Synthesis of three trisaccharide congeners to investigate frame shifting of β1,2-mannan homo-oligomers in an antibody binding site

Article abstract of DOI:10.1016/j.carres.2012.03.019

Homopolysaccharides such as the protective β1,2-mannan present in the cell wall of Candida albicans have the capability to bind to antibody in numerous frame shifted modes. A protective monoclonal antibody C3.1 binds this antigen and exhibits a unique binding profile where di and trisaccharides are the most potent inhibitors, while the intrinsic affinities of tetrasaccharide and larger oligomers dramatically decrease with increasing chain length. The design, synthesis and inhibitory activity of three β1,2-linked trisaccharide congeners is reported. Selective functional group modification was introduced at the terminal reducing or non-reducing mannose residues so that each trisaccharide would be capable of binding to antibody in only one of the possible frame shifted modes. Inhibition data show that C3.1 has the highest affinity for internally situated disaccharide epitopes but can bind the terminal non-reducing disaccharide of a trisaccharide epitope.

Full text of DOI:10.1016/j.carres.2012.03.019

Carbohydrate Research 357 (2012) 7–15
Contents lists available at SciVerse ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Synthesis of three trisaccharide congeners to investigate frame shifting
of b1,2-mannan homo-oligomers in an antibody binding site
⇑
Casey Costello, David R. Bundle
Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2G2
a r t i c l e i n f o
a b s t r a c t
Article history:
Homopolysaccharides such as the protective b1,2-mannan present in the cell wall of Candida albicans
have the capability to bind to antibody in numerous frame shifted modes. A protective monoclonal anti-
body C3.1 binds this antigen and exhibits a unique binding profile where di and trisaccharides are the
most potent inhibitors, while the intrinsic affinities of tetrasaccharide and larger oligomers dramatically
decrease with increasing chain length. The design, synthesis and inhibitory activity of three b1,2-linked
trisaccharide congeners is reported. Selective functional group modification was introduced at the termi-
nal reducing or non-reducing mannose residues so that each trisaccharide would be capable of binding to
antibody in only one of the possible frame shifted modes. Inhibition data show that C3.1 has the highest
affinity for internally situated disaccharide epitopes but can bind the terminal non-reducing disaccharide
of a trisaccharide epitope.
Received 19 January 2012
Received in revised form 12 March 2012
Accepted 19 March 2012
Available online 27 April 2012
D.R.B. wishes to dedicate this manuscript to
Dr. Malcom Perry on the occasion of his
retirement from the National Research
Council of Canada
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Mapping an anti-Candida albicans antibody
Methyl b-D-mannopyranosyl-(1?2)-b-D-
mannopyranosyl-(1?2)-b-D-
mannopyranoside
Preference for internal or external
disaccharide epitopes
Frame shifted disaccharide epitopes
Synthesis of terminal mono-deoxy and
mono-O-methyl congeners
1. Introduction
We have attributed this observation to a relatively well organized
solution conformation for the b1,2-oligomannan which precludes
Candida albicans is a fungal commensal that can cause serious
infections in individuals with a compromised immune system or
for those in a deliberately immunosuppressed condition such as
individuals undergoing solid organ transplant or certain cancer
therapies.1,2 The incidence of hospital acquired infection continues
to rise in North America and passive and active immunizations are
treatment options that are beginning to be considered.2–5 Two cell
wall carbohydrate antigens have been the focus of vaccine re-
search. One is the b-glucan4,5 and the other is the b-mannan which
is a minor component of the cell wall phosphomannan.6–10 Mono-
clonal antibodies that recognize the b-mannan confer protection in
passive transfer experiments.11,12
binding of larger structures.13 We imagine that oligosaccharide
segments of larger oligomers must protrude from the binding site
and those parts adjacent to the site likely make unfavourable con-
tacts with the antibody surface. Since the oligosaccharide is some-
what rigid an energetic penalty will be associated with altered
conformations that avoid these contacts and consequently will re-
sult in lower intrinsic affinity. To explore these unique binding char-
acteristics, in the absence of a solved crystal structure of the
antibody and bound ligand, we have mapped the fine specificity of
the binding site of C3.1 with functional group replacement14 and
NMR methods.15 Surprisingly the primary polar contacts between
a disaccharide epitope and antibody involve O-3 and O-4 of the ter-
minal reducing residue and only one hydroxyl group at C-40 of the
non-reducing residue makes an important polar contact at the
periphery of the binding site.14
One of these antibodies C3.1, an IgG3j has been the subject of
detailed chemical mapping and conformational studies.13,14 It
shows a unique dependence on oligosaccharide size. Di and tri-sac-
charides of b1,2 linked mannohexosides are optimal inhibitorswhile
larger oligosaccharides exhibit increasingly diminished activity.13
The structure of the glycan component of C. albicans phospho-
mannan has been elucidated by Suzuki’s group16–20 and reveals
an
incorporate two types of b-mannan chains. One is linked glycosid-
ically to the 1,2-linked side chains. The second is attached to an
a1,6-mannan backbone with a1,2-linked side chains that
⇑
Corresponding author. Tel.: +1 780 492 8808; fax: +1 780 492 7705.
E-mail address: dave.bundle@ualberta.ca (D.R. Bundle).
a
0008-6215/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.carres.2012.03.019

8
C. Costello, D. R. Bundle / Carbohydrate Research 357 (2012) 7–15
(b)
(a)
Non-Reducing
OH
O
Non-Reducing
Epitope
OH
O
HO
HO
HO
HO
Epitope
HO
O
O
HO
HO
HO
HO
HO
HO
HO
O
O
O
O
HO
HO
HO
HO
HO
HO
O
O
Reducing
Epitope
O
O
Reducing
Epitope
HO
HO
HO
HO
O
O
HO
HO
O
O
O
HO
HO
HO
O
P
OH
O
O
O
HO
HO
O
O
Figure 1. Two forms of b1,2-mannan trisaccharide epitopes found in the cell wall of Candida albicans. If an antibody recognizing a b1,2-mannan disaccharide is capable of
frame shifting along the homo-oligomeric antigen chain both forms, an internal disaccharide epitope (blue) and external disaccharide (red) can be bound by antibodies. The
b1,2-mannan trisaccharide may be attached (a) to the phosphomannan side chain residues via a phosphodiester or (b) the same trisaccharide can be attached to side chain
residues via a glycosidic bond.
a
-mannose residue through a phosphodiester. The precise lengths
binding to synthetic b-mannan glycoconjugate coated EIA plates.14
The C-40 monodeoxy disaccharide congener was also inactive, while
the C-40 mono-O-methyl ether had an inhibitory potency of 5% rel-
ative to the native b-mannan disaccharide. Therefore if we con-
structed trisaccharides with a C-400 monodeoxy residue (Fig. 2,
compound 1) and C-4 monodeoxy or C-4 mono-O-methyl groups
(Fig. 2, compounds 2 and 3), we anticipated that these trisaccharides
would be forced into a single binding motif. Thus 1 should be bound
only via its internal disaccharide, while 2 and 3 prevent binding of
the reducing disaccharide and require the non-reducing
disaccharide to be recognized.
of the b1,2-linked mannose oligosaccharides are dependent on
growth conditions and have been variously reported to range
between 1 and 4 residues to as high as 14.21
Our mapping data are decisive with respect to the involvement
of the terminal reducing residue of the disaccharide as the primary
recognition element.14 This is surprising since many protective
antibodies bind to the most exposed residues at the distal end of
cell surface antigens.22,23 These results pose an important question.
When the b-mannan of the cell wall antigen is larger than a disac-
charide, which disaccharide element is bound by the antibody?
Does the antibody always bind to the terminal reducing disaccha-
ride or can it frame shift and bind a non-reducing disaccharide?
The two binding modes are illustrated for the two types of C. albi-
cans oligosaccharide epitopes present in the cell wall
phosphomannan as acid labile phosphodiester linked b-mannan
Chemical synthesis of trisaccharides 1–3 (Fig. 2) incorporating
these functional group replacements that abrogate binding to
internal or external disaccharide elements was undertaken.
2.1. Synthesis of 400-deoxy trisaccharide analogue 1
or the b-mannan linked glycosidically to the
residues (Fig. 1).
a-(1?2) mannose
Disaccharide 4 prepared by a published procedure24 was gly-
cosylated by the 4-deoxy thioglycoside 514 to give trisaccharide 6
in 69% yield. Debenzoylation gave the 4-deoxy derivative 7.
Epimerization at C-200 to give the 4-deoxy derivative 8 was accom-
plished by two reactions involving oxidation to give the C-200
uloside (not isolated) which was immediately reduced with good
stereoselectivity by L-selectrideÒ.24 Hydrogenolysis of 8 using
10% Pd/C as a catalyst afforded the globally deprotected 400-
deoxy-trisaccharide analogue 1 in 80% yield (Scheme 1). Purifica-
2. Results and discussion
In order to identify whether frame shifted disaccharide epitopes
are bound by C3.1 we utilized the findings of chemical mapping
studies.13,14 The binding site was mapped by monodeoxy and
mono-O-methyl congeners. The C-3 and C-4 monodeoxy or mono-
O-methyl ether analogues were all inactive in inhibition of C3.1
OH
O
OH
O
OH
O
HO
HO
HO
HO
HO
HO
O
O
O
HO
HO
HO
HO
HO
HO
HO
HO
HO
HO
HO
O
O
O
O
O
O
HO
HO
HO
HO
O
O
O
HO
MeO
OMe
OMe
OMe
HO
HO
1
2
3
Figure 2. Trisaccharide congener 1 lacking a crucial hydroxyl group in the terminal non-reducing residue is designed to force antibody recognition via the internal
disaccharide epitope. Trisaccharides 2 and 3 lack a crucial hydroxyl group in the terminal reducing residue and should force recognition of the external disaccharide epitope.

C. Costello, D. R. Bundle / Carbohydrate Research 357 (2012) 7–15
9
tion of the final compound was accomplished by HPLC with 70%
CH3CN—30% H2O as eluent on a TSK-GEL Amide-80 column.
(Scheme 3). Employing a similar set of transformations, transe-
sterification afforded 20 and 21. Then the C-200 hydroxyl groups
of these two trisaccharides were oxidized and the keto deriva-
tives reduced to afford the manno derivatives 22 and 23. Cat-
alytic hydrogenation of 22 and 23 afforded the desired 4-deoxy
and 4-O-methyl trisaccharide analogues 2 and 3. Purification of
2 and 3 was accomplished by HPLC, using the same conditions
as described for 1.
2.2. Synthesis of 4-deoxy and 4-O-methyl trisaccharide
analogues 2 and 3
Two different monosaccharides 9 and 10 were required as the
starting point for the synthesis of trisaccharide analogues 2 and 3.
Starting from methyl 2-O-benzoyl-3-O-benzyl-4,6-O-benzylidene
b-
D
-glucopyranoside both glycosyl acceptors the 4-deoxy 9 and
2.3. Enzyme immunoassay evaluation of the inhibitory power of
trisaccharides 1–3
the 4-O-methyl 10 were synthesized via the selectively protected
methyl 2-O-benzoyl-3,6-di-O-benzyl b-D-glucopyranoside accord-
ing to a previous publication from our group.24
Competitive ELISA was used to compare the inhibitory activity
The acceptors 9 and 10 were glycosylated by trichloroacetim-
idate donor 1125 to give disaccharides 12 and 13 (Scheme 2).
Deacetylation provided disaccharides 14 and 15 and as described
previously epimerization at C-20 was achieved by an oxidation
and reduction sequence using Ac2O/DMSO and then L-selectrideÒ
to give the disaccharide acceptors 16 and 17, the synthesis of
which had been performed as part of our disaccharide mapping
studies.24
of trisaccharides 1–3 and the native trisaccharide, methyl b-D-
mannopyranosyl-(1?2)-b-D-mannopyranosyl-(1?2)-b-D-manno-
pyranoside (Fig. 3).
Native (1?2)-b-D-mannopyranose trisaccharide conjugated
to BSA in PBS was used to coat 96-well ELISA plates.14,25 Solu-
tions of mAb IgG C3.1 mixed with serial dilutions of inhibitor
were assayed in triplicate. Bound C3.1 monoclonal antibody
was detected by a goat anti-mouse IgG antibody conjugated
to the enzyme horseradish peroxidase. Percent inhibition was
plotted against inhibitor concentration (Fig. 3) and IC50 values
Disaccharide acceptors 16 and 17 were glycosylated by tri-
chloroacetimidate 1125 to provide trisaccharides 18 and 19
BnO
O
BnO
O
SPh
BnO
BnO
O
OBz
OBz
a
BnO
BnO
BnO
O
OH
BnO
O
O
BnO
O
BnO
BnO
BnO
O
BnO
OMe
BnO
O
BnO
6
OMe
BnO
b
4
BnO
BnO
OH
O
BnO
O
O
BnO
O
OH
c
BnO
BnO
BnO
BnO
BnO
BnO
O
O
45%
O
O
BnO
BnO
O
O
BnO
BnO
OMe
OMe
BnO
BnO
8
7
d
OH
O
HO
HO
O
HO
HO
O
O
HO
HO
O
HO
OMe
HO
1
Scheme 1. Reagents and conditions: (a) NIS, AgOTf, CH2Cl2, 4 Å MS, ꢀ30 °C, 69%; (b) NaOMe, MeOH, CH2Cl2, 85%; (c) (i) Ac2O, DMSO, (ii) L-Selectride, THF, ꢀ78 °C, 45%; (d) H2,
Pd/C, MeOH, CH2Cl2, 80%.

10
C. Costello, D. R. Bundle / Carbohydrate Research 357 (2012) 7–15
BnO
O
BnO
BnO
O
CCl3
BnO
BnO
BnO
OAc
O
11
NH
O
a
OH
O
OAc
BnO
R
BnO
O
OMe
BnO
R
OMe
BnO
9 R = H
12 R = H
10 R = OMe
13 R = OMe
b
BnO
BnO
BnO
OH
O
BnO
BnO
BnO
O
O
OH
O
c
BnO
BnO
O
O
R
R
OMe
BnO
OMe
BnO
16 R = H
17 R = OMe
14 R = H
15 R = OMe
Scheme 2. Reagents and conditions: (a) 11, TMSOTf, CH2Cl2, 4 Å MS, ꢀ40 °C, 9 + 11?12, 81%, 10 + 11?12, 83%; (b) NaOMe, MeOH, CH2Cl2 12?14, 98%, 13?15, 92%; (c) (i)
Ac2O, DMSO, (ii) L-Selectride, THF, ꢀ78 °C, 14?16, 58%, 15?17, 79%.
BnO
O
OAc
BnO
O
OH
O
BnO
BnO
BnO
BnO
O
BnO
BnO
BnO
a
O
BnO
O
O
R
BnO
OMe
BnO
O
R
OMe
BnO
16 R = H
18 R = H
17 R = OMe
19 R = OMe
b
BnO
BnO
OH
BnO
BnO
BnO
O
OH
O
O
BnO
O
c
BnO
BnO
BnO
BnO
BnO
O
O
O
O
BnO
BnO
BnO
O
O
R
R
BnO
20 R = H
OMe
OMe
BnO
22 R = H
23 R = OMe
21 R = OMe
d
OH
O
HO
HO
O
HO
HO
HO
HO
O
O
HO
O
R
HO
OMe
2 R
= H
3 R
= OMe
Scheme 3. Reagents and conditions: (a) 11, TMSOTf, CH2Cl2, 4 Å MS, ꢀ30 °C 16 + 11?18, 61%, 17 + 11?19, 80%, (b) NaOMe, MeOH, CH2Cl2 18?20, 85%, 19?21, 93%; (c) (i)
Ac2O, DMSO, (ii) L-Selectride, THF, ꢀ78 °C, 20?22, 82%, 21?23, 66%, (d) H2, Pd/C, MeOH, CH2Cl2, 22?2, 42%, 23?3, 47%.

C. Costello, D. R. Bundle / Carbohydrate Research 357 (2012) 7–15
11
described were employed. If we consider the opposite end of the
binding site, which would accommodate the reducing mannose res-
idue of compounds 2 and 3 we see that these compounds are
approximately sixfold weaker inhibitors than 1. Our NMR and
homology model of the antibody binding site suggest b-mannose
residues at this end of the groove type binding site encounter
increasingly unfavourable interactions with the antibody surface.15
We therefore attribute the weaker inhibitory power of compounds
2 and 3 to unfavourable antibody–trisaccharide contacts.
3. Experimental
3.1. General methods
All chemical reagents were of analytical grade and used as
obtained from commercial sources unless otherwise indicated.
Solvents used in water-sensitive reactions were purified by
successive passage through columns of alumina and copper un-
der nitrogen, except for DMSO, which was distilled under vac-
uum and collected over 4 Å molecular sieves. Unless otherwise
noted, reactions were carried out at room temperature, and
water-sensitive reactions were performed under an atmosphere
of argon. Molecular sieves were flame dried and then allowed
to cool to room temperature under argon before use. Reactions
were monitored by analytical thin-layer chromatography (TLC)
performed on Silica Gel 60-F254 (E. Merck). Plates were visual-
ized under UV light, and/or by treatment with 5% sulfuric acid
in ethanol followed by heating. Organic solvents were removed
under vacuum at <40 °C. Medium-pressure chromatography
was conducted using silica gel (230–400 mesh, Silicycle, Mon-
Figure 3. Competitive ELISA. Inhibition of Mab C3.1 binding to antigen coat plates
by methyl b- -mannopyranosyl-(1?2)-b- -mannopyranosyl-(1?2)-b- -manno-
D
D
D
pyranoside and trisaccharides congeners j 1, 2,
3.
Table 1
IC50 and relative free energy for native trisaccharide and trisaccharide congeners 1–3
Trisaccharide
IC50
(
lM)
Relative potency
D
(D )
G)a (kcal molꢀ1
Native
25
79
189 11
139
1
4
100
32
13
0
0.68
1.2
1
2
3
9
18
1.0
treal) at flow rates between
5 . Following
and 10 mL minꢀ1
a
The calculated standard error on all three values of
0.04 kcal molꢀ1
D(DG) was less than
.
deprotection, final compounds were purified by HPLC (flow
rate = 3.0 mL/min, diameter = 7.8 mm) with 70% CH3CN—30%
H2O as eluent on a TSK-GEL Amide-80 column, concentrated
under reduced pressure to remove acetonitrile, and then lyoph-
ilized. 1H NMR spectra were recorded at 500 or 600 MHz, and
chemical shifts, reported in d (ppm), were referenced to inter-
nal residual protonated solvent signals or to external acetone
(0.1% ext. acetone @ d 2.225 ppm) in the case of D2O. 13C
NMR spectra were recorded at 125 MHz, and chemical shifts
are referenced to internal CDCl3 (d 77.23) or external acetone
(d 31.07). First order chemical shifts of 1H and 13C are reported
to the second and first decimal place, respectively. High reso-
lution mass spectra were obtained on a Micromass Zabspec
TOF-mass spectrometer by analytical services in the depart-
ment. Optical rotations were determined with a Perkin-Elmer,
model 241, polarimeter at room temperature using the sodium
D-line and are reported in units of deg mL gꢀ1 dmꢀ1. Elemental
analysis was performed by analytical services of this
department.
were used to determine the potency of inhibition and the rel-
ative change in free energy of binding (Table 1).
2.4. Conclusion
Inhibition data demonstrate that antibody C3.1 has a preference
for binding the reducing disaccharide element of a trisaccharide,
since analogue 1 is bound approximately twofold more tightly
than either 2 or 3. If the reducing end was exclusively preferred
over the non-reducing end, a modification of the C-4 hydroxyl
group (compounds 2 and 3) would result in a complete loss of inhi-
bition. This was not observed, as compounds 2 and 3 were both
active. This clearly establishes that the non-reducing disaccharide
element can also bind in the C3.1 binding site. The possibility of
a frame shift to bind the non-reducing end disaccharide element
at least for a trisaccharide epitope is thus confirmed. This is impor-
tant since Cutler has reported that di and trisaccharide sequences
are heavily expressed on growing C. albicans cultures26 and C3.1
would clearly be capable of binding to either of these structures.
It is interesting to note that although compound 1 exhibits the
preferred recognition element, there was a difference in binding en-
ergy between the native trisaccharide and 1. The potency of 1 is only
32% of that of the native trisaccharide, a difference in free energy of
0.68 kcal molꢀ1. It is possible that the replacement of the hydroxyl
group by a hydrogen at C-400 leads to unfavourable interactions with
the antibody, at the periphery of the binding site. We favour the
interpretation that 400-OH is likely involved in a hydrogen bonding
network with water at the periphery of the binding site. A 400-deoxy
trisaccharide analogue would have reduced affinity for binding, due
to the loss of this hydrogen bonding capability. Relatively small
changes in binding activity of this type have been seen in other de-
tailed studies of congener binding to antibodies and lectins27 when
the functional group modifications similar to those we have
3.2. Enzyme linked immunosorbent inhibition assay
The mAb C3.1 was produced as a cell culture harvest superna-
tant (8.9 mg/mL) and was diluted 1:5000 for ELISA. A solution of
synthetic C. albicans (1?2)-b-
jugated to BSA in PBS (5 g/mL) was used to coat the 96-well ELISA
plate (100 L) overnight at 4 °C. Plates were washed five times
with PBST buffer. Solutions of mAb IgG C3.1 mixed with inhibitor
in PBST at concentrations ranging from 0.316 g/mL up to of
D-mannopyranose trisaccharide con-
l
l
l
1 mg/mL were added to the microtitre plates in triplicate and incu-
bated at room temperature for 2 h. Goat anti-mouse IgG antibody
conjugated to the horseradish peroxidase (diluted 1:5000, Kirkeg-
aard & Perry Laboratories) in PBST was added and incubated at
room temperature for 30 min. The plates were washed five times

12
C. Costello, D. R. Bundle / Carbohydrate Research 357 (2012) 7–15
with PBST buffer. The enzyme substrate 3,30,5,50-tetramethylbenz-
idine (100 L, Kirkegaard & Perry Laboratories) was added and
incubated at room temperature. After 15 min, the colour reaction
was stopped by the addition of 1 M H3PO4 (100 L). The absor-
bance of the solution was measured at 450 nm. Percent inhibition
was calculated from the absorbance readings of the sample in
relative to those of wells containing antibody without inhibitor.
9 H, ArH), 5.10 (d, 1H, Jgem = 11.1 Hz, PhCH2O), 5.04 (d, 1H,
Jgem = 12.1 Hz, PhCH2O), 5.00 (d, 1H, Jgem = 11.1 Hz, PhCH2O), 4.97 (d,
1H, Jgem = 12.6 Hz, PhCH2O), 4.85 (s, 1H, H-10), 4.78 (d, 1H,
Jgem = 11.9 Hz, PhCH2O), 4.63–4.59 (m, 2H, H-100, PhCH2O), 4.56–4.51
(m,3H,PhCH2O),4.51–4.43(m,5H,H-2,H-20,PhCH2O),4.43–4.35(m,
3H,PhCH2O),4.26(s,1H,H-1),4.18(t,1H,J3,4 = J4,5 = 9.6 Hz,H-4),3.87
(dd, 1H, J5,6a = 4.5 Hz, Jgem = 10.6 Hz, H-6a), 3.82–3.72 (m, 3H, H-6b,
H-40, H-6a00), 3.66–3.61 (m, 2H, H-200, H-6b00), 3.56–3.52 (m, 2H, H-30,
H-6a0), 3.52(s,3H,OCH3),3.50–3.46(m, 2H,H-3,H-500), 3.42–3.34(m,
l
l
3.2.1. Methyl 2-O-benzoyl-3,6-di-O-benzyl-4-deoxy-b-
hexopyranosyl-(1?2) 3,4,6-tri-O-benzyl-b- -mannopyranosyl-
(1?2)-3,4,6-tri-O-benzyl-b- -mannopyranoside (6)
D-xylo-
4H, H-5, H-50, H-6b0, H-300), 1.84 (dd, 1H, J3 ,4eq = J4eq ,5 = 5.2 Hz,
Jgem = 12.3 Hz, H-4eq00), 1.49 (q, 1H, Jgem = 12.0 Hz, H-4ax00). 13C NMR
(125 MHz, CDCl3): d 139.2 (Ar), 138.9 (Ar), 138.6 (Ar), 138.4 (Ar),
138.3 (Ar), 138.1(4) (Ar), 138.1(0) (Ar), 138.0 (Ar), 128.3(4) (Ar),
128.3(1)(Ar),128.2(4)(Ar),128.2(3)(Ar),128.1(7)(Ar),128.1(4)(Ar),
128.0(8) (Ar), 128.0(4) (Ar), 127.9 (Ar), 127.8 (Ar), 127.7(3) (Ar),
127.7(2) (Ar), 127.6 (Ar), 127.5(7) (Ar), 127.5(2) (Ar), 127.4(9) (Ar),
127.4(3)(Ar),127.4(0)(Ar),127.3(4)(Ar),127.3(0)(Ar),127.2(9)(Ar),
127.1(Ar),105.5(C-100),102.3(C-1),99.9(C-10),80.1(C-30),79.8(C-3),
79.6 (C-300), 75.7, 75.5 (C-5, C-200), 75.4 (PhCH2O), 75.1 (C-500), 75.1
(PhCH2O), 74.7 (C-40), 74.3 (C-20), 73.8 (PhCH2O), 73.4 (PhCH2O), 73.3
(PhCH2O), 72.7, 72.6(C-60, PhCH2O), 70.9, 70.8(C-2, C-50), 70.3(C-600),
69.9 (C-6), 69.8 (PhCH2O), 57.5 (OCH3), 33.5 (C-400). HRESIMS: calcd
for C75H82O15Na (M+Na): 1245.5546, found 1245.5532. Anal. Calcd
forC75H82O15:C,73.63;H,6.76.Found:C,73.61;H,6.77.
00
00
00 00
D
D
Disaccharide acceptor 424 (0.050 g, 0.056 mmol) and thioglyco-
side donor 514 (0.040 g, 0.074 mmol) were dried together in a pear
shaped flask (50 mL) under vacuum overnight. The contents of the
flask were dissolved in CH2Cl2 (1.5 mL) and activated 4 Å molecular
sieves were added. The solution was cooled to ꢀ30 °C (dry ice–ace-
tone bath). NIS (0.017 g, 76 mmol) was added. After 20 min, AgOTf
(0.011 g, 43 mmol) was added. After 1 h, the reaction mixture was
neutralized with Et3N and filtered through celite. The filtrate was
washed with saturated aqueous Na2S2O3, saturated aqueous NaH-
CO3, distilled water and brine. The organic phase was dried
(Na2SO4) and concentrated under reduced pressure. Purification
by silica gel chromatography (19:1, toluene–EtOAc) gave
6
(0.051 g, 69%) as a clear and colourless syrup. Rf 0.26 (1:2, hex-
anes–EtOAc); [
a
]
D ꢀ34.5 (c 1.3, CHCl3); 1H NMR (500 MHz, CDCl3):
d 8.22–8.20 (m, 2H, ArH), 7.42–7.00 (m, 43H, ArH), 5.52 (d, 1H,
3.2.3. Methyl 3,6-di-O-benzyl-4-deoxy-b-
(1?2)-3,4,6-tri-O-benzyl-b- -mannopyranosyl-(1?2)-3,4,6-tri-
O-benzyl-b- -mannopyranoside (8)
D-lyxo-hexopyranosyl-
00
J1 ,2 = 8.1 Hz, H-100), 5.35 (dd, 1H, J1 ,2 = 8.1 Hz, J2 ,3 = 9.2 Hz, H-
200), 4.95–4.88 (m, 3H, PhCH2O), 4.70–4.66 (m, 2H, H-20, PhCH2O),
4.63–4.58 (m, 2H, PhCH2O), 4.58–4.52 (m, 4H, H-10, PhCH2O),
4.51–4.41 (m, 4H, PhCH2O), 4.34 (d, 1H, Jgem = 10.9 Hz, PhCH2O),
4.22 (s, 1H, H-1), 4.18 (d, 1H, Jgem = 12.1 Hz, PhCH2O), 4.08–4.04
(m, 2H, H-2, PhCH2O), 3.85 (m, 1H, H-500), 3.74–3.68 (m, 4H, H-4,
H-6a, H-300, H6a00), 3.60–3.49 (m, 4H, H-3, H-6b, H-40, H6a0, H6b00),
D
00
00 00
00 00
D
Acetic anhydride (2.5 mL, 26 mmol) was added to a solution of
trisaccharide 7 (0.159 g, 0.130 mmol) in DMSO (5.0 mL, 70 mmol).
The reaction was stirred overnight. The mixture was concentrated
under reduced pressure and the residue was redissolved in THF
(5 mL). This solution was cooled to ꢀ78 °C (dry ice–acetone bath).
A 1.0 M solution of L-SelectrideÒ in THF (0.52 mL, 0.52 mmol) was
added dropwise. The reaction was stirred at ꢀ78 °C for 3 h and
then warmed to room temperature over 1 h. The reaction was
quenched with MeOH and diluted with CH2Cl2. It was washed with
10% aqueous H2O2, 1 M aqueous NaOH, distilled water and brine.
The organic phase was dried (Na2SO4) and concentrated under re-
duced pressure. Purification by silica gel chromatography (2:1,
hexanes–EtOAc) gave 8 (0.071 g, 45%) as a clear and colourless syr-
3.46 (dd, 1H, J2 ,3 = 2.9 Hz, J3 ,4 = 9.3 Hz, H-30), 3.43 (m, 1H, H-5),
0
0
0
0
0
0
3.41–3.38 (m, 4H, H-5, CH3O), 3.12 (dd, 1H, J5 ,6 = 6.9 Hz,
Jgem = 10.7 Hz, H-6b0), 2.12 (ddd, 1H, J3 ,4eq = 5.3 Hz, Jgem = 12.7 Hz,
00
00
J4eq ,5 = 1.8 Hz, H-4eq00), 1.67 (q, 1H, Jgem = 11.7 Hz, H-4ax00). 13C
00 00
NMR (125 MHz, CDCl3): d 166.5 (C@O), 138.7 (Ar), 138.6 (Ar),
138.5 (Ar), 138.4(09) (Ar), 138.4(05) (Ar), 138.3(9) (Ar), 138.3
(Ar), 138.2 (Ar), 134.5 (Ar), 133.5 (Ar), 134.5 (Ar), 132.2 (Ar),
131.3 (Ar), 130.3 (Ar), 128.5 (Ar), 128.3(5) (Ar), 128.3(4) (Ar),
128.3(3) (Ar), 128.2(4) (Ar), 128.2(0) (Ar), 128.1(5) (Ar), 128.1(4)
(Ar), 128.1(3) (Ar), 128.1(2) (Ar), 128.0 (Ar), 127.9 (Ar), 127.7
(Ar), 127.6(3) (Ar), 127.6(1) (Ar), 127.5(8) (Ar), 127.5(5) (Ar),
127.5(3) (Ar), 127.4(9) (Ar), 127.4(2) (Ar), 127.3(4) (Ar), 127.3(0)
(Ar), 127.2 (Ar), 103.3 (C-10), 101.8 (C-1), 99.7 (C-100), 80.1, 80.0
(C-30, C-3), 76.4 (C-300), 76.0, 75.7, 75.5 (C-50, C-2, C-5), 75.2, 75.1
(PhCH2O, C-6), 75.1, 75.0 (C-200, C-40), 74.5 (C-20), 73.4 (PhCH2O),
73.3 (PhCH2O), 73.1 (PhCH2O), 72.8 (C-600), 70.8 (C-500), 70.7, 70.5
(C-60, PhCH2O), 70.3 (C-4), 70.0 (PhCH2O), 69.8 (PhCH2O), 69.7
(PhCH2O), 56.6 (CH3O), 33.8 (C-400). HRESIMS: calcd for
up. Rf 0.57 (1:1, hexanes–EtOAc); [
a
]
D ꢀ76.5 (c 1.0, CHCl3); 1H NMR
(600 MHz, CDCl3): d 7.51–7.48 (m, 2H, ArH), 7.41–7.38 (m, 2H,
ArH), 7.36–7.20 (m, 29 H, ArH), 7.15–7.12 (m, 3H, ArH), 7.08–
7.04 (m, 4H, ArH), 5.02 (s, 1H, H-10), 5.01–4.94 (m, 3H, PhCH2O),
4.92 (s, 1H, H-100), 4.86 (s, 1H, Jgem = 10.4 Hz, PhCH2O), 4.67–4.62
(m, 2H, H-20, PhCH2O), 4.59 (d, 1H, J2,3 = 3.4 Hz, H-2), 4.56–4.34
(m, 9 H, PhCH2O), 3.87 (t, 1H, J3 ,4 = J4 ,5 = 9.5 Hz, H-40), 3.82 (dd,
1H, Jgem = 10.6 Hz, J5,6a = 4.4 Hz, H-6a), 3.77–3.80 (m, 2H, H-6b,
H-6a0), 3.72–3.66 (m, 2H, H-6b0, H-4), 3.61–3.56 (m, 3H, H-3, H-
30, H-6a00), 3.56–3.50 (m, 5H, H-50, H-6b00, CH3O), 3.46–3.40 (m,
0
0
0
0
2H, H-5, H-500), 3.35 (ddd, 1H, J2 ,3 = 2.8 Hz, J3 ,4ax = 11.6 Hz,
00 00
00
00
C82H86O16Na (M+Na): 1349.5808, found 1349.5795. Anal. Calcd
J 3 ,4eq = 4.8 Hz, H-300), 1.78 (q, 1H, Jgem = 12.0 Hz, H-4ax00), 1.64
00
00
for C82H86O16: C, 74.19; H, 6.53. Found: C, 73.75; H, 6.75.
(dd, 1H, Jgem = 11.0 Hz, J3 ,4eq = 4.8 Hz H-4eq00). 13C NMR
(125 MHz, CDCl3): d 138.6 (Ar), 138.4 (Ar), 138.2(7) (Ar), 138.2(2)
(Ar), 138.1(94) (Ar), 138.1(66) (Ar), 138.1(45) (Ar), 138.1(27) (Ar),
129.0 (Ar), 128.4 (Ar), 128.3(5) (Ar), 128.3(4) (Ar), 128.3(2) (Ar),
128.2(7) (Ar), 128.2(2) (Ar), 128.1(6) (Ar), 128.1(2) (Ar), 128.0(8)
(Ar), 128.0(6) (Ar), 127.8 (Ar), 127.7(9) (Ar), 127.7(8) (Ar),
127.7(4) (Ar), 127.7(2) (Ar), 127.6(7) (Ar), 127.6 (Ar), 127.5 (Ar),
127.4 (Ar), 127.3 (Ar), 127.2 (Ar), 102.8 (C-1), 100.8 (C-100), 100.0
(C-10), 80.3(2), 80.2(7) (C-3, C-30), 76.6 (C-300), 75.4 (PhCH2O),
75.3, 75.2 (C-5, C-50), 75.1 (PhCH2O), 74.7, 74.5 (C-4, C-40), 73.6
(PhCH2O), 73.4(5) (PhCH2O), 73.4(3) (PhCH2O), 73.0 (PhCH2O),
71.8 (C-500), 71.2 (C-20), 70.3 (C-600), 69.9 (C-60), 69.6 (PhCH2O),
69.4(4) (C-6), 69.4(1) (C-2), 69.3 (PhCH2O), 66.4 (C-200), 57.4
00
00
3.2.2. Methyl 3,6-di-O-benzyl-4-deoxy-b-
(1?2)-3,4,6-tri-O-benzyl-b- -mannopyranosyl-(1?2)-3,4,6-tri-
O-benzyl-b- -mannopyranoside (7)
Trisaccharide6(0.204 g,0.154 mmol)wasdissolvedinamixtureof
MeOH(4 mL)andCH2Cl2(2 mL).A1 MsolutionofNaOMe/MeOH(1 mL,
1 mmol)wasadded.Thereactionwasstirredfor24 h.Thereactionmix-
turewasneutralizedwithAmberliteIR120andfilteredtoremovethe
resin.Thefiltratewasconcentratedunderreducedpressure.Purifica-
tion by silica gel chromatography (4:1, toluene–EtOAc) gave 7
D-xylo-hexopyranosyl-
D
D
(0.159 g, 85%) as a white film. Rf 0.54 (1:1, hexanes–EtOAc); [a]
D
ꢀ54.5 (c 1.0, CHCl3); 1H NMR (600 MHz, CDCl3): d 7.42–7.38 (m, 2H,
ArH), 7.38–7.33(m, 4H, ArH), 7.33–7.20(m, 25H, ArH), 7.20–7.14(m,

C. Costello, D. R. Bundle / Carbohydrate Research 357 (2012) 7–15
13
(CH3O), 28.6 (C-400). HRESIMS: calcd for C75H82O15Na (M+Na):
1245.5546, found 1245.5545. Anal. Calcd for C75H82O15: C, 73.63;
H, 6.76. Found: C, 73.62; H, 6.72.
J3,4eq = J4eq,5 = 4.5 Hz, Jgem = 12.5 Hz, H-4eq). 13C NMR (125 MHz,
CDCl3): d 170.5 (C@O), 138.7 (Ar), 138.5 (Ar), 138.4(4) (Ar),
138.3(8) (Ar), 138.2(5) (Ar), 138.2(3) (Ar), 138.1(9) (Ar), 128.4
(Ar), 128.3 (Ar), 128.2(9) (Ar), 128.2(7) (Ar), 128.2(4) (Ar),
128.1(8) (Ar), 128.1(3) (Ar), 128.0(9) (Ar), 128.0(0) (Ar), 127.8(6)
(Ar), 127.8(4) (Ar), 127.6(9) (Ar), 127.6(4) (Ar), 127.6(1) (Ar),
127.5(6) (Ar), 127.5(2) (Ar), 127.4(9) (Ar), 127.4(5) (Ar), 127.4(4)
(Ar), 127.4(3) (Ar), 127.3(9) (Ar), 127.3(1) (Ar), 102.6 (C-1), 101.9
(C-10), 100.9 (C-100), 83.0 (C-300), 80.0 (C-30), 78.3 (C-500), 75.5 (C-
3), 75.1 (PhCH2O), 74.9 (PhCH2O), 74.8 (C-40), 74.6 (C-20), 74.4
(PhCH2O), 74.2 (C-50), 73.7 (C-2), 73.4 (PhCH2O), 73.3(4) (PhCH2O),
73.2(7) (C-200), 73.2(0) (PhCH2O), 73.1 (PhCH2O), 72.0 (C-5), 71.9
(C-400), 70.7 (C-60), 69.8 (C-600), 69.5 (C-6), 68.8 (PhCH2O), 56.8
(OCH3), 29.6 (C-4), 21.2 (C(O)CH3). HRESIMS: calcd for C77H84O16Na
(M+Na): 1287.5652, found 1287.5639. Anal. Calcd for C77H84O16: C,
73.08; H, 6.69. Found: C, 72.83; H, 6.66.
3.2.4. Methyl 4-deoxy-b-
D-lyxo-hexopyranosyl-(1?2)-b-
D-
mannopyranosyl-(1?2)-b-
D
-mannopyranoside (1)
Trisaccharide 8 (0.073 g, 0.060 mmol) was dissolved in a mixture
of CH2Cl2 (5 mL) and MeOH (5 mL). 10% Pd/C (0.050 g) was added
and the reaction was evacuated and purged with H2(g). The mixture
was stirred overnight under a hydrogen atmosphere. The catalyst
was separated by filtration through celite and the filtrate was con-
centrated under reduced pressure. Purification of the product by
HPLC gave 1 (0.024 g, 80%) as a white solid. Rf 0.02 (9:1, CH2Cl2–
MeOH); [
a
]
D ꢀ60.0 (c 0.9, H2O). 1H NMR (600 MHz, D2O): d 4.86 (s,
1H, H-10), 4.75 (s, 1H, H-100), 4.61 (s, 1H, H-1), 4.31 (d, 1H,
J2 ,3 = 3.1 Hz, H-20), 4.20 (d, 1H, J2,3 = 3.2 Hz, H-2), 4.01 (d, 1H,
0
0
J2 ,3 = 2.9 Hz, H-200), 3.91 (dd, 1H, Jgem = 4.7 Hz, J5,6a = 2.2 Hz, H-6a),
00 00
3.89 (dd, 1H, Jgem = 4.7 Hz, J5 ,6a = 2.2 Hz, H-6a0), 3.86 (ddd, 1H,
3.2.7. Methyl 2-O-acetyl-3,4,6-tri-O-benzyl-b-
(1?2)-3,4,6-tri-O-benzyl-b- -mannopyranosyl-(1?2)-3,6-di-O-
benzyl-4-O-methyl-b- -mannopyranoside (19)
D-glucopyranosyl-
0
0
J2 ,3 = 3.1 Hz, J3 ,4ax = 11.9 Hz, J3 ,4eq = 5.1 Hz, H-300), 3.74 (t, 1H,
D
00 00
00
00
00
00
Jgem = J5 ,6b = 6.2 Hz, H-6b0), 3.71 (t, 1H, Jgem = J5,6b = 5.9 Hz, H-6b),
3.67 (m, 1H, H-3), 3.66–3.58 (m, 4H, H-30, H-500, H-6a00, H-6b00), 3.57
(m, 1H, H-40), 3.52 (s, 3H, CH3O), 3.47 (t, 1H, J3,4 = J4,5 = 9.8 Hz, H-
D
0
0
Disaccharide acceptor 1724 (0.6755 g, 0.8228 mmol) and mono-
saccharide donor 1124 (0.786 g, 1.23 mmol) were dried together in
a pear shaped flask (50 mL) under vacuum overnight. The contents
of the flask were dissolved in CH2Cl2 (5 mL) and activated 4 Å
molecular sieves were added. The solution was cooled to ꢀ35 °C
4), 3.37–3.33 (m, 2H, H-5, H-50), 1.64 (dd, 1H, J3 ,4eq = 5.0 Hz,
Jgem = 11.3 Hz, H-4eq00), 1.50 (q, 1H, Jgem = 11.9 Hz, H-4ax00). 13C NMR
(125 MHz, D2O): d 102.1(8) (C-1), 102.1(7) (C-100), 101.5 (C-10),
79.0(6) (C-20), 79.0(1) (C-2), 77.2, 77.1 (C-5, C-50), 73.7, 73.1 (C-30,
C-500), 72.9 (C-3), 69.9 (C-200), 68.8 (C-300), 68.2 (C-4), 67.9 (C-40),
64.8 (C-600), 63.3 (C-60), 61.6 (C-6), 57.8 (CH3O), 29.4 (C-400). HRE-
SIMS: calcd for C19H34O15Na (M+Na): 525.179, found 525.1786.
00
00
(dry ice–acetone bath). TMSOTf (37 lL, 0.20 mmol) was added
and the reaction was stirred from ꢀ35 °C to ꢀ25 °C over 2 h. The
mixture was neutralized with Et3N, filtered and concentrated un-
der reduced pressure. Purification by silica gel chromatography
(4:1, hexanes–EtOAc) gave 19 (0.856 g, 80%) as a white solid. Rf
3.2.5. 2-O-Acetyl-3,4,6-tri-O-benzyl-
D
-glucopyranosyl trichloro
0.32 (2:1, hexanes–EtOAc); [
a]
ꢀ40.0 (c 1.5, CHCl3); 1H NMR
D
acetimidate (11)25
(500 MHz, CDCl3): d 7.38–7.34 (m, 4H, ArH), 7.31–7.16 (m, 34H,
ArH), 7.10–7.07 (m, 2H, ArH), 5.20–5.14 (m, 2H, H-100, H-200), 4.95
(d, 1H, Jgem = 11.0 Hz, PhCH2O), 4.92 (d, 1H, Jgem = 12.0 Hz,
PhCH2O), 4.80 (d, 2H, Jgem = 11.5 Hz, PhCH2O), 4.74–4.68 (m, 3H,
PhCH2O), 4.62 (d, 1H, Jgem = 12.9 Hz, PhCH2O), 4.59 (s, 1H, H-10),
4.59–4.49 (m, 5H, PhCH2O), 4.49–4.40 (m, 4H, H-20, PhCH2O),
4.20 (s, 1H, H-1), 4.10 (d, 1H, J2,3 = 2.8 Hz, H-2), 3.84 (m, 1H, H-
300), 3.78–3.68 (m, 6H, H-6a, H-6b, H-400, H-500, H-6a00, H-6b00),
3.68–3.56 (m, 4H, H-4, H-40, H-6a0, H-6b0), 3.52 (s, 3H, 4-OCH3),
3.48–3.42 (m, 2H, H-30, H-50), 3.41 (s, 3H, 1-OCH3), 3.39 (dd, 1H,
J2,3 = 2.8 Hz, J3,4 = 9.6 Hz, H-3), 3.26 (m, 1H, H-5), 1.95 (s, 3H,
C(O)CH3). 13C NMR (125 MHz, CDCl3): d 170.5 (C=O), 138.7(32)
(Ar), 138.7(28) (Ar), 138.5 (Ar), 138.4(09) (Ar), 138.4(03) (Ar),
138.3 (Ar), 138.2 (Ar), 128.2(6) (Ar), 128.2(5) (Ar), 128.2(3) (Ar),
128.2(1) (Ar), 128.1(8) (Ar), 128.1(6) (Ar), 128.1(5) (Ar), 128.0(3)
(Ar), 127.9 (Ar), 127.7 (Ar), 127.6 (Ar), 127.5(5) (Ar), 127.5(3)
(Ar), 127.5(1) (Ar), 127.4(6) (Ar), 127.4(1) (Ar), 127.4(0) (Ar),
127.3(8) (Ar), 127.3(7) (Ar), 127.3(5) (Ar), 127.3(0) (Ar), 102.1 (C-
10), 102.0 (C-1), 101.0 (C-100), 83.4 (C-300), 80.3 (C-30), 79.9 (C-3),
78.4 (C-4), 76.0 (C-2), 75.4(0), 75.3(6), 75.0 (C-5, C-50, C-500), 75.1
(PhCH2O), 74.8 (PhCH2O), 74.6, 74.5 (C-40, C-400), 74.6 (PhCH2O),
73.3 (C-200), 73.2(9) (PhCH2O), 73.2 (PhCH2O), 73.0 (PhCH2O), 71.9
(C-20), 70.8 (C-600), 69.8 (PhCH2O), 69.7 (C-60), 69.5 (PhCH2O), 69.3
(C-6), 61.0 (4-CH3O), 56.9 (1-CH3O), 22.6 (C(O)CH3). HRESIMS:
calcd for C78H86O17Na (M+Na): 1317.5757, found 1317.5758. Anal.
Calcd for C78H86O17: C, 72.31; H, 6.69. Found: C, 72.26; H, 6.67.
DBU (1.1 mL, 7.4 mmol) was added dropwise to a solution of 2-
O-Acetyl-3,4,6-tri-O-benzyl- -glucopyranose (13.90 g,
D
28.22 mmol) and Cl3CCN (20.0 mL, 199 mmol) in CH2Cl2
(100 mL). The reaction was stirred for 3 h. The solution was
washed with saturated aqueous NH4Cl. The organic phase was
dried (Na2SO4), and concentrated under reduced pressure. Purifica-
tion by silica gel chromatography (2:1, hexanes–EtOAc with 1%
Et3N) gave 11 (17.91 g, 97%) as a yellow syrup.
3.2.6. Methyl 2-O-acetyl-3,4,6-tri-O-benzyl-b-
(1?2)-3,4,6-tri-O-benzyl-b- -mannopyranosyl-(1?2)-3,6-di-O-
benzyl-4-deoxy-b- -lyxo-hexopyranoside (18)
D-glucopyranosyl-
D
D
Disaccharide acceptor 1624 (0.097 g, 0.12 mmol) and monosac-
charide donor 1125 (0.117 g, 0.184 mmol) were dried together in
a pear shaped flask (50 mL) under vacuum overnight. The contents
of the flask were dissolved in CH2Cl2 (2 mL) and activated 4 Å
molecular sieves were added. The solution was cooled to ꢀ30 °C
(dry ice–acetone bath). TMSOTf (6 lL, 0.03 mmol) was added and
the reaction was stirred for 1 h. The mixture was neutralized with
Et3N, filtered and concentrated under reduced pressure. Purifica-
tion by silica gel chromatography (19:1, toluene–EtOAc) gave 18
(94 mg, 61%) as a white syrup. Rf 0.41 (2:1, hexanes–EtOAc); [a]
D
ꢀ55.5 (c 1.4, CHCl3); 1H NMR (500 MHz, CDCl3): d 7.42–7.16 (m,
40 H, ArH), 5.22 (d, 1H, J1 ,2 = 8.1 Hz, H-100), 5.18 (t, 1H,
00 00
J1 ,2 = J2 ,3 = 8.5 Hz, H-200), 4.98 (d, 1H, Jgem = 10.9 Hz, PhCH2O),
4.95 (d, 1H, Jgem = 12.1 Hz, PhCH2O), 4.86 (d, 1H, Jgem = 11.0 Hz,
PhCH2O), 4.80 (d, 1H, Jgem = 11.9 Hz, PhCH2O), 4.75–4.73 (m, 2H,
PhCH2O), 4.71 (s, 1H, H-10), 4.63 (d, 1H, Jgem = 12.3 Hz, PhCH2O),
4.61–4.58 (m, 2H, PhCH2O), 4.58–4.55 (m, 2H, PhCH2O), 4.55–
4.46 (m, 6H, PhCH2O), 4.17 (s, 1H, H-1), 4.14 (d, 1H, J2,3 = 2.8 Hz,
H-2), 3.80–3.76 (m, 3H, H-6a, H-6a0, H-300), 3.75–3.69 (m, 2H, H-
6b, H-500), 3.67–3.60 (m, 5H, H-40, H-6b0, H-400, H-6a00, H-6b00), 3.57
(m, 1H, H-5), 3.53–3.46 (m, 3H, H-3, H-30, H-50), 3.45 (s, 3H,
CH3O), 1.97 (s, 3H, C(O)CH3), 1.92 (m, 1H, H-4ax), 1.75 (dd, 1H,
00 00
00 00
3.2.8. Methyl 3,4,6-tri-O-benzyl-b-
3,4,6-tri-O-benzyl-b- -mannopyranosyl-(1?2)-3,6-di-O-benzyl-
4-deoxy-b- -lyxo-hexopyranoside (20)
D-glucopyranosyl-(1?2)-
D
D
Trisaccharide 18 (0.085 g, 0.067 mmol) was dissolved in a mix-
ture of MeOH (4 mL) and CH2Cl2 (2 mL). A 1 M solution of NaOMe/
MeOH (1 mL, 1 mmol) was added. The reaction was stirred for
24 h. The reaction mixture was neutralized with Amberlite IR
120 and filtered to remove the resin. The filtrate was concentrated.

14
C. Costello, D. R. Bundle / Carbohydrate Research 357 (2012) 7–15
Purification by silica gel chromatography (4:1, toluene–EtOAc)
gave 20 (0.070 g, 85%) as a clear and colourless syrup. Rf 0.41
70.2, 70.0 (C-6, PhCH2O), 69.6(9), 69.6(6) (C-600, PhCH2O), 61.0 (4-
CH3O), 57.4 (1-CH3O). HRESIMS: calcd for C76H84O16Na (M+Na):
1275.5652, found 1275.5648. Anal. Calcd for C76H84O16: C, 72.82;
H, 6.75. Found: C, 72.72; H, 6.69.
(2:1, hexanes–EtOAc);
[
a
]
ꢀ65.0 (c 1.0, CHCl3); 1H NMR
D
(500 MHz, CDCl3): d 7.42–7.22 (m, 36H, ArH), 7.21–7.15 (m, 4H,
ArH), 5.07 (d, 1H, Jgem = 11.4 Hz, PhCH2O), 4.99 (d, 1H,
Jgem = 10.8 Hz, PhCH2O), 4.98 (d, 1H, Jgem = 12.6 Hz, PhCH2O), 4.91
(s, 1H, H-10), 4.89 (d, 1H, Jgem = 12.0 Hz, PhCH2O), 4.87 (d, 1H,
Jgem = 10.9 Hz, PhCH2O), 4.79 (d, 1H, Jgem = 11.2 Hz, PhCH2O), 4.77
3.2.10. Methyl 3,4,6-tri-O-benzyl-b-
3,4,6-tri-O-benzyl-b- -mannopyranosyl-(1?2)-3,6-di-O-benzyl-
4-deoxy-b- -lyxo-hexopyranoside (22)
D-mannopyranosyl-(1?2)-
D
D
(d, 1H, J1 ,2 = 7.9 Hz, H-100), 4.62 (d, 1H, Jgem = 12.0 Hz, PhCH2O),
Acetic anhydride (1.0 mL, 11 mmol) was added to a solution of
trisaccharide 20 (0.045 g, 0.037 mmol) in DMSO (2.0 mL,
28 mmol). The reaction was stirred overnight. The mixture was
concentrated under reduced pressure and the residue was redis-
solved in THF (2 mL). This solution was cooled to ꢀ78 °C. A solution
of L-SelectrideÒ in THF (0.15 mL, 0.15 mmol) was added dropwise.
The reaction was stirred at ꢀ78 °C for 2.5 h and was then warmed
to room temperature over 2 h. The reaction was quenched with
MeOH and concentrated under reduced pressure. The residue
was dissolved in CH2Cl2, where it was washed with 10% aqueous
H2O2, 1 M aqueous NaOH, distilled water, and brine. The organic
phase was dried (Na2SO4) and concentrated under reduced pres-
sure. Purification by silica gel chromatography (2:1, hexanes–
EtOAc) gave 20 (0.037 g, 82%) as a clear and colourless syrup. Rf
00 00
4.56 (d, 1H, Jgem = 12.0 Hz, PhCH2O), 4.55–4.44 (m, 6H, H-20,
PhCH2O), 4.42 (d, 1H, Jgem = 12.1 Hz, PhCH2O), 4.41 (d, 1H,
Jgem = 12.0 Hz, PhCH2O), 4.37 (d, 1H, J2,3 = 2.8 Hz, H-2), 4.18 (s,
1H, H-1), 3.81 (t, 1H, J3 ,4 = J4 ,5 = 9.7 Hz, H-40), 3.77 (t, 1H,
0
0
0
0
J1 ,2 = J2 ,3 = 8.5 Hz, H-200), 3.76–3.67 (m 3H, H-40, H-50, H-6a0),
3.67–3.61 (m, 4H, H-6a, H-6b, H-6b0, H-300), 3.61–3.52 (m, 4H, H-
5, H-30, H-6a0, H-6b0), 3.50 (s 3H, OCH3), 3.49–3.44 (m, 2H, H-3,
H-50), 1.96 (q, 1H, Jgem = 12.1 Hz, H-4ax), 1.74 (ddd, 1H,
J3,4eq = 4.6 Hz, Jgem = 12.5 Hz, J4eq,5 = 1.8 Hz, H-4eq). 13C NMR
(125 MHz, CDCl3): d 139.2 (Ar), 138.6 (Ar), 138.4 (Ar), 138.3(4)
(Ar), 138.2(7) (Ar), 138.1(4) (Ar), 138.1(3) (Ar), 128.4 (Ar),
128.3(1) (Ar), 128.2(9) (Ar), 128.2(7) (Ar), 128.2(5) (Ar), 128.2(0)
(Ar), 128.1(6) (Ar), 128.1(2) (Ar), 128.1(0) (Ar), 127.9(9) (Ar),
127.9(7) (Ar), 127.8 (Ar), 127.7(5) (Ar), 127.7(2) (Ar), 127.6 (Ar),
127.5 (Ar), 127.4(9) (Ar), 127.4(8) (Ar), 127.3(4) (Ar), 127.3(2)
(Ar), 104.6 (C-100), 102.7 (C-1), 99.9 (C-10), 85.8 (C-300), 80.1 (C-30),
77.2 (C-50), 75.3(5), 75.2(9) (C-3, C-20), 75.1 (PhCH2O), 75.0
(PhCH2O), 74.8, 74.6 (C-40, C-200), 74.5 (PhCH2O), 74.0, 73.8 (C-400,
C-500), 73.6 (PhCH2O), 73.4 (PhCH2O), 73.3 (PhCH2O), 73.0 (C-600),
72.1 (C-5), 70.1 (C-60), 70.0 (C-2), 69.8 (C-6), 69.7 (PhCH2O), 69.2
(PhCH2O), 57.3 (OCH3), 29.7 (C-4). HRESIMS: calcd for C75H82O15Na
(M+Na): 1245.5546, found 1245.5539. Anal. Calcd for C75H82O15: C,
73.63; H, 6.76. Found: C, 73.35; H, 6.69.
00 00
00 00
0.06 (4:1, toluene–EtOAc); [
a
]
ꢀ35.5 (c 1.4, CHCl3); 1H NMR
D
(600 MHz, CDCl3): d 7.48–7.46 (m, 2H, ArH), 7.40–7.37 (m, 2H,
ArH), 7.31–7.16 (m, 36H, ArH), 5.05 (s, 1H, H-10), 5.01–4.95 (m,
4H, H-100, PhCH2O), 4.90 (d, 1H, Jgem = 10.7 Hz, PhCH2O), 4.61 (d,
1H, J2 ,3 = 3.2 Hz, H-20), 4.57–4.54 (m, 2H, H-1, PhCH2O), 4.54–
4.51 (m, 2H, PhCH2O), 4.50–4.47 (m, 2H, PhCH2O), 4.48–4.45 (m,
2H, PhCH2O), 4.45–4.43 (m, 2H, H-200, PhCH2O), 4.40 (d, 1H,
Jgem = 12.0 Hz, PhCH2O), 4.31 (d, 1H, Jgem = 12.1 Hz, PhCH2O), 4.27
(d, 1H, Jgem = 9.9 Hz, PhCH2O), 4.23 (s, 1H, H-1), 4.04 (d, 1H,
0
0
Jgem = 12.2 Hz, PhCH2O), 3.89 (d, 1H, J3 ,4 = J4 ,5 = 9.5 Hz, H-40),
0
0
0
0
3.82 (d, 1H, J3 ,4 = J4 ,5 = 9.5 Hz, H-400), 3.78 (dd, 1H, J5 ,6a = 1.7 Hz,
00 00
00 00
0
0
Jgem = 10.5 Hz, H-6a0), 3.73 (dd, 1H, J5 ,6a = 1.9 Hz, Jgem = 10.4 Hz, H-
6a00), 3.71–3.68 (m, 2H, H-6b0, H-6b00), 3.62–3.59 (m, 2H, H-5, H-30),
3.55–3.50 (m, 3H, H-3, H-6a, H-50), 3.49 (s, 3H, OCH3), 3.45–3.37
(m, 3H, H-6b, H-300, H-500), 1.64 (ddd, 1H, J3,4eq = 4.5 Hz,
Jgem = 12.4 Hz, J4eq,5 = 1.8 Hz, H-4eq), 1.46 (q, 1H, Jgem = 12.2 Hz, H-
00
00
3.2.9. Methyl 3,4,6-tri-O-benzyl-b-
3,4,6-tri-O-benzyl-b- -mannopyranosyl-(1?2)-3,6-di-O-benzyl-
4-O-methyl-b- -mannopyranoside (21)
D
-glucopyranosyl-(1?2)-
D
D
Trisaccharide 19 (0.856 g, 0.661 mmol) was dissolved in a mix-
ture of MeOH (6 mL) and CH2Cl2 (3 mL). A 1 M solution of
NaOMe/MeOH (1 mL, 1 mmol) was added. The reaction was stirred
for 24 h. The reaction mixture was neutralized with Amberlite IR
120 and filtered to remove the resin. The filtrate was concentrated.
Purification by silica gel chromatography (4:1, toluene–EtOAc) gave
21 (0.7666 g, 93%) as a white solid. Rf 0.64 (1:1, hexanes–EtOAc);
4
ax). 13C NMR (125 MHz, CDCl3): d 138.6 (Ar), 138.5 (Ar), 138.3(7)
(Ar), 138.3(2) (Ar), 138.2 (Ar), 138.1(6) (Ar), 138.1(3) (Ar), 138.0
(Ar), 129.2 (Ar), 128.3(7) (Ar), 128.3(1) (Ar), 128.2(7) (Ar),
128.2(4) (Ar), 128.2(2) (Ar), 128.2(1) (Ar), 128.2(0) (Ar), 128.0(6)
(Ar), 128.0(4) (Ar), 127.9(8) (Ar), 127.8(7) (Ar), 127.7(9) (Ar),
127.7(1) (Ar), 127.6(6) (Ar), 127.6(2) (Ar), 127.5(9) (Ar), 127.5(1)
(Ar), 127.4(9) (Ar), 127.3(6) (Ar), 127.3(5) (Ar), 127.2(6) (Ar),
103.2 (C-1), 100.7 (C-100), 99.6 (C-10), 81.4 (C-300), 80.1 (C-30), 75.3
(PhCH2O), 75.2 (PhCH2O), 75.0(7), 75.0(6) (C-50, C-500), 74.6 (C-3),
74.2(7) (C-40), 74.2(0) (C-400), 73.5 (PhCH2O), 73.4 (PhCH2O), 73.3
(PhCH2O), 72.7(0), 72.6(8) (C-6, C-20), 71.9 (C-5), 70.2 (C-60), 69.9
(C-6), 69.5 (PhCH2O), 69.4 (PhCH2O), 69.2 (PhCH2O), 67.6 (C-2),
66.8 (C-200), 57.2 (OCH3), 30.2 (C-4). HRESIMS: calcd for
[
a
]
ꢀ58.0 (c 1.6, CHCl3); 1H NMR (500 MHz, CDCl3): d 7.45–7.42
D
(m, 2H, ArH), 7.39–7.35 (m, 4H, ArH), 7.35–7.21 (m, 29 H, ArH),
7.21–7.14 (m, 5H, ArH), 5.02–4.97 (m, 4H, PhCH2O), 4.88–4.84 (m,
2H, H-10, PhCH2O), 4.72 (d, 1H, J1 ,2 = 7.8 Hz, H-100), 4.70–4.65 (m,
00 00
2H, PhCH2O), 4.61 (d, 1H, Jgem = 12.2 Hz, PhCH2O), 4.54–4.46 (m,
4H, H-20, PhCH2O), 4.46–4.41 (m, 4H, H-2, PhCH2O), 4.38 (d, 1H,
Jgem = 12.0 Hz, PhCH2O), 4.37 (d, 1H, Jgem = 12.1 Hz, PhCH2O), 4.25
(s, 1H, H-1), 3.82–3.70 (m, 6H, H-4, H-6a, H-6b, H-6a0, H-200, H-500),
3.68–3.60 (m, 4H, H-40, H-6b0, H-300, H-6a00), 3.55–3.52 (m, 3H, H-
30, H-400, H-6b00), 3.50 (s, 3H, 4-OCH3), 3.49 (s, 3H, 1-OCH3), 3.48
(m, 1H, H-50), 3.40 (dd, 1H, J2,3 = 3.3 Hz, J3,4 = 9.3 Hz, H-3), 3.29
(ddd, 1H, J4,5 = 9.8 Hz, J5,6a = 2.0 Hz, J5,6b = 5.0 Hz, H-5). 13C NMR
(125 MHz, CDCl3): d 139.2 (Ar), 138.6 (Ar) 138.4(1) (Ar), 138.3(9)
(Ar), 138.2(4) (Ar), 138.2(0), 138.1(4) (Ar), 138.0(8) (Ar), 129.0
(Ar), 128.6 (Ar), 128.4 (Ar), 128.3 (Ar), 128.2(64) (Ar), 128.2(56)
(Ar), 128.2(4) (Ar), 128.2(2) (Ar), 128.1(9) (Ar), 128.0(8) (Ar),
127.9(7) (Ar), 127.9(4) (Ar), 127.8(7) (Ar), 127.8(1) (Ar), 127.7
(Ar), 127.6 (Ar), 127.5 (Ar), 127.4(8) (Ar), 127.4(7) (Ar), 127.4(3)
(Ar), 127.3(7) (Ar), 127.3 (Ar), 127.0 (Ar), 125.3 (Ar), 105.1 (C-100),
102.1 (C-1), 99.7 (C-10), 86.5 (C-40), 80.1, 80.0 (C-3, C-30), 77.2 (C-
200), 75.6 (C-5), 75.3 (C-50), 75.1(9), 75.1(6) (C-20, C-500), 75.1(2)
(PhCH2O), 74.9, 74.8 (C-60, PhCH2O), 74.7, 74.6, 74.5 (C-2, C-4, C-
400), 73.6 (PhCH2O), 73.4 (PhCH2O), 73.2 (PhCH2O), 70.8 (C-300),
C
75H82O15Na (M+Na): 1245.5546, found 1245.5539.
3.2.11. Methyl 3,4,6-tri-O-benzyl-b-
3,4,6-tri-O-benzyl-b- -mannopyranosyl-(1?2)-3,6-di-O-benzyl-
4-O-methyl-b- -mannopyranoside (23)
D-mannopyranosyl-(1?2)-
D
D
Acetic anhydride (2.5 mL, 26 mmol) was added to a solution of tri-
saccharide 21 (0.2829 g, 0.2257 mmol) in DMSO (5.0 mL, 70 mmol).
The reaction was stirred overnight. The mixture was concentrated
under reduced pressure and the residue was redissolved in THF
(5 mL). This solution was cooled to ꢀ78 °C. A solution of L-SelectrideÒ
in THF (0.90 mL, 0.90 mmol) was added dropwise. The reaction was
stirred at ꢀ78 °C for 2 h and was then warmed to room temperature
overnight. The reaction was quenched with MeOH and concentrated
underreducedpressure. Theresidue was dissolved inCH2Cl2, where it
was washed with 10% aqueous H2O2, 1 M aqueous NaOH, distilled

C. Costello, D. R. Bundle / Carbohydrate Research 357 (2012) 7–15
15
water, and brine. The organic phase was dried (Na2SO4) and concen-
trated under reduced pressure. Purification by silica gel chromatogra-
phy (2:1, hexanes–EtOAc) gave 23 (0.188 g, 66%) as a clear and
H2(g). The mixture was stirred overnight under a hydrogen atmo-
sphere. The catalyst was separated by filtration through celite and
the filtrate was concentrated under reduced pressure. The crude
product was dissolved in water and lyophilized. Purification of
colourless syrup. Rf 0.20 (4:1, toluene–EtOAc); [a D
]
ꢀ53.0 (c 2.2,
CHCl3); 1H NMR (600 MHz, CDCl3): d 7.52–7.49 (m, 2H, ArH), 7.42–
7.39 (m, 2H, ArH), 7.32–7.20 (m, 31H, ArH), 7.19–7.15 (m, 5H, ArH),
5.06 (s, 1H, H-100), 5.05 (s, 1H, H-10), 4.99 (d, 1H, Jgem = 11.8 Hz,
PhCH2O), 4.98 (d, 1H, Jgem = 10.9 Hz, PhCH2O), 4.94–4.90 (m, 2H,
the product by HPLC gave 3 (0.018 g, 47%) as a white solid. [a]
D
ꢀ53.0 (c 1.0, CHCl3); 1H NMR (500 MHz, D2O): d 4.93 (s, 1H,
H-100), 4.89 (s, 1H, H-10), 4.57 (s, 1H, H-1), 4.34 (d, 1H, J2 ,3 = 3.1 Hz,
0
0
H-20), 4.20 (d, 1H, J2,3 = 3.0 Hz, H-2), 4.18 (d, 1H, J2 ,3 = 3.2 Hz, H-
00 00
PhCH2O), 4.67 (d, 1H, J2 ,3 = 3.1 Hz, H-20), 4.63 (d, 1H, Jgem = 12.2 Hz,
PhCH2O), 4.58 (d, 1H, J2,3 = 3.4 Hz, H-2), 4.57–4.52 (m, 2H, PhCH2O),
4.52–4.47 (m, 4H, PhCH2O), 4.45–4.40 (m, 2H, PhCH2O), 4.32 (d, 1H,
Jgem = 9.9 Hz, PhCH2O), 4.29 (s, 1H, H-1), 4.28–4.25 (m, 2H, H-200,
PhCH2O), 4.05 (d, 1H, Jgem = 12.2 Hz, PhCH2O), 3.89 (t, 1H,
200), 3.93–3.88 (m, 3H, H-6a, H-6a00, H-6b00), 3.69 (m, 4H, H-3, H-
6b, H-6a0, H-6b0), 3.65–3.60 (m, 2H, H-30, H-300), 3.60–3.55 (m,
2H, H-40, H-400), 3.53 (s, 3H, 4-OCH3), 3.51 (s, 3H, 1-OCH3), 3.40–
3.33 (m, 3H, H-5, H-50, H-500), 3.26 (t, 1H, J3,4 = J4,5 = 9.7 Hz). 13C
NMR (125 MHz, D2O): d 102.1 (C-1), 101.7 (C-100), 101.5 (C-10),
79.1 (C-2), 78.9 (C-20), 78.2 (C-4), 77.1(4), 77.1(2) (C-50, C-500),
76.0 (C-5), 73.8 (C-300), 73.0 (C-30), 72.7 (C-3), 71.4 (C-200), 67.8,
67.7 (C-40, C-400), 62.0 (C-6), 61.6 (C-60, C-600), 61.2 (4-OCH3), 58.0
(1-OCH3). HRESIMS: calcd for C20H36O16Na (M+Na): 555.1896,
found 555.1895.
0
0
J3 ,4 = J4 ,5 = 9.5 Hz, H-40), 3.87 (t, 1H, J3 ,4 = J4 ,5 = 9.4 Hz, H-400),
0
0
0
0
00 00
00 00
3.79 (dd, 1H, J5 ,6a = 1.6 Hz, Jgem = 10.5 Hz, H-6a0), 3.73–3.70 (m, 2H,
0
0
H-6a, H-6b0), 3.70–3.66 (m, 2H, H-6a00, H-6b00), 3.64 (dd, 1H,
0
0
J5,6b = 5.0 Hz, Jgem = 10.5 Hz, H-6b), 3.61 (dd, 1H, J2 ,3 = 3.2 Hz,
J3 ,4 = 9.3 Hz, H-30), 3.53 (ddd, 1H, J4 ,5 = 9.6 Hz, J5 ,6a = 1.7 Hz,
0
0
0
0
0
0
J5 ,6b = 6.0 Hz, H-50), 3.48 (s, 3H, 1-OCH3), 3.47 (m, 1H, H-500), 3.46–
3.42 (m, 2H, H-3, H-300), 3.31 (ddd, 1H, J4,5 = 9.8 Hz, J5,6a = 1.6 Hz,
J5,6b = 5.0 Hz, H-5), 3.25 (t, 1H, J3,4 = J4,5 = 9.4 Hz, H-4), 3.21 (s, 3H,
4-OCH3). 13C NMR (125 MHz, CDCl3): d 138.5(9) (Ar), 138.5(6) (Ar),
138.4(0) (Ar), 138.3(7) (Ar), 138.3(0) (Ar), 138.2(3) (Ar), 138.1(7)
(Ar), 138.0 (Ar), 129.1 (Ar), 138.3(4) (Ar), 128.3(2) (Ar), 128.2(6) (Ar),
128.2(5) (Ar), 128.2(1) (Ar), 128.2(0) (Ar), 128.1 (Ar), 128.0(8) (Ar),
128.0(2) (Ar), 127.9(8) (Ar), 127.8 (Ar), 127.7(2) (Ar), 127.6(7)
(Ar), 127.5(4) (Ar), 127.5(3) (Ar), 127.5(1) (Ar), 127.4(9) (Ar),
127.3(6) (Ar), 127.2 (Ar), 127.1(5) (Ar), 102.6 (C-1), 100.0 (C-100),
99.8 (C-10). 82.5 (C-300), 80.4 (C-3), 80.2 (C-30), 76.1 (C-4), 75.2(7),
75.2(6) (C-5, C-50), 75.2(1) (PhCH2O), 75.0 (PhCH2O), 74.9(7) (C-500),
74.4(5), 74.4(2) (C-40, C-400), 73.5 (PhCH2O), 73.4 (PhCH2O), 73.3
(PhCH2O), 71.4 (C-20), 70.2 (C-60), 69.8 (C-600), 69.8 (PhCH2O),
69.5(2) (PhCH2O), 69.4(8) (PhCH2O), 69.3 (C-6), 68.9 (C-2), 67.1
(C-200), 60.8 (4-OCH3), 57.3 (4-OCH3). HRESIMS: calcd for C76H84
O16Na (M+Na): 1275.5652, found 1275.5641. Anal. Calcd for C76H84
O16: C, 72.82; H, 6.75. Found: C, 72.83; H, 6.77.
0
0
Acknowledgements
We thank Mrs. Joanna Sadowska for technical assistance in
acquiring the inhibition data. The research was made possible by
the Natural Science and Engineering Research Council of Canada
Discovery grant to D. R. Bundle.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.carres.2012.03.
019.
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D-
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the product by HPLC gave 2 (0.0064 g, 42%) as a white solid. [a]
D
1
ꢀ41.0 (c 0.6, H2O); H NMR (600 MHz, D2O): d 4.95 (s, 1H, H-10),
4.90 (s, 1H, H-100), 4.48 (s, 1H, H-1), 4.35 (d, 1H, J2 ,3 = 3.4 Hz, H-
00 00
200), 4.15 (d, 1H, J2 ,3 = 3.0 Hz, H-20), 4.09 (d, 1H, J2,3 = 2.7 Hz, H-2),
3.93–3.88 (m, 3H, H-3, H-6a0, H-6a00), 3.75–3.67 (m, 3H, H-6a, H-
6b0, H-6b00), 3.66–3.59 (m, 3H, H-5, H-6b, H-300), 3.59–3.54 (m,
3H, H-30, H-40, H-400), 3.53 (s, 3H, OCH3), 3.40–3.34 (m, 2H, H-50,
H-500), 1.73 (dd, 1H, J3,4eq = 4.3 Hz, Jgem = 11.8 Hz, H-4eq), 1.41 (q,
1H, Jgem = 12.0 Hz, H-4ax). 13C NMR (125 MHz, D2O): d 102.7 (C-
1), 101.7 (C-10), 101.6 (C-100), 79.1 (C-200), 77.9 (C-2), 77.2(3),
77.2(1) (C-50, C-500), 73.9, 73.7 (C-5, C-30), 73.1 (C-300), 71.3 (C-20),
68.1 (C-3), 68.0, 67.7 (C-40, C-400), 64.8 (C-6), 62.0, 61.7 (C-60,
C-600), 57.9 (OCH3), 30.9 (C-4). HRESIMS: calcd for C19H34O15Na
(M+Na): 525.179, found 525.1789.
0
0
3.2.13. Methyl b-
D-mannopyranosyl-(1?2)-b-
D-
mannopyranosyl-(1?2)-4-O-methyl-b-
D-mannopyranoside (3)
Trisaccharide 23 (0.089 g, 0.071 mmol) was dissolved in a mix-
ture of CH2Cl2 (5 mL) and MeOH (5 mL). 10% Pd/C (50 mg) was
added and the reaction vessel was evacuated and purged with