2-Oxabutane as a substitute for internal monomer units of oligosaccharides to create lectin ligands

Article abstract of DOI:10.1039/c1ob05775h

The synthesis of bioactive oligosaccharides is too tedious to scale up for commercialization. However, structurally simplified glycomimetics are commercializable, if they can be synthesized much more easily than the oligosaccharides while having a compara

Full text of DOI:10.1039/c1ob05775h

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Organic &
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PAPER
2-Oxabutane as a substitute for internal monomer units of oligosaccharides to
create lectin ligands†
Li-Ying Yang, Yuki Kawada, Lina Bai, Daijiro Kubota and Hideya Yuasa*
Received 18th May 2011, Accepted 7th July 2011
DOI: 10.1039/c1ob05775h
The synthesis of bioactive oligosaccharides is too tedious to scale up for commercialization. However,
structurally simplified glycomimetics are commercializable, if they can be synthesized much more easily
than the oligosaccharides while having a comparable bioactivity. In this study, we propose a
2-oxabutane (OB) structure as an imitation of the internal monosaccharide units in oligosaccharides.
Two trimannoside and three pentamannoside OB-glycomimics were synthesized in remarkably short
steps. Among them, Mana1-OB-2Man 10, a trimannoside mimic, showed eight-fold affinity toward
concanavalin A (ConA) relative to methyl mannoside in latex agglutination lectin assay and equilibrium
dialysis assay (EDA), while the other mimics showed three- to four-fold affinities. EDA indicated that
the bindings between each mimic molecule and a ConA subsite were all in one-to-one stoichiometry
and thus these mimics were monovalent ligands, excluding multivalence effect for the high affinities.
The strong affinity of 10 could be explained by the occupation of two mannose binding sites of a ConA
subsite by its two mannose units. Mimic 10 proved to be even a better ligand for ConA than the natural
disaccharide Mana1,2Man, while been much more easy to synthesize, thereby illustrating the potential
of the approach here presented.
is a potential agent for photodynamic diagnosis and therapy,
respectively, of cancer tissues,7 because the mannose ligand would
Introduction
Lectins,1 a class of carbohydrate-binding proteins, play important
roles in infectious diseases. For example, lectins on the surface
of bacteria and viruses bind to specific oligosaccharide epitopes
on the host cells prior to invasion, causing targeted infections.2 A
small molecule incorporating a structure of these oligosaccharide
epitopes, therefore, could be a blocking agent against the infection.
Lectin-oligosaccharide interactions are also involved in immune
systems.3 For example, selectins, the sialyl Lewisa (sLea) and sialyl
Lewisx (sLex) binding lectins, mediate the interaction between
leukocytes and the endothelial cells at the inflammatory sites,
helping leukocytes reach the inflammatory sites.4 Cancer cells in
turn exploit this recognition system and pretend to be leukocytes,
escaping from the immune system and metastasizing to other
organs.5 The antagonists of selectins, therefore, are potential
anti-cancer agents. Another aspect of cancer cells perverting the
immune system is the recruitment of macrophage 2, an atypical
macrophage, to promote the growth of cancer tissues as opposed to
the phagocytosis expected for a typical macrophage.6 Macrophage
2 expresses high level of mannose receptor and therefore the
mannose ligand conjugated with a fluorophore or photosensitizer
be trapped by the macrophage 2 accumulated in cancer tissues.8
As mentioned above, lectin anatagonists have a potential of
becoming new bioactive products. However, the synthesis of
oligosaccharides is generally tedious. Although oligosaccharide
mimics could be more easily synthesized than the original oligosac-
carides, the design of them requires trial and error even with
the aid of sophisticated computer modeling based on the crystal
structures of lectin-oligosaccharide complexes.9 Thus, efforts have
been made for the facile syntheses of oligosaccharide mimics using
linkers, e.g. disulfide, amide, and triazole segments, to connect
monosaccharides.10
Here we propose the use of a 2-oxabutane-1,4-diyl (OB: -
CH2OCH2CH2-) unit as an imitation of inner monosaccharide
units in oligosaccharides (Fig. 1). Since the OB linker can be a
Department of Life Science, Graduate School of Bioscience and Biotech-
nology, Tokyo Institute of Technology, J2-10, 4259 Nagatsutacho, Mi-
doriku, Yokohama, 226-8501, Japan. E-mail: hyuasa@bio.titech.ac.jp;
Fax: (+81)45-924-5850
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c1ob05775h
Fig. 1 A structure and synthetic plan of an oxabutane glycomimics.
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common imitation of internal glycosides, we would need no trial
and error for the mimic design. We named these oligosaccharide
mimics “oxabutane (OB)-glycomimics”. In this study, we focused
on OB-glycomimics for oligomannosides, since oligomannosides
are recognized by a lot of biologically important lectins,1 such as
FimH of Escherichia coli,11 mannose receptor on macrophages,12
DC-SIGN of dendric cells,13 and mannose-binding protein induc-
ing complement activation.14 The OB skeleton imitates the internal
mannosides with 1→4 and 1→6 linkages and these mimicry
linkages could be synthesized by the coupling reaction between
a protected 2-methylthiomethoxyethyl mannoside and a partially
protected mannoside; e.g., MTM-Et-Man and 2-OH-Man (Fig. 2).
In addition, the OB linker can afford a separation length similar
to that of the internal mannosides with 1→2 linkages (the broken
circle in Fig. 2), and thus we regard this as the mimic of 1→2
mannosides hereafter. The reaction center of this substitution
reaction is a primary carbon atom as opposed to a secondary one
in glycosidation reactions, which means that the coupling reaction
is non-stereogenic and would probably proceed efficiently, solving
some difficulties inherent in glycosidation reactions.
for their synthesis would become exponentially greater as the
size of oligosaccharides increases. Although the trisaccharide,
Mana1,6(Mana1,3)Man, have no internal mannose units to meet
the concept of OB-glycomimics, we were interested in the effcts of
inserting OB unit in the glycosidic linkages of this oligosaccharide.
We thus synthesized Mana1-OB-6(Mana1-OB-3)Man. We also
synthesized
Mana1-OB-6Man,
Mana1-OB-3(Mana1-OB-
2)Man and Mana1-OB-6(Mana1-OB-2)Man for comparison.
We synthesized an OB glycomimic donor 4 in three steps from
per-O-acetyl mannose 1 (Scheme 1). The reaction of compound
1 with mono-benzyl protected ethylene glycol in the presence of
SnCl4 gave 2-benzyloxyethyl glycoside 2, which was subsequently
debenzylated to give 2-hydroxyethyl glycoside 3 in 49%. Direct
mannosylation of ethylene glycol yielded 3 in 15%,16 accompanied
by a divalent mannoside in 11% yield. We thus took the former
method to synthesize compound 4 in large scale. Compound 4
was synthesized by Pummerer-type methylthiomethylation17 of the
hydroxyl group of 3 in 58%.
Scheme 1 a) HO(CH2)2OBn, SnCl4/CH2Cl2; b) H2, Pd–C/CH3OH
(49%); c) DMSO-Ac2O–AcOH 7 : 5 : 1 (58%).
Compound 4 was coupled to a 6-free acceptor 518 in the usual
glycosidation conditions for thioglycosides promoted with methyl
triflate19 to give the coupled product 6 in 33% (Scheme 2). The low
yield was probably due to decomposition of MTM group, which is
much more reactive than thioglycosides. Therefore, we needed to
find milder yet effective conditions to improve the yield. The frozen
conditions for glycosidations developed by Ito et al.20 matched our
demand and the coupling reaction conducted in frozen p-xylene
afforded 56% yield. Deacetylation of compound 6 gave Mana1-
OB-6Man (7) in 70%.
Fig. 2 The structure and synthetic plan of an oxabutane glycomimic of
an oligomannoside.
Results and discussion
We chose concanavalin A (ConA) as a representative mannose-
binding lectin to assess the effectiveness of OB-glycomimics as a
substitute for natural oligomannoside ligands, since ConA is best
studied among commercially available lectins, being abundant
with the available binding constants toward oligosaccharide
ligands.15 The affinity of typical small oligomannosides for
Scheme 2 a) 4, CH3OTf/CH2Cl2, -60 ◦C (33%); b) 4, CH3OTf/2,4,6-tri-
tert-butylpyridine, p-xylene, -78 ◦C → 4 ◦C (55%); c) CH3ONa/CH3OH
(70%).
ConA decreases in the order: Mana1,6(Mana1,3)Man
Mana1,2Mana1,2Man > Mana1,2Man > Mana1,4Man ª
Mana1,3Man Mana1,6Man MeMan. Among the
>
The coupling of compound 4 to a 2-free acceptor 821 was also
conducted by two methods, i.e., the normal and frozen conditions
(Scheme 3). The results were similar to those of the previous 1,6-
mimic and the frozen conditions improved the yield of compound
9 from 27% for the normal conditions to 54%. We used only
the frozen conditions for the other coupling reactions hereafter.
Debenzylation and deacetylation of 9 gave Mana1-OB-2Man (10)
in 61%.
>
ª
above small oligomannosides, the linear trimannoside,
Mana1,2Mana1,2Man, has a structure mimickable by OB-
glycomimics: the internal mannose unit can be substituted with
OB unit to build a dumbbell-shaped mimic, Mana1-OB-2Man.
The OB-glycomimics would be more effectively applied for larger
oligosaccharides, because the time, cost, and effort required
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Scheme 3 a) 4, CH3OTf/CH2Cl2, -60 ◦C (27%); b) 4, CH3OTf/2,4,6-tri-
tert-butylpyridine, p-xylene, -78 ◦C → 4 ◦C (54%); c) H2, Pd–C/CH3OH
then CH3ONa/CH3OH (61%).
A branched OB-glycomimic, Mana1-OB-6(Mana1-OB-3)Man
(13), was synthesized by coupling of compound 4 to a 3,6-
free acceptor 1122 (Scheme 4). The coupling intermediate was
acetylated for structure elucidation to give the fully-acetylated
mimic 12, which was deacetylated to give 13 in 39% yield.
Another branched OB-glycomimic, Mana1-OB-2(Mana1-OB-
3)Man (18), was synthesized in a similar manner from a 2,3-
free acceptor 1423 in 19% total yield (Scheme 5). In the coupling
reaction, mono-functionalized mannoside 16 was accompanied in
9% yield, whose structure was elucidated by 1H NMR analysis of
the per-acetylated derivative 17. A third branched OB-glycomimic,
Mana1-OB-6(Mana1-OB-2)Man (23), was synthesized from a
2,6-free acceptor 19.24 The coupling of compounds 4 and 19
then deprotection gave compound 23 (Scheme 6). In the cou-
Scheme 6 a) 4, CH3OTf/2,4,6-tri-tert-butylpyridine, p-xylene, -78 ◦C →
4 ◦C (52% + 39% of 21); b) tetra-butylammonium fluoride (TBAF), THF;
Ac2O–Py (83%); c) CH3ONa/CH3OH (84%).
pling reaction, the mono-functionalized derivative 21 was also
produced. Compound 21 was derivatizable to compound 6 by
desilylation and deacetylation. The branched OB-glycomimics 13,
18, and 23 are pentasaccharide mimics if we regard the OB linker
as a substitute for a mannose unit, and the synthesis of these
mimics was extremely simple compared to that of the original
pentasaccharides. It should be noted here that the 2-oxabutane
linker contains a very acid sensitive acyclic acetal linker. It can be
expected that the use of acid labile protecting groups is prohibited
during the synthesis of this type of glycomimetics.
Scheme 4 a) 4, CH3OTf/2,4,6-tri-tert-butylpyridine, p-xylene, -78 ◦C →
4
◦C; CH3ONa/CH3OH; Ac2O–Py (44%); b) CH3ONa/CH3OH (89%).
The affinity of the synthesized glycomimics toward ConA
was evaluated by latex agglutination lectin assay (LALA)25 and
enzyme-linked lectin assay (ELLA)26 in IC50 and by equilibrium
dialysis assay (EDA)27 in Kd (Table 1). In LALA, aggregation
Table 1 Affinity of oxabutane (OB) glycomimics toward ConA
IC50/mMa
EDA
Ligand
LALA
ELLA
Kd/mMa
n
MeMan
Mana1,2Man
7
10
13
18
23
165 (1)
94 (3)
74 (2)
37 (5)
96 (2)
48 (3)
ND
529 (1)
148 (4)
506 (1)
125 (4)
183 (3)
127 (4)
ND
205 (1)
57 (4)
62 (3)
26 (8)
54 (4)
57 (4)
83 (3)
0.96
0.98
0.97
0.95
0.99
0.96
1.00
Scheme 5 a) 4, CH3OTf/2,4,6-tri-tert-butylpyridine, p-xylene, -78 ◦C
→ 4 ◦C; b) 80% AcOH, 45 ◦C (20% + 9% of 16); c) Ac2O–Py; d)
CH3ONa/CH3OH (94%).
a Numbers in parentheses are relative potency.
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of the polystyrene beads coated with mannosylated bovine
serum albumin in the presence of ConA was observed. IC50
is the concentration of a compound to inhibit the aggregation
by 50%. In ELLA, the horseradish peroxidase-labeled ConA
bound on yeast mannan-coated wells of a microtiter plate after
several washings was quantified by absorption at 415 nm derived
from the peroxidase-catalyzed oxidation of 2,2¢-azino diethyl-
benzothiazoline sulfonic acid. IC50 is the concentration of a com-
pound to inhibit the binding by 50%. In EDA, a ligand solution in
buffer was divided by dialysis membrane into two compartments
and ConA was added into one of two compartments so that ConA
was restricted in one compartment and the ligand could freely
pass through the membrane. The free ligand concentration after
equilibrium was determined by phenol-H2SO4 quantification of
the sugar in the compartment containing only the ligand. The
unbound ligand concentrations were determined for several runs
with varied initial ligand concentrations and the Kd values and
n, the number of bound ligands per subsite, were deduced by
Scatchard plots. There are limited numbers of methods with which
n of binding can be deduced, especially when there are no labels
on either lectin or sugar to evaluate the concentration. EDA is a
classical method but still a convenient and the most inexpensive
method to determine both Kd and n.
Fig.
3
Proposed binding modes of
a
natural trisaccharide
Mana1,6(Mana1,3)Man and Mana1-OB-2Man (10) toward ConA.
showed almost the same affinity as those of the disaccharide
Mana1,2Man and OB glycomimic 18. The almost unity n, the
number of bound ligands per subsite, suggested that the better
affinities of these OB glycomimics than that of MeMan are not
because of multivalent binding of mannose residues to different
subsites.
Conclusions
OB-glycomimics, the mimics with the oxabutane linkers imitating
internal monosaccharide units of oligosaccharides, were synthe-
sized much more straightforwardly than the original oligomanno-
sides are supposed to be. They showed good affinities for ConA
comparable to those of the original oligomannosides. These good
affinities are likely due to occupation of two mannose-binding
sites in a ConA subsite by the mannose units of OB glycomimics.
Among the synthesized OB-glycomimics, Mana1-OB-2Man (10)
showed the best affinity probably owing to the appropriate spacing
between two mannose units. These OB-glycomimics could be
further developed for the antagonists of biologically important
lectins and for the tags of a drug delivery system. It should
be noted that the flexibility of the OB linker as opposed to
pyranosides may be sometimes detrimental to the overall affinity
of this glycomimetic.
The affinity of the ligands toward ConA was consistent between
LALA and EDA, the relative binding potency being in the order:
Mana1-OB-2Man (10) > the others > MeMan, the affinity of 10
being distinctive from others. ELLA showed a slightly different
tendency: the others > Mana1-OB-6Man (7) = MeMan. The
difference might be due to the low sensitivity of ELLA, which
produces more reliable data for multivalent ligand bindings.28
ConA is tetramer above pH
7
and the monomer
has a subsite primarily recognizing the core trisaccharide,
Mana1,6(Mana1,3)Man.1 On the other hand, a trisaccharide
Mana1,2Mana1,2Man has the same magnitude of affinity for
ConA as that of the core trisaccharide.15 The affinity of the
disaccharide Mana1,2Man to ConA, though 2.7-fold less than
that of the trisaccharide Mana1,2Mana1,2Man, has shown to
be at least 4-fold higher than those of the other disaccharides
with different glycosidic linkages. These distinctive affinities of
a1,2-linked oligomannosides toward ConA were found to be
mainly due to enhanced entropy effect, which was ascribed to
increased number of binding modes due to sliding of the mannose
residues with strongly binding 3-, 4-, 6- hydroxyl groups at the
monosaccharide-binding site. In our case, the trisaccharide mimic
Mana1-OB-2Man (10) increased the affinity to ConA by 2-fold
over the dimannoside Mana1,2Man, a similar enhancement as
has been observed for the trimannoside Mana1,2Mana1,2Man
(2.7-fold). However, the enhanced affinity of 10 is unlikely due to
the increased number of binding modes, because the analog 10 has
the same number of hydroxyl groups as that of the dimannoside.
Probably, two mannose residues of Mana1-OB-2Man (10) are
appropriately separated so as to fit the two mannose binding sites
supposedly occupied by two nonreducing mannose residues of
the high affinity core trisaccharide Mana1,6(Mana1,3)Man (Fig.
3). This binding model is supported by the fact that addition
of a further branch on the OB glycomimic 10; i.e., Mana1-OB-
2(Mana1-OB-3)Man (18) and Mana1-OB-2(Mana1-OB-6)Man
(23), resulted in diminished affinity to ConA. The extended
core trimannoside mimic, Mana1-OB-6(Mana1-OB-3)Man (13),
Experimental
General methods
All solvents and reagents were reagent-grade, and in cases in
which further purification was required, standard procedures
were followed.29 Reactions were monitored by TLC (thin-layer
chromatography). TLC was performed on pre-coated silica gel
Merck 60-F254 plates (Art 5715) and visualized by the quenching
of fluorescence and/or by charring after spraying with 1% CeSO4-
1.5% (NH4)6Mo7O24·4H2O-10% H2SO4. Flash column chromatog-
raphy was performed on Merck Kieselgel 60 (Art 7734), Wako gel
C-300 or Kanto Silica gel 60 N (spherical, neutral) with the solvent
systems specified. Optical rotations were determined with a Horiba
SEPA-200 polarimeter using a 1 dm length cell. 1H NMR spectra
were recorded at 600 (Bruker AV-600), 400 (Varian Unity-400) or
270 MHz (JEOL EX-270). Internal tetramethylsilane (d 0 ppm) in
CDCl3 or a DOH peak (d 4.8 ppm) in D2O was used as a standard.
Chemical shifts were expressed in ppm referenced to the standard.
The multiplicity of signals was abbreviated as follows: s = singlet,
d = doublet, dd = doublet of doublets, t = triplet, dt = doublet of
triplets, ddd = doublet of doublets of doublets, br = broad signal,
and m = multiplet. Protons on the sugars at non-reducing ends
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were designated as H-1¢, H-1¢¢, etc. 13C NMR spectra were recorded
at 67.8 MHz (JEOL JNM-EX-270) or 100.6 MHz (Varian Unity-
500), and a solvent peak (d 77.0 ppm) in CDCl3 or internal acetone
(d 30.8 ppm) in D2O was used as a standard. High-resolution mass
spectra (HRMS) were recorded on a Mariner Biospectrometry
Workstation ESI-TOF mass spectrometer. The reagents for lectin
assay, i.e. latex beads (10% suspension, 10 mL diameter), ConA
(C2010, powder), were obtained from Sigma. DVS was purchased
from Aldrich. LALA was carried out using the method of Yuasa
and coworkers.24 ELLA was carried out using the method of Page´
and coworkers.25 Optical absorbance at 405 nm in ELLA was
measured on a Model 550 Microplate Reader (Bio-Rad).
s, OCH3), 2.16 (6H, s, COCH3), 2.11 (3H, s, COCH3), 2.05 (6H,
s, COCH3), 1.99 (6H, s, COCH3); dC (67.8 MHz, CDCl3, 25 ◦C)
170.6, 170.2, 169.99, 169.95, 169.8, 169.8, 169.7, 98.5, 97.7, 95.7,
76.5, 69.5, 69.3, 68.9, 68.4, 67.2, 66.5, 66.3, 66.0, 62.3, 55.2, 20.92,
20.86, 20.8, 20.71, 20.68, 20.64; HRMS (ESI): m/z calcd for
C30H44O20+Na, 747.2325; found, 747.2297.
Methyl 6-O-(a-D-mannnopyranosyl-oxyethoxymethyl)-a-D-ma-
nnopyranoside (7). To a stirred solution of 6 (60 mg, 83 mmol) in
dry MeOH (3 mL) was added 0.1 M NaOMe (100 mL). After
30 min, the solution was neutralized with Dowex50 (H+) and
evaporated to give 7 (25 mg, 70%) as syrup. Rf 0.32 (iPrOH-H2O–
NH3 8 : 3 : 1). [a]2D5 +95.5 (c 1.0 in H2O); dH (600 MHz, D2O, 60 ◦C)
4.88 (1H, d, J1¢,2¢ 1.6 Hz, H-1¢), 4.87–4.78 (2H, m, OCH2O), 4.73
(1H, d, J1,2 1.5 Hz, H-1), 3.95 (1H, dd, J1¢,2¢ 1.6, J2¢,3¢ 3.4 Hz, H-2¢),
3.92–3.62 (16H, m, H-2, H-3, H-3¢, H-4, H-4¢, H-5, H-5¢, H-6a,
H-6¢a, H-6b, H-6¢b, OCH2CH2O), 3.39 (3H, s, OCH3); dC (67.8
MHz, D2O, 25 ◦C, acetone) 101.6, 100.5, 95.8, 73.4, 71.9, 71.1,
70.6, 70.5, 67.6, 67.4, 67.3, 67.1, 61.6, 55.5; HRMS (ESI): m/z
calcd for C16H30O13+Na, 453.1585; found, 453.1557.
Methylthiomethoxyethyl 2,3,4,6-tetra-O-aectyl-a-D-mannopyra-
noside (4). 2¢-Hydroxyethyl 2,3,4, 6-tetra-O-acetyl-a-D-
mannopyranoside (3)16 (153 mg, 390 mmol) was dissolved in
DMSO-Ac2O–AcOH (7 : 5 : 1, 6.5 mL) at RT. After 24 h, the
solution was diluted with EtOAc and washed with sat. NaHCO3.
The organic layer was dried over MgSO4, filtered, and evaporated.
The residue was chromatographed on siliga gel (hexane–EtOAc
2 : 1) to give 4 (103 mg, 58%). Rf 0.64 (Hexane–EtOAc 1 : 2); [a]D25
+41.5 (c 1.00 in CHCl3); dH (600 MHz, CDCl3, 25 ◦C, Me4Si) 5.37
(1H, dd, J2,3 3.4, J3,4 9.9 Hz, H-3), 5.29 (1H, t, J3,4 = J4,5 9.9 Hz,
H-4), 5.28 (1H, dd, J1,2 1.7, J2,3 3.4 Hz, H-2), 4.89 (1H, d, J1,2 1.7
Hz, H-1), 4.69 (1H, d, J 11.6 Hz, OCHHS), 4.66 (1H, d, J 11.6
Hz, OCHHS), 4.29 (1H, dd, J5,6a 5.2, J6a,6b 12.0 Hz, H-6a), 4.12
(1H, dd, J5,6b 2.4, J6a,6b 12.2 Hz, H-6b), 4.07 (1H, ddd, J5,6b 2.4,
J5,6a 5.2, J4,5 9.9 Hz, H-5), 3.87–3.69 (4H, m, OCH2CH2O), 2.16,
2.11, 2.05, 2.00 (15H, each s, COCH3 ¥ 4, SCH3); dC (67.8 MHz,
CDCl3, 25 ◦C, Me4Si) 170.5, 169.9, 169.7, 169.6, 97.5, 75.3, 69.4,
68.9, 68.3, 66.9, 66.3, 66.0, 62.3, 20.8, 20.7, 20.59, 20.57, 13.6;
HRMS (ESI): m/z calcd for C18H28O11S+Na, 475.1251; found,
475.1246.
Methyl 2-O-(2,3,4,6-tetra-O-acetyl-a-D-mannnopyranosyl-oxye-
thoxymethyl)-3,4,6-tri-O-benzyl-a-D-mannopyranoside
(9). A
mixture methyl 3,4,6-tri-O-benzyl-a-D-mannopyranoside (8)21
(54 mg, 116 mmol), compound 4 (81 mg, 179 mmol), TTBP (45 mg,
182 mmol) and activated molecular sieves 4A (100 mg) in p-xylene
(2.6 mL) was stirred under N2 at RT for 30 min. Immediately after
the addition of MeOTf (40 mL, 353 mmol), the mixture was rapidly
mixed and frozen over dry ice in acetone. The mixture was stored
in refrigerator at 4 ◦C for 48 h and defrosted at RT. Et3N (50 mL)
was added to deactivate MeOTf and the mixture was diluted
with EtOAc and filtered through Celite. The filtrate was washed
with brine, dried over MgSO4, and evaporated. The residue was
chromatographed on silica gel (hexane–EtOAc 4 : 3→1:2) to give
9 (55 mg, 54%). Rf 0.20 (hexane–EtOAc 1 : 1); [a]2D5 +58.0 (c 0.66
in CHCl3); dH (600 MHz, CDCl3, 25 ◦C, Me4Si) 7.36–7.15 (15H,
m, Ph), 5.35 (1H, dd, J2¢,3¢ 3.5, J3¢,4¢ 10.0 Hz, H-3¢), 5.29–5.26 (2H,
m, H-2¢, H-4¢), 4.88 (1H, d, J 10.8 Hz, PhCHH), 4.84 (3H, m,
OCH2O, H-1¢), 4.80 (1H, d, J1,2 1.7 Hz, H-1), 4.71 (1H, d, J 11.7
Hz, PhCHH), 4.68 (1H, d, J 11.7 Hz, PhCHH), 4.64 (1H, d, J
12.1 Hz, PhCHH), 4.54 (1H, d, J 12.1 Hz, PhCHH), 4.50 (1H,
d, J 10.8 Hz, PhCHH), 4.28 (1H, dd, J5¢,6¢a 5.1, J6¢a,6¢b 12.3 Hz,
H-6¢a), 4.08 (1H, dd, J5¢,6¢b 2.3, J6¢a,6¢b 12.3 Hz, H-6¢b), 4.01 (1H,
ddd, J5¢,6¢b 2.3, J5¢,6¢a 5.1, J4¢,5¢ 10.0 Hz, H-5¢), 3.99 (1H, dd, J1,2
1.7, J2,3 2.7 Hz, H-2), 3.93–3.55 (9H, m, H-3, H-4, H-5, H-6a,
H-6b, OCH2CH2O), 3.35 (3H, s, OCH3), 2.15 (3H, s, COCH3),
2.09 (3H, s, COCH3), 2.03 (3H, s, COCH3), 1.99 (3H, s, COCH3);
dC (67.8 MHz, CDCl3) 170.6, 170.0, 169.8, 169.7, 138.4, 138.2,
128.4, 128.3, 127.8, 127.7, 127.6, 127.55, 127.48, 99.6, 97.6, 95.7,
79.6, 76.5, 75.1, 74.7, 73.9, 73.3, 72.2, 71. 4, 69.4, 69.0, 68.3, 67.0,
66.7, 66.1, 62.4, 54.8, 20.9, 20.72, 20.66, 20.64; HRMS (ESI): m/z
calcd for C45H56O17+Na, 891.3417; found, 891.3420.
Methyl 6-O-(2,3,4,6-tetra-O-acetyl-a-D-mannnopyranosyl-oxye-
thoxymethyl)-2,3,4-tri-O-acetyl-a-D-mannopyranoside
(6). A
mixture of methyl 2,3,4-tri-O-acetyl-a-D-mannopyranoside
(5)18 (50 mg, 156 mmol), 4 (92 mg, 203 mmol), 2,4,6-tri-tert-
butylpyridine (TTBP) (116 mg, 467 mmol) and activated
molecular sieves 4A (100 mg) in p-xylene (2.6 mL) was stirred
under N2 at RT for 30 min. Immediately after the addition
of MeOTf (51 mL, 451 mmol), the mixture was rapidly mixed
and frozen over dry ice in acetone. The mixture was stored in
refrigerator at 4 ◦C for 48 h and defrosted at RT. Et3N (60 mL)
was added to deactivate MeOTf and the mixture was diluted
with EtOAc and filtered through Celite. The filtrate was washed
with brine, dried over MgSO4, and evaporated. The residue was
chromatographed on silica gel (hexane–EtOAc 4 : 3 → 1 : 2) to
give 6 (60 mg, 53%). Rf 0.34 (hexane–EtOAc 1 : 2). [a]2D5 +55.3 (c
0.40 in CHCl3); dH (600 MHz, CDCl3, 25 ◦C, Me4Si) 5.35 (1H,
dd, J2¢,3¢ 3.4, J3¢,4¢ 9.9 Hz, H-3¢), 5.34–5.31 (2H, m, H-3, H-4),
5.29 (1H, t, J3¢,4¢ = J4¢,5¢ 9.9 Hz, H-4¢), 5.27 (1H, dd, J1¢,2¢ 1.6,
J2¢, 3.4 Hz, H-2¢), 5.24 (1H, dd, J1,2 1.6, J2,3 3.0 Hz, H-2), 4.88
3¢
(1H, d, J1¢,2¢ 1.6, H-1¢), 4.75 (1H, d, J 6.8 Hz, OCHHO), 4.73
(1H, d, J 6.8 Hz, OCHHO), 4.72 (1H, d, J1,2 1.6 Hz, H-1), 4.29
(1H, dd, J5¢,6¢a 5.1, J6¢a,6¢b 12.3 Hz, H-6¢a), 4.11 (1H, dd, J5¢,6¢b 2.3,
J6¢a,6¢b 12.3 Hz, H-6¢b), 4.05 (1H, ddd, J4¢,5¢ 9.9, J5¢,6¢b 2.3, J5¢,6¢a 5.1
Hz, H-5¢), 3.92 (1H, ddd, J5,6b 2.6, J5,6a 4.8, J4,5 9.6 Hz, H-5),
3.84–3.66 (4H, m, OCH2CH2O), 3.70 (1H, dd, J5,6a 4.8, J6a,6b 11.3
Hz, H-6a), 3.64 (1H, dd, J5,6b 2.6, J6a,6b 11.3 Hz, H-6b), 3.41 (3H,
Methyl 2-O-(a-D-mannnopyranosyl-oxyethoxymethyl)-a-D-ma-
nnopyranoside (10). A mixture of 9 (74 mg, 85 mmol) and 10%
Pd–C (18 mg) in dry MeOH (1 mL) was stirred under 1 atm H2
for 1 day at RT. The catalyst was filtered off and washed throughly
with MeOH. After evaporation of the filtrate, the residue was
dissolved in dry MeOH (3 mL) and 0.5 M NaOMe (100 mL) was
added to the solution. After 30 min, the solution was neutralized
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with Dowex50(H+) and evaporated to give 10 (16.5 mg, 45%). Rf
0.36 (iPrOH-H2O–NH3 8 : 3 : 1); [a]2D5 +67.9 (c 0.28 in H2O); dH
(600 MHz, D2O, 60 ◦C) 4.89 (1H, d, J1,2 1.7 Hz, H-1), 4.88 (1H, d,
J 7.5 Hz, OCHHO), 4.86 (1H, d, J 7.5 Hz, OCHHO), 4.85 (1H,
d, J1,2 1.6 Hz, H-1¢), 3.96 (1H, dd, J1,2 1.7, J2,3 3.5 Hz, H-2), 3.92
(1H, dd, J1¢,2¢ 1.6, J2¢,3¢ 3.5 Hz, H-2¢), 3.91–3.58 (14H, m, H-3, H-3¢,
H-4, H-4¢, H-5, H-5¢, H-6a, H-6¢a, H-6b, H-6¢b, OCH2CH2O),
3.40 (3H, s, OCH3); dC (150.92 MHz, D2O, 25 ◦C, acetone) 100.0,
99.6, 96.2, 77.3, 72.9, 72.7, 70.7, 70.3, 70.2, 67.3, 67.2, 67.0, 66.5,
61.14, 61.12, 55.0; HRMS (ESI): m/z calcd for C16H30O13+Na,
453.1585; found, 453.1548.
1.7 Hz, H-1¢), 5.11 (1H, d, J 6.9 Hz, OCHHO), 5.09 (1H, d, J 6.9
Hz, OCHHO), 5.04 (1H, d, J1,2 1.6 Hz, H-1), 4.36 (1H, dd, J1,2
1.6, J2,3 3.0 Hz, H-2), 4.25 (1H, dd, J1¢¢,2¢¢ 1.6, J2¢¢,3¢¢ 3.0 Hz, H-2¢¢),
4.24 (1H, dd, J1¢,2¢ 1.6, J2¢,3¢ 3.0 Hz, H-2¢), 4.22–3.93 (23H, m, H-3,
H-3¢, H-3¢¢, H-4, H-4¢, H-4¢¢, H-5, H-5¢, H-5¢¢, H-6a, H-6¢a, H-
6¢¢a, H-6b, H-6¢b, H-6¢¢b, OCH2CH2O ¥ 2), 3.70 (3H, s, OCH3);
dC (67.8 MHz, D2O, 25 ◦C, acetone) 101.4, 100.5, 95.7, 95.3, 78.0,
73.3, 71.8, 71.1, 70.6, 69.0, 67.33, 67.26, 67.03, 66.96, 66.2, 61.5,
55.5; HRMS (ESI): m/z calcd for C25H46O20+Na, 689.2482; found,
689.2481.
Methyl 2,3-di-O-(2,3,4,6-tetra-O-acetyl-a-D-mannnopyranosyl-
oxyethoxymethyl)- a-D-mannopyranoside (15). A mixture of
methyl 4,6-O-benzylidene-a-D-mannopyranos-ide (14)23 (50 mg,
180 mmol), 4 (172 mg, 380 mmol), TTBP (220 mg, 889 mmol),
and activated molecular sieves 4A (180 mg) in p-xylene (4.5 mL)
was stirred under N2 at RT for 30 min. Immediately after
the addition of MeOTf (60 mL, 530 mmol), the mixture was
rapidly mixed and frozen over dry ice in acetone. The mixture
was stored in refrigerator at 4 ◦C for 48 h and defrosted at
RT. Et3N (80 mL) was added to the mixture to deactivate
MeOTf. The mixture was diluted with EtOAc and filtered
through Celite. The filtrate was washed with brine, dried over
MgSO4, and evaporated. The residue was dissolved in 80%
AcOH (3 mL) at 40 ◦C. After 6 h, the solution was evaporated
and chromatographed on silica gel to give 15 (23 mg, 13%)
and methyl 3-O-(2,3,4,6-tetra-O-acetyl-a-D-mannnopyranosyl-
oxyethoxy-methyl)-a-D-mannopyranoside (16) (10 mg, 9%). Com-
pound 16 was acetylated in pyridine-acetic anhydride (1 : 1, 2 mL)
to give methyl 3-O-(2,3,4,6-tetra-O-acetyl-a-D-mannnopyranosyl-
oxyethoxymethyl)-2,4,6-tri-O-acetyl-a-D-mannopyranoside (17).
Compound 15: Rf 0.31(CHCl3–MeOH 20 : 1); [a]2D5 +51.0 (c 0.96
in CHCl3); dH (600 MHz, CDCl3, 25 ◦C, Me4Se) 5.38 (1H, dd, J2¢,3¢
3.4, J3¢,4¢ 10.0 Hz, H-3¢), 5.35 (1H, dd, J2¢¢,3¢¢ 3.4, J3¢¢,4¢¢ 10.0 Hz, H-
3¢¢), 5.34 (1H, d, J2¢,3¢ 3.4 Hz, H-2¢), 5.29 (2H, t, J3¢,4¢ = J4¢,5¢ 10.0
Hz, H-4¢, H-4¢¢), 5.28 (1H, d, J2¢¢,3¢¢ 3.4 Hz, H-2¢¢), 4.89 (1H, s,
H-1¢¢), 4.87 (1H, d, J 7.1 Hz, OCHHO), 4.86 (1H, s, H-1¢), 4.85
(1H, d, J 7.1 Hz, OCHHO), 4.82 (2H, d, J 7.1 Hz, OCHHO ¥ 2),
4.80 (1H, s, H-1), 4.30 (1H, dd, J5¢¢,6¢¢a 5.2, J6¢¢a,6¢¢b 12.2 Hz, H-6¢¢a),
4.28 (1H, dd, J5¢,6¢a 5.1, J6¢a,6¢b 12.1 Hz, H-6¢a), 4.12 (1H, dd, J5¢¢,6¢¢b
1.7, J6¢¢a,6¢¢b 12.2 Hz, H-6¢¢b), 4.10 (1H, dd, J5¢,6¢b 1.9, J6¢a,6¢b 12.1 Hz,
H-6¢b), 4.05–4.02 (2H, m, H-5¢, H-5¢¢), 3.97–3.81 (6H, m, H-2,
H-4, OCH2CH2O ¥ 2), 3.78 (1H, dd, J2,3 3.1, J3,4 9.3 Hz, H-3),
3.73–3.66 (4H, m, OCH2CH2O ¥ 2), 3.62 (1H, d, J4,4OH 1.4 Hz,
4OH), 3.57 (1H, dt, J4,5 9.5, J5,6a = J5,6b 4.2 Hz, H-5), 3.37 (3H,
s, OCH3), 2.53 (1H, t, J6a,6OH = J6b,6OH 6.4 Hz, 6OH), 2.16 (6H, s,
COCH3 ¥ 2), 2.10 (6H, s, COCH3 ¥ 2), 2.06 (3H, s, COCH3), 2.05
(3H, s, COCH3), 1.997 (3H, s, COCH3), 1.990 (3H, s, COCH3);
dC (67.8 MHz, CDCl3, 25 ◦C) 170.7, 170.1, 170.05, 170.00, 169.84,
169.77, 169.7, 99.7, 97.9, 97.6, 96.2, 81.1, 77.2, 76.0, 72.4, 70.1,
69.5, 69.3, 69.03, 68.98, 68.5, 68.4, 67.2, 67.1, 66.9, 66.8, 66.1,
66.0, 62.6, 62.4, 54.8, 20.9, 20.73, 20.68; HRMS (ESI): m/z calcd
for C41H62O28+Na, 1025.3327; found, 1025.3376.
Methyl 3,6-di-O-(2,3,4,6-tetra-O-acetyl-a-D-mannnopyranosyl-
oxyethoxymethyl)- 2,4-di-O-acetyl-a-D-mannopyranoside (12).
A
mixture of methyl 2,4-di-O-benzoyl-a-D-mannopyranoside (11)22
(50 mg, 124 mmol), 4 (225 mg, 497 mmol), TTBP (123 mg,
496 mmol), and activated molecular sieves 4A (160 mg) in p-
xylene (4 mL) was stirred under N2 at RT for 30 min. Immediately
after the addition of MeOTf (110 mL, 972 mmol), the mixture was
rapidly mixed and frozen over◦dry ice in acetone. The mixture
was stored in refrigerator at 4 C for 48 h and defrosted at RT.
Et3N (140 mL) was added to deactivate MeOTf and the mixture
was diluted with EtOAc and filtered through Celite. The filtrate
was washed with brine, dried over MgSO4, and evaporated. The
residue was dissolved in 3 mL MeOH and to the solution was
added 0.5 M NaOMe (100 mL). After 30 min, the solution was
neutralized with Dowex50 (H+) and evaporated. The residue was
dissolved in pyridine–acetic anhydride (1 : 1, 2 mL). After 3 h, the
solution was evaporated and the residue was chromatographed on
silica gel (hexane–EtOAc 1 : 3) to give 12 (27 mg, 20%). Rf 0.26
(hexane–EtOAc 2 : 5); [a]D25 +49.2 (c 0.90 in CHCl3); dH (600 MHz,
CDCl3, 25 ◦C, Me4Si) 5.35 (1H, dd, J2¢¢,3¢¢ 3.4, J3¢¢,4¢¢ 10.1 Hz, H-
3¢¢), 5.33 (1H, dd, J2¢,3¢ 3.2, J3¢,4¢ 10.4 Hz, H-3¢), 5.31–5.23 (3H,
m, H-4, H-4¢, H-4¢¢), 5.29 (1H, dd, J1¢¢,2¢¢ 1.4, J2¢¢,3¢¢ 3.4 Hz, H-2¢¢),
5.27 (1H, dd, J1¢,2¢ 1.5, J2¢,3¢ 3.2 Hz, H-2¢), 5.24 (1H, dd, J1,2 1.5,
J2,3 3.4 Hz, H-2), 4.90 (1H, d, J1¢¢,2¢¢ 1.4 Hz, H-1¢¢), 4.88 (1H, d,
J1¢,2¢ 1.5 Hz, H-1¢), 4.75 (1H, d, J 7.3 Hz, OCHHO), 4.74 (2H, s,
OCH2O), 4.70 (1H, d, J1,2 1.5 Hz, H-1), 4.64 (1H, d, J 7.3 Hz,
OCHHO), 4.30 (1H,dd, J5¢¢,6¢¢a 5.0, J6¢¢a,6¢¢b 12.2 Hz, H-6¢¢a), 4.29
(1H, dd, J5¢,6¢a 5.1, J6¢a,6¢b 12.2 Hz, H-6¢a), 4.13-4.08 (3H, m, H-3,
H-6¢b, H-6¢¢b), 4.05 (1H, ddd, J4¢¢,5¢¢ 9.8, J5¢¢,6¢¢a 5.0, J5¢¢,6¢¢b 2.3 Hz,
H-5¢¢), 4.02 (1H, ddd, J4¢,5¢ 9.8, J5¢,6¢a 5.1, J5¢,6¢b 2.4 Hz, H-5¢), 3.86–
3.60 (11H, m, H-5, H-6a, OCH2CH2O ¥ 2), 3.61 (1H, dd, J5,6b 2.7,
J6a,6b 11.3 Hz, H-6b), 3.39 (3H, s, OCH3), 2.15 (9H, s, COCH3 ¥
3), 2.11 (9H, s, COCH3 ¥ 3), 2.00 (6H, s, COCH3 ¥ 2), 1.99 (6H, s,
COCH3 ¥ 2); dC (67.8 MHz, CDCl3, 25 ◦C) 170.70, 170.68, 170.4,
170.03, 169.96, 169.82, 169.80, 169.7, 98.6, 97.8, 97.7, 95.8, 94.6,
76.5, 72.8, 69.7, 69.55, 69.49, 69.4, 69.05, 69.00, 68.4, 67.7, 67.3,
66.9, 66.8, 66.5, 66.1, 62.4, 60.4, 55.1, 21.1, 21.0, 20.9, 20.8, 20.76,
20.71, 20.68, 14.2; HRMS (ESI): m/z calcd for C45H66O30+Na,
1109.3539; found, 1109.3559.
Methyl 3,6-di-O-(a-D-mannnopyranosyl-oxyethoxymethyl)-a-D-
mannopyranoside (13). To a stirred solution of 12 (27 mg,
25 mmole) in dry MeOH (3 mL) was added 0.5 M NaOMe
(100 mL). After 30 min, the solution was neutralized with Dowex50
(H+) and evaporated to give 13 (15 mg, 91%). Rf 0.22 (iPrOH-
H2O–NH3 8 : 3 : 1); [a]D25 +67.9 (c 0.72 in H2O); dH (600 MHz,
Compound 17: Rf 0.14 (CH3Cl–MeOH 20 : 1); [a]2D5 +58.0 (c
0.05 in CH3Cl); dH (600 MHz, CDCl3, 25 ◦C, Me4Si) 5.33 (1H, dd,
J2¢,3¢ 3.1, J3¢,4¢ 10.1 Hz, H-3¢), 5.30 (1H, d, J2¢,3¢ 3.1 Hz, H-2¢), 5.29
(1H, t, J3¢,4¢ = J4¢,5¢ 10.1 Hz, H-4¢), 5.25 (1H, d, J2,3 3.1 Hz, H-2),
5.23 (1H, t, J3,4 = J4,5 10.1 Hz, H-4), 4.90 (1H, s, H-1¢), 4.75 (1H,
d, J 7.2 Hz, OCHHO), 4.71(1H, s, H-1), 4.64 (1H, d, J 7.2 Hz,
◦
D2O, 60 C) 5.19 (1H, d, J 11.6 Hz, OCHHO), 5.19 (1H, d, J1,2
1.7 Hz, H-1¢¢), 5.17 (1H, d, J 11.6 Hz, OCHHO), 5.17 (1H, d, J1,2
6584 | Org. Biomol. Chem., 2011, 9, 6579–6586
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OCHHO), 4.30 (1H, dd, J5¢,6¢a 5.0, J6a¢,6¢b 12.4 Hz, H-6¢a), 4.27 (1H,
dd, J5,6a 5.5, J6a,6b 12.2 Hz, H-6a), 4.13–4.09 (3H, m, H-3, H-6b,
H-6¢b), 4.02 (1H, ddd, J5¢,6¢a 5.0, J5¢,6¢b 2.2, J4¢,5¢ 10.1 Hz, H-5¢), 3.92
(1H, ddd, J5,6a 5.5, J5,6b 2.3, J4,5 10.1 Hz, H-5), 3.82–3.65 (4H, m,
OCH2CH2O), 3.39 (3H, s, OCH3), 2.18 (6H, m, COCH3 ¥ 2), 2.09
(9H, m, COCH3 ¥ 3), 2.03 (3H, s, COCH3), 1.98 (s, 3H, COCH3);
dC (150.92 MHz, CDCl3, 25 ◦C) 170.7, 170.6, 170.2, 169.9, 169.73,
169.71, 169.7, 98.6, 97.8, 94.4, 72.4, 69.6, 69.3, 69.0, 68.5, 68.4,
67.3, 66.9, 66.7, 66.1, 62.7, 62.4, 55.1, 21.0, 20.84, 20.81, 20.72,
20.69, 20.66, 20.61; HRMS (ESI): m/z calcd for C30H44O20+Na,
747.2325; found, 747.2344.
s, COCH3), 2.04 (6H, s, COCH3 ¥ 2), 1.99 (6H, s, COCH3 ¥ 2),
1.25–0.96 (28H, m, CH(CH3)2); dC (150.92 MHz, CDCl3, 25 ◦C):
170.6, 170.0, 169.8, 169.7, 100.2, 97.8, 97.7, 96.2, 95.8, 75.9, 74.4,
72.1, 70.7, 69.54, 69.48, 69.0, 68.4, 67.24, 67.21, 67.16, 66.6, 66.2,
66.1, 62.4, 54.8, 20.9, 20.74, 20.67, 17.5, 17.4, 17.3, 17.2, 12.9,
12.7, 12.2, 12.1; HRMS (ESI): m/z calcd for C53H88O29Si2+Na,
1267.4851; found, 1267.4869.
Methyl 2,6-di-O-(2,3,4,6-tetra-O-acetyl-a-D-mannnopyranosyl-
oxyethoxy-methyl)-3,4-di-O-acetyl-a-D-mannopyranoside
(22).
To a stirred solution of 20 (28 mg, 22.5 mmol) in THF (0.5 mL) was
added 1 M TBAF in THF (50 mL, 50 mmol). After 12 h at RT, the
solution was evaporated and the residue was chromatographed on
silica gel (CHCl3–MeOH 10 : 1). The product was acetylated with
pyridine–Ac2O (1 : 1, 1 mL) for 6 h. The reaction was quenched
by adding MeOH at 0 ◦C and the solution was evaporated.
The residue was chromatographed on siliga gel to give 22 (20
mg, 82%). Rf 0.23 (hexane–EtOAc 1 : 3); [a]2D5 +52.8 (c 0.29 in
CHCl3); dH (600 MHz, CDCl3, 25 ◦C, Me4Si) 5.37–5.32 (3H,
m, H-3¢, H-3¢¢, H-4), 5.30–5.26 (5H, m, H-2¢, H-2¢¢, H-3, H-4¢,
H-4¢¢), 4.87 (1H, s, H-1¢, H-1¢¢), 4.80–4.77 (3H, m, -OCH2O-,
H-1), 4.73 (1H, d, J 6.8 Hz, -OCHHO-), 4.73 (1H, d, J 7.0 Hz,
-OCHHO-), 4.30–4.26 (2H, m, H-6¢a, H-6¢¢a), 4.12–4.10 (2H,
m, H-6¢b, H-6¢¢b), 4.06–4.02 (2H, m, H-5¢, H-5¢¢), 4.00 (1H, t,
J1,2 = J2,3 1.7 Hz, H-2), 3.86 (1H, ddd, J5,6a 5.2, J5,6b 2.4, J4,5 9.9
Hz, H-5), 3.83–3.64 (9H, m, H-6a, -OCH2CH2O- ¥ 2), 3.61 (1H,
dd, J5,6b 2.3, J6a,6b 11.2 Hz, H-6b), 3.40 (3H, s, OCH3), 2.16 (6H,
s, COCH3 ¥ 2), 2.11 (6H, s, COCH3 ¥ 2), 2.06–2.03 (12H, m,
COCH3 ¥ 4), 1.99 (6H, s, COCH3 ¥ 2); dC (150.9 MHz, CDCl3,
25 ◦C) 170.6, 170.1, 170.0, 169.8, 99.5, 97.7, 95.73, 95.68, 74.4,
71.0, 69.5, 69.4, 69.0, 68.4, 67.2, 67.0, 66.8, 66.5, 66.3, 66.1, 62.42,
62.36, 55.1, 20.9, 20.8, 20.72, 20.67, 20.64; HRMS (ESI): m/z
calcd for C45H66O30+Na, 1109.3539; found, 1109.3556.
Methyl 2,3-di-O-(a-D-mannnopyranosyl-oxyethoxymethyl)-a-D-
mannopyranoside (18). To a stirred solution of 15 (30 mg,
30 mmol) in dry MeOH (2 mL) was added 0.5 M NaOMe (100 mL).
After 30 min, the solution was neutralized with Dowex50 (H+)
and evaporated to give 18 (19 mg, 95%). Rf 0.21 (iPrOH-H2O–
NH3 8 : 3 : 1); [a]2D5 +86.3 (c 0.40 in H2O); dH (600 MHz, D2O,
70 ◦C) 5.39–5.33 (7H, m, H-1, H-1¢, H-1¢¢, OCH2O ¥ 2), 4.50
(1H, dd, J1,2 1.8, J2,3 3.0 Hz, H-2), 4.43 (2H, dd, J1¢,2¢ 1.8, J2¢,3¢ 4.4
Hz, H-2¢, H-2¢¢), 4.38–4.08 (23H, m, H-3, H-3¢, H-3¢¢, H-4, H-4¢,
H-4¢¢, H-5, H-5¢, H-5¢¢, H-6a, H-6¢a, H-6¢¢a, H-6b, H-6¢b, H-6¢¢b,
OCH2CH2O ¥ 2), 3.70 (3H, s, OCH3); dC (67.8 MHz, D2O, 25 ◦C,
acetone) 100.6, 100.5, 99.8, 96.3, 95.4, 77.1, 75.9, 73.4, 73.4, 73.2,
71.2, 70.6, 67.7, 67.5, 67.4, 67.0, 66.9, 66.7, 61.6, 55.5; HRMS
(ESI): m/z calcd for C25H46O20+Na, 689.2482; found, 689.2447.
Methyl 2,6-di-O-(2,3,4,6-tetra-O-acetyl-a-D-mannnopyranosyl-
oxyethoxymethyl)-3, 4-O-(1,1,3,3-tetraisopropyl-1,3-disiloxane-
1,3-diyl)-a-D-mannopyranoside (20). A mixture of methyl 3,
4-O-(1,1,3,3-tetraisopropyl-1,3-disiloxane-1,3-diyl)-a-D-manno-
pyranoside (19)24 (27 mg, 62 mmol), 4 (64 mg, 141 mmol), TTBP
(78 mg, 315 mmol), and activated molecular sieves 4A (100 mg)
in p-xylene (2.5 mL) was stirred under N2 at RT for 30 min.
Immediately after the addition of MeOTf (21 mL, 186 mmol), the
mixture was rapidly mixed and frozen over dry ice in acetone.
The mixture was stored in refrigerator at 4 ◦C for 48 h and
defrosted at RT. Et3N (30 mL) was added to the mixture to
deactivate MeOTf. The mixture was diluted with EtOAc and
filtered through Celite. The filtrate was washed with brine, dried
over MgSO4, and evaporated. The residue was chromatographed
on silica gel (Hexane–EtOAc 4 : 3→1:3) to give 20 (41 mg, 53%)
and methyl 6- O-(2,3,4,6-tetra-O-acetyl-a-D-mannnopyranosyl-
oxyethoxymethyl)-3,4-O-(1,1,3,3-tetraisopropyl-1,3-disiloxane-
1,3-diyl)-a-D-mannopyranoside (21) (21 mg, 40%).
Methyl 2,6-di-O-(a-D-mannnopyranosyl-oxyethoxymethyl)-a-D-
mannopyranoside (23). To a stirred solution of 22 (20 mg,
19 mmole) in dry MeOH (2 mL) was added 0.5 M NaOMe
(100 mL). After 30 min, the solution was neutralized with Dowex50
(H+) and evaporated to give 23 (11 mg, 86%). Rf 0.18 (iPrOH-
H2O–NH3 8 : 3 : 1); [a]D25 +66.1 (c 0.40 in H2O); dH (D2O, 600
MHz, 40 ◦C) 5.09–4.99 (7H, m, H-1, H-1¢, H-1¢¢, -OCH2O- ¥
2), 4.16–3.84 (26H, m, H-2, H-2¢, H-2¢¢, H-3, H-3¢, H-3¢¢, H-4,
H-4¢, H-4¢¢, H-5, H-5¢, H-5¢¢, H-6a, H-6a¢, H-6a¢¢, H-6b, H-6b¢,
H-6b¢¢, -OCH2CH2O- ¥ 2), 3.60 (3H, s, OCH3); dC (150.92 MHz,
D2O, 25 ◦C, acetone) 100.5, 100.1, 96.6, 95.8, 77.7, 73.4, 71.2,
70.6, 67.6, 67.4, 67.1, 67.0, 61.6, 55.6; HRMS (ESI): m/z calcd for
C25H46O20+Na, 689.2482; found, 689.2478.
Compound 20: Rf 0.32 (hexane–EtOAc 1 : 2); [a]2D5 +53.8 (c 0.47
in CHCl3); dH (600 MHz, CDCl3, 25 ◦C, Me4Si) 5.36 (1H, dd,
J
2¢¢,3¢¢ 3.6, J3¢¢,4¢¢ 10.0 Hz, H-3¢¢), 5.35 (1H, dd, J2¢,3¢ 3.6, J3¢,4¢ 10.0 Hz,
H-3¢), 5.29 (1H, t, J3¢¢,4¢¢ = J4¢¢,5¢¢ 10.0 Hz, H-4¢¢), 5.285 (1H, d, J2¢¢,3¢¢
3.6 Hz, H-2¢¢), 5.28 (1H, t, J3¢,4¢ = J4¢,5¢ 10.0 Hz, H-4¢), 5.275 (1H,
d, J2¢,3¢ 3.6 Hz, H-2¢), 4.97 (1H, d, J 7.0 Hz, OCHHO), 4.88 (2H,
s, H-1¢, H-1¢¢), 4.78 (2H, d ¥ 2, J 7.0, 6.7 Hz, OCHHO ¥ 2), 4.75
(1H, d, J 6.7 Hz, OCHHO), 4.67 (1H, s, H-1), 4.30 (1H, dd, J5¢¢,6¢¢a
5.3, J6¢¢a,6¢¢b 12.0 Hz, H-6¢¢a), 4.29 (1H, dd, J5¢,6¢a 5.3, J6¢a,6¢b 12.0 Hz,
H-6¢a), 4.09 (2H, dd, J5¢,6¢b 2.2, J6¢¢a,6¢¢b 12.0 Hz, H-6¢b, H-6¢¢b), 4.05
(1H, ddd, J4¢¢,5¢¢ 10.0, J5¢¢,6¢¢a 5.3, J5¢¢,6¢¢b 2.2 Hz, H-5¢), 4.045 (1H, ddd,
J4¢,5¢ 10.0, J5¢,6¢a 5.3, J5¢,6¢b 2.2 Hz, H-5¢), 3.98 (1H, dd, J2,3 3.3, J3,4
8.9 Hz, H-3), 3.91–3.86 (3H, m, H-2, H-4, H-6a), 3.84-3.62 (10H,
m, H-5, H-6b, OCH2H2O ¥ 2), 3.38 (3H, s, OCH3), 2.160 (3H, s,
COCH3), 2.156 (3H, s, COCH3), 2.11 (3H, s, COCH3), 2.09 (3H,
Equlibrium dialysis assay. We used a clear acrylic multi-sample
dialysis cell (Sanplatec Corp. EB-0), which consists of a pair of
bars (L 300 ¥ W 35 ¥ H 15 mm) each having 8 cylinders (ø 12 ¥
D 7 mm) in a row. By tightly sandwiching a dialysis membrane
sheet (L 300 ¥ W 35 mm) on the bored surface of the bars using
nine screws in a row, one can make eight chambers with a 1 mL
volume bisected by the dialysis membrane. Each chamber has
two needle holes for the access to two rooms with a syringe. An
assay solution (1 mL), which is bisected by a dialysis membrane,
includes a sugar ligand with an appropriate concentration, NaCl
(0.15 M), CaCl2 (5 mM), MnCl2 (5 mM), and MgCl2 (5 mM) in
0.01 M phosphate-buffered saline (pH 7.3). One of the two bisected
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solutions is prepared so as to include 62.5 mM ConA. The ConA
concentration was determined by the Lowry method. The dialysis
cell was slowly shaken on a bath thermostated at 30 ◦C. After
7 h, the 100 mL solution free of ConA was diluted 2-fold with
water, which was then subjected to phenol-sulfuric acid assay for
sugar concentration. The calibration curves were made for all the
synthesized sugar ligands. The free sugar ligand concentrations
thus obtained were used to determine the Kd and n values using
Scatchard plots.
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This work was supported by a grant from the Global COE Pro-
gram and a Grant-in-Aid for Scientific Research (No. 19655056)
from the Japanese Ministry of Education, Culture, Sports, Science
and Technology. We thank Mr Satoshi Ganaha for his help in the
large scale synthesis of compound 4.
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