCH), 4.76 (d, 1H, J =
1.0 Hz, H-1>), 4.59 (d, 1H, J = 11.5 Hz, Bn), 4.44 (d, 1H,
J = 11.5 Hz, Bn), 4.31 (dd, 1H, J = 1.4, 12.4 Hz, H-6a), 4.25
(“d”, 1H, J = 3.1 Hz, H-4), 4.12 (d, 1H, J = 7.8 Hz, H-1),
4.05 (dd, 1H, J = 1.7, 12.4 Hz, H-6b), 3.94 (m, 1H, H-2>),
3.65 (dd, 1H, J = 3.9, 10.5 Hz, H-3), 3.53 (s, 3H, OMe),
3.48 (m, 2H, H-4> +H-5>), 3.41 (m, 1H, H-5), 3.26 (dd, 1H,
J = 7.9, 10.4 Hz, H-2), 2.43 (d>t>, 1H, J = 3.9, 13.7 Hz, H-3e>),
1.52 (m, 1H, H-3a>), 1.32 (d, 3H, J = 5.7 Hz, H-6>). HR-ES-
MS m/e calcd. for C27H35O8N (MH+): 502.24409; found:
502.244418.
Methyl 2-acetamido-3-O-(3,6-dideoxy-ꢀ-D-arabino-
hexopyranosyl)-2-deoxy-ꢀ-D-galactopyranoside (3)
Compound 29 (25 mg, 0.046 mmol) was deprotected in a
similar fashion as compound 5 and disaccharide 3 (14.7 mg,
88% yield) was purified by reverse-phase HPLC using a gra-
dient of H2O–MeOH (0 J 30%) as eluent, [ꢁ]D –32.4 (c 0.5,
1
MeOH). H NMR (D2O) ꢂ: 4.68 (d, 1H, J = 0.7 Hz, H-1>),
4.44 (d, 1H, J = 8.6 Hz, H-1), 4.12 (“d”, 1H, J = 3.1 Hz,
H-4), 4.01 (dd, 1H, J = 8.6, 10.8 Hz, H-2), 3.89 (m, 1H, H-2>),
3.89 (dd, 1H, J = 3.1, 10.8 Hz, H-3), 3.82 (dd, 1H, J = 7.9,
11.7 Hz, H-6a), 3.77 (dd, 1H, J = 4.2, 11.7 Hz, H-6b), 3.70
(m, 1H, H-5), 3.55 (ddd, 1H, J = 4.8, 9.3, 11.4 Hz, H-4>),
3.52 (s, 3H, OMe), 3.43 (dq, 1H, J = 6.2, 9.5 Hz, H-5>), 2.17
(ddd, 1H, J = 3.5, 4.8, 14.1 Hz, H-3e>), 2.02 (s, 3H, Ac),
1.65 (ddd, 1H, J = 3.1, 11.5, 14.3 Hz, H-3a>), 1.27 (d, 3H,
J = 6.2 Hz, H-6>). HR-ES-MS m/e calcd. for C15H27O9N
(MNa+): 388.15835; found: 388.158854.
Methyl 2-amino-3-O-(3,6-dideoxy-ꢀ-D-arabino-
hexopyranosyl)-2-deoxy-ꢀ-D-galactopyranoside (4)
With the help of a dry ice – acetone condenser, liquid am-
monia (25 mL) was collected in a flask containing the amine
28 (35 mg, 0.07 mmol). A small piece of sodium (100 mg)
was added, and reaction was continued for 4 h. During this
time, the reaction mixture remained dark blue. The con-
denser was removed and MeOH (0.5 mL) was added. The
colour of the reaction mixture turned white and the ammonia
© 2002 NRC Canada

1154
Can. J. Chem. Vol. 80, 2002
(dd, 1H, J = 4.2, 11.7 Hz, H-6b), 3.70 (m, 1H, H-5), 3.56
(m, 1H, H-2>), 3.52 (s, 3H, OMe), 3.42–3.48 (m, 2H, H-4>,
H-5>), 3.41 (s, 3H, OMe), 2.37 (d>t>, 1H, J = 3.7, 14.1 Hz,
H-3e>), 2.03 (s, 3H, Ac), 1.54 (ddd, 1H, J = 2.8, 11.2,
14.1 Hz, H-3a>), 1.26 (d, 3H, J = 5.7 Hz, H-6>). HR-ES-MS
m/e calcd. for C16H29O9N (MNa+): 402.17400; found: 402.174693.
Methyl 2-acetamido-3-O-(3,6-dideoxy-ꢀ-D-arabino-hex-
opyranosyl)-2-deoxy-6-O-methyl-ꢀ-D-galactopyranoside (10)
A mixture of tetraol 3 (31.6 mg, 0.0865 mmol) and
dibutyltin oxide (26 mg, 0.104 mmol) in anhydrous metha-
nol (4 mL) was refluxed for 2 h. The mixture was concen-
trated and coevaporated with toluene (2 × 15 mL), and dried
under high vacuum. The mixture was dissolved in anhyd
DMF (2 mL). CsF (40 mg, 0.259 mmol) and MeI (16 L,
0.259 mmol) were added, and the reaction was heated to
50°C overnight. The solution was concentrated under re-
duced pressure and the desired 6-O-methyl disaccharide 10
was obtained by reverse-phase HPLC using a gradient of
H2O–MeOH (0 J 20%) as eluent (14.5 mg, 44% yield),
[ꢁ]D –34.8 (c 0.3, MeOH). Starting material 3 (18 mg) was
Methyl 2-acetamido-3-O-(4-O-benzyl-3,6-dideoxy-2-O-
imidazoylthiocarbonyl-ꢀ-D-arabino-hexopyranosyl)-4,6-
O-benzylidene-2-deoxy-ꢀ-D-galactopyranoside (31)
To a solution of disaccharide 29 (30 mg, 0.055 mmol) in
anhydrous toluene (2.5 mL), was added 1,1>-thiocarbonyldi-
imidazole (27 mg, 0.15 mmol), and the mixture was heated
to 90°C for 17 h. After cooling, the mixture was concen-
trated, and disaccharide 31 was obtained following chroma-
tography on silica gel using 2% MeOH–CH2Cl2 as eluent
1
also recovered. H NMR (D2O) ꢂ: 4.68 (“s”, 1H, H-1>), 4.45
(d, 1H, J = 8.6 Hz, H-1), 4.10 (“d”, 1H, J = 3.2 Hz, H-4),
4.01 (dd, 1H, J = 8.6, 10.8 Hz, H-2), 3.89 (m, 1H, H-2>),
3.87 (dd, 1H, J = 3.1, 10.8 Hz, H-3), 3.83 (m, 1H, H-5),
3.72 (dd, 1H, J = 7.7, 10.8 Hz, H-6a), 3.68 (dd, 1H, J = 4.0,
11.0 Hz, H-6b), 3.55 (ddd, 1H, J = 4.6, 9.2, 11.4 Hz, H-4>),
3.52 (s, 3H, OMe), 3.44 (dq, 1H, J = 6.0, 9.3 Hz, H-5>), 3.42
(s, 3H, OMe), 2.17 (ddd, 1H, J = 3.7, 4.6, 13.9 Hz, H-3e>),
2.03 (s, 3H, Ac), 1.65 (ddd, 1H, J = 3.1, 11.5, 14.3 Hz, H-3a>),
1.28 (d, 3H, J = 6.2 Hz, H-6>). HR-ES-MS m/e calcd. for
C16H29O9N (MNa+): 402.17400; found: 402.174490.
1
(35.3 mg, 98% yield), [ꢁ]D +21.5 (c 0.3, CHCl3). H NMR
(CDCl3) ꢂ: 8.31 (s, 1H, imidazole), 7.20–7.44 (m, 11H, Ar),
6.71 (s, 1H, imidazole), 5.88 (d, 1H, J = 6.5 Hz, NH), 5.66
(m, 1H, H-2>), 5.51 (s, 1H, PhCH), 4.95 (d, 1H, J =
8.2 Hz, H-1), 4.87 (“s”, 1H, H-1>), 4.75 (dd, 1H, J = 3.4,
11.1 Hz, H-3), 4.54 (d, 1H, J = 11.5 Hz, Bn), 4.42 (d, 1H,
J = 11.5 Hz, Bn), 4.33 (“d”, 1H, J = 3.3 Hz, H-4), 4.30 (dd,
1H, J = 1.2, 12.5 Hz, H-6a), 4.05 (dd, 1H, J = 1.4, 12.4 Hz,
H-6b), 3.58 (m, 1H, H-5>), 3.50 (m, 4H, H-5, OMe), 3.40
(m, 1H, H-2), 3.25 (m, 1H, H-4), 2.63 (ddd, 1H, J = 3.9, 3.9,
14.5 Hz, H-3e>), 1.95 (s, 3H, Ac), 1.70 (ddd, 1H, J = 3.0,
11.1, 14.1 Hz, H-3a>), 1.34 (d, 3H, J = 6.1 Hz, H-6>). Anal.
calcd. for C33H39O9N3S: C 60.63, H 6.01, N 6.43; found:
C 60.37, H 6.13, N 6.34.
Methyl 2-acetamido-3-O-(4-O-benzyl-3,6-dideoxy-2-O-
methyl-ꢀ-D-arabino-hexopyranosyl)-4,6-O-benzylidene-2-
deoxy-ꢀ-D-galactopyranoside (30)
Alcohol 29 (42 mg, 0.077 mmol) was dissolved in anhyd
DMF (1 mL) at 0°C, NaH (9 mg, 0.38 mmol) was added,
and the mixture was stirred for 15 min. MeI (5.2 L,
0.085 mmol) was added, and the reaction was continued for
another 1 h. Methanol (100 L) was added, and the mixture
was concentrated. Chromatography on silica gel using 5%
MeOH–CH2Cl2, gave the desired disaccharide 30 (16 mg,
Methyl 2-acetamido-3-O-(4-O-benzyl-2,3,6-trideoxy-ꢀ-D-
erythro-hexopyranosyl)-4,6-O-benzylidene-2-deoxy-ꢀ-D-
galactopyranoside (32)
To a refluxing solution of disaccharide 31 (35.3 mg,
0.054 mmol) in anhydrous toluene (2 mL) was added
1
dropwise
a
solution of tributyltin hydride (45
L,
37% yield), [ꢁ]D +42.6 (c 0.5, CHCl3). H NMR (CDCl3) ꢂ :
0.16 mmol) in toluene (0.75 mL), and a catalytic amount of
AIBN (~2 mg) was added, the reaction was refluxed for 8 h.
After concentration, the mixture was chromatographed on
silica gel using 2% MeOH–CH2Cl2 as eluent to give
disaccharide 32 (27 mg, 95% yield), [ꢁ]D +68.9 (c 0.6,
7.44–7.53 (m, 2H, Ar), 7.21–7.33 (m, 8H, Ar), 5.78 (d, 1H,
J = 6.4 Hz, NH), 5.55 (s, 1H, PhCH), 5.02 (d, 1H, J =
8.2 Hz, H-1), 4.67 (br d, 1H, J ~ 1 Hz, H-1>), 4.59 (dd, 1H,
J = 3.3, 11.1 Hz, H-3), 4.55 (d, 1H, J = 11.6 Hz, Bn), 4.44
(d, 1H, J = 11.6 Hz, Bn), 4.34 (d, 1H, J = 3.1 Hz, H-4), 4.30
(dd, 1H, J ~ 1, 12.4 Hz, H-6a), 4.06 (dd, 1H, J = 1.4,
12.3 Hz, H-6b), 3.35–3.58 (m, 5H, H-5>, H-2>, H-4> H-2, H-
5), 3.50 (s, 3H, OMe), 3.40 (s, 3H, OMe), 2.35 (ddd, 1H, J =
4.4, 4.4, 13.5 Hz, H-3e>), 1.93 (s, 3H, Ac), 1.43 (dd, 1H, J =
3.0, 10.3, 13.3 Hz, H-3a>), 1.31 (d, 3H, H-6>). Anal. calcd.
for C30H39O9N: C 64.62, H 7.05, N 2.51; found: C 64.32,
H 7.15, N 2.59.
1
CHCl3). H NMR (CDCl3) ꢂ: 7.46–7.52 (m, 2H, Ar), 7.20–
7.38 (m, 8H, Ar), 5.85 (br, 1H, NH), 5.53 (s, 1H, PhCH),
5.06 (d, 1H, J = 8.2 Hz, H-1), 4.56–4.67 (m, 3H, H-3, Bn,
H-1>), 4.43 (d, 1H, Bn), 4.33 (d, 1H, J = 3.2 Hz, H-4), 4.29
(dd, 1H, J ~ 1, 12.5 Hz, H-6a), 4.05 (dd, 1H, J = 1.4,
12.5 Hz, H-6b), 3.50 (m, 4H, OMe, H-5), 3.34–3.46 (m, 2H,
H-2, H-5>), 3.04 (m, 1H, H-4>), 2.15 (m, 1H, H-3e>), 1.89 (s,
3H, Ac), 1.84 (m, 1H, H-3a>), 1.30–1.62 (m, 2H, H-2a>,
H-2b>), 1.27 (d, 3H, J = 6.1 Hz, H-6>). Anal. calcd. for
C29H37O8N: C 66.02, H 7.07, N 2.66; found: C 66.38, H 7.30,
N 2.68.
Methyl 2-acetamido-3-O-(3,6-dideoxy-2-O-methyl-ꢀ-D-
arabino-hexopyranosyl)-2-deoxy-ꢀ-D-galactopyranoside (12)
Disaccharide 30 (16 mg) was deprotected in a similar
fashion as compound 5 and disaccharide 12 (10.1 mg, 93%
yield) was obtained by flash chromatography on reverse-
phase silica gel followed by reverse-phase HPLC using a
gradient of H2O–MeOH (0 J 30%) as eluent. [ꢁ]D –36.5 (c
0.2, MeOH). 1H NMR (D2O) ꢂ: 4.73 (“s”, 1H, H-1>), 4.44 (d,
1H, J = 8.6 Hz, H-1), 4.13 (“d”, 1H, J = 3.1 Hz, H-4), 4.01
(dd, 1H, J = 8.6, 10.8 Hz, H-2), 3.83 (dd, 1H, J = 7.9,
12.1 Hz, H-6a), 3.81 (dd, 1H, J = 3.1, 10.8 Hz, H-3), 3.77
Methyl 2-acetamido-3-O-(2,3,6-trideoxy-ꢀ-D-erythro-
hexopyranosyl)-2-deoxy-ꢀ-D-galactopyranoside (11)
Compound 32 (25 mg, 0.047 mmol) was deprotected in a
similar fashion as compound 5 and disaccharide 11
(15.7 mg, 95% yield) was obtained by reverse-phase HPLC
using a gradient of H2O–MeOH (0 J 30%) as eluent. [ꢁ]D
1
–29.0 (c 0.2, MeOH). H NMR (D2O) ꢂ: 4.69 (dd, 1H, J =
© 2002 NRC Canada

Zhang et al.
1155
2.0, 9.5 Hz, H-1>), 4.44 (d, 1H, J = 8.6 Hz, H-1), 4.09 (“d”,
1H, J = 3.3 Hz, H-4), 3.98 (dd, 1H, J = 8.8, 10.8 Hz, H-2),
3.83 (dd, 1H, J = 3.1, 10.8 Hz, H-3), 3.82 (dd, 1H, J = 8.1,
11.2 Hz, H-6a), 3.76 (dd, 1H, J = 4.4, 11.9 Hz, H-6b), 3.69
(m, 1H, H-5), 3.52 (s, 3H, OMe), 3.42 (dq, 1H, J = 6.2,
9.2 Hz, H-5>), 3.27 (m, 1H, H-4>), 2.04 (m, 1H, H-3e>), 2.03
(s, 3H, Ac), 1.84 (m, 1H, H-2e>), 1.45–1.59 (m, 2H, H-2a>,
H-3a>), 1.26 (d, 3H, J = 6.2 Hz, H-6>). HR-ES-MS m/e
calcd. for C15H27O8N (MNa+): 372.16344; found:
372.163591.
Methyl 2-acetamido-3-O-(2-O-benzoyl-3,6-dideoxy-4-O-
imidazoylthiocarbonyl-ꢀ-D-arabino-hexopyranosyl)-4,6-
O-benzylidene-2-deoxy-ꢀ-D-galactopyranoside (35)
To a solution of disaccharide 34 (30 mg, 0.055 mmol) in
anhydrous toluene (3 mL), was added 1,1>-thiocarbonyl-
diimidazole (35 mg, 0.18 mmol), and the mixture was
heated to reflux for 8 h. After cooling, the mixture was con-
centrated, and the disaccharide 35 was obtained by chroma-
tography on silica gel using 2.5 J 5% MeOH – CH2Cl2 as
eluent (32.9 mg, 92% yield), [ꢁ]D +5.2 (c 0.5, CHCl3).
1H NMR (CDCl3) ꢂ : 8.29 (t, 1H, J = 0.8 Hz, imidazole),
8.03 (m, 2H, Bz), 7.56 (t, 1H, 1.6 Hz, imidazole), 7.51 (m,
1H, Bz), 7.13–7.38 (m, 7H, Ar), 7.03 (dd, 1H, J = 0.8,
1.6 Hz, imidazole), 6.00 (d, 1H, J = 7.1 Hz, NH), 5.56 (m,
1H, H-4>), 5.53 (s, 1H, PhCH), 5.43 (m, 1H, H-2>), 4.96 (d,
1H, J = 1.5 Hz, H-1>), 4.93 (d, 1H, J = 8.2 Hz, H-1), 4.69
(dd, 1H, J = 3.4, 11.2 Hz, H-3), 4.27–4.36 (m, 2H, H-4,
H-6a), 4.06 (dd, 1H, J = 1.6, 12.4 Hz, H-6b), 3.94 (dq,
1H, J = 6.1, 9.0 Hz, H-5>), 3.53 (m, 1H, H-2), 3.49 (m, 1H,
H-5), 3.48 (s, 3H, OMe), 2.67 (d>t>, 1H, J = 4.5, 13.8 Hz,
H-3e>), 1.93 (s, 3H, Ac), 1.84 (ddd, 1H, 3.4, 10.5, 13.8 Hz,
H-3a>), 1.37 (d, 1H, J = 6.2 Hz, H-6>). Anal. calcd. for
C33H37O10N3S: C 59.36, H 5.59, N 6.29; found: C 59.01,
H 5.33, N 6.24.
Methyl 2-acetamido-3-O-(2-O-benzoyl-4-O-benzyl-3,6-
dideoxy-ꢀ-D-arabino-hexopyranosyl)-4,6-O-benzylidene-
2-deoxy-ꢀ-D-galactopyranoside (33)
Disaccharide 29 (50 mg, 0.092 mmol) was dissolved in a
1:1 mixture of pyridine–CH2Cl2 (3 mL) at –30°C, benzoyl
chloride (12 L, 0.101 mmol) was added and the reaction
was stirred at ambient temperature for 7 h. TLC revealed
that the starting material was not completely consumed. The
reaction mixture was cooled to –30°C and more benzoyl
chloride (8 L, 0.068 mmol) was added. The reaction was
allowed to warm to –10°C over 30 min, at which point TLC
showed the starting material to be completely transformed.
Methanol (200 L) was added, and the mixture was concen-
trated under reduced pressure. Chromatography on silica gel
using 2% MeOH–CH2Cl2 as eluent gave compound 33
Methyl 2-acetamido-3-O-(2-O-benzoyl-3,4,6-trideoxy-ꢀ-D-
threo-hexopyranosyl)-4,6-O-benzylidene-2-deoxy-ꢀ-D-
galactopyranoside (36)
1
(52 mg, 87% yield), [ꢁ]D +44.3 (c 0.7, CHCl3). H NMR
(CDCl3) ꢂ: 7.98 (m, 2H, Bz), 7.46 (m, 1H, Bz), 7.08–7.36
(m, 7H, Ar), 5.88 (d, 1H, J = 6.8 Hz, NH), 5.51 (s, 1H,
PhCH), 5.34 (m, 1H, H-2>), 5.05 (d, 1H, J = 8.2 Hz, H-1),
4.81 (s, 1H, H-1>), 4.68 (dd, 1H, J = 3.4, 11.1 Hz, H-3), 4.55
(d, 1H, J = 11.5 Hz, Bn), 4.41 (d, 1H, J = 11.5 Hz, Bn), 4.33
(d, 1H, J = 3.1 Hz, H-4), 4.29 (dd, 1H, J = 1.2, 12.3 Hz, H-
6a), 4.04 (dd, 1H, J = 1.6, 12.3 Hz, H-6b), 3.57 (m, 1H, H-5>),
3.48 (s, 1H, H-5>), 3.47 (s, 4H, OMe, H-5), 3.31–3.46 (m,
2H, H-4>, H-2), 2.46 (d>t>, 1H, J = 4.0,, 14.0 Hz, H-3e>),
1.95 (s, 3H, Ac), 1.70 (ddd, 1H, 3.2, 11.2, 14.1 Hz, H-3a>),
1.36 (d, 1H, J = 6.1 Hz, H-6>). Anal. calcd. for C36H41O10N:
C 66.76, H 6.38, N 2.16; found: C 66.38, H 6.28, N 2.05.
To a refluxing solution of disaccharide 35 (32.9 mg,
0.061 mmol) in anhydrous toluene (2 mL) was added
dropwise a solution of tributyltin hydride (41 L, 0.15 mmol)
in toluene (0.75 mL), and a catalytic amount of AIBN
(~2 mg) was added, the reaction was continued to reflux for
3 h. After concentration, the mixture was chromatographed
on silica gel using 2% MeOH – CH2Cl2 as eluent to give
disaccharide 36 (22 mg, 83% yield), [ꢁ]D +36.2 (c 0.6,
CHCl3). 1H NMR (CDCl3) ꢂ: 8.01 (m, 2H, Bz), 7.46 (m, 1H,
Bz), 7.09–7.36 (m, 7H, Ar), 5.85 (d, 1H, J = 6.9 Hz, NH),
5.53 (s, 1H, PhCH), 5.16 (m, 1H, H-2>), 5.03 (d, 1H, J =
8.2 Hz, H-1), 4.71 (d, 1H, J = 1.1 Hz, H-1>), 4.63 (dd, 1H, J =
3.4, 11.2 Hz, H-3), 4.35 (d, 1H, J = 3.3 Hz, H-4), 4.29 (dd,
1H, J = 1.5, 12.3 Hz, H-6a), 4.04 (dd, 1H, J = 1.4, 12.3 Hz,
H-6b), 3.64 (m, 1H, H-5>), 3.47 (m, 4H, H-5+ OMe), 3.41
(m, 1H, H-2), 2.07 (m, 1H, H-3e>), 1.96 (s, 3H, Ac), 1.72
(m, 1H, H-3a>), 1.30–1.68 (m, 2H, H-4a>, H4e>), 1.27 (d,
3H, J = 6.2 Hz, H-6>). Anal. calcd. for C29H35O9N: C 64.31,
H 6.51, N 2.59; found: C 64.00, H 6.42, N 2.56.
Methyl 2-acetamido-3-O-(2-O-benzoyl-3,6-dideoxy-ꢀ-D-
arabino-hexopyranosyl)-4,6-O-benzylidene-2-deoxy-ꢀ-D-
galactopyranoside (34)
A mixture of disaccharide 33 (52 mg, 0.08 mmol),
HCO2NH4 (25 mg, 0.40 mmol) and 5% Pd-C (34 mg) in
MeOH (10 mL) was heated to reflux for 1 h. The catalyst
was filtered off and the filtrate was concentrated.
Disaccharide 34 was obtained by chromatography on silica
gel using 2% MeOH – CH2Cl2 as eluent (35.2 mg, 78%
Methyl 2-acetamido-3-O-(3,4,6-trideoxy-ꢀ-D-threo-
1
hexopyranosyl)-2-deoxy-ꢀ-D-galactopyranoside (13)
Compound 36 (22 mg, 0.0406 mmol) was dissolved in
anhyd MeOH (3 mL), and a solution of NaOMe in MeOH
(1.5 M, 400 L) was added. After 1 h, the mixture was neu-
tralized with Amberlite IR-120 (H+), the solution was fil-
tered and evaporated to dryness. The residue was
hydrogenated in a similar fashion as compound 5 and
disaccharide 13 (12.5 mg, 88% yield) was purified by re-
verse-phase HPLC using a gradient of H2O–MeOH (0 J
yield), [ꢁ]D +32.6 (c 0.5, CHCl3). H NMR (CDCl3) ꢂ: 7.98
(m, 2H, Bz), 7.45 (m, 1H, Bz), 7.07–7.34 (m, 7H, Ar), 6.08
(d, 1H, J = 7.1 Hz, NH), 5.50 (s, 1H, PhCH), 5.31 (m, 1H,
H-2>), 5.00 (d, 1H, J = 8.2 Hz, H-1), 4.78 (“s”, 1H, H-1>),
4.65 (dd, 1H, J = 3.3, 11.1 Hz, H-3), 4.10 (d, 1H, J =
3.3 Hz, H-4), 4.27 (dd, 1H, J ~ 1.0, 12.3 Hz, H-6a), 4.02
(dd, 1H, J = 1.3, 12.3 Hz, H-6b), 3.61 (m, 1H, H-4>), 3.46
(s, 4H, OMe, H-5), 3.34ꢃ3.45 (m, 2H, H-5>, H-2), 2.29 (d>t>,
1H, J = 5.1, 14.0 Hz, H-3e>), 1.96 (s, 3H, Ac), 1.66 (ddd,
1H, 3.2, 11.2, 14.1 Hz, H-3a>), 1.34 (d, 1H, J = 6.1 Hz,
H-6>). Anal. calcd. for C29H35O10N: C 62.47, H 6.33, N 2.51;
found: C 62.26, H 6.34, N 2.33.
1
30%) as eluent. [ꢁ]D –34 (c 0.5, MeOH). H NMR (D2O) ꢂ:
4.60 (d, 1H, J = 0.9 Hz, H-1>), 4.45 (d, 1H, J = 8.6 Hz, H-1),
4.12 (“d”, 1H, J = 3.1 Hz, H-4), 4.02 (dd, 1H, J = 8.6,
© 2002 NRC Canada

1156
Can. J. Chem. Vol. 80, 2002
10.8 Hz, H-2), 3.87 (dd, 1H, J = 3.3, 10.8 Hz, H-3), 3.83
(dd, 1H, J = 7.9, 11.9 Hz, H-6a), 3.78 (dd, 1H, J = 4.2,
11.7 Hz, H-6b), 3.69–3.72 (m, 3H, H-2>, H-5>, H-5), 3.52 (s,
3H, OMe), 2.03 (s, 3H, Ac), 1.89 (m, 1H, H-3e>), 1.75 (m,
1H, H-3a>), 1.42–1.47 (m, 2H, H-4e>, H-4a>), 1.21 (d, 3H,
J = 6.2 Hz, H-6>). HR-ES-MS m/e calcd. for C15H27O8N
(MNa+): 372.16344; found: 372.164136.
18 h. The catalyst was filtered off and the filtrate was con-
centrated. Diol 39 was purified by chromatography on silica
gel using 5% MeOH – CH2Cl2 as eluent (40 mg, 89% yield),
[ꢁ]D –48.1 (c 0.3, CHCl3). 1H NMR (CDCl3) ꢂ: 8.05 (m, 2H,
Bz), 7.98 (m, 2H, Bz), 7.53–7.61 (m, 2H, Bz), 7.40–7.49 (m,
4H, Bz), 5.94 (br, 1H, NH), 5.46 (m, 1H, H-2>), 5.10 (m,
1H, H-4>), 5.03 (d, 1H, J = 8.2 Hz, H-1), 4.86 (br s, 1H, H-1>),
4.62 (dd, 1H, J = 3.1, 10.6 Hz, H-3), 4.10 (d, 1H, J =
2.5 Hz, H-4), 3.94 (dd, 1H, J = 6.5, 11.6 Hz, H-6a), 3.75–
3.88 (m, 2H, H-6b, H-5>), 3.62 (m, 1H, H-5), 3.47 (s, 3H,
OMe), 3.05 (m, 1H, H-2), 2.55 (d>t>, 1H, J = 3.7, 14.1 Hz,
H-3e>), 2.03 (s, 3H, Ac), 1.92 (ddd, 1H, J = 3.2, 10.5,
14.1 Hz, H-3a>),1.34 (d, 3H, J = 6.1 Hz, H-6>). Anal. calcd.
for C29H35O11N: C 60.72, H 6.15, N 2.44; found: C 60.38,
H 6.05, N 2.66.
Methyl 2-acetamido-3-O-(3,6-dideoxy-ꢀ-D-arabino-hexo-
pyranosyl)-4,6-O-benzylidene-2-deoxy-ꢀ-D-galactopy-
ranoside (37)
To a solution of disaccharide 29 (243 mg, 0.45 mmol) in
MeOH (35 mL), was added 10% Pd-C (250 mg) and
HCO2NH4 (243 mg, 3.86 mmol), and the mixture was
heated to reflux for 1 h. The catalyst was filtered off, and the
filtrate was concentrated. The syrupy mixture was purified
by chromatography on silica gel using 10% J 20% MeOH –
CH2Cl2 as eluent to give first disaccharide 37 (160 mg, 79%
yield) and then disaccharide 1 (18 mg, 11% yield), [ꢁ]D
+30.3 (c 0.4, MeOH). 1H NMR (CDCl3) ꢂ: 7.48 (m, 2H, Ph),
7.29–7.35 (m, 3H, Ph), 5.81 (br, 1H, NH), 5.53 (s, 1H,
PhCH), 5.01 (d, 1H, J = 8.3 Hz, H-1), 4.77 (dd, 1H, J = 3.2,
11.1 Hz, H-3), 4.60 (“s”, 1H, H-1>), 4.32 (“d”, 1H, J =
3.2 Hz, H-4), 4.30 (dd, 1H, J = 1.0, 12.2 Hz, H-6a), 4.06
(dd, 1H, J = 1.1, 12.2 Hz, H-6b), 3.82 (m, 1H, H-2>), 3.65
(ddd, 1H, J = 4.4, 9.2, 10.8 Hz, H-4>), 3.51 (s, 4H, OMe,
H-5), 3.44 (m, 1H, H-2), 3.30 (dq, 1H, J = 6.1, 9.0 Hz,
H-5>), 2.24 (d>t>, 1H, J = 3.8, 14.1, H-3e>), 1.44 (ddd, 1H,
J = 2.9, 11.0, 13.7 Hz, H-3a>), 1.31 (d, 3H, J = 6.1 Hz, H-6>).
Anal. calcd. for C22H31O9N: C 58.27, H 6.89, N 3.09;
found: C 57.84, H 6.90, N 3.08.
Methyl 2-acetamido-3-O-(3,6-dideoxy-ꢀ-D-arabino-
hexopyranosyl)-2,6-dideoxy-ꢀ-D-galactopyranoside (9)
Tolunesulfonyl chloride (12.5 mg, 0.066 mmol) was
added to a solution of compound 39 (34.7 mg, 0.06 mmol)
in anhydrous pyridine (2 mL), and the reaction was stirred at
room temperature for 3 h. Water (0.3 mL) was added to
quench the reaction. The mixture was diluted with EtOAc
(50 mL), washed with brine (1 × 25 mL), dried and concen-
trated. Lithium bromide (34.2 mg, 0.39 mmol) and KI
(6 mg, 0.036 mmol) were added to a solution of the residue
in DMF (1.5 mL), and the resulting solution was heated to
100°C for 1 h. The reaction mixture was diluted with EtOAc
(75 mL), washed with brine (sat., 2 × 25 mL), dried and
evaporated. Tributyltin hydride (330 L, 0.12 mmol) and
AIBN (5 mg, 0.03 mmol) were added to a refluxing solution
of bromide 41 (25 mg, 0.04 mmol) in anhydrous toluene
(2 mL). After 1 h, the reaction was cooled to room tempera-
ture and concentrated. The syrupy mixture was dissolved in
anhyd MeOH (5 mL), and a solution of MeONa in MeOH
(1.5 M, 50 L) was added. After stirring for 1 h, the reaction
was neutralized with Amberlite IR-120 (H+) resin, filtered
and concentrated. Disaccharide 9 was obtained by reverse-
phase HPLC using a gradient of H2O–MeOH (0 J 30%) as
eluent (10.5 mg, 49% yield), [ꢁ]D –38.4 (c 0.5, MeOH).
1H NMR (D2O) ꢂ: 4.68 (d, 1H, J = 0.9 Hz, H-1>), 4.42 (d,
1H, J = 8.6 Hz, H-1), 3.97 (dd, 1H, J = 8.6, 10.8 Hz, H-2),
3.94 (“d”, 1H, J = 3.1 Hz, H-4), 3.89 (m, 1H, H-2>), 3.86
(dd, 1H, J = 3.1, 10.8 Hz, H-3), 3.80 (dq, 1H, J = 0.7,
6.6 Hz, H-5), 3.55 (ddd, 1H, J = 4.8, 9.3, 11.5 Hz, H-4>),
3.49 (s, 3H, OMe), 3.43 (dq, 1H, J = 6.1, 9.3 Hz, H-5>), 2.17
(ddd, 1H, J = 3.5, 4.8, 14.1 Hz, H-3e>), 2.02 (s, 3H, Ac),
1.65 (ddd, 1H, J = 2.9, 11.5, 14.3 Hz, H-3e>), 1.29 (d, 3H,
J = 6.4 Hz, H-6), 1.27 (d, 3H, J = 6.0 Hz, H-6>). HR-ES-MS
m/e calcd. for C15H27O8N (MNa+): 372.16344; found:
372.163372.
Methyl 2-acetamido-3-O-(2,4-di-O-benzoyl-3,6-dideoxy-
ꢀ-D-arabino-hexopyranosyl)-4,6-O-benzylidene-2-deoxy-
ꢀ-D-galactopyranoside (38)
Benzoic anhydride (112 mg, 0.534 mmol) and DMAP
(10 mg, 0.0818 mmol) were added to diol 37 (80.9 mg,
0.178 mmol) was dissolved in anhydrous pyridine (3 mL).
After stirring for 18 h at ambient temperature, H2O (0.5 mL)
was added to destroy the excess anhydride, and the mixture
was concentrated to dryness. Dibenzoate 38 was obtained by
chromatography on silica gel using 3% MeOH – CH2Cl2 as
eluent (105 mg, 89% yield), [ꢁ]D +25.2 (c 0.8, CHCl3).
1H NMR (CDCl3) ꢂ: 8.03 (m, 2H, Bz), 7.98 (m, 2H, Bz),
7.12–7.60 (m, 11H, Ar), 5.78 (d, 1H, J = 7.0 Hz, NH), 5.54
(s, 1H, PhCH), 5.43 (m, 1H, H-2>), 5.08 (m, 1H, H-4>), 4.99
(d, 1H, J = 8.3 Hz, H-1), 4.91 (d, 1H, J = 1.3 Hz, H-1>),
4.70 (dd, 1H, J = 3.4, 11.2 Hz, H-3), 4.36 (d, 1H, J =
3.3 Hz, H-4), 4.30 (dd, 1H, J = 1.3, 12.4 Hz, H-6a), 4.06
(dd, 1H, J = 1.6, 12.4 Hz, H-6b), 3.81 (dq, 1H, J = 6.2,
9.0 Hz, H-5>), 3.49 (m, 1H, H-5), 3.48 (s, 3H, OMe), 3.47
(m, 1H, H-2), 2.54 (d>t>, 1H, J = 4.4, 13.9 Hz, H-3e>), 1.97
(s, 3H, Ac), 1.85 (ddd, 1H, J = 3.3, 10.5, 13.9 Hz, H-3a>),1.35
(d, 3H, J = 6.1 Hz, H-6>). Anal. calcd. for C36H39O11N:
C 65.35, H 5.94, N 2.12; found: C 65.55, H 5.93, N 1.95.
3,6-Dideoxy-4-O-methyl-1,2-O-propylidene-ꢁ-D-ribo-
hexopyranose (44)
Sodium hydride (60% in mineral oil, 218 mg, 5.445 mmol)
was added to a solution of compound 43 (38) (205 mg,
1.089 mmol) in anhyd DMF, followed after 10 min by MeI
(136 L, 2.178 mmol), and the reaction was stirred at ambi-
ent temperature for 2 h. MeOH (0.5 mL) was added to
quench the reaction, and the reaction mixture was diluted
with EtOAc (75 mL), washed with brine (sat., 2 × 30 mL),
Methyl 2-acetamido-3-O-(2,4-di-O-benzoyl-3,6-dideoxy-
ꢀ-D-arabino-hexopyranosyl)-2-deoxy-ꢀ-D-galactopyranoside
(39)
A solution of compound 38 (52 mg, 0.078 mmol) in
MeOH (30 mL) was hydrogenated over 10% Pd(OH)2 for
© 2002 NRC Canada

Zhang et al.
1157
dried and evaporated. The compound 44 was obtained by
chromatography on silica gel using 20% EtOAc – hexane as
eluent (201 mg, 92% yield), [ꢁ]D +44.6 (c 0.5, CHCl3).
1H NMR (CDCl3) ꢂ: 5.30 (d, 1H, J = 5.2 Hz, H-1), 4.73 (t,
1H, J = 4.9 Hz, CH3CH2CH), 4.01 (m, 1H, H-2), 3.80 (dq,
1H, J = 6.1, 9.3 Hz, H-5), 3.31 (s, 3H, OMe), 3.08 (m, 1H,
H-4), 2.17 (ddd, 1H, J = 1.7, 2.6, 15.5 Hz, H-3e), 1.86 (ddd,
1H, J = 3.6, 7.6, 15.9 Hz, H-3a), 1.77 (m, 2H, CH3CH2),
1.23 (d, 3H, J = 6.3 Hz, H-6), 0.98 (t, 3H, J = 7.6 hz,
CH3CH2). Anal. calcd. for C10H18O4: C 59.39, H 8.97;
found: C 59.63, H 8.88.
2H, Bz), 7.57 (m, 1H, Bz), 7.46 (m, 2H, Bz), 7.26 (m, 5H,
Ph), 4.90 (d>t>, 1H, J = 4.8, 9.9 Hz, H-21), 4.80 (d, 1H, J =
9.9 Hz, H-1), 3.42 (dq, 1H, J = 6.2, 9.2 Hz, H-5), 3.36 (s,
3H, OMe), 3.02 (m, 1H, H-4), 2.82 (d>t>, 1H, J = 4.5,
12.0 Hz, H-3e), 1.53 (d>t>, 1H, J = 12.1, 12.1 Hz, H-3a),
1.36 (d, 3H, J = 6.2 Hz, H-6). Anal. calcd. for C20H22O4S: C
67.02, H 6.19; found: C 67.34, H 6.16.
Methyl 2-azido-3-O-(2-O-benzoyl-3,6-dideoxy-4-O-
methyl-ꢀ-D-ribo-hexopyranosyl)-4,6-O-benzylidene-2-
deoxy-ꢀ-D-galactopyranoside (48)
A
mixture containing thioglycoside 47 (140 mg,
1,2-Di-O-benzoyl-3,6-dideoxy-4-O-methyl-ꢁ,ꢀ -D-ribo-
hexopyranose (46)
0.40 mmol), alcohol 24 (39) (95 mg, 0.31 mmol) and 4 Å
molecular sieves (500 mg) in CH2Cl2 was stirred for 30 min
and N-iodosuccinimide (146 mg, 0.62 mmol) was added. Af-
ter the reaction mixture had been cooled to –60°C, TfOH (6
L, 0.067 mmol) was added, and the reaction was continued
for 3 h. Triethylamine (0.5 mL) was added to quench the re-
action, and disaccharide 48 was obtained in a similar fashion
to that described above (134.7 mg, 78% yield), [ꢁ]D –14.7 (c
The 1,2-O-propylidene derivative 44 (112 mg, 0.55 mmol)
was dissolved in THF (5 mL), and a 10% aqueous solution
of H2SO4 (1 mL) was added, and the reaction was stirred at
65°C for 2 h. The mixture was diluted with CH2Cl2
(100 mL), washed with aq NaHCO3 (sat., 2 × 30 mL), dried,
and evaporated to afford the crude diol 45 that was used di-
rectly to the next step. Benzoyl chloride (314.9 L,
1.21 mmol) was added to an ice-cold solution of the crude
diol 45 in a mixture of anhyd CH2Cl2 (1 mL) and pyridine
(0.5 mL), and the reaction was continued for 1 h. Methanol
(0.5 mL) was added to quench the reaction. The reaction
mixture was diluted with CH2Cl2 (50 mL), the solution ex-
tracted with H2O, then dried, and evaporated. Dibenzoate 46
was obtained as a ꢁ,ꢀ-mixture by chromatography on silica
gel using 10% EtOAc – hexane as eluent (ꢁ/ꢀ: 1:1, 197 mg,
1
0.8, CHCl3). H NMR (CDCl3) ꢂ: 8.02–8.08 (m, 2 H, Bz),
7.20–7.57 (m, 8H, Ar), 5.50 (s, 1H, PhCH), 4.82–4.96 (m,
2H, H-1> + H-2>), 4.30 (dd, 1H, J = 1.6, 12.4 Hz, H-6a), 4.23
(br d, 1H, J = 3.1 Hz, H-4), 4.21 (d, 1H, J = 8.0 Hz, H-1),
4.04 (dd, 1H, J = 1.6, 12.4 Hz, H-6b), 3.77 (dd, 1H, J = 8.0,
10.6 Hz, H-2), 3.55 (s, 3H, OMe), 3.51 (dd, 1H, J = 3.5,
10.6 Hz, H-3), 3.43 (dq, 1H, J = 6.1, 9.0 Hz, H-5>), 3.39 (m,
1H, H-5), 3.35 (s, 3H, OMe), 3.04 (ddd, 1H, J = 4.5, 9.0,
10.8 Hz, H-4>), 2.79 (d>t>,1H, J = 4.4, 12.1 Hz, H-3e>), 1.45
(d>t>, 1H, J = ~11.5, 11.5 Hz, H-3a>), 1.31 (d, 3H, J =
6.1 Hz, H-6>). Anal. calcd. for C28H33O9N3: C 60.53, H 5.99,
N 7.56; found: C 60.64, H 6.00, N 7.32.
1
96% yield). H NMR (CD3OD) for ꢁ-anomer ꢂ: 8.10 (m,
2H, Bz), 7.90 (m, 2 H, Bz), 7.30–7.63 (m, 3H, Bz), 6.52 (d,
1H, J = 3.3 Hz, H-1), 5.27 (m, 1H, H-2), 3.89 (dq, 1H, J =
6.4, 9.3 Hz, H-5), 3.43 (s, 3H, OMe), 3.17 (m, 1H, H-4),
2.66 (dd>t>, 1H, J = 0.9, 4.4, 11.4 Hz, H-3e), 2.05 (d>t>,
11.5, 11.5 Hz, H-3a), 1.29 (d, 3H, J = 6.1 Hz, H-6);
ꢀ-anomer ꢂ: 8.02 (m, 2H, Bz), 7.96 (m, 2 H, Bz), 7.30–7.63
(m, 3H, Bz), 6.06 (d, 1H, J = 8.2 Hz, H-1), 5.24 (m, 1H, H-
2), 3.67 (dq, 1H, J = 6.1, 9.0 Hz, H-5), 3.40 (s, 3H, OMe),
3.12 (m, 1H, H-4), 2.83 (d>t>, 1H, J = 4.9, 12.1 Hz, H-3e),
1.66 (d>t>, 11.9, 11.9 Hz, H-3a), 1.35 (d, 3H, J = 6.1 Hz,
H-6). Anal. calcd. for C21H22O6: C 68.10, H 5.99; found:
C 68.43, H 5.95.
Methyl 2-azido-3-O-(3,6-dideoxy-4-O-methyl-ꢀ-D-ribo-
hexopyranosyl)-4,6-O-benzylidene-2-deoxy-ꢀ-D-
galactopyranoside (49)
Compound 48 (140 mg, 0.25 mmol) was transesterified as
described above to give the disaccharide 49 (107 mg, 94%
1
yield), [ꢁ]D +22.7 (c 1.1, CHCl3). H NMR (CDCl3) ꢂ: 7.51
(m, 2H, Ph), 7.31–7.38 (m, 3H, Ph), 5.52 (s, 1H, PhCH),
4.43 (d, 1H, J = 7.3 Hz, H-1>), 4.31 (dd, 1H, J = 1.5,
12.4 Hz, H-6a), 4.24 (“d”, 1H, J = 3.4 Hz, H-4), 4.22 (d, 1H,
J = 8.0 Hz, H-1), 4.04 (dd, 1H, J = 1.7, 12.4 Hz, H-6b), 3.85
(dd, 1H, J = 8.1, 10.5 Hz, H-2), 3.58 (s, 3H, OMe), 3.53 (dd,
1H, J = 3.5, 10.6 Hz, H-3), 3.52 (dq, 1H, J = 6.1, 9.0 Hz,
H-2>), 3.32–3.42 (m, 5H, H-5, H-5>, OMe), 3.35 (s, 3H,
OMe), 2.92 (ddd, 1H, J = 4.4, 8.8, 10.9 Hz, H-4>), 2.49
(d>t>, 1H, J = 4.7, 12.3 Hz, H-3e>), 1.36 (d>t>, 1H, J ~ 11.5,
11.5 Hz, H-3a>), 1.28 (d, 3H, J = 6.1 Hz, H-6>). Anal. calcd.
for C21H29O8N3: C 55.87, H 6.48, N 9.31; found: C 55.81,
H 6.36, N 9.40.
Phenyl 2-O-benzoyl-3,6-dideoxy-4-O-methyl-1-thio-ꢁ,ꢀ-
D-ribo-hexopyranoside (47)
A solution of 46 (185 mg, 0.50 mmol) in anhydrous tolu-
ene (3 mL) was stirred with molecular sieves 4 Å (400 mg)
for 30 min at 0°C. Me3SiSPh (244 L, 1.25 mmol) was
added, followed by TMSOTf (97 L, 0.5 mmol). Reaction
was continued at room temperature overnight. Triethylamine
(0.5 mL) was added, and the insoluble material was filtered
off. After evaporation, the thioglycoside 47 was obtained by
chromatography of the residue on silica gel using 5% EtOAc
– hexane as eluent (ꢁ/ꢀ 4:6, 160 mg, 89%). 1H NMR
(CDCl3) for ꢁ-anomer ꢂ: 8.09 (m, 2H, Bz), 7.57 (m, 1H,
Bz), 7.45 (m, 2H, Bz), 7.17–7.28 (m, 5H, Ph), 5.75 (d,
1H, J = 5.1 Hz, H-1), 5.29 (d>t>, 1H, J = 5.1, 12.4 Hz, H-2),
4.18 (dq, 1H, J = 6.2, 9.2 Hz, H-5), 3.40 (s, 3H, OMe), 3.07
(ddd, 1H, J = 4.5, 9.3, 10.9 Hz, H-4), 2.52 (dd>t>, 1H, J =
0.7, 4.5, 11.9 Hz, H-3e), 1.87 (d>t>, 1H, J = 12.1, 12.1 Hz,
H-3a), 1.26 (d, 3H, J = 6.2 Hz, H-6); ꢀ-anomer ꢂ: 8.04 (m,
Methyl 2- acetamido-3-O-(3,6-dideoxy-4-O-methyl-ꢀ-D-
arabino-hexopyranosyl)-4,6-O-benzylidene-2-deoxy-ꢀ-D-
galactopyranoside (51)
The disaccharide 49 (96.3 mg, 0.21 mmol) was oxidized
by a mixture of Ac2O (1 mL) and anhydrous DMSO (2 mL)
at room temperature for 18 h to give the intermediate ketone
50, after purification by column chromatography on silica
gel using 50% EtOAc – hexane as eluent. Ketone 50
(66.2 mg, 0.147 mmol) was dissolved in anhyd THF
© 2002 NRC Canada

1158
Can. J. Chem. Vol. 80, 2002
(2.5 mL) at 0°C, a solution of L-selectride in THF (1 M,
500 L, 0.50 mmol) was added, and the reaction was continued
for 1 h. Water (0.5 mL) was added to quench the reaction,
and Ac2O (0.5 mL) was added. After stirring for 30 min, the
reaction mixture was concentrated to dryness. Disaccharide
51 was obtained by chromatography on silica gel using 5%
MeOH – CH2Cl21as eluent (52.6 mg, 76% yield), [ꢁ]D +28.3
(c 0.7, CHCl3). H NMR (CD3OD) ꢂ: 7.45–7.51 (m, 2 H,
Ph), 7.28–7.35 (m, 3H, Ph), 5.97 (d, 1H, J = 4.6 Hz, NH),
5.51 (s, 1H, PhCH), 5.01 (d, 1H, J = 8.2 Hz, H-1), 4.73 (dd,
1H, J = 3.4, 11.3 Hz, H-3), 4.56 (br “s”, 1H, H-1>), 4.26–
4.36 (m, 2H, H-6a, H-4), 4.04 (dd, 1H, J = 1.5, 12.4 Hz, H-
6b), 3.81 (m, 1H, H-2>), 3.28–3.56 (m, 9H, 2 × OMe, H-5,
H-2, H-5>), 3.18 (m, 1H, H-4>), 2.32 (d>t>, 1H, J = 4.1,
13.6 Hz, H-3e>), 1.84 (s, 3H, Ac), 1.22–1.34 (m, 4H, H-6>,
H3a>). Anal. calcd. for C23H33O9N: C 59.09, H 7.12, N
3.00; found: C 59.19, H 7.11, N 2.99.
4 h. The mixture was diluted with EtOAc (75 mL), washed
with H2O (1 × 30 mL), dried and evaporated to dryness. The
protected glycoside 54 was obtained by chromatography on
silica gel using 15% EtOAc – hexane as eluent (401 mg,
1
94% yield), [ꢁ]D –15.4 (c 0.6, CHCl3). H NMR (CDCl3) ꢂ:
8.11 (m, 2H, Bz), 8.02 (m, 2H, Bz), 7.53–7.64 (m, 2H, Bz),
7.40–7.52 (m, 4H, Bz), 5.00 (dd, 1H, J = 2.9, 10.8 Hz, H-3),
4.58 (dd, 1H, J = 6.4, 11.1 Hz, H-6a), 4.49 (dd, 1H, J = 6.8,
11.1 Hz, H-6b), 4.31 (d, 1H, J = 8.0 Hz, H-1), 3.82 (dd, 1H,
J = 8.1, 10.7 Hz, H-2), 3.91 (m, 1H, H-5), 3.82 (br d, 1H,
J = 2.9 Hz, H-4), 3.59 (s, 3H, OMe), 3.50 (s, 3H, OMe).
Anal. calcd. for C22H23O7N3: C 59.86, H 5.25, N 9.52;
found: C 59.83, H 5.31, N 9.28.
Methyl 2-azido-2-deoxy-4-O-methyl-ꢀ-D-galactopyranoside (55)
Dibenzoate 54 (350 mg, 0.79 mmol) was debenzoylated
as described above to give the selectively methylated
monosaccharide 55 (179 mg, 97% yield), [ꢁ]D –12.2 (c 0.7,
1
CHCl3). H NMR (CDCl3) ꢂ: 4.15 (m, 1H, H-1, high order),
Methyl 2-acetamido-3-O-(3,6-dideoxy-4-O-methyl-ꢀ-D-
arabino-hexopyranosyl)-2-deoxy-ꢀ-D-galactopyranoside (14)
Compound 51 (42 mg, 0.09 mmol) was hydrogenated in a
similar fashion as compound 5 and disaccharide 14 (32 mg,
94% yield) was obtained by reverse-phase HPLC using a
gradient of H2O–MeOH (0 J 30%) as eluent. [ꢁ]D –24.5
(c 0.4, MeOH). 1H NMR (D2O) ꢂ: 4.69 (“s”, 1H, H-1>), 4.44
(d, 1H, J = 8.6 Hz, H-1), 4.12 (“d”, 1H, J = 3.1 Hz, H-4),
4.01 (dd, 1H, J = 8.2, 10.6 Hz, H-2), 3.91 (m, 1H, H-2>),
3.86 (dd, 1H, J = 3.3, 10.8 Hz, H-3), 3.82 (dd, 1H, J = 7.9,
11.9 Hz, H-6a), 3.77 (dd, 1H, J = 4.2, 11.7 Hz, H-6b), 3.70
(m, 1H, H-5), 3.52 (s, 3H, OMe), 3.49 (dq, 1H, J = 6.2,
9.2 Hz, H-5>), 3.39 (s, 3H, OMe), 3.25 (ddd, 1H, J = 4.6,
9.3, 11.2 Hz, H-4>), 2.38 (d>t>, 1H, J = 4.2, 13.9 Hz, H-3e>),
2.02 (s, 3H, Ac), 1.54 (ddd, 1H, J = 2.9, 11.4, 14.1 Hz, H-3a>),
1.29 (d, 3H, J = 6.2 Hz, H-6>). HR-ES-MS m/e calcd. for
C16H29O9N (MNa+): 402.17400; found: 402.173637.
3.94 (dd, 1H, J = 7.4, 11.2 Hz, H-6a), 3.94 (dd, 1H, J = 7.4,
11.2 Hz, H-6a), 3.77 (dd, 1H, J = 4.9, 11.2Hz, H-6b), 3.56
(s, 3H, OMe), 3.55 (s, 3H, OMe), 3.45–3.51 (m, 4H, H-2,
H-3, H-4, H-5). Anal. calcd. for C8H15O5N3: C 41.20,
H 6.48, N 18.02; found: C 41.53, H 6.53, N 18.33.
Methyl 2-azido-6-O-tert-butyldimethylsilyl-2-deoxy-4-O-
methyl-ꢀ-D-galactopyranoside (56)
Diol 55 (108 mg, 0.46 mmol) was dissolved in anhydrous
pyridine (4 mL), t-BuMe2SiCl (98 mg, 0.65 mmol) was
added, and the reaction was stirred at ambient temperature
for 3 h. Water (0.3 mL) was added and the mixture was con-
centrated. The syrup was chromatographed on silica gel us-
ing 15% EtOAc – hexane as eluent to yield 56 (135.1 mg,
1
84% yield), [ꢁ]D –5.0 (c 0.6, CHCl3). H NMR (CDCl3) ꢂ:
4.11 (m, 1H, H-1, high order), 3.79 (dd, 1H, J = 8.6, 9.6 Hz,
H-6a), 3.73 (dd, 1H, J = 5.6, 9.8 Hz, H-6a), 3.61 (br, 1H, H-
4), 3.59 (s, 3H, OMe), 3.52 (s, 3H, OMe), 3.37–3.47 (m, 3H,
H-2, H-3, H-5), 0.88 (s, 9H, tert-BuSi), 0.06 (s, 6H, SiMe2).
Anal. calcd. for C14H29O5N3Si: C 48.39, H 8.41, N 12.09;
found: C 48.70, H 8.59, N 11.73.
Methyl 2-azido-3,6-di-O-benzoyl-2-deoxy-ꢀ-D-galactopy-
ranoside (53)
A solution of triol 52 (39) (860 mg, 3.9 mmol) and
bis(tributyltin)oxide in anhydrous toluene (60 mL) was
refluxed for 4.5 h with azeotropic removal of water. The so-
lution was cooled to room temperature, and BzCl (1.0 mL,
8.97 mmol) was added. After stirring for 2.5 h, the mixture
was diluted with EtOAc (75 mL), washed with brine (1 ×
30 mL), dried and evaporated. Dibenzoate 53 was obtained
by chromatography on silica gel using 15% EtOAc – hexane
Methyl 2-azido-3-O-(2-O-benzoyl-4-O-benzyl-3,6-dideoxy-
ꢀ-D-ribo-hexopyranosyl)-6-O-tert-butyldimethylsilyl-2-
deoxy-4-O-methyl-ꢀ-D-galactopyranoside (57)
A solution of acceptor 56 (40 mg, 0.115 mmol),
thioglycoside 22 (55 mg, 0.127 mmol), and 4 Å molecular
sieves (500 mg) in anhyd CH2Cl2 (3 mL) was stirred for 1 h,
cooled to –40°C and NIS (54 mg, 0.23 mmol) and TfOH (4
L) were added. After 1 h, the reaction was quenched with
Et3N. The mixture was diluted with EtOAc (75 mL), filtered,
washed with a 10% aqueous solution of Na2S2O3, dried and
evaporated. Disaccharide 57 was obtained by chromatogra-
phy on silica gel using 8% EtOAc – hexane as eluent
1
as eluent (1.46 g, 86% yield), [ꢁ]D +29.8 (c 0.5, CHCl3). H
NMR (CDCl3) ꢂ: 8.08 (m, 2H, Bz), 8.01 (m, 2H, Bz), 7.53–
7.63 (m, 2H, Bz), 7.39–7.49 (m, 4H, Bz), 4.98 (dd, 1H, J =
3.2, 10.8 Hz, H-3), 4.66 (dd, 1H, J = 6.8, 11.4 Hz, H-6a),
4.51 (dd, 1H, J = 6.2, 11.3 Hz, H-6b), 4.34 (d, 1H, J =
8.0 Hz, H-1), 4.20 (“d”, 1H, J = 2.7 Hz, H-4), 3.93 (dd, 1H,
J = 8.0, 10.8 Hz, H-2), 3.91 (m, 1H, H-5), 3.61 (s, 3H,
OMe), 2.53 (br s, 1H, OH-4). Anal. calcd. for C21H21O7N3:
C 59.01, H 4.95, N 9.83; found: C 58.75, H 4.85, N 9.60.
1
(55 mg, 71%), [ꢁ]D –3.4 (c 0.6, CHCl3). H NMR (CDCl3)
ꢂ: 8.07 (m, 2H, Bz), 7.53 (m, 1H, Bz), 7.42 (m, 2H, Bz),
7.24–7.38 (m, 5H, Bn), 4.90 (m, 1H, H-2>), 4.77 (d, 1H, J =
7.9 Hz, H-1>), 4.63 (d, 1H, J = 11.5 Hz, Bn), 4.45 (d, 1H, J =
11.5 Hz, Bn), 4.10 (d, 1H, J = 7.9 Hz, H-1), 3.73–3.83 (m,
2H, H-6a, H-4), 3.66 (dd, 1H, J = 5.3, 9.4 Hz, H-6b), 3.45–
3.63 (m, 8H, 2 × OMe, H-5>, H-2), 3.35–3.45 (m, 2H, H-3,
H-5), 3.26 (m, 1H, H-4>), 2.80 (d>t>, 1H, J = 4.8, 12.0 Hz,
Methyl 2-azido-3,6-di-O-benzoyl-2-deoxy-4-O-methyl-ꢀ-
D-galactopyranoside (54)
A solution of dibenzoate 53 (416 mg, 0.973 mmol),
MeOTf (330 L, 2.91 mmol), and 2,6-di-tert-butyl-4-
methylpyridine in anhyd CH2Cl2 (15 mL) was refluxed for
© 2002 NRC Canada

Zhang et al.
1159
H-3e>), 1.60 (d>t>, 1H, J = 11.5, 11.5 Hz, H-3a>), 1.30 (d,
3H, J = 6.1 Hz, H-6>), 0.88 (s, 9H, t-BuSi), 0.06 (s, 6H,
Me2Si). Anal. calcd. for C34H49O9N3Si: C 60.78, H 7.35,
N 6.25; found: C 60.57, H 7.17, N 5.83.
(ddd, 1H, J = 3.5, 4.8, 13.9 Hz, H-3e>), 2.02 (s, 3H, Ac),
1.65 (ddd, 1H, J = 3.1, 11.7, 14.3 Hz, H-3a>), 1.29 (d, 3H,
J = 6.2 Hz, H-6>). HR-ES-MS m/e calcd. for C16H29O9N
(MH+): 402.17400; found: 402.174200.
Methyl 2-azido-3-O-(4-O-benzyl-3,6-dideoxy-ꢀ-D-ribo-
hexopyranosyl)-6-O-tert-butyldimethylsilyl-2-deoxy-4-O-
methyl-ꢀ-D-galactopyranoside (58)
Methyl 2-acetamido-3-O-benzoyl-4,6-O-benzylidene-2-
deoxy-ꢀ-D-glucopyranoside (61)
Benzoyl chloride (646 L, 5.57 mmol) was added
dropwise to an ice-cold solution of alcohol 17 (45) (1.5 g,
4.6 mmol) in a mixture of anhyd CH2Cl2 (10 mL) and
pyridine (8 mL), and the mixture was stirred at room tem-
perature for 5 h. Methanol (1 mL) was added, the mixture
was concentrated and dissolved in CH2Cl2 (100 mL),
washed with 5% NaHCO3 (2 × 50 mL), saturated brine (1 ×
50 mL), dried and evaporated. The benzoylated acetal 61
was obtained by chromatography on silica gel using 5%
MeOH–CH2Cl2 as eluent (1.90 g, 96%), [ꢁ]D –99.2 (c 0.8,
CHCl3). 1H NMR (CDCl3) ꢂ: 8.03 (m, 2H, Bz), 7.56 (m, 1H,
Bz), 7.43 (m, 2H, Bz), 7.40 (m, 2H, Ph), 7.26–7.32 (m, 3H,
Ph), 6.05 (d, 1H, J = 9.5 Hz, NH), 5.58 (t, 1H, J = 9.8 Hz,
H-3), 5.53 (s, 1H, PhCH), 4.47 (d, 1H, J = 8.4 Hz, H-1),
4.37 (dd, 1H, J = 5.0, 10.5 Hz, H-6a), 4.32 (d>t>, 1H, J =
10.0 Hz, 10.0 Hz, H-2), 3.86 (t, 1H, J = 9.4 Hz, H-4), 3.83
(t, 1H, J = 10.3 Hz, H-6b), 3.67 (m, 1H, H-5), 3.42 (s, 3H,
OMe), 1.89 (s, 3H, Ac). Anal. calcd. for C23H25O7N: C 64.63,
H 5.90, N 3.28; found: C 64.56, H 5.88, N 3.21.
A solution of 57 (50 mg, 0.0744 mmol) in anhyd MeOH
(5 mL) at 0°C was de-acylated by addition of a solution of
NaOMe in MeOH (1.5 M, 200 L). After stirring for 5 h at
0°C, the mixture was neutralized with Amberlite IR-120
(H+) resin and evaporated. The residue was chromato-
graphed on silica gel using 15% EtOAc – hexane as eluent
to give 58 (41.5 mg, 98% yield), [ꢁ]D –11.6 (c 0.7, CHCl3).
1H NMR (CDCl3) ꢂ: 7.25–7.36 (m, 5H, Bn), 4.63 (d, 1H, J =
11.6 Hz, Bn), 4.45 (d, 1H, J = 11.6 Hz, Bn), 4.35 (d, 1H, J =
7.5 Hz, H-1>), 4.11 (d, 1H, J = 7.9 Hz, H-1), 3.74–3.82 (m,
2H, H-6a, H-4), 3.65–3.73 (m, 2H, H-3, H-6b), 3.54 (s, 3H,
OMe), 3.36–3.53 (m, 7H, OMe, H-2>, H-5>, H-2, H-5), 3.13
(m, 1H, H-4>), 2.53 (d>t>, 1H, J = 4.7, 12.2 Hz, H-3e>), 1.47
(d>t>, 1H, J = 11.8, 11.8 Hz, H-3a>), 1.33 (d, 3H, J = 6.1 Hz,
H-6>), 0.87 (s, 9H, t-BuSi), 0.05 (s, 6H, Me2Si). Anal. calcd.
for C27H45O8N3Si: C 57.12, H 7.99, N 7.40; found: C 57.00,
H 7.83, N 7.45.
Methyl 2-acetamido-3-O-(4-O-benzyl-3,6-dideoxy-ꢀ-D-
arabino-hexopyranosyl)-6-O-tert-butyldimethylsilyl-2-
deoxy-4-O-methyl-ꢀ-D-galactopyranoside (60)
Methyl 2-acetamido-3-O-benzoyl-6-O-benzyl-2-deoxy-ꢀ-
D-glucopyranoside (62)
Alcohol 58 (55 mg, 0.097 mmol) was oxidized using
DMSO (2 mL) and Ac2O (1 mL) as described above. The in-
termediate ketone 59 was reduced using a solution of L-
selectride in THF (1.0 M, 200 L) and acetylated using
Ac2O as described above to give the disaccharide 60 (43 mg,
To a mixture containing compound 61 (1.0 g, 2.34 mmol),
NaBH3CN (1.8 g, 28 mmol), and 4 Å molecular sieves
(1.5 g) in anhyd THF (20 mL), was added a trace amount of
methyl orange (~1 mg). A saturated solution of HCl in anhy-
drous ether (30 mL) was added dropwise until the color of
the reaction became slightly pink. The reaction was contin-
ued for 2 h, the mixture was diluted with EtOAc (50 mL),
and filtered. After concentration, the residue was dissolved
in EtOAc (120 mL), and the solution was washed with 2 M
HCl (2 × 50 mL), 10% NaHCO3 aqueous solution (2 ×
50 mL), dried, and evaporated. The alcohol 62 was obtained
by chromatography on silica gel using 7.5% MeOH –
CH2Cl2 as eluent (950 mg, 95%), [ꢁ]D –13.2 (c 0.3, CHCl3).
1H NMR (CDCl3) ꢂ: 8.03 (m, 2H, Bz), 7.57 (m, 1H, Bz),
7.44 (m, 2H, Bz), 7.40 (m, 2H, Ph), 7.25–7.36 (m, 5H, Ph),
5.56 (d, 1H, J = 9.2 Hz, NH), 5.27 (dd, 1H, J = 9.0, 10.7 Hz,
H-3), 4.64 (d, 1H, J = 11.9 Hz, Bn), 4.58 (d, 1H, J =
11.9 Hz, Bn), 4.48 (d, 1H, J = 8.3 Hz, H-1), 4.11 (d>t>,
1H, J = 8.8, 10.7 Hz, H-2), 3.90 (t, 1H, J = 9.3 Hz, H-4),
3.86 (dd, 1H, J = 4.9, 10.3 Hz, H-6a), 3.83 (dd, 1H, J = 4.8,
10.3 Hz, H-6b), 3.62 (m, 1H, H-5), 3.50 (s, 3H, OMe), 1.86
(s, 3H, Ac). Anal. calcd. for C23H27O7N: C 64.32, H 6.34,
N 3.26; found: C 64.16, H 6.12, N 3.01.
1
76% yield), [ꢁ]D +4.0 (c 0.6, CHCl3). H NMR (CDCl3) ꢂ:
7.22–7.36 (m, 5H, Bn), 6.05 (d, 1H, J = 6.9 Hz, NH), 4.85
(d, 1H, J = 8.2 Hz, H-1), 4.54–4.66 (m, 3H, H-3, H-1>, Bn),
4.43 (d, 1H, J = 11.5 Hz, Bn), 3.91 (br d, 1H, J = 2.7 Hz, H-
4), 3.87 (m, 1H, H-2>), 3.62–3.80 (m, 2H, H-6a, H-6b),
3.37–3.60 (m, 9H, 2 × OMe, H-5, H-4>, H-5>), 3.25 (m, 1H,
H-2), 2.85 (br s, 1H, OH-2>), 2.41 (d>t>, 1H, J = 3.1,
13.6 Hz, H-3e>), 1.49 (m, 1H, H-3a>), 1.28 (d, 3H, J =
5.2 Hz, H-6>), 0.88 (s, 9H, t-BuSi), 0.05 (s, 6H, Me2Si).
Anal. calcd. for C29H49O9NSi: C 59.66, H 8.46, N 2.40;
found: C 59.96, H 8.78, N 2.27.
Methyl 2-acetamido-3-O-(3,6-dideoxy-ꢀ-D-arabino-hexo-
pyranosyl)-2-deoxy-4-O-methyl-ꢀ-D-galactopyranoside (8)
Compound 60 (35 mg, 0.06 mmol) was hydrogenated in a
similar fashion as compound 5. After evaporation, the resi-
due was dissolved in anhyd THF (2 mL), a solution of
TBAF in THF (1.0 M, 50 L) was added and reaction was
continued for 5 h. After concentration, disaccharide 8 was
obtained by reverse-phase HPLC using a gradient of H2O–
MeOH (0 J 30%) as eluent (20 mg, 88% yield), [ꢁ]D ꢃ47.6
Methyl 2-acetamido-3-O-benzoyl-6-O-benzyl-2-deoxy-4-
imidazoylthiocarbonyl-ꢀ-D-glucopyranoside (63)
1
(c 0.2, MeOH). H NMR (D2O) ꢂ: 4.62 (d, 1H, J = 0.6 Hz,
1,1>-Thiocarbonyldiimidazole (538 mg, 2.72 mmol) was
added to a solution of compound 62 (585 mg, 1.36 mmol) in
anhydrous toluene (10 mL) and the mixture was heated to
90°C overnight. The solution was diluted with EtOAc
(75 mL), washed with brine (1 × 25 mL), dried and concen-
trated to dryness to give 63 (691 mg, 94%), [ꢁ]D –21.0 (c
H-1>), 4.41 (d, 1H, J = 8.6 Hz, H-1), 3.97 (dd, 1H, J = 8.4,
10.8 Hz, H-2), 3.87 (dd, 1H, J = 3.1, 11.0 Hz, H-3), 3.86 (m,
2H, H-2>, H-3), 3.82 (“d”, 1H, J = 3.1 Hz, H-4), 3.77–3.81
(m, 2H, H-6a, H-6b), 3.67 (m, 1H, H-5), 3.55 (m, 1H, H-4>),
3.50 (s, 3H, OMe), 3.42 (dq, 1H, J = 6.2, 9.2 Hz, H-5>), 2.17
© 2002 NRC Canada

1160
Can. J. Chem. Vol. 80, 2002
1
0.8, CHCl3). H NMR (CDCl3) ꢂ: 8.13 (s, 1H, imidazole),
7.90 (m, 2H, Bz), 7.62 (s, 1H, imidazole), 7.51 (m, 1H, Bz),
7.17–7.40 (m, 7H, Ar), 7.02 (s, 1H, imidazole), 6.08 (t, 1H,
J = 9.5 Hz, H-4), 5.86 (d, 1H, J = 8.8 Hz, NH), 5.78 (t, 1H,
J = 10.5 Hz, H-3), 4.76 (d, 1H, J = 8.4 Hz, H-1), 4.46 (s,
2H, Bn), 4.08 (m, 1H, H-2), 3.96 (m, 1H, H-5), 3.71 (dd,
1H, J = 3.8, 10.6 Hz, H-6a), 3.65 (dd, 1H, J = 5.0, 10,8 Hz,
H-6b), 3.54 (s, 3H, OMe), 1.86 (s, 3H, Ac), Anal. calcd. for
C27H29O7N3S: C 60.10, H 5.42, N 7.79; found: C 60.21, H 5.33,
N 7.72.
gel using 5% MeOH – CH2Cl2 to afford the disaccharide 66
(91 mg, 54% yield) in pure form, [ꢁ]D +38.2 (c 0.5, CHCl3).
1H NMR (CDCl3) ꢂ: 8.10 (m, 2H, Bz), 7.57 (m, 1H, Bz),
7.44 (m, 2H, Bz), 7.25–7.33 (m, 10H, Bn), 5.41 (d, 1H, J =
6.7 Hz, NH), 4.96 (m, 1H, H-2>), 4.90 (d, 1H, J = 8.2 Hz,
H-1), 4.64 (d, 1H, J = 7.6 Hz, H-1>), 4.61 (d, 1H, J =
11.5 Hz, Bn), 4.57 (d, 1H, J = 12.1 Hz, Bn), 4.53 (d, 1H, J =
12.1 Hz, Bn), 4.46 (m, 1H, H-3), 4.45 (d, 1H, J = 11.7 Hz,
Bn), 3.72 (m, 1H, H-5), 3.56 (dd, 1H, J = 5.9, 10.1 Hz,
H-6a), 3.45–3.55 (m, 2H, H-5>, H-6b), 3.43 (s, 3H, OMe),
3.24 (m, 1H, H-4>), 2.76 (m, 1H, H-2), 2.65 (d>t>, 1H,
J = 4.9, 12.0 Hz, H-3e>), 2.24 (m, 1H, H-4e), 1.62 (d>t>, 1H,
J = 11.7, 11.7 Hz, H-3a>), 1.56 (s, 3H, Ac), 1.50 (d>t>, 1H,
J = 11.6, 11.6 Hz, H-4a), 1.30 (d, 3H, J = 6.1 Hz, H-6>).
HR-ES-MS m/e calcd. for C36H43O9N (MNa+): 656.28355;
found: 656.283345.
Methyl 2-acetamido-3-O-benzoyl-6-O-benzyl-2,4-
dideoxy-ꢀ-D-xylo-hexopyranoside (64)
A solution of 63 (321.4 mg, 0.60 mmol) in anhydrous to-
luene (15 mL) was heated to reflux, a solution of tributyltin
hydride (535 L, 1.93 mmol) in anhydrous toluene (5 mL)
and a catalytic amount of AIBN (~10 mg) were added. After
4 h, the deoxygenation was complete. Compound 64 was ob-
tained by chromatography on silica gel using 3% MeOH –
CH2Cl2 as eluent (217 mg, 88% yield), [ꢁ]D –8.2 (c 0.7,
Methyl 2-acetamido-3-O-(4-O-benzyl-3,6-dideoxy-ꢀ-D-
ribo-hexopyranosyl)-6-O-benzyl-2,4-dideoxy-ꢀ-D-xylo-
hexopyranoside (67)
1
CHCl3). H NMR (CDCl3) ꢂ: 7.99 (m, 2H, Bz), 7.54 (m 1H,
Disaccharide 66 (70 mg, 0.11 mmol) was dissolved in
anhyd MeOH (15 mL), and a solution of NaOMe in MeOH
(1.5 M, 200 L) was added, the reaction was stirred at room
temperature overnight. After neutralization with Amberlite
IR-120 (H+), the mixture was concentrated to dryness. The
alcohol 67 was obtained (53 mg, 91% yield) by chromatog-
raphy on silica gel using 7.5% MeOH – CH2Cl2 as eluent,
Bz), 7.41 (m, 2H, Bz), 7.24–7.35 (m, 5H, Bn), 5.42 (br, 1H,
NH), 4.57 (s, 2H, Bn), 4.43 (d, 1H J = 8.4 Hz, H-1), 4.02
(d>t>, 1H, J = 8.9, 10.3 Hz, H-2), 3.80 (m, 1H, H-5), 3.63
(dd, 1H, J = 5.7, 10.2 Hz, H-6a), 3.54 (dd, 1H, J = 4.6,
10.2 Hz, H-6b), 3.50 (s, 3H, Ome), 2.22 (ddd, 1H, J = 1.8,
5.1, 12.6 Hz, H-4e), 1.87 (s, 3H, Ac), 1.72 (d>t>, 1H, J =
11.5, 11.5 Hz, H-3a). Anal. calcd. for C23H27O6N: C 66.81, H
6.58, N 3.39; found: C 66.59, H 6.41, N 3.02.
1
[ꢁ]D +3.3 (c 0.6, CHCl3). H NMR (CDCl3) ꢂ: 7.22–7.34
(m, 10H, Bn), 5.58 (d, 1H, J = 7.9 Hz, NH), 4.57 (d, 1H, J =
11.3 Hz, Bn), 4.56 (d, 1H, J = 12.1 Hz, Bn), 4.53 (d, 1H, J =
12.1 Hz, Bn), 4.43 (d, 1H, J = 9.3 Hz, H-1), 4.41 (d, 1H, J =
11.8 Hz, Bn), 4.21 (d, 1H, J = 7.5 Hz, H-1>), 3.88 (m, 1H,
H-3), 3.68 (m, 1H, H-5), 3.59 (dd, 1H, J = 6.0 Hz, 10.1 Hz,
H-6a), 3.45–3.55 (m, 5H, H-2, H-6b, OMe), 3.34–3.42 (m,
2H, H-2>, H-5>), 3.10 (m, 1H, H-4>), 2.46 (d>t>, 1H, J = 4.6,
12.2 Hz, H-3e>), 2.19 (ddd, 1H, J = 1.8, 5.2, 13.4 Hz, H-4e),
1.99 (s, 3H, Ac), 1.57 (d>t>, 1H, J = 11.5, 11.4 Hz, H-4a),
1.38 (d>t>, 1H, J = 11.6, 11.6 Hz, H-3a>), 1.25 (d, 3H, J =
6.1 Hz, H-6>). HR-ES-MS m/e calcd. for C29H39O8N
(MNa+): 552.25734; found: 552.257502.
Methyl 2-acetamido-6-O-benzyl-2,4-dideoxy-ꢀ-D-xylo-
hexopyranoside (65)
A solution of NaOMe in MeOH (1.5 M, 200 L) was
added to a solution of compound 64 (400 mg, 0.967 mmol)
in anhyd MeOH (50 mL), and the reaction was stirred at
room temperature for 2 h. After neutralization with
Amberlite IR-120 (H+), the organic solution was evaporated,
and the acceptor 65 was obtained by chromatography on sil-
ica gel using 5% MeOH – CH2Cl2 as eluent (280 mg, 94%
yield), [ꢁ]D –63.3 (c 0.5, CHCl3). 1H NMR (CDCl3) ꢂ: 7.23–
7.34 (m, 5H, Bn), 6.08 (d, 1H, J = 4.6 Hz, NH), 4.57 (d, 1H,
J = 12.1 Hz, Bn), 4.52 (d, 1H, J = 12.1 Hz, Bn), 4.21 (d, 1H,
J = 8.1 Hz, H-1), 3.77 (m, 1H, H-3), 3.65 (m, 1H, H-5), 3.58
(dd, 1H, J = 6.0, 10.3 Hz, H-6a), 3.51 (dd, 1H, J = 4.3,
10.2 Hz, H-6b), 3.48 (s, 3H, OMe), 3.30 (m, 1H, H-2), 2.05
(m, 1H, H-4e), 2.02 (s, 3H, Ac), 1.46 (d>t>, 1H, J = 11.6,
11.6 Hz, H-4a). Anal. calcd. for C16H23O5N: C 62.12,
H 7.49, N 4.53; found: C 61.87, H 7.37, N 4.29.
Methyl 2-acetamido-3-O-(3,6-dideoxy-ꢀ-D-arabino-
hexopyranosyl)-2,4-dideoxy-ꢀ-D-xylo-hexopyranoside (7)
A solution of alcohol 67 (25 mg, 0.047 mmol) in anhyd
DMSO (2 mL) and Ac2O (1 mL) was stirred at 0°C for
30 min, and the reaction was left at room temperature over-
night. After evaporation, the residue was partitioned in a
mixture of EtOAc – 10% aqueous NaCl solution, and the or-
ganic solution was evaporated and dissolved in anhyd THF
(3 mL) at 0°C. A solution of L-selectride in THF (1.0 M,
200 L, 0.20 mmol) was added and the reaction was left at
0°C for 1 h. Water (0.3 mL) was added and the mixture was
diluted with EtOAc (50 mL), washed with Na2S2O3, brine,
dried and concentrated to give crude 69. Liquid ammonia
(25 mL) was collected in a flask containing compound 69
(15 mg, 0.028 mmol) equipped with a dry ice condenser. So-
dium (50 mg, 0.97 mmol) was added to this solution, and re-
action was continued for 4 h. Methanol (0.5 mL) was added,
and the condenser was removed to allow ammonia to evapo-
rate. The residue was dissolved in H2O (3 mL), and neutral-
ized with a saturated aqueous solution of NH4Cl and
Methyl 2-acetamido-3-O-(2-O-benzoyl-4-O-benzyl-3,6-
dideoxy-ꢀ-D-ribo-hexopyranosyl)-6-O-benzyl-2,4-
dideoxy-ꢀ-D-xylo-hexopyranoside (66)
N-Iodosuccinimide (125 mg, 0.52 mmol) was added to a
mixture of donor 22 (230 mg, 0.52 mmol), acceptor 65
(82 mg, 0.26 mmol), and 4 Å molecular sieves (350 mg) in
anhyd CH2Cl2 (5 mL) The reaction was cooled to –60°C,
and TfOH (5.0 L) was added. After 3 h, the reaction was
neutralized with Et3N (0.5 mL), diluted with EtOAc
(75 mL), and filtered. The organic solution was washed with
a 10% aqueous solution of Na2S2O3 (1 × 30 mL), and the
mixture was purified by repeated chromatography on silica
© 2002 NRC Canada

Zhang et al.
1161
15. L.A. Ellis, A.J. Reason, H.R. Morris, A. Dell, R. Iglesias,
F.M. Ubeira, and J.A. Appleton. Glycobiology, 4, 585 (1994).
16. L.A. Ellis, C.S. McVay, M.A. Probert, J. Zhang, D.R. Bundle,
and J.A. Appleton. Glycobiology, 7, 383 (1997).
17. O. Lüderitz. Angew. Chem. Intl. Ed. Engl. 9, 649 (1970).
18. Y. Zheng, M.Sc. Thesis, University of Alberta, 1998.
19. M.A. Probert, J. Zhang, and D.R. Bundle. Carbohydr. Res.
296, 149 (1996).
20. M. Nitz and D.R. Bundle. J. Org. Chem. 65, 3064 (2000).
21. M. Nitz. Ph.D. Thesis, University of Alberta, 2001.
22. M.A. Probert, J. Zhang, and D.R. Bundle. Carbohydr. Res.
296, 149 (1996).
evaporated. The desired disaccharide 7 was obtained by re-
verse-phase HPLC using a gradient of H2O–MeOH (0 J
30%) as eluent (4.0 mg, 23%), [ꢁ]D –55.5 (c 0.4, MeOH). 1H
NMR (D2O) ꢂ: 4.67 (“s”, 1H, H-1>), 4.40 (d, 1H, J = 8.6 Hz,
H-1), 3.89 (ddd, 1H, J = 5.1, 10.6, 10.6 Hz, H-3), 3.84 (m,
1H, H-2>), 3.63–3.72 (m, 3H, H-5, H-6a, H-6b), 3.62 (dd,
1H, J = 8.6, 10.1 Hz, H-2), 3.56 (ddd, 1H, J = 4.6, 9.3,
11.4 Hz, H-4>), 3.51 (s, 3H, OMe), 3.43 (dq, 1H, J = 6.1,
9.3 Hz, H-5>), 2.13–2.18 (m, 2H, H-3e>, H-4e), 2.02 (s, 3H,
Ac), 1.64 (ddd, 1H, J = 3.1, 11.5, 14.3 Hz, H-3a>), 1.50
(d>t>, 1H, J = 11.5, 11.5 Hz, H-4a), 1.27 (d, 3H, J = 6.2 Hz,
H-6>). HR-ES-MS m/e calcd. for C15H27O8N (MNa+):
372.16344; found: 372.163817.
23. J. Zhang, A. Otter, and D.R. Bundle. Bioorg. Med. Chem. 4,
1989 (1996).
24. J.A. Appleton, A. Dell, M. Nitz, and D.R. Bundle. Trends in
Glycoscience and Glycotechnology, 13, 481 (2002).
25. D.R. Bundle, E. Eichler, M.A.J. Gidney, M. Meldal, A. Ragauskas,
B.W. Sigurskjold, B. Sinnott, D.C. Watson, M. Yaguchi, and
N.M. Young. Biochemistry, 33, 5172 (1994).
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27. D.R. Bundle. Pure Appl. Chem. 61, 1171 (1989).
28. C.P.J. Glaudemans. Chem. Rev. 91, 25 (1991).
29. F.-I. Auzanneau, H.R. Hanna, and D.R. Bundle. Carbohydr.
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Acknowledgements
We thank Mrs. Joanna Sadowska for invaluable technical
support. Financial support was provided by research grants
to D.R. Bundle from the Natural Sciences and Engineering
Research Council of Canada (NSERC) and the University of
Alberta. Work in the laboratory of J.A. Appleton was sup-
ported by a grant from the National Institutes of Health
(AI14490).
30. F.-I. Auzanneau and D.R. Bundle. Carbohydr. Res. 247, 195
(1993).
31. K. Bock and B.W. Sigurskjold. In Studies in natural products
chemistry. Vol. 7. Edited by Atta-ur-Rahman. Elsevier, Am-
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© 2002 NRC Canada