Synthesis of monomeric and dimeric repeating units of the zwitterionic type 1 capsular polysaccharide from Streptococcus pneumoniae

Article abstract of DOI:10.1002/chem.200902460

Zwitterionic polysaccharides (ZPSs) from Bacteroides fragilis and Streptococcus pneumoniae display unique T-cell activities. The first synthesis of a hexasaccharide representing two repeating units of the zwitterionic capsular polysaccharide from S. pneum

Full text of DOI:10.1002/chem.200902460

DOI: 10.1002/chem.200902460
Synthesis of Monomeric and Dimeric Repeating Units of the Zwitterionic
Type 1 Capsular Polysaccharide from Streptococcus pneumoniae
Xiangyang Wu,[b] Lina Cui,[c] Tomasz Lipinski,[a] and David R. Bundle*[a]
Abstract: Zwitterionic polysaccharides
(ZPSs) from Bacteroides fragilis and
rates a 6-O-acetyl group, which may
contribute to the high a selectivity in
glycosylation. After assembly of the
fully protected hexasaccharide from
five monosaccharide synthons 2–4, 24
and 25, selective deprotection of the
primary hydroxyl groups of the four
galactose residues followed by oxida-
tion to the corresponding uronic acids
provides hexasaccharide 19. The trisac-
charide counterpart 1 was synthesized
in similar fashion from three synthons,
2–4. This approach employed both con-
ventional and dehydrative glycosyla-
tion methodologies and avoids the use
of poorly reactive uronic acid derived
glycosyl donors and acceptors.
Streptococcus
pneumoniae
display
unique T-cell activities. The first syn-
thesis of a hexasaccharide representing
two repeating units of the zwitterionic
capsular polysaccharide from S. pneu-
moniae type 1 (Sp1) is reported. Key
elements of the approach are stereose-
lective construction of 1,4-cis-a-galac-
tose linkages based on a reactive tri-
chloroacetimidate donor that incorpo-
Keywords: glycosylation
tion polysaccharides
activation · uronic acid
·
oxida-
·
· T-cell
Introduction
(abTCR) proteins. These zwitterionic polysaccharides
(ZPSs) from Bacteroides fragilis induce a variety of T-cell
specific responses such as cell proliferation, cytokine secre-
tion, and regulation of antibody production.[2] ZPSs activate
CD4+ T cells by a recently described mechanism of process-
ing and presentation by antigen-presenting cells that re-
quires nitric oxide-mediated degradation of these polymers
in the MHCII pathway.[3]
Active ZPSs share a common structural motif: a high den-
sity of positively charged amino and negatively charged car-
boxyl or phosphate groups. Functional group modification
established that the zwitterionic motif was essential for ac-
tivity since conversion of positively charged amines to neu-
tral acetamido groups eliminated the activity of ZPSs.[4]
Fragmentation of native capsular polysaccharides has fur-
ther established that large oligosaccharides with molecular
weight around 8 kDa retain biological activity.[3,5] However,
successful synthesis and deprotection of oligosaccharide
fragments, or isolation of single repeating units or oligomers
containing the minimum structural element for biological
activity has yet to be achieved. Efficient syntheses of the re-
peating units of ZPSs are a prerequisite for establishing
their minimal size for activity and an appreciation of precise
structure-function relationships.
The immune system has evolved the ability for T cells to
recognize nearly any biological polymer, including peptides,
protein superantigens, and glycolipids through presentation
by the major histocompatibility complex (MHC) proteins
such as MHC class I (MHCI), MHC class II (MHCII), and
CD1. However, polysaccharides are poorly immunogenic, T-
cell-independent (TI-2) antigens[1] and are not recognized by
MHC proteins. Recent and unexpected additions to the list
of biopolymers recognized by T-cells are zwitterionic capsu-
lar polysaccharide (ZPS). These bacterial molecules utilize
MHCII presentation to activate T cells by ab T cell receptor
[a] Dr. T. Lipinski, Prof. Dr. D. R. Bundle
Department of Chemistry, University of Alberta
Alberta Ingenuity Centre for Carbohydrate Science
Edmonton, AB T6G2G2 (Canada)
E-mail: dave.bundle@ualberta.ca
[b] Dr. X. Wu
Molecular Pharmacology and Chemistry
Memorial Sloan Kettering Cancer Center
1275 York Avenue, New York, NY 10065 (USA)
[c] Dr. L. Cui
719 Latimer Hall, Department of Chemistry
University of California, Berkeley, CA 94720-1460 (USA)
The structure of the type 1 capsular polysaccharide of
Streptococcus pneumoniae (Sp1)[6] is less complex than
either the polysaccharides A and B of Bacteroides fragilis
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.200902460.
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FULL PAPER
(Figure 1) and exhibits similar T-cell activity,[7] rendering the
synthesis of Sp1 repeating units more tractable. Sp1 is a
linear polymer of trisaccharide repeating units, containing
two galacturonic acids (GalA, residues a and c) and a 2-
acetamido-4-amino-2,4,6-trideoxygalactose (residue b), (in
Figure 1). All anomeric linkages have the a-configuration.
Each repeating unit of Sp1 contains one positively charged
amine and two negatively charged carboxyl groups. The in-
herent challenges confronting the synthesis of Sp1 repeating
units are the low reactivity of uronic acid derivatives in gly-
cosylation reactions and the synthesis of a-galactopyrano-
sides with high stereoselectivity. There have been attempts
to synthesize the PS-A of B. fragilis, however, the complete
repeating unit was not reached nor was the oligosaccharide
deprotected.[8] In other work a blocked but not deprotected
trisaccharide repeating unit of Sp1 has been reported.[9]
Here we report the first completed chemical synthesis and
deprotection of a zwitterionic polysaccharide trisaccharide
repeating unit 1 and the corresponding hexasaccharide 19
containing two repeating units.
tation of this approach requires the stereoselective construc-
tion of a1,3 and a1,4 galactopyranosyl linkages. Typically
these require glycosyl donors possessing a non-participating
functionality at the C2 position, which may lead to anomeric
mixtures. Although there are numerous methods for a-gly-
coside synthesis,[14–16] direct synthesis of the a-galactopyra-
nosyl linkage with high stereoselectivity remains a challenge.
Traditionally, reasonable yields of a-glycosides could be
achieved by extensive optimization of reaction conditions,
such as solvent, temperature, promoter, as well as leaving
group and protecting-group pattern.[17] Conformationally
locked N-benzyl 2,3-trans-oxazolidione derivatives described
by Ito and co-workers exhibit good a-selectivity for amino
sugar glycoside formation.[18] However, when considering
the synthesis of a 2,4-diamino-2,4,6-trideoxy-a-d-hexopyran-
AHCTUNGTREGoNNNU side, that approach required more steps than the adoption
of 2-azido-2-deoxy-hexose donors for the construction of
1,2-cis glycosides.[17] In this work stereoselective formation
of the 1,4-cis-a-galactose linkage of the diamino-dideoxy-
hexose residue could be achieved using an azido moiety at
C-2 as a nonparticipating group combined with an O-6 ace-
tate. Although Crich et al. have established that there is no
evidence for long range neighboring group participation for
such esters,[19a] there is empirical evidence that a 6-O-acetate
may contribute to stereoselective a-glycosylation.[19b,c–20]
Based on the above principles, retrosynthesis of trisac-
charide 1 starting from the reducing terminus led to three
different building blocks 2, 3 and 4 (Scheme 1). Building
blocks 2[21] and 3[22] were readily synthesized according to
published literature. Utilization of the p-methoxybenzyl
(PMB) group as a temporary protecting group at C-6 in
compound 3 was intended to enhance the nucleophilicity of
the 4-hydroxyl group, and facilitate its glycosylation by
donor 4. The choice of 4-chlorothiophenol as leaving group
for the donor 2 avoids strong thiophenol odors. For the effi-
cient construction of 1,4-cis glycoside, we introduced an
azido moiety at C-2 as a nonparticipating group.[17]
Synthesis of building block 4 started from commercially
available d-glucosamine hydrochloride 5 (Scheme 2). Diazo
transfer from triflyl azide to d-glucosamine hydrochloride
Figure 1. Chemical structures of the repeating units of various zwitterion-
ic polysaccharides. PS A1 and PS B are capsules from B. fragilis. Sp1 is
the capsule from S. pneumoniae type 1.
Results and Discussion
A key issue for the synthesis of the target molecules is the
availability of high-yielding glycosylation strategies, especial-
ly in light of the low reactivity of uronic acid derivatives
which generally result in poor glycosylation yields.[10–13] The
alternative strategy adopted here is to create uronic acid
units by oxidation of the hydroxymethyl group of appropri-
ately prepared substrate oligosaccharides.[11–12,34] Implemen-
Scheme 1. Retrosynthesis of target trisaccharide 1.
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3477

D. R. Bundle et al.
5[23] with cupric sulfate as catalyst, followed by acetylation
furnished 6 in 86% yield in two steps. Selective removal of
the anomeric acetate with hydrazinium acetate in DMF
gave the desired intermediate which upon treatment with
thexyldimethylsilyl chloride (TDSCl) in DMF afforded com-
pound 7 in excellent yield.[24] The utilization of a very bulky
TDS protecting group at the anomeric position gave the
pure b-isomer and thus simplified the purification. Deacety-
lation by transesterification, followed by reaction with benz-
fide (H2S) under basic conditions afforded the free amine
intermediate followed by acetylation with acetic anhydride
in pyridine to give the desired disaccharide 11 in 74% yield
over two steps. Transesterification of the acetate group of 11
followed by selective mesylation gave intermediate 12, and
subsequent reduction with sodium borohydride in DMSO at
858C furnished the 6-deoxy disaccharide 13 in good yield
while leaving the N-acetyl group intact.[28] Triflation of 13
with triflic anhydride (Tf2O) and pyridine at ꢀ308C gave
the unstable 4-O-triflate which was immediately treated
with sodium azide (NaN3) in DMF at room temperature to
produce the required 2-acetamido-4-azido-galactopyranose
disaccharide 14 in 57% yield.[29] Selective removal of the p-
methoxybenzyl group with trifluoroacetic acid (TFA), fol-
lowed by acetylation gave 15. Subsequent PdCl2-catalyzed
deallylation[30] afforded the selectively deprotected building
block 16 in moderate yield. Coordination of the azido group
with PdCl2 may account for the low yield of the deallylation
step.
ACHTUNGTRENNUNGaldehyde dimethyl acetal in the presence of catalytic
amount of toluenesulfonic acid (TsOH) in acetonitrile, and
subsequent allylation afforded compound 8 in an excellent
yield. Selective cleavage of the 4,6-O-benzylidene group[25]
with ethanethiol in the presence of TsOH as catalyst in di-
chloromethane, followed by acetylation gave the desired
compound 9. Removal of the O-silyl group with tetrabutyl-
ACHTUNGTRENNUNG
ammonium fluoride (TBAF)[26] under acidic conditions and
then reaction with trichloroacetonitrile in the presence of
1,8-diazabicyclohexylcarbodiimide (DBU)[27] as base fur-
nished the desired building block 4 in good yield.
Scheme 2. a) TfN3, CH2Cl2, K2CO3, H2O, CuSO4, MeOH; b) Ac2O, pyri-
dine, 86% over two steps; c) NH2NH2·HOAc, DMF, 86%; d) TDSCl,
CH2Cl2, imidazole, 93%; e) NaOMe, MeOH, 100%; f) PhCHACHTNUTRGNEUNG(OMe)2,
TsOH, CH3CN, 80%; g) AllBr, DMF, NaH, 90%; h) EtSH, TsOH,
CH2Cl2, 85%; i) Ac2O, pyridine, 100%; j) TBAF, AcOH, THF, 74%; k)
CCl3CN, DBU, CH2Cl2, 80%.
Scheme 3. a) TMSOTf, CH2Cl2, ꢀ158C ! RT, 60 %; b) H2S, pyridine,
H2O, NEt3; c) Ac2O, pyridine, 74% over two steps; d) NaOMe, MeOH,
100%; e) MsCl, pyridine, ꢀ158C, 71%; f) DMSO, NaBH4, 858C, 80%;
g) Tf2O, pyridine, CH2Cl2, ꢀ308C; h) NaN3, DMF, RT, 57%; i) TFA
(1%) in CH2Cl2; j) Ac2O, pyridine, 80%; k) PdCl2, NaOAc, AcOH, H2O,
53%.
With the required building blocks at our disposal, the
multistep synthesis of trisaccharide
1
was initiated
(Scheme 3). Activation of donor 4 by trimethylsilyl trifluoro-
methane sulfonate (TMSOTf) was employed for the glyco-
sylation of acceptor 3 to give disaccharide 10 in 60% yield.
Excellent diastereoselectivity was observed in this strategy
since 1H NMR detected none of the b-glucopyranosyl
The glycosylation[31] of disaccharide 16 by thiogalactoside
2 in the presence of N-iodosuccinimide (NIS) and trifluoro-
methane sulfonic acid (TfOH) at ꢀ308C in dichloromethane
(CH2Cl2) furnished the required trisaccharide 17 in 73%
yield (Scheme 4). Only trace amounts of b-linked galactose
3
anomer. The NMR data and J1,2 coupling constant values
indicated the presence of the 1,2-cis-glucopyranosidic link-
age (d=4.91 ppm, 1b-H, J1,2 =3.62 Hz; d=3.34 ppm, 2b-H,
1
J
2,1 =3.62, J2,3 =10.2 Hz), which was also confirmed by its
were detectable by H NMR. The heteronuclear one-bond
1JCꢀH value (174 Hz). The high a selectivity suggested a pos-
sible remote stereoelectronic effect,[19b,c,20] while the relative-
ly low yield (60%) was ascribed to the low reactivity and
coupling constant (1JC–H =174 Hz) unambiguously estab-
lished the a-anomeric configuration of the newly introduced
glycosidic bond.[32] Consistent with the findings of oth-
ers,[19b,c,20] we attribute the a selectivity to remote stereoelec-
tronic effects associated with the 6-O-acetyl group. In order
steric hindrance at the 4-O position of the galactopy
ACHUTGTNRNEUGrN anHCATUNGTRNEoNUGN side
acceptor 3. Reduction of compound 10 with hydrogen sul-
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Polysaccharide Synthesis
FULL PAPER
to successfully transform two primary alcohol groups to car-
boxylic acids simultaneously, an efficient oxidation condi-
tions was highly desirable. Jones oxidation[33] requires strong
acidic conditions which are not compatible with sensitive
functional groups, such as electron-rich aromatic rings, acid
labile isopropylidene ketals and glycosidic linkages, and pre-
cludes its application for the synthesis of complex oligosac-
charides. Recently, Huang[34] and co-workers developed a
novel two-step, one-pot procedure for the conversion of pri-
mary alcohols to carboxylic acids under very mild conditions
in good yields. Using a similar procedure employing sodium
hypochlorite
and
2,2,6,6-tetramethylpiperidine-oxyl
(TEMPO), the two hydroxymethyl groups after selective de-
acetylation of trisaccharide 17 were concurrently oxidized to
the corresponding uronic acids, which were protected as
benzyl ethers[12,35] for ease of purification to give the trisac-
charide 18. Final hydrogenolysis of the fully protected trisac-
charide 18 afforded the target molecular 1 in 58% yield.
However, biological evaluation showed that trisaccharide
1 did not activate T-cells, so we turned our attention to the
synthesis of hexasaccharide 19 containing two repeating
units. Based on the synthetic strategy described above, ret-
rosynthetic analysis of hexasaccharide 19 and its protected
form 20 led to two key trisaccharide building blocks, accept-
or 21 and trisaccharide donor 23 (Scheme 5). The terminal
reducing trisaccharide acceptor 21 could be readily synthe-
sized by glycosylation of disaccharide 16 with monosaccha-
Scheme 4. a) NIS, TfOH, CH2Cl2, ꢀ30 ! RT, 73%; b) NaOMe, MeOH,
100%; c) TEMPO, KBr, NaOCl; d) BnBr, CsF, DMF, 51% over three
steps; e) H2, HOAc/H2O, Pd/C (10%), 58%.
yield. The glycosylation of disacchaACTHNUTRGNEUNGride acceptor 16 with
donor 24 in the presence of NIS and silver triflate (AgOTf)
gave trisaccharide 22 in 79% yield without affecting the p-
methoxybenzyl group.[31] Cleavage of the p-meth
ACHTUNGTRENNUNG
AHCTUNGTRENNUNG
ACHTUNGTRENNUNGride 24 using similar conditions
to those described above. Tri-
saccharide donor 23 (shown in
Scheme 5) can be assembled
from building blocks 2, 4 and
25.
In order to synthesize trisac-
charide acceptor 21, we re-
quired a temporary protecting
group for the 3’’-hydroxyl
group, which is compatible with
the glycosylation conditions and
acetyl group removal.
methoxybenzyl (PMB) group
fulfilled this requirement
A p-
ACHTUNGTRENNUNG
(Scheme 5). The synthesis of
monosaccharide 24 could be
achieved starting from the
known 4,6-O-benzylidene ac
26 (Scheme 6).[35] p-Meth
oxy-
benzylation of 26 with p-meth-
oxylbenzyl chloride (PMBCl)
ACTHUNGERTNNUNGeCAHTUNGTRENtNUNG al
AHCTUNGTRENNUNG
ACHTUNGTRENNUNG
ACHTUNGTRENNUNG
and sodium hydride (NaH) in
DMF followed by selective
ring-opening[21] of the 4,6-O-
benzylidene group with dibutyl
boranetriflate (Bu2BOTf) and
BH3·THF and subsequent acet-
ACHTUNGTRENNUNGylation in pyridine provided the
desired product 24 in excellent Scheme 5. Retrosynthetic analysis of hexasaccharide 19.
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D. R. Bundle et al.
H2O gave trisaccharide accept-
or 21 in 73% yield (Scheme 6).
In an attempt to improve the
coupling efficiency of glycosyla-
tion, careful adjustment of
donor and acceptor reactivity
via protecting group strategy is
crucial. In the construction of
trisaccharide trichloroacetimi-
date donor 23, it is highly desir-
able that the temporary pro-
tecting group at the anomeric
position of the terminal re-
A
galactose
residue
(Scheme 7) is orthogonal to
other protecting groups and the
glycosylation conditions without
dampening the reactivity of ac-
ceptor. After a few attempts,
we were delighted to find that
the TDS group could satisfy
these requirements.[25a] Silyla-
Scheme 7. a) TDSCl, imidazole, CH2Cl2, 95%; b) NaOMe, MeOH; c) MeO-C6H4-CHACHTNUGRTENUNG(OMe)2, 70%; d) BnBr,
NaH, DMF, 95%;[21] e) NaCNBH3, HCl in Et2O, 65%; f) TMSOTf, CH2Cl2, 08C, 30%; g) Ph2SO, Tf2O, TTBP,
CH2Cl2, ꢀ258C, 73%; h) H2S, pyridine/H2O/Et3N; i) Ac2O, pyridine, 75% over two steps; j) NaOMe, MeOH;
k) MsCl, pyridine, ꢀ15 ! 08C, 95%.
ceptor 25 by donor 30 performed in the presence of phenyl
sulfoxide, triflic anhydride and tri-tert-butylpyrimidine
(TTBP) gave disaccharide 29 in 73% yield.[37] Selective re-
duction of the azide group of disaccharide 29 with hydrogen
sulfide (H2S) under basic conditions was followed by N-acet-
AHCTUNGTRENNUNG
AHCTUNGTRENNUNG
AHCTUNGTRENNUNG
dures to those described for compound 16 provided the de-
sired disaccharide 36 via derivatives 33–35 (Scheme 8). Gly-
cosylation of disaccharide 36 with donor 2 using N-iodosuc-
cinimide (NIS) and silver triflate (AgOTf) provided trisac-
charide 37 in 66% yield. Desilylation[25a] of trisaccharide 37
with tetrabutylammonium fluoride (TBAF) under acidic
conditions gave the reducing trisaccharide 38 in 92% yield
without cleaving the acetyl groups, and subsequent reaction
with trichloroacetonitrile using DBU provided the trisac-
charide trichloroacetimidate donor 23 in 71% yield.[27]
The glycosidation of trisaccharide donor 23 with trisac-
charide acceptor 21 was performed in the presence of
TMSOTf at room temperature to give hexasaccharide 39 in
85% yield (Scheme 9). Careful tuning of the reaction tem-
perature and the equivalents of donor and Lewis acid
(TMSOTf) was crucial to achieving this high yield.
Scheme 6. a) PMBnCl, NaH, DMF, 90%; b) Bu2BOTf, BH3·THF, 70%;
c) Ac2O, pyridine, 100%; d) NIS/AgOTf, CH2Cl2, ꢀ208C, 79%; e)
CH2Cl2, H2O, DDQ, 73%.
tion of starting material 27[35b] with TDSCl under basic con-
ditions followed by deacetylation with sodium methoxide
and subsequent reaction with anisaldehyde dimethyl acetal
under acidic conditions provided 28 in excellent yield. Ben-
zylation of 28 with benzyl bromide (BnBr) followed by
sodium cyanoborohydride (NaCNH3) reductive ring-open-
ing[36] of the 4,6-O-p-meth
ACTHNUTRGENNoUG xyACHTUNGTRNENbUGN enzylidene acetal group with
HCl in Et2O and subsequent acetylation provided acceptor
25 in good yield (Scheme 7).
In an initial attempt to perform the glycosylation of ac-
ceptor 25 with donor 4 using TMSOTf as catalyst, the de-
sired disaccharide 29 was obtained in only 30% yield. The
low yield was probably due to steric hindrance at the axial
4-OH of galactopyranoside 25, which was accentuated by
the bulky TDS group. However, using the dehydration strat-
egy developed by Gin and co-workers,[37] glycosylation of ac-
Transesterification of hexasaccharide 39 gave tetraol 40
quantitatively (Scheme 10). The TEMPO/NaClO procedure
used to prepare trisaccharide 1 failed to oxidize hexasac-
charide 40. However, by using Huangꢁs oxidation proce-
dure,[34] all four hydroxyl groups of the hexasaccharide 40
could be oxidized by vigorous stirring of a heterogeneous
mixture of water/organic solvents containing TEMPO/
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Polysaccharide Synthesis
FULL PAPER
were also recorded for these
salts and compared (Figure 2)
with the published spectrum of
the Sp1 polysaccharide.[6] The
internal anomeric proton reso-
nances of the hexasaccharide
such as a’, c, begin to adopt
chemical shifts that are similar
to those of the Sp1 polysaccha-
ride. However, it is clear that
residues a, b, b’ are quite dis-
tinct in their chemical shifts
when compared to the corre-
sponding resonances of the
polysaccharide suggesting that
the conformation of even the
hexasaccharide should be dis-
tinct from the average confor-
mation for the repeating unit as
it occurs in the polysaccharide.
Initial biological assays indicate
that there is only very low if
any activity for even the hexa-
saccharide, consistent with the
inference of solution conforma-
tional preferences.
Scheme 8. a) NaBH4, DMSO, 858C, 85%; b) Tf2O, pyridine, CH2Cl2, ꢀ308C; c) NaN3, DMF, 62% over two
steps; d) DDQ, CH2Cl2, H2O; e) Ac2O, py. 80% over two steps; f) PdCl2, NaOAc, HOAc, 66%; g) NIS/
AgOTf, CH2Cl2, 66%; h) TBAF, HOAc, THF, 92%; i) CCl3CN, DBU, CH2Cl2, 71%.
Fragmentation of zwitterionic
polysaccharides established that large oligosaccharides of
8 kDa molecular weight retain biological activity.[3,5] This
would correspond to an oligosaccharide of approximately
10–15 repeating units. Clearly given the synthetic challenges
in preparing two repeating units, compound 19, the resolu-
tion of the unresolved issue of the minimal size oligosac-
charide for biological activity is unlikely to be addressed by
oligosaccharide synthesis.
Scheme 9. a) TMSOTf, CH2Cl2, 85%.
NaClO. Immediate benzylation with benzyl bromide under
slightly basic conditions provided the desired hexasaccharide
20 smoothly in 52% yield over three steps. Hydrogenolysis
of hexasaccharide 20 was challenging owing to different po-
larity of the reactant and its product, therefore a solvent
system that could provide good solubility of both was criti-
cal to the hydrogenolysis reaction. Here we used a mixture
of CH2Cl2/MeOH/H2O 1:3:0.5 for the hydrogenolysis of
hexasaccharide 20 using palladium hydroxide [Pd(OH)2] on
charcoal to give after gel permeation chromatography pure
hexasaccharide 19 in 53% yield.
Trisaccharide 1 and hexasacharide 19 were purified from
lower molecular weight side products by gel-permeation
chromatography as their ammonium salts. The NMR spectra
Figure 2. A comparison of the 1H NMR spectra of A) polysaccharide
Sp1, B) trisaccharide 1 and C) hexasaccharide 19.
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D. R. Bundle et al.
furic acid in ethanol. All commercial
reagents were used as supplied.
Column chromatography used silica
gel (SiliCycle, 230–400 mesh, 60 ꢂ),
and solvents were distilled. High-per-
formance
liquid
chromatography
(HPLC) was performed using
a
Waters HPLC system that consisted of
a Waters 600S controller, 626 pump,
and 486 tunable absorbance detector.
HPLC separations were performed on
a Beckmann C18 semi preparative re-
versed-phase column with a combina-
tion of methanol and water as eluents.
Photoadditions were carried out using
a
spectroline model ENF-260C UV
lamp and cylindrical quartz vessels.
1H NMR spectra were recorded at
either 400, 500, or 600 MHz, and are
referenced to the residual protonated
solvent peaks; dH 7.24 ppm for solu-
tions in CDCl3, and 0.1% external
acetone (dH 2.225) for solutions in
D2O. Optical rotations were measured
with a Perkin–Elmer 241 polarimeter
at 228C. Mass spectrometric analysis
was performed by positive-mode elec-
trospray ionization on
a Micromass
ZabSpec Hybrid Sector-TOF mass
spectrometer. MALDI mass spectro-
metric analysis was performed on a
Voyager-Elite system from Applied
Biosystems.
Scheme 10. a) NaOMe, MeOH; b) TBABr, NaHCO3, TEMPO, CH2Cl2, NaOCl; c) HCl, tBuOH, 2-methylbut-
2-ene, NaClO2, NaH2PO4; d) CsF, BnBr, DMF, 52% over three steps; e) H2, Pd(OH)2, CH2Cl2, MeOH, H2O,
53%.
Dimethylthexylsilyl 3-O-allyl-2-azido-4,6-O-benzylidene-2-deoxy-b-d-glu-
copyranoside (8): Allyl bromide (0.34 mL, 3.59 mmol) was added to a
stirred solution of dimethylthexylsilyl 2-azido-4,6-O-benzylidene-2-deoxy-
b-d-glucopyranoside[24] (1.2 g, 2.76 mmol) in DMF (15 mL). After 15 min
at 08C sodium hydride (190 mg, 4.75 mmol) was added to the solution
under argon. The resulting mixture was warmed to room temperature
and stirred for two hours. TLC indicated the consumption of starting ma-
terial, and the reaction mixture was diluted with ethyl acetate (30 mL),
and excess NaH was neutralized with methanol. The resulting solution
was washed with brine and dried over magnesium sulfate and concentrat-
ed to yield a yellow oil. The residue was purified by flash chromatogra-
phy (hexane/ethyl acetate 6:1) to afford 8 (1.18 g, 90%) as a white oil.
[a]D = ꢀ65.58 (c = 1.0, CHCl3); 1H NMR (600 MHz, CDCl3): d=7.5–
7.37 (m, 5H, Ar), 5.93 (m, 1H, OCH2CH=CH2), 5.56 (s, 1H, CHPh),
5.33–5.19 (m, 2H, OCH2CH=CH2), 4.57 (d, J1,2 =7.7 Hz, 1H, 1-H), 4.43–
4.39 (m, 1H, OCH2CH=CH2), 4.31–4.24 (m, 2H, OCH2CH=CH2, 6-H),
Conclusion
A fully synthetic strategy was developed to synthesize the
trisaccharide and hexasacharide repeating units of the zwit-
terionic capsular polysaccharide antigen of Streptococcus
pneumoniae type 1. The creation of trisaccharide and hexa-
saccharide target compounds containing, 2- and 4-a-linked
galacturonic acids residues, respectively, was accomplished
by first creating the corresponding a-galactopyranosides.
This avoids the inherently low reactivity of uronic acid gly-
cosyl donors. Careful simultaneous oxidation of two and
four primary hydroxymethyl groups gave the trisaccharide 1
and hexasaccharide 19 could be obtained in respectable
yields. Excellent stereocontrol of a-galactosylation was ac-
complished by employing the very reactive trichloroacetimi-
date anomeric activating group in combination with a donor
6-O-acetyl group. The utilization of a 6-O-p-methoxybenzyl
group as a temporary protecting group enhanced the nucleo-
philicity of the 4-O position of the galactose acceptor, there-
by improving the glycosylation yield. Initial immunological
evaluation of hexasaccharide 19 suggests that structures con-
taining more than two repeating units will be necessary to
achieve significant biological activity.
3
3
3.78 (t, J=10.3 Hz, 1H, 6’-H), 3.64 (t, J=9.2 Hz, 1H, 4-H), 3.44 (t, 3J=
9.2, 9.6 Hz, 1H, 3-H), 3.38 (m, 1H, 5-H), 3.33 (dd, 3J=7.7, 9.6 Hz, 1H,
2-H), 1.69 (m, 1H, CHACTHNUTRGNEUNG(CH3)2), 0.92 (m, 12H, 4ꢃCH3), 0.21 ppm (m, 6H,
2ꢃCH3); HRMS (ESI): m/z: calcd for C24H37N3O5SiNa: 498.2395; found:
498.2397 [M+Na+].
Dimethylthexylsilyl 4,6-di-O-acetyl-3-O-allyl-2-azido-2-deoxy-b-d-gluco-
pyranoside (9): To a solution of 8 (0.57 g, 1.13 mmol) in CH2Cl2 (10 mL)
at room temperature was added TsOH (30 mg, 0.23 mmol), followed by
addition of ethanethiol (494 mL, 6.78 mmol). The reaction mixture was
stirred for 30 min, and then evaporated at low pressure, followed by addi-
tion of acetic anhydride (5 mL) and pyridine (5 mL). The resulting mix-
ture was stirred overnight and the resulting solution was concentrated to
slightly yellow oil. The residue was purified by flash chromatography
(hexanes/ethyl acetate 4:1) to afford 9 (228 mg, 85%) as a white solid.
[a]D = ꢀ29.58 (c=1.0, CHCl3); 1H NMR (600 MHz, CDCl3): d=5.88–
5.84 (m, 1H, OCH2CH=CH2), 5.27–5.17 (m, 2H, OCH2CH=CH2), 4.93
(dd, 3J=9.1, 10.1 Hz, 1H, 4-H), 4.50 (d, 3J=7.6 Hz, 1H, 1-H), 4.29–4.26
(m, 1H, OCH2CH=CH2), 4.14–4.08 (m, 3H, OCH2CH=CH2, 6-H, 6’-H),
3.53 (m, 1H, 5-H), 3.34 (dd, 3J=7.6, 9.8 Hz, 1H, 2-H), 3.27 (dd, 3J=9.1,
Experimental Section
Analytical thin-layer chromatography (TLC) was performed on silica gel
60-F254 (Merck). TLC detection was achieved by charring with 5% sul-
9.8 Hz, 1H, 3-H), 2.07 (m, 6H, 2ꢃCH3CO), 1.66 (m, 1H, CHACHTUNGTRENNUNG(CH3)2),
3482
www.chemeurj.org
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2010, 16, 3476 – 3488

Polysaccharide Synthesis
FULL PAPER
0.89 (m, 12H, 4ꢃCH3), 0.19 ppm (m, 6H, 2ꢃCH3); (HRMS) EMS: m/z:
calcd for C21H37N3O7SiNa: 494.2293; found: 494.2294 [M+Na+].
51.8 ppm; (HRMS) EMS: m/z: calcd for C44H55NO14Na: 844.3515; found:
844.3515 [M+Na+].
Methyl 2-acetamido-3-O-allyl-6-O-methanesulfonyl-2-deoxy-a-d-gluco-
pyranosyl-(1!4)-2,3-di-O-benzyl-6-O-methoxybenzyl-a-d-galactopyrano-
side (12): Compound 11 (30 mg, 0.037 mmol) was dissolved in methanol
(3 mL), and NaOMe in methanol (0.54 mL, 0.1m) was added. After one
hour, the reaction mixture was neutralized with Amberlite IR120 (H+
form), and filtered. The resulting solution was evaporated at low pressure
to give the crude diol which was used in the next step without purifica-
tion. The residue in pyridine (3 mL) was treated with methanesulfonyl
chloride (MsCl) (4.5 mL, 0.056 mmol) under argon at ꢀ158C and stirred
for 30 min. Then the reaction mixture was slowly warmed to 08C and
kept at this temperature for 3 h. TLC indicated that the starting material
had completely reacted. The reaction mixture was quenched with MeOH
(1 mL), concentrated and the residue was purified by flash chromatogra-
4,6-Di-O-acetyl-3-O-allyl-2-azido-2-deoxy-a-d-glucopyranosyl trichloro-
ACHTUNGTRENNUNGacetimidate (4): Acetic acid (0.3 mL) was added to a solution of 9
(360 mg, 0.72 mmol) in THF (10 mL) at room temperature, followed by
slow addition of TBAF (1 mL, 1.01 mmol). The reaction was stirred for
2 h at which point TLC check indicated that the starting material had re-
acted. The solution was diluted with ethyl acetate (30 mL), and neutral-
ized by extraction with saturated sodium bicarbonate solution (10 mL).
The aqueous layer was extracted with ethyl acetate (3ꢃ20 mL) and the
combined organic layers were dried over magnesium sulfate and concen-
trated in vacuo. The residue was purified by flash chromatography (hex-
anes/ethyl acetate 3:1) to afford 4,6-di-O-acetyl-3-O-allyl-2-azido-2-
deoxy-d-glucopyranose as a mixture of anomers (190 mg, 74%). This glu-
copyranose (170 mg, 0.476 mmol) was dissolved in a mixture of CH2Cl2
(5 mL) and Cl3CCN (0.11 mL) under argon, and then one drop of DBU
was added at room temperature. After 2 h, the resulting mixture was con-
centrated at low pressure. The residue was purified by flash chromatogra-
phy (n-hexane/acetone 3:2) to afford the 12 (22 mg, 71%). [a]D
=
+65.08 (c=1.0, CHCl3); 1H NMR (600 MHz, CDCl3): d=7.44–6.85 (m,
14H, Ar), 5.92–5.84 (m, 1H, OCH2CH=CH2), 5.73 (d, 1H, NH), 5.26–
5.15 (m, 2H, OCH2CH=CH2), 4. 87–4.63 (m, 6H, 1a-H, 1b-H, 2ꢃ
CH2Ph), 4.39 (m, 2H, CH2Ph), 4.12–4.23 (m, 5H, 4a-H, OCH2CH=CH2,
6b-H, 5b-H), 3.96–3.82 (m, 5H, 6’b-H, 2a-H, 2b-H, 3a-H, 3b-H), 3.81 (s,
3H, OCH3), 3.68–3.61 (m, 2H, 5a-H, 4b-H), 3.44–3.38 (m, 6H, 6a-H,
6a’-H, 5a-H, OCH3), 2.88 (s, 3H, CH3), 2.58 (d, 1H, OH), 1.98 ppm (s,
3H, Ac); 13C NMR (125 MHz, CDCl3): d=135.6, 129.5–127.8, 116.8,
114.2, 98.6, 98.2, 79.8, 77.5, 75.8, 74.4, 73.8, 73.6, 69.8, 68.8, 68.6, 68.0,
66.8, 60.6 ppm; (HRMS) EMS: m/z: calcd for C41H53NO14Na: 838.3079;
found: 838.3083 [M+Na+].
phy (hexanes/ethyl acetate 4:1) to afford 4 (190 mg, 80%). [a]D
=
+60.48 (c=1.0, CHCl3); 1H NMR (600 MHz, CDCl3): d=8.77 (s, 1H,
NH), 6.41 (d, 3J=3.5 Hz, 1H, 1-H), 5.92–5.85 (m, 1H, OCH2CH=CH2),
5.29–5.18 (m, 2H, OCH2CH=CH2), 5.13 (dd, 3J=9.5, 10.0 Hz, 1H, 4-H),
4.30 (m, 1H, OCH2CH=CH2), 4.21–4.15 (m, 2H, OCH2CH=CH2, 6-H),
5.07 (m, 2H, 5-H, 6’-H), 3.90 (t, 3J=9.5, 9.9 Hz, 1H, 3-H), 3.72 (dd, 3J=
3.5, 10.1 Hz, 1H, 2-H), 2.10–2.03 ppm (m, 6H, 2ꢃCH3CO); (HRMS)
EMS: m/z: calcd for C15H19N4O7Cl3Na: 495.0211; found: 495.0214
[M+Na+].
Methyl 2-acetamido-3-O-allyl-2,6-dideoxy-a-d-glucopyranosyl-(1!4)-2,3-
di-O-benzyl-6-O-p-methoxybenzyl-a-d-galactopyranoside (13): Sodium
borohydride (230 mg, 6.13 mmol) was added to a solution of methyl 2-
acetamido-3-O-allyl-6-O-methanesulfonyl-2-deoxy-a-d-glucopyranosyl-
(1!4)-2,3-di-O-benzyl-6-O-methoxybenzyl-a-d-galactopyranoside
Methyl
(1!4)-2,3-di-O-benzyl-6-O-methoxybenzyl-a-d-galactopyranoside (10):
Glycosyl donor
(50 mg, 0.1 mmol), monosaccharide acceptor 3[22]
4,6-di-O-acetyl-3-O-allyl-2-azido-2-deoxy-a-d-glucopyranosyl-
4
(50 mg, 0.1 mmol) and activated 4 ꢂ molecular sieves (20 mg) were dried
together under vacuum for one hour in a pear-shaped flask (5 mL). The
contents of the flask were then dissolved in CH2Cl2 (3 mL). The suspen-
sion was stirred for 10 min at room temperature under argon, and then
the temperature was reduced to ꢀ158C, and TMSOTf (50 mL, 0.1m in
CH2Cl2) was added dropwise and the reaction mixture was slowly
warmed to room temperature. After 30 min, the reaction mixture was
neutralized with triethylamine and concentrated in vacuum. The residue
was purified by flash chromatography (hexanes/ethyl acetate 6:1!3:1) to
(250 mg, 0.306 mmol) in DMSO (6 mL) under argon at 858C and the so-
lution was stirred overnight. The resulting mixture was quenched with
methanol at room temperature and diluted with ethyl acetate (20 mL).
This solution was washed with water (10 mL). The aqueous wash was ex-
tracted with ethyl acetate (3ꢃ20 mL) and the combined organic extracts
were dried over MgSO4 and concentrated in vacuum. The residue was
purified by flash chromatography (n-hexane/acetone 5:2) to afford 13
(184 mg, 80%). [a]D
=
+47.08 (c=1.0, CHCl3); 1H NMR (600 MHz,
afford 10 (50 mg, 60%). [a]D
=
+78.08 (c=1.0, CHCl3); 1H NMR
CDCl3): d=7.41–6.85 (m, 14H, Ar), 5.93–5.85 (m, 1H, OCH2CH=CH2),
5.76 (d, 1H, NH), 5.27–5.14 (m, 2H, OCH2CH=CH2), 4.88–4.71 (m, 5H,
1b-H, 2ꢃCH2Ph), 4.66 (d, 3J=2.8 Hz, 1H, 1a-H), 4.43–4.36 (m, 2H,
CH2Ph), 4.26–4.08 (m, 5H, OCH2CH=CH2, 2b-H, 5b-H, 3a-H), 3.87–3.80
(m, 6H, 2a-H, 6a-H, 6a’-H, OCH3), 3.46–3.34 (m, 5H, 3b-H, 5a-H,
OCH3), 3.28 (t, 3J=9.6 Hz, 1H, 4b-H), 2.26 (m, 1H, OH), 1.93 (s, 3H,
COCH3), 1.04 ppm (d, 3H, CH3); 13C NMR (125 MHz, CDCl3): d=135.2,
129.6–127.4, 116.8, 114.2, 98.8, 98.2, 80.2, 77.5, 75.7, 75.5, 73.6, 73.4, 73.2,
72.6, 72.1, 68.8, 68.6, 67.2 ppm; (HRMS) EMS: m/z: calcd for
C40H51NO11Na: 744.3354; found: 744.3353 [M+Na+].
(600 MHz, CDCl3): d=7.41–6.89 (m, 14H, Ar), 5.92–5.85 (m, 1H,
OCH2CH=CH2), 5.28–5.16 (m, 2H, OCH2CH=CH2), 5.04 (t, 3J=9.5,
10.2 Hz, 1H, 4b-H), 4.91 (d, 3J=3.6 Hz, 1H, 1b-H), 4.83–4.70 (m, 5H,
1a-H, 2CH2Ph), 4.51–4.42 (m, 2H, CH2Ph), 4.38 (m, 1H, 5b-H), 4.25 (m,
1H, OCH2CH=CH2), 4.16 (m, 1H, 4a-H), 4.08 (m, 1H, OCH2CH=CH2),
3.91–3.81 (m, 5H, 2a-H, 3a-H, 5a-H, 6a-H, 6b-H), 3.80 (s, 3H, OCH3),
3.72 (t, 3J=9.6, 9.9 Hz, 1H, 3b-H), 3.49–3.45 (m, 2H, 6a’-H, 6b’-H), 3.37
3
(s, 3H, OCH3), 3.31 (dd, J=3.5, 10.2 Hz, 1H, 2b-H), 2.1–2.0 (m, 6H, 2ꢃ
COCH3); (HRMS) EMS: m/z: calcd for C42H51N3O13Na: 828.3314;
found: 828.3313 [M+Na+].
Methyl 2-acetamido-3-O-allyl-4-azido-2,4,6-trideoxy-a-d-galactopyrano-
syl-(1!4)-2,3-di-O-benzyl-6-O-methoxybenzyl-a-d-galactopyranoside
(14): Pyridine (25 mL, 0.287 mmol) and Tf2O (30 mL, 0.191 mmol) were
added to a stirred to a solution of 13 (65 mg, 0.096 mmol) in CH2Cl2
(6 mL) at ꢀ308C under argon. After 2.0 h, TLC indicated that the start-
ing material had been consumed. The reaction mixture was diluted with
Methyl 2-acetamido-4,6-di-O-acetyl-3-O-allyl-2-deoxy-a-d-glucopyrano-
syl-(1!4)-2,3-di-O-benzyl-6-O-methoxybenzyl-a-d-galactopyranoside
(11): H2S was bubbled into a solution of 10 (54 mg, 0.066 mmol) in a mix-
ture of pyridine/H2O/NEt3 (10:1:0.3) (5 mL) for 3 h. The reaction mixture
was co-evaporated with toluene to give a residue which was dissolved in
pyridine (3 mL) and acetic anhydride (1 mL). The resulting solution was
stirred overnight, concentrated and the residue was purified by flash
CH2Cl2 (10 mL), and extracted with
a saturated NaHCO3 solution
(10 mL). The aqueous layer was extracted with ethyl acetate (3ꢃ10 mL)
and the combined organic layers were dried over magnesium sulfate and
concentrated in vacuum. The crude triflate was dissolved in dry DMF
(2 mL) under argon, NaN3 (123 mg, 1.914 mmol) was added and the reac-
tion mixture was stirred overnight. The reaction mixture was diluted with
ethyl acetate (5 mL), and then washed with water (5 mL). The aqueous
layer was extracted with ethyl acetate (3ꢃ5 mL) and the combined or-
ganic extracts were dried over MgSO4 and concentrated in vacuo. The
residue was purified by flash chromatography (n-hexane/acetone 3:1!
chromatography (n-hexane/acetone 2:1) to afford 11 (41 mg, 74%). [a]D
=
+66.78 (c=1.0, CHCl3); 1H NMR (600 MHz, CDCl3): d=7.44–6.84 (m,
14H, Ar), 5.83–5.74 (m, 1H, OCH2CH=CH2), 5.71 (d, 1H, NH), 5.21–
5.10 (m, 2H, OCH2CH=CH2, 4b-H), 4.94 (d, 3J=3.6 Hz, 1H, 1b-H),
4.86–4.67 (m, 5H, 1a-H, CH2Ph), 4.43–4.29 (m, 4H, 2b-H, 5b-H, CH2Ph),
4.19 (m, 1H, 4a-H), 4.06–3.97 (m, 2H, OCH2CH=CH2), 3.88–3.77 (m,
7H, 2a-H, 3a-H, 5a-H, 6b-H, OCH3), 3.62–3.55 (m, 2H, 3b-H, 6’b-H),
3.42 (m, 2H, 6a-H, 6’a-H), 3.36 (s, 3H, OCH3), 2.08–1.94 ppm (m, 9H,
3ꢃCOCH3); 13C NMR (125 MHz, CDCl3): d=138.1–126.8, 116.4, 114.1,
98.4, 98.1, 76.6, 76.4, 75.8, 73.2, 70.8, 68.8, 68.2, 66.9, 61.5, 60.4, 55.4, 55.1,
1
2:1) to afford 14 (32 mg, 57%). [a]D = +86.18 (c=1.0, CHCl3); H NMR
(600 MHz, CDCl3): d=7.42–6.84 (m, 14H, Ar), 5.94–5.84 (m, 1H,
Chem. Eur. J. 2010, 16, 3476 – 3488
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
3483

D. R. Bundle et al.
OCH2CH=CH2), 5.57 (d, 1H, NH), 5.31–5.18 (m, 2H, OCH2CH=CH2),
4.87–4.72 (m, 6H, 1a-H, 1b-H, 2ꢃCH2Ph), 4.87 (d, 3J=3.6 Hz, 1H,
1b-H), 4.53 (ddd, 3J=3.6, 10.7 Hz, 1H, 2b-H), 4.45–4.31 (m, 3H, 5b-H,
CH2Ph), 4.22–4.15 (m, 2H, 3a-H, OCH2CH=CH2), 3.99–3.92 (m, 1H,
OCH2CH=CH2), 3.87–3.76 (m, 6H, 2a-H, 4a-H, 6a-H, OCH3), 3.71 (m,
1H, 4b-H), 3.58–3.53 (dd, 3J=3.3, 10.8 Hz, 1H, 3b-H), 3.42 (m, 2H,
5a-H, 6’a-H), 3.37 (s, 3H, OCH3), 1.95 (s, 3H, COCH3), 0.98 ppm (d, 3H,
CH3); 13C NMR (125 MHz, CDCl3): d=135.2, 129.6–127.4, 116.8, 114.2,
98.4, 98.2, 77.0, 76.8, 73.4, 73.3, 73.1, 70.5, 68.8, 67.2, 65.3, 63.3 ppm;
(HRMS) EMS: m/z: calcd for C40H50N4O10Na: 769.3419; found: 769.3421
[M+Na+].
73%). [a]D = +42.48 (c=1.0, CHCl3); 1H NMR (600 MHz, CDCl3): d=
7.44–7.21 (m, 15H, Ar), 5.72 (d, 1H, NH), 4.97 (m, 2H, CH2Ph), 4.86–
4.70 (m, 9H, 1a-H, 1b-H, 1c-H, 3ꢃCH2Ph), 4.69–4.57 (m, 2H, CH2Ph),
4.56–4.50 (m, 1H, 2b-H), 4.32 (m, 1H, 6a-H), 4.19–4.03 (m, 5H, 2c-H,
4b-H, 5b-H, 6b-H, 6’b-H), 4.00 (m, 1H, 4a-H), 3.89–3.79 (m, 5H, 2a-H,
3a-H, 3c-H, 5a-H, 6’a-H), 3.67–3.62 (m, 2H, 3b-H, 4b-H), 3.35 (s, 3H,
CH3), 2.05–1.89 (m, 9H, 3ꢃCOCH3), 0.89 ppm (d, 3H, CH3); 13C NMR
(125 MHz, CDCl3): d=128.4–127.8, 101.2, 98.6, 98.2, 79.5, 79.1, 77.2, 76.8,
76.4, 76.0, 75.4, 74.8, 74.5, 74.3, 73.3, 73.0, 69.4, 68.3, 65.9 ppm; (HRMS)
EMS: m/z: calcd for C60H70N4O16Na: 1125.4679; found: 1125.4672
[M+Na+].
Methyl 2-acetamido-3-O-allyl-4-azido-2,4,6-trideoxy-a-d-galactopyrano-
syl-(1!4)-6-O-acetyl-2,3-di-O-benzyl-a-d-galactopyranoside (15): Tri-
fluoroacetic acid (1%) (20 mL) was added to a solution of disaccharide
14 (70 mg, 0.094 mmol) in CH2Cl2 (3 mL) and the mixture was stirred for
two hours. TLC check indicated that the starting material had reacted
completely. The reaction mixture was neutralized with triethylamine
(0.1 mL) and concentrated. The residue was dissolved in a mixture of
pyridine (4 mL) and Ac2O (1 mL) and the solution was stirred for 18 h.
Concentration gave the crude product and this residue was purified by
flash chromatography (n-hexane/acetone 3:1!3:2) to afford 15 (50 mg,
80%). [a]D = +75.08 (c=1.0, CHCl3); 1H NMR (600 MHz, CDCl3): d=
7.42–7.25 (m, 10H, Ar), 5.94–5.83 (m, 1H, OCH2CH=CH2), 5.52 (d, 1H,
NH), 5.31–5.19 (m, 2H, OCH2CH=CH2), 4.86 (d, 3J=3.8 Hz, 1H, 1b-H),
4.81–4.72 (m, H, 1a-H, 2ꢃCH2Ph), 4.47 (m, 1H, 2b-H), 4.39 (t, 1H,
6a-H), 4.27 (t, 1H, 5b-H), 4.19 (m, 1H, OCH2CH=CH2), 4.01 (m, 1H,
4a-H), 3.95 (m, 1H, OCH2CH=CH2), 3.88–3.75 (m, 4H, 3a-H, 2a-H,
5a-H, 6’a-H), 3.71 (m, 1H, 4b-H), 3.57 (dd, 3J=3.3, 10.9 Hz, 1H, 3b-H),
3.37 (s, 3H, CH3), 2.05–1.98 (m, 6H, 2COCH3), 1.00 ppm (d, 3H, CH3);
13C NMR (125 MHz, CDCl3): d=138.2, 134.3, 128.4–127.2, 117.3, 98.8,
98.2, 76.4, 76.2, 75.1, 73.4, 73.1, 68.4, 65.8, 63.2 ppm; (HRMS) EMS: m/z:
calcd for C34H44N4O10Na: 691.2949; found: 691.2951 [M+Na+].
Methyl [benzyl 2,3,4-tri-O-benzyl-a-d-galactopyranosiduronate]-(1!3)-
2-acetamido-4-azido-2,4,6-trideoxy-a-d-galactopyranosyl-(1!4)-[benzyl
2,3-O-dibenzyl-a-d-galactopyranosiduronate (18): Trisaccharide 17
(11 mg, 0.01 mmol) was dissolved in dry MeOH (2 mL), followed by ad-
dition of 0.5m NaOMe in MeOH (0.01 mmol, 20 mL), and the reaction
mixture was stirred for one hour. The resulting mixture was neutralized
with Amberlite IR120 (H+ form), filtered and concentrated to give the
trisaccharide diol. This diol (10.2 mg, 0.01 mmol) was dissolved in an ace-
tone (1 mL) and NaHCO3 solution (5%) (0.5 mL) and KBr (2.5 mg,
0.02 mmol) and TEMPO (3.8 mg, 0.014 mmol) were added at 08C. After
5 min, NaOCl (13%, 42 mL) was added and the mixture was stirred for
10 min at 08C. The reaction mixture was diluted with ethyl acetate
(15 mL), washed with brine and dried over MgSO4, and then the result-
ing organic phase was concentrated. The residue was dissolved in DMF
(2 mL), and CsF (20 mg) and BnBr (16 mL) were added under argon, and
the mixture was stirred overnight. The reaction mixture was diluted with
ethyl acetate (15 mL), washed with brine, dried over MgSO4, and then
the resulting organic phase was concentrated. The residue was purified
by flash chromatography (n-hexane/acetone 5:2) to afford 18 (6.3 mg,
51%). [a]D = +61.58 (c=1.0, CHCl3); 1H NMR (600 MHz, CDCl3): d=
7.41–7.17 (m, 35H, Ar), 5.70 (d, 1H, NH), 5.17 (m, 2H, CH2Ph), 5.00–
4.87 (m, 6H, 1a-H, 1c-H, 2ꢃCH2Ph), 4.82–4.71 (m, 6H, 3ꢃCH2Ph), 4.63
(m, 2H, 1/2ꢃCH2Ph, 5c-H), 4.56 (d, 1H, 3J=3.84 Hz, 1H, 1b-H), 4.46–
4.40 (m, 2H, 1/2ꢃCH2Ph, 2b-H), 4.32 (m, 1H, 4c-H), 4.28 (m, 1H, 3a-H),
4.25 (m, 1H, 4a-H), 4.20 (dd, 3J=3.84, 10.1 Hz, 1H, 3c-H), 4.13 (dd, 3J=
3.52, 10.2 Hz, 1H, 2c-H), 4.04 (m, 1H, 5b-H), 3.81 (m, 2H, 2a-H, 5a-H),
3.60 (m, 1H, 4b-H), 3.54 (dd, 3J=3.2, 10.8 Hz, 1H, 3b-H), 3.38 (s, 3H,
CH3), 1.83 (s, 3H, COCH3), 0.84 ppm (d, 3H, CH3); (HRMS) EMS:
m/z: calcd for C70H74N4O16Na: 1249.4992; found: 1249.4995 [M+Na+].
Methyl 2-acetamido-4-azido-2,4,6-trideoxy-a-d-galactopyranosyl-(1!4)-
6-O-acetyl-2,3-di-O-benzyl-a-d-galactopyranoside (16):
A solution of
NaOAc (29 mg, 0.036 mmol) in a mixture of HOAc/H2O 20:1 (3 mL) was
degassed under argon for 30 min. Compound 15 (30 mg, 0.045 mmol) was
dissolved in this degassed solution under an atmosphere of argon. PdCl2
(30 mg, 0.135 mmol) was added and the reaction mixture was stirred until
TLC indicated that starting material had reacted. The reaction mixture
was passed through a filter (0.2 mm) to give a yellow solution, which was
diluted with ethyl acetate (10 mL), and extracted with
a
saturated
Methyl a-d-galactopyranosiduronate-(1!3)-2-acetamido-4-amino-2,4,6-
trideoxy-a-d-galactopyranosyl-(1!4)-a-d-galactopyranosiduronate (1): A
solution of 18 (11 mg, 9 mmol) in aqueous acetic acid (90%) (5 mL) was
hydrogenated at 400 kPa over Pd/C (10%) (50 mg) for 18 h. The reaction
mixture was passed through a filter (0.2 mm) and a reverse phase column
(Sep-PaK) to give a yellow solution. Gel filtration of the yellow solution
on a column (2.5ꢃ85 cm) of Biogel P-2 eluting with ammonium acetate
(100 mm) gave pure product 1 (3 mg, 58%) as a white powder after re-
NaHCO3 solution (10 mL). The aqueous layer was extracted with ethyl
acetate (3ꢃ10 mL) and the combined organic layers were dried over
MgSO4 and concentrated. The residue was purified by flash chromatogra-
phy (n-hexane/acetone 3:1!3:2) to afford 16 (12 mg, 53%). [a]D
=
+88.28 (c=1.0, CHCl3); 1H NMR (600 MHz, CDCl3): d=7.41–7.25 (m,
10H, Ar), 6.01 (d, 1H, NH), 4.84–4.78 (m, 2H, CH2Ph), 4.77 (d, 3J=
3.95 Hz, 1H, 1b-H), 4.74–4.69 (m, 3H, 1a-H, CH2Ph), 4.41–4.27 (m, 3H,
2b-H, 5b-H, 6a-H), 4.09 (m, 1H, 4a-H), 3.93–3.80 (m, 4H, 2a-H, 3b-H,
5a-H, 6’a-H), 3.59 (m, 1H, 4b-H), 3.37 (s, 3H, CH3), 2.07 (m, 6H, 2ꢃ
COCH3), 0.97 ppm (d, 3H, CH3); 13C NMR (125 MHz, CDCl3): d=135.2,
129.6–127.4, 98.2, 97.8, 76.8, 76.1, 73.8, 73.2, 71.5, 68.2, 66.4, 63.8 ppm;
(HRMS) EMS: m/z: calcd for C31H40N4O10Na: 651.2637; found: 651.2639
[M+Na+].
1
peated lyophilization. [a]D = +35.68 (c=0.5, D2O); H NMR (600 MHz,
3
3
D2O): d=5.06 (d, J=3.4 Hz, 1H, 1c-H), 4.97 (d, J=3.96 Hz, 1H, 1b-H),
4.92 (d, 3J=3.84 Hz, 1H, 1a-H), 4.75 (m, 1H, 5b-H), 4.35 (d, 3J=3.2 Hz,
1H, 4a-H), 4.26 (m, 1H, 4c-H), 4.23 (m, 2H, 3b-H, 5a-H), 4.13 (dd, 3J=
3.95 Hz, 1H, 2b-H), 4.10 (m, 1H, 5c-H), 3.98 (dd, 3J=3.2, 10.6 Hz, 1H,
3a-H), 3.89 (dd, 3J=3.84, 10.5 Hz, 1H, 2a-H), 3.85–3.82 (m, 2H, 2c-H,
3c-H), 3.74 (m, 1H, 4b-H), 2.05 (s, 3H, COCH3), 1.30 ppm (d, 3H, CH3);
(HRMS) EMS: m/z: calcd for C21H33N2O16ꢀ: 569.1836; found: 569.1806
[MꢀHꢀ].
Methyl 2,3,4-tri-O-benzyl-6-O-acetyl-a-d-galactopyranosyl-(1!3)-2-acet-
amido-4-azido-2,4,6-trideoxy-a-galactopyranosyl-(1!4)-6-O-acetyl-2,3-di-
O-benzyl-a-d-galactopyranoside (17): NIS (10.2 mg, 0.045 mmol) was
added to
a
solution of 2[21] (37.2 mg, 0.06 mmol) and 16 (19 mg,
Phenyl 6-O-acetyl-2,4-di-O-benzyl-3-O-p-methoxybenzyl-1-thio-b-d-gal-
actopyranoside (24): Compound 26[35] (427 mg, 0.95 mmol) was dissolved
in dry DMF (20 mL) under argon, followed by addition of p-methoxy-
benzyl chloride (157 mg, 1.0 mmol). The resulting mixture was treated
with NaH (60%) (57 mg, 1.43 mmol) at 08C, and slowly warmed to room
temperature. After one hour, the reaction mixture was quenched with
methanol, and extracted with ethyl acetate (2ꢃ20 mL), then washed with
brine. The resulting organic phase was evaporated at low pressure to give
crude phenyl 2,4-di-O-benzyl-4,6-O-benzylidene-3-O-p-methoxybenzyl-1-
thio-b-d-galactopyranoside, which was used without purification. This
residue was dissolved CH2Cl2 (10 mL) and BH3·THF in THF (1.0m)
0.03 mmol) containing 4 ꢂ molecular sieves (50 mg) in CH2Cl2 (3 mL)
under argon at ꢀ308C and the mixture was stirred for 15 min. The result-
ing mixture was treated with TfOH (1 mg, 0.006 mmol) at ꢀ308C and the
reaction mixture was slowly warmed to room temperature. After 30 min,
TLC indicated that the starting material had been consumed. The reac-
tion mixture was quenched with triethylamine, diluted with CH2Cl2
(10 mL), and this solution was washed with saturated NaHCO3 solution,
Na2S2O3 solution (10%) and brine. The organic phase was dried over
MgSO4, concentrated to give the crude product and this residue was puri-
fied by flash chromatography (n-hexane/acetone 5:2) to afford 17 (24 mg,
3484
www.chemeurj.org
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2010, 16, 3476 – 3488

Polysaccharide Synthesis
FULL PAPER
(9.5 mmol, 9.5 mL) was added. The reaction mixture was stirred for
10 min followed by dropwise addition of Bu2BOTf (0.95 mL, 0.95 mmol)
at 08C. TLC indicated that the starting material had disappeared after
2 h. The resulting mixture was neutralized with triethylamine and excess
borane was quenched with methanol. The solution was concentrated and
the residue was dissolved in a mixture of pyridine (10 mL) and acetic an-
hydride (5 mL) and stirred overnight. Purification by chromatography
gave 24 (436 mg, 75%) as a colorless oil. [a]D = +45.68 (c=1.0, CHCl3);
1H NMR (500 MHz, CDCl3): d=7.59–6.87 (m, 14H, Ar), 5.02 (d, 2J=
11.2 Hz, 1H, 1/2ꢃCH2Ph), 4.84–4.76 (dd, 2H, CH2Ph), 4.71 (s, 2H,
CH2Ph), 4.60 (m, 2H, 1/2ꢃCH2Ph, 1-H), 4.29–4.26 (m, 1H, 6-H), 4.13–
4.10 (m, 1H, 6’-H), 3.94 (t, 3J=9.5 Hz, 1H, 2-H), 3.82 (m, 4H, CH3,
4-H), 3.58 (m, 2H, 3H, 5-H), 2.01 ppm (s, 3H, CH3CO); 13C NMR
(125 MHz, CDCl3): d=138.3, 134.1–127.2, 113.9, 87.8, 83.9, 76.8, 76.0,
75.7, 74.3, 73.4, 72.8 ppm; (HRMS) EMS: m/z: calcd for C36H38O7SNa:
637.2231; found: 637.2234 [M+Na+].
phy (hexanes/ethyl acetate 4:1) to afford 25 (117 mg, 65%). [a]D =
+11.28 (c=1.0, CHCl3); 1H NMR (600 MHz, CDCl3): d=7.38–6.38 (m,
14H, Ar), 4.93 (d, 2J=11.2 Hz, 1H, 1/2ꢃCH2Ph), 4.76 (d, 2J=11.2 Hz,
1H, 1/2ꢃCH2Ph), 4.71 (m, 2H, CH2Ph), 4.61 (d, 3J=7.4 Hz, 1H, 1-H),
4.52 (s, 2H, CH2Ph), 4.02 (m, 1H, 4-H), 3.82 (s, 3H, CH3), 3.78 (m, 1H,
3
3
6-H), 3.68 (m, 1H, 6’-H), 3.59 (dd, J=7.4, 9.4 Hz, 1H, 2-H), 3.54 (t, J=
5.9 Hz, 1H, 5-H), 3.49 (dd, 3J=3.4, 9.4 Hz, 1H, 3-H), 2.51 (brs, 1H,
OH), 1.71 (m, 1H, CHACTHNUTRGNE(UNG CH3)2), 0.91 (m, 12H, 4ꢃCH3), 0.21 ppm (m, 6H,
2ꢃCH3); (HRMS) EMS: m/z: calcd for C36H50O7SiNa: 645.3218; found:
645.3219 [M+Na+].
Dimethylthexylsilyl 4,6-di-O-acetyl-3-O-allyl-2-azido-2-deoxy-a-d-gluco-
pyranosyl-(1!4)-2,3-di-O-benzyl-6-O-methoxybenzyl-a-d-galactopyrano-
side (29):
A mixture of 30 (197 mg, 0.6 mmol), diphenylsulfoxide
(242 mg, 1.2 mmol) and tri-tert-butylpyrimidine (446 mg, 1.8 mmol) in
CH2Cl2 (8 mL) were stirred over flame dried 4 ꢂ molecule sieves
(200 mg) for 30 min. Then the reaction mixture was cooled to ꢀ608C,
Tf2O (120 mL, 0.72 mmol) was added and the mixture was first brought to
ꢀ408C and then slowly warmed to ꢀ208C over a period of approximate-
ly 1.5 h. After the reaction was cooled to ꢀ258C, a solution of the accept-
or 25 (302 mg, 0.485 mmol) in CH2Cl2 (4 mL) was added dropwise. The
resulting mixture was warmed very slowly to room temperature (typically
overnight), after which time the reaction was quenched with triethyl-
Methyl 2,4-di-O-benzyl-3-O-p-methoxybenzyl-6-O-acetyl-a-d-galactopyr-
anosyl-(1!3)-2-acetamido-4-azido-2,4,6-trideoxy-a-galactopyranosyl-(1!
4)-6-O-acetyl-2,3-di-O-benzyl-a-d-galactopyranoside (22): NIS (30 mg,
0.134 mmol) was added to a solution of 24 (86 mg, 0.134 mmol) and 16
(42 mg, 0.067 mmol) containing 4 ꢂ molecular sieves (200 mg) in CH2Cl2
(5 mL) under argon at ꢀ308C. The mixture was stirred for 15 min and
then AgOTf (2.5 mg) and slowly warmed to room temperature. After
one hour, TLC indicated that the starting material had been completely
consumed. The reaction mixture was quenched with triethylamine, dilut-
ed with CH2Cl2 (10 mL), and the solution was washed with saturated
NaHCO3 solution, Na2S2O3 solution (10%) and brine. The organic phase
was dried over MgSO4, concentrated and the residue was purified by
flash chromatography (n-hexane/acetone 5:2) to afford 22 (58 mg, 79%).
[a]D = +41.58 (c=1.0, CHCl3); 1H NMR (600 MHz, CDCl3): d=7.41–
6.85 (m, 24H, Ar), 5.75 (d, 1H, NH), 4.97 (m, 2H, CH2Ph), 4.83–4.57 (m,
11H, 1a-H, 1b-H, 1c-H, 4ꢃCH2Ph), 4.54 (m, 1H, 2b- H), 4.32 (m, 1H,
6a-H), 4.16 (m, 1H, 5b-H), 4.13–4.03 (m, 5H, 2c-H, 3c-H, 5c-H, 6c-H,
6’c-H), 4.00 (m, 1H, 4a-H), 3.87–3.79 (m, 8H, 2a-H, 3a-H, 4c-H, 5a-H,
6’a-H, CH3), 3.68–3.63 (m, 2H, 3b-H, 4b-H), 3.35 (s, 3H, CH3), 2.03–1.92
(m, 9H, 3ꢃCOCH3), 0.90 ppm (d, 3H, CH3); 13C NMR (125 MHz,
CDCl3): d=138.6–127.6, 113.8, 101.3, 98.8, 98.3, 79.3, 78.7, 76.6, 76.0,
75.4, 74.8, 74.7, 74.4, 73.2, 73.0, 72.9, 69.4, 68.5, 65.7, 64.0, 62.7, 61.9 ppm;
(HRMS) EMS: m/z: calcd for C61H72N4O17Na: 1155.4785; found:
1155.4782 [M+Na+].
AHCTUNGERTGaNNUN mine and concentrated. The residue was purified by flash chromatogra-
phy (n-hexane/acetone 4:1) to afford 29 (330 mg, 73%). [a]D = +53.38
(c=1.0, CHCl3); 1H NMR (600 MHz, CDCl3): d=7.67–6.89 (m, 14H,
Ar), 5.88–5.95 (m, 1H, OCH2CH=CH2), 5.18–5.31 (m, 2H, OCH2CH=
CH2), 5.04 (t, 3J=10.3 Hz, 1H, 4b-H), 4.94 (m, 2H, 1b-H, 1/2ꢃCH2Ph),
4.82 (d, 2J=11.2 Hz, 1H, 1/2ꢃCH2Ph), 4.69 (m, 2H, CH2Ph), 4.61 (d,
3J=7.2 Hz, 1H, 1a-H), 4.46 (s, 2H, CH2Ph), 4.34–4.28 (m, 2H, 5b-H,
OCH2CH=CH2), 4.14 (m, 1H, OCH2CH=CH2), 4.08 (d, 3J=3.1 Hz, 1H,
4a-H), 3.90–3.83 (m, 3H, 3b-H, 6b-H, 6’b-H), 3.81 (s, 3H, CH3), 3.60–3.43
(m, 4H, 2a-H, 5a-H, 6a-H, 6’a-H), 3.38 (dd, 3J=3.1, 10.0 Hz, 1H, 3a-H),
3
3.30 (dd, J=3.5, 10.3 Hz, 1H, 2b-H), 2.08–2.00 (m, 6H, 2ꢃCH3CO), 1.69
(m, 1H, CHACHTNUTRGNE(UNG CH3)2), 0.91 (m, 12H, 4ꢃCH3), 0.19 ppm (m, 6H, 2ꢃCH3);
13C NMR (125 MHz, CDCl3): d=138.6–124.8, 117.3, 113.9, 113.8, 98.6,
98.5, 80.9, 79.9, 74.7, 74.4, 73.4, 73.1, 73.0, 72.7, 69.7, 67.9, 66.9, 63.3,
61.3 ppm; (HRMS) EMS: m/z: calcd for C49H67N3O13SiNa: 956.4335;
found: 956.4330 [M+Na+].
Dimethylthexylsilyl 2-acetamido-4,6-di-O-acetyl-3-O-allyl-2-deoxy-a-d-
glucopyranosyl-(1!4)-2,3-di-O-benzyl-6-O-p-methoxybenzyl-a-d-galacto-
Methyl
2,4-di-O-benzyl-6-O-acetyl-a-d-galactopyranosyl-(1!3)-2-acet-
pyranoside (31): H2S was bubbled into a solution of 29 (330 mg,
amido-4-azido-2,4,6-trideoxy-a-galactopyranosyl-(1!4)-2,3-di-O-benzyl-
6-O-acetyl-a-d-galactopyranoside (21): DDQ (26 mg, 0.115 mmol) was
added a solution of 22 (26 mg, 0.023 mmol) in a mixture of CH2Cl2 and
H2O (10:1, 5 mL) and this mixture was stirred for 2 h. After dilution with
CH2Cl2 (10 mL) the organic phase was washed with NaHCO3 solution,
dried over MgSO4 and concentrated. The residue was purified by flash
chromatography (n-hexane/acetone 2:1) to afford 21 (17 mg, 73%). [a]D
0.355 mmol) in a mixture of pyridine/H2O/NEt3 10:1:0.3 (10 mL) for 3 h.
TLC indicated that the starting material had disappeared and the reac-
tion mixture was co-evaporated with toluene at to give the amino form
of the disaccharide. The residue was dissolved in pyridine (10 mL) and
acetic anhydride (4 mL) and resulting solution was stirred overnight and
then concentrated to give a residue that was purified by flash chromatog-
raphy (n-hexane/acetone 3:1) to afford 31 (255 mg, 75%). [a]D = +61.28
(c=1.0, CHCl3); 1H NMR (600 MHz, CDCl3): d=7.38–6.87 (m, 14H,
Ar), 5.85–5.77 (m, 2H, NH, OCH2CH=CH2), 5.24–5.10 (m, 3H, 4b-H,
OCH2CH=CH2), 4.93 (d, 2J=11.2 Hz, 1H, 1/2ꢃCH2Ph), 4.92 (d, 3J=
3.7 Hz. 1H, 1b-H), 4.84 (d, 2J=11.2 Hz, 1H, 1/2ꢃCH2Ph), 4.74–4.66 (m,
2H, CH2Ph), 4.63 (d, 3J=7.2 Hz, 1H, 1a-H), 4.44–4.30 (m, 4H, 2b-H,
5b-H, CH2Ph), 4.20 (d, 3J=2.95 Hz, 1H, 4a-H), 4.10–4.00 (m, 2H,
OCH2CH=CH2), 3.82–3.76 (m, 4H, 6b-H, CH3), 3.71 (t, 3J=10.5, 9.8 Hz,
1H, 3b-H), 3.60–3.56 (m, 2H, 2a-H, 6’b-H), 3.52–3.44 (m, 3H, 5a-H,
6a-H, 6’a-H), 3.41 (dd, 3J=9.7, 3.0 Hz, 1H, 3a-H), 2.15–2.03 (m, 9H, 3ꢃ
=
+21.58 (c=1.0, CHCl3); 1H NMR (600 MHz, CDCl3): d=7.41–6.85
(m, 20H, Ar), 5.72 (d, 1H, NH), 4.91–4.62 (m, 11H, 1a-H, 1b-H, 1c-H,
4ꢃCH2Ph), 4.54 (m, 1H, 2b-H), 4.32 (m, 1H, 6c-H), 4.42–4.00 (m, 5H,
4a-H, 5a-H, 5b-H, 6a-H, 6’a-H), 3.92–3.80 (m, 5H, 2a-H, 3a-H, 4c-H,
5c-H, 6’c-H), 3.71 (dd, 3J=3.2, 11.0 Hz, 1H, 3b-H), 3.53 (m, 1H, 4b-H),
3.35 (s, 3H, CH3), 2.03–1.93 (m, 9H, 3ꢃCH3CO), 0.94 ppm (d, 3H, CH3);
13C NMR (125 MHz, CDCl3): d=138.3–127.6, 98.6, 98.5, 98.4, 76.9, 76.8,
76.6, 75.9, 75.7, 75.2, 74.5, 73.9, 73.4, 73.1, 70.0, 69.0, 68.4, 65.8, 63.6, 62.8,
61.7 ppm; (HRMS) EMS: m/z: calcd for C53H64N4O16Na: 1035.4209;
found: 1035.4214 [M+Na+].
CH3CO), 1.70 (m, 1H, CHACHTUNRGTNE(UNG CH3)2), 0.91 (m, 12H, 4ꢃCH3), 0.19 ppm (m,
6H, 2ꢃCH3); 13C NMR (125 MHz, CDCl3): d=138.4–127.2, 116.8, 114.0,
98.4, 97.7, 81.2, 79.9, 76.8, 74.9, 73.2, 72.7, 72.6, 71.6, 70.7, 68.6, 68.2, 66.8,
61.4, 60.3 ppm; (HRMS) EMS: m/z: calcd for C51H71NO14SiNa: 972.4536;
found: 972.4550 [M+Na+].
Dimethylthexylsilyl
2,3-di-O-benzyl-6-O-p-methoxybenzyl-b-d-galacto-
pyranoside (25): A solution of dimethylthexylsilyl 2,3-di-O-benzyl-4,6-O-
p-methoxybenzylidene-b-d-galactopyranoside[21] (180 mg, 0.29 mmol) and
molecule sieve 4 ꢂ (100 mg) in dry THF (6 mL) was cooled to 08C, and
NaCNBH3 (183 mg, 2.9 mmol) was added, followed by dropwise addition
of hydrogen chloride solution in Et2O (1m, 1 mL). The reaction was con-
tinued until evolution gas ceased. After 30 min, the reaction mixture was
diluted with CH2Cl2 (10 mL), and extracted with saturated NaHCO3 solu-
tion and brine. The organic phase was dried over MgSO4 and evaporated
at low pressure to give a residue, that was purified by flash chromatogra-
Dimethylthexylsilyl 2-acetamido-3-O-allyl-6-O-methanesulfonyl-2-deoxy-
a-d-glucopyranosyl-(1!4)-2,3-di-O-benzyl-6-O-methoxybenzyl-a-d-gal-
actopyranoside (32): Compound 31 (512 mg, 0.54 mmol) was dissolved in
methanol (20 mL), and sodium methoxide (87 mg, 1.62 mmol) was
added. After one hour, the reaction mixture was neutralized with Amber-
lite IR120 (H+ form) and filtered. The resulting solution was evaporated
Chem. Eur. J. 2010, 16, 3476 – 3488
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
3485

D. R. Bundle et al.
at low pressure. The crude disaccharide diol was used directly in the next
step. Methansulfonyl chloride (62 mL, 0.81 mmol) in pyridine (30 mL)
under argon at ꢀ158C was added to the diol and the solution was stirred
for 30 min, then slowly warmed to 08C and kept at this temperature for
3 h. The reaction mixture was quenched with MeOH (5 mL) and concen-
trated. The residue was purified by flash chromatography (n-hexane/ace-
tone 3:1) to afford 32 (483 mg, 95%). [a]D = +35.18 (c=1.0, CHCl3);
1H NMR (600 MHz, CDCl3): d=7.38–6.87 (m, 14H, Ar), 5.94–5.86 (m,
1H, OCH2CH=CH2), 5.84 (d, 1H, NH), 5.31–5.18 (m, 2H, OCH2CH=
CH2), 4.94 (d, 2J=11.2 Hz, 1H, 1/2ꢃCH2Ph), 4.84–4.81 (m, 2H, 1b-H,
1/2ꢃCH2Ph), 4.74 (d, 2J=11.2 Hz, 1H, 1/2ꢃCH2Ph), 4.65–4.62 (m, 2H,
1a-H, 1/2ꢃCH2Ph), 4.39 (m, 2H, CH2Ph), 4.27–4.14 (m, 5H, OCH2CH=
CH2, 2b-H, 5b-H, 4a-H), 3.95 (m, 1H, 6b-H), 3.82 (s, 3H, CH3), 3.66 (m,
2H, 4b-H, 6’b-H), 3.58–3.45 (m, 5H, 2a-H, 3b-H, 5a-H, 6a-H, 6’a-H), 3.41
(dd, 3J=9.78, 2.86 Hz, 1H, 3a-H), 2.90 (s, 3H, CH3), 1.96 (s, 3H, CH3),
CH3), 0.92 (m, 12H, 4ꢃCH3), 0.19 ppm (m, 6H, 2ꢃCH3); 13C NMR
(125 MHz, CDCl3): d=134.4–127.3, 117.4, 113.9, 98.4, 97.9, 81.1, 80.3,
76.4, 74.9, 73.2, 72.9, 72.3, 70.9, 70.5, 66.9, 65.2, 63.4 ppm; (HRMS) EMS:
m/z: calcd for C47H66N4O10SiNa: 897.4440; found: 897.4436 [M+Na+].
Dimethylthexylsilyl 2-acetamido-3-O-allyl-4-azido-2,4,6-trideoxy-a-d-gal-
actopyranosyl-(1!4)-6-O-acetyl-2,3-di-O-benzyl-a-d-galactopyranoside
(35): Compound 34 (45 mg, 0.051 mmol) was dissolved in a mixture of
CH2Cl2 and H2O solvents (10:1, 5 mL), followed by addition of DDQ
(47 mg, 0.206 mmol) and the reaction mixture was stirred for 2 h. The re-
action mixture was diluted with CH2Cl2 (10 mL), and washed with
NaHCO3 solution the organic phase was dried over MgSO4 and then con-
centrated. The crude produce was dissolved in pyridine (4 mL) and acetic
anhydrate (2 mL), and stirred overnight. The reaction mixture was con-
centrated and the residue was purified by flash chromatography (n-
hexane/acetone 3:1) to afford 35 (32 mg, 80%). [a]D = +43.48 (c=1.0,
CHCl3); 1H NMR (600 MHz, CDCl3): d=7.41–7.24 (m, 10H, Ar), 5.98–
5.84 (m, 1H, OCH2CH=CH2), 5.77 (d, 1H, NH), 5.38–5.20 (m, 2H,
OCH2CH=CH2), 4.94 (d, 1H, 1/2ꢃCH2Ph), 4.83–4.76 (m, 2H, 1b-H, 1/2ꢃ
CH2Ph), 4.71 (m, 2H, CH2Ph), 4.61 (d, 2J=7.1 Hz, 1H, 1a-H), 4.53 (m,
1H, 2b-H), 4.43–4.36 (m, 1H, 6a-H), 4.31 (m, 1H, 5b-H), 4.26–4.18 (m,
1H, OCH2CH=CH2), 4.06–3.96 (m, 1H, OCH2CH=CH2), 3.90 (m, 1H,
6’a-H), 3.71–3.67 (m, 2H, 3b-H, 4b-H), 3.54–3.48 (m, 2H, 2a-H, 5a-H),
3.39 (dd, 3J=2.8, 9.8 Hz, 1H, 3a-H), 2.02 (m, 6H, 2ꢃCH3CO), 1.69 (m,
1.69 (m, 1H, CHACHTUNGTRENNUNG(CH3)2), 0.92 (m, 12H, 4ꢃCH3), 0.19 ppm (m, 6H, 2ꢃ
CH3); 13C NMR (125 MHz, CDCl3): d=138.4–127.4, 117.2, 114.0, 98.5,
98.0, 81.5, 80.5, 79.5, 77.4, 77.1, 76.9, 75.1, 73.3, 73.1, 72.7, 72.6, 71.9, 69.8,
68.9, 67.9, 66.8 ppm; (HRMS) EMS: m/z: calcd for C48H69NO14SiNa:
966.4100; found: 966.4101 [M+Na+].
Dimethylthexylsilyl 2-acetamido-3-O-allyl-2,6-dideoxy-a-d-glucopyrano-
syl-(1!4)-2,3-di-O-benzyl-6-O-p-methoxybenzyl-a-d-galactopyranoside
(33): Sodium borohydride (230 mg, 6.13 mmol) was added to a solution
of 32 (200 mg, 0.21 mmol) in DMSO (10 mL) under argon at 858C, and
the solution was stirred overnight. The resulting mixture was quenched
with methanol at room temperature, and the mixture was diluted with
ethyl acetate (15 mL), and the resultant mixture was washed with water
(10 mL). The aqueous layer was extracted with ethyl acetate (3ꢃ15 mL)
and the combined organic extracts were dried over MgSO4 and concen-
trated. The residue was purified by flash chromatography (n-hexane/ace-
tone 3:1) to afford 33 (153 mg, 85%). [a]D = +45.28 (c=1.0, CHCl3);
1H NMR (600 MHz, CDCl3): d=7.37–6.87 (m, 14H, Ar), 5.95–5.88 (m,
2H, NH, OCH2CH=CH2), 5.32–5.18 (m, 2H, OCH2CH=CH2), 4.90–4.82
(m, 2H, CH2Ph), 4.79–4.76 (m, 2H, 1b-H, 1/2ꢃCH2Ph), 4.63–4.61 (m,
2H, 1a-H, 1/2ꢃCH2Ph), 4.40 (m, 2H, CH2Ph), 4.27–2.12 (m, 5H,
OCH2CH=CH2, 2b-H, 4a-H, 5b-H), 3.82 (s, 3H, CH3), 3.55 (dd, 3J=7.2,
9.8 Hz, 1H, 2a-H), 3.50 (t, 3J=9.1 Hz, 1H, 3b-H), 3.48 (m, 3H, 5a-H,
6a-H, 6’b-H), 3.40 (dd, 3J=2.9, 9.7 Hz, 1H, 3a-H), 3.28 (t, 3J=9.3 Hz,
1H, CHACHTUNRTGENUNG(CH3)2), 1.00 (d, 3H, CH3), 0.92 (m, 12H, 4ꢃCH3), 0.20 ppm (m,
6H, 2ꢃCH3); 13C NMR (125 MHz, CDCl3): d=138.3–127.3, 98.5, 97.3,
80.9, 79.8, 75.2, 72.8, 72.4, 72.1, 71.6, 66.5, 65.7, 61.9 ppm; (HRMS) EMS:
m/z: calcd for C41H60N4O10SiNa: 819.3971; found: 819.3973 [M+Na+].
Dimethylthexylsilyl 2-acetamido-4-azido-2,4,6-trideoxy-a-d-galactopyra-
nosyl-(1!4)-6-O-acetyl-2,3-di-O-benzyl-a-d-galactopyranoside (36):
A
solution of NaOAc (26 mg, 0.32 mmol) in a mixture of HOAc/H2O (20:1)
(4 mL) was degassed under argon for 30 min. Compound 35 (32 mg,
0.04 mmol) was dissolved in the degassed above solution and PdCl2
(27 mg, 0.12 mmol) was added, and mixture was stirred under argon until
the starting material had reacted. The reaction mixture was passed
through a filter (0.2 mm) to give a yellow solution, followed by dilution
with ethyl acetate (10 mL), and then extracted with saturated NaHCO3
solution (10 mL). The aqueous layer was extracted with ethyl acetate (3ꢃ
10 mL) and the combined organic layers were dried over MgSO4 and
concentrated. The residue was purified by flash chromatography (n-
hexane/acetone 3:1!2:1) to afford 36 (20 mg, 66%). [a]D = +51.58 (c=
1.0, CHCl3); 1H NMR (600 MHz, CDCl3): d=7.37–7.25 (m, 10H, Ar),
6.22 (d, 1H, NH), 4.92 (d, 2J=11.2 Hz, 1H, 1/2ꢃCH2Ph), 4.79–4.76 (m,
2H, 1b-H, 1/2ꢃCH2Ph), 4.71 (s, 2H, CH2Ph), 4.62 (d, 2J=7.25 Hz, 1H,
1a-H), 4.45 (m, 1H, 6a-H), 4.36–4.30 (m, 2H, 2b-H, 5b-H), 4.06 (m, 1H,
4a-H), 4.00 (dd, 3J=3.5, 10.5 Hz, 1H, 3b-H), 3.90 (m, 1H, 6’a-H), 3.60
(m, 1H, 4b-H), 3.57–3.53 (m, 2H, 2a-H, 5a-H), 3.42 (dd, 3J=9.67,
1H, 4b-H), 1.95 (s, 3H, CH3CO), 1.69 (m, 1H, CHACHTUNTRGNEUNG(CH3)2), 1.05 (d, 3H,
CH3), 0.91 (m, 12H, 4ꢃCH3), 0.18 ppm (m, 6H, 2ꢃCH3); 13C NMR
(125 MHz, CDCl3): d=138.6–127.3, 117.2, 114.0, 98.4, 98.0, 81.2, 80.3,
80.1, 75.2, 75.1, 73.3, 72.9, 72.0, 71.9, 70.9, 68.0, 67.0 ppm; (HRMS) EMS:
m/z: calcd for C47H67NO11SiNa: 872.4376; found: 872.4375 [M+Na+].
Dimethylthexylsilyl 2-acetamido-3-O-allyl-4-azido-2,4,6-trideoxy-a-d-gal-
actopyranosyl-(1!4)-2,3-di-O-benzyl-6-O-p-methoxybenzyl-a-d-galacto-
pyranoside (34): Pyridine (42 mL, 0.494 mmol) and Tf2O (52 mL,
0.33 mmol) were added to a solution of 33 (140 mg, 0.165 mmol) in
CH2Cl2 (10 mL) at ꢀ308C, and the reaction mixture was stirred at this
temperature under argon for two hours. The reaction mixture was diluted
with CH2Cl2 (10 mL), and extracted with saturated NaHCO3 solution
(10 mL). The aqueous layer was extracted with ethyl acetate (3ꢃ10 mL)
and the combined organic layers were dried over MgSO4 and concentrat-
ed in vacuum. The crude product was dissolved in dry DMF (4 mL)
under argon, and NaN3 (123 mg, 1.914 mmol) was added. The reaction
mixture was stirred overnight. Then the reaction mixture was diluted
with ethyl acetate (10 mL), and washed with water (10 mL). The aqueous
layer was extracted with ethyl acetate (3ꢃ10 mL) and the combined or-
ganic extracts were dried over MgSO4 and concentrated. The residue was
purified by flash chromatography (n-hexane/acetone 3:1!2:1) to afford
34 (90 mg, 62%). [a]D = +32.48 (c=1.0, CHCl3); 1H NMR (600 MHz,
CDCl3): d=7.39–6.86 (m, 14H, Ar), 5.95–5.87 (m, 1H, OCH2CH=CH2),
5.69 (d, 1H, NH), 5.35–5.23 (m, 2H, OCH2CH=CH2), 4.93 (d, 2J=
11.2 Hz, 1H, 1/2ꢃCH2Ph), 4.85–4.79 (m, 2H, 1b-H, 1/2ꢃCH2Ph), 4.74 (d,
1H, 1/2ꢃCH2Ph), 4.65–4.62 (m, 2H, 1a-H, 1/2ꢃCH2Ph), 4.52 (m, 1H,
2b-H), 4.44–4.33 (m, 3H, 5b-H, CH2Ph), 4.25–4.20 (m, 1H, OCH2CH=
CH2), 4.18 (m, 1H, 4a-H), 4.05–4.00 (m, 1H, OCH2CH=CH2), 3.82 (s,
3H, CH3), 3.71 (m, 1H, 4b-H), 3.66 (dd, 3J=3.3, 10.7 Hz, 1H, 3b-H),
3.52–3.45 (m, 4H, 2a-H, 5a-H, 6a-H, 6’a-H), 3.39 (dd, 3J=2.9, 9.8 Hz,
2.96 Hz, 1H, 3a-H), 2.07 (m, 6H, 2ꢃCH3CO), 1.69 (m, 1H, CHACHTUNGTRENNUNG(CH3)2),
0.99 (d, 3H, CH3), 0.91 (m, 12H, 4ꢃCH3), 0.18 ppm (m, 6H, 2ꢃCH3);
13C NMR (125 MHz, CDCl3): d=138.3–127.3, 98.5, 97.3, 80.9, 79.8, 75.2,
72.8, 72.4, 72.1, 71.6, 66.5, 65.7, 61.9, 50.7 ppm; (HRMS) EMS: m/z: calcd
for C38H56N4O10SiNa: 779.3658; found: 779.3658 [M+Na+].
Dimethylthexylsilyl 6-O-acetyl-2,3,4-tri-O-benzyl-a-d-galactopyranosyl-
(1!3)-2-acetamido-4-azido-2,4,6-trideoxy-a-galactopyranosyl-(1!4)-6-
O-acetyl-2,3-di-O-benzyl-a-d-galactopyranoside (37): NIS (12 mg,
0.053 mmol)was added to a solution of containing acceptor 36 (20 mg,
0.026 mmol), donor 2 (33 mg, 0.053 mmol) and 4 ꢂ molecular sieves
(200 mg) in CH2Cl2 (5 mL) under argon at ꢀ308C. The mixture was
stirred at that temperature for 15 min, then AgOTf (1 mg) was added at
ꢀ308C and the reaction mixture was slowly warmed to room tempera-
ture. After 1 h, the reaction was complete as judged by TLC. The reac-
tion mixture was quenched with triethylamine, diluted with CH2Cl2
(5 mL), and this solution was extracted with saturated NaHCO3 solution,
Na2S2O3 solution (10%) and brine. The organic phase was dried with
MgSO4 and concentrated. The residue was purified by flash chromatogra-
phy (n-hexane/acetone 7:2) to afford 37 (21 mg, 66%). [a]D = +23.48
(c=1.0, CHCl3); 1H NMR (600 MHz, CDCl3): d=7.37–7.25 (m, 25H,
Ar), 5.90 (d, 1H, NH), 4.98–4.93 (m, 3H, 3/2ꢃCH2Ph), 4.88 (d, 3J=
3.1 Hz, 1H, 1c-H), 4.85–4.72 (m, 5H, 1b-H, 2ꢃCH2Ph), 4.67 (m, 2H,
1H, 3a-H), 1.96 (s, 3H, CH3CO), 1.71 (m, 1H, CHACHTUNTRGNEUNG(CH3)2), 1.00 (d, 3H,
3486
www.chemeurj.org
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2010, 16, 3476 – 3488

Polysaccharide Synthesis
FULL PAPER
CH2Ph), 4.59 (m, 2H, 1a-H, 1/2ꢃCH2Ph), 4.55 (m, 1H, 2b-H), 4.35 (m,
1H, 6a-H), 4.16 (m, 1H, 5b-H), 4.13–4.08 (m, 5H, 2c-H, 3c-H, 5c-H,
6c-H, 6’c-H), 3.97 (m, 1H, 4a-H), 3.92–3.88 (m, 2H, 4c-H, 6’a-H), 3.73
(dd, 3J=3.2, 10.8 Hz, 1H, 3b-H), 3.67 (m, 1H, 4b-H), 3.54–3.48 (m, 2H,
2a-H, 5a-H), 3.37 (dd, 3J=10.2, 3.0 Hz, 1H, 3a-H), 2.04–1.96 (m, 9H, 3ꢃ
solution of NaHCO3 (62 mL) were added to a solution of crude 40
(10 mg, 5 mmol) in CH2Cl2 (0.5 mL) and H2O (85 mL) in an ice-water
bath. An aqueous solution of NaOCl (available chlorine 10–15%, 75 mL)
was added and the reaction mixture was continuously stirred for 80 min.
As the temperature increased from 08C to room temperature, the reac-
tion was neutralized with HCl (0.5 1m HCl, about 20 mL) to pH 6–7.
After neutralization, tBuOH (0.35 mL), 2-methylbut-2-ene in THF (2m,
0.7 mL) and a solution of NaClO2 (25 mg) and NaH2PO4 in water
(100 mL) were added. The reaction mixture was kept at room tempera-
ture for two hours. The reaction mixture was diluted with saturated aque-
ous NaH2PO4 solution (5 mL), and extracted with ethyl acetate (3ꢃ
10 mL). The organic layers were combined and dried over MgSO4. The
crude product from the oxidation reaction was dissolved in DMF (1 mL),
followed by addition of CsF (10 mg) and BnBr (40 mL). The reaction
mixture was stirred overnight and the resulting mixture was diluted with
ethyl acetate (5 mL) and extracted with saturated NaHCO3 solution. The
aqueous layer was extracted with ethyl acetate (3ꢃ5 mL) and the com-
bined organic layers were dried over MgSO4 and concentrated in
vacuum. The residue was purified by flash chromatography (n-hexane/
acetone 3:1!12:1) to afford 20 (6 mg, 52%). [a]D = +25.48 (c=1.0,
CHCl3); 1H NMR (600 MHz, CDCl3): d=4.91 (1a-H), 3.82 (2a-H), 3.82
(3a-H), 4.33 (4a-H), 4.25 (5a-H); 4.61 (1b-H), 4.38 (2b-H), 3.52 (3b-H),
3.35 (4b-H), 4.06 (5b-H); 4.86 (1c-H), 3.97 (2c-H), 4.23 (3c-H), 4.31
CH3CO), 1.68 (m, 1H, CHACHTNUGTRNEUNG(CH3)2), 0.9 (m, 15H, 5ꢃCH3), 0.18 ppm (m,
6H, 2ꢃCH3); 13C NMR (125 MHz, CDCl3): d=138.7–127.4, 102.0, 98.7,
98.5, 80.6, 80.5, 79.8, 79.0, 76.3, 74.9, 74.7, 74.5, 74.3, 73.2, 72.9, 72.8, 72.5,
69.9, 65.6, 64.3, 62.4, 61.8 ppm; (HRMS) EMS: m/z: calcd for
C67H86N4O16SiNa: 1253.5700; found: 1253.5698 [M+Na+].
6-O-Acetyl-2,3,4-tri-O-benzyl-a-d-galactopyranosyl-(1!3)-2-acetamido-
4-azido-2,4,6-trideoxy-a-galactopyranosyl-(1!4)-6-O-acetyl-2,3-O-diben-
zyl-a/b-d-galactopyranose (38): HOAc (70 mL) and TBAF (0.7 mL,
0.62 mmol) were added to a solution of 37 (306 mg, 0.249 mmol) in THF
(10 mL) and the solution was then stirred for two hours. The resulting
mixture was diluted with ethyl acetate (20 mL) and this solution was ex-
tracted with saturated NaHCO3 solution. The aqueous layer was extract-
ed with ethyl acetate (3ꢃ20 mL) and the combined organic layers were
dried over MgSO4 and concentrated. The residue was purified by flash
chromatography (n-hexane/acetone 2:1!1:1) to afford 38 (250 mg, 92%)
directly for next step.
6-O-Acetyl-2,3,4-tri-O-benzyl-a-d-galactopyranosyl-(1!3)-2-acetamido-
4-azido-2,4,6-trideoxy-a-galactopyranosyl-(1!4)-2,3-O-dibenzyl-6-O-
acetyl-a-d-galactopyranosyl trichloroacetimidate (23): Compound 38
(108 mg, 0.1 mmol) was dissolved in a mixture of CH2Cl2 (5 mL) and
Cl3CCN (0.11 mL) under argon, and then one drop of DBU was added at
room temperature. After 2 h, the resulting mixture was concentrated.
The residue was purified by flash chromatography (hexanes/ethyl acetate
AHCTUNGTRENNUNG
AHCTUNGTRENNUNG
AHCTUNGTRENNUNG
98.9, 98.4, 95.7, 82.3, 81.9, 78.6, 76.3, 76.0, 75.9, 75.3, 74.8, 74.6, 74.5, 73.8,
73.5, 73.2, 73.1, 72.4, 72.0, 71.7, 70.6, 69.5, 67.5, 67.2, 66.9, 65.7, 65.4, 64.4,
64.2 ppm; (HRMS) EMS: m/z: calcd for C132H138N8O31Na: 2353.9360;
found: 2353.9347 [M+Na+].
1
2:1) to afford 23 (87 mg, 71%). [a]D = +35.28 (c=1.0, CHCl3); H NMR
(600 MHz, CDCl3): d=8.58 (s, 1H, NH), 7.42–7.23 (m, 25H, Ar), 6.55 (d,
3J=3.5 Hz, 1H, 1a-H), 5.67 (d, 1H, NH), 4.98 (m, 2H, CH2Ph), 4.84 (m,
2H, 1b-H, 1c-H), 4.81–4.60 (m, 8H, 4ꢃCH2Ph), 4.55 (m, 1H, 2b-H), 4.28
(m, 1H, 6a-H), 4.20 (m, 1H, 5b-H), 4.15 (m, 1H, 6c-H), 4.11–4.05 (m,
Glycoside 19: Compound 20 (6 mg, 2.57 mmol) was dissolved in a mixture
of CH3OH and CH2Cl2 and H2O (3:1:0.5, 4.5 mL), and Pd(OH)2 (20 mg)
were also added to the reaction mixture. The reaction mixture was hydro-
genated overnight and then passed through a filter (0.2 mm) and reverse
phase column (Sep-Pak) to give a yellow solution. Gel filtration of the
yellow solution on a column (2.5ꢃ85 cm) of Biogel P-2 eluting with am-
monium acetate (100 mm) gave the pure product 19 (1.5 mg, 53%) as a
3
6H, 2c-H, 4c-H, 5c-H, 6’c-H, 4a-H, 6’a-H), 4.02 (dd, J=10.2, 3.5 Hz, 1H,
2a-H), 3.92–3.88 (m, 3H, 3a-H, 3c-H, 5a-H), 3.67 (m, 2H, 3b-H, 4b-H),
2.02 (m, 9H, 3ꢃCH3CO), 0.95 ppm (d, 1H, CH3); 13C NMR (125 MHz,
CDCl3): d=138.6–127.6, 100.9, 98.7, 94.2, 78.9, 78.9, 75.9, 75.6, 74.8, 74.7,
74.4, 74.3, 73.7, 73.3, 73.1, 72.6, 71.4, 69.5, 65.8, 63.9, 62.7, 61.4 ppm;
(HRMS) EMS: m/z: calcd for C61H68Cl3N5O16Na: 1254.3727; found:
1254.3725 [M+Na+].
white powder after repeated lyophilization. [a]D
D2O); 1H NMR (600 MHz, D2O): d=4.91 (1a-H), 3.89 (2a-H), 3.98
AHCTUNGERTG(NNUN 3a-H), 4.36 (4a-H), 4.24 (5a-H); 4.97 (1b-H), 4.13 (2b-H), 4.28 (3b-H),
= +83.98 (c=0.4,
Glycoside 39: Glycosyl donor 23 (173 mg, 0.1 mmol), monosaccharide ac-
ceptor 21 (95 mg, 0.094 mmol) and activated 4 ꢂ molecular sieves
(200 mg) were dried together under vacuum for 1 h in a pear-shaped
flask (25 mL). The contents of the flask were then dissolved in CH2Cl2
(10 mL). The suspension was stirred for 10 min at room temperature
under argon. Then the temperature was reduced to ꢀ158C, and TMSOTf
(47 mL, 0.1m in CH2Cl2) was added dropwise and the mixture was slowly
warmed to room temperature. After 30 min, the reaction mixture was
neutralized with triethylamine and concentrated. The residue was puri-
fied by flash chromatography (toluene/acetone 10:1) to afford 39
3.84 (4b-H), 4.79 (5b-H); 5.06 (1c-H), 3.94 (2c-H), 4.02 (3c-H), 4.46 (4c-
H), 4.13 (5c-H); 5.22 (1d-H), 3.98 (2d-H), 4.08 (3d-H), 4.39 (4d-H), 4.61
(5d-H); 4.99 (1e-H), 4.13 (2e-H), 4.25 (3e-H), 3.79 (4e-H), 4.77 (5e-H);
5.08 (1f-H), 3.88 (2f-H), 3.86 (3f-H), 4.27 (4f-H), 4.13 ppm (5f-H);
(HRMS) EMS: m/z: calcd for C41H64N4O31: 1108.3406 [Mꢀ2Hꢀ]; found:
553.1703 [Mꢀ2Hꢀ2].
(166 mg, 85%). [a]D
CDCl3): d=4.70 (1a-H), 3.78 (2a-H), 3.82 (3a-H), 4.00 (4a-H); 4.82
(1b-H), 4.55 (2b-H), 3.66 (3b-H, 4b-H), 4.18 (5b-H); 4.86 (1c-H), 4.12
(2c-H, 3c-H), 3.96 (4c-H); 5.28 (2J=3.4 Hz, 1d-H), 3.88 (2d-H), 3.81
(3d-H), 3.94 (4d-H), 4.04 (5d-H), 3.72 (6d-H), 4.28 (6’d-H), 4.81 (1e-H),
4.48 (2e-H), 3.62 (3e-H), 3.53 (4e-H), 4.16 (5e-H), 4.73 (1f-H), 3.97
(2f-H), 4.14 (3f-H), 4.09 (4f-H), 3.89 (5f-H), 3.81 (6f-H), 3.83 ppm
(6’f-H); 13C NMR (125 MHz, CDCl3): d=138.6–127.7, 101.2, 99.07, 98.7,
=
+45.28 (c=1.0, CHCl3); 1H NMR (600 MHz,
Acknowledgements
ACHTUNGTRENNUNG
We thank Dr. Dennis Kasper Harvard medical school for preliminary
assays to test for T-cell activity in compounds 1 and 19. These studies
were supported by a Discovery grant from the Natural Science and Engi-
neering Research Council of Canada (NSERC) and funds from the Al-
berta Ingenuity Foundationꢁs Centreꢁs program.
ACHTUNGTRENNUNG
ACHTUNGTRENNUNG
ACHTUNGTRENNUNG
98.3, 97.3, 79.3, 78.9, 76.8, 76.6, 76.3, 76.1, 76.0, 75.7, 75.6, 74.8, 74.7, 74.6,
74.5, 74.4, 73.8, 73.3, 73.2, 73.1, 72.3, 69.5, 69.1, 68.4, 65.7, 64.1, 62.7, 62.6,
61.7, 61.2, 55.3, 48.9, 48.8 ppm; (HRMS) EMS: m/z: calcd for
C112H130N8O31Na: 2105.8734; found: 2105.8721.
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Glycoside 20: Compound 39 (166 mg, 0.08 mmol) was dissolved in metha-
nol (5 mL), and NaOMe (21.6 mg, 0.4 mmol) was added. After 1 h, the
reaction mixture was neutralized with Amberlite IR120 (H+ form), and
filtered. The resulting solution was evaporated at low pressure to give
the crude 40. An aqueous solution of NaBr (1m, 12.5 mL), an aqueous so-
lution of TBABr (1m, 25 mL), TEMPO (1.5 mg) and a saturated aqueous
Chem. Eur. J. 2010, 16, 3476 – 3488
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
3487

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Received: September 6, 2009
Published online: February 9, 2010
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