Synthesis of a Candida albicans tetrasaccharide spanning the β1,2-mannan phosphodiester α-mannan junction

Article abstract of DOI:10.1039/c2ob26355f

The cell wall phosphomannan of Candida species is a complex N-linked glycoprotein with a glycan chain containing predominantly an α-linked mannose backbone with α-mannose branches. A minor β-mannan component is attached to the branches either via a glycos

Full text of DOI:10.1039/c2ob26355f

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PAPER
Synthesis of a Candida albicans tetrasaccharide spanning the β1,2-mannan
phosphodiester α-mannan junction†
Anh-Thu Dang, Margaret A. Johnson and David R. Bundle*
Received 13th July 2012, Accepted 30th August 2012
DOI: 10.1039/c2ob26355f
The cell wall phosphomannan of Candida species is a complex N-linked glycoprotein with a glycan chain
containing predominantly an α-linked mannose backbone with α-mannose branches. A minor β-mannan
component is attached to the branches either via a glycosidic bond (acid stable β-mannan) or a
phosphodiester bond (acid-labile β-mannan). The α-mannan residues of the cell wall phosphomannan do
not afford protective antibody, while the β-mannan portion is a protective antigen and has become an
attractive target as the key epitope of a conjugate vaccine. We report the first synthesis of a tetrasaccharide
1 consisting of a β1,2-mannopyranosyl trisaccharide linked via a phosphodiester to methyl
α-mannopyranoside. This encompasses the attachment site of the acid labile β-mannan to the α-mannan
component of the cell wall phosphomannan. The trisaccharide was formed by an iterative process to first
create a β-glucopyranoside linkage and then epimerize the C-2 center via an oxidation–reduction
sequence. The phosphate diester linkage was accessed via an anomeric H-phosphonate. The binding of
phosphomannan fragment 1 with the protective antibody C3.1 has been evaluated and compared with a
β-mannotrioside in hapten inhibition experiments. The observed activities are rationalized with a model
for 1 docked in the binding site of C3.1.
β-mannan, while strains of serotype A have both acid-stable and
Introduction
acid-labile β-mannan epitopes.8,9
Candida albicans, the most common etiologic agent of candidia-
sis,1 commonly affects immunocompromised patients and indi-
viduals undergoing long term antibiotic treatment or major
invasive surgery.2 Therapeutics to combat infection have been
limited to triazole derivatives and amphotericin B.3 Despite anti-
fungal therapy mortality due to Candida infections remains
high,4 which highlights the need for alternative treatment sol-
utions.5 Conjugate vaccines composed of microbial polysacchar-
ides or oligosaccharides covalently linked to immunogenic
proteins have been shown to be an attractive and cost effective
strategy.6 The cell wall phosphomannan antigen has received the
greatest attention as it is highly immunogenic.1 The structure of
this complex N-linked glycoprotein is an extended (1 → 6)-α-D-
mannopyranan backbone containing (1 → 2)-α-D-mannopyranan
branches to which are attached (1 → 2)-β-D-mannopyranan oli-
gomers either via glycosidic bonds (acid-stable β-mannan) or
phosphodiester bonds (acid-labile β-mannan) (Fig. 1).7–9 C. albi-
cans of serotype B is defined by the presence of acid-labile
The α-mannan component of the Candida cell wall phospho-
mannan is not a protective antigen, whereas, the (1 → 2)-
β-mannan oligosaccharides attached to the α-mannan side chains
have been shown to produce protective antibodies.10 Conse-
quently, antigens composed of this epitope have become an
attractive component of a conjugate vaccine.11 Immunochemical
studies of two protective monoclonal antibodies with synthetic
Alberta Glycomics Centre, Department of Chemistry, University of
Alberta, Edmonton, Canada, T6G 2G2. E-mail: dave.bundle@ualberta.ca;
Fax: (+1) 780-492-7705; Tel: (+1) 780-492-8808
Fig. 1 Structure of the cell wall phosphomannan antigen. Acid labile
β-mannan is linked via a phosphodiester and acid stable β-mannan is the
glycosidically linked moiety.
†Electronic supplementary information (ESI) available: 1H and 13C
NMR spectra are provided. See DOI: 10.1039/c2ob26355f
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synthesize a variety of β-mannopyranosyl oligomers including
1,2-linked β-mannopyranosyl oligomers.16 A low temperature is
essential for complete stereoselective formation of β-mannopyra-
nosides. The second approach, which involves β-glucopyranosyl
linkage formation and C-2 epimerization via an oxidation-
reduction sequence, has been employed by several authors since
1974.17 We reported the related but simplified approach based on
a trichloroacetimidate glucopyranosyl donor that was especially
well suited to the construction of (1 → 2)-β-mannan oligomers
on a multigram-scale.13b,e This approach had to be adapted to
synthesize the trisaccharide component of 1. To avoid the known
problem of aglycon transfer when glycosyl imidates are
employed to glycosylate a thioglycoside18 a glucopyranosyl
chloride donor was used.
Construction of the phosphodiester linkage requires control of
the anomeric configuration of an oligosaccharide 1-phosphate.
Synthetically, this linkage is in general constructed using the
H-phosphonate19 or the amidite20 approach developed for oligonu-
cleotide synthesis, in which the former approach has become the
more powerful. Two synthetic pathways can be considered for
elaborating this crucial linkage. The first involves the coupling
between an anomeric H-phosphonate monoester (donor) and a
mannopyranoside alcohol (acceptor)21 while the second pathway
regards a “normal” glycosylation between a H-phosphonate
monoester of methyl mannopyranoside (acceptor) and a glycosyl
(donor).22 In the synthesis of 1, we preferred the first approach
since an α-phosphonate monoester should be formed selectively.
The synthesis of disaccharide and trisaccharide was accom-
plished as outlined in Scheme 1. Building block 223 was syn-
thesized according to literature procedures, while 3 was prepared
by a modification of a literature report.24 The glycosyl chloride
donor 2 and thioglycoside acceptor 3 were reacted at −40 °C
under promotion by silver trifluoromethanesulfonate (AgOTf)
(2.0 equiv.) in toluene and CH2Cl2, in the presence of molecular
sieves,25 affording the desired disaccharide 4 in 74% yield.
Transesterification of 4 gave the selectively protected disacchar-
ide alcohol 5 (96%) which was oxidized to the corresponding
ketone by the Swern reaction, followed by selective reduction
with L-selectride at −78 °C in THF to afford the mannobiose 6
in 73% yield. The iterative process involving glucosylation,
saponification, oxidation, and reduction was repeated to build
mannose trisaccharide 9 from donor 2 and disaccharide acceptor
6. Trisaccharides 7, 8, and 9 were obtained in relatively good
yields of 71%, 92%, and 65%. Acetylation of 9 using acetic
anhydride in pyridine gave protected trisaccharide 10 in low
43% yield over 5 days. However, a higher yield (83%) and
shorter reaction time (22 h) was achieved with acetic anhydride,
triethylamine, and DMAP in CH2Cl2.26 Hydrolysis of thioglyco-
side 10 using N-iodosuccinimide in acetone–water gave hemi-
acetal 11 as an anomeric mixture (α : β 6 : 1) in 82% yield (87%
based on recovery of unreacted starting material).21f
Fig. 2 Tetrasaccharide 1 encompasses the essential elements of the
phosphodiester linkage region where the β-mannan is attached to the
α-mannan side chains of the cell wall phosphomannan complex. The tri-
saccharide glycoside, propyl β-D-mannopyranosyl-(1 → 2)-β-D-manno-
pyranosyl-(1 → 2)-β-D-mannopyranoside is the most active inhibitor of
the monoclonal antibody C3.1.
di- to hexasaccharide glycosides of (1 → 2)-β-D-mannopyranans
revealed that the maximum activity was reached with di- and tri-
saccharides and diminished significantly for larger structures.12
To date the most active inhibitor of the protective antibody C3.1
is propyl β-D-mannopyranosyl-(1 → 2)-β-D-mannopyranosyl-
(1 → 2)-β-D-mannopyranoside (Fig. 2). Our attention has been
focused on the synthesis and immunochemical studies of acid-
stable β-mannan epitopes composed of (1 → 2)-β di- and tri-
mannopyranosides as oligosaccharide glycosides and as conju-
gates to protein carriers.13 However, a C. albicans serotype B
acid-labile β-mannan epitope that incorporates the phosphodi-
ester has yet to be synthesized. Here, we investigated the
synthesis of the C. albicans phosphodiester epitope consisting of
what we imagine to be the important elements of 1,2-β-mannan
linked via a phosphodiester to the first residue of the α-mannan
backbone (Fig. 2, also that portion highlighted in Fig. 1).
Binding of this structure with the protective monoclonal anti-
body C3.114 should allow us to determine whether the phosphate
linkage plays any role in antibody recognition.
Results and discussion
Synthesis of (1 → 2)-β-linked disaccharide and trisaccharide
One of the most challenging tasks in the synthesis of compound
1 is the elaboration of the (1 → 2)-β-mannanpyranosyl linkage.
Several methodologies have been developed.15 Recent successes
have focused on two approaches: (1) direct and general for-
mation of the β-mannopyranosyl linkage; (2) via a β-glycopyra-
nosyl linkage with subsequent C-2 epimerization. Crich and co-
workers have successfully employed 4,6-O-benzylidene-
protected mannopyranosyl sulfoxide as a glycosyl donor to
Excellent diastereoselectivity was observed for the selectride
reduction of the di and trisaccharide 2-uloside precursors of 6
and 9. No β-gluco disaccharide epimer could be detected by H
NMR and for trisaccharide 9 only trace amounts of the β-gluco
trisaccharide epimer were observed in crude products. However,
di- and tri-saccharide sulphoxide by-products were formed as
minor side products in Swern oxidation reactions (S-methyl ether
was not observed, but SPh was oxidized to S(O)Ph, confirmed
1
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Scheme 2 Synthesis of anomeric H-phosphonate monoester 12.
Table 1 Conditions for synthesis of phosphonate 12
PCl3
Imidazole Et3N
11
Entry (equiv.) (equiv.)
(equiv.) 12 (recovered) 12 α : βa
1
2
3
4
4
4
5
5
12
8
6
12
8
6
68
7
6 : 1
8 : 1
33.1
100 : 0
43 18
54 13
38 39
5.5
5.9
a α : β ratios were determined by integration of 31P NMR resonances
from spectra recorded with continuous broadband hydrogen decoupling.
Ratios of anomers were confirmed by integration of the α and β H-2
resonances of residue D.
The H-phosphonate 12 was formed as an anomeric mixture
(α : β 6 : 1, 31P NMR analysis) in 68% yield along with recov-
ered starting material 11 (7%). An attempt was made to selec-
tively hydrolyze or anomerize the β-anomer of 12 by exposing
12 to H3PO3 (17 equiv.).28,29 We had hoped that the β-glycosyl
H-phosphonate would be more reactive, yielding either the
α-linked isomer or the easily separable hemiacetal derivative 11.
Unfortunately, both α- and β-linked 12 were still observed with
hemiacetal 11 after 8 days (tlc analysis). Since we were unable to
obtain the α-anomerically pure 12 by this approach or to resolve
the mixture by column chromatography, we attempted phospho-
nylation reaction in acidic conditions. It is possible that
β-anomer formed under these phosphonylation conditions may
either hydrolyze or perhaps anomerize to the more thermodyna-
mically stable α product.
Reactions were carried out for 1 h (from 0 °C to rt) using the
reagents shown in Table 1. The acidic environment was
increased by decreasing the amounts of imidazole and triethyl-
amine. This resulted in incomplete transformation of 12 and
increased yield of starting material 11. The yield of 12 dropped
from 68% to 43% and the amount of 11 rose from 7% to 18%
(Entries 1 and 2). However, the ratio of α-anomer to β-anomer
increased from 6 : 1 to 8 : 1 using PCl3 : imidazole : Et3N ratio of
1 : 2 : 2. Better α-selectivity (α : β 33 : 1) was observed using a
reagent ratio of 1 : 1.2 : 1.2 (Entry 3) and the α-anomer was
obtained exclusively at a reagent ratio of 1 : 1.1 : 1.8 (Entry 4).
We presume the yield of H-phosphonate 12 was decreased due
to either incomplete phosphonylation reaction or hemiacetal 11
formation via acid-catalyzed cleavage of the H-phosphonate
group.
Scheme 1 Synthesis of (1 → 2)-β-linked mannose disaccharide and
trisaccharide 11.
by MS). Attempts to employ milder oxidation reagents such as
Dess–Martin periodinane to minimize the amount of sulphoxide
by-product were unsuccessful. Specifically, although the Dess–
Martin reagent had excellent performance in oxidation of the di-
saccharide alcohol 5 with only trace amount of sulphoxide by-
product (1H NMR detected), it failed completely to react with
trisaccharide alcohol 8. Although future investigation of oxi-
dation reagents for di- and trisaccharide alcohol in the presence
of a thioglycoside group may be of interest, the Swern oxidation-
L-selectride reduction sequence in this synthesis strategy is
reliable and proceeds in good yield. Most importantly, all reac-
tions could be performed on a multigram scale. Heteronuclear
one-bond coupling constants (1JC–H) were used to unambigu-
ously establish the anomeric configuration of each mannopyrano-
syl residue.27
Synthesis of the anomeric H-phosphonate monoester
The H-phosphonate monoester 12 was prepared in a one-pot
reaction from hemiacetal 11 by treatment with triimidazolylpho-
sphine (generated in situ from imidazole, phosphorus trichloride,
and triethylamine), followed by basic hydrolysis (Scheme 2).
In brief, the anomerically pure α-linked 12 was obtained, but
in low yield (38%). We speculated that the α-phosphodiester 1,
which is more stable than the β-anomer, might be formed using
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Table 2 Ratio of anomers of phosphodiester 1 derived from α/β
mixtures of phosphonate 12
an anomeric mixture of the H-phosphonate 12 rather than
restricting this step to the use of pure α-anomer.
12
16
17
1
Synthesis of the anomeric phosphodiester
Entry
α : β
α : β
%
α : β
%
α : β
%
The synthesis of phosphodiester 1 is illustrated in Scheme 3.
Sequential treatment of methyl mannopyranoside 13 with trityl
chloride in pyridine and then acetic anhydride gave 14. The
selectively protected monosaccharide 1530 was obtained after the
trityl group was removed using iodine monobromide in CH2Cl2
and methanol31 affording 15 in 76% yield.
The anomeric phosphate diester 16 was formed from H-phos-
phonate 12 and monosaccharide 15 using pivaloyl chloride as
coupling reagent followed by I2/H2O oxidation of the newly-
formed H-phosphonate diester. Transesterification removed
acetyl groups and benzyl groups were cleaved under hydrogeno-
lysis conditions to give the desired product 1.
1
2
6 : 1
33 : 1
7 : 1
50 : 1
73
67
7 : 1
100 : 0
81
94
20 : 1
100 : 0
45
40
To determine whether the mixture of H-phosphonate anomers
12 could lead to the anomerically pure α-phosphodiester 1, we
used two batches of 12, composed of different anomeric mix-
tures α : β 6 : 1 and 33 : 1 (Table 2). The anomeric mixture (α : β
6 : 1), after coupling, gave mixture phosphodiester 16 (α : β 7 : 1)
in 73% yield (Entry 1). Deacetylation of 16 afforded compound
17 in 81% yield with the same α : β ratio. Phosphodiester 1 was
obtained as an anomeric mixture (α : β 20 : 1) in 45% yield via
hydrogenolysis of 17. Identical reaction conditions were applied
for the mixture α : β 33 : 1 (Entry 2). This gave 16 with an
improved α : β ratio of 50 : 1 in 67% yield. Subsequent
deprotection resulted in the isolation of 17 in 94% yield as a
single anomer. The final product 1 (40%) was obtained α-anom-
erically pure from 17. It seems likely that the relatively small
amount of β-anomer is reduced by successive chromatographic
steps as the synthesis progresses from compound 12 via 16 and
17 to 1.
Overall, the four-step reaction sequence to construct the
desired phosphodiester 1 from H-phosphonate 12 occurred in
good yields. Anomerically pure phosphodiester 1 was obtained
from the mixture of H-phosphonate 12 (α : β 33 : 1). This consti-
tutes a practical approach as the mixture of H-phosphonate can
be synthesized more readily and in higher yield than the pure
α-anomer.
Fig. 3 Competitive ELISA showing the determination of IC50 values
for 1 (solid blue circles) and propyl β-mannotrioside (solid green
triangles) binding to the mAb C3.1.
Immunochemistry studies
To date the most active inhibitor of the protective monoclonal
antibody C3.1 has been the propyl β-mannotrioside (Fig. 2).12
We compared the inhibitory activity of tetrasaccharide 1 with
Scheme 3 Synthesis of anomeric phosphodiester 1.
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Fig. 4 Wall-eye stereo views of modeled binding modes of oligosaccharides in the binding site of the C3.1 antibody. In panels a and b, the antibody
is shown as an all-atom molecular surface, and the ligand is shown in a stick representation. In panel c, oligosaccharide and protein are shown as stick
representations. (a) β-Mannohexoside. The central four residues (purple carbons) are accommodated well and make energetically favourable inter-
actions with the antibody, but extending to longer oligosaccharides (end residues, white carbons) causes steric clashes with the antibody, as seen by
the overlap of these residues with the antibody surface. (b) and (c) Tetrasaccharide 1. Residues A–D of 1 and selected residues of the antibody are
labeled. Predicted hydrogen bonds are shown as dashed lines. Colour scheme is by atom type: oxygen, red; hydrogen, white; nitrogen, blue; sulfur,
yellow. In panels b and c, antibody carbons are green and oligosaccharide carbons are purple.
this trisaccharide in an ELISA. Cell wall phosphomannan was
used to coat 96 well microtitre plates and the binding of C3.1
was measured in the absence and presence of increasing concen-
trations of tetrasaccharide 1 and the trisaccharide, methyl β-D-
mannopyranosyl (1 → 2)-β-D-mannopyranosyl-(1 → 2)-β-D-
mannopyranoside.
The two inhibition curves (Fig. 3) show that 1 and the β-man-
notrioside possess essentially identical IC50 values. This implies
that the phosphodiester does not play a significant role in inter-
action with this protective antibody.
32, the comparative model of the C3.1 binding site revealed a
groove that is unable to accommodate β-mannan oligosacchar-
ides larger than a β-mannotetroside without significant, energeti-
cally unfavorable conformational adjustments to avoid steric
clashes with the antibody binding site (Fig. 4a). In contrast, the
α-linkage of residue B in 1 allows clashes with the narrowest
part of the binding groove to be avoided (Fig. 4b and c), while
the phosphodiester and the α-linked monosaccharide residue,
residue A, provide additional shape complementarity to the
binding site. The residues C and D of 1 bind in a similar orien-
tation and conformation (Table 3) to the residues A and B of a
β-mannobioside or β-mannotrioside, and are predicted to partici-
pate in similar intermolecular interactions on the basis of our
model. In addition, residue A of 1 is predicted to form two
hydrogen bonds, to Asp H101 and Ser L56. The configuration of
1 thus allows this molecule to contact a greater area of the
binding site compared to molecules with exclusively β-linkages
(Fig. 4b and c).
Modeling studies
We modeled the binding mode of 1 based on solution NMR,
chemical mapping and molecular modeling studies of β-manno-
sides in the C3.1 binding site.32 The results suggested an expla-
nation for the inhibition data reported above. As described in ref.
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Table 3 Dihedral angles (°) in the modeled binding mode of 1 and of
a β-mannotrioside
Experimental
General methods
1
Tri
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 suc-
cessive passage through columns of alumina and copper under
nitrogen, except for DMSO, which was distilled under vacuum
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, Montreal)
at flow rates between 5 and 10 mL min−1. Following deprotec-
tion, final compounds were purified by HPLC (Flow rate =
3.0 mL min−1, 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 lyophi-
φa
ψ
ψ2
φ
ψ
A-OMe
Phosphate-B
B–C
−48
94
46
69
4
12
−154
C–D
38
48
3
−1
6
a Dihedral angle conventions: for angles involving the phosphate group,
we adopted the conventions of Tvaroska, André and Carver (ref. 37), i.e.
φ = O5–C1–O1–P, ψ = C1–O1–P–O6, ψ2 = O1–P–O6–C6. For all other
angles, we used standard definitions of φH and ψH for carbohydrates, i.e.
φ = H1–C1–O1–C2 and ψ = C1–O1–C2–H2.
No specific interactions are predicted for the phosphate group,
although one tyrosine hydroxyl group (Tyr H32) is at a distance
of less than 4 Å from a phosphate oxygen, which at the level of
resolution of homology modeling, might indicate a possible
interaction. That the phosphate does not contribute significant
strong interactions (e.g. salt bridges or strong hydrogen bonds) is
consistent with our inhibition data.
Since tetrasaccharide 1 makes more favourable contacts than
the β-mannotriose it is surprising that 1 is only as active as the
β-mannotriose inhibitor (Fig. 3). There is precedent for net zero
binding energy changes when complementary charged groups
are organized at the periphery of the binding site.33
It is intriguing to investigate which compound will yield a
more promising hapten for use in conjugate vaccines. Modeling
shows that 1 is more complementary to the C3.1 binding site
and contacts a more diverse set of amino acid residues than
β-mannosides, suggesting that 1 could more faithfully elicit
C3.1-like antibodies. However, the efficiency of synthesis,
chemical and possibly conformational stability,12,32 and our
recent immunological studies13f,g suggest the primary antibody-
antigen contacts are restricted to the β-mannan. With this in
mind, a β-1,2-mannotriose linked as its α-anomer to a tether
would still provide the specificity to β-1,2-mannans but would
allow the tether to exit the binding site unrestricted (Fig. 4c).
1
lized. H NMR spectra were recorded at 500 or 600 MHz, and
chemical shifts, reported in δ (ppm), were referenced to internal
residual protonated solvent signals or to external acetone (0.1%
ext. acetone @ δ 2.225 ppm) in the case of D2O. 13C NMR
spectra were recorded at 125 MHz, and chemical shifts are refer-
enced to internal CDCl3 (δ 77.23) or external acetone (δ 31.07).
First order chemical shifts of 1H and 13C are reported to the
second and first decimal place, respectively. Proton decoupled
31P NMR spectra were recorded at 201.6 MHz and are refer-
enced to 85% H3PO4 via external TMS using the absolute reson-
ance frequency ratio Ξ.38 High resolution mass spectra were
obtained on a Micromass Zabspec TOF-mass spectrometer by
analytical services in the Department of Chemistry, University of
Alberta. 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 g−1 dm−1. Elemental
analysis was performed by analytical services of the Department
of Chemistry, University of Alberta.
Conclusions
Phenyl 3,4,6-tri-O-benzyl-1-thio-α-D-mannopyranoside (3).24
Sodium methoxide in methanol (3 mL, 0.2 M) was added to a
solution of phenyl 2-O-acetyl-3,4,6-tri-O-benzyl-1-thio-α-D-
mannopyranoside24 (10.83 g, 18.52 mmol) in tetrahydrofuran
(15 mL) and methanol (100 mL). The reaction mixture was
stirred at room temperature for 4 h, neutralized with Amberlite
IR-120 (H+) resin and filtered. Solvents were removed under
reduced pressure and the residue was co-evaporated with
toluene. Column chromatography (80 : 20 Hexane : EtOAc) of
the crude product yielded 3 (9.51 g, 95%) as a colourless oil: Rf
= 0.45 (70 : 30 Hexane : EtOAc); [α]D +169 (c 0.18, CHCl3);
Lit.24b [α]D +182.5 (c 1.26, CHCI3); IR ν 3427 (br), 3062 (m),
3030 (m), 2922 (s), 2870 (s), 1584 (w), 1496 (m), 1480 (m),
1453 (s), 1439 (m), 1365 (m), 1208 (m), 1097 (s), 1027 (s), 769
The tetrasaccharide 1 of the C. albicans phosphomannan
complex (PMC) spanning acid labile β-1,2-mannan and its phos-
phodiester attachment to α-mannan has been synthesized
employing a strategy suitable for synthetic antigen synthesis. A
comparison of the relative inhibitory power of 1 and the β-1,2-
mannotriose suggests that the phosphodiester linkage is not
crucial to binding with the protective monoclonal antibody C3.1.
The data when rationalized with a model of 1 in the C3.1
binding site suggest an optimal design for a Candida albicans
conjugate vaccine consisting of a β-1,2-mannotriose linked as its
α-anomer to a tether. A 2-(2-azidoethoxy)ethyl glycoside similar
to 1 has been synthesized to provide a functional group suitable
for conjugation to protein. Evaluation of the ability of the result-
ing conjugate to evoke an antibody response to the C. albicans
cell wall phosphomannan is in progress.
1
(m), 740 (s), 697 (s) (cm−1); H NMR (600 MHz, CDCl3) δ
7.51–7.49 (m, 2H, ArH), 7.41–7.23 (m, 18H, ArH), 5.64 (d, 1H,
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J1B,2B = 1.3 Hz, H-1B), 4.86 (d, 1H, J = 10.8 Hz, PhCH2O),
4.74 (dd, 2H, J = 11.5 Hz, PhCH2O), 4.64 (d, 1H, J = 12.0 Hz,
PhCH2O), 4.56 (d, 1H, J = 10.8 Hz, PhCH2O), 4.48 (d, 1H, J =
12.0 Hz, PhCH2O), 4.34–4.32 (m, 1H, H-5B), 4.28 (dd, 1H,
(C-2C), 72.3 (C-5B), 70.8 (PhCH2O), 70.0 (C-6C), 69.5 (C-6B);
HRMS [ESI] calcd for C62H64O11SNa 1039.4062, found
1039.4058.
J1B,2B = 1.6 Hz, J2B,3B = 3.2 Hz, H-2B), 3.97 (t, 1H, J3B,4B
J4B,5B ≈ 9.4 Hz, H-4B), 3.91 (dd, 1H, J2B,3B = 3.1 Hz, J3B,4B
9.1 Hz, H-3B), 3.83 (dd, 1H, J5B,6aB = 4.7 Hz, J6aB,6bB
≈
=
=
Phenyl 3,4,6-tri-O-benzyl-β-D-glucopyranosyl-(1 → 2)-3,4,6-
tri-O-benzyl-1-thio-α-D-mannopyranoside (5). Sodium metal
(33.4 mg, 1.45 mmol) was added in a portion to a solution of 4
(5.28 g, 5.19 mmol) in tetrahydrofuran (5 mL) and methanol
(30 mL). The reaction mixture was stirred at room temperature
for 2 d, neutralized with Amberlite IR-120 (H+) resin and
filtered. Solvents were removed under reduced pressure and the
residue was co-evaporated with toluene. Column chromato-
graphy (90 : 10 to 85 : 15 Hexane : EtOAc) of the crude product
yielded 5 (4.87 g, 96%) as a colourless oil (solidified after 2
weeks): Rf = 0.62 (70 : 30 Hexane : EtOAc); [α]D +41.7 (c 0.2,
CHCl3); IR ν 3477 (w, br), 3087 (w), 3062 (m), 3030 (m), 2867
(m), 1584 (w), 1497 (m), 1480 (w), 1434 (s), 1363 (m), 1208
10.9 Hz, H-6aB), 3.72 (dd, 1H, J5B,6bB = 1.9 Hz, J6bB,6aB = 10.9
Hz, H-6bB), 2.70 (s, br, 1H, OH); 13C NMR (125 MHz, CDCl3)
δ 138.3 (Ar), 138.2 (Ar), 137.6 (Ar), 133.9 (Ar), 131.4 (Ar),
129.0 (Ar), 128.6 (Ar), 128.4 (Ar), 128.3 (Ar), 128.1 (Ar), 128.0
(Ar), 127.9 (Ar), 127.86 (Ar), 127.7 (Ar), 127.6 (Ar), 127.4
(Ar), 87.3 (JC–1B,H–1B = 167 Hz, C-1B), 80.3 (C-3B), 75.2
(PhCH2O), 74.5 (C-4B), 73.4 (PhCH2O), 72.3 (C-5B), 72.2
(PhCH2O), 69.9 (C-2B), 68.8 (C-6B); HRMS [ESI] calcd for
C33H34O5SNa 565.2019, found 565.2016.
1
Phenyl 2-O-acetyl-3,4,6-tri-O-benzyl-β-D-glucopyranosyl-(1 →
2)-3,4,6-tri-O-benzyl-1-thio-α-D-mannopyranoside (4). A solu-
tion of 2-O-acetyl-3,4,6-tri-O-benzyl-D-glucopyranosyl chloride
(2)23 (2.67 g, 5.23 mmol) and acceptor 3 (1.85 g, 3.41 mmol) in
CH2Cl2 (50 mL) was stirred with activated 4 Å molecular sieves
(2 g) at room temperature for 30 min. A suspension of silver
triflate (2.68 g, 10.5 mmol) in toluene (40 mL) was also stirred
with activated 4 Å molecular sieves (2 g) at −40 °C for 30 min.
The mixture of donor and acceptor was added dropwise over
30 min to the flask containing promoter at −40 °C, and the
mixture was stirred for a further 15 min. The reaction mixture
was quenched with Et3N (1 mL) and saturated NaHCO3
(40 mL). After 15 min, the mixture was filtered through celite,
and rinsed with CH2Cl2. The organic layer was separated,
washed with brine, dried over Na2SO4, and filtered. Solvent was
removed under reduced pressure. Column chromatography
(95 : 5 to 90 : 10 Hexane : EtOAc) of the crude product yielded 4
(2.58 g, 74%) as colourless oil: Rf = 0.65 (70 : 30 Hexane :
EtOAc); [α]D +49.6 (c 0.2, CHCl3); IR ν 3062 (w), 3030 (w),
2866 (m), 1750 (s), 1583 (w), 1497 (w), 1454 (m), 1365 (m),
(m), 1100 (s), 1083 (s), 1028 (s), 739 (s), 698 (s) (cm−1); H
NMR (600 MHz, CDCl3) δ 7.47–7.46 (m, 2H, ArH), 7.44–7.43
(m, 2H, ArH), 7.40–7.39 (m, 2H, ArH), 7.37–7.24 (m, 27H,
ArH), 7.22–7.20 (m, 2H, ArH), 5.63 (d, 1H, J1B,2B = 1.7 Hz,
H-1B), 4.99 (d, 1H, J = 11.2 Hz, PhCH2O), 4.92 (d, 1H, J =
10.9 Hz, PhCH2O), 4.88 (d, 1H, J = 10.8 Hz, PhCH2O), 4.85 (d,
1H, J = 11.5 Hz, PhCH2O), 4.76 (d, 1H, J = 11.4 Hz, PhCH2O),
4.70 (d, 1H, J = 11.4 Hz, PhCH2O), 4.63 (d, 1H, J = 11.8 Hz,
PhCH2O), 4.57 (d, 1H, J = 10.8 Hz, PhCH2O), 4.54 (d, 1H, J =
10.7 Hz, PhCH2O), 4.53 (d, 1H, J = 10.7 Hz, PhCH2O),
4.50–4.44 (m, 4H, PhCH2O, H-2B, H-1C), 4.28–4.25 (m, 1H,
H-5B), 4.15 (t, 1H, J3B,4B ≈ J4B,5B ≈ 9.5 Hz, H-4B), 3.93 (dd,
1H, J2B,3B = 2.9 Hz, J3B,4B = 9.2 Hz, H-3B), 3.83 (dd, 1H,
J5B,6aB = 3.7 Hz, J6aB,6bB = 10.7 Hz, H-6aB), 3.73 (dd, 2H,
J5B,6bB ≈ J5C,6aC ≈ 1.8 Hz, J6bB,6aB ≈ J6aC,6bC ≈ 10.7 Hz,
H-6bB, H-6aC), 3.71–3.67 (m, 2H, H-6bC, H-2C), 3.62 (t, 1H,
J2C,3C ≈ J3C,4C ≈ 8.6 Hz, H-3C), 3.57 (t, 1H, J4C,5C = 8.5 Hz,
H-4C), 3.55–3.52 (m, 1H, H-5C); 13C NMR (125 MHz, CDCl3)
δ 139.0 (Ar), 138.4 (Ar), 138.2 (Ar), 138.16 (Ar), 138.1 (Ar),
137.5 (Ar), 134.1 (Ar), 131.5 (Ar), 129.1 (Ar), 128.6 (Ar), 128.5
(Ar), 128.38 (Ar, 2C), 128.36 (Ar), 128.35 (Ar), 128.3 (Ar),
128.0 (Ar), 127.98 (Ar), 127.97 (Ar), 127.92 (Ar), 127.8 (Ar),
127.72 (Ar), 127.70 (Ar), 127.68 (Ar), 127.66 (Ar), 127.6 (Ar),
127.5 (Ar), 127.47 (Ar), 100.9 (JC–1C,H–1C = 158 Hz, C-1C,),
86.2 (JC–1B,H–1B = 168 Hz, C-1B), 84.2 (C-3C), 78.7 (C-3B),
77.2 (C-4C), 75.7 (C-5C), 75.2 (PhCH2O), 75.1 (PhCH2O), 74.9
(PhCH2O), 74.7 (C-4B), 73.9 (C-2B), 73.64 (C-2C), 73.63
(PhCH2O), 73.4 (PhCH2O), 72.5 (C-5B), 72.2 (PhCH2O), 69.5
(C-6C), 68.8 (C-6B); HRMS [ESI] calcd for C60H62O10SNa
997.3956, found 997.3953.
1
1229 (s), 1071 (s), 1028 (m), 740 (m), 698 (s) (cm−1); H NMR
(500 MHz, CDCl3) δ 7.56–7.54 (m, 2H, ArH), 7.44–7.42 (m,
2H, ArH), 7.35–7.22 (m, 29H, ArH), 7.21–7.20 (m, 2H, ArH),
5.46 (d, 1H, J1B,2B = 1.9 Hz, H-1B), 5.17–5.13 (m, 1H, H-2C),
4.92 (d, 1H, J = 11.0 Hz, PhCH2O), 4.87 (d, 1H, J = 11.5 Hz,
PhCH2O), 4.83 (d, 1H, J = 11.0 Hz, PhCH2O), 4.79 (d, 1H, J =
11.5 Hz, PhCH2O), 4.69 (d, 1H, J = 11.4 Hz, PhCH2O),
4.59–4.45 (m, 8H, PhCH2O, H-1C), 4.38 (t, 1H, J2B,3B
2.6 Hz, H-2B), 4.36–4.33 (m, 1H, H-5B), 3.87 (dd, 1H, J2B,3B
=
=
3.1 Hz, J3B,4B = 8.8 Hz, H-3B), 3.80–3.75 (m, 3H, H-6aC,
H-6aB, H-4B), 3.72–3.65 (m, 4H, H-6bC, H-6bB, H-3C),
3.57–3.54 (m, 1H, H-5C), 1.90 (s, 3H, CH3); 13C NMR
(125 MHz, CDCl3) δ 169.4 (CvO), 138.5 (Ar), 138.48 (Ar),
138.2 (Ar), 138.0 (Ar), 137.98 (Ar), 137.8 (Ar), 134.6 (Ar),
131.7 (Ar), 129.0 (Ar), 128.5 (Ar), 128.4 (Ar, 2C), 128.38 (Ar),
128.34 (Ar), 128.32 (Ar), 128.3 (Ar), 128.2 (Ar), 128.0 (Ar),
127.9 (Ar), 127.88 (Ar), 127.75 (Ar), 127.74 (Ar), 127.69 (Ar),
127.66 (Ar), 127.59 (Ar), 127.56 (Ar), 127.5 (Ar), 127.4 (Ar),
99.7 (JC–1C,H–1C = 158 Hz C-1C), 85.4 (JC–1B,H–1B = 167 Hz,
C-1B), 82.9 (C-4C), 78.2 (C-3B), 77.9 (C-3C), 75.5 (C-2B),
75.3 (C-5C), 75.14 (PhCH2O), 75.11 (PhCH2O), 74.9
(PhCH2O), 75.0 (C-4B), 73.6 (PhCH2O), 72.9 (PhCH2O), 72.8
Phenyl 3,4,6-tri-O-benzyl-β-D-mannopyranosyl-(1 → 2)-3,4,6-
tri-O-benzyl-1-thio-α-D-mannopyranoside (6). Oxalyl chloride
(348 mg, 2.74 mmol) in CH2Cl2 (2.0 mL) was added dropwise
to a solution of 5 (1.34 g, 1.37 mmol) and dimethyl sulfoxide
(428 mg, 5.48 mmol) in CH2Cl2 (50 mL) at −78 °C . The reac-
tion mixture was stirred at −78 °C for 45 min, and triethylamine
(555 mg, 5.48 mmol) was added. Stirring was continued until
the temperature reached 0 °C (approx. 2 h), and the reaction was
quenched with a 1 M aqueous solution of HCl. The mixture was
washed with a saturated solution of NaHCO3, dried over
MgSO4, and filtered. Solvent was removed under reduced
8354 | Org. Biomol. Chem., 2012, 10, 8348–8360
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pressure, and the residue was dried under vacuum pump to
afford the desired ketone as light yellow oil: Rf = 0.44 (70 : 30
Hexane : EtOAc). The crude product of ketone (1.33 g,
1.37 mmol, assumed 100%) was dissolved in THF (50 mL),
cooled to −78 °C, and a 1 M solution of L-selectride in THF
(5.5 mL, 5.48 mmol) was added dropwise. The reaction mixture
was stirred for 50 min at −78 °C, and quenched with methanol.
After 5 min, the mixture was diluted with CH2Cl2, washed with
a 10% aqueous solution of H2O2, a saturated solution of
NaHCO3, H2O, brine, dried over Na2SO4, and filtered. Solvent
was removed under reduced pressure. Column chromatography
(90 : 10 to 85 : 15 Hexane : EtOAc) of the crude product yielded
6 (981 mg, 73% for 2 steps) as a colourless oil: Rf = 0.44
(70 : 30 Hexane : EtOAc); [α]D +9.1 (c 0.5, CHCl3); IR ν 3451
(w, br), 3087 (w), 3062 (m), 3030 (m), 2868 (m), 1584 (w),
1497 (m), 1479 (w), 1454 (m), 1365 (m), 1208 (w), 1100 (s),
layer was separated, washed with brine, dried over Na2SO4, and
filtered. Solvent was removed under reduced pressure. Column
chromatography (80 : 20 Hexane : EtOAc) of the crude product
yielded 7 (669 mg, 71%) as white foam: Rf = 0.45 (98 : 2
CH2Cl2 : EtOAc); [α]D +4.5 (c 0.2, CHCl3); IR ν 3087 (w),
3062 (w), 3030 (w), 2908 (m), 2865 (m), 1748 (m), 1584 (w),
1497 (m), 1454 (m), 1367 (m), 1316 (m), 1232 (s), 1100 (s),
1067 (s), 1028 (s), 737 (s), 698 (s) (cm−1); 1H NMR (500 MHz,
CDCl3) δ 7.49–7.47 (m, 2H, ArH), 7.38–7.36 (m, 4H, ArH),
7.32–7.21 (m, 32H, ArH), 7.20–7.15 (m, 8H, ArH), 7.14–7.12
(m, 2H, ArH), 7.08–7.06 (m, 2H, ArH), 5.50 (d, 1H, J1B,2B
=
1.8 Hz, H-1B), 5.19–5.13 (m, 2H, H-2D, H-1D), 4.98 (d, 1H, J
= 10.5 Hz, PhCH2O), 4.93 (d, 1H, J = 11.3 Hz, PhCH2O), 4.92
(d, 1H, J = 11.9 Hz, PhCH2O), 4.88 (d, 1H, J = 12.0 Hz,
PhCH2O), 4.78 (d, 1H, J = 11.1 Hz, PhCH2O), 4.69–4.45 (m,
12H, PhCH2O, H-1C, H-2B), 4.41 (d, 1H, J = 12.0 Hz,
PhCH2O), 4.37 (d, 1H, J2C,3C = 3.0 Hz, H-2C), 4.29–4.26 (m,
1H, H-5B), 4.08 (t, 1H, J3B,4B = 9.5 Hz, H-4B), 3.86–3.61 (m,
11H, H-3B, H-6aB, H-3D, H-6aC, H-4D, H-5D, H-6bB, H-6aD,
1
1028 (m), 739 (s), 698 (s) (cm−1); H NMR (600 MHz, CDCl3)
δ 7.53–7.51 (m, 2H, ArH), 7.43–7.42 (m, 2H, ArH), 7.41–7.39
(m, 2H, ArH), 7.37–7.26 (m, 27H, ArH), 7.22–7.20 (m, 2H,
ArH), 5.63 (d, 1H, J1B,2B = 1.9 Hz, H-1B), 4.96 (d, 1H, J = 10.9
Hz, PhCH2O), 4.89 (t, 2H, J = 10.4 Hz, PhCH2O), 4.81 (d, 1H,
J = 12.0 Hz, PhCH2O), 4.71 (t, 1H, J2B,3B = 2.5 Hz, H-2B),
4.68–4.64 (m, 3H, PhCH2O, H-1C), 4.61 (d, 1H, J = 10.9 Hz,
PhCH2O), 4.56 (d, 1H, J = 11.2 Hz, PhCH2O), 4.53–4.48 (m,
2H, PhCH2O), 4.46 (d, 1H, PhCH2O), 4.35–4.32 (m, 1H,
H-5B), 4.22 (d, 1H, J2C,1C = 2.9 Hz, H-2C), 4.00–3.98 (m, 2H,
H-4B, H-4C), 3.94 (dd, 1H, J2B,3B = 3.2 Hz, J3B,4B = 9.0 Hz, H-3B),
3.83 (dd, 1H, J5B,6aB = 5.0 Hz, J6aB,6bB = 10.9 Hz, H-6aB), 3.78
(dd, 1H, J5C,6bC = 2.0 Hz, J6bC,6aC = 10.8 Hz, H-6bC), 3.75 (dd,
1H, J5B,6bB = 1.9 Hz, J6bB,6aB = 10.9 Hz, H-6bB), 3.71 (dd, 1H,
J5C,6aC = 5.5 Hz, J6aC,6bC = 10.7 Hz, H-6aC), 3.57 (dd, 1H,
J2C,3C = 3.0 Hz, J3C,4C = 8.9 Hz, H-3C), 3.51–3.48 (m, 1H,
H-5C); 13C NMR (125 MHz, CDCl3) δ 138.4 (Ar), 138.3 (Ar),
138.2 (Ar), 138.17 (Ar), 138.0 (Ar), 137.9 (Ar), 134.3 (Ar),
131.6 (Ar), 129.0 (Ar), 128.5 (Ar), 128.39 (Ar, 2C), 128.38
(Ar), 128.35 (Ar), 128.33 (Ar), 128.32 (Ar), 128.1 (Ar), 128.0
(Ar), 127.96 (Ar), 127.8 (Ar), 127.79 (Ar), 127.74 (Ar), 127.70
(Ar), 127.69 (Ar), 127.6 (Ar), 127.59 (Ar), 127.5 (Ar), 96.6
(JC-1C,H-1C = 161 Hz, C-1C), 85.4 (JC-1B,H-1B = 168 Hz, C-1B),
81.1 (C-3C), 78.5 (C-3B), 75.4 (C-5C), 75.1 (PhCH2O), 75.0
(PhCH2O), 74.5 (C-4B), 74.1 (C-4C), 73.4 (PhCH2O), 73.3
(PhCH2O), 72.5 (C-2B), 72.4 (C-5B), 71.1 (PhCH2O), 70.8
(PhCH2O), 69.6 (C-6C), 69.1 (C-6B), 68.2 (C-2C); HRMS
[ESI] calcd for C60H62O10SNa 997.3956, found 997.3945.
H-6bD, H-6bC, H-4C), 3.50 (dd, 1H, J2C,3C = 3.1 Hz, J3C,4C
=
9.2 Hz, H-3C), 3.48–3.45 (m, 1H, H-5C), 1.88 (s, 3H, CH3);
13C NMR (125 MHz, CDCl3) δ 170.4 (CvO), 138.5 (Ar),
138.47 (Ar), 138.3 (Ar), 138.23 (Ar), 138.15 (Ar), 138.1 (Ar),
138.0 (Ar), 134.2 (Ar), 132.1 (Ar), 129.1 (Ar), 128.4 (Ar),
128.28 (Ar), 128.36 (Ar), 128.33 (Ar), 128.32 (Ar), 128.31 (Ar),
128.30 (Ar), 128.28 (Ar), 128.22 (Ar), 128.19 (Ar), 128.17 (Ar),
128.16 (Ar), 128.0 (Ar), 127.9 (Ar), 127.8 (Ar), 127.74 (Ar),
127.71 (Ar), 127.69 (Ar), 127.60 (Ar), 127.58 (Ar), 127.56 (Ar),
127.54 (Ar), 127.51 (Ar), 127.47 (Ar), 127.46 (Ar), 127.3 (Ar),
100.8 (JC-1D,H-1D = 165 Hz, C-1D), 99.0 (JC-1C,H-1C = 153 Hz,
C-1C), 86.2 (JC-1B,H-1B = 166 Hz, C-1B), 83.7 (C-3D), 80.1
(C-3C), 78.6 (C-3B), 78.2 (C-4D), 76.53 (PhCH2O), 76.51
(C-2B), 75.6 (C-5C), 75.4 (PhCH2O), 75.3 (C-5D), 75.26
(PhCH2O), 74.9 (C-4B), 74.88 (PhCH2O), 74.80 (PhCH2O),
74.78 (C-4C), 74.3 (C-2D), 73.5 (PhCH2O), 73.3 (PhCH2O),
73.2 (PhCH2O), 72.4 (C-5B), 71.7 (C-2C), 70.45 (PhCH2O),
70.39 (C-6C), 69.7 (C-6D), 68.8 (C-6B), 21.1 (COCH3); HRMS
[ESI] calcd for C89H92O16SNa 1471.5998, found 1471.5992.
Phenyl 3,4,6-tri-O-benzyl-β-D-glucopyranosyl-(1 → 2)-3,4,6-
tri-O-benzyl-β-D-mannopyranosyl-(1 → 2)-3,4,6-tri-O-benzyl-1-
thio-α-D-mannopyranoside (8). Sodium metal (14.5 mg,
630 μmol) was added in a portion to a solution of 7 (175 mg,
121 μmol) in tetrahydrofuran (1 mL) and methanol (4 mL). The
reaction mixture was stirred at room temperature for 5 d, neutral-
ized with Amberlite IR-120 (H+) resin and filtered. Solvents
were removed under reduced pressure and the residue was co-
evaporated with toluene. Column chromatography (90 : 10
Hexane : EtOAc) of the crude product yielded 8 (156 mg, 92%)
as a colourless oil: Rf = 0.60 (70 : 30 Hexane : EtOAc); [α]D
−2.8 (c 0.8, CHCl3); IR ν 3530 (w, br), 3087 (w), 3062 (w),
3030 (m), 2866 (m), 1604 (w), 1584 (w), 1497 (m), 1453 (m),
1367 (m), 1315 (w), 1208 (w), 1105 (s), 1068 (s), 1028 (s), 737
Phenyl 2-O-acetyl-3,4,6-tri-O-benzyl-β-D-glucopyranosyl-(1 →
2)-3,4,6-tri-O-benzyl-β-D-mannopyranosyl-(1
→ 2)-3,4,6-tri-O-
benzyl-1-thio-α-D-mannopyranoside (7). A solution of monosac-
charide donor (2)23 (869 mg, 1.70 mmol) and disaccharide
acceptor 6 (635 mg, 0.65 mmol) in CH2Cl2 (10 mL) was stirred
with activated 4 Å molecular sieves (1.0 g) at room temperature
for 30 min. A suspension of silver triflate (873 mg, 3.40 mmol)
in toluene (10 mL) was stirred with activated 4 Å molecular
sieves (1 g) at −40 °C for 30 min. The mixture of donor and
acceptor was added dropwise to the flask containing promoter at
−40 °C over 20 min, and the mixture was stirred for a further
20 min. The reaction mixture was quenched with Et3N (0.3 mL)
and saturated NaHCO3 (20 mL). After 15 min, the mixture was
filtered through celite, and rinsed with CH2Cl2. The organic
1
(s), 698 (s) (cm−1); H NMR (600 MHz, CDCl3) δ 7.49–7.47
(m, 2H, ArH), 7.42–7.40 (m, 2H, ArH), 7.38–7.37 (m, 2H,
ArH), 7.36–7.34 (m, 2H, ArH), 7.29–7.20 (m, 28H, ArH),
7.19–7.12 (m, 12H, ArH), 7.00–6.99 (m, 2H, ArH), 5.62 (d, 1H,
J1B,2B = 1.5 Hz, H-1B), 5.02 (d, 1H, J = 10.9 Hz, PhCH2O),
4.99–4.39 (m, 4H, PhCH2O), 4.86 (d, 1H, J = 10.0 Hz,
This journal is © The Royal Society of Chemistry 2012
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PhCH2O), 4.73 (d, 1H, J = 12.1 Hz, PhCH2O), 4.71 (d, 1H,
J1D,2D = 7.2 Hz, H-1D), 4.63 (dd, 1H, J1B,2B = 1.8 Hz, J2B,3B
J1B,2B = 1.5 Hz, H-1B), 5.17 (s, 1H, H-1D), 4.99 (d, 1H, J =
10.9 Hz, PhCH2O), 4.96 (d, 1H, J = 11.6 Hz, PhCH2O),
4.90–4.88 (m, 2H, PhCH2O), 4.78 (d, 1H, J = 10.4 Hz,
PhCH2O), 4.76–4.75 (m, 1H, H-2B), 4.69 (d, 1H, J = 12.1 Hz,
PhCH2O), 4.66 (s, 1H, H-1C), 4.61–4.39 (m, 10H, H-2C,
PhCH2O), 4.30–4.28 (m, 2H, H-2D, H-5B), 4.27 (d, 1H, J =
10.6 Hz, PhCH2O), 4.24 (d, 1H, J = 10.4 Hz, PhCH2O), 4.06 (d,
1H, J = 11.7 Hz, PhCH2O), 3.99–3.88 (m, 3H, H-4C, H-3B,
H-4D), 3.84–3.68 (m, 6H, H-4B, H-6aB, H-6aC, H-6bC,
=
3.2 Hz, H-2B), 4.61 (d, 1H, J = 11.1 Hz, PhCH2O), 4.54–4.40
(m, 9H, H-1C, PhCH2O), 4.38–4.34 (m, 3H, PhCH2O, H-2C),
4.30 (t, 1H, J3B,4B = 9.6 Hz, H-4B), 4.27–4.24 (m, 1H, H-5B),
3.91 (dd, 1H, J5B,6aB = 4.1 Hz, J6aB,6bB = 10.6 Hz, H-6aB), 3.86
(t, 1H, J3C,4C = 9.6 Hz, H-4C), 3.82–3.77 (m, 2H, H-3B, H-2D),
3.72–3.65 (m, 4H, H-3D, H-6bB, H-6aC, H-6bC), 3.62–3.52
(m, 4H, H-6aD, H-6bD, H-5D, H-4D), 3.48 (dd, 1H, J2C,3C
=
3.2 Hz, J3C,4C = 9.4 Hz, H-3C), 3.44–3.41 (m, 1H, H-5C); 13C
NMR (125 MHz, CDCl3) δ 139.1 (Ar), 138.6 (Ar), 138.5 (Ar),
138.4 (Ar), 138.1 (Ar), 138.05 (Ar), 138.04 (Ar), 138.0 (Ar),
137.8 (Ar), 134.4 (Ar), 131.2 (Ar), 129.1 (Ar), 128.5 (Ar), 128.4
(Ar), 128.3 (Ar), 128.29 (Ar), 128.28 (Ar), 128.27 (Ar), 128.26
(Ar), 128.20 (Ar), 128.19 (Ar), 128.15 (Ar), 128.03 (Ar), 128.0
(Ar), 127.9 (Ar), 127.83 (Ar), 127.80 (Ar), 127.7 (Ar), 127.58
(Ar), 127.57 (Ar), 127.56 (Ar), 127.55 (Ar), 127.53 (Ar), 127.51
H-6aD, H-6bD), 3.64 (dd, 1H, J5B,6bB = 2.0 Hz, J6bB,6aB =
10.6 Hz, H-6bB), 3.60–3.54 (m, 2H, H-3C, H-5D), 3.50–3.46
(m, 2H, H-3D, H-5C), 2.62 (s, br, 1H, OH); 13C NMR
(125 MHz, CDCl3) δ 138.6 (Ar), 138.5 (Ar), 138.2 (Ar), 138.17
(Ar), 138.15 (Ar), 138.12 (Ar), 138.07 (Ar), 138.05 (Ar), 137.7
(Ar), 134.4 (Ar), 131.1 (Ar), 129.1 (Ar), 129.0 (Ar), 128.5 (Ar),
128.4 (Ar), 128.38 (Ar), 128.34 (Ar), 128.32 (Ar), 128.31 (Ar),
128.29 (Ar), 128.26 (Ar), 128.2 (Ar), 128.1 (Ar), 128.0 (Ar),
127.9 (Ar), 127.8 (Ar), 127.74 (Ar), 127.73 (Ar), 127.62 (Ar),
127.59 (Ar), 127.47 (Ar), 127.45 (Ar), 127.42 (Ar), 127.26 (Ar),
100.0 (JC–1D,H–1D = 163 Hz, C-1D), 97.3 (JC–1C,H–1C = 155 Hz,
C-1C), 84.53 (JC–1B,H–1B = 166 Hz, C-1B), 83.1 (C-3D), 79.9
(C-3C), 78.4 (C-3B), 75.6 (C-5C), 75.4 (PhCH2O), 75.3 (C-5D),
75.17 (PhCH2O), 75.12 (PhCH2O), 74.9 (C-4B), 74.3 (C-4D),
74.1 (C-4C), 73.54 (PhCH2O), 73.52 (PhCH2O), 73.4
(PhCH2O), 71.9 (C-5B), 71.6 (C-2B), 71.4 (C-2C), 70.3
(PhCH2O), 70.2 (C-6C), 69.8 (C-6D), 69.7 (PhCH2O), 68.5
(C-6B), 67.4 (C-2D); HRMS [ESI] calcd for C87H90O15SNa
1429.5893, found 1429.5885.
(Ar), 127.46 (Ar), 127.38 (Ar), 127.2 (Ar), 105.3 (JC–1D,H–1D
=
161 Hz, C-1D), 97.5 (JC–1C,H–1C = 155 Hz, C-1C), 86.7 (C-3D),
85.5 (JC–1B,H–1B = 168 Hz, C-1B), 79.5 (C-3C), 78.3 (C-3B),
77.2 (C-4D), 75.6 (PhCH2O), 75.5 (C-5C), 75.4 (C-2D), 75.2
(PhCH2O), 75.0 (C-5D), 74.96 (PhCH2O), 74.95 (PhCH2O),
74.9 (C-2C), 74.3 (C-4C), 74.0 (C-4B), 73.7 (PhCH2O), 73.4
(PhCH2O), 73.35 (PhCH2O), 73.1 (C-2B), 72.2 (C-5B), 70.5
(PhCH2O), 69.9 (C-6D), 69.8 (PhCH2O), 69.7 (C-6C), 68.9
(C-6B); HRMS [ESI] calcd for C87H90O15SNa 1429.5893,
found 1429.5889.
Phenyl 3,4,6-tri-O-benzyl-β-D-mannopyranosyl-(1 → 2)-3,4,6-
tri-O-benzyl-β-D-mannopyranosyl-(1 → 2)-3,4,6-tri-O-benzyl-1-
thio-α-D-mannopyranoside (9). Solution of oxalyl chloride
(32 μL, 382 μmol) in CH2Cl2 (1.0 mL) was added dropwise to a
solution of 8 (269 mg, 191 μmol) and dimethyl sulfoxide
(54.0 μL, 764 μmol) in CH2Cl2 (20 mL), at −78 °C a. The reac-
tion mixture was stirred at −78 °C for 40 min, and triethylamine
(106 μL, 764 μmol) was added. Stirring was continued until
temperature reached 0 °C (approx. 1.5 h), and the reaction was
quenched with a 1 M aqueous solution of HCl. The mixture was
washed with a saturated solution of NaHCO3, dried over
MgSO4, and filtered. Solvent was removed under reduced
pressure, and the residue was dried under vacuum to afford the
desired ketone as a light yellow oil: Rf = 0.28 (70 : 30 Hexane :
EtOAc). The crude product of ketone (268 mg, 191 μmol) was
dissolved in THF (24 mL), cooled to −78 °C, and a 1 M solution
of L-selectride in THF (400 μL, 764 μmol) was added dropwise.
The reaction mixture was stirred for 30 min at −78 °C and
quenched with methanol. After 5 min, the mixture was diluted
with CH2Cl2, washed with a 10% aqueous solution of H2O2, a
saturated solution of NaHCO3, H2O, brine, dried over Na2SO4,
and filtered. Solvent was removed under reduced pressure.
Column chromatography (85 : 15 to 80 : 20 Hexane : EtOAc) of
the crude product yielded 9 (176 mg, 65% for 2 steps) as a col-
ourless oil: Rf = 0.28 (30 : 70 EtOAc : hexanes); [α]D −10.5 (c
0.2, CHCl3); IR ν 3516 (w, br), 3087 (w), 3062 (m), 3030 (m),
2905 (m), 2864 (s), 1605 (w), 1584 (w), 1497 (m), 1453 (s),
1366 (m), 1316 (m), 1208 (m), 1100 (s), 1028 (s), 737 (s), 698
Phenyl 2-O-acetyl-3,4,6-tri-O-benzyl-β-D-mannopyranosyl-(1
→ 2)-3,4,6-tri-O-benzyl-β-D-mannopyranosyl-(1 → 2)-3,4,6-tri-
O-benzyl-1-thio-α-D-mannopyranoside (10). A mixture of 9
(38.8 mg, 27.6 μmol), triethylamine (80 μL, 577 μmol),
4-dimethylaminopyridine (4.01 mg, 32.7 μmol), and acetic
anhydride (26 μL, 275 μmol) in CH2Cl2 (1.0 mL) was stirred at
room temperature for 22 h. The reaction mixture was quenched
with MeOH and solvents were removed under reduced pressure.
Column chromatography (99 : 1 CH2Cl2 : EtOAc) of the crude
product yielded 10 (33.3 mg, 83%) as a colourless oil: Rf = 0.48
(98 : 2 CH2Cl2 : EtOAc); [α]D −7.0 (c 1.5, CHCl3); IR ν 3088
(w), 3062 (w), 3030 (m), 2925 (m), 2863 (m), 1744 (s), 1604
(w), 1584 (w), 1496 (m), 1454 (s), 1367 (s), 1316 (m), 1238 (s),
1
1102 (s), 1027 (s), 739 (s), 698 (s) (cm−1); H NMR (600 MHz,
CDCl3) δ 7.55–7.54 (m, 2H, ArH), 7.51–7.50 (m, 2H, ArH),
7.44–7.42 (m, 2H, ArH), 7.39–7.36 (m, 2H, ArH), 7.35–7.12
(m, 38H, ArH), 7.10–7.09 (m, 2H, ArH), 7.03–7.01 (m, 2H,
ArH), 5.73 (d, 1H, J1D,2D = 3.2 Hz, H-2D), 5.68 (d, 1H, J1B,2B
=
1.5 Hz, H-1B), 5.52 (s, 1H, H-1D), 4.97 (d, 1H, J = 10.7 Hz,
PhCH2O), 4.92–4.86 (m, 3H, PhCH2O), 4.82 (d, 1H, J = 10.5
Hz, PhCH2O), 4.78–4.77 (m, 1H, H-2B), 4.70 (s, 1H, H-1C),
4.69 (d, 1H, J2C,3C = 3.0 Hz, H-2C), 4.65 (d, 1H, J = 12.2 Hz,
PhCH2O), 4.60 (d, 1H, J = 12.0 Hz, PhCH2O), 4.55 (d, 1H, J =
11.0 Hz, PhCH2O), 4.54 (d, 1H, J = 10.9 Hz, PhCH2O),
4.51–4.43 (m, 5H, PhCH2O), 4.39 (d, 1H, J = 12.2 Hz,
PhCH2O), 4.38 (d, 1H, J = 11.3 Hz, PhCH2O), 4.31 (d, 1H, J =
10.5 Hz, PhCH2O), 4.30–4.28 (m, 1H, H-5B), 3.98 (d, 1H, J =
1
(s) (cm−1); H NMR (600 MHz, CDCl3) δ 7.51–7.47 (m, 4H,
11.2 Hz, PhCH2O), 3.94 (dd, 1H, J2B,3B = 3.4 Hz, J3B,4B =
ArH), 7.41–7.39 (m, 2H, ArH), 7.32–7.12 (m, 40H, ArH),
7.10–7.08 (m, 2H, ArH), 6.99–6.97 (m, 2H, ArH), 5.68 (d, 1H,
9.1 Hz, H-3B), 3.89 (t, 1H, J3D,4D ≈ J4D,5D ≈ 9.5 Hz, H-4D),
3.86 (t, 1H, J3,4 ≈ J4,5 ≈ 9.4 Hz, H-4B), 3.80–3.78 (m, 4H,
8356 | Org. Biomol. Chem., 2012, 10, 8348–8360
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H-6aB, H-6bC, H-6aD, H-6bD), 3.74 (t, 1H, J3C,4C ≈ J4C,5C
≈
138.73 (Ar), 138.71 (Ar), 138.45 (Ar), 138.42 (Ar), 138.4 (Ar),
138.37 (Ar), 138.34 (Ar), 138.0 (Ar), 137.77 (Ar), 128.75 (Ar),
128.8 (Ar), 128.5 (Ar), 128.4 (Ar), 128.36 (Ar), 128.33 (Ar),
128.3 (Ar), 128.23 (Ar), 128.22 (Ar), 128.2 (Ar), 128.1 (Ar),
128.0 (Ar), 127.98 (Ar), 127.9 (Ar), 127.8 (Ar), 127.69 (Ar),
127.67 (Ar), 127.56 (Ar), 127.5 (Ar), 127.46 (Ar), 127.45 (Ar),
127.36 (Ar), 98.2 (JC-1C,H-1C = 155 Hz, C-1C), 97.3 (JC-1D,H-1D
= 164 Hz, C-1D), 92.3 (JC-1B,H-1B = 170 Hz, C-1B), 80.9
(C-3D), 80.3 (C-3C), 77.5 (C-3B), 75.65 (C-5B), 75.6 (C-5D),
75.3 (PhCH2O), 75.1 (PhCH2O), 74.9 (PhCH2O), 74.89 (C-4D),
74.6 (C-4C), 74.58 (C-4B), 73.6 (PhCH2O), 73.5 (PhCH2O),
73.4 (PhCH2O), 70.93 (PhCH2O), 70.92 (C-5C), 70.3
(PhCH2O), 70.2 (C-6B), 70.18 (C-2B), 69.6 (PhCH2O), 69.3
(C-2D), 69.0 (C-6C), 68.9 (C-2C), 68.2 (C-2D), 21.1 (COCH3);
HRMS [ESI] calcd for C83H88O17Na 1379.5914, found
1379.5900.
9.4 Hz, H-4C), 3.69 (dd, 1H, J5C,6aC = 6.7 Hz, J6aC,6bC = 10.8
Hz, H-6aC), 3.66–3.63 (m, 1H, H-5D), 3.61 (dd, 1H, J5B,6aB
2.0 Hz, J6bB,6aB = 10.7 Hz, H-6bB), 3.59 (dd, 1H, J2C,3C
=
=
2.5 Hz, J3C,4C = 9.1 Hz, H-3C), 3.54–3.50 (m, 2H, H-5C,
H-3D), 2.03 (s, 3H, COCH3); 13C NMR (125 MHz, CDCl3) δ
170.8 (CvO), 140.1 (Ar), 138.7 (Ar), 138.4 (Ar), 138.39 (Ar),
138.3 (Ar), 138.26 (Ar), 138.2 (Ar), 137.74 (Ar), 137.72 (Ar),
134.3 (Ar), 131.3 (Ar), 129.1 (Ar), 128.8 (Ar), 128.6 (Ar),
128.38 (Ar), 128.33 (Ar), 128.32 (Ar), 128.3 (Ar), 128.25 (Ar),
128.24 (Ar), 128.2 (Ar), 128.18 (Ar), 128.15 (Ar), 127.9 (Ar),
127.89 (Ar), 127.82 (Ar), 127.7 (Ar), 127.66 (Ar), 127.53 (Ar),
127.5 (Ar), 127.46 (Ar), 127.43 (Ar), 127.3 (Ar), 127.1 (Ar),
124.5 (Ar), 121.3 (Ar), 112.0 (Ar), 97.51 (JC-1D,H-1D = 164 Hz,
C-1D), 97.46 (JC-1C,H-1C = 154 Hz, C-1C), 84.4 (JC-1B,H-1B
=
168 Hz, C-1B), 80.9 (C-3D), 80.2 (C-3C), 78.4 (C-3B), 75.7
(C-5C), 75.68 (C-5D), 75.5 (PhCH2O), 75.1 (PhCH2O), 75.0
(PhCH2O), 74.9 (C-4B), 74.56 (C-4D), 74.53 (C-4C), 73.6
(PhCH2O), 73.45 (PhCH2O), 73.41 (PhCH2O), 71.9 (C-5B),
71.5 (C-2B), 71.0 (PhCH2O), 70.3 (PhCH2O), 70.29 (PhCH2O),
69.6 (C-6C), 69.4 (C-6D), 69.1 (C-2C), 68.5 (C-6B), 68.2
(C-2D), 21.1 (COCH3); HRMS [ESI] calcd for C89H92O16SNa
1471.5998, found 1471.5997.
Triethylammonium 2-O-acetyl-3,4,6-tri-O-benzyl-β-D-manno-
pyranosyl-(1 → 2)-3,4,6-tri-O-benzyl-β-D-mannopyranosyl-(1 →
2)-3,4,6-tri-O-benzyl-α-D-mannopyranosyl hydrogenphosphonate
(12). A solution of acetonitrile (4.0 mL), imidazole (24.8 mg,
364 μmol), and triethylamine (50 μL, 360 μmol) was cooled to
0 °C and phosphorus trichloride (26 μL, 299 μmol) was added.
The cloudy resulting mixture was stirred at 0 °C for 15 min. The
solution of 10 (α : β 6 : 1) (80.5 mg, 59.3 μmol) in acetonitrile
(4.0 mL) was added dropwise at 0 °C for 30 min, then stirred at
RT for further 30 min. Triethylammonium bicarbonate buffer
(TEAB) (1.0 M, pH 8.0–8.5) (1.0 mL) was added and stirred for
15 min to give a clear solution. This solution was co-evaporated
with a mixture of pyridine : Et3N 4 : 1 (2 × 5.0 mL) to give a
white solid which was then dissolved in CHCl3 and washed with
cold solution of 0.5 M TEAB (4 × 10 mL). The organic phase
was filtered through a plug of tissue paper and concentrated.
Column chromatography (92 : 7 : 1 to 90 : 9 : 1 EtOAc : MeOH :
Et3N) of the crude product yielded mixture of H-phosphonate 12
[α : β 33 : 1 δP 0.76 (Pα) and 1.25 (Pβ)] (48.8 mg, 54%, 62%
based on recovery of starting material) as a colorless semi-solid:
Rf = 0.20 (92 : 7 : 1 CH2Cl2 : MeOH : Et3N); [α]D −32.3 (c 2.8,
CHCl3); spectroscopy data of α-anomer was reported: IR ν 3088
(w), 3062 (m), 3030 (m), 2928 (s), 2865 (s), 2606 (w), 2464
(w), 2355 (w), 1744 (s), 1604 (w), 1585 (w), 1497 (m), 1454
(s), 1397 (m), 1366 (s), 1238 (s), 1100 (s, br), 1028 (s), 955 (s),
2-O-Acetyl-3,4,6-tri-O-benzyl-β-D-mannopyranosyl-(1
→ 2)-
3,4,6-tri-O-benzyl-β-D-mannopyranosyl-(1 2)-3,4,6-tri-O-
→
benzyl-α,β-D-mannopyranose (11). N-iodosuccinimide (104 mg,
42.6 μmol) was added in one portion to a solution of 10
(440 mg, 304 μmol) in acetone (8.0 mL) and water (80 μL). The
yellow resulting reaction mixture was stirred at RT for 5 h. The
reaction was quenched with 1 M solution of Na2S2O3 (10 mL)
and extracted with CH2Cl2. The organic layer was washed with
H2O, brine, dried over MgSO4, and filtered. Solvent was
removed under reduced pressure. Column chromatography
(96 : 4 to 80 : 20 toluene : acetone) of the crude product yielded
mixture of 11 (α : β 6 : 1) (337 mg, 82%, 87% based on recovery
of starting material) as a colourless foam: Rf = 0.18 (9 : 1
toluene : acetone); IR ν 3377 (w, br), 3088 (w), 3063 (m), 3030
(m), 2907 (m), 2864 (m), 1744 (s), 1605 (w), 1586 (w), 1497
(m), 1454 (s), 1398 (w), 1366 (s), 1315 (m), 1239 (s), 1102 (s),
1064 (s), 1028 (s), 915 (w), 736 (s), 698 (s) (cm−1); Isomer α:
1H NMR (600 MHz, CDCl3) δ 7.51–7.50 (m, 2H, ArH),
7.42–7.40 (m, 2H, ArH), 7.34–7.09 (m, 37H, ArH), 7.07–7.04
1
837 (m), 796 (m), 751 (s), 699 (s) (cm−1); H NMR (600 MHz,
(m, 2H, ArH), 6.99–6.98 (m, 2H, ArH), 5.68 (d, 1H, J2D,3D
=
CDCl3) δ 7.52–7.51 (m, 2H, ArH), 7.43–7.41 (m, 2H, ArH),
7.33–7.16 (m, 29H, ArH), 7.15–7.10 (m, 8H, ArH), 7.06–7.05
(m, 2H, ArH), 7.00–6.99 (m, 2H, ArH), 6.42 (s, 1H, H-phospho-
nate), 5.77 (dd, 1H, J1B,2B = 1.3 Hz, JH–1B,P = 8.3 Hz, H-1B),
5.72 (d, 1H, J2D,3D = 3.3 Hz, H-2D), 5.54 (s, 1H, H-1D), 4.96
(d, 1H, J = 10.8 Hz, PhCH2O), 4.89 (d, 1H, J = 10.0 Hz,
PhCH2O), 4.87 (d, 1H, J = 11.3 Hz, PhCH2O), 4.84 (d, 1H, J =
10.9 Hz, PhCH2O), 4.82 (d, 1H, J = 10.8 Hz, PhCH2O), 4.74 (s,
1H, H-1C), 4.68 (d, 1H, J2C,3C = 2.9 Hz, H-2C), 4.61 (d, 1H, J
= 12.0 Hz, PhCH2O), 4.60 (d, 1H, J = 12.5 Hz, PhCH2O),
4.53–4.51 (m, 3H, H-2B, PhCH2O), 4.44–4.37 (m, 6H,
PhCH2O), 4.34 (d, 1H, J = 11.1 Hz, PhCH2O), 4.30 (d, 1H, J =
3.1 Hz, H-2D), 5.49 (s, 1H, H-1D), 5.36–5.35 (m, 1H, H1-B),
4.93 (d, 1H, J = 10.8 Hz, PhCH2O), 4.88–4.83 (m, 4H,
PhCH2O), 4.80 (d, 1H, J = 10.6 Hz, PhCH2O), 4.67 (d, 1H,
J2C,3C = 3.1 Hz, H-2C), 4.65 (s, 1H, H-1C), 4.58 (d, 1H, J =
11.9 Hz, PhCH2O), 4.57 (d, 1H, J = 12.4 Hz, PhCH2O),
4.54–4.53 (m, 1H, H-2B), 4.51 (d, 1H, J = 11.0 Hz, PhCH2O),
4.47–4.41 (m, 6H, PhCH2O), 4.33 (d, 1H, J = 11.1 Hz,
PhCH2O), 4.28 (d, 1H, J = 10.6 Hz, PhCH2O), 4.00 (dd, 1H,
J2B,3B = 3.4 Hz, J3B,4B = 9.7 Hz, H-3B), 3.98–3.96 (m, 1H,
H-5C), 3.93 (d, 1H, J = 11.3 Hz, PhCH2O), 3.82 (t, 1H, J3D,4D
≈ J4D,5D ≈ 9.5 Hz, H-4D), 3.78–3.76 (m, 3H, H-6abB, H-6aD),
3.70–3.64 (m, 4H, H-4B, H-4C, H-6bD, H6aC), 3.60–3.56 (m,
3H, H-5D, H-6bC, H-3C), 3.52–3.49 (m, 1H, H-5B), 3.46 (dd,
J2D,3D = 3.4 Hz, J3D,4D = 9.3 Hz), 2.68 (s, br, 1H, OH), 1.99 (s,
3H, COCH3); 13C NMR (125 MHz, CDCl3) δ 170.8 (CvO),
10.7 Hz, PhCH2O), 4.07 (dd, 1H, J2B,3B = 3.5 Hz, J3B,4B
=
9.4 Hz, H-3B), 4.01–3.98 (m, 1H, H-5B), 3.94 (d, 1H, J =
11.3 Hz, PhCH2O), 3.88 (t, 1H, J3D,4D ≈ J4D,5D ≈ 9.5 Hz,
H-4D), 3.81–3.65 (m, 7H, H-4B, H-6aD, H-6bD, H-6aB, H-4C,
This journal is © The Royal Society of Chemistry 2012
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mannopyranosyl)]-6-(triethylammonium phosphate (16). H-
phosphonate 12 (α : β 33 : 1) (48.8 mg, 32.0 μmol) and monosac-
charide 13 (10.3 mg, 32.2 μmol) were co-evaporated with pyri-
dine (4 × 2.0 mL) and dried under vacuum for 48 h. The residue
was dissolved in pyridine (2.0 mL) and pivaloyl chloride
(12.0 μL, 97.0 μmol) was added. The reaction mixture was
stirred at RT for 1 h, then it was cooled to −40 °C and the
freshly prepared solution of iodine (8.93 mg, 35.2 μmol) in
pyridine : water (95 : 5, 1.0 mL) was added. The resulting yellow
reaction mixture was stirred at −40 °C to 0 °C for 1 h. When the
temperature reached 0 °C, the reaction mixture was diluted with
CH2Cl2, washed with 1 M solution of Na2S2O3 (10 mL), water
(2 × 10 mL), and brine. The organic layer was dried over
Na2SO4 and filtered. Solvent was removed under reduced
pressure. Column chromatography (92 : 7 : 1 to 91 : 8 : 1 EtOAc :
MeOH : Et3N) of the crude product yielded mixture of 16 (α : β
50 : 1, δP −2.19 (Pα) and −2.54 (Pβ)] (39.4 mg, 67%) as color-
less solid: Rf = 0.11 (91 : 9 : 1 EtOAc : MeOH : Et3N); [α]D
−16.9 (c 0.4, CHCl3); spectroscopy data of α-anomer was
reported: IR ν 3385 (w, br), 3080 (w), 3062 (m), 3030 (m), 2933
(s), 2867 (s), 2739 (m), 2677 (m), 2624 (m), 2604 (m), 2493
(m), 1747 (s), 1605 (w), 1586 (w), 1497 (m), 1454 (s), 1398
(m), 1368 (s), 1244 (s), 1090 (s, br), 1028 (s), 871 (m), 799 (m),
741 (s), 698 (s) (cm−1); 1H NMR (600 MHz, CDCl3) δ
7.53–7.52 (m, 2H, ArH), 7.42–7.41 (m, 2H, ArH), 7.33–7.16
(m, 29H, ArH), 7.15–7.08 (m, 8H, ArH), 7.05–7.03 (m, 2H,
ArH), 6.96–6.95 (m, 2H, ArH), 5.73 (m, 2H, H-2D, H-1B), 5.54
(s, 1H, H-1D), 5.28 (dd, 1H, J2A,3A = 3.0 Hz, J3A,4A = 10.0 Hz,
H-3A), 5.19 (dd, 1H, J1A,2A = 1.7 Hz, J2A,3A = 3.5 Hz, H-2A),
5.15 (t, 1H, J3A,4A = 10.0 Hz, H-4A), 4.97 (d, 1H, J = 10.6 Hz,
PhCH2O), 4.88 (d, 1H, J = 9.7 Hz, PhCH2O), 4.87 (d, 1H, J =
10.6 Hz, PhCH2O), 4.83 (d, 1H, J = 11.0 Hz, PhCH2O), 4.80 (d,
1H, J = 10.6 Hz, PhCH2O), 4.73 (s, 1H, H-1C), 4.65 (d, 1H,
J2C,3C = 2.9 Hz, H-2C), 4.62 (d, 1H, J = 12.6 Hz, PhCH2O),
H-6aC, H-6bC), 3.63–3.60 (m, 1H, H-5D), 3.60–3.56 (m, 2H,
H-3C, H-6bB), 3.54–3.50 (m, 2H, H-5C, H-3D), 2.97–2.93 (m,
6H, CH2CH3), 2.00 (s, 3H, COCH3), 1.26 (t, 9H, J = 7.3 Hz,
CH3); 13C NMR (125 MHz, CDCl3) δ 170.8 (CvO), 138.9
(Ar), 138.8 (Ar), 138.6 (Ar), 138.54 (Ar), 138.51 (Ar), 138.49
(Ar), 138.43 (Ar), 138.0 (Ar), 137.8 (Ar), 128.7 (Ar), 128.5
(Ar), 128.4 (Ar), 128.33 (Ar), 128.31 (Ar), 128.30 (Ar), 128.25
(Ar), 128.24 (Ar), 128.2 (Ar), 128.16 (Ar), 128.15 (Ar), 128.12
(Ar), 128.11 (Ar), 128.0 (Ar), 127.8 (Ar), 127.7 (Ar), 127.65
(Ar), 127.62 (Ar), 127.54 (Ar), 127.51 (Ar), 127.4 (Ar), 127.38
(Ar), 127.37 (Ar), 127.34 (Ar), 127.3 (Ar), 127.28 (Ar), 127.24
(Ar), 98.3 (JC-1C,H-1C = 154 Hz, C-1C), 97.3 (JC-1D,H-1D = 164
Hz, C-1D), 92.50–92.46 (m, JC-1B,H-1B = 178 Hz, C-1B), 81.0
(C-3D), 80.3 (C-3C), 77.6 (C-3B), 76.5 (C-5B), 75.6 (C-5D),
75.5 (C-5C), 75.2 (PhCH2O), 75.1 (PhCH2O), 74.9 (PhCH2O),
74.7 (C-4B), 74.6 (C-4C), 74.5 (C-4D), 73.6 (PhCH2O), 73.4
(PhCH2O), 73.37 (PhCH2O), 71.9 (C-2B), 70.9 (PhCH2O), 70.6
(C-6C), 70.0 (PhCH2O), 69.54 (C-6D), 69.4 (PhCH2O), 69.1
(C-2C), 68.7 (C-6B), 68.3 (C-2D), 45.5 (CH2CH3), 21.1
(COCH3), 8.6 (CH2CH3); 31P NMR (201 MHz) δ 0.76; HRMS
[ESI] calcd for C83H89O19P 1419.5663 (M-Et3NH)−, found
1419.5657.
Methyl 2,3,4-tri-O-acetyl-α-D-mannopyranoside (15).30 Trityl
chloride (3.64 g, 13.06 mmol) was added to a suspension of
methyl-α-D-mannopyranoside (2.29 g, 11.79 mmol) in pyridine
(35 mL) at 0 °C. The reaction was stirred at RT and 40 °C for 2
days. The reaction was cooled to 0 °C and acetic anhydride
(15 mL) was added dropwise. The resulting reaction mixture was
stirred at RT for further 2 days. Solvent and reagent was evapor-
ated under reduced pressure. Column chromatography (80 :
20 hexanes : EtOAc) of the crude product gave protected com-
pound 14 (5.52 g, 83%).
Compound 14 (157.3 mg, 0.280 mmol) was dissolved in
CH2Cl2–methanol (9 : 1, 2 mL) and iodine monochloride (1 M
in CH2Cl2) (0.60 mL, 0.60 mmol) was added. The reaction
mixture was stirred at RT for 5 h, diluted with CH2Cl2, washed
with 1 M aqueous solution of Na2S2O3 (10 mL), saturated
NaHCO3, brine, and dried over MgSO4. Solvent was removed
under reduced pressure. Column chromatography (1 : 1 EtOAc :
hexanes) of the crude product yielded 15 (67.8 mg, 76%) as a
colourless oily solid: Rf = 0.15 (50 : 50 EtOAc : hexanes); [α]D
+45 (c 0.22, CHCl3). Lit.30 [α]D +50 (c 1.0, CHCl3); IR ν 3512
(w, br), 2939 (w), 2841 (w), 1753 (s), 1434 (w), 1371 (s), 1227
4.59 (d, 1H, J = 12.0 Hz, PhCH2O), 4.57 (d, 1H, J1A,2A
=
1.4 Hz, H-1A), 4.53–4.50 (m, 3H, H-2B, PhCH2O), 4.44–4.33
(m, 7H, PhCH2O), 4.28 (d, 1H, J = 10.6 Hz, PhCH2O), 4.04
(dd, 1H, J2B,3B = 3.4 Hz, J3B,4B = 9.4 Hz, H-3B), 4.02–3.92 (m,
5H, H-6aA, H-6bA, H-5B, H-5A, PhCH2O), 3.88 (t, 1H, J3D,4D
≈ J4D,5D ≈ 9.5 Hz, H-4D), 3.82 (t, 1H, J3B,4B ≈ J4B,5B ≈ 9.7 Hz,
H-4B), 3.77–3.70 (m, 5H, H-6aD, H-6bD, H-6aC, H-6aB,
H-4C), 3.67–3.64 (m, 1H, H-6bC), 3.62–3.61 (m, 1H, H-5D),
3.58–3.55 (m, 2H, H-3C, H-6bB), 3.53–3.49 (m, 2H, H-3D,
H-5C), 3.28 (s, 3H, OCH3), 2.96–2.92 (m, 6H, CH2CH3), 2.03
(s, 3H, COCH3), 2.00 (s, 3H, COCH3), 1.96 (s, 3H, COCH3),
1.93 (s, 3H, COCH3), 1.26 (t, 9H, J = 7.3 Hz, CH3); 13C NMR
(125 MHz, CDCl3) δ 170.7 (CvO), 170.1 (CvO), 169.9
(CvO), 169.88 (CvO), 139.0 (Ar), 138.8 (Ar), 138.7 (Ar),
138.6 (Ar), 138.5 (2C, Ar), 138.46 (Ar), 138.0 (Ar), 137.8 (Ar),
128.7 (Ar), 128.4 (Ar), 128.36 (Ar), 128.32 (Ar), 128.30 (Ar),
128.29 (Ar), 128.26 (Ar), 128.24 (Ar), 128.23 (Ar), 128.16 (Ar),
128.14 (Ar), 128.1 (Ar), 128.06 (Ar), 127.9 (Ar), 127.8 (Ar),
127.7 (Ar), 127.66 (Ar), 127.63 (Ar), 127.61 (Ar), 127.55 (Ar),
127.53 (Ar), 127.4 (Ar), 127.37 (Ar), 127.35 (Ar), 127.33 (Ar),
127.25 (Ar), 127.21 (Ar), 98.4 (JC-1A,H-1A = 174 Hz, C-1A),
98.2 (JC-1C,H-1C = 157 Hz, C-1C), 97.4 (JC-1D,H-1D = 163 Hz,
C-1D), 93.4–93.36 (m, JC-1B,H-1B = 176 Hz, C-1B), 81.0
(C-3D), 80.3 (C-3C), 77.7 (C-3B), 76.5 (C-5D), 75.6 (C-5C),
75.5 (C-4D), 75.2 (PhCH2O), 75.1 (PhCH2O), 74.9 (PhCH2O),
1
(s), 1137 (s), 1087 (s), 1064 (s), 937 (m), 757 (w) (cm−1); H
NMR (600 MHz, CDCl3) δ 5.40 (dd, 1H, J2,3 = 3.5 Hz, J3,4
10.2 Hz, H-3), 5.27–5.23 (m, 2H, H-2, H-4), 4.73 (d, 1H, J1,2
=
=
1.7 Hz, H-1), 3.78–3.70 (m, 2H, H-5, H-6a), 3.66–3.62 (m, 1H,
H-6b), 3.41 (s, 3H, OCH3), 2.36 (dd, 1H, JOH,6a = 5.6 Hz,
JOH,6b = 8.6 Hz, OH), 2.15 (s, 3H, COCH3), 2.08 (s, 3H,
COCH3), 2.01 (s, 3H, COCH3); 13C NMR (125 MHz, CDCl3) δ
170.8 (CvO); 170.1 (CvO), 169.9 (CvO), 98.6 (C-1), 70.5
(C-5), 69.6 (C-2), 68.9 (C-3), 66.5 (C-4), 61.3 (C-6), 55.3
(OCH3), 20.9 (COCH3), 20.73 (COCH3), 20.70 (COCH3); MS
[ESI] calcd for C13H20O9Na 343.1 (M + Na)+, found 343.1.
Methyl 2,3,4-tri-O-acetyl-α-D-mannopyranoside [(2-O-acetyl
3,4,6-tri-O-benzyl-β-D-mannopyranosyl) (1 → 2) (3,4,6-tri-O-
benzyl-β-D-mannopyranosyl) (1 → 2) (3,4,6-tri-O-benzyl-α-D-
8358 | Org. Biomol. Chem., 2012, 10, 8348–8360
This journal is © The Royal Society of Chemistry 2012

View Article Online
74.7 (C-4C), 74.5 (C-4B), 74.4 (C-5B), 73.6 (PhCH2O), 73.5
(PhCH2O), 73.4 (PhCH2O), 71.8 (C-2B), 70.9 (PhCH2O), 70.6
(C-6C), 70.0 (PhCH2O), 69.8 (C-5A), 69.7 (C-2A), 69.55
(C-6D), 69.5 (C-2C), 69.3 (PhCH2O), 69.2 (C-3A), 68.6 (C-6B),
68.3 (C-2D), 66.6 (C-4A), 64.7–64.6 (m, C-6A), 55.1 (OCH3),
45.5 (CH2CH3), 21.1 (COCH3), 20.85 (COCH3), 20.83
(COCH3), 20.7 (COCH3), 8.6 (CH2CH3); 31P NMR (201 MHz)
δ −2.19; HRMS [ESI] calcd for C96H106O28P 1737.6614
(M − H)−, found 1737.6604.
= 5.2 Hz, C-6A), 62.1 (C-6D), 61.6 (C-6C), 61.0 (C-6B), 55.8
(OCH3), 47.6 (CH2CH3), 9.2 (CH2CH3); 31P NMR (201 MHz) δ
−1.68; HRMS [ESI] calcd for C25H44O24P 759.1966 (M − H)−,
found 759.1968.
Oligosaccharide inhibition
A solution (100 μL) of synthetic β-mannan trisaccharide-BSA
conjugate13b in PBS (5 μg mL−1) was added to wells of an
ELISA plate (Maxisorb, NUNC) and left overnight at 4 °C.
Serial dilutions of monoclonal antibody C3.1 in PBS (100 μL)
were added to successive wells and a titration curve was con-
structed. The antibody dilution in the linear portion of the titra-
tion curve giving an OD of 1.5 in the absence of inhibitor was
identified and used for all inhibition experiments.
In a separate 96 well plate (Corning 3641 – Non-Binding
Surface, Flat Bottom) a solution of inhibitor (2 mg mL−1) in
PBS was diluted in serial √0.1 steps. The effective range of
inhibitor concentrations was 0.1 μM to 1 mM. Equal volumes of
inhibitor solution (175 μL) and C3.1 antibody solution (175 μL)
were mixed. The antigen coated ELISA plate was washed 5
times with PBST (PBS containing Tween 20, 0.05% v/v) and ali-
quots of inhibitor-antibody solution (100 μL) were added in tri-
plicate. Antibody solution diluted with an equal volume of
PBST was used as the reference to calculate percent inhibition.
Background controls used wells filled with PBST.
The mixture of antibody-inhibitor was incubated in the
antigen coated ELISA plate for 2 h at RT, then washed 5 times
with PBST. Peroxidase labelled goat anti mouse IgG (Kirkegaard
& Perry Laboratories Inc.) was diluted (1 : 5000 from 1 mg
mL−1) in PBST containing 0.1% BSA and this solution (100 μL)
was added to each well. After 30 min incubation at RT the plate
was washed 5 times with PBST and colour was developed by
addition of 3,3′,5,5′-tetramethylbenzidine (TMB, 100 μL, Kirke-
gaard and Perry Lab). Colour development was stopped by the
addition of 1 M phosphoric acid (100 μL). Absorbance was read
at 450 nm.
Methyl-α-D-mannopyranoside [β-D-mannopyranosyl-(1 → 2)-
β-D-mannopyranosyl-(1 → 2)-α-D-mannopyranosyl)]-6-triethyl-
ammonium phosphate (1). A mixture of phosphodiester 16
(α : β 50 : 1, 38.3 mg, 20.8 μmol) in 0.03 M solution of NaOMe
(11.5 mg Na/15 mL of MeOH) was stirred at RT until the start-
ing material was consumed completely (84 h). The reaction
mixture was neutralized with Amberlite IR120 (H+) and filtered
to the flask containing Et3N (2.0 mL). Solvents were removed
under reduced pressure. Column chromatography (90 : 9 : 1 to
88 : 12 : 1 EtOAc : MeOH : Et3N) of the crude product yielded
α-anomer 17 (32.8 mg, 94%) as colourless semi-solid: Rf = 0.15
(80 : 20 : 1 EtOAc : MeOH : Et3N). This intermediate was dis-
solved in the mixture of solvents MeOH : CH2Cl2 : H2O 4 : 4 : 1
(2.7 mL) and the flask was purged with H2 for 5 min before
adding Pd(OH)2/C (50.1 mg) in one portion. The reaction
mixture was stirred under H2 bubbling and H2 atmosphere for
48 h. A portion of Pd(OH)2/C (36.1 mg) was added and the reac-
tion mixture was stirred with H2 bubbling for further 24 h. The
reaction mixture was filtered through Hydrophilic PVDF
0.45 μm filter and was washed with MeOH and H2O. Methanol
was removed under reduced pressure and water was removed via
lyophilization. Purification of the final product using ion
exchange column (8 × 0.5 cm) of Protein-ParkTM DEAE 8HR
−
(HCO3 form) (Waters) eluted with a linear gradient of aq.
Et3NHHCO3 (0 → 0.3 M) in H2O at 1 cm3 min−1 to afford the
α-anomer of tetrasaccharide phosphodiester 1 (6.8 mg, 40%) as
an amorphous solid: [α]D −12.4 (c 0.5, water); IR ν 3265 (s, br),
2923 (m), 1654 (w, br), 1442 (w), 1407 (w), 1365 (w), 1210
Percent inhibition was calculated as follows using mean OD
values determined from triplicate assays:
(OD of Reference Wells − Inhibitor OD) ÷ OD of Reference
Wells × 100%.
(m), 1132 (m), 1060 (s), 967 (s), 881 (m), 807 (m), 713 (w)
1
(cm−1); H NMR (600 MHz, CDCl3) δ 5.53 (dd, 1H, J1B,2B
=
1.9 Hz, JH-1B,P = 7.7 Hz, H-1B), 4.90 (s, 1H, H-1C), 4.84 (s,
1H, H-1D), 4.75 (s, 1H, H-1A), 4.28 (d, 1H, J2C,3C = 3.3 Hz,
H-2C), 4.18–4.14 (m, 3H, H-2B, H-2D, H-6aA), 4.10–4.06 (m,
1H, H-6bA), 3.96 (dd, 1H, J2B,3B = 3.2 Hz, J3B,4B = 9.8 Hz,
H-3B), 3.92–3.89 (m, 3H, H-2A, H-6aC, H-6aD), 3.86–3.83 (m,
1H, H-6aB), 3.80–3.69 (m, 8H, H-6bB, H-5B, H-6bC, H-3A,
H-4A, H-6bD, H-5A, H-4B), 3.67 (dd, 1H, J2C,3C = 3.4 Hz,
Modelling
Tetrasaccharide 1 was built and energy-minimized using the
InsightII (Accelrys, Inc.) software package. β-Linkages were
built in the previously determined solution global minimum con-
formation12 and ϕ angles of −60°, consistent with the exo-
anomeric effect, were assigned to the α-linkages. The binding
mode was modelled by interactively superimposing residues C
and D of 1 on residues A and B of a β-mannotrioside model32
and optimized by a short (2 ps) molecular dynamics refinement
at 300 K, with Amber 1134 and Glycam06 (revision g)35 in the
presence of explicit TIP3P water. After separate structure refine-
ment of the protein model, 1 was again repositioned interactively
as a final step. Simulations of 1 for 1 ns or longer, with either an
implicit solvent model36 or explicit water and sodium ions were
carried out as a preliminary investigation of conformational pre-
ferences in the absence of antibody.
J3C,4C = 9.9 Hz, H-3C), 3.63 (dd, 1H, J2D,3D = 3.3 Hz, J3D,4D
=
9.7 Hz, H-3D), 3.60 (t, 1H, J3C,4C ≈ J4C,5C ≈ 9.8 Hz, H-4C),
3.55 (t, 1H, J3D,4D ≈ J4D,5D ≈ 9.7 Hz, H-4D), 3.42–3.39 (m, 4H,
H-5C, OCH3), 3.36–3.33 (m, 1H, H-5D), 3.18–3.15 (q, 6H, J =
7.3 Hz, CH2CH3), 1.25 (t, 9H, J = 7.3 Hz, CH2CH3); 13C NMR
(125 MHz, CDCl3) δ 101.96 (JC-1A,H-1A = 173 Hz, C-1A),
101.92 (JC-1D,H-1D = 163 Hz, C-1D), 99.8 (JC-1C,H-1C = 162 Hz,
C-1C), 95.10–98.08 (m, JC-1B,H-1B = 178 Hz, C-1B), 79.6
(C-2C), 79.03 (d, J = 8.5 Hz, C-2B), 77.26 (C-5D), 77.23
(C-5C), 74.8 (C-5B), 73.8 (C-3D), 73.1 (C-3C), 72.41 (C-5A),
72.4 (C-3A), 71.4 (C-2D), 70.8 (C-2A), 69.9 (C-3B), 67.9
(C-4C), 67.7 (C-4D), 67.6 (C-4B), 67.2 (C-4A), 65.7 (d, JC-6A,P
This journal is © The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 8348–8360 | 8359

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Acknowledgements
We thank Mrs Joanna Sadowska for performing the ELISA
experiments and Mr Jonathan Cartmell for assistance with the
molecular dynamics studies. This work was funded by a Natural
Science and Engineering Research Council Discovery Grant to
DRB and by the Alberta Glycomics Centre.
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