Synthesis of hexa- to tridecasaccharides related to Shigella dysenteriae type 1 for incorporation in experimental vaccines

Article abstract of DOI:10.1016/j.carres.2006.04.006

Hexa- to tridecasaccharides corresponding to the O-specific polysaccharide (O-SP) of the Gram-negative bacterium Shigella dysenteriae type 1 were synthesized in solution phase. The syntheses utilized tetra-, octa-, and dodecasaccharide intermediates that

Full text of DOI:10.1016/j.carres.2006.04.006

Carbohydrate Research 341 (2006) 1408–1427
Synthesis of hexa- to tridecasaccharides related to Shigella
dysenteriae type 1 for incorporation in experimental vaccines
Vince Pozsgay,* Go¨ran Ekborg and Srinivasa-Gopalan Sampathkumar
Laboratory of Developmental and Molecular Immunity, National Institute of Child Health and Human Development,
National Institutes of Health, 31 Center Dr. Rm. 2A29, MSC 2423, Bethesda, MD 20892-2423, USA
Received 11 February 2006; received in revised form 27 March 2006; accepted 5 April 2006
Available online 2 May 2006
Abstract—Hexa- to tridecasaccharides corresponding to the O-specific polysaccharide (O-SP) of the Gram-negative bacterium Shi-
gella dysenteriae type 1 were synthesized in solution phase. The syntheses utilized tetra-, octa-, and dodecasaccharide intermediates
that represent one to three contiguous tetrasaccharide repeating units of the O-SP [Synlett 2003, 743]. These compounds were gly-
cosylated with mono-, di-, and trisaccharide trichloroacetamidates, which were synthesized in this study. The excellent stereodirect-
ing effect of 4,6-O-benzophenone ketals in glycosylation reactions of 2-azido-2-deoxy-glucopyranosyl donors was demonstrated.
The free oligosaccharides were characterized by 1H and 13C NMR spectroscopy and by high-resolution mass spectrometry. The oligo-
saccharides described herein contain the 5-(methoxycarbonyl)pentyl aglycon for eventual attachment to immunogenic carriers using
a recently published protocol [J. Org. Chem. 2005, 70, 6987].
ꢀ 2006 Elsevier Ltd. All rights reserved.
Keywords: Benzophenone ketal; Oligosaccharide; Protecting groups; Synthesis; Conjugation; Antibacterial; Vaccine
1. Introduction
saccharides of enteric bacteria kill the inoculum of the
pathogen, thus preventing infection. We hypothesized
that a glycoconjugate vaccine could be constructed
against S. dysenteriae type 1 from chemically defined,
extended oligosaccharide fragments of the O-SP2 of this
organism, when covalently bound to an immunogenic
carrier protein.3,4 The biological repeating unit of the
O-SP5,6 is:
Shigella dysenteriae type 1 is a human pathogen that can
cause endemic and epidemic diarrhea and dysentery
worldwide; these infections are characterized by enteric
and extraintestinal complications that can cause hemo-
lytic uremic syndrome with high rates of morbidity
and mortality.1 All strains of Shigellae develop increas-
[→3)-α-D-Rhap-(1→3)-α-L-Rhap-(1→2)-α-D-Galp-(1→3)-α-D-GlcpNAc-(1]
1
n
ing resistance to most available antibiotics. Despite its
discovery more than hundred years ago, there is still
no licensed vaccine against S. dysenteriae type 1.
A consensus on the mechanism of protection against
enteric bacteria is still missing. Robbins and co-workers1
advanced the idea that serum IgG antibodies to the O-
specific polysaccharide (O-SP) portion of the lipopoly-
We have reported that serum albumin conjugates of
frame-shifted tetra-, octa-, dodeca-, and hexadecasac-
charide fragments (2–5) of the O-SP, corresponding to
one to four tetrasaccharide repeating units, elicited O-
SP-specific IgG antibodies in mice.4 It was found that
the IgG antibody levels were related to both the chain
length of the hapten and the saccharide loading on the
protein, the latter in a non-linear fashion. Typically,
human serum albumin conjugates of dodeca- and hexa-
decasaccharides gave the highest antibody response
*
Corresponding author. Tel.: +1 301 402 0036; fax: +1 301 402
9108; e-mail addresses: pozsgayv@mail.nih.gov; vipo@helix.nih.gov
0008-6215/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2006.04.006

V. Pozsgay et al. / Carbohydrate Research 341 (2006) 1408–1427
1409
when the average loading was about 10 saccharide
chains per conjugate. Another variable in the construc-
tion of a synthetic carbohydrate–protein vaccine, which
has not been studied before, is the identity of the non-
reducing end residue. In the reported study, this residue
was invariably L-rhamnose.4 Although earlier studies7,8
indicated that several monoclonal antibodies raised
against the O-SP appeared to be groove-binders that
bind the saccharide along its chain, that is, the single res-
idue at the end of the saccharide chain might not play an
important role in its immunogenicity, a direct assess-
ment was missing.
approach to the target structures, the highest yields are
predictably obtained when the 3-hydroxyl group of a
rhamnopyranosyl moiety is the acceptor site.
Our approach to the spacer-equipped target oligosac-
charides 6–11 was based on previously published tetra-
21, octa- 44, and dodecasaccharides 48 (vide infra) that
were extended, at their reducing ends, with mono- 43,
di- 32, and trisaccharides 41, the latter two representing
the partial sequences a-D-GlcpNAc-(1!3)-a-L-Rhap
and a-D-Galp-(1!3)-a-D-GlcpNAc-(1!3)-a-L-Rhap, res-
pectively. For example, hexasaccharide 6 was assembled
from the disaccharide donor 32 and the lipidated tetra-
saccharide acceptor12 21. The presence of lipid protect-
ing groups in 21 makes this, and other lipidated
derivatives, amenable to purification by reverse phase
chromatography on a C-18-coated matrix while also
allowing the use of conventional separation on silica
gel if necessary.12,13
O(CH2)5CONHNH2
HO
HO
CH2OH
2
3
4
5
n = 1
n = 2
n = 3
n = 4
H3C
HO
O
O
O
O
OH
AcHN
O
The synthesis of compound 21 was reported,12 using 2-
azido-4,6-di-O-acetyl-3-O-bromoacetyl-2-deoxy-gluco-
pyranosyl bromide as the precursor of the 2-acetamido-
glucopyranose unit.11,12 The unsatisfactory yield in its
reaction with acceptor 15 prompted us to develop an im-
proved alternative. After having examined several possi-
ble donors having a variety of protecting groups, we
have found that benzophenone ketal 14 is the azido-
glucopyranose donor of choice. While not unprece-
dented, benzophenone ketals have found only a few appli-
cations as protecting moieties14 and there are only
sporadic reports on the use of this protecting group in car-
bohydrates15,16 and none describing benzophenone ketal
protected glycosyl donors. The rationale for our choice
was the anticipation that the one of the aromatic rings
of the ketal moiety in 14, which is axial in the ground state,
might sterically hinder an approaching nucleophile in the
transition state leading to the predominant formation of
the required a-anomer. Compound 14 was prepared
from triol17,18 12 in 96% yield by transacetalization with
benzophenone dimethyl acetal19 in the presence of cam-
phorsulfonic acid, followed by reaction with monochloro-
acetic anhydride in the presence of 2,4,6-tri-tert-butyl-
pyrimidine20 (Scheme 1). The three-bond, homonuclear
coupling constants in the 1H NMR spectra of compounds
13 and 14 agree with the corresponding values for 12
within less than 1 Hz, indicating no major conformational
change of the pyranose conformation.
H3C
HO
O
HO
O
CH2OH
O
n
OH
H
The eventual purpose of our studies is the develop-
ment of a synthetic oligosaccharide-based glycoconju-
gate vaccine against S. dysenteriae type 1.9,10 As part
of our endeavors, we have asked ourselves the question
whether the identity of the terminal residue has an effect
on anti-polysaccharide immunogenicity. In an attempt
to answer this question, we have synthesized a range
of oligosaccharides (6–11), corresponding to fragments
of the O-SP that are equipped with a spacer as the agly-
con for covalent attachment to immunogenic carriers.
The glycoconjugates so synthesized will be used in
immunization experiments in mice to evaluate the con-
tribution of the terminal sugar unit to the level of poly-
saccharide-specific antibody pool.
2. Results and discussion
The synthesis of the target oligosaccharides followed a
convergent, blockwise approach, relying on intermedi-
ates either described by us earlier11 or developed in this
study. The decision to use tetrasaccharide building
blocks was based entirely on the fact that it is the
Rhap-Galp-GlcpNAc-Rhap sequence that can be oligo-
merized with the highest degree of a-anomeric selectiv-
ity. This is because formation of a-rhamnopyranosyl
linkages is, essentially, completely stereoselective, which
is rarely the case with either the galactopyranosyl or the
glucopyranosaminyl components of the targets. Addi-
tionally, the 3-hydroxyl group of the rhamnopyranosyl
residue is more nucleophilic in glycosylation reactions
than either the 2-hydroxyl group of the galactopyrano-
syl or the 3-hydroxyl group of the glucopyranosaminyl
moieties. Consequently, when using the block synthetic
Next, azido-glucopyranose donor 14 was condensed
with a rhamnopyranose acceptor containing a (meth-
oxycarbonyl)pentyl aglycone (15)12 using the NIS/
TMSOTf promoter21,22 to furnish disaccharide 16 in
an excellent yield with less than 5% (estimated by
NMR) of the unwanted, b-linked anomer (Scheme 2).
The 1,2-cis configuration of the interglycosidic linkage
was ascertained by the characteristic one bond, C-1,H-
1 coupling constant whose value was 173 Hz.23 The ben-
zophenone ketal moiety was cleaved by treatment with

1410
V. Pozsgay et al. / Carbohydrate Research 341 (2006) 1408–1427
OMe
O
O
HO
HO
CH2OH
O
H3C
HO
O
O
OH
O
AcNH
O
H3C
HO
O
HO
6
O
CH2OH
O
OH
H3C
HO
O
O
OH
OMe
AcNH
O
O
HO
HO
CH2OH
O
H3C
HO
O
HO
O
CH2OH
HO
H3C
O
OH
O
AcNH
O
HO
7
O
O
HO
CH2OH
O
OH
CH2OH
HO
HO
H3C
HO
O
O
O
OH
AcNH
HO
O
HO
O
CH2OH
OMe
O
O
CH2OH
HO
HO
H3C
HO
OMe
O
O
O
O
CH2OH
HO
HO
O
OH
H3C
HO
O
AcHN
O
O
O
HO
H3C
HO
O
O
OH
O
AcHN
O
CH2OH
O
HO
O
H3C
HO
OH
2
O
O
H3C
HO
CH2OH
O
O
OH
2
O
H3C
HO
OH
O
AcHN
HO
HO
HO
OH
O
9
8
CH2OH
OMe
O
O
OMe
CH2OH
O
HO
HO
O
H3C
HO
O
O
CH2OH
HO
HO
H3C
HO
O
O
O
OH
AcHN
O
O
O
O
OH
H3C
HO
AcHN
O
HO
O
O
HO
CH2OH
H3C
HO
O
O
O
OH
2
CH2OH
O
CH2OH
HO
HO
H3C
HO
O
O
OH
3
H3C
HO
O
O
OH
AcHN
HO
HO
O
HO
OH
O
11
10
CH2OH
Ph
Ph
Ph
O
HO
HO
Ph
O
O
O
a
b
O
O
O
SPh
SPh
SPh
CAO
HO
HO
N3
N3
N3
12
14
13
Scheme 1. Reagents and conditions: (a) 2.3 equiv of benzophenone dimethyl ketal, camphorsulfonic acid, DMF, 45–50 ꢁC, 8 h, reduced pressure,
96%; (b) 1.3 equiv of chloroacetic anhydride, 2,4,6-tri-tert-butylpyrimidine, CH2Cl2, 96%.

V. Pozsgay et al. / Carbohydrate Research 341 (2006) 1408–1427
OR
1411
OR
OR
H3C
StO
H3C
StO
H3C
StO
O
O
O
OR
b,c
d
a
H3C
StO
O
O
O
O
OBz
OBz
17
OBz
18
N3
14
+
N3
N3
CAO
O
O
CAO
AcO
HO
HO
OBz
AcO
O
O
O
16
15
Ph
CH2OAc
CH2OAc
Ph
R = (CH2)5CO2Me
St = C17H35CO
Scheme 2. Reagents and conditions: (a) 2 equiv of 14, NIS, CH2Cl2, TMSOTf (cat), 0 ! 22 ꢁC, 96%; (b) CF3CO2H, aq HCl, 22 ꢁC; (c) Ac2O,
C5H5N, DMAP, 22 ꢁC, 20 min, 70%; (d) CH4N2S, DMF, C5H5N, 22 ꢁC, 2 h, 90%.
TFA/HCl followed by acetylation with Ac2O/C5H5N to
afford disaccharide 17 from which acceptor 18 was
obtained after treatment with thiourea, in 90% yield.
Subsequently, disaccharide 18 was transformed into
tetrasaccharide acceptor 21 using the galactopyranosyl
and rhamnopyranosyl donors, 1912 and 20,12 respec-
tively, as described previously.11
precursors for glycosyl donors with various leaving
groups at their reducing-end anomeric center.24
The disaccharide donor 32 was prepared as follows
(Scheme 3). First, triol 22 was treated with 2,2-dimeth-
oxypropane in the presence of camphorsulfonic acid to
afford acetonide derivative 23 followed by benzylation
with benzyl bromide/NaH to furnish 24. Acid-catalyzed
hydrolytic cleavage of the isopropylidene acetal, fol-
lowed by regioselective benzoylation with trimethyl
orthobenzoate via the formation of a 2,3-orthoester,
afforded glycosyl acceptor 25 in a high overall yield.
The presence of the benzoyl group at O-2 in 25 was sup-
ported by the characteristic doublet of doublets in its 1H
NMR spectrum at 5.31 ppm with three-bond coupling
constants of 1.7 and 3.5 Hz, respectively.
NH
CH2OLau
O
LauO
LauO
O
CCl3
H3C
StO
O
Cl
OBn
CAO
OBz
19
20
Lau = C12H25CO
Reaction of thioglucoside 14 with acceptor 25 under
NIS/TMSOTf activation21,22 as described above for
the preparation of stearoylated disaccharide 16 afforded
compound 26 in 89% yield (Scheme 4). The a-intergly-
cosidic linkage in 26 was indicated by the characteristic
3JH-1,H-2 coupling constant of the H-1 proton of the glu-
copyranose moiety (5.29 ppm), which is 3.8 Hz. The
benzophenone ketal moiety was cleaved by treatment
with AcOH/HCl yielding a diol (not characterized) fol-
lowed by acetylation with Ac2O/C5H5N to afford disac-
charide 27. The disaccharide alcohol 28 was obtained in
92% yield from 27 after treatment with thiourea. Next,
the free hydroxyl group in 28 was acetylated (giving
29) and the azido group was converted to an acetamido
group by hydrogenation over Pd/C in the presence of
triethylamine followed by treatment with Ac2O. Com-
pound 30 so obtained was exposed to a 4:1 mixture of
trifluoroacetic acid and CH2Cl2 to afford hemiacetal
OR
LauO
LauO
CH2OLau
O
H3C
StO
O
O
OBz
O
O
21
AcNH
H3C
StO
O
AcO
O
CH2OAc
HO
OBz
R = (CH2)5CO2Me
Having developed an efficient route to tetrasaccharide
acceptor 21, our attention was focused on the prepara-
tion of di- and trisaccharide donors 32 and 41, respec-
tively. The synthesis of both donors relied on the well-
documented property of the 2-(trimethylsilyl)ethyl
glycosides to tolerate most conditions employed in
oligosaccharide synthesis and on their sensitivity to
CF3CO2H leading to hemiacetals, which are excellent
OSE
OSE
OSE
OSE
H3C
HO
H3C
HO
O
H3C
BnO
O
a
b,c
H3C
BnO
O
d,e
O
O
HO
HO
O
O
OH
OBz
O
Me
Me
23
Me
Me
24
22
25
Scheme 3. Reagents and conditions: (a) 2,2-dimethoxypropane, 22 ꢁC, 4 h, 98%; (b) BnBr, NaH, DMF, 1 h, 99%; (c) AcOH, H2O, 65 ꢁC, 2 h;
(d) PhC(OMe)3, CSA, 2 h, 22 ꢁC; (e) AcOH, H2O, 87% for two steps.

1412
V. Pozsgay et al. / Carbohydrate Research 341 (2006) 1408–1427
OSE
OSE
OSE
H3C
BnO
H3C
BnO
H3C
BnO
O
O
O
e
b,c
d
a
O
O
O
OBz
14
25
OBz
OBz
28
+
N3
N3
N3
CAO
O
O
CAO
AcO
HO
AcO
O
O
O
27
26
Ph
CH2OAc
CH2OAc
Ph
OSE
O
CCl3
NH
OSE
OH
H3C
BnO
O
H3C
BnO
H3C
BnO
H3C
BnO
O
O
O
h
f,g
i
O
O
O
OBz
O
OBz
OBz
OBz
AcNH
AcNH
N3
AcNH
AcO
AcO
AcO
AcO
AcO
AcO
AcO
AcO
O
O
O
O
CH2OAc
32
CH2OAc
CH2OAc
CH2OAc
29
30
31
Scheme 4. Reagents and conditions: (a) 1.5 equiv of 14, NIS, TMSOTf, 0 ! 22 ꢁC, 5 h, 82%; (b) AcOH, aq HCl, 22 ꢁC, 20 min; (c) Ac2O, C5H5N,
DMAP, 22 ꢁC, 1 h, 68%; (d) CH4N2S, DMF, C5H5N, 22 ꢁC, 2 h, 92%), (e) Ac2O, C5H5N, DMAP, 22 ꢁC, 1 h, 95%; (f) H2, Pd/C, Et3N, EtOH; (g)
Ac2O, C5H5N, DMAP, 98% for two steps; (h) CF3CO2H, CH2Cl2, 22 ꢁC, 4 h, 92%; (i) CCl3CN, DBU, CH2Cl2, 22 ꢁC, 10 min, 85%.
31 from which imidate 32 was obtained by treatment
with trichloroacetonitrile and DBU.25
residue.11 We surmise that this is due to conforma-
tional mismatch26 between 21 and various glycosyl
donors because the corresponding hydroxyl group in
an octasaccharide representing two consecutive repeat-
ing units is highly reactive as suggested by the high
yields obtained with them.3,12 The structure of 33 is sup-
ported by high-resolution mass spectrometry and by the
NMR data catalogued in the Experimental. Particular
support derives from the presence of three resonances
(17.6–17.9 ppm) in the 13C NMR spectrum correspond-
ing to the C-6 atoms of the three rhamnose residues, six
resonances (98.9–93.7 ppm) corresponding to the six
anomeric carbon atoms and 14 carbonyl resonances
(173.9–165.2 ppm). The 1H NMR spectrum exhibits
Condensation of the disaccharide donor (32) with the
tetrasaccharide acceptor 21 under promotion by
TMSOTf afforded the protected hexasaccharide 33 in
33% yield, from which conventional removal of the
protecting groups afforded the target hexasaccharide
glycoside 6 (Scheme 5). While this yield is less than
satisfactory, it is not surprising in the light of earlier
experiences. Elongation of the partially protected tetra-
saccharide 21 or its analogues having different protect-
ing groups was similarly low-yielding, a finding that
suggests unusually low nucleophilicity of 3-hydroxyl
group of the non-reducing terminal rhamnopyranose
32
+
21
a
OMe
O
LauO
LauO
CH2OLau
O
H3C
StO
O
O
O
OBz
O
AcNH
O
H3C
StO
O
AcO
OBz
O
CH2OAc
O
H3C
BnO
O
33
O
OBz
AcNH
AcO
AcO
b,c
O
CH2OAc
6
Scheme 5. Reagents and conditions: (a) 5 equiv of 32, CH2Cl2, TMSOTf (cat), 0 ! 22 ꢁC, 2 h, 33%; (b) NaOCH3, CH2Cl2, CH3OH, 22 ꢁC; (c) H2,
Pd/C, 22 ꢁC, 150–200 psi, 48 h.

V. Pozsgay et al. / Carbohydrate Research 341 (2006) 1408–1427
1413
seven singlets (each representing three protons) that cor-
respond to the methyl groups of the seven acetyl groups.
It is well documented that glycosylation with a rhamno-
pyranosyl donor having a ‘participating’ group at O-2
proceeds in high a-1,2-trans-selectivity. Therefore, at
this stage no attempts were made for the verification
of the stereochemistry of the newly formed interglycosi-
dic linkage. This was performed on the deprotected oligo-
saccharides 6–11.
trimethylsilylethyl aglycon in a subsequent step, thus
exposing the 2-hydroxyl group of the galactopyranose
moiety, it was replaced by acetate ester in a two-step
process involving oxidative removal of the methoxyben-
zyl moiety using ammonium cerium(IV) nitrate followed
by O-acetylation to afford glycoside 39. A partial proof
for its proposed structure comes from the presence of
four three-proton singlets in its 1H NMR spectrum
between 2.1 and 1.5 ppm corresponding to four acetyl
groups. In the next step, the trimethylsilylethyl aglycon
in 39 was selectively cleaved by trifluoroacetic acid in
dichloromethane and the resulting hemiacetal 40 was
converted to imidate 41 using CCl3CN and DBU. Cou-
pling of tetrasaccharide 21 with the trisaccharide donor
41 in the presence of TMSOTf afforded the fully pro-
tected heptasaccharide 42 (Scheme 7). Conventional re-
moval of the protecting groups by sodium methoxide
mediated transacetylation followed by hydrogenolytic
cleavage of the benzyl protecting groups afforded hepta-
saccharide methyl ester 7.
Synthesis of nonasaccharide 45 was carried out by
condensation of octasaccharide alcohol 44 available
from an earlier study12 with the rhamnopyranosyl
donor3 43 as shown in Scheme 8. Conventional removal
of the protecting groups in two steps as described for the
preparation of 7 afforded the spacer-linked unprotected
nonasaccharide 8.
Alternatively, treatment of a solution of octasaccharide
alcohol12 44 and disaccharide donor 32 in CH2Cl2 with a
catalytic amount ofTMSOTfafforded the protected deca-
saccharide 46 from which the protecting groups were
removed by sequential treatment with sodium methoxide
in methanol followed by catalytic hydrogenolysis to
afford the unprotected decamer 9 (Scheme 9). Likewise,
undecasaccharide 47 was assembled from trisaccharide
donor 41 and octasaccharide alcohol12 44 as shown in
Scheme 10 using TMSOTf as the promoter.
Conventional removal of the protecting groups from
33 afforded the targeted hexasaccharide glycoside 6.
1
For 6, the one-bond JC-1,H-1 heteronuclear coupling
constants for the anomeric carbon atoms are within
the range of 170–173 Hz, indicating the a-stereochemis-
try of all glycosidic linkages. We note that similar argu-
ments were used for the structural verification of the
higher membered protected oligosaccharides, that is,
42, 45–47, and 49.
Heptasaccharide 7 was synthesized by coupling the
lipidated tetrasaccharide 21 with the trisaccharide donor
41. The preparation of the latter is shown in Scheme 6.
Thus, phenylthio galactopyranoside 34, available from
an earlier work,12 was hydrolyzed in the presence of
N-bromosuccinimide to afford hemiacetal 35, which
was transformed, in a quantitative yield, to imidate 36
using trichloroacetonitrile and DBU. Compound 36
was used immediately in condensation reactions with
disaccharide 28 under TMSOTf activation to afford tri-
saccharide 37 in 97% yield. The presence of two a-inter-
glycosidic linkages in 37 was indicated by two doublets
at 5.35 and 5.00 ppm in its 1H NMR spectrum with cou-
pling constants of 3.9 and 3.5 Hz, respectively. Sub-
sequently, the azido group was converted to an
acetamido moiety by reduction with triphenylphosphine
followed by N-acetylation with Ac2O to afford trisac-
charide 38. Because the 4-methoxybenzyl group would
not have survived the acid-mediated removal of the
OSE
CH2OBn
O
BnO
BnO
H3C
BnO
O
CH2OBn
O
CH2OBn
O
CH2OBn
O
BnO
BnO
BnO
BnO
BnO
BnO
a
b
c
O
NH
CCl3
OBz
R1
SPh
R2O
O
OH
O
MBnO
MBnO
MBnO
AcO
O
CH2OAc
34
35
36
37 R1 = N3
R2 = MBn
OR
CH2OBn
O
d
BnO
BnO
H3C
BnO
O
40 R = H
41 R = C(NH)CCl3
38 R1 = NHAc R2 = MBn
39 R1 = NHAc R2 = Ac
f
O
e
g
OBz
AcHN
AcO
O
AcO
O
CH2OAc
Scheme 6. Reagents and conditions: (a) NBS, THF, H2O, 0 ꢁC, 10 min, 94%; (b) CCl3CN, DBU, CH2Cl2, 22 ꢁC, 15 min, 95%; (c) 28, 4 equiv of 36,
CH2Cl2, TMSOTf (cat), ꢀ55 ꢁC ! ꢀ25 ꢁC, 97%; (d) PPh3, CH2Cl2, reflux, 12 h, then H2O and NH4OH, reflux 8 h, then Ac2O, 90%; (e) 2.3 equiv of
CAN, MeCN, H2O, 22 ꢁC, 1 h, then NaHSO3, NaHCO3, followed by Ac2O, C5H5N, DMAP, 22 ꢁC; (f) CF3CO2H, CH2Cl2, 22 ꢁC, 4 h, 85%; (g)
CCl3CN, DBU, CH2Cl2, 92%.

1414
V. Pozsgay et al. / Carbohydrate Research 341 (2006) 1408–1427
41
+
21
a
OMe
O
LauO
LauO
CH2OLau
O
O
H3C
StO
O
O
OBz
O
O
AcO
AcNH
H3C
StO
O
O
CH2OAc
O
OBz
CH2OBn
O
BnO
BnO
H3C
BnO
O
O
OBz
AcHN
42
AcO
O
AcO
O
b,c,d
CH2OAc
7
Scheme 7. Reagents and conditions: (a) 3 equiv of 41, TMSOTf (cat), 0 ꢁC, 8 h, 61%; (b) CH3ONa, CH3OH; (c) CH2N2; (d) H2/Pd–C, EtOH, AcOH.
OMe
O
O
CH2OOCC11H23
C11H23COO
H3C
O
O
C17H35COO
C11H23COO
O
OBz
AcHN
O
O
NH
H3C
O
AcO
O
C
17H35COO
CH2OAc
O
CCl3
O
OBz
BnO CH2OBn
O
H3C
BnO
H3C
BnO
O
O
+
BnO
O
O
CAO
OBz
OBz
AcHN
O
H3C
BnO
O
AcO
O
44
CH2OAc
43
HO
OBz
a
OMe
O
O
CH2OOCC11H23
C11H23COO
H3C
O
O
C17H35COO
C11H23COO
O
OBz
AcHN
O
O
H3C
O
AcO
O
C17H35COO
CH2OAc
O
OBz
BnO CH2OBn
O
H3C
BnO
O
BnO
O
O
OBz
AcHN
O
H3C
O
AcO
O
BnO
CH2OAc
O
OBz
45
H3C
O
BnO
CAO
OBz
b,c,d
8
Scheme 8. Reagents and conditions: (a) 6 equiv of 43, TMSOTf (cat), 0 ! 22 ꢁC, 20 min, 65%; (b) CH3ONa, CH3OH; (c) CH2N2; (d) H2/Pd–C,
EtOH, AcOH.

V. Pozsgay et al. / Carbohydrate Research 341 (2006) 1408–1427
1415
32
44
+
a
OMe
O
O
CH2OOCC11H23
C11H23COO
H3C
O
O
C17H35COO
C11H23COO
O
OBz
AcHN
O
O
H3C
O
AcO
O
C
17H35COO
CH2OAc
O
OBz
BnO CH2OBn
O
H3C
BnO
O
BnO
O
O
OBz
AcHN
O
H3C
BnO
O
AcO
O
46
CH2OAc
O
OBz
H3C
BnO
O
b,c,d
O
OBz
AcHN
AcO
AcO
O
9
CH2OAc
Scheme 9. Reagents and conditions: (a) 6 equiv of 32, CH2Cl2, TMSOTf (cat), 0 ! 22 ꢁC, 1 h, 72%; (b) CH3ONa, CH3OH; (c) CH2N2; (d) H2/Pd–C,
EtOH, AcOH.
41
44
+
a
OMe
O
O
CH2OOCC11H23
C11H23COO
H3C
O
O
C17H35COO
C11H23COO
O
OBz
AcHN
O
O
H3C
O
AcO
O
C17H35COO
CH2OAc
O
OBz
BnO CH2OBn
O
H3C
BnO
O
BnO
O
O
OBz
AcHN
O
H3C
BnO
O
AcO
O
47
CH2OAc
O
OBz
CH2OBn
O
BnO
BnO
H3C
BnO
O
b,c,d
O
OBz
AcHN
AcO
O
AcO
O
10
CH2OAc
Scheme 10. Reagents and conditions: (a) 4.8 equiv of 41, CH2Cl2, TMSOTf (cat), 0 ! 22 ꢁC, 4 h, 72%; (b) CH3ONa, CH3OH; (c) CH2N2; (d) H2/
Pd–C, EtOH, AcOH.
Similar to the foregoing examples, TMSOTf-mediated
condensation of the previously reported dodecasaccha-
ride alcohol12 48 and rhamnopyranosyl donor3 43
(Scheme 11) afforded the fully protected tridecasaccha-
ride 49 in 84% yield. Conventional removal of the
protecting groups from undeca- 47 and tridecasaccha-
rides 49 afforded unprotected oligosaccharides 10 and
11, respectively.
All the intermediates and the unprotected target
oligosaccharides were fully characterized by NMR

1416
V. Pozsgay et al. / Carbohydrate Research 341 (2006) 1408–1427
OMe
O
O
CH2OOCC11H23
C11H23COO
H3C
O
O
C17H35COO
C11H23COO
O
OBz
NH
CCl3
AcHN
O
O
O
H3C
O
AcO
O
H3C
BnO
C
17H35COO
CH2OAc
O
+
O
OBz
CAO
OBz
BnO CH2OBn
O
H3C
BnO
O
43
BnO
O
O
OBz
AcHN
O
H3C
BnO
O
AcO
O
48
CH2OAc
O
H
OBz
2
a
OMe
O
O
CH2OOCC11H23
C11H23COO
H3C
O
O
C17H35COO
C11H23COO
O
OBz
AcHN
O
O
H3C
O
AcO
O
C
17H35COO
CH2OAc
O
OBz
BnO CH2OBn
O
H3C
O
BnO
BnO
O
O
OBz
AcHN
O
H3C
BnO
O
AcO
O
49
CH2OAc
O
OBz
2
H3C
BnO
O
CAO
OBz
b,c
11
Scheme 11. Reagents and conditions: (a) 10 equiv of 43, TMSOTf (cat), 0 ! 22 ꢁC, 3 h, 72%; (b) CH3ONa, CH3OH; (c) CH2N2; (d) H2/Pd–C,
EtOH, AcOH.
spectroscopy and MS spectrometry, and their purities
have been established by elemental analytical and
spectroscopic methods as catalogued in detail in the
Experimental. The stereochemistry of the interglycosidic
linkages was verified by measuring their one-bond,
JC-1,H-1 coupling constants, which were all in the
170 Hz range, unequivocally establishing their 1,2-cis
(a) configurations.
attachment to immunogenic proteins. We have already
shown that the immunogenicity of such conjugates
depends on the length of the saccharide and on their
density on the protein, in a non-linear fashion. The
glycoconjugates to be prepared will be used to map
the role of the non-reducing terminal saccharide unit
on the anti-O-SP immunogenicity. These studies are ex-
pected to further refine our understanding of the struc-
tural factors that influence the immunogenicity and
will be reported in due course.
3. Conclusion
In summary, we have synthesized spacer-linked oligo-
saccharide fragments of the O-SP of the Gram-negative
human pathogen Shigella dysenteriae type 1 ranging
from hexa- to tridecasaccharides. These constructs are
currently being transformed into intermediates having
a reactive group in their aglyconic portion for covalent
4. Experimental
4.1. General methods
All chemicals were of commercial grade and were used
without purification. Anhydrous solvents were obtained

V. Pozsgay et al. / Carbohydrate Research 341 (2006) 1408–1427
1417
from Aldrich (St. Louis, MO). Column chromatography
was performed on Silica Gel 60 (0.040–0.063 mm) and
on dimethyloctadecylsilyl-bonded amorphous silica (C-
172 Hz), 100.1 (1JC-1,H-1 = 174 Hz), 98.5 (1JC-1,H-1 = 172
Hz), 95.1 (1JC-1,H-1 = 172 Hz), 95.9 (1JC-1,H-1 = 173
Hz), 78.9, 78.0, 75.9, 75.8, 75.5, 74.6, 72.7, 72.5, 72.1,
71.9, 71.7, 71.6, 71.3, 71.1, 70.4, 70.3, 70.1 (2C), 70.0,
69.8 (2C), 69.4, 69.3, 68.6, 67.6, 67.5, 63.3, 61.5, 61.3,
60.9, 60.8, 52.9, 52.8, 52.7, 34.4, 28.9, 25.7, 24.8, 22.90,
22.86, 17.6, 17.49, 17.47; ESIMS m/z calcd for
[C53H90N2O35+H]+: 1315.5402. Found: 1315.5385.
˚
18 adsorbent, 125 A, Waters, MA). Melting points were
taken on a Meltemp capillary melting point apparatus
and are uncorrected. NMR spectra were obtained using
either a Varian Mercury 300 spectrometer or a Bruker
DRX-500 spectrometer at 300 K. Internal references:
1
TMS (0.000 ppm for H for solutions in CDCl3), ace-
1
tone (2.225 ppm for H and 31.07 ppm for 13C for solu-
4.4. 5-(Methoxycarbonyl)pentyl a-L-rhamnopyranosyl-
(1!3)-a-L-rhamnopyranosyl-(1!2)-a-D-galactopyran-
osyl-(1!3)-2-acetamido-2-deoxy-a-D-glucopyranosyl-
(1!3)-a-L-rhamnopyranosyl-(1!3)-a-L-rhamnopyran-
osyl-(1!2)-a-D-galactopyranosyl-(1!3)-2-acetamido-2-
deoxy-a-D-glucopyranosyl-(1!3)-a-L-rhamnopyranoside
(8)
tions in D2O), MeOH (3.358 ppm for 1H and 49.68 ppm
for 13C for solution in CD3OD), and CDCl3 (77.00 ppm
for 13C for solutions in CDCl3). Coupling constants are
given in hertz. Mass spectra were recorded at the Labo-
ratory of Analytical Chemistry, NIDDK, NIH, Bethes-
da, MD. Elemental analyses were performed by Atlantic
Microlab, Inc., Norcross, GA.
1H NMR (D2O): d 5.61 (br d, 1H), 5.60 (br d, 1H), 5.11
(br s, 2H), 5.07–5.03 (m, 4H), 4.99 (d, 1H, J = 3.6 Hz),
4.82 (br s, 1H), 4.24 (br d, 1H), 4.17 (m, 2H), 3.69 (s,
3H), 3.46 (t, 1H, J = 9.6 Hz), 2.40 (t, 2H, J = 7.6 Hz),
2.060 (s, 3H), 2.055 (s, 3H), 1.66–1.57 (m, 4H), 1.44–
1.33 (m, 2H), 1.35–1.28 (m, 15H); 13C NMR (D2O): d
4.2. 5-(Methoxycarbonyl)pentyl 2-acetamido-2-deoxy-a-
D-glucopyranosyl-(1!3)-a-L-rhamnopyranosyl-(1!3)-a-
L-rhamnopyranosyl-(1!2)-a-D-galactopyranosyl-(1!3)-
2-acetamido-2-deoxy-a-D-glucopyranosyl-(1!3)-a-L-
rhamnopyranoside (6)
178.4, 174.9, 103.1 (1JC-1,H-1 = 171 Hz), 102.8 (1JC-1,H-1
=
=
1H NMR (D2O): d 5.60 (d, 1H, J = 3.7 Hz), 5.10 (d, 1H,
J = 1.6 Hz), 5.06 (d, 1H, J = 3.6 Hz), 5.05 (d, 1H,
J = 1.5 Hz), 4.99 (d, 1H, J = 3.7 Hz), 4.81 (br s, 1H),
4.19 (dd, 1H, J = 1.8 Hz, J = 2.6 Hz), 4.17 (dd,
1H, J = 1.9 Hz, J = 3.2 Hz), 4.14 (dd, 1H, J = 3.9 Hz,
J = 11.1 Hz), 3.69 (s, 3H), 3.65 (dd, 1H, J = 4.0 Hz,
J = 11.5 Hz), 2.41 (t, 2H, J = 7.8 Hz), 2.06 (s, 6H),
1.63 (m, 4H), 1.38 (m, 2H), 1.34 (d, 3H, J = 6.2 Hz),
1.31 (d, 3H, J = 6.2 Hz), 1.30 (d, 3H, J = 6.0 Hz); 13C
1
170 Hz), 102.3 (2C, JC-1,H-1 = 173 Hz), 100.3 (1JC-1,H-1
1
170 Hz), 98.5 (2C, JC-1,H-1 = 175 Hz), 94.9 (2C,
1JC-1,H-1 = 172 Hz), 78.9, 75.9, 75.6, 74.6, 72.9, 72.7,
71.9, 71.7, 71.0, 70.4, 70.3, 70.0, 69.8, 69.4, 68.6, 67.5,
91.5, 60.9, 52.9, 52.8, 34.4, 28.9, 25.7, 24.8, 22.9, 17.6,
17.5; ESIMS m/z calcd for [C65H110N2O43+H]+:
1607.6561. Found: 1607.6611.
NMR (D2O): d 178.4, 175.3, 174.9, 102.8 (1JC-1,H-1
=
4.5. 5-(Methoxycarbonyl)pentyl 2-acetamido-2-deoxy-a-
D-glucopyranosyl-(1!3)-a-L-rhamnopyranosyl-(1!3)-a-
L-rhamnopyranosyl-(1!2)-a-D-galactopyranosyl-(1!3)-
2-acetamido-2-deoxy-a-D-glucopyranosyl-(1!3)-a-L-
rhamnopyranosyl-(1!3)-a-L-rhamnopyranosyl-(1!2)-a-
D-galactopyranosyl-(1!3)-2-acetamido-2-deoxy-a-D-
glucopyranosyl-(1!3)-a-L-rhamnopyranoside (9)
172 Hz), 102.3 (1JC-1,H-1 = 171 Hz), 100.3 (1JC-1,H-1 = 172
Hz), 98.5 (1JC-1,H-1 = 171 Hz), 95.1 (1JC-1,H-1 = 170
Hz), 94.9 (1JC-1,H-1 = 173 Hz), 78.9, 76.1, 75.9, 75.6,
74.6, 72.7 (2C), 72.1, 71.9, 71.7 (2C), 71.18, 71.1, 70.5,
70.4, 70.3, 70.14, 70.0, 69.8, 69.4, 68.6, 67.7, 67.5, 61.5,
61.1, 60.9, 54.4, 52.9, 52.8, 34.4, 28.9, 25.7, 24.8, 22.9,
22.7, 17.6, 17.48, 17.46; ESIMS m/z calcd for
[C47H80N2O30+H]+: 1153.4874. Found: 1153.4889.
1H NMR (D2O): d 5.61 (br d, 1H), 5.60 (br d, 1H), 5.11
(br s, 2H), 5.07–5.03 (m, 4H), 4.99 (d, 1H, J = 3.6 Hz),
4.82 (br s, 1H), 4.23 (m, 1H), 4.20 (m, 1H), 3.69 (s, 3H),
2.39 (t, 2H, J = 7.0 Hz), 2.06 (s, 3H), 2.055 (s, 3H),
2.054 (s, 3H), 1.67–1.54 (m, 4H), 1.38 (m, 2H), 1.36–
1.27 (m, 15H); 13C NMR (D2O): d 178.4, 175.3, 174.9,
4.3. 5-(Methoxycarbonyl)pentyl a-D-galactopyranosyl-
(1!3)-2-acetamido-2-deoxy-a-D-glucopyranosyl-(1!3)-
a-L-rhamnopyranosyl-(1!3)-a-L-rhamnopyranosyl-
(1!2)-a-D-galactopyranosyl-(1!3)-2-acetamido-2-
deoxy-a-D-glucopyranosyl-(1!3)-a-L-rhamnopyranoside
(7)
1
1
102.8 (2C, JC-1,H-1 = 171 Hz), 102.2 (2C, JC-1,H-1
=
173 Hz), 100.4 (1JC-1,H-1 = 170 Hz), 98.5 (2C,
1JC-1,H-1 = 175 Hz), 95.1 (1JC-1,H-1 = 171 Hz), 94.9 (2C,
1JC-1,H-1 = 172 Hz), 78.9, 76.1, 75.9, 75.7, 75.6, 74.6,
72.7, 72.1, 71.9, 71.7, 71.2, 71.1, 70.6, 70.4, 70,3. 70.1,
70.0, 69.8, 69.5, 68.6, 67.7, 67.5, 61.5, 61.1, 60.9, 54.4,
52.9, 52.8, 34.4, 28.9, 25.7, 24.8, 22.9, 22.8, 17.6, 17.5;
ESIMS m/z calcd for [C73H123N3O48+H]+: 1810.735.
Found: 1810.7308.
1H NMR (D2O): d 5.59 (br d, 1H), 5.43 (br d, 1H), 5.10
(br s, 1H), 5.05 (br s, 2H), 4.99 (br d, 1H), 4.80 (br s,
1H), 3.69 (s, 3H), 2.40 (br t, 2H), 2.06 (s, 3H), 2.05 (s,
3H), 1.62 (m, 4H), 1.43–1.28 (m, 11H); 13C NMR
(D2O):
d
178.4, 175.0, 174.9, 102.8 (1JC-1,H-1
=
=
173 Hz), 102.3 (1JC-1,H-1 = 171 Hz), 100.3 (1JC-1,H-1

1418
V. Pozsgay et al. / Carbohydrate Research 341 (2006) 1408–1427
4.6. 5-(Methoxycarbonyl)pentyl a-D-galactopyranosyl-
(1!3)-2-acetamido-2-deoxy-a-D-glucopyranosyl-(1!3)-
a-L-rhamnopyranosyl-(1!3)-a-L-rhamnopyranosyl-
(1!2)-a-D-galactopyranosyl-(1!3)-2-acetamido-2-
deoxy-a-D-glucopyranosyl-(1!3)-a-L-rhamnopyranosyl-
(1!3)-a-L-rhamnopyranosyl-(1!2)-a-D-galactopyran-
osyl-(1!3)-2-acetamido-2-deoxy-a-D-glucopyranosyl-
(1!3)-a-L-rhamnopyranoside (10)
(300 mL) was added camphorsulfonic acid (4.0 g) at
22 ꢁC. The flask containing the reaction mixture was
attached to a rotary evaporator under vacuum (approx
20 mmHg) and was warmed to 45–50 ꢁC. After 8 h,
the mixture was treated with Et3N followed by the addi-
tion of aq NaHCO3. The mixture was concentrated
under vacuum at 30 ꢁC to a volume of approx 50 mL.
The residue so obtained was purified by column chro-
matography using a 20:1 ! 4:1 hexanes/EtOAc gradient
to afford amorphous 13 (66.8 g, 96%); 1H NMR
(CDCl3): d 7.20–7.55 (m, 15H), 4.51 (d, 1H, J =
10.2 Hz), 4.18 (dd, 1H, J = 5.1 Hz, 10.5 Hz), 3.83 (dt,
1H, J = 3.1 Hz, J 9.1 Hz), 3.81 (t, 1H, J = 10.4 Hz),
3.63 (t, 1H, J = 9.3 Hz), 3.56 (ddd, 1H, J = 5.0 Hz,
J = 9.7 Hz), 3.28 (dd, 1H, J = 9.0 Hz, J 10.2 Hz), 2.81
(d, 1H, J = 3.2 Hz); 13C NMR (CDCl3): d 143.0,
137.9, 133.2, 131.2, 129.1, 129.0, 128.5, 128.4, 128.3,
128.0, 127.5, 125.7, 102.6, 89.0, 74.6, 74.4, 71.1, 65.4,
63.4. Anal. Calcd for C25H23N3O4SÆ0.25 H2O: C,
64.43; H, 5.08. Found: C, 64.45; H, 5.21.
1H NMR (D2O): d 5.62–5.58 (m 2H), 5.41 (br d, 1H,
J = 3.6 Hz), 5.13–5.09 (m, 2H), 5.08–5.02 (m, 3H),
4.99 (br d, 1H), 4.83–4.78 (m, 2H), 3.69 (s, 3H), 2.40
(t, 2H, J = 7.0 Hz), 2.06 (s, 3H), 2.05 (s, 3H), 1.98–
1.53 (m, 4H), 1.37–1.28 (m, 15H); 13C NMR (D2O): d
1
178.4, 175.0, 174.9, 102.8 (2C, JC-1,H-1 = 171 Hz),
1
102.4 (2C, JC-1,H-1 = 172 Hz), 100.3 (1JC-1,H-1
=
=
169 Hz), 100.1 (1JC-1,H-1 = 173 Hz), 98.5 (2C, JC-1,H-1
1
175 Hz),
95.1
(1JC-1,H-1 = 172 Hz),
94.9
(2C,
1JC-1,H-1 = 172 Hz), 79.9, 78.0, 75.9, 75.6, 74.6, 72.7,
72.5, 72.1, 71.9, 71.7, 71.3, 71.2, 70.4, 70.3, 70.1, 70.0,
69.8, 69.5, 69.4, 68.6, 67.5, 61.5, 61.3, 60.9, 52.9, 52.78,
52.74, 34.4, 28.9, 25.7, 24.8, 22.91, 22.88, 17.7, 17.5;
ESIMS m/z calcd for [C79H129N3O53+2H++: 984.8825.
Found: 984.8765.
4.9. Phenyl 2-azido-3-O-chloroacetyl-2-deoxy-4,6-O-
diphenylmethylidene-1-thio-b-D-glucopyranoside (14)
To a stirred solution of 13 (49.0 g, 106 mmol), 4-dimeth-
ylaminopyridine (5.0 g), and 2,4,6-tri-tert-butylpyrimi-
dine20 (49.0 g) in CH2Cl2 (270 mL) was added
chloroacetic anhydride (24.0 g, 140 mmol) at 0 ꢁC. The
reaction mixture was removed from the ice bath. After
1 h, the solution was treated with CH3OH (10 mL) fol-
lowed by satd aq NaHCO3 (20 mL). The organic layer
was separated, was washed with H2O and was concen-
trated under reduced pressure. Column chromato-
graphic purification of the residue, using a 20:1 ! 4:1
hexanes/EtOAc gradient afforded amorphous 14
(55.2 g, 96%): 1H NMR (CDCl3): d 7.52–7.18 (m,
15H), 5.33 (t, 1H, J = 9.4 Hz, H-3), 4.59 (d, 1H,
J = 10.1 Hz, H-1), 4.23 (s, 2H), 4.22 (dd, 1H,
J = 5.0 Hz, J = 10.4 Hz, H-6), 3.85 (t, 1H, J = 10.2,
H-60), 3.74 (t, 1H, J = 10.3 Hz, H-4), 3.64 (dt, 1H,
J = 5.0 Hz, J = 9.8 Hz, H-5), 3.39 (t, 1H, J = 9.8 Hz,
H-2); 13C NMR (CDCl3): d 166.1, 142.7, 138.4, 133.6,
130.7, 129.2, 128.8, 128.4, 128.1, 127.1, 125.1, 102.3,
87.3, 75.1, 72.2, 71.4, 63.5, 40.5. Anal. Calcd for
C27H24ClN3O5S: C, 60.28; H, 4.50. Found: C, 60.40;
H, 4.29.
4.7. 5-(Methoxycarbonyl)pentyl a-L-rhamnopyranosyl-
(1!3)-a-L-rhamnopyranosyl-(1!2)-a-D-galactopyran-
osyl-(1!3)-2-acetamido-2-deoxy-a-D-glucopyranosyl-
(1!3)-a-L-rhamnopyranosyl-(1!3)-a-L-rhamnopyran-
osyl-(1!2)-a-D-galactopyranosyl-(1!3)-2-acetamido-2-
deoxy-a-D-glucopyranosyl-(1!3)-a-L-rhamnopyranosyl-
(1!3)-a-L-rhamnopyranosyl-(1!2)-a-D-galactopyran-
osyl-(1!3)-2-acetamido-2-deoxy-a-D-glucopyranosyl-
(1!3)-a-L-rhamnopyranoside (11)
1H NMR (D2O): d 5.62–5.58 (m, 3H), 5.11 (br s, 2H),
5.08 (br s, 1H), 5.02–5.07 (m, 5H), 4.99 (br d, 1H),
4.81 (br d, 1H), 4.23 (m, 2H), 4.17 (m, 3H), 3.69
(s, 3H), 3.46 (t, 1H, J = 9.7 Hz), 2.40 (t, 1H), 2.06
(s, 2H), 2.05 (s, 3H), 1.66–1.59 (m, 4H), 1.38
(m, 2H), 1.36–1.26 (m, 21H); 13C NMR (D2O): d
178.4, 174.9, 103.3 (1JC-1,H-1 = 170 Hz), 103.0 (2C,
1
1JC-1,H-1 = 171 Hz), 102.30 (3C, JC-1,H-1 = 172 Hz),
1
100.3 (1JC-1,H-1 = 170 Hz), 98.5 (3C, JC-1,H-1 = 175 Hz),
1
96.8 (3C, JC-1,H-1 = 172 Hz), 78.9, 78.8, 75.9, 75.7,
75.58, 75.56, 74.6, 72.9, 72.7, 72.12, 72.08, 72.0, 71.2,
71.1, 71.0, 70.9, 70.4, 70.3, 70.1, 70.0, 69.9, 69.8, 68.6,
67.5, 61.5, 60.9, 34.4, 28.9, 25.7, 24.8, 22.9, 17.6, 17.50,
17.48; ESIMS m/z calcd for [C91H153N3O61+2Li]++
:
4.10. 5-(Methoxycarbonyl)pentyl 2-azido-3-O-chloro-
acetyl-2-deoxy-4,6-O-diphenylmethylidene-a-D-glucopyran-
osyl-(1!3)-2-O-benzoyl-4-O-stearoyl-a-L-rhamnopyran-
oside (16)
1138.964. Found: 1138.9617.
4.8. Phenyl 2-azido-2-deoxy-4,6-O-diphenylmethylidene-
1-thio-b-D-glucopyranoside (13)
To a stirred solution of 14 (47.5 g, 88 mmol), 1512
(29.0 g, 44 mmol), and N-iodosuccinimide (50.0 g,
222 mmol) in anhydrous CH2Cl2 (300 mL) was added
To a solution of triol17,18 12 (45.0 g, 150 mmol) and ben-
zophenone dimethyl ketal19 (78.0 g, 340 mmol) in DMF

V. Pozsgay et al. / Carbohydrate Research 341 (2006) 1408–1427
1419
TMSOTf (400 lL) at 0 ꢁC. The reaction mixture was
allowed to reach 22 ꢁC over 1 h. At this time, TLC
(8:1, hexanes/EtOAc) indicated the complete disappear-
ance of 14. The reaction mixture was cooled to 0 ꢁC. An
aq solution of NaHSO3 (200 mL) was added followed
by solid NaHSO3 until the color of iodine disappeared.
To the mixture was added solid NaHCO3 until efferves-
cence stopped. The mixture was extracted with CHCl3.
The organic layer was concentrated and the residue
was purified by chromatography using 19:4 ! 2:1 hex-
anes/EtOAc as the eluant to afford 16 (42.4 g, 89%) as
an amorphous material: 1H NMR (CDCl3): d 8.13–
8.11 (m, 2H), 7.55–7.16 (m, 13H), 5.55 (dd, 1H,
J = 1.8 Hz, J = 3.2 Hz), 5.42 (t, 1H, J = 10.0 Hz), 5.30
(t, 1H, J = 10.9 Hz), 5.16 (d, 1H, J = 3.5 Hz), 4.87 (d,
1H, J = 1.6 Hz), 4.25 (dd, 1H, J = 1.3 Hz, J = 3.4 Hz),
4.18 (dd, 1H), 4.16 (m, 2H), 4.07 (ddd, 1H,
J = 4.9 Hz, J = 9.9 Hz), 3.87 (dq, 1H, J = 6.3 Hz),
3.81 (t, 1H, J = 10.3 Hz), 3.75 (t, 1H, J = 9.7 Hz), 3.68
(s, 2H), 3.45 (m, 1H), 3.10 (dd, 1H, J = 3.5 Hz,
J = 10.1 Hz), 2.34 (t, 2H, J = 7.4 Hz), 1.70–1.62 (m),
1.55–0.96 (m), 0.88 (m, 3H); 13C NMR (CDCl3): d
172.6, 166.0, 165.8, 142.9, 138.8, 133.5, 130.0, 129.2,
128.4, 128.3, 128.0, 127.9, 127.1, 125.0, 102.3, 97.6,
94.5, 73.1, 71.3, 70.9, 67.9, 67.7, 66.7, 63.8, 63.6, 60.9,
51.5, 40.5, 34.3, 33.9, 31.9, 29.7, 29.4, 29.3, 29.0, 28.9,
25.6, 24.7, 22.7, 17.6, 14.1. Anal. Calcd for
C59H80ClN3O14: C, 64.97; H, 7.39. Found: C, 65.07;
H, 7.49.
61.5, 60.4, 40.3, 34.2, 33.9, 31.9, 29.7, 29.5, 29.3, 29.0,
25.6, 24.8, 24.6, 22.7, 20.6, 20.5, 17.7, 14.1. Anal. Calcd
for C50H76ClN3O16: C, 59.42; H, 7.58. Found: C, 59.63;
H, 7.61.
4.12. 5-(Methoxycarbonyl)pentyl 4,6-di-O-acetyl-2-
deoxy-a-D-glucopyranosyl-(1!3)-2-O-benzoyl-4-O-
stearoyl-a-L-rhamnopyranoside (18)
To a stirred solution of compound 17 (14.5 g, 14 mmol)
in DMF (55 mL) and pyridine (5 mL) was added thio-
urea (3 g) at 22 ꢁC. After 2 h the solution was concen-
trated under reduced pressure. The residue was stirred
with CHCl3 followed by filtration. The solids were dis-
carded and the filtrate was concentrated. The residue
so obtained was purified by column chromatography
using 2:1 hexanes/EtOAc as the eluant to afford 18
(12.1 g, 90%) as an amorphous material: 1H NMR
(CDCl3): d 8.10 (m, 2H), 7.58 (m, 1H), 7.49 (m, 2H),
5.57 (dd, 1H, J = 1.9 Hz, J = 3.5 Hz), 5.14 (t, 1H,
J = 9.8 Hz), 5.14 (d, 1H, J = 3.5 Hz), 4.85 (d, 1H),
4.84 (dd, 1H), 4.18–4.22 (m, 2H), 4.11 (dd, 1H,
J = 2.3 Hz, J = 12.3 Hz), 3.90–3.82 (m, 3.69 (m, 1H),
3.67 (s, 3H), 3.45 (m, 1H), 3.21 (dd, 1H, J = 3.5 Hz,
J = 10.3 Hz), 2.34 (m, 4H), 2.08 (s, 3H), 2.04 (s, 3H),
1.70–1.60 (m, 6H), 1.43–1.37 (m, 3H), 1.36–1.22 (m,
28H), 0.88 (m, 3H); 13C NMR (CDCl3): d 173.9,
172.4, 170.4, 170.2, 165.8, 133.3, 129.8, 129.2, 128.3,
97.5, 93.7, 71.8, 71.3, 70.6, 69.6, 67.9, 67.7, 66.3, 62.9,
61.8, 51.4, 34.1, 33.8, 31.8, 29.6, 29.4, 29.2, 28.9, 25.5,
24.7, 24.5, 22.6, 20.6, 20.5, 17.5, 14.0. Anal. Calcd for
C48H75N3O15Æ2H2O: C, 59.43; H, 8.21. Found: C,
59.47; H, 7.92.
4.11. 5-(Methoxycarbonyl)pentyl 2-azido-4,6-di-O-acet-
yl-2-deoxy-3-O-chloroacetyl-a-D-glucopyranosyl-(1!3)-
2-O-benzoyl-4-O-stearoyl-a-L-rhamnopyranoside (17)
To a solution of 16 (10.0 g, 9.1 mmol) in CF3CO2H
(200 mL) was added concd aq HCl (4 mL) at 22 ꢁC. After
15 min, most of the volatiles were evaporated under
reduced pressure. The residual syrup was dissolved in
Ac2O (40 mL). To the solution so obtained were added
pyridine (40 mL) and 4-dimethylaminopyridine (0.2 g)
at 22 ꢁC. After 20 min, the volatiles were removed under
vacuum and the residue was purified by column chroma-
tography using 8:1 ! 4:1 hexanes/EtOAc as the eluant to
afford syrupy 17 (6.4 g, 70%): 1H NMR (CDCl3): d 8.12–
8.10 (m, 2H), 7.59–7.56 (m, 1H), 7.43–7.44 (m, 2H), 5.59
(dd, 1H, J = 1.8 Hz, J = 3.3 Hz), 5.28 (t, 1H, J =
9.8 Hz), 5.26 (d, 1H, J = 3.6 Hz), 5.24 (dd, 1H, J =
9.4 Hz, J = 10.4 Hz), 5.04 (dd, 1H, J = 9.4 Hz,
J = 10.1 Hz), 4.89 (d, 1H, J = 1.6 Hz), 4.13 (dd, 1H,
J = 2.2 Hz, J = 12.4 Hz), 1.87 (m, 2H), 3.85 (m, 1H),
3.65 (s, 3H), 3.47 (m, 1H), 3.34 (dd, 1H, J = 3.6 Hz,
J = 10.6 Hz), 2.39–2.34 (m, 4H), 2.10 (s, 3H), 2.00 (s,
3H), 1.75–1.60 (m, 8H), 1.07 (m, 3H); 13C NMR (CDCl3):
d 173.9, 172.2, 170.4, 169.5, 166.2, 165.9, 133.5, 130.0,
129.2, 128.5, 97.6, 93.7, 72.2, 71.6, 71.5, 68.0, 67.7, 66.6,
4.13. 2-(Trimethylsilyl)ethyl 2,3-O-isopropylidene-a-L-
rhamnopyranoside (23)
To a solution of 22 (94 g, 356 mmol) in 2,2-dimethoxy-
propane (400 mL) was added camphorsulfonic acid
(4 g) at 22 ꢁC. After 4 h, the mixture was treated with
Et3N (4 mL) followed by concentration under reduced
pressure. A solution of the residue in CHCl3 was
extracted with water. The organic layer was concen-
trated and the residue so obtained was purified by col-
umn chromatography using 6:1 ! 4:1 hexanes/EtOAc
1
as the eluant to afford 23 (98.3 g, 98%) as a syrup: H
NMR (CDCl3):
d 4.96 (s, 1H), 4.11 (dd, 1H,
J = 0.5 Hz, J = 5.7 Hz), 4.09 (t, 1H, J = 5.7 Hz,
J = 12.5 Hz), 3.80 (dq, 1H), 3.76 (dq, 1H), 3.73 (dq,
1H), 3.40 (m, 1H), 2.84 (d, 1H, J = 4.5 Hz), 1.53 (s,
3H), 1.36 (s, 3H), 1.29 (d, 1H, J = 6.3 Hz), 0.98 (m,
1H), 0.92 (m, 1H), 0.03 (s, 9H); 13C NMR (CDCl3): d
109.4, 100.2, 96.5, 78.4, 76.0, 74.3, 66.0, 64.9, 27.9,
26.1, 17.9, 17.6, ꢀ1.4. Anal. Calcd for C14H28O5Si: C,
55.23; H, 9.27. Found: C, 55.35; H, 9.33.

1420
V. Pozsgay et al. / Carbohydrate Research 341 (2006) 1408–1427
4.14. 2-(Trimethylsilyl)ethyl 4-O-benzyl-2,3-O-isopropyl-
idene-a-L-rhamnopyranoside (24)
4.16. 2-(Trimethylsilyl)ethyl 2-azido-3-O-chloroacetyl-2-
deoxy-4,6-O-diphenylmethylidene-a-D-glucopyranosyl-
(1!3)-2-O-benzoyl-4-O-benzyl-a-L-rhamnopyranoside
(26)
To a stirred solution of 23 (110.0 g, 362 mmol) in anhy-
drous DMF (300 mL) was added NaH (60% suspension
in oil) in portions. After 30 min, benzyl bromide (57 mL,
480 mmol) was added dropwise. After 1 h, the solution
was cooled in ice-water and was treated with CH3OH
(excess). The solution was concentrated under vacuum
at <30 ꢁC. The residue so obtained was purified by col-
umn chromatography using 20:1 ! 10:1 hexanes/
EtOAc gradient containing 0.01% Et3N to afford 24
To a stirred solution of 25 (2.9 g, 6.3 mmol), 14 (4.9 g,
9.1 mmol), and N-iodosuccinimide (5.5 g) in anhydrous
CH2Cl2 (30 mL) was added TMSOTf (17 lL) at 0 ꢁC.
The solution was allowed to reach 22 ꢁC. After 5 h, the
solution was cooled in an ice bath followed by the addi-
tion of Et3N (0.2 mL). The solution was treated with a
10% aq solution of NaHSO3 (approx 300 mL) followed
by satd aq NaHCO3 until effervescence stopped. The or-
ganic layer was washed with H2O twice, and was then
concentrated. Column chromatographic purification of
the residue in 2:1 hexanes/EtOAc afforded syrupy 26
1
(141.0 g, 99%) as a syrup: H NMR (CDCl3): d 7.36–
7.26 (m, 5H), 4.96 (d, 1H, J = 0.6 Hz), 4.90 (d, 1H,
J = 11.6 Hz), 4.62 (d, 1H, J = 11.6 Hz), 4.26 (t, 1H,
J = 7.0 Hz), 4.11 (dd, 1H, J = 0.6 Hz, J = 5.4 Hz),
3.76 (dq), 3.69 (dq, 1H), 3.49 (dq, 1H), 3.21 (dd, 1H,
J = 7.0 Hz, J 9.7 Hz), 1.60 (s, 3H), 1.37 (s, 3H), 1.27
(d, 1H, 6.3 Hz), 0.01 (s, 9H); 13C NMR (CDCl3): d
128.2, 128.0, 127.6, 109.1, 96.4, 81.2, 78.7, 76.3, 72.9,
64.7, 64.4 28.0, 26.3, 17.9, ꢀ1.4. Anal. Calcd for
C21H34O5SiÆ0.25 C6H14: C, 64.94; H, 9.08. Found: C,
64.71; H, 9.08.
1
(4.6 g, 82%): H NMR (CDCl3): d 8.13–8.11 (m, 2H),
7.61–7.58 (m, 1H), 7.52–7.23 (m, 14H), 7.09 (m, 1H),
6.86 (m, 2H), 5.72 (t, 1H, J = 9.9 Hz), 5.56 (dd, 1H,
J = 1.8, J = 3.2), 5.29 (d, 1H, J = 3.8), 4.95 (d, 1H
J = 10.1), 4.86 (d, 1H, J = 1.8 Hz), 4.64 (d, 1H J =
10.1 Hz), 4.32 (dd, 1H, J = 3.3 Hz, J = 9.6 Hz), 4.26
(ddd, 1H), 4.21 (d, 2H), 4.11 (dd, 1H, J = 5.0 Hz,
J = 10.2 Hz), 3.85 (dq, 1H, J = 6.3 Hz, J = 9.6 Hz),
3.81 (t, 1H, J = 9.7 Hz), 3.76 (t, 1H, J = 10.3 Hz), 3.67
(t, 1H, J = 9.6 Hz), 3.51 (m, 1H), 3.10 (dd, 1H,
J = 3.6 Hz, J = 10.2), 1.43 (d, 1H, J = 6.3 Hz), 0.96 (m,
1H), 0.89 (m, 1H), 0.01 (s, 9H); 13C NMR (CDCl3): d
166.3, 166.0, 143.2, 138.9, 137.1, 133.5, 132.4, 130.0,
129.9, 129.3, 129.2, 128.7, 128.6, 128.5, 128.4, 128.3,
128.0, 127.9, 127.8, 127.6, 127.1, 125.3, 102.3, 97.0,
93.7, 79.9, 73.1, 72.8, 71.0, 67.9, 67.3, 65.3, 63.5, 61.0,
40.5, 18.0, ꢀ1.32. Anal. Calcd for C46H52ClN3O11Si: C,
62.33; H, 5.91. Found: C, 61.99; H, 5.93.
4.15. 2-(Trimethylsilyl)ethyl 2-O-benzoyl-4-O-benzyl-a-
L-rhamnopyranoside (25)
A solution of 24 (141 g, 358 mmol) in AcOH (700 mL)
and H2O (240 mL) was kept at 65 ꢁC for 2 h. The vola-
tiles were removed under reduced pressure. Toluene
(100 mL) was added to and evaporated from the residue
thrice. The residue so obtained was purified by column
chromatography using 6:1 ! 4:1 hexanes/EtOAc as
the eluant to afford 120 g of an intermediate as a semi-
solid. A stirred solution of this material in CH2Cl2
(450 mL) and trimethylorthobenzoate (75 mL) was trea-
ted with camphorsulfonic acid (2 g). After 2 h, a mixture
of AcOH (240 mL) and H2O (25 mL) was added. After
10 min, a satd aq solution of NaHCO3 (50 mL) was
added followed by removal of the volatiles under re-
duced pressure. The residue was equilibrated between
CHCl3 and H2O. The organic layer was concentrated
and the residue was purified by chromatography using
10:1 ! 6:1 hexanes/EtOAc as the eluant to yield 25
(141 g, 87% for two steps) as a syrup: 1H NMR
(CDCl3): d 8.06 (m, 1H), 7.59–7.35 (m, 9H), 5.31 (dd,
1H, J = 1.7 Hz, J = 3.5 Hz), 4.87 (d, 1H, J = 1.7 Hz),
4.85 (d, 1H, J = 10.2 Hz), 4.75 (d, 1H, J = 10.2 Hz),
4.23 (m, 1H), 3.82 (dq), 3.79 (dq, 1H), 3.51 (dq, 1H),
3.46 (t, 1H, J = 9.5 Hz), 1.39 (d, 1H, J = 6.3 Hz),
1.00–0.88 (m, 2H), 0.02 (s, 9H); 13C NMR (CDCl3): d
166.4, 138.2, 133.3, 129.8, 128.5, 128.4, 128.0, 127.9,
96.9, 81.8, 75.1, 73.6, 70.6, 67.4, 65.2, 18.1, 17.9, ꢀ1.4.
Anal. Calcd for C25H34O6Si: C, 65.47; H, 7.47. Found:
C, 65.54; H, 7.60.
4.17. 2-(Trimethylsilyl)ethyl 4,6-di-O-acetyl-2-azido-2-
deoxy-3-O-chloroacetyl-a-D-glucopyranosyl-(1!3)-2-O-
benzoyl-4-O-benzyl-a-L-rhamnopyranoside (27)
To a solution of 26 (22.0 g, 24.8 mmol) in AcOH
(110 mL) was added concd aq HCl (4.0 mL) at 22 ꢁC.
After 20 min, the solution was concentrated under re-
duced pressure at <30 ꢁC to a thin syrup. To the residue
were added Ac2O (40 mL), pyridine (60 mL) and 4-di-
methylaminopyridine (0.5 g) at 22 ꢁC. After 1 h, the vola-
tiles were removed under vacuum and the residue was
purified by column chromatography using 8:1 ! 5:1 hex-
anes/EtOAc gradient as the eluant to afford 27 (13.5 g,
1
68%): H NMR (CDCl3): d 8.09 (m 2H), 7.57 (m, 1H),
7.46–7.23 (m, 7H), 5.56 (dd, 1H, J = 2.0 Hz,
J = 3.2 Hz), 5.41 (dd, 1H, J = 9.4 Hz, J = 10.5 Hz),
5.34 (d, 1H, J = 3.6 Hz), 5.01 (t, 1H, J = 9.5 Hz), 4.85
(br s, 1H), 4.84 (d, 1H, J = 11.1 Hz), 4.76 (d, 1H,
J = 11.1 Hz), 4.26 (dd, 1H, J = 3.3 Hz, J = 9.6 Hz),
3.97 (s, 1H), 3.96 (s, 1H), 3.91 (dd, 1H, J = 2.3 Hz,
J = 12.4 Hz), 3.86 (dq, 1H), 3.78 (m, 1H), 3.60 (t, 1H,

V. Pozsgay et al. / Carbohydrate Research 341 (2006) 1408–1427
1421
J = 9.8 Hz), 3.51 (m, 1H), 3.27 (dd, 1H, J = 3.6 Hz,
J = 10.6 Hz), 2.02 (s, 3H), 1.84 (s, 3H), 1.40 (d, 1H,
J = 6.3 Hz), 0.98 (m, 1H), 0.91 (m, 1H), 0.01 (s, 9H);
13C NMR (CDCl3): d 170.4, 169.4, 166.5, 165.8, 137.8,
129.9, 129.4, 128.3, 127.7, 127.4, 97.0, 92.1, 79.6, 75.7,
72.9, 72.1, 67.9, 67.6, 67.4, 67.3, 65.4, 61.2, 60.4, 40.3,
20.6, 20.4, 18.0, 17.9, ꢀ1.4. Anal. Calcd for C37H48-
ClN3O13Si: C, 55.11; H, 6.00. Found: C, 55.15; H, 6.11.
J = 6.3 Hz), 3.79 (dt, 1H, J = 6.0 Hz, J = 10.5 Hz),
3.63 (t, 1H, J = 9.6 Hz), 3.53 (m, 1H), 3.27 (dd, 1H,
J = 3.6 Hz, J = 10.6 Hz), 2.05 (s, 3H), 2.01 (s, 3H),
1.88 (s, 3H), 1.59 (s, 3H), 1.43 (d, 3H, J = 6.3 Hz),
0.97 (m, 1H), 0.92 (m, 1H), 0.03 (s, 9H); 13C NMR
(CDCl3): d 170.5, 169.8, 169.5, 165.9, 137.9, 133.3,
129.9, 129.5, 128.4, 127.7, 127.6, 97.1, 93.0, 79.6, 75.7,
72.9, 70.2, 68.0, 67.9, 67.5, 67.3, 65.4, 61.3, 60.6, 20.7,
20.6, 20.5, 18.0, 17.9, ꢀ1.4. Anal. Calcd for
C37H49N3O13Si: C, 57.57; H, 6.40. Found: C, 57.66; H,
6.32.
4.18. 2-(Trimethylsilyl)ethyl 4,6-di-O-acetyl-2-azido-2-
deoxy-a-D-glucopyranosyl-(1!3)-2-O-benzoyl-4-O-benz-
yl-a-L-rhamnopyranoside (28)
4.20. 2-Trimethylsilylethyl 2-acetamido-3,4,6-tri-O-acet-
yl-2-deoxy-a-D-glucopyranosyl-(1!3)-2-O-benzoyl-4-O-
benzyl-a-L-rhamnopyranoside (30)
To a stirred solution of compound 27 (33.8 g, 42 mmol)
in DMF (175 mL) and pyridine (16 mL) was added thio-
urea (16 g) at 22 ꢁC. After 2 h, the solution was concen-
trated under reduced pressure. The residue was stirred
with CHCl3 followed by filtration. The solids were dis-
carded and the filtrate was concentrated. The residue
so obtained was purified by column chromatography
using 5:1 ! 2:1 hexanes/EtOAc gradient to afford 28
(28.2 g, 92%) as a syrup: 1H NMR (CDCl3): d 8.13
(m, 2H), 7.60 (m, 1H), 7.47 (m, 2H), 7.36–7.32 (m,
5H), 5.57 (dd, 1H), 5.26 (d, 1H, J = 3.6 Hz), 4.87 (d,
1H, J = 1.7 Hz), 4.84 (t, 1H, J = 9.7 Hz), 4.80 (d, 1H,
J = 11.2 Hz), 4.72 (d, 1H, J = 11.2 Hz), 4.28 (dd, 1H,
J = 3.4 Hz, J = 9.6 Hz), 3.87 (dq, 1H, J = 6.3 Hz),
3.54 (dt, 1H), 3.59 (t, 1H, J = 9.5 Hz), 3.52 (dd, 1H,
J = 5.9 Hz), J = 10.2 Hz), 3.21 (dd, 1H, J = 3.6 Hz,
J = 10.2 Hz), 2.06 (s, 3H), 1.97 (s, 3H), 1.38 (d, 3H,
J = 6.3 Hz), 0.98 (m, 1H), 0.92 (m, 1H), 0.03 (s, 9H);
13C NMR (CDCl3): d 170.8, 170.6, 165.8, 137.8, 133.3,
129.8, 129.5, 128.3, 127.7, 127.4, 97.0, 93.0, 79.6, 75.4,
72.8, 70.7, 70.1, 67.7, 67.5, 67.3, 65.3, 62.9, 61.6, 20.6,
18.0, 17.9, ꢀ1.4. Anal. Calcd for C35H47N3O12Si: C,
57.60; H, 6.49. Found: C, 57.53; H, 6.55.
A mixture of 29 (2.7 g, 3.5 mmol), EtOH (25 mL), Et3N
(2 mL), and 10% Pd/C (0.5 g) was stirred under hydro-
gen at 200 psi at 22 ꢁC for 12 h. The mixture was filtered
and the solids were washed with CH2Cl2. The combined
organic layer was concentrated under reduced pressure.
To a solution of the residue in pyridine (7.5 mL) was
added Ac2O (7.5 mL) followed by a catalytic amount
of 4-dimethylaminopyridine at 22 ꢁC. After 2 h, the vol-
atiles were removed under vacuum. Column chromato-
graphic purification of the residue using 1:1 hexanes/
EtOAc as the eluant afforded amorphous 30 (2.70 g,
98%): 1H NMR (CDCl3): d 8.09 (m, 2H), 7.65
(m, 1H), 7.53 (m, 2H), 7.40–7.25 (m, 5H), 5.43 (dd,
1H, J = 1.8 Hz, J = 3.1 Hz), 5.36 (d, 1H, J = 9.7 Hz),
5.18 (dd, 1H, J = 9.7 Hz, J = 10.6 Hz), 5.11 (t,
1H, J = 9.7 Hz), 5.06 (d, 1H, J = 3.7 Hz), 4.89 (d, 1H,
J = 11.8 Hz), 4.84 (d, 1H, J = 1.8 Hz), 4.79 (d, 1H,
J = 11.8 Hz), 4.38 (dt, 1H, J = 3.7 Hz, J = 9.8 Hz),
4.30 (dd, 1H, J = 3.2 Hz, J = 10.7 Hz), 4.11 (m, 1H),
3.86–3.92 (m, 3H), 3.78 (dq, 1H), 3.58 (t, 1H,
J = 9.6 Hz), 3.52 (d, 1H), 2.04 (s, 3H), 1.95 (s, 3H),
1.86 (s, 3H), 1.45 (d, 3H, J = 6.3 Hz), 1.04–0.88 (m,
2H), 0.03 (s, 9H); 13C NMR (CDCl3): d 171.3, 170.6,
169.8, 169.0, 165.4, 137.7, 133.8, 129.8, 129.3, 128.8,
128.4, 127.8, 127.3, 96.9, 93.6, 79.7, 75.5, 73.1, 71.6,
68.7, 68.1, 67.6, 67.5, 65.5, 61.2, 51.1, 22.4, 20.7, 20.5,
18.1, 17.9. Anal. Calcd for C39H53N3O12Si: C, 59.45;
H, 6.78. Found: C, 59.23; H, 6.82.
4.19. 2-(Trimethylsilyl)ethyl 3,4,6-tri-O-acetyl-2-azido-
2-deoxy-a-D-glucopyranosyl-(1!3)-2-O-benzoyl-4-O-
benzyl-a-L-rhamnopyranoside (29)
To a stirred solution of 28 (2.9 g, 4.0 mmol) in pyridine
(10 mL) and Ac2O (10 mL) was added a catalytic
amount of 4-dimethylaminopyridine at 22 ꢁC. After
1 h, the volatiles were removed under vacuum. The res-
idue was purified by column chromatography using
5:1 ! 3:1 hexanes/EtOAc gradient to yield amorphous
29 (2.9 g, 95%): 1H NMR (CDCl3): d 8.13 (m, 2H),
7.59 (m, 1H), 7.46 (m, 2H), 7.42 (m, 2H), 7.34 (m,
2H), 7.28 (m, 2H), 5.58 (dd, 1H, J = 1.8 Hz,
J = 3.1 Hz), 5.42 (dd, J = 9.4 Hz, J = 10.6 Hz), 5.33
(d, 1H, J = 3.7 Hz), 5.01 (t, 1H, J = 9.6 Hz), 4.89 (d,
1H J = 11.2 Hz), 4.88 (d, 1H, J = 1.8 Hz), 4.78 (d, 1H,
J = 11.2 Hz), 4.29 (dd, 1H, J = 3.3 Hz, J = 9.6 Hz),
4.12 (m, 1H), 4.06 (dd, 1H, J = 3.6 Hz, J = 12.4 Hz),
3.92 (dd, 1H, J = 2.2 Hz, J = 12.4 Hz), 3.88 (dq, 1H,
4.21. 2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-a-D-gluco-
pyranosyl-(1!3)-2-O-benzoyl-4-O-benzyl-a,b-L-rhamno-
pyranose (31)
To a stirred solution of 30 (2.75 g, 3.5 mmol) in CH2Cl2
(10 mL) was added trifluoroacetic acid (40 mL) at 22 ꢁC.
After 4 h, the mixture was concentrated. Toluene was
added to and evaporated from the residue several times.
Column chromatographic purification of the residue
using 2:1 ! 1:1 hexanes/EtOAc as the eluant afforded
1
31 (2.2 g, 92%) as a solid material: mp 82–85 ꢁC; H

1422
V. Pozsgay et al. / Carbohydrate Research 341 (2006) 1408–1427
NMR (CDCl3): d (mixture of anomers a/b ꢁ9/1) 8.20–
7.20 (m, 10H), 5.64 (br s, 0.1H, J = 2.7 Hz), 5.49 (dd,
1H, J = 2.6, 2.2 Hz), 5.44 (d, 1H, J = 9.6 Hz), 5.24
(br s, 0.9H), 5.18 (t, 1H, J = 9.6 Hz), 5.09 (d,
1H, J = 3.5 Hz), 5.08 (t, 1H, J = 9.6 Hz), 4.88 (d, 1H,
J = 11.0 Hz), 4.79 (d, 1H, J = 11.0 Hz), 4.39 (dd,
1H J = 3.1 Hz, J = 9.4 Hz), 4.37 (dt, 1H, J = 3.1 Hz,
J = 9.3 Hz), 4.23 (br s, 1H), 4.18–4.09 (m, 2H), 4.02–
3.83 (m, 2H), 3.59 (t, 1H, J = 9.6 Hz), 2.03 (s, 3H),
1.95 (s, 3H), 1.85 (s, 3H), 1.43 (s, 3H), 1.43 (d, 3H,
J = 6.0 Hz); 13C NMR (CDCl3): d 171.3, 170.7, 170.2,
169.1, 165.9, 165.4, 93.4, 92.9, 92.0, 79.6, 78.9, 75.5,
75.4, 74.9, 72.6, 72.1, 71.5, 71.3, 69.2, 69.0, 67.9, 67.7,
67.5, 61.2, 51.5, 51.0, 22.4, 20.6 (2C), 20.4, 18.2; FABMS
m/z [M+H]+: 688. Anal. Calcd for C34H41NO14: C,
59.38; H, 6.01. Found: C, 59.33; H, 5.94.
satd aq NaHCO3. The organic layer was concentrated
under reduced pressure. The residue was purified by
chromatography on a C-18 column prepared in CH3OH
that was eluted with a gradient of CH3OH/EtOH. The
product was eluted with a 2:1 mixture of CH3OH and
EtOH. The fractions containing the product were pooled
followed by concentration under reduced pressure. The
residue was further purified by silica gel chromatography
using 2:1 ! 3:2 hexanes/EtOAc as the eluant to afford
amorphous 33 (430 mg, 33%): 1H NMR (CDCl3): d
8.0–8.1 (m, 5H), 7.65–7.05 (m, 15H), 5.46 (dd, 1H,
J = 1.7 Hz, J = 3.2 Hz), 5.38 (dd, 1H), 5.30 (d, 1H,
J = 9.8 Hz), 5.27 (m, 1H), 5.26 (m, 1H), 4.96 (d,
1H, J = 3.4 Hz), 4.87 (d, 1H, J = 3.6 Hz), 4.83 (d, 1H,
J = 1.4 Hz), 4.45 (dt, 1H), 4.16 (dd, 1H), 4.11 (dd, 1H),
3.68 (s, 3H), 2.01 (s, 3H), 1.99 (s, 3H), 1.98 (s, 3H),
1.91 (s, 3H), 1.76 (s, 3H), 1.73 (s, 3H), 1.39 (s, 3H); 13C
NMR (CDCl3): d 173.9, 173.3, 172.9, 172.7, 172.6 (2C),
171.2, 170.6 (2C), 170.0, 169.7, 169.0, 168.6, 166.2,
165.5, 165.2, 137.7, 133.8, 133.6, 130.1, 129.8, 129.4,
129.1, 128.8, 128.7, 128.1, 127.5, 126.8, 98.9, 98.5, 97.6,
97.0, 96.8, 93.7, 79.0, 75.2, 74.4, 74.3, 72.6, 72.5, 72.1,
71.9, 71.7, 70.5, 69.5, 69.3, 68.9, 68.6, 68.5, 68.4, 67.4,
67.3, 67.0, 66.4, 66.1, 61.4, 61.0, 60.2, 51.5, 51.4, 51.0,
34.4, 34.2, 34.0, 33.9, 33.8, 31.9, 29.7, 29.65, 29.6,
29.56, 29.5, 29.48, 29.3, 29.2, 29.1, 29.0, 25.6, 25.0,
24.9, 24.87, 27.76, 24.6, 24.4, 22.7, 22.6, 22.4, 20.9,
20.7, 20.63, 20.56, 20.4, 17.9, 17.7, 17.6, 14.1; ESIMS:
calcd for [C157H242N2O43+2H]++: m/z 1422.848. Found:
m/z 1422.8583. Anal. Calcd for C157H242N2O43: C, 66.27;
H, 8.57. Found: C, 66.51; H, 8.64.
4.22. 2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-a-D-gluco-
pyranosyl-(1!3)-2-O-benzoyl-4-O-benzyl-a/b-L-rhamno-
pyranosyl trichloroacetimidate (32)
To a solution of 31 (6.6 g, 9.8 mmol) in anhyd CH2Cl2
(25 mL) at 0 ꢁC were added CCl3CN (3 mL) and DBU
(cat). After 10 min, the mixture was concentrated. Purifi-
cation of the residue by column chromatography in 2:1
EtOAc/hexanes afforded 5 (6.8 g, 85%, a/b 10/1) as a
crystalline solid: mp 88–90 ꢁC; 1H NMR (CDCl3): d
8.77 (s, 0.9H), 8.67 (s, 0.1H), 8.12–7.29 (m, 10H), 6.31
(d, 0.9H, J = 1.1 Hz), 5.99 (d, 0.1H, J = 2.4 Hz), 5.69
(dd, 1H, J = 2.4, J = 2.3 Hz), 5.40 (d, 1H, J = 9.4 Hz),
5.21 (t, 1H, J = 9.9 Hz), 5.11 (d, 1H, J = 3.5 Hz), 5.10
(t, 1H, J = 9.4 Hz), 4.92 (d, 1H, J = 11.0 Hz), 4.80 (d,
1H, J = 11.0 Hz), 4.39 (dt, 1H, J = 3.5 Hz, J = 9.3 Hz),
4.37 (dd, 1H, J = 9.9, 3.0 Hz), 4.15–4.05 (m, 2H, ),
3.96–3.85 (m, 2H), 3.70 (t, 1H, J = 9.4 Hz), 2.04 (s,
3H), 1.96 (s, 3H), 1.87 (s, 3H), 1.45 (s, 3H), 1.49 (d,
3H, J = 5.9 Hz); 13C NMR (CDCl3): d 171.1, 170.9,
170.4, 169.7, 168.9, 165.0, 159.8, 94.8, 93.7, 90.5, 78.8,
75.7, 72.7, 71.2, 71.0, 67.8, 67.4, 66.7, 66.1, 61.2, 60.2,
51.0, 22.3, 20.9, 20.6 (2C), 20.3, 18.1, 14.1; FABMS
m/z [M+Cs]+: 963. Anal. Calcd for C36H41Cl3N2O14:
C, 51.96; H, 4.97. Found: C, 51.79; H, 4.99.
4.24. 3,4,6-Tri-O-benzyl-2-O-p-methoxybenzyl-a,b-D-
galactopyranose (35)
To a stirred solution of 34 (9.7 g, 14.6 mmol) in THF
(50 mL) and H2O (10 mL) at 0 ꢁC was added N-bromo-
succinimide (6.7 g, 37.6 mmol). After 10 min, the mix-
ture was treated sequentially with 10% aq NaHSO3
(4 mL), satd aq NaHCO3 (20 mL), and water
(300 mL). The solution was extracted with chloroform
(2 · 100 mL). The organic layer was concentrated. Col-
umn chromatography (2:1, hexanes/EtOAc) of the resi-
1
4.23. 5-(Methoxycarbonyl)pentyl 2-acetamido-3,4,6-tri-
O-acetyl-2-deoxy-a-D-glucopyranosyl-(1!3)-2-O-benz-
oyl-4-O-benzyl-a-L-rhamnopyranosyl-(1!3)-2-O-benz-
oyl-4-O-stearoyl-a-L-rhamnopyranosyl-(1!2)-3,4,6-tri-
O-stearoyl-a-D-galactopyranosyl-(1!3)-2-acetamido-
4,6-di-O-acetyl-2-deoxy-a-D-glucopyranosyl-(1!3)-2-O-
benzoyl-4-O-stearoyl-a-L-rhamnopyranoside (33)
due gave 35 (7.9 g, 94%) as a syrup: H NMR (CDCl3):
d 7.40–6.80 (m, 19H), 5.21 (d, 1H, J = 3.5 Hz), 4.93 (d,
1H, J = 11.0 Hz), 4.91 (d, 1H, J = 11.0 Hz), 4.82 (d, 1H,
J = 11.0 Hz), 4.78 (d, 1H, J = 11.0 Hz), 4.59 (d, 1H, J =
11.0 Hz), 4.55 (d, 1H, J = 11.0 Hz), 4.46 (d, 1H, J =
11.0 Hz), 4.38 (d, 1H, J = 11.0 Hz), 4.14 (t, 1H, J = 6.1
Hz), 4.00 (dd, 1H, J = 9.6, 3.6 Hz), 3.93–3.85 (m, 1H),
3.77 (s, 3H), 3.77–3.71 (m, 1H), 3.61–3.44 (m, 2H); 13C
NMR (CDCl3): d 97.7, 91.9, 82.2, 80.3, 78.7, 76.2,
74.7, 74.6, 74.5, 73.6, 73.5, 73.4, 73.1, 72.9, 69.4, 68.9,
68.8, 55.2; FABMS m/z [M+Na]+: 593. Anal. Calcd
for C35H38O7Æ0.5H2O: C, 72.52; H, 6.78. Found: C,
72.73; H, 6.65.
To a stirred solution of 21 (540 mg, 0.25 mmol) and 32
(1.15 g, 1.38 mmol) in anhydrous CH2Cl2 (7 mL) was
added TMSOTf (8 lL) at 0 ꢁC. The solution was allowed
to reach 22 ꢁC. After 2 h, the mixture was diluted with
CHCl3 and the solution so obtained was washed with

V. Pozsgay et al. / Carbohydrate Research 341 (2006) 1408–1427
1423
4.25. 3,4,6-Tri-O-benzyl-2-O-p-methoxybenzyl-a,b-D-
galactopyranosyl trichloroacetimidate (36)
was continued with vigorous stirring for 11 h. A 28%
solution of NH4OH (2 mL) was added and heating at
reflux was continued for another 8 h. The solution was
cooled to 22 ꢁC and was washed with H2O
(3 · 300 mL). The solution was concentrated to a vol-
ume of approx. 150 mL followed by treatment with
Ac2O (20 mL). The volatiles were removed under vac-
uum. Toluene (100 mL) was added to and evaporated
from the residue thrice. The residue was purified by col-
umn chromatography using 5:1 ! 2:1 hexanes/EtOAc
gradient to afford 38 (25.6 g, 90%) as a syrup: 1H
NMR (CDCl3): d 8.15 (m, 2H), 7.57 (m, 1H), 7.43 (m,
2H), 7.37–7.27 (m, 13H), 7.24–7.20 (m, 3H), 7.18–7.15
(m, 6H), 6.57 (m, 2H), 5.70 (d, 1H, J = 9.5 Hz), 5.44
(dd, 1H), 5.19 (t, 1H, J = 9.6 Hz), 5.08 (d, 1H, J =
3.5 Hz), 4.96 (d, 1H, J = 3.6 Hz), 4.82 (d, 1H, J =
11.4 Hz), 4.81 (d, 1H, J = 1.5 Hz), 4.68 (d, 1H,
11.5 Hz), 4.64 (d, 1H, J = 11.2 Hz), 4.62 (d, 1H, J = 11.5
Hz), 4.58 (s, 2H), 4.44 (d, 1H, J = 11.4 Hz), 4.39 (dt,
J = 3.5 Hz, J = 9.8 Hz), 3.74 (s, 3H), 3.41 (dd, 1H, J =
6.8 Hz, J = 9.3 Hz), 3.25 (dd, 1H, J = 6.1 Hz,
J = 9.3 Hz), 2.04 (s, 3H), 1.69 (s, 3H), 1.49 (s, 3H),
1.41 (d, 3H, J = 6.3 Hz), 0.97 (m, 1H), 0.92 (m, 1H),
0.02 (s, 9H); 13C NMR (CDCl3): d 170.7, 169.8, 168.9,
165.3, 159.1, 138.6, 138.5, 137.7, 133.6, 130.4, 129.8,
129.7, 129.5, 128.7, 128.4, 128.3, 128.2, 128.1, 127.8,
127.7, 127.6, 127.5, 127.4, 127.3, 113.5, 99.2, 96.9,
94.2, 79.9, 79.0, 75.8, 75.4, 75.3, 75.1, 74.6, 73.3, 73.2,
70.3, 69.8, 69.0, 68.0, 67.8, 65.5, 61.7, 55.2, 51.7, 22.6,
20.8, 20.7, 18.1, 17.9. Anal. Calcd for C72H87NO19Si:
C, 66.60; H, 6.75. Found: C, 66.43; H, 6.48.
To a solution of 35 (10.1 g, 17.7 mmol) in CCl3CN
(10 mL) in anhydrous CH2Cl2 (20 mL) was added
DBU (100 lL) at 0 ꢁC. After 15 min, the mixture was
concentrated. Column chromatography (4:1, hexanes/
EtOAc containing 1% Et3N) of the residue gave 36
1
(11.9 g, 94%) as a syrup: H NMR (CDCl3): d (mixture
of anomers a/b 3:7) 8.62 (s, 0.7H), 8.51 (s, 0.3H), 7.32–
6.79 (m, 19H), 6.48 (d, 0.3H, J = 3.1 Hz), 5.73 (d, 0.7H,
J = 7.9 Hz), 4.98–4.57 (m, 6H), 4.49–4.37 (m, 2H), 4.24–
3.85 (m, 3H), 3.78 (s, 0.9H), 3.77 (s, 2.1H), 3.77–3.52 (m,
3H); 13C NMR (CDCl3): d 161.4, 161.2, 159.2, 159.0,
98.7, 95.2, 82.1, 77.9, 77.7, 75.5, 74.9, 74.7, 74.4, 73.4,
73.0, 72.5, 72.1, 68.2, 68.0, 55.2; FABMS m/z
[M+NH4]+: 731. Anal. Calcd for C37H38Cl3NO7Æ0.5-
CH2Cl2: C, 59.46; H, 5.19. Found: C, 59.50; H, 5.20.
4.26. 2-(Trimethylsilyl)ethyl 3,4,6-tri-O-benzyl-2-O-p-
methoxybenzyl-a-D-galactopyranosyl-(1!3)-4,6-di-O-
acetyl-2-azido-2-deoxy-a-D-glucopyranosyl-(1!3)-2-O-
benzoyl-4-O-benzyl-a-L-rhamnopyranoside (37)
To a solution of disaccharide 27 (28.0 g, 34 mmol) and
imidate 36 (approx 85 g, 119 mmol) in anhydrous
CH2Cl2 (300 mL) was added TMSOTf (55 lL) at
ꢀ55 ꢁC over 4.5 h while the temperature was allowed
to rise to ꢀ25 ꢁC. Et3N (1 mL) was added followed by
aq NaHCO3. Extractive work-up using CHCl3 followed
by column chromatographic purification using
20:1 ! 5:1 hexanes/EtOAc gradient afforded 37 (43 g,
1
97%) as an amorphous material: H NMR (CDCl3): d
4.28. 2-(Trimethylsilyl)ethyl 2-O-acetyl-3,4,6-tri-O-benz-
yl-a-D-galactopyranosyl-(1!3)-2-acetamido-4,6-di-O-
acetyl-2-deoxy-a-D-glucopyranosyl-(1!3)-2-O-benzoyl-
4-O-benzyl-a-L-rhamnopyranoside (39)
8.14 (m, 2H), 7.54 (m, 1H), 7.43 (m, 2H), 7.4–7.1
(m, 22H), 6.70 (m, 2H), 5.59 (dd, 1H), 5.36 (d,
1H, J = 3.9 Hz), 5.15 (t, 1H, J = 10.0), 5.00 (d, 1H,
J = 3.5 Hz), 3.74 (s, 3H), 3.42 (dd, 1H, J = 5.1 Hz,
J = 9.0 Hz), 3.29 (dd, 1H, J = 3.8 Hz, J = 10.1), 2.06
(s, 3H), 1.71 (s, 3H), 1.39 (d, 1H, J = 6.3 Hz), 0.97 (m,
1H), 0.90 (m, 1H), 0.03 (s, 9H); 13C NMR (CDCl3): d
170.6, 169.2, 165.8, 159.1, 138.8, 138.7, 138.1, 137.9,
133.3, 130.3, 129.9, 129.8, 129.6, 128.4, 128.3, 128.1,
127.8, 127.6, 127.5, 127.4, 113.6, 98.8, 97.2, 93.0, 79.6,
79.0, 75.5, 75.0, 74.8, 73.3, 73.2, 72.8, 72.6, 69.9, 69.8,
68.4, 67.8, 67.5, 67.3, 65.4, 62.2, 61.6, 55.2, 20.8, 18.0,
17.9, ꢀ1.4. Anal. Calcd for C70H83N3O18SiÆH2O: C,
64.65; H, 6.59. Found: C, 64.83; H, 5.45.
To a stirred solution of 38 (25.0 g, 19.3 mmol) in CH3CN
(300 mL) and H2O (40 mL) was added ammonium
cerium(IV) nitrate (24 g, 43.8 mmol) in portions at
22 ꢁC. After 30 min, the solution was cooled with ice-
water and was treated with 10% aq NaHSO3 until the dis-
appearance of the iodine color. The mixture was neutral-
ized by the addition of satd aq NaHCO3. To this
mixture was added CH2Cl2 and the mixture was filtered
through a layer of Celite. The organic layer was washed
with brine and was then concentrated. Purification by
column chromatography using 2:1 ! 3:2 hexanes/EtOAc
as the eluant afforded 22.0 g of a syrupy material. To the
solution of 2.2 g of this syrup in CH2Cl2 (10 mL) were
added pyridine (1 mL), Ac2O (1 mL), and a catalytic
amount of 4-dimethylaminopyridine at 22 ꢁC. After 1 h,
the volatiles were evaporated under reduced pressure. Col-
umn chromatographic purification of the residue using 2:1
hexanes/EtOAc as the eluant afforded amorphous 39
4.27. 2-(Trimethylsilyl)ethyl 3,4,6-tri-O-benzyl-2-O-p-
methoxybenzyl-a-D-galactopyranosyl-(1!3)-2-acet-
amido-4,6-di-O-acetyl-2-deoxy-a-D-glucopyranosyl-(1!3)-
2-O-benzoyl-4-O-benzyl-a-L-rhamnopyranoside (38)
A solution of 37 (28.0 g, 21 mmol) and PPh3 (28.0 g,
107 mmol) in anhydrous CH2Cl2 was heated at reflux
for 12 h. Water (15 mL) was added and heating at reflux
1
(2.02 g): H NMR (CDCl3): d 5.90 (d, 1H, J = 9.6 Hz),

1424
V. Pozsgay et al. / Carbohydrate Research 341 (2006) 1408–1427
5,46 (dd, 1H, J = 2.1 Hz), 3.8 Hz), 5.23 (dd, 1H, J =
4.1 Hz, J = 9.5 Hz), 5.19 (dd, 1H, J = 4.1 Hz), 5.11 (dd,
1H, J = 9.2 Hz, J = 10.2 Hz), 5.09 (d, 1H, J = 3.5 Hz),
4.41 (d, 1H, J = 11.8 Hz), 4.40 (m, 1H), 4.26 (dd, 1H,
J = 3.2 Hz, J = 9.7 Hz), 3.40 (dd, 1H, J = 7.9 Hz,
J = 10.1), 3.08 (dd, 1H, J = 4.2 Hz, J = 9.9 Hz), 2.04 (s,
3H), 2.00 (s, 3H), 1.79 (s, 3H), 1.53 (s, 3H), 1.42 (d, 3H,
J = 6.3 Hz), 0.98–0.87 (m, 2H), 0.02 (s, 9H); 13C NMR
(CDCl3): d 171.1, 170.6, 169.9, 169.4, 165.2, 138.0, 137.7,
137.2, 133.5, 129.7, 128.6, 128.4, 128.2, 128.1, 127.8,
127.6, 127.5, 127.4, 126.9, 97.0, 96.4, 93.2, 79.8, 76.9,
75.3, 74.6, 74.2, 73.5, 73.3, 73.1, 72.5, 71.0, 70.3, 69.7,
68.5, 68.0, 67.6, 65.5, 61.4, 51.2, 22.5, 20.9, 20.7, 18.1,
17.9, ꢀ1.4. Anal. Calcd for C66H81NO19SiÆEtOAc: C,
64.25; H, 6.86. Found: C, 64.22; H, 6.71.
(100 lL) at 0 ꢁC. After 10 min, the mixture was concen-
trated. Column chromatography of the residue using 1:1
hexanes/EtOAc with 0.01% Et3N as the eluant gave 41
(2.8 g, 92%) as a crystalline solid: 79–81 ꢁC; [a]D +73
(c 1.0, CHCl3); 1H NMR (CDCl3): d 8.80 (s, 1H),
8.20–7.00 (m, 25H), 6.27 (d, 1H, J = 1.8 Hz), 5.99 (d,
1H, J = 9.6 Hz), 5.71 (dd, 1H, J= 2.4 Hz, J = 3.0 Hz),
5.24 (dd, 1H, J = 3.6 Hz, J = 10.2 Hz), 5.21 (d,
1H, J = 3.6 Hz), 5.15 (d, 1H, J = 3.3 Hz), 5.11 (t, 1H,
J = 9.3 Hz), 4.81 (d, 1H, J = 11.0 Hz), 4.80 (d, 1H,
J = 11.0 Hz), 4.66 (d, 1H, J = 11.0 Hz), 4.63 (d,
1H, J = 11.0 Hz), 4.60 (d, 1H, J = 11.0 Hz), 4.42 (d,
1H, J = 11.0 Hz), 4.40–4.32 (m, 2H), 4.18 (s, 2H),
4.15–3.98 (m, 5H), 3.94–3.77 (m, 3H), 3.63 (dd, 1H,
J = 1.8 Hz, J = 3.0 Hz), 3.58 (t, 1H, J = 9.3 Hz), 3.42
(dd, 1H, J = .1 Hz, J = 9.9 Hz), 3.06 (dd, 1H,
J = 3.6 Hz, J = 9.9 Hz), 2.03 (s, 3H), 2.01 (s, 3H), 1.81
(s, 3H), 1.56 (s, 3H), 1.45 (d, 3H, J = 6.6 Hz); 13C
NMR (CDCl3): d 171.1, 170.5, 169.9, 164.8, 159.9,
96.4, 94.9, 93.6, 90.6, 79.1, 76.9, 75.6, 74.6, 74.2, 73.5,
73.3, 72.8, 72.2, 71.1, 70.9, 70.2, 69.8, 69.7, 67.9, 66.6,
61.5, 60.3, 22.5, 21.0, 20.9, 20.7, 18.1; FABMS m/z
[M+Cs]+: 1397; HR-MS calcd for C63H69Cl3N2O19Cs
1395.2614. Found 1395.2574.
4.29. 2-O-Acetyl-3,4,6-tri-O-benzyl-a-D-galactopyran-
osyl-(1!3)-2-acetamido-4,6-di-O-acetyl-2-deoxy-a-D-
glucopyranosyl-(1!3)-2-O-benzoyl-4-O-benzyl-1-thio-
a,b-L-rhamnopyranose (40)
To a stirred solution of 39 (2.15 g, 1.8 mmol) in CH2Cl2
(10 mL) was added trifluoroacetic acid (40 mL) at 22 ꢁC.
After 4 h, the mixture was concentrated. Toluene was
added to and evaporated from the residue several times.
Column chromatographic purification of the residue
using 2:1 ! 1:1 hexanes/EtOAc as the eluant afforded
40 (1.68 g, 85%) as a crystalline solid: mp 93–96 ꢁC;
[a]D +109 (c 0.9, CHCl3); 1H NMR (CDCl3): d (mixture
of anomers a/b 9:1) 8.10–7.00 (m, 25H), 5.96 (d, 1H,
J = 9.7 Hz), 5.64 (br d, 0.1H), 5.52 (dd, 0.9H, J =
2.2 Hz, J = 2.7 Hz), 5.23 (s, 0.9H), 5.22 (dd, 1H,
J = 9.7, 3.5 Hz), 5.18 (d, 1H, J = 3.9 Hz), 5.13 (d, 1H,
J = 3.5 Hz), 5.08 (dd, 1H, J = 9.2 Hz, J = 10.2 Hz),
4.94 (br d, 0.1H), 4.81 (d, 1H, J = 11.0 Hz), 4.78 (d,
1H, J = 11.0 Hz), 4.65 (d, 1H, J = 11.0 Hz), 4.62
(d, 1H, J = 11.0 Hz), 4.60 (d, 1H, J = 11.0 Hz), 4.56
(d, 1H, J = 11.0 Hz), 4.39–4.15 (m, 3H), 4.14 (s, 2H),
4.12–3.74 (m, 6H), 3.62 (d, 1H, J = 2.2 Hz), 3.58–3.36
(m, 3H), 3.06 (dd, 1H, J = 9.7, 4.0 Hz), 2.04 (s, 3H),
2.00 (s, 3H), 1.18 (s, 3H), 1.56 (s, 3H), 1.41 (d, 3H,
J = 6.2 Hz); 13C NMR (CDCl3): d 171.1, 170.7, 170.3,
170.2, 169.5, 165.2, 96.5, 93.2, 93.1, 92.3, 79.8, 79.1,
75.3, 74.6, 74.3, 73.5, 73.3, 73.1, 72.8, 72.0, 71.1, 70.3,
69.8, 69.7, 69.1, 68.8, 68.2, 67.6, 61.6, 51.4, 22.6, 20.9,
20.7 (2C), 18.2; FABMS m/z [M+H]+: 1120. Anal.
Calcd for C61H69NO19Æ0.5H2O: C, 64.88; H, 6.25.
Found: C, 64.70; H, 6.20.
4.31. 5-(Methoxycarbonyl)pentyl 2-O-acetyl-3,4,6-tri-O-
benzyl-a-D-galactopyranosyl-(1!3)-2-acetamido-3,4,6-
tri-O-acetyl-2-deoxy-a-D-glucopyranosyl-(1!3)-2-O-
benzoyl-4-O-benzyl-a-L-rhamnopyranosyl-(1!3)-2-O-
benzoyl-4-O-stearoyl-a-L-rhamnopyranosyl-(1!2)-3,4,6-
tri-O-stearoyl-a-D-galactopyranosyl-(1!3)-2-acetamido-
4,6-di-O-acetyl-2-deoxy-a-D-glucopyranosyl-(1!3)-2-O-
benzoyl-4-O-stearoyl-a-L-rhamnopyranoside (42)
A stirred solution of compound 21 (590 mg, 0.27 mmol)
and imidate 41 (1.19 g, 0.9 mmol) in anhydrous CH2Cl2
(5 mL) was treated with TMSOTf (2 lL) at 0 ꢁC. After
8 h, the mixture was treated with Et3N (0.1 mL) fol-
lowed by concentration under reduced pressure. The res-
idue was purified by C-18 chromatography using a
mixture of CH3OH and EtOH. The product eluted in
1:1 CH3OH/EtOH. The fractions containing the
product were pooled and concentrated. The residue so
obtained was further purified by silica gel chromato-
graphy using 2:1 hexanes/EtOAc as the eluant to give
unreacted 21 (254 mg, 43%) followed by 42 (307 mg,
61 based on recovered 21) as an amorphous substance:
1H NMR (CDCl3) (partial): d 6.26 (d, 1H, J = 9.5 Hz),
5.76 (d, 1H, J = 9.6 Hz), 5.46 (dd, 1H), 5.37 (dd, 1H),
5.29 (m, 1H), 5.27 (m, 1H), 4.98 (d, 1H), 4.96 (d,
1H, J = 3.6 Hz), 4.89 (d, 1H, J = 3.6 Hz), 4.83 (d, 1H,
J = 1.6 Hz), 3.34 (m, 2H), 3.07 (dd, 1H, J = 4.3 Hz,
J = 9.8 Hz), 2.00 (s, 3H), 1.98 (s, 3H), 1.97 (s, 3H),
1.72 (s, 3H), 1.69 (s, 3H), 1.51 (s, 3H), 2.51 (d, 3H,
J = 6.3 Hz); 13C NMR (CDCl3): d 173.3, 172.8, 172.7,
4.30. 2-O-Acetyl-3,4,6-tri-O-benzyl-a-D-galactopyran-
osyl-(1!3)-2-acetamido-4,6-di-O-acetyl-2-deoxy-a-D-
glucopyranosyl-(1!3)-2-O-benzoyl-4-O-benzyl-a-L-
rhamnopyranosyl trichloroacetimidate (41)
To a solution of 40 (2.7 g, 2.4 mmol) and CCl3CN
(3 mL) in anhydrous CH2Cl2 (15 mL) was added DBU

V. Pozsgay et al. / Carbohydrate Research 341 (2006) 1408–1427
1425
172.5, 171.1, 170.6, 170.0, 169.9, 169.3, 168.6, 166.2,
165.4, 164.9, 138.3, 138.1, 137.7, 137.3, 133.7, 130.1,
129.8, 129.4, 129.1, 128.6, 128.4, 128.2, 127.8, 127.6,
127.5, 127.4, 126.5, 98.9, 98.5, 97.7, 96.9, 96.6, 96.4,
93.3, 79.2, 75.2, 74.7, 74.3, 73.3, 72.1, 71.9, 70.6, 70.2,
69.6, 69.4, 68.8, 68.6, 68.3, 68.0, 67.5, 67.3, 67.0, 66.4,
66.1, 61.4, 61.0, 60.2, 60.1, 51.5, 51.4, 51.1, 34.4, 34.2,
33.9, 33.8, 31.9, 29.7, 29.6, 29.3, 29.2, 25.6, 25.0, 24.9,
24.8, 24.6, 24.4, 22.7, 20.9, 20.7, 17.9, 17.6, 17.5,
79.3, 79.0, 78.2, 75.3, 75.1, 74.4, 74.3, 74.0, 73.8, 73.6,
73.4, 73.3, 72.6, 72.4, 72.3, 72.1, 71.84, 71.75, 71.5,
70.5, 70.4, 69.9, 69.6, 69.3, 69.2, 68.9, 68.6, 68.3, 68.0,
67.9, 67.5, 67.2, 66.9, 66.4, 66.0, 61.4, 60.8, 60.2, 51.4,
51.34, 51.26, 40.6, 34.3, 34.1, 34.0, 33.9, 33.8, 33.7,
31.9, 31.8, 29.6, 29.65, 29.62, 29.6, 29.55, 29.50, 29.47,
29.4, 29.30, 29.65, 29.1, 29.0, 28.96, 25.5, 25.0, 24.9,
24.8, 24.7, 24.5, 24.3, 22.6, 22.5, 20.9, 20.8, 20.5,
20.4, 17.9, 17.6, 17.5, 14.0. Anal. Calcd for C224H309
-
14.1; ESIMS m/z calcd for [C184H270N2O48+2H]++
1638.945. Found: 1638.9482. Anal. Calcd for
184H270N2O48: C, 67.42; H, 8.30. Found: C, 67.44; H,
:
ClN2O58: C, 67.37; H, 7.80. Found: C, 67.26; H,
7.85.
C
8.37.
4.33. 5-(Methoxycarbonyl)pentyl 2-acetamido-3,4,6-tri-
O-acetyl-2-deoxy-a-D-glucopyranosyl-(1!3)-2-O-benz-
oyl-4-O-benzyl-a-L-rhamnopyranosyl-(1!3)-2-O-benz-
oyl-4-O-benzyl-a-L-rhamnopyranosyl-(1!2)-3,4,6-tri-
O-benzyl-a-D-galactopyranosyl-(1!3)-2-acetamido-
4,6-di-O-acetyl-2-deoxy-a-D-glucopyranosyl-(1!3)-2-
O-benzoyl-4-O-benzyl-a-L-rhamnopyranosyl-(1!3)-2-
O-benzoyl-4-O-stearoyl-a-L-rhamnopyranosyl-(1!2)-
3,4,6-tri-O-stearoyl-a-D-galactopyranosyl-(1!3)-2-acet-
amido-4,6-di-O-acetyl-2-deoxy-a-D-glucopyranosyl-
(1!3)-2-O-benzoyl-4-O-stearoyl-a-L-rhamnopyranoside
(46)
4.32. 5-(Methoxycarbonyl)pentyl 2-O-benzoyl-4-O-
benzyl-3-O-chloroacetyl-a-L-rhamnopyranosyl-(1!3)-
2-O-benzoyl-4-O-benzyl-a-L-rhamnopyranosyl-(1!2)-
3,4,6-tri-O-benzyl-a-D-galactopyranosyl-(1!3)-2-acet-
amido-4,6-di-O-acetyl-2-deoxy-a-D-glucopyranosyl-
(1!3)-2-O-benzoyl-4-O-benzyl-a-L-rhamnopyranosyl-
(1!3)-2-O-benzoyl-4-O-stearoyl-a-L-rhamnopyranosyl-
(1!2)-3,4,6-tri-O-stearoyl-a-D-galactopyranosyl-(1!3)-
2-acetamido-4,6-di-O-acetyl-2-deoxy-a-D-glucopyran-
osyl-(1!3)-2-O-benzoyl-4-O-stearoyl-a-L-rhamnopyran-
oside (45)
To a stirred solution of 32 (890 mg, 1.07 mmol) and 44
(520 mg, 0.15 mmol) in anhydrous CH2Cl2 was added
TMSOTf (4 lL) at 0 ꢁC. The solution was allowed to
reach 22 ꢁC. After 1 h, Et3N (0.2 mL) was added fol-
lowed by concentration of the solution under vacuum.
Column chromatography of the residue using 3:2 hex-
A solution of 43 (1.10 g, 1.9 mmol) and 44 (1.00 g,
0.28 mmol) in anhydrous CH2Cl2 (10 mL) was treated
with TMSOTf (2 lL) at 0 ꢁC. The solution was allowed
to reach 22 ꢁC. After 20 min, Et3N (0.2 mL) was added
and the solution was concentrated. Column chromato-
graphic purification of the residue using 3:1 ! 5:2 hex-
anes/EtOAc gradient afforded 45 (0.95 g, 85%) as an
1
anes/EtOAc afforded amorphous 46 (445 mg, 72%): H
NMR (CDCl3): d 8.08 (m, 2H), 8.06–7.98 (m, 7H),
7.67–7.04 (m, 44H), 6.96 (m, 2H), 6.08 (d, 1H,
J = 9.5 Hz), 5.66 (d, 1H, J = 10.0 Hz), 5.55 (m, 1H),
5.46 (m, 2H), 5.42 (d, 1H, J = 9.5 Hz), 5.38 (br d, 1H),
5.35 (m, 1H), 5.19 (t, 1H, J = 9.3 Hz), 5.07 (br s, 2H),
4.96 (d, 1H, J = 3.5 Hz), 4.91 (d, 1H, J = 3.4 Hz), 4.81
(d, 1H, J = 3.6 Hz), 3.67 (s, 3H), 3.23 (t, 1H,
J = 9.4 Hz), 3.09 (m, 1H), 1.99 (s, 3H), 1.98 (s, 3H),
1.97 (s, 3H), 1.91 (s, 3H), 1.76 (s, 3H), 1.75 (s, 3H),
1.73 (s, 3H), 1.71 (s, 3H), 1.55 (s, 3H), 1.47 (s, 3H),
1.09 (d, 3H, J = 6.4 Hz); 13C NMR (CDCl3): d 173.9,
173.3, 172.8, 172.6, 172.4, 171.0, 170.54, 170.50, 170.0,
169.9, 169.5, 169.0, 168.8, 168.6, 166.1, 165.5, 165.4,
165.05, 165.03, 138.3, 138.0, 137.8, 137.6, 133.7, 133.5,
133.2, 130.0, 129.93, 129.85, 129.7, 129.3, 129.22,
129.17, 129.1, 128.7, 128.6, 128.5, 128.4, 128.3, 128.2,
128.1, 128.0, 127.7, 127.6, 127.44, 127.41, 126.7, 126.6,
9.2, 98.9, 98.4, 98.2, 98.0, 97.6, 96.9, 96.6, 94.0, 80.2,
79.4, 79.0, 77.7, 75.4, 75.3, 75.1, 74.4, 74.1, 73.5, 73.4,
73.3, 73.2, 72.6, 72.3, 72.0, 71.9, 71.7, 71.5, 70.6, 69.9,
69.6, 69.4, 69.2, 69.1, 68.9, 68.6, 68.0, 67.9, 67.5, 67.3,
66.9, 66.4, 66.0, 61.4, 61.0, 60.7, 60.2, 51.5, 51.4, 51.2,
50.9, 34.4, 34.2, 34.0, 33.9, 33.8, 33.7, 31.9, 31.8, 29.74,
1
amorphous solid: H NMR (CDCl3): d 8.11–7.95 (m,
8H), 7.64–7.49 (5H), 7.50–7.39 (m, 9H), 7.37–7.32 (m,
4H), 7.31–7.02 (m, 27H), 7.06–6.94 (m, 2H), 6.29 (d,
2H, J = 10.0 Hz), 5.66 (d, 1H, J = 10.0 Hz), 5.54 (dd,
1H), 5.51 (dd, 1H J = 1.8 Hz, J = 3.4 Hz), 5.46 (m,
2H), 5.38 m, 1H), 5.33 (dd, 1H, J = 3.1 Hz,
J = 9.7 Hz), 5.24 (m, 3H), 5.19 (1H, J = 9.1 Hz), 5.07
(br s, 1H), 5.03 (d, 1H, J = 3.3 Hz), 4.81 (d, 1H,
J = 3.6 Hz), 4.83 (br s, 1H), 4.66 (d, 1H, J = 11.6 Hz),
4.12 (dd, 1H, J = 3.8 Hz, J = 6.6 Hz), 4.10 (dd, 1H,
J = 3.8 Hz, J = 6.6 Hz), 3.67 (s, 3H), 3.23 (t, 1H,
J = 9.4 Hz), 3.10 (dd, 1H, J = 5.5 Hz, J = 9.7 Hz),
1.98 (s, 3H), 1.97 (s, 3H), 1.77 (s, 3H), 1.73 (s, 3H),
1.69 (s, 3H), 1.65 (s, 6H), 1.47 (s, 3H), 1.08 (d, 6H, J
6.2 Hz); 13C NMR (CDCl3): d 173.8, 173.3, 172.8,
172.6, 172.5, 172.4, 170.6, 170.5, 170.0, 169.5, 168.8,
168.5, 166.2, 166.1, 165.6, 165.5, 165.2, 165.0, 138.3,
138.1, 138.0, 137.6, 133.7, 133.5, 133.3, 133.1, 133.0,
129.9, 129.8, 129.7, 129.6, 129.5, 129.3, 129.2, 129.1,
128.7, 128.6, 128.5, 128.4, 128.3, 128.22, 128.18, 128.1,
128.04, 128.02, 127.8, 127.6, 127.5, 127.4, 127.3, 126.6,
99.3, 98.9, 98.4, 98.1, 97.9, 97.6, 96.9, 96.6, 94.1, 79.8,

1426
V. Pozsgay et al. / Carbohydrate Research 341 (2006) 1408–1427
29.69, 29.64, 29.62, 29.58, 29.56, 29.54, 29.5, 29.46,
29.45, 29.32, 29.29, 29.3, 29.2, 29.06, 28.99, 25.6, 25.0,
24.9, 24.8, 24.7, 24.6, 24.4, 22.7, 22.6, 22.5, 20.92,
20.90, 20.6, 20.5, 20.42, 20.37, 18.0, 17.9, 17.8, 17.7,
17.6, 14.1. Anal. Calcd for C237H327N3O65: C, 66.85;
H, 7.74. Found: C, 66.69; H, 7.84.
4.35. 5-(Methoxycarbonyl)pentyl 2-O-benzoyl-4-O-benz-
yl-3-O-chloroacetyl-a-L-rhamnopyranosyl-(1!3)-2-O-
benzoyl-4-O-benzyl-a-L-rhamnopyranosyl-(1!2)-3,4,6-
tri-O-benzyl-a-D-galactopyranosyl-(1!3)-2-acetamido-
4,6-di-O-acetyl-2-deoxy-a-D-glucopyranosyl-(1!3)-2-O-
benzoyl-4-O-benzyl-a-L-rhamnopyranosyl-(1!3)-2-O-
benzoyl-4-O-benzyl-a-L-rhamnopyranosyl-(1!2)-3,4,6-
tri-O-benzyl-a-D-galactopyranosyl-(1!3)-2-acetamido-
4,6-di-O-acetyl-2-deoxy-a-D-glucopyranosyl-(1!3)-2-O-
benzoyl-4-O-benzyl-a-L-rhamnopyranosyl-(1!3)-2-O-
benzoyl-4-O-stearoyl-a-L-rhamnopyranosyl-(1!2)-3,4,6-
tri-O-stearoyl-a-D-galactopyranosyl-(1!3)-2-acetamido-
4,6-di-O-acetyl-2-deoxy-a-D-glucopyranosyl-(1!3)-2-O-
benzoyl-4-O-stearoyl-a-L-rhamnopyranoside (49)
4.34. 5-(Methoxycarbonyl)pentyl 2-O-acetyl-3,4,6-tri-O-
benzyl-a-D-galactopyranosyl-(1!3)-2-acetamido-4,6-di-
O-acetyl-2-deoxy-a-D-glucopyranosyl-(1!3)-2-O-benz-
oyl-4-O-benzyl-a-L-rhamnopyranosyl-(1!3)-2-O-benz-
oyl-4-O-benzyl-a-L-rhamnopyranosyl-(1!2)-3,4,6-tri-
O-benzyl-a-D-galactopyranosyl-(1!3)-2-acetamido-4,6-
di-O-acetyl-2-deoxy-a-D-glucopyranosyl-(1!3)-2-O-benz-
oyl-4-O-benzyl-a-L-rhamnopyranosyl-(1!3)-2-O-benz-
oyl-4-O-stearoyl-a-L-rhamnopyranosyl-(1!2)-3,4,6-tri-
O-stearoyl-a-D-galactopyranosyl-(1!3)-2-acetamido-
4,6-di-O-acetyl-2-deoxy-a-D-glucopyranosyl-(1!3)-2-O-
benzoyl-4-O-stearoyl-a-L-rhamnopyranoside (47)
A stirred solution of 43 (0.5 g, 0.85 mmol) and 48
(400 mg, 0.08 mmol) in anhydrous CH2Cl2 (5 mL) was
treated with TMSOTf (3 lL) at 0 ꢁC. The solution was
allowed to reach 22 ꢁC. After 3 h, the solution was trea-
ted with Et3N (0.2 mL), followed by concentration and
chromatographic purification of the residue using 3:2
hexanes/EtOAc as the eluant to afford amorphous 49
(365 mg, 84%): 1H NMR (CDCl3): d 8.06–7.95 (m),
7.62–6.94 (m), 6.55 (d, 1H, J = 10.0 Hz), 5.74 (d, 1H,
J = 9.5 Hz), 5.65 (d, 1H, J = 10.0 Hz), 5.55 (m, 1H),
5.52 (m, 2H), 5.45 (m, 3H), 3.67 (s, 3H), 2.04 (s, 3H),
2.03 (s, 3H), 1.95 (s, 3H), 1.75 (s, 3H), 1.73 (s, 3H),
1.71 (s, 3H), 1.69 (s, 3H), 1.62 (s, 3H), 1.47 (s, 3H);
13C NMR (CDCl3): d 174.0, 173.6, 172.8, 172.7, 172.6,
172.4, 170.5, 170.0, 169.9, 169.5, 168.6, 166.2, 165.6,
165.5, 165.2, 165.0, 164.9, 161.8, 138.5–137.6, 133.7–
133.1, 129.8–126.6, 99.3, 99.0, 98.2, 98.1, 97.6, 97.0,
94.6, 94.1, 80.1, 79.8, 79.3, 79.0, 78.2, 75.7, 75.4, 74.4,
73.8, 73.6, 73.4, 72.7, 71.9, 71.4, 70.7, 70.4, 69.9, 69.6,
69.4, 69.2, 68.9. 68.7, 68.3, 67.9, 67.4, 67.3, 67.0, 66.4,
66.1, 61.3, 60.8, 60.7, 60.4, 51.6, 51.5, 51.3, 40.6, 34.4,
34.2, 34.0, 33.8, 31.9, 29.7, 29.6, 29.5, 29.3, 29.1, 29.0,
25.6, 25.2, 25.1, 24.9, 24.8, 24.7, 24.4, 22.9, 22.8, 22.7,
21.0, 20.9, 20.5, 20.4, 20.3, 17.8, 17.4, 14.1. Anal. Calcd
for C303H394ClN3O80: C, 67.27; H, 7.34. Found: C,
67.06; H, 7.66.
To a stirred solution of 41 (650 mg, 0.51 mmol) and 44
(420 mg, 0.12 mmol) in anhydrous CH2Cl2 (5 mL) was
added TMSOTf (4 lL) at 0 ꢁC. After 4 h, Et3N
(0.2 mL) was added and the solution was concentrated.
Purification on a silica gel column using 2:1 hexanes/
EtOAc as the eluant afforded 47 (440 mg, 80%) as an
1
amorphous substance: H NMR (CDCl3): d 8.11–8.06
(m, 2H), 8.06–7.96 (m, 8H), 7.62–7.50 (m, 5H), 7.48–
7.36 (m, 10H), 7.36–7.20 (m, 23H), 7.20–7.16 (m, 3H),
7.16–7.09 (m, 8H), 7.09–7.05 (m, 6H), 7.04–7.00 (m,
3H), 7.00–6.93 (m, 2H), 6.29 (d, 1H, J = 10.0 Hz), 5.83
(d, 1H, J = 9.8 Hz), 5.66 (d, 1H, J = 10.0 Hz), 5.53 (m,
1H), 5.47 (m, 1H), 5.03 (m, 1H), 4.98 (m, 2H), 5.25
(m, 2H), 5.25 (m, 2H), 5.03 (d, 1H, J = 3.5 Hz), 4.98
(m, 2H), 4.96 (d, 1H, J = 3.6 Hz), 4.84 (br s, 2H), 4.75
(d, 1H, J = 2.6 Hz), 4.73 (d, 1H, J = 2.2 Hz), 4.62 (br
s, 1H), 3.67 (s, 3H), 3.10 (m, 2H), 1.99 (s, 3H), 1.97 (s,
9H), 1.75 (s, 3H), 1.73 (s, 3H), 1.71 (s, 3H), 1.67 (s,
3H), 1.63 (s, 3H), 1.62 (s, 3H), 1.47 (s, 3H), 1.09 (d,
3H, J 6.2 Hz); 13C NMR (CDCl3): d 173.9, 173.3,
172.8, 172.6, 172.4 (2C), 171.1, 170.5 (3C), 170.1,
170.0, 169.5, 169.3, 168.8, 168.5, 166.1, 165.5, 165.4,
165.0, 164.8, 138.2–126.4, 99.3, 98.9, 98.4, 98.3, 98.1,
97.6, 96.9, 96.6, 96.4, 94.1, 93.7, 80.2, 79.3, 79.1, 79.0,
77.6, 77.2, 75.4, 75.3, 75.1, 74.7, 74.4, 74.3, 74.2, 74.0,
73.6, 73.5, 73.4, 73.3, 73.2, 72.5, 72.3, 72.1, 71.9, 70.8,
70.6, 70.3, 69.9, 69.6, 69.5, 69.4, 69.3, 69.0, 68.9, 68.6,
68.0, 67.9, 67.4, 67.2, 66.9, 66.4, 66.0, 61.4, 61.0, 60.7,
60.2, 51.4, 51.3, 51.2, 51.0, 34.3, 34.1, 34.0, 33.9, 33.8,
33.7, 31.9, 29.71, 29.65, 29.6, 29.55, 29.51, 29.5, 29.47,
29.44, 29.30, 29.27, 29.1, 29.0, 28.9, 25.5, 25.0, 24.9,
24.8, 24.7, 24.5, 24.3, 22.7, 22.6, 22.5, 20.9, 20.7, 20.6,
20.5, 20.4, 18.0, 17.9, 17.8, 17.6, 17.5, 14.1. Anal. Calcd
for C263H355N3O70: C, 67.52; H, 7.65. Found: C, 67.33;
H, 7.71.
4.36. General procedure for the removal of the protecting
groups from oligosaccharides 33, 42, 45, 46, 47, and 49
To an approx. 20% solution of the oligosaccharide in
anhydrous CH2Cl2 was added an equal volume of anhy-
drous CH3OH followed by NaOCH3 in CH3OH at
22 ꢁC until the pH of the mixture reached 12 as deter-
mined by a wet indicator paper. After 48 h, the solution
was treated with Dowex 50 (H+) ion exchange resin until
the solution became slightly acidic. This solution was
treated with an excess of diazomethane. The solution
was concentrated and the residue was purified by chro-
matography using 98:2 ! 97:3 EtOAc/CH3OH as the
eluant. The fractions containing the product were

V. Pozsgay et al. / Carbohydrate Research 341 (2006) 1408–1427
1427
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pooled and concentrated. An approx. 2% solution of the
residue in EtOH containing approx. 5% AcOH and 10%
Pd/C was stirred under hydrogen at a pressure of 150–
200 psi for 48 h. The mixture was filtered and the filtrate
was concentrated under reduced pressure. The residue
was purified on a BioGel P-2 column using 0.05 M
pyridinium acetate in water as the eluant. The fractions
were monitored with a UV detector. The fractions
containing the product were pooled and freeze-dried.
Residual pyridine and AcOH were removed by repeated
dissolution of the residue in water followed by
lyophilization.
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Acknowledgement
This work was supported by the Intramural Research
Program of the National Institute of Child Health and
Human Development.
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