Synthetic study and structural analysis of the antifreeze agent xylomannan from Upis ceramboides

Article abstract of DOI:10.1021/ja208528c

The novel antifreeze factor, xylomannan, first isolated from the freeze-tolerant Alaskan beetle Upis ceramboides, demonstrates a high degree of thermal hysteresis, comparable to that of the most active insect antifreeze proteins. Although the presence of a lipid component in this factor has not yet been verified, it has been proposed that the glycan backbone consists of a β-d-mannopyranosyl-(1→4)-β-d-xylopyranose-disaccharide-repeating structure according to MS and NMR analyses. In this contribution, we report the stereoselective synthesis of the tetrasaccharide β-d-mannopyranosyl- (1→4)-β-d-xylopyranosyl-(1→4)-β-d-mannopyranosyl-(1→4) -d-xylopyranoside, a structural component of xylomannan. Our synthesis features the use of 2-naphthylmethyl (NAP)-ether-mediated intramolecular aglycon delivery (IAD) as the key reaction in obtaining β-mannopyranoside stereoselectively. Various donors for NAP-IAD were tested to determine the most suitable for the purposes of this synthesis. Fragment coupling between a disaccharyl fluoride and a disaccharide acceptor obtained from a common β-d-mannopyranosyl-(1→4)-β-d-xylopyranoside derivative was successfully carried out to afford the desired tetrasaccharide in the presence of Cp₂HfCl₂-AgClO₄. Structural analysis of the resulting synthetic tetrasaccharide using NMR techniques and molecular modeling was performed in order to demonstrate the presence of the proposed xylomannan linkages in this molecule.

Full text of DOI:10.1021/ja208528c

ARTICLE
pubs.acs.org/JACS
Synthetic Study and Structural Analysis of the Antifreeze Agent
Xylomannan from Upis ceramboides
Akihiro Ishiwata,*,† Ayaka Sakurai,† Yoshiyuki Nishimiya,‡ Sakae Tsuda,‡ and Yukishige Ito*,†,§
†RIKEN Advanced Science Institute, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan,
‡Research Institute of Genome-based Biofactory, National Institute of Advanced Industrial Science and Technology,
2-17-2-1 Tsukisamu-Higashi, Toyohira, Sapporo 062-8517, Japan, and
§ERATO Glycotrilory Project, Japan Science and Technology Agency (JST), 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
S Supporting Information
b
ABSTRACT: The novel antifreeze factor, xylomannan, first iso-
lated from the freeze-tolerant Alaskan beetle Upis ceramboides,
demonstrates a high degree of thermal hysteresis, comparable to
that of the most active insect antifreeze proteins. Although the
presence of a lipid component in this factor has not yet been
verified, it has been proposed that the glycan backbone consists of
a β-D-mannopyranosyl-(1f4)-β-D-xylopyranose-disaccharide-re-
peating structure according to MS and NMR analyses. In this
contribution, we report the stereoselective synthesis of the tetra-
saccharide β-D-mannopyranosyl-(1f4)-β-D-xylopyranosyl-(1f4)-β-D-mannopyranosyl-(1f4)-D-xylopyranoside, a structural com-
ponent of xylomannan. Our synthesis features the use of 2-naphthylmethyl (NAP)-ether-mediated intramolecular aglycon delivery
(IAD) as the key reaction in obtaining β-mannopyranoside stereoselectively. Various donors for NAP-IAD were tested to determine the
most suitable for the purposes of this synthesis. Fragment coupling between a disaccharyl fluoride and a disaccharide acceptor obtained
from a common β-D-mannopyranosyl-(1f4)-β-D-xylopyranoside derivative was successfully carried out to afford the desired
tetrasaccharide in the presence of Cp2HfCl2ꢀAgClO4. Structural analysis of the resulting synthetic tetrasaccharide using NMR
techniques and molecular modeling was performed in order to demonstrate the presence of the proposed xylomannan linkages in this
molecule.
’ INTRODUCTION
is considered to consist of a β-D-mannopyranosyl-(1f4)-
β-D-xylopyranose-disaccharide-repeating unit, according to MS
and NMR analyses (Figure 1).
Antifreeze substances such as antifreeze proteins (AFPs) and
glycoproteins (AFGPs) are vitally important for the survival of
plants, insects, collembola, bacteria, and fungi, especially in the
Arctic and Antarctic and their subregions.1,2 Both AFP and
AFGP are capable of inducing thermal hysteresis (TH) and thus
protect the cell membranes of cold-tolerant organisms from
damage due to extracellular ice-crystal formation at low temper-
atures. Such TH factors (THF) have not been precisely charac-
terized structurally, although it has been suggested that they
function by binding to the surface of ice crystals to prevent
further ice formation,3 for which an anchored clathrate mech-
anism4 has been recently proposed. Synthetic studies on various
artificial antifreeze substances, especially those having a glyco-
protein moiety, have also been reported.5
In 2009, Walters et al. reported the isolation of the novel
antifreeze xylomannan from the freeze-tolerant Alaskan beetle
Upis ceramboides.6 This substance was subsequently also identi-
fied in other diverse taxa.7 This THF shows 3.7 ( 0.3 °C of TH at
a concentration of 5 mg/mL, which is comparable to that of
the most active insect AFP, although it has been suggested that
it contains little or no protein. The presence of lipid com-
ponents in this THF has not been verified, butits glycan backbone
In this study, we achieved the stereoselective synthesis of a
structural portion of xylomannan; the tetrasaccharide, β-D-man-
nopyranosyl-(1f4)-β-D-xylopyranosyl-(1f4)-β-D-mannopyra-
nosyl-(1f4)-D-xylopyranoside (Man2Xyl2). Our synthesis
features the use of 2-naphthylmethyl (NAP)-ether-mediated
intramolecular aglycon delivery (IAD)8 as the key reaction to
produce β-mannopyranoside stereoselectively (Figure 2). Var-
ious mannopyranose donors for NAP-IAD were examined to
determine their suitability for this purpose. Detailed structural
analysis of the resulting tetrasaccharide has been carried out to
support the presence of the proposed xylomannan linkages.6
’ RESULTS AND DISCUSSION
The stereoselective synthesis of 1,2-cis-glycosides such as
β-mannopyranoside is potentially problematic,9 and a number
of strategies to accomplish this type of synthesis have been
explored.10 Among these, approaches based on intramolecular
Received: September 9, 2011
Published: October 26, 2011
r
2011 American Chemical Society
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aglycon delivery (IAD)11 are especially promising, since they are
predicted to result in the exclusive formation of 1,2-cis-glyco-
sides. The concept of IAD was first proposed by Baressi and
Hindsgaul,12 who employed isopropylidene mixed acetals as a
tether to make a temporary linkage between donor and acceptor
as a mixed acetal. Subsequent work by Stork et al.13 explored the
use of silaketal for similar purposes. Following these pioneering
reports, newer versions of IAD have been developed using
various tethers including the p-methoxybenzylidene acetal uti-
lized in our work.14 Efficiency of the p-methoxybenzyl-ether-
assisted β-mannopyranosylation was optimized by the introduc-
tion of a 4,6-O-cyclic protective group15 especially in reactions
with challenging acceptors such as chitobiose derivatives. We
recently found that 2-naphthylidene acetal is highly effective as a
tether for IAD and that NAP ether-mediated IAD could be
applied to the formation of various types of 1,2-cis-glycosides8
such as β-D-mannopyranosides, β-D-arabinofuranosides,8c
α-D-glucopyranosides, and β-L-rhamnopyranosides,8b resulting
in high yields with complete selectivity. The NAP-protected
donor was cleanly converted to the mixed acetal upon oxidative
activation with DDQ. This was followed by the subsequent
activation of the thioglycosidic linkage to initiate rearrangement
of an aglycon from the 2-naphthylidene acetal moiety, affording
1,2-cis-glycosides (Figure 2).
obtained from a common mannopyranosylxylopyranoside
(ManXyl)1 fragment, allowing fragment couplings to produce
a xylosyl-mannoside linkage with a 1,2-trans-β-configuration.
For the fragment coupling, fluoride was chosen as the leaving
group, due to its high reactivity16 and its use in standardized syn-
thetic procedures involving the application of 4-methoxyphenyl
glycoside.17 Synthesis of a common (ManXyl)1 fragment was to
be achieved by the stereoselective introduction of β-mannopyr-
anose residues by using our NAP-protected mannopyranose
donors with xylopyranose acceptor via IAD.
Synthesis of Monosaccharide Fragments. NAP-protected
mannopyranosyl donors for the synthesis of xylomannan frag-
ment were prepared from methyl 4,6-O-benzylidene-1-thio-α-D-
mannopyranoside 1518 (Figure 2, Scheme 1). Regioselective
protection of diol 15 gave the 3-O-triisopropylsilyl (TIPS) ether
16, which was then equipped with a 2-O-NAP group to give 17.
The TIPS ether was then converted to a benzyl ether through 18.
Reductive ring-opening of the resultant 19 with trifluoroacetic
acid (TFA) and triethylsilane (TESH)19 gave 3,6-di-O-benzyl
derivative 20 in good yield. A triethylsilyl (TES) group was
introduced to 20 by treatment with TESCl to give 1, while
conversion to the TIPS ether 2 required more vigorous reaction
conditions and the use of 5 equiv of TIPSOTf. Tri-O-benzyl-
protected methyl thioglycoside 3 was also synthesized from 20.
Our synthetic scheme for xylomannan is depicted in Figure 1.
It employs a disaccharide (ManXyl)1 donor and acceptor
Scheme 1. Synthesis of 2-O-NAP-Ether-Protected Mannose
donorsa
a Reagents and conditions: (a) TIPSCl, imidazole, DMF, 70%; (b)
NAPBr, NaH, DMF, 95%; (c) TBAF, 90%; (d) BnBr, NaH, DMF, 92%
(19), 96% (3); (e) TESH, TFA, CH2Cl2, 86%; (f) TESCl, imidazole,
DMF, 87%; (g) TIPSOTf, 2,6-lutidine, 85%.
Figure 1. Retrosynthetic analysis of xylomannan.
Figure 2. NAP-ether-mediated intramolecular aglycon delivery.
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Xylopyranose acceptor 5 was synthesized from peracetylated
xylopyranose 2120 as depicted in Scheme 2. Reaction of 21 with
4-methoxyphenol in the presence of TMSOTf unexpectedly gave
the α-glycoside 22α (JH1,H2 = 3.7 Hz) exclusively. The same
reaction at lower temperature in the presence of triethylamine
(TEA)21 resulted in β-selective glycoside formation, albeit in low
yield. Since β-glycoside 22β (JH1,H2 = 6.4 Hz) was observed to
isomerize readily to 22α, we decided to use α-glycoside to avoid
any complications caused by isomerization at a later stage in the
synthesis. Deprotection of the acetate followed by regioselective
acetonide formation of the triol 23 with 2-methoxypropene22
gave 24. Temporary TIPS protection of 24 followed by depro-
tection of the acetonide of 25, benzoylation of resultant diol 26
and deprotection of the TIPS ether of 27 gave a xylose acceptor
5, whose regioselective formation was confirmed by 1H NMR of
5 (d 5.31 ppm for 2-H and 5.76 ppm for 3-H).
β-Mannnopyranosylation through NAP-Ether-Mediated
IAD. In order to achieve the synthesis of xylomannan disaccharide
fragments, we examined the use of β-mannopyranosylation
through NAP-ether-mediated intramolecular aglycon delivery
(IAD).8 Although the importance of 4,6-O-cyclic protection
in achieving the highest possible efficiency in IAD has been
reported,15,23 we experimented with the use of the 3,6-di-O-
benzyl-protected flexible donors 1 and 2. While IAD of 1 with 5
under standardized conditions gave 53% yield of the desired
β-mannopyranoside 8 (Table 1, entry 1),24 concomitant forma-
tion of dimeric 2-naphthaldehyde bis(disaccharyl)-acetal was
evident by MALDI TOF MS analysis ([M + Na]+ calcd for
C115H126O26Si2Na1, 2001.80, found 2001.99). Accordingly, we
carried out acidic workup of the crude IAD mixture followed by
acetylation to give diacetate 9 in 93% yield (entry 2). In order
to preserve a silyl group at the 4-position, the TIPS protected
donor 2 was employed, which gave mannopyranosylxylopyranoside
(ManXyl)1 as the 2-O-acetate 10 in good yield (entry 3), which
was then used as the key intermediate in subsequent work.
Application of the 3,6-di-O-benzyl-protected donors 2 and 3
in the context of the synthesis of the core structures of N-glycan
was also examined using acceptors 625 and 7.26 With the gluco-
samine acceptor 6, both 2 and 3 gave the desired β-mannopyra-
nosides (11, 12) cleanly (entries 4 and 6), with little or no
reduction of yield compared to the previously reported 4,6-O-
cyclohexylidene-protected donor 48a which gave 138a (entry 7).
Even reaction with a chitobiose acceptor 7 (entry 5), which has
been a challenging acceptor for IAD without the use of a cyclic
acetal-protected donor,15 proceeded without difficulty, giving the
desired core trisaccharide derivative (Man1GlcNAc2, 14) in a
highly satisfactory yield of 83%.27
Scheme 2. Synthesis of Xylose Acceptor 5a
a Reagents and conditions: (a) 4-methoxyphenol, TMSOTf, CH2Cl2,
90% (α); (b) MeONa, MeOH, 95%; (c) 2-methoxypropene, CSA, 74%;
(d) TIPSCl, imidazole, DMF; (e) THFꢀAcOHꢀH2O; (f) BzCl,
DMAP, pyridine, 79% in three steps; (g) TBAFꢀAcOH, 72%.
Table 1. β-Mannosylation Using Various Mannopyranose
Donors through NAP-IADa
Synthesis of the Tetrasaccharide and Structural Analysis.
The common disaccharide fragment 10 was diverged to a dis-
accharide acceptor 30 and a disaccharide donor 32 as shown in
Scheme 3. After conversion of acetate and benzoate groups
to benzyl ethers through triol 28, the TIPS ether of 29 was
deprotected to give 30 in good yield.28 Deprotection of the
4-methoxyphenyl glycoside of 10 with CAN followed by fluori-
nation of 31 with DAST gave the fluoride 32 in good yield.
The fragment coupling of 30 with 32 was carried out under
Suzuki’s Cp2HfCl2ꢀAgClO4 conditions29 in benzene16b to afford
the tetrasaccharide (ManXyl)2 derivative 33 in good yield. Con-
figuration of the newly generated β-xyloside moiety was assigned
entry
Db
Ab
MAb (%)
product (%)
JC1,H1 (Hz)
1
2
3
4
5
6
7
1
1
2
2
2
3
4
5
5
5
6
7
6
6
93
ꢀ
8 (55, β)
9 (93, β)
10 (93, β)
11 (90, β)
14 (90, β)
12 (85, β)
13 (90, β)
ꢀ
161.2
157.4
160.2
163.1
161.2
161.7
95
96
92
92
quant.
a Reagents and conditions: (a) DDQ, MS4A, CH2Cl2; (b) i) MeOTf,
DTBMP, MS4A, (CH2Cl)2; ii) for entries 2ꢀ7: TFA, CH2Cl2, then
Ac2O, pyridine. b D: donor, A: acceptor, MA: mixed acetals.
1
by H NMR (JH1,H2 = 6.4 Hz). Conventional three-step
Scheme 3. Synthesis of Xylomannan Tetrasaccharide Fragment 36a
a Reagents and conditions: (a) MeONa, MeOHꢀTHF (1:1); (b) NaH, BnBr, DMF; (c) TBAF, THF (85% in three steps); (d) CAN, CH3CNꢀH2O;
(e) DAST, CH2Cl2, 67% in two steps (α:β = 84:16); (f) Cp2HfCl2, AgClO4, PhH, 88% (β); (g) TBAF, THF; (h) MeONa, MeOH; (i) Pd(OH)2, H2,
MeOHꢀH2O (2:1), 70% in three steps.
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1
1
Table 2. 13C and 1H NMR Chemical Shifts and 1Hꢀ H and 13Cꢀ H Spin-Couplings of Saccharide Signals for Reported
Xylomannan and Synthetic Man2Xyl2, 36, and Standard β-Glycosides
chemical shifts (ppm)
C1 (H1)
C2 (H2)
C3 (H3)
C4 (H4)
C5 (H5, H50)
J-coupling (Hz)
C6 (H6, H60)
Man:
xylomannana
β-Man2-b
4)-β-Man1-b
Δδ (ppm)c
β-D-Man-OMed
1JC1,H1
∼160
162.1
163.9
100.2 (4.92)
98.5 (4.94)
98.6 (4.97)
ꢀ1.6 (+0.05)
101.3 (4.66)
70.1 (4.30)
71.0 (4.15)
70.2 (4.20)
+0.1 (ꢀ0.10)
70.6 (4.07)
71.6 (3.93)
73.1 (3.81)
71.7 (3.90)
+0.1 (ꢀ0.03)
73.3 (3.72)
76.6 (3.96)
67.0 (3.75)
76.6 (3.95)
(0.0 (ꢀ0.01)
67.1 (3.63)
75.1 (3.74)
76.6 (3.56)
75.4 (3.70)
+0.3 (ꢀ0.04)
76.6 (3.46)
60.6 (4.09, 3.93)
61.3 (4.12, 3.91)
60.6 (4.18, 4.00)
( 0.0 (+0.09, +0.07)
61.4 (4.02, 3.83)
159.5
Xyl:
xylomannana
4)-β-Xyl2-b
Δδ (ppm)c
4)-α-Xyl1-OMPb
β-D-Xyl-OMed
3JH1,H2
7.8
101.7 (4.66)
103.5 (4.60)
+1.8 (ꢀ0.06)
98.6 (5.68)
72.8 (3.48)
73.1 (3.51)
+0.3 (+0.03)
71.8 (3.93)
74.0 (3.35)
73.8 (3.73)
74.1 (3.79)
+0.3 (+0.06)
77.0 (4.17)
76.9 (3.53)
76.6 (4.00)
76.5 (4.05)
ꢀ0.1 (+0.05)
72.2 (4.10)
70.4 (3.71)
63.0 (4.28, 3.55)
63.2 (4.30, 3.56)
+0.2 (+0.02, +0.01)
59.8 (4.03, 3.89)
66.3 (4.06, 3.42)
8.0
4.0
7.8
105.1 (4.42)
a Data from ref 6. b Data of residues from synthetic 36 measured in pH 7.5 phosphate buffer at 40 °C. c The differences from reported xylomannan. d Data
from ref 32.
deprotection afforded (ManXyl)2 derivative 36. The synthetic
tetrasaccharide derivative exhibited neither TH nor recrystalliza-
tion inhibition activities.6,7,30,31 These results are not unexpected,
because suppression of TH activity after degradation of xylo-
mannan by β-(1f4)xylosidase has been reported.6
single helix (Figure 3c).33,36 In addition, calculations were per-
formed on the basis of the planar model structure of cellulose,35
which has two constrained torsion angles (∼98° and 140°
for O5ꢀC1ꢀO1ꢀC40 and C1ꢀO1ꢀC40ꢀC50, respectively)
(Figure 3d). The analysis suggested that, due to the absence of
HO-C6H2 and HO-C2 in the xylose and mannose residues,
respectively, the interaction between each planar structure of
xylomannan chains via intermolecular hydrogen bonding35 is
likely far less significant than in the cellulose three-dimensional
planar structure. In the planar structure of xylomannan, all
the axial HO-C2 groups of mannoside direct to the same phase,
resulting in a difference in hydrophobicity between the two
phases of the xylomannan chain. The repeat spacing of the
oxygen atoms of water molecules in an ice lattice on the primary
prism plane (7.35, 4.52 Å) and the basal plane (7.83, 4.52 Å),37
however, did not correlate well with the distance between the
axial HO-C2 groups of mannose residues (10.7 Å).33
NMR analysis of synthetic (ManXyl)2 36 was carried out at
40 °C to allow comparison with reported 13C and 1H chemical
1
1
1
shifts and Hꢀ H and 13Cꢀ H spin-couplings of xylomannan
(Table 2).32 Both chemical shifts and spin-couplings of the
internal β-mannopyranoside (Man1) and β-xylopyranoside
(Xyl2) of 36 were in good agreement with those of the core
(ManXyl)n structure of xylomannan, especially for carbon reso-
nance of the 4-position of the pyranoside. Although the 13C
signal at 103.5 ppm assignable to C1 of Xyl2 appeared was
discernibly different from that reported for xylomannan (101.7
ppm), our results generally support the proposed xylomannan
structure as consisting of repeating f4)-β-Man and f4)-β-Xyl
linkages.
All 1-Hꢀ4-H0 cross peaks related to both Man-(1f4)-β-Xyl
and Xyl-(1f4)-β-Man linkages (linkage A and linkage B, respec-
tively) were detected during ROESY analysis of 36 (Figure 3a).
Molecular modeling of (ManXyl)n (n = 2, 1033) using Macro-
Model, ver 8.1,34 supported the ROESY results concerning both
the 1,4-linkages (Figure 3b). Both NMR and modeling studies
suggested that the 1,4-linkages result in the formation of intra-
and inter-residual hydrogen bonding, the stabilizing effects of
which cause slight conformational differences between these
two linkages. Specifically, Man-(1f4)-β-Xyl (A) forms the
’ CONCLUSION
In this study we achieved the stereoselective synthesis of the
tetrasaccharide β-D-mannopyranosyl-(1f4)-β-D-xylopyranosyl-
(1f4)-β-D-mannopyranosyl-(1f4)-D-xylopyranoside, proposed
as a structural component of the novel natural antifreeze
xylomannan. As the key step, we employed the use of 3,6-di-O-
benzyl-protected open-type mannopyranose donors for NAP-
ether-mediated intramolecular aglycon delivery. This procedure
was applied to the synthesis of various β-mannopyranoside-
containing oligosaccharides including the xylomannan fragment
and core trisaccharide of N-glycans. Structural analysis via NMR
and molecular modeling of the tetrasaccharide obtained through
fragment coupling of a disaccharyl fluoride and a disaccharide
acceptor supported the proposed structure. Although some
details of the structure of xylomannan are still unknown (includ-
ing its molecular weight and the regularity of the repeating
disaccharide) and its mode of action is not entirely clear, the
proposed helical and/or biphasic nature of this molecule might
account for its utility in preventing the growth of ice crystals.38
inter-residual ring C5ꢀO HꢀOꢀC30 as well as intra-residual
3 3 3
C5ꢀO HꢀOꢀC6 hydrogen bonds, as has previously been
3 3 3
proposed for cellulose, the most common β-(1f4)-glycan
which has a planar structure in the solid state.35 Since xylose,
unlike glucose, lacks the HO-C6H2 group, only the presence of
the inter-residual ring C5ꢀO HꢀOꢀC30 contributes to the
3 3 3
formation of a three-dimensional hydrogen-bond network, re-
sulting in a smaller degree of conformational turn as compared to
the Man-(1f4)-β-Xyl linkage. The angles between two manno-
side units is on the order of 70°, causing (ManXyl)5 to form a
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HOD (4.80), or CD3OD (3.30 ppm). 13C NMR spectra were recorded at
100 MHz on the same instrument, and chemical shifts are referred to internal
CDCl3 (77.0 ppm), C6D6 (128.0 ppm), CD3OD (49.0 ppm), (CD3)2CO
(34.1 ppm), or native scale. MALDI-TOF mass spectra were recorded
on a SHIMADZU Kompact MALDI AXIMA-CFR spectrometer with
2,5-dihydroxybenzoic acid as the matrix. ESI-TOF mass spectra were
recorded on a JEOL AccuTOF JMS-T700LCK (RIKEN Advanced
Science Institute) with CF3CO2Na as the internal standard.
Methyl 3,6-Di-O-benzyl-2-O-(2-naphthylmethyl)-1-thio-4-O-trieth-
ylsilyl-α-D-mannopyranoside (1). To a solution of 20 (500.0 mg, 0.95
mmol) and 2,6-lutidine (0.335 mL, 2.85 mmol) in CH2Cl2 (5.0 mL) was
added dropwise TESOTf (0.474 mL, 2.09 mmol) at 0 °C under Ar
atmosphere, and the mixture was stirred for 4 d at room temperature.
The reaction was quenched with triethylamine, extracted with CHCl3,
washed with sat. NaHCO3 aq and brine, dried over Na2SO4, and
evaporated in vacuo. The residue was purified by silica gel column
chromatography using a gradient solvent system (toluene/ethyl acetate =
100/1 to 50/1 to 20/1 to 10/1) to give the title compound 1 (534.4 mg,
87%). 1: [α]25D 46.9° (c 1.0, CHCl3); 1H NMR (400 MHz, CDCl3): δ
0.43ꢀ0.60 (m, TES, 6H), 0.82ꢀ0.88 (m, TES, 9H), 2.10 (s, SMe, 3H),
3.63 (dd, J = 8.7, 2.8 Hz, 3-H, 1H), 3.70ꢀ3.76 (m, 6a-H, 6b-H, 2H), 3.81
(dd, J = 2.8, 1.8 Hz, 2-H, 1H), 3.96ꢀ4.01 (m, 5-H, 1H), 4.05 (t, J =
9.2 Hz, 4-H, 1H), 4.52 (s, CH2Ph, 2H), 4.59 (s, CH2Ph, 2H), 4.66 (d, J =
12.8 Hz, CH2Ph, 1H), 4.77 (d, J = 12.8 Hz, CH2Ph, 1H), 5.29 (d, J =
1.8 Hz, 1-H, 1H), 7.17ꢀ7.79 (m, Ar, 17H); 13C NMR (100 MHz, CDCl3):
δ 5.08, 6.93, 13.6, 68.4, 69.7, 71.6, 72.1, 73.3, 73.5, 75.9, 80.6, 83.2, 125.68,
125.73, 126.0, 126.2, 127.3, 127.4, 127.59, 127.63, 127.9, 128.0, 128.2, 132.9,
133.2, 125.8, 138.35, 138.44; MALDI-TOF MS: [M + Na]+ calcd for
C38H48O5S1Si1Na1, 667.29, found 667.77. ESI-TOF MS: [M + Na]+
calcd for C38H48O5S1Si1Na1, 667.29, found 667.21; HRMS ESI-TOF:
[M + Na]+ calcd for C38H48O5S1Si1Na1, 667.2889, found 667.2871.
Methyl 3,6-Di-O-benzyl-2-O-(2-naphthylmethyl)-1-thio-4-O-triiso-
propylsilyl-α-D-mannopyranoside (2). To a solution of 20 (1.6023 g,
3.02 mmol) and 2,6-lutidine (2.665 mL, 22.65 mmol) in CH2Cl2
(30 mL) was added dropwise TIPSOTf (4.1 mL, 15.1 mmol) at 0 °C
under Ar atmosphere, and the mixture was stirred for 24 h at room tem-
perature. The reaction was quenched with triethylamine, extracted with
CHCl3, washed with sat. NaHCO3 aq and brine, dried over Na2SO4, and
evaporated in vacuo. The residue was purified by silica gel column
chromatography using a gradient solvent system (toluene/ethyl acetate =
100/1 to 50/1 to 20/1 to 10/1) to give the title compound 2 (1.7587 g,
85%). 2: [α]25D 39.8° (c 1.0, CHCl3); 1H NMR (400 MHz, CDCl3): δ
1.02ꢀ1.04 (m, TIPS, 21H), 1.97 (s, SMe, 3H), 3.89ꢀ4.00 (m, 2-H, 3-H,
6a-H, 6b-H, 4H), 4.18 (d, J = 11.5 Hz, CH2Ph, 1H), 4.37ꢀ4.43 (m, 5-H,
CH2Ph ꢁ 2, 3H), 4.57ꢀ4.63 (m, 4-H, CH2Ph ꢁ 3, 4H), 5.43 (s, 1-H, 1H),
7.04ꢀ7.35 (m, Ar, 12H), 7.44 (d, J = 8.7 Hz, Ar, 1H), 7.58ꢀ7.61
(m, Ar, 2H), 7.65 (d, J = 7.3 Hz, Ar, 1H), 7.78 (s, Ar, 1H); 13C NMR
(100 MHz, CDCl3): δ 12.9, 13.5, 18.1, 18.2, 68.8, 69.8, 71.1, 71.9, 73.2,
74.0, 75.3, 80.6, 82.4, 125.5, 125.7, 125.9, 126.1, 127.3, 127.4, 127.5,
127.6, 127.8, 127.9, 128.1, 128.2, 132.9, 133.6, 135.7, 137.2, 138.3;
MALDI-TOF MS: [M + Na]+ calcd for C41H54O5S1Si1Na1, 709.34,
found 709.14. ESI-TOF MS: [M + Na]+ calcd for C41H54O5S1Si1Na1,
709.34, found 709.25; HRMS ESI-TOF: [M + Na]+ calcd for C41H54-
O5S1Si1Na1, 709.3359, found 709.3386.
Figure 3. Analysis of β-1,4-glycan structure of xylomannan. (a) ROESY
correlation of two linkages (linkage A: β-Man-(1f4)-Xyl and linkage B:
β-Xyl-(1f4)-Man) using tetrasaccharide 36. (b) Global minimum
structure of tetrasaccharide β-Man-(1f4)-β-Xyl-(1f4)-β-Man-
(1f4)-β-Xyl in H2O. Manno- and xylopyranosides are colored gray
and green, respectively. Dotted lines indicate possible hydrogen bond-
ings. (c) Possible hydrogen-bond network of the two linkages calculated
by MacroModel, ver 8.1. The single- and double-dotted squares indicate
the absence of functionality in mannoside and xyloside from cellulose,
respectively. (d) Global minimum structure of the planar model struc-
ture of tetrasaccharide β-Man-(1f4)-β-Xyl-(1f4)-β-Man-(1f4)-β-
Xyl in H2O using the parameter for that of cellulose which has two con-
strained torsion angles (∼98° and 140° for O5ꢀC1ꢀO1ꢀC40 and C1ꢀ
O1ꢀC40 ꢀC50, respectively) with force constants of 300 kJ/mol Å2.
3
The results of antifreeze activity measurements of the resulting
tetrasaccharide indicate that a longer repeating motif of xylo-
mannan should be required for these activities. Further synthetic
and structural studies are underway to clarify these intriguing
issues.
’ EXPERIMENTAL SECTION
General Procedure. All reactions sensitive to air and/or moisture
were carried out under nitrogen or argon atmosphere with anhydrous
solvents. Substrates of glycosylations were dried by azeotropic removal
with toluene. Column chromatography was performed on silica gel 60N,
100ꢀ210 mesh (Kanto Kagaku Co., Ltd.). Preparative thin layer chro-
matography (PTLC) was performed on silica gel 60 F254,0.5mm(E.Merck).
Gel filtration was performed on Sephadex LH-20 (Pharmacia). All other
reagents were purchased from Wako Pure Chemical Industries Ltd.,
Kanto Chemicals Co. Inc., Tokyo Kasei Kogyo Co., Ltd. and Aldrich
Chemical Co.. Optical rotations were measured with a JASCO DIP 370
polarimeter. The melting point was determined with a B€uchi 510
melting point apparatus. 1H NMR spectra were recorded at 400 MHz
on a JEOL JNM-AL 400 or ECX 400 spectrometer, and chemical shifts
are referred to internal tetramethylsilane (0 ppm), CDCl3 (7.24 ppm),
Methyl 3,4,6-Tri-O-benzyl-2-O-(2-naphthylmethyl)-1-thio-α-D-manno-
pyranoside (3). 3 was synthesized according to the procedure for the
synthesis of 19 except using 20 instead of 18 as starting material (96%).
3: [α]27D 51.8° (c 1.1, CHCl3); 1H NMR (400 MHz, CDCl3): δ 2.08
(s, SMe, 3H), 3.72 (dd, J = 11.2, 2.0 Hz, 6-H, 1H), 3.81 (dd, J = 11.2,
4.8 Hz, 6-H, 1H), 3.38ꢀ3.87 (m, 2-H, 3-H, 2H), 4.04 (dd, J = 10.0,
8.4 Hz, 4-H, 1H), 4.07ꢀ4.18 (m, 5-H, 1H), 4.50 (d, J = 10.8 Hz, CH2Ar,
1H), 4.52 (d, J = 12.4 Hz, CH2Ar, 1H), 4.53 (d, J = 11.8 Hz, CH2Ar, 1H),
4.58 (d, J = 11.8 Hz, CH2Ar, 1H), 4.65(d, J = 12.4 Hz, CH2Ar, 1H),
4.79 (d, J = 12.8 Hz, CH2Ar, 1H), 4.87 (d, J = 12.8 Hz, CH2Ar, 1H), 4.87
19528
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Journal of the American Chemical Society
ARTICLE
(d, J = 10.8 Hz, CH2Ar, 1H), 5.32 (s, 1-H, 1H), 7.15ꢀ7.81 (m, Ar, 22H);
13C NMR (100 MHz, CDCl3): δ 13.7 (q), 69.0 (t, C6), 71.8 (d, C5),
71.9 (t), 72.1 (t), 73.2 (t), 74.9 (d, C4), 75.0 (t), 76.0 (d, C2), 80.2
(d, C3), 83.1 (C1, JC,H = 168.81 Hz), 125.8, 125.9, 126.0, 126.5, 127.41,
127.49, 127.54, 127.58, 127.66, 127.8, 128.1, 128.2, 128.3, 132.9, 133.1,
135.4, 138.1, 138.2, 138.3. ESI-TOF MS: [M + Na]+ calcd for C39H40-
O5S1Na1, 643.25, found 643.24; HRMS ESI-TOF: [M + Na]+ calcd for
C39H40O5S1Na1, 643.2494, found 643.2469.
(1+5) (92.0 mg, 83.1 mmol) were added DTBMP (104.3 mg, 497 μmol)
and MS4A (100 mg/mL) in dry (CH2Cl)2 (8.5 mL) at room temperature
under AratmosphereThenMeOTf (42.3 μL, 374 μmol) was added to the
mixture, and the mixture was stirred for 20 h at 40 °C. The reaction
mixture was quenched with triethylamine, diluted with ethyl acetate, and
filtered through Celite. The filtrate was washed with sat. NaHCO3 aq and
brine. The washed organic layer was dried over Na2SO4 and evaporated in
vacuo. To the mixture in CH2Cl2 (10.0 mL) was added TFA (1.0 mL) at
0 °C, and themixture was stirredfor 30min atthe sametemperature. After
evaporation, to the mixture in pyridine (0.5 mL) was added Ac2O
(0.1 mL), and the mixture was stirred for 1 h at room temperature. After
evaporation, the residue was diluted with ethyl acetate, washed with sat.
NaHCO3 aq and brine, dried over Na2SO4, and evaporated in vacuo. The
crude product was purified by gel filtration chromatography (SX-3, ethyl
acetate/toluene = 1/1) togivethe product (68.6mg, 93%, β) asacetate. 9:
[α]29D 27.6° (c1.0, CHCl3);1H NMR (400 MHz, C6D6):δ1.65 (s, Ac, 3H),
4-Methoxyphenyl 3,6-Di-O-benzyl-4-O-triethylsilyl-β-D-mannopy-
ranosyl-(1f4)-2,3-di-O-benzoyl-α-D-xylopyranoside (8). General Pro-
cedure for the Synthesis of Mixed Acetal (MA). To a mixture of acceptor
5 (46.4 mg, 100 μmol), donor 1 (67.7 mg, 105 μmol), and dried
powdered MS4A (100 mg) in dry CH2Cl2 (1.0 mL) was added DDQ
(27.8 mg, 120 μmol) at 0 °C under Ar atmosphere. The mixture was
stirred for 4 h at room temperature, quenched with aqueous ascorbate
buffer (L-ascorbic acid (0.7 g), citricacidmonohydrate (1.2 g), and NaOH
(0.92 g) in H2O (100 mL)), and then was filtered through Celite. The
filtrate was extracted with CHCl3 and washed with sat. NaHCO3 aq
and brine. The combined organic layers were dried over Na2SO4 and
evaporated in vacuo. The crude product was purified by gel filtration
chromatography (SX-3, ethyl acetate/toluene = 1/1) to give the mixed
acetal (103.2 mg, 93%). Mixed acetal (1+5): 1H NMR (400 MHz, C6D6):
δ 0.56ꢀ0.65 (m, TES, 6H), 0.90ꢀ0.94 (m, TES, 9H), 2.11 (s, SMe, 3H),
3.19 (s, OMe, 3H), 3.73 (dd, J = 11.0, 1.4 Hz, 6a-HMan, 1H), 3.77 (dd, J =
9.6, 3.2 Hz, 3-HMan, 1H), 3.90 (dd, J = 11.0, 5.0 Hz, 6b-HMan, 1H),
4.10ꢀ4.33 (m, 2-HMan, 5-HMan, 4-HXyl, 5a-HXyl, 5b-HXyl, CH2Ph ꢁ
2, 7H), 4.44ꢀ4.52 (m, 4-HMan, CH2Ph, 2H), 4.60 (d, J = 12.4 Hz,
CH2Ph, 1H), 5.32 (dd, J = 10.5, 3.6 Hz, 2-HXyl, 1H), 5.72 (d, J = 1.4 Hz,
1-HMan, 1H), 5.81 (d, J = 2.3 Hz, 1-HXyl, 1H), 5.82 (s, >CHNaph, 1H),
6.50ꢀ6.52 (m, Ar, 2H), 6.71 (dd, J = 10.1, 9.6 Hz, 3-HXyl, 1H), 6.82ꢀ6.93
(m, Ar, 5H), 6.98ꢀ7.20 (m, Ar, 11H), 7.26ꢀ7.30 (m, Ar, 2H), 7.35ꢀ7.37
(m, Ar, 2H), 7.50ꢀ7.66 (m, Ar, 4H), 8.10ꢀ8.17 (m, Ar, 5H); MALDI-
TOF MS: [M + Na]+ calcd for C64H70O13S1Si1Na1, 1129.42, found
1129.71. ESI-TOF MS: [M + Na]+ calcd for C64H70O13S1Si1Na1,
1129.42, found 1129.31; HRMS ESI-TOF: [M + Na]+ calcd for
C64H70O13S1Si1Na1, 1129.4204, found 1129.4240.
1.72 (s, Ac, 3H), 3.23 (s, OMe, 3H), 3.28ꢀ3.46 (m, 3-HMan, 5-HMan
,
6-H2Man, 4H), 3.75 (dd, J = 11.4, 5.9 Hz, 5a-HXyl, 1H), 4.03 (dd, J = 11.5,
11.4 Hz, 5b-HXyl, 1H), 4.20 (s, 1-HMan, 1H), 4.20ꢀ4.32 (m, 4-HXyl, 1H),
4.29 (s, CH2Ph, 2H), 4.38 (d, J = 12.4 Hz, CH2Ph, 1H), 4.70 (d, J = 12.4
Hz, CH2Ph, 1H), 5.39 (dd, J = 10.1, 9.6 Hz, 4-HMan, 1H), 5.60 (dd, J =
10.6, 3.6 Hz, 2-HXyl, 1H), 5.63 (d, J = 2.8 Hz, 2-HMan, 1H), 5.94 (d, J =
3.7 Hz, 1-HXyl, 1H), 6.55 (t, J = 10.1 Hz, 3-HXyl, 1H), 6.58ꢀ6.61 (m, Ar,
2H), 6.85ꢀ7.42 (m, Ar, 17H), 8.18ꢀ8.20 (m, Ar, 5H); 13C NMR
(100 MHz, C6D6): 20.1 (Ac), 20.6 (Ac), 55.0 (OMe), 60.2 (C5Xyl),
67.4 (C2Man), 69.2 (C4Man), 70.7 (C6Man), 70.96 (Bn), 70.99 (C3Xyl),
72.2 (C2Xyl), 73.6 (Bn), 73.8 (C4Xyl), 73.9 (C5Man), 76.8 (C3Man), 96.3
(C1Xyl, JC,H = 177.4 Hz), 96.8 (C1Man, JC,H = 161.2 Hz), 114.9, 118.7,
127.7ꢀ128.7 (Ar, overlapped with C6D6), 129.5, 130.06, 130.13, 130.85,
132.9, 133.4, 138.3, 138.7, 150.8, 155.9, 166.1, 166.2, 169.3, 170.3; MALDI-
TOF MS: [M + Na]+ calcd for C50H50O15Na1, 913.30, found 913.88. ESI-
TOF MS: [M + Na]+ calcd for C50H50O15Na1, 913.30, found 913.20; HRMS
ESI-TOF: [M + Na]+ calcd for C50H50O15Na1, 913.3047, found 913.3013.
4-Methoxyphenyl 2-O-Acetyl-3,6-di-O-benzyl-4-O-triisopropylsilyl-
β-D-mannopyranosyl-(1f4)-2,3-di-O-benzoyl-α-D-xylopyranoside (10).
The title compound was synthesized from 2 and 5 according to the pro-
cedure for the synthesis of the mixed acetal (1+5) (95%) followed by the
procedure for the synthesis of 9 (93%, β). Mixed acetal (2+5): 1H NMR
(400 MHz, C6D6): δ 1.02ꢀ1.06 (m, TIPS, 21H), 1.95 (s, SMe, 3H),
3.18 (s, OMe, 3H), 3.78 (dd, J = 9.2, 2.3 Hz, 3-HMan, 1H), 3.83 (dd, J =
10.6, 1.8 Hz, 6a-HMan, 1H), 3.92 (dd, J = 10.6, 5.5 Hz, 6b-HMan, 1H),
4.00ꢀ4.03 (m, 5-HMan, 1H), 4.16ꢀ4.19 (m, 4-HMan, CH2Ph, 2H),
4.26ꢀ4.37 (m, 2-HMan, 5-HMan, 5-HXyl, CH2Ph, 4H), 4.49ꢀ4.59
(m, 4-HMan, CH2Ph, 3H), 5.31 (d, J = 10.6, 3.7 Hz, 2-HXyl, 1H), 5.74
General Procedure for Intramolecular Aglycon Delivery 1. To the
mixed acetal (1+5) (103.2 mg, 93.0 μmol) were added DTBMP (116.9
mg, 558 μmol) and MS4A in dry (CH2Cl)2 (10 mL) at room tem-
perature under Ar atmosphere Then MeOTf (47.4 μL, 419 μmol) was
added to the mixture, and the mixture was stirred for 48 h at 40 °C. After
the mixture was cooled down, the reaction mixture was quenched with
triethylamine, diluted with ethyl acetate, and filtered through Celite. The
filtrate was washed with sat. NaHCO3 aq and brine. The washed organic
layer was dried over Na2SO4 and evaporated in vacuo. The crude prod-
uct was purified by gel filtration chromatography (SX-3, ethyl acetate/
(s, >CHNaph, 1H), 5.80 (s, 1-HMan, 1H), 5.82 (d, J = 3.6 Hz, 1-HXyl
,
1H) 6.51ꢀ6.53 (m, Ar, 2H), 6.72 (dd, J = 10.1, 9.6 Hz, 3-HXyl, 1H),
6.82ꢀ7.20 (m, Ar, 16H), 7.28ꢀ7.31 (m, Ar, 2H), 7.35ꢀ7.37 (m, Ar,
2H), 7.53ꢀ7.62 (m, Ar, 3H), 7.69ꢀ7.72 (m, Ar, 1H), 8.11ꢀ8.15 (m, Ar,
1
toluene = 1/1) to give the product (46.9 mg, 55%, β). 8: H NMR
(400 MHz, CDCl3): δ 0.40ꢀ0.51 (m, TES, 6H), 0.86ꢀ0.84 (m, TES,
9H), 2.52 (br s, OH, 1H), 2.87 (dd, J = 10.5, 7.3 Hz, 6a-HMan, 1H), 3.20
5H); MALDI-TOF MS: [M + Na]+ calcd for C68H80O13S1Si1Na1,
(dd, J = 9.2, 3.2 Hz, 3-HMan, 1H), 3.27 (ddd, J = 9.2, 7.3, 1.8 Hz, 5-HMan
,
,
1187.50, found 1187.77. 10: [α]29 36.9° (c 2.0, CHCl3); H NMR
1
D
1H), 3.51 (dd, J = 10.5, 1.8 Hz, 6b-HMan, 1H), 3.58 (t, J = 9.2 Hz, 4-HMan
(400 MHz, C6D6): δ 0.99ꢀ1.05 (m, TIPS, 21H), 1.84 (s, Ac, 3H), 3.15
(dd, J = 9.2, 3.2 Hz, 3-HMan, 1H), 3.25 (s, OMe, 3H), 3.25ꢀ3.30
(m, 5-HMan, 1H), 3.59 (dd, J = 11.0, 6.4 Hz, 6a-HMan, 1H), 3.77ꢀ3.80
(m, 6b-HMan, 1H), 3.87ꢀ3.91 (m, 5-HXyl, 1H), 3.98 (dd, J = 9.7, 9.2 Hz,
4-HMan, 1H), 4.03ꢀ4.14 (m, 5-HXyl, CH2Ph, 2H), 4.36 (s, 1-HMan, 1H),
4.40 (ddd, J = 11.0, 6.0, 5.0 Hz, 4-HXyl, 1H), 4.46ꢀ4.56 (m, CH2Ph,
1H), 3.72 (s, OMe, 3H), 3.86 (dd, J = 11.4, 6.0 Hz, 5a-HXyl, 1H), 3.90ꢀ
4.00 (m, 2-HMan, 5b-HXyl, 2H), 4.24 (d, J = 12.4 Hz, CH2Ph, 1H),
4.30ꢀ4. Twenty-four (m, 4-HXyl, CH2Ph, 2H), 4.48ꢀ4.55 (m, 1-HMan
,
CH2Ph, 2H), 4.63 (d, J = 11.9 Hz, CH2Ph, 1H), 5.37 (dd, J = 10.1, 3.7 Hz,
2-HXyl, 1H), 5.71 (d, J = 3.7 Hz, 1-HXyl, 1H), 6.03 (dd, J = 10.1, 9.6 Hz,
3-HXyl, 1H), 6.76ꢀ8.02 (m, Ar, 24H); 13C NMR (100 MHz, CDCl3): δ
5.0, 6.8, 55.6, 59.9, 67.4, 67.6, 69.8, 70.8, 71.1 (ꢁ2), 76.8, 81.4, 95.6, 97.0,
114.6, 118.1, 127.5, 127.9, 128.2, 128.3, 128.4, 129.0, 129.9, 130.2, 132.9,
133.3, 137.8, 138.4, 150.4, 155.3, 165.9, 167.0; MALDI-TOF MS:
[M + Na]+ calcd for C52H60O13Si1Na1, 943.37, found 943.56.
2H), 4.85 (d, J = 10.1 Hz, CH2Ph, 1H), 5.62 (dd, J = 10.5, 3.7 Hz, 2-HXyl
,
1H), 5.65 (d, J = 3.2 Hz, 2-HMan, 1H), 5.96 (d, J = 3.6 Hz, 1-HXyl, 1H),
6.57ꢀ6.63 (m, 3-HXyl, Ar, 3H), 6.87ꢀ7.20 (m, Ar, 11H), 7.25ꢀ7.29 (m,
Ar, 2H), 7.33ꢀ7.39 (m, Ar, 4H), 8.18ꢀ8.23 (m, Ar, 4H); 13C NMR
(100 MHz, C6D6): δ 13.3, 18.4, 18.5, 20.3, 55.1, 60.3, 67.2, 68.8, 69.9,
70.9, 71.2, 72.4, 72.5, 73.7, 77.7, 81.0, 96.4 (C1Xyl, JC,H = 176.4 Hz), 96.6
(C1Man, JC,H = 157.4 Hz), 115.0, 118.8, 127.8ꢀ128.7 (Ar, overlapped
withC6D6), 129.7, 130.15, 130.17, 130.9, 132.8, 133.3, 138.0, 139.1, 150.9,
4-Methoxyphenyl 2,4-Di-O-acetyl-3,6-di-O-benzyl-β-D-mannopy-
ranosyl-(1f4)-2,3-di-O-benzoyl-α-D-xylopyranoside (9). General Pro-
cedure for Intramolecular Aglycon Delivery 2. To the mixed acetal
19529
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Journal of the American Chemical Society
ARTICLE
155.9, 166.11, 166.19, 170.1; MALDI-TOF MS: [M + Na]+ calcd for
C57H68O14Si1Na1, 1027.43, found 1027.59. ESI-TOF MS: [M + Na]+
calcd for C57H68O14Si1Na1, 1027.43, found 1027.33; HRMS ESI-TOF:
[M + Na]+ calcd for C57H68O14Si1Na1, 1027.4276, found 1027.4232.
4-Methoxyphenyl 2-O-Acetyl-3,6-di-O-benzyl-4-O-triisopropylsilyl-
β-D-mannopyranosyl-(1f4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-
β-D-glucopyranoside (11). The title compound was synthesized from 2
and 6 according to the procedure for the synthesis of the mixed acetal
(1+5) (96%) followed by the procedure for the synthesis of 9 (90%, β).
Mixed acetal (2+6): MALDI-TOF MS: [M + Na]+ calcd for C76H85N1-
O13Si1Na1, 1302.54, found 1303.01. ESI-TOF MS: [M + Na]+ calcd
for C76H85N1O13Si1Na1, 1302.54, found 1302.43; HRMS ESI-TOF:
[M + Na]+ calcd for C76H85N1O13Si1Na1, 1302.5409, found 1302.5371.
11: [α]24D 33.6° (c 1.0, CHCl3); 1H NMR (400 MHz, C6D6): δ 1.01ꢀ
1H), 3.19 (s, OMe, 3H), 3.24 (ddd, J = 9.6, 5.5, 3.6 Hz, 5-HMan, 1H),
3.41 (m, 5-HGlcNAc, 1H), 3.57 (dd, J = 11.9, 1.8 Hz, 6a-HGlcNAc, 1H), 3.72ꢀ
3.80 (m, 6a-HMan, 6b-HGlcNAc, 2H), 3.85ꢀ3.88 (m, 6b-HMan, 1H), 3.93
(d, J = 10.1 Hz, CH2Ph, 1H), 4.22 (dd, J = 9.6, 9.2 Hz, 4-HMan, 1H), 4.32
(d, J = 11.9 Hz, CH2Ph, 1H), 4.45 (dd, J = 9.6, 9.2 Hz, 4-HGlcNAc, 1H),
4.50ꢀ4.55 (m, CH2Ph, 2H), 4.72 (dd, J = 10.6, 9.2 Hz, 3-HGlcNAc, 1H),
4.80ꢀ4.88 (m, 1-HMan, CH2Ph, 3H), 5.03 (dd, J = 11.0, 8.7 Hz,
2-HGlcNAc, 1H), 5.32 (d, J = 12.8 Hz, CH2Ph, 1H), 5.78 (d, J = 2.7 Hz,
2-HMan, 1H), 6.06 (d, J = 8.7 Hz, 1-HGlcNAc, 1H), 6.55ꢀ6.59 (m, Ar, 2H),
6.67ꢀ6.85 (m, Ar, 5H), 6.97ꢀ7.00 (m, Ar, 2H), 7.10ꢀ7.31 (m, Ar, 14H),
7.38ꢀ7.45 (m, Ar, 5H); 13C NMR (100 MHz, C6D6): δ 13.4, 18.4, 18.6,
20.7, 55.0, 56.5, 67.9, 68.5, 68.9, 69.5, 70.9, 73.5, 73.6, 75.1, 77.9, 78.0, 79.6,
81.7, 98.3 (C1GlcNAc, JC,H = 169.77 Hz), 99.6 (C1Man, JC,H = 160.23 Hz),
114.8, 119.0, 123.2, 125.6, 127.2, 127.5ꢀ128.7 (Ar, overlapped with
C6D6), 132.2, 133.4, 138.2, 138.6, 139.2, 139.5, 151.5, 156.0, 168.0, 168.2,
170.1. MALDI-TOF MS: [M + Na]+ calcd for C66H77N1O14Si1Na1,
1158.50, found 1158.82. ESI-TOF MS: [M + Na]+ calcd for C66H77N1-
O14Si1Na1, 1158.50, found 1158.39; HRMS ESI-TOF: [M + Na]+ calcd
for C66H77N1O14Si1Na1, 1158.5011, found 1158.5016.
and 7 according to the procedure for the synthesis of the mixed acetal
(1+5) (92%) followed by the procedure for the synthesis of 9 (90%, β).
Mixed acetal (2+7): MALDI-TOF MS: [M + Na]+ calcd for C104H110
-
N2O19Si1Na1, 1773.71, found 1774.25. ESI-TOF MS: [M + Na]+
calcd for C104H110N2O19Si1Na1, 1773.71, found 1774.63; HRMS ESI-
TOF: [M + Na]+ calcd for C104H110N2O19Si1Na1, 1773.7090, found
1773.7060. 14: [α]24 12.0° (c 1.0, CHCl3); H NMR (400 MHz,
1
D
C6D6): δ 1.04ꢀ1.09 (m, TIPS, 21H), 1.90 (s, Ac, 3H), 3.15ꢀ3.17
(m, 3-HMan, OMe, 4H), 3.27ꢀ3.29 (m, 5-HGlcNAc2, 1H), 3.41ꢀ 3.45
(m, 6-HGlcNAc2, 5-HMan, 2H), 3.50 (dd, J = 11.5, 3.7 Hz, 6-HGlcNAc2, 1H),
3.56ꢀ3.59 (m, 6-HGlcNAc1, 1H), 3.65 (dd, J = 12.4, 2.8 Hz, 6-HGlcNAc1
,
,
,
1H), 3.74 (dd, J = 11.0, 5.0 Hz, 6a-HMan, 1H), 3.87ꢀ 3.93 (m, 6b-HMan
1H), 4.22 (dd, J = 9.6, 9.2 Hz, 4-HMan, 1H), 4.40ꢀ4.63 (m, 4-HGlcNAc1
3-HGlcNAc2, 4-HGlcNAc2, CH2Ph, 7H), 4.67ꢀ4.95 (m, 2-HGlcNAc1
,
1.11 (m, TIPS, 21H), 1.91 (s, Ac, 3H), 3.12 (dd, J = 9.2, 3.2 Hz, 3-HMan
,
3-HGlcNAc1, 2-HGlcNAc2, CH2Ph, 10H), 4.97 (s, 1-HMan, 1H), 5.10
(d, J =13.3Hz, CH2Ph, 1H),5.36(d, J= 12.8 Hz, CH2Ph, 1H), 5.62(d, J=
8.2 Hz, 1-HGlcNAc1, 1H), 5.79 (d, J = 3.2 Hz, 2-HMan, 1H), 5.83 (d, J =
8.7 Hz, 1-HGlcNAc2, 1H), 6.50ꢀ7.60 (m, Ar, 42H); 13C NMR
(100 MHz, C6D6): δ 13.4, 18.4, 18.6, 20.7, 54.9, 56.4, 57.3, 67.8, 67.9,
68.4, 68.6, 69.5, 70.9, 73.1, 73.2, 73.6, 74.9, 75.1, 76.1, 77.0, 77.6, 78.1,
79.3, 81.8, 97.5 (C1GlcNAc1, JC,H = 166.6 Hz), 98.0 (C1GlcNAc2, JC,H
=
175.5 Hz), 99.2 (C1Man, JC,H = 163.1 Hz), 114.6, 119.2, 123.1, 123.5,
127.5ꢀ128.7 (Ar, overlapped with C6D6), 132.2, 133.3, 133.5, 133.9,
138.2, 138.7, 138.9, 139.2, 139.3, 139.7, 151.5, 155.8, 167.8, 168.5, 170.0;
MALDI-TOF MS: [M + Na]+ calcd for C94H102N2O20Si1Na1, 1629.67,
found 1629.87. ESI-TOF MS: [M + Na]+ calcd for C94H102N2O20Si1-
Na1, 1629.67, found 1629.59; HRMS ESI-TOF: [M + Na]+ calcd for
C94H102N2O20Si1Na1, 1629.6693, found 1629.6723.
Methyl 4,6-O-Benzylidene-1-thio-3-O-triisopropylsilyl-α-D-manno-
pyranoside (16). To a solution of 15 (15.8 g, 0.053 mol) and imidazole
(9.02 g, 0.133 mol) in DMF (50 mL) was added dropwise TIPSCl
(12.5 mL, 0.058 mol) at 0 °C under Ar atmosphere, and the mixture was
stirred for 18 h at room temperature. The reaction was quenched with
sat. NaHCO3 aq, extracted with CHCl3, washed with brine, dried over
Na2SO4, and evaporated in vacuo. The residue was purified by silica gel
column chromatography using a gradient solvent system (hexane/ethyl
acetate = 50/1 to 20/1 to 10/1 to 5/1) to give the title compound 16
(17.04 g, 70%). 16: [α]25D 90.4° (c 1.0, CHCl3); 1H NMR (400 MHz,
CDCl3): δ 1.01ꢀ1.14 (m, TIPS, 21H), 2.13 (s, SMe, 3H), 3.00 (br s,
OH, 1H), 3.84 (t, J = 9.6 Hz, 6a-H, 1H), 3.90 (t, J = 9.6 Hz, 4-H, 1H),
4.01 (d, J = 3.7 Hz, 2-H, 1H), 4.12ꢀ4.24 (m, 3-H, 5-H, 6b-H, 3H),
5.27 (s, 1-H, 1H), 5.50 (s, >CHPh, 1H), 7.30ꢀ7.46 (m, Ar, 5H);
13C NMR (100 MHz, CDCl3): δ 12.2, 13.5, 17.88, 17.92, 63.7, 68.7,
70.2, 73.2, 79.5, 85.3, 102.3, 126.3, 128.0, 129.0, 137.3; MALDI-TOF
MS: [M + Na]+ calcd for C23H38O5S1Na1, 477.21, found 477.55. ESI-
TOF MS: [M + Na]+ calcd for C23H38O5S1Si1Na1, 477.21, found
477.14; HRMS ESI-TOF: [M + Na]+ calcd for C23H38O5S1Si1Na1,
477.2107, found 477.2123.
Methyl 4,6-O-Benzylidene-2-O-(2-naphthylmethyl)-1-thio-3-O-trii-
sopropylsilyl-α-D-mannopyranoside (17). After azeotropic removal of
water by toluene, to a solution of 16 (1.20 g, 2.64 mmol) in DMF
(50 mL) was added NaH (60%, 95.04 mg, 3.96 mmol) and 2-NAPBr
(668.8 mg, 2.90 mmol) at 0 °C under Ar atmosphere, and the mixture
was stirred for 3 h at room temperature. The reaction was quenched
with triethylamine and brine, extracted with CHCl3, washed with sat.
NaHCO3 aq and brine, dried over Na2SO4, and evaporated in vacuo.
The residue was purified by silica gel column chromatography using a
gradient solvent system (hexane/ethyl acetate = 50/1 to 25/1 to 10/1 to
4-Methoxyphenyl 2-O-Acetyl-3,4,6-tri-O-benzyl-β-D-mannopyrano-
syl-(1f4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-β-D-glucopyrano-
side (12). The title compound was synthesized from 3 and 6 according
to the procedure for the synthesis of the mixed acetal (1+5) (92%)
followed by the procedure for the synthesis of 9 (85%, β). 12: [α]25
D
39.7° (c 0.59, CHCl3); 1H NMR (400 MHz, CDCl3): δ 2.09 (s, Ac, 3H),
3.28 (ddd, J = 10.4, 4.8, 2.0 Hz, 5-HMan, 1H), 3.38 (dd, J = 9.2, 3.2 Hz,
3-HMan, 1H), 3.58 (dd, J = 11.6, 4.8 Hz, 6-HMan, 1H), 3.62ꢀ3.71
(m, 4-HMan, 6-HMan, 5-HGlcNAc, 6-HGlcNAc, 4H), 3.63 (s, OMe, 3H), 3.75
(dd, J = 11.2, 3.6 Hz, 6-HGlcNAc, 1H), 4.13 (dd, J = 9.6, 8.4 Hz, 4-HGlcNAc
,
1H), 4.27 (dd, J = 11.6, 8.4 Hz, 3-HGlcNAc, 1H), 4.29 (dd, J = 11.6 Hz,
CH2Ph, 1H), 4.34 (dd, J = 11.6, 8.0 Hz, 2-HGlcNAc, 1H), 4.34ꢀ4.47 (m,
CH2Ph, 5H), 4.56 (dd, J = 11.6 Hz, CH2Ph, 1H), 4.63 (s, 1-HMan, 1H),
4.64 (dd, J = 12.8 Hz, CH2Ph, 1H), 4.76 (dd, J = 11.6 Hz, CH2Ph, 1H),
4.89 (dd, J = 12.8 Hz, CH2Ph, 1H), 5.42 (d, J = 3.2 Hz, 2-HMan, 1H), 5.53
(d, J = 8.0 Hz, 1-HGlcNAc, 1H), 6.59ꢀ7.59 (m, Ar, 33H); 13C NMR
(100 MHz, CDCl3): δ 21.2 (q, Ac), 55.6 (q, OMe), 55.7 (d, C2GlcNAc),
68.4 (d, C2Man), 68.6 (t, C6GlcNAc), 68.9 (t, C6Man), 71.5 (t, Bn), 73.3
(t, Bn), 73.4 (t, Bn), 74.1 (d, C4Man), 74.5 (d, C5GlcNAc), 74.8(t, Bn), 75.0
(t, Bn), 75.7 (d, C5Man), 77.3 (d, C3GlcNAc), 78.7 (d, C4GlcNAc), 80.5
(d, C3Man), 97.6(d, C1GlcNAc, JC,H =174.5Hz),99.1(d, C1Man, JC,H = 161.2
Hz), 114.3, 118.6, 123.3, 127.0, 127.3, 127.5, 127.6, 127.6, 127.72, 127.75,
127.79, 127.82, 127.9, 128.0, 128.1, 128.27, 128.31, 128.4, 128.5, 133.7,
137.7, 137.9, 138.4, 138.7, 150.8, 155.3, 167.9, 170.5. ESI-TOF MS: [M +
Na]+ calcd for C64H63N1O14Na1, 1092.41, found 1092.42; HRMS ESI-
TOF: [M + Na]+ calcd for C64H63N1O14Na1, 1092.4146, found 1092.4102.
4-Methoxyphenyl 2-O-Acetyl-3,6-di-O-benzyl-4-O-triisopropylsilyl-
β-D-mannopyranosyl-(1f4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-
β-D-glucopyranosyl-(1f4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-β-
D-glucopyranoside (14). The title compound was synthesized from 2
5/1 to 3/1) to give the title compound 17 (1.492 g, 95%). 17: [α]25
D
1
44.6° (c 1.0, CHCl3); H NMR (400 MHz, CDCl3): δ 0.92ꢀ1.11
(m, TIPS, 21H), 2.10 (s, SMe, 3H), 3.87 (t, J = 10.1 Hz, 6a-H, 1H), 3.92
(dd, J = 3.2, 1.6 Hz, 2-H, 1H), 4.09ꢀ4.21 (m, 4-H, 5-H, 6b-H, 3H), 4.30
(dd, J = 9.2, 3.2 Hz, 3-H, 1H), 4.84 (d, J = 12.4, CH2Ph, 1H), 4.99 (d, J =
12.4, CH2Ph, 1H), 5.23 (d, J = 1.6 Hz, 1-H, 1H), 5.54 (s, >CHPh, 1H),
19530
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ARTICLE
7.27ꢀ7.39 (m, Ar, 3H), 7.47ꢀ7.57 (m, Ar, 5H), 7.84ꢀ7.86 (m, Ar, 3H);
13C NMR (100 MHz, CDCl3): δ 12.5, 13.9, 18.0, 18.1, 64.9 (C5), 68.7
(C6), 70.9 (C3), 73.9 (Bn), 79.5 (C4), 81.4 (C2), 85.3 (C1), 102.4,
125.9, 126.1, 126.4, 126.5, 127.7, 127.9, 128.0, 128.1, 128.9, 133.0, 133.2,
135.7, 137.5; MALDI-TOF MS: [M + Na]+ calcd for C34H46O5S1Si1-
Na1, 617.27, found 617.11. ESI-TOF MS: [M + Na]+ calcd for C34H46-
O5S1Si1Na1, 617.27, found 617.18; HRMS ESI-TOF: [M + Na]+ calcd
for C34H46O5S1Si1Na1, 617.2733, found 617.2758.
to 25/1 to 10/1 to 5/1) to give the title compound 20 (3.61 g, 86%). 20:
1
[α]28 21.4° (c 1.0, CHCl3); H NMR (400 MHz, CDCl3): δ 2.14
D
(s, SMe, 3H), 2.66 (s, OH, 1H), 3.72 (dd, J = 8.7, 3.2 Hz, 3-H, 1H),
3.80ꢀ3.86 (m, 6a-H, 6b-H, 2H), 3.91 (dd, J = 3.2, 1.8 Hz, 2-H, 1H),
4.10ꢀ4.19 (m, 4-H, 5-H, 2H), 4.47ꢀ4.67 (m, CH2Ph, 4H), 4.72 (d, J =
12.8 Hz, CH2Ph, 1H), 4.87 (d, J =12.4Hz, CH2Ph,1H), 5.38 (s, 1-H,1H),
7.24ꢀ7.36 (m, Ar, 10H), 7.46ꢀ7.53 (m, Ar, 3H), 7.75ꢀ7.85 (m, Ar, 4H);
13C NMR (100 MHz, CDCl3): δ 13.7, 67.7, 70.0, 71.6, 71.7, 72.0, 73.4,
75.3, 77.2, 79.7, 83.2, 125.9, 126.0, 126.6, 127.4, 127.5, 127.6, 127.8, 127.9,
128.1, 128.2, 128.4, 132.4, 132.9, 133.1, 135.3, 137.8, 138.2; MALDI-TOF
MS: [M + Na]+ calcd for C32H34O5S1Na1, 553.20, found 552.90. ESI-
TOF MS: [M + Na]+ calcd for C32H34O5S1Na1, 553.20, found 553.12;
HRMS ESI-TOF: [M + Na]+ calcd for C32H34O5S1Na1, 553.2025, found
553.2003.
4-Methoxyphenyl 2,3,4-Tri-O-acetyl-D-xylopyranoside (22α). After
azeotropic removal of H2O with toluene, to a solution of 21 (synthesized
from xylose (100 g 0.67 mol) treated with Ac2O in pyridine), 4-meth-
oxyphenol (91.49 g, 737 mmol) and MS4A in CH2Cl2 was added
TMSOTf (12.31 mL, 67 mmol) at 0 °C. After stirring for 12 h at room
temperature, further TMSOTf (12.31 mL, 67 mmol) was added to the
mixture at 0 °C. After stirring for 12 h at room temperature, the reaction
mixture was quenched with sat. NaHCO3 aq and extracted with CHCl3.
The combined organic layers were washed with brine, dried over Na2SO4,
and evaporated in vacuo to give unexpected α-isomer (230.6 g, 90%, α).
22α: [α]27D 125.6° (c 1.0, CH3OH); 1H NMR (400 MHz, CDCl3): δ
2.02 (s, Ac, 3H), 2.05 (s, Ac, 6H), 3.74 (dd, J = 11.0, 9.6 Hz, 5a-H, 1H),
3.75 (s, OMe, 3H), 3.83 (dd, J = 11.4, 6.4 Hz, 5b-H, 1H), 4.92 (dd, J =
10.6, 3.7 Hz, 2-H, 1H), 5.03 (ddd, J = 10.1, 9.6, 6.4 Hz, 4-H, 1H), 5.50
(d, J = 3.7 Hz, 1-H, 1H), 5.66 (t, J = 10.1 Hz, 3-H, 1H), 6.78ꢀ6.83
(m, Ar, 2H), 6.93ꢀ7.00 (m, Ar, 2H); 13C NMR (100 MHz, CDCl3): δ
20.7, 20.8, 55.7, 58.9, 69.2, 69.5, 70.8, 95.1, 114.6, 117.8, 150.2, 150.2,
170.0, 170.1, 170.3; MALDI-TOF MS: [M + Na]+ calcd for C18H22-
O9Na1, 405.12, found 405.62. ESI-TOF MS: [M + Na]+ calcd for
C18H22O9Na1, 405.12, found 405.06; HRMS ESI-TOF: [M + Na]+
calcd for C18H22O9Na1, 405.1162, found 405.1181.
Methyl 4,6-O-Benzylidene-2-O-(2-naphthylmethyl)-1-thio-α-D-man-
nopyranoside (18). After azeotropic removal of water by toluene, to a
solution of 17 (10.01 g, 16.8 mmol) in THF (20 mL) was added 1.0 M
solution of TBAF in THF (25.2 mL, 25.2 mmol) at 0 °C under Ar
atmosphere, and the mixture was stirred for 18 h at room temperature.
The reaction was quenched with sat. NaHCO3 aq, extracted with ethyl
acetate, washed with brine, dried over Na2SO4, and evaporated in vacuo.
The residue was purified by silica gel column chromatography using a
gradient solvent system (hexane/ethyl acetate = 50/1 to 25/1 to 5/1 to
3/1) to give the title compound 18 (6.636 g, 90%). 18: [α]25 64.6°
D
(c 1.0, CHCl3); 1H NMR (400 MHz, CDCl3): δ 2.10 (s, SMe, 3H), 2.43
(d, J = 8.2, OH, 1H), 3.97 (dd, J = 10.1, 9.6 Hz, 6a-H, 1H), 3.98 (d, J = 3.6
Hz, 2-H, 1H), 4.07 (ddd, J = 10.1, 8.2, 3.6 Hz, 3-H, 1H), 4.15 (d, J = 10.1,
5.0 Hz, 6b-H, 1H), 4.33 (ddd, J = 11.4, 10.1, 5.0 Hz, 5-H, 1H), 4.78
(d, J = 12.4, CH2Ph, 1H), 4.91 (d, J = 11.9, CH2Ph, 1H), 5.31 (s, 1-H,
1H), 5.57 (s, >CHPh, 1H), 7.32ꢀ7.36 (m, Ar, 3H), 7.47ꢀ7.50 (m, Ar,
5H), 7.81ꢀ7.86 (m, Ar, 4H); 13C NMR (100 MHz, CDCl3): δ 13.7,
63.9, 68.6, 69.0, 73.2, 79.7, 79.8, 83.9, 102.2, 125.7, 126.2, 126.3, 126.9,
127.7, 127.9, 128.3, 128.5, 129.1, 133.1, 133.2, 134.7, 137.2; MALDI-
TOF MS: [M + Na]+ calcd for C25H26O5S1Na1, 461.14, found 462.34.
ESI-TOF MS: [M + Na]+ calcd for C25H26O5S1Na1, 461.14, found
461.07; HRMS ESI-TOF: [M + Na]+ calcd for C25H26O5S1Na1,
461.1399, found 461.1393.
Methyl 3-O-Benzyl-4,6-O-benzylidene-2-O-(2-naphthylmethyl)-
1-thio-α-D-mannopyranoside (19). After azeotropic removal of water
by toluene, to a solution of 18 (6.636 g, 15.1 mmol) in DMF (10 mL)
was added NaH (60%, 846 mg, 21.1 mmol) and BnBr (2.21 mL, 18.12
mmol) at 0 °C under Ar atmosphere, and the mixture was stirred for 3 h
at room temperature. The reaction was quenched with triethylamine and
brine, extracted with CHCl3, washed with sat. NaHCO3 aq and brine,
dried over Na2SO4, and evaporated in vacuo. The residue was purified
by silica gel column chromatography using a gradient solvent system
(hexane/ethyl acetate = 50/1 to 25/1 to 10/1 to 5/1) to give the title
compound 19 (7.10 g, 92%). 19: [α]25D 73.8° (c 1.0, CHCl3); 1H NMR
(400 MHz, CDCl3): δ 2.07 (s, SMe, 3H), 3.89ꢀ3.95 (m, 2-H, 3-H, 6a-H,
3H), 4.13ꢀ4.19 (m, 5-H, 1H), 4.23 (dd, J = 10.1, 5.0 Hz, 6b-H, 1H),
4.31 (td, J = 9.6, 1.2 Hz, 4-H, 1H), 4.60 (d, J = 12.4 Hz, CH2Ph, 1H), 4.79
(d, J = 12.4 Hz, CH2Ph, 1H), 4.86 (d, J = 13.2 Hz, CH2Ph, 1H), 4.91
(d, J = 13.2 Hz, CH2Ph, 1H), 5.23 (s, 1-H, 1H), 5.64 (s, >CHPh, 1H),
7.25ꢀ7.84 (m, Ar, 17H); 13C NMR (100 MHz, CDCl3): δ 13.8, 64.6,
68.7, 73.0, 73.2, 76.4, 77.8, 79.2, 84.9, 101.5, 126.0, 126.1, 126.9, 127.56,
127.63, 127.93, 128.16, 128.22, 128.29, 128.84, 133.0, 133.2, 135.2,
137.6, 138.4; MALDI-TOF MS: [M + Na]+ calcd for C32H32O5S1Na1,
551.19, found 551.33. ESI-TOF MS: [M + Na]+ calcd for C32H32O5-
S1Na1, 551.19, found 551.11; HRMS ESI-TOF: [M + Na]+ calcd for
C32H32O5S1Na1, 551.1868, found 551.1880.
Methyl 3,6-Di-O-benzyl-2-O-(2-naphthylmethyl)-1-thio-α-D-manno-
pyranoside (20). After azeotropic removal of water by toluene, to a
solution of 19 (4.073 g, 7.94 mmol) in CH2Cl2 (5.0 mL) was added
TESH (6.34 mL, 39.7 mmol) at 0 °C under Ar atmosphere, and TFA
(2.95 mL, 39.7 mmol) was added dropwise to the mixture at the same
temperature. The mixture was stirred for 20 h at room temperature, and
then the reaction was quenched with triethylamine, extracted with ethyl
acetate, washed with sat. NaHCO3 aq and brine, dried over Na2SO4, and
evaporated in vacuo. The residue was purified by silica gel column chro-
matography using a gradient solvent system (hexane/ethyl acetate = 50/1
4-Methoxyphenyl 2,3,4-Tri-O-acetyl-β-D-xylopyranoside (22β). 22β
was obtained according to the procedure for 22α except 2.4 equiv of
BF3 OEt was used instead of TMSOTf in the presence of 0.7 equiv of
3
triethylamine (20%, β as the major product). 22β was isomerized
completely to 22α in the presence of 0.1 equiv of TMSOTf in CDCl3
within 26 h. 22β: [α]26D ꢀ33.6° (c 1.0, CHCl3); 1H NMR (400 MHz,
CDCl3): δ 2.06 (s, Ac, 3H), 2.07 (s, Ac, 3H), 2.09 (s, Ac, 3H), 3.46
(dd, J = 12.4, 8.7 Hz, 5a-H, 1H), 3.76 (s, OMe, 3H), 4.20 (dd, J =
11.9, 5.0 Hz, 5b-H, 1H), 4.98ꢀ5.05 (m, 4-H, 1H), 5.02 (d, J = 6.4 Hz,
1-H, 1H), 5.14 (dd, J = 8.2, 6.4 Hz, 2-H, 1H), 5.22 (t, J = 8.2 Hz, 3-H,
1H), 6.79ꢀ6.84 (m, Ar, 2H), 6.92ꢀ7.00 (m, Ar, 2H); 13C NMR
(100 MHz, CDCl3): δ 20.7, 55.6, 61.9, 68.5, 70.3, 70.9, 99.7, 114.5,
114.7, 115.9, 117.8, 118.4, 150.6, 155.2, 169.4, 169.8, 170.0; MALDI-
TOF MS: [M + Na]+ calcd for C18H22O9Na1, 405.12, found 405.33.
4-Methoxyphenyl α-D-Xylopyranoside (23). To a solution of 22α
(230.56 g, 0.603 mol) in dry methanol (500 mL) was added NaOMe to
justify to alkaline pH, indicated by phenolphthalein, at room tempera-
ture under Ar atmosphere, and the mixture was stirred for 16 h at
the same temperature. Amberlist 15H+ was added to the mixture to
quench excess NaOMe. The resin was filtered off, and the mixture was
concentrated. The residue was purified by recrystallization in hexane/
ethyl acetate to give the title compound 23 (146.79 g, 95%). 23: [α]25
D
172.4° (c 1.0, CH3OH); mp 152ꢀ154 °C; 1H NMR (400 MHz,
CD3OD): δ 3.49 (dd, J = 9.3, 3.7 Hz, 2-H, 1H), 3.51ꢀ3.60 (m, 4-H,
5a-H, 5b-H, 3H), 3.71 (s, OMe, 3H), 3.74 (dd, J = 10.1, 8.2 Hz, 3-H,
1H), 5.28 (d, J = 3.7 Hz, 1-H, 1H), 6.79ꢀ6.83 (m, Ar, 2H), 6.99ꢀ7.03
(m, Ar, 2H); 13C NMR (100 MHz, CD3OD): δ 56.0, 63.5, 71.4, 73.4,
75.1, 100.1, 115.5, 119.3, 152.4, 156.7; MALDI-TOF MS: [M + Na]+
calcd for C12H16O6Na1, 279.08, found 279.14. ESI-TOF MS: [M + Na]+
19531
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Journal of the American Chemical Society
ARTICLE
calcd for C12H16O6Na1, 279.08, found 279.04; HRMS ESI-TOF: [M +
Na]+ calcd for C12H16O6Na1, 279.0845, found 279.0836.
1H), 5.81 (dd, J = 10.1, 9.2 Hz, 3-H, 1H), 6.78ꢀ8.02 (m, Ar, 14H); 13
C
NMR (100 MHz, CDCl3): δ 55.6, 55.7, 62.3, 69.6, 70.8, 74.9, 95.9,
114.7, 118.2, 128.5, 128.99, 129.03, 129.8, 129.9, 133.4, 133.6, 150.5,
155.3, 165.9, 167.8; MALDI-TOF MS: [M + Na]+ calcd for C26H24-
O8Na1, 487.14, found 487.67. ESI-TOF MS: [M + Na]+ calcd for
C26H24O8Na1, 487.14, found 487.06; HRMS ESI-TOF: [M + Na]+
calcd for C26H24O8Na1, 487.1369, found 487.1372.
4-Methoxyphenyl 2,3-O-Isopropylidene-α-D-xylopyranoside (24).
To a mixture of 23 (1.0027 g, 3.90 mmol) and 2-methoxypropene
(0.41 mL, 4.29 mmol) in dry DMF was added CSA (90.6 mg, 0.39
mmol) at room temperature, and the mixture was stirred for 18 h at
room temperature. The reaction was quenched by sat. NaHCO3 aq and
extracted with CHCl3. Combined solutions were washed with brine and
dried over Na2SO4. After concentration, the residue was purified by silica
gel column chromatography using a gradient solvent system (hexane/
ethyl acetate = 40/0 to 20/1 to 10/1 to 5/1) to give the title compound
4-Methoxyphenyl 2,3,6-Tri-O-benzyl-β-D-mannopyranosyl-(1f4)-
2,3-di-O-benzyl-α-D-xylopyranoside (30). To a solution of 10 (35.0 mg,
34.8 μmol) in MeOHꢀTHF (1:1, 2.0 mL) was added 1 M solution of
NaOMe in MeOH (10.0 μL) at 0 °C. After stirring for 4 h at room
temperature, the reaction was quenched by Amberlyst H+ resin, filtered,
and concentrated in vacuo to give a crude, deacylated compound 28
(MALDI-TOF MS: [M + Na]+ calcd for C41H58O11Si1Na1, 777.36,
found 777.62). To a solution of crude 28 in dry DMF (1.0 mL) were
added NaH (60%, 6.3 mg, 157.5 μmol) and BnBr (13.7 μL, 115.2 μmol)
at 0 °C, and the mixture was stirred for 2 h at the same temperature. The
reaction was quenched by triethylamine and brine, extracted with
CHCl3, dried over Na2SO4, and evaporated in vacuo to give crude 29
(MALDI-TOF MS: [M + Na]+ calcd for C62H76O11Si1Na1, 1047.51,
found 1047.51). To a solution of the crude 29 in THF (1.0 mL) was
added 1.0 M THF solution of TBAF (0.1 mL, 100 μmol), and the mix-
ture was stirred for 3 h at room temperature. After evaporation the resi-
due was purified by PTLC (hexane/ethyl acetate = 1/1) to give the title
compound 30 (25.6 mg, 85% in three steps). 30: [α]27D ꢀ2.4° (c 1.0,
CHCl3); 1H NMR (400 MHz, CDCl3): δ 2.70 (br s, OH, 1H), 3.24 (dd,
(855.2 mg, 74%). 24: [α]25 170.6° (c 1.0, CH3OH); 1H NMR
D
(400 MHz, C6D6): δ 1.41 (s, CH3, 3H), 1.49 (s, CH3, 3H), 2.05
(d, J = 4.6 Hz, OH, 1H), 3.26 (s, OMe, 3H), 3.43 (dd, J = 9.6, 3.2 Hz,
2-H, 1H), 3.52 (t, J = 11.0 Hz, 5a-H, 1H), 3.66 (dd, J = 11.4, 5.5 Hz,
5b-H, 1H), 3.80 (ddd, J = 11.0, 10.1, 5.5, 4-H, 1H), 4.29 (dd, J = 10.1,
9.6 Hz, 3-H, 1H), 5.54 (d, J = 3.2 Hz, 1-H, 1H), 6.66ꢀ6.71 (m, Ar, 2H),
7.03ꢀ7.07 (m, Ar, 2H); 13C NMR (100 MHz, C6D6): δ 26.7, 27.1, 55.1,
63.6, 70.3, 76.2, 77.7, 97.0, 110.7, 114.9, 118.9, 151.3, 155.9; MALDI-
TOF MS: [M + Na]+ calcd for C15H20O6Na1, 319.12, found 319.56.
ESI-TOF MS: [M + Na]+ calcd for C15H20O6Na1, 319.12, found
319.07; HRMS ESI-TOF: [M + Na]+ calcd for C15H20O6Na1,
319.1158, found 319.1133.
4-Methoxyphenyl 2,3-Di-O-benzoyl-α-D-xylopyranoside (27). To a
solution of 24 (7.74 g, 26.13 mmol) and imidazole (6.60 g, 96.9 mmol)
in DMF (50 mL) was added dropwise TIPSCl (9.2 mL, 43.0 mmol) at
0 °C under Ar atmosphere, and the mixture was stirred for 18 h at room
temperature. The reaction was quenched with sat. NaHCO3 aq,
extracted with CHCl3, washed with brine, dried over Na2SO4, and eva-
porated in vacuo to give crude 4-methoxyphenyl 2,3-O-isopropylidene-
4-O-triisopropyl-silyl-α-D-xylopyranoside (25) which was used in the
next reaction without further purifications. To a solution of 25 in isopro-
panol (20 mL) was added PPTS (667 mg, 2.6 mmol) at room tem-
perature under Ar atmosphere, and the mixture was stirred for 3 d at the
same temperature. The reaction was quenched with triethylamine, diluted
with ethylacetate, washed with sat. NaHCO3 aq and brine, dried over
Na2SO4, and evaporated in vacuo to give crude 4-methoxyphenyl 4-O-
triisopropylsilyl-α-D-xylopyranoside (26) which was used in the next
reaction without further purifications. To a solution of 26 in pyridine
(10 mL) were added BzCl (6.64 mL, 57.2 mmol) and DMAP (318 mg,
2.6 mmol) at 0 °C under Ar atmosphere, and the mixture was stirred for
1.5 h at room temperature. The reaction was quenched with MeOH and
evaporated in vacuo. The residue was diluted with CH2Cl2 and washed
with 1 N HCl aq, water, sat. NaHCO3 aq, water, and brine, dried over
MgSO4, and evaporated in vacuo. The residue was purified by silica gel
column chromatography using a gradient solvent system (hexane/ethyl
acetate = 50/1 to 20/1 to 10/1 to 5/1) to give 4-methoxyphenyl 2,3-di-
O-benzoyl-4-O-triisopropylsilyl-α-D-xylo-pyranoside (27) (13.04 g, 79%).
27: MALDI-TOF MS: [M + Na]+ calcd for C35H44O8Si1Na1, 643.27,
found 643.85. ESI-TOF MS: [M + Na]+ calcd for C35H44O8Si1Na1,
643.27, found 643.27; HRMS ESI-TOF: [M + Na]+ calcd for C35H44-
O8Si1Na1, 643.2703, found 643.2686. After azeotropic removal of water
by toluene, to a solution of 27 (8.91 g, 14.36 mmol) in THF (100 mL)
were added acetic acid (1.972 mL, 34.46 mmol) and 1.0 M solution of
TBAF in THF (34.46 mL, 34.46 mmol) at 0 °C under Ar atmosphere,
and the mixture was stirred for 18 h at room temperature. The reaction
was quenched with sat. NaHCO3 aq, extracted with ethyl acetate,
washed with brine, dried over Na2SO4, and evaporated in vacuo. The
residue was purified by silica gel column chromatography using a gra-
dient solvent system (hexane/ethyl acetate = 50/1 to 25/1 to 5/1 to
3/1) to give the title compound 5 (4.80 g, 72%). 5: [α]26D 163.4° (c 1.0,
J = 10.0, 2.8 Hz, 3-HMan, 1H), 3.32 (dt, J = 10.0, 4.8 Hz, 5-HMan, 1H),
Xyl
3.49 (dd, J = 9.2, 3.6 Hz, 2-HXyl, 1H), 3.47ꢀ3.65 (m, 6-HMan, 5-H2
,
3H), 3.70ꢀ3.73 (m, 6-HMan, 1H), 3.71 (s, OMe, 3H), 3.76 (d, J = 2.8
Hz, 2-HMan, 1H), 3.91ꢀ4.02 (m, 4-HMan, 3-HXyl, 4-HXyl, 3H), 4.37
(d, J = 12.4 Hz, CH2Ph, 1H), 4.42 (d, J = 12.4 Hz, CH2Ph, 1H),
4.46 (s, 1-HMan, 1H), 4.47 (d, J = 12.0 Hz, CH2Ph, 1H), 4.48 (d, J =
12.4 Hz, CH2Ph, 1H), 4.60 (d, J = 12.4 Hz, CH2Ph, 1H), 4.70 (d, J =
12.4 Hz, CH2Ph, 1H), 4.73 (d, J= 10.8 Hz, CH2Ph, 1H), 4.75 (d, J= 12.0 Hz,
CH2Ph, 1H), 4.82 (d, J = 12.4 Hz, CH2Ph, 1H), 5.03 (d, J = 10.8 Hz,
CH2Ph, 1H), 5.18 (d, J = 3.6 Hz, 1-HXyl, 1H), 6.75ꢀ7.30 (m, Ar, 24H);
13C NMR (100 MHz, CDCl3): δ 55.6 (q, OMe), 59.8 (t, C5Xyl), 68.5
(d, C4Man), 70.9 (t, C6Man), 71.4 (t, Bn), 73.6 (t, Bn), 73.7 (t, Bn), 74.2
(t, Bn), 74.3 (d, C2Man), 75.2 (d, C5Man and t, Bn), 75.9 (d, C4Xyl), 78.8
(d, C2Xyl), 80.0 (d, C3Xyl), 81.4 (d, C3Man), 96.8 (d, C1Xyl), 99.6
(d, C1Man), 114.5, 118.3, 127.2, 127.4, 127.5, 127.6, 127.7, 127.8, 127.8,
128.0, 128.1, 128.3, 128.4, 128.5, 137.9, 138.0, 138.1, 138.5, 139.1, 150.8,
155.1; MALDI-TOF MS: [M + Na]+ calcd for C53H56O11Na1, 891.37,
found 891.21; ESI-TOF MS: [M + Na]+ calcd for C53H56O11Na1,
891.37, found 891.45; HRMS ESI-TOF: [M + Na]+ calcd for C53H56-
O11Na1, 891.3720, found 891.3741.
2-O-Acetyl-3,6-di-O-benzyl-4-O-triisopropylsilyl-β-D-mannopyrano-
syl-(1f4)-2,3-di-O-benzoyl-D-xylopyranosyl Fluoride (32). To a solu-
tion of 10 (41.8 mg, 41.6 μmol) in CH3CN (2.0 mL) and H2O (0.2 mL)
was added CAN (68.4 mg, 125 μmol) at 0 °C under Ar atmosphere. The
reaction mixture was stirred for 18 h at room temperature, diluted with
ethyl acetate and washed with sat. NaHCO3 aq and brine, dried over
Na2SO4, and evaporated in vacuo. The crude product was purified by
florisil column chromatography using a gradient solvent system (hexane/
ethyl acetate =20/1 to 10/1 to 5/1 to 2/1 to 1/1) to give 2-O-acetyl-3,
6-di-O-benzyl-4-O-triisopropylsilyl-β-D-mannopyranosyl-(1f4)-2,3-di-O-
benzoyl-D-xylopyranose (31). 31: MALDI-TOF MS: [M + Na]+ calcd
for C50H62O13Si1Na1, 921.39, found 921.32. To a solution of a mixture
of 31 (27.1 mg, 29.4 μmol) in dry CH2Cl2 (1.0 mL) was added DAST
(7.8 μL, 58.8 μmol) at ꢀ40 °C under Ar atmosphere. The reaction
mixture was stirred for 2 h at the same temperature and then quenched
with MeOH and sat. NaHCO3 aq. The reaction mixture was extracted
with CHCl3, and the combined organic layers were washed with sat.
NaHCO3 aq and brine, dried over Na2SO4, and evaporated in vacuo.
1
CHCl3); H NMR (400 MHz, CDCl3): δ 2.99 (br s, OH, 1H), 3.73
(s, OMe, 3H), 3.86ꢀ3.96 (m, 5a-H, 5b-H, 2H), 4.10 (td, J = 9.2, 6.8 Hz,
4-H, 1H), 5.37 (dd, J = 10.1, 4.1 Hz, 2-H, 1H), 5.69 (d, J = 4.1 Hz, 2-H,
19532
dx.doi.org/10.1021/ja208528c |J. Am. Chem. Soc. 2011, 133, 19524–19535

Journal of the American Chemical Society
ARTICLE
The crude product was purified by silica gel flash column chromatogra-
phy using a gradient solvent system (hexane/ethyl acetate = 50/1 to 20/1
to 10/1 to 5/1to 3/1to 1/1) to give 32 (25.8 mg, 67%in twosteps, α:β =
84:16). 32: α-isomer (major): 1H NMR (400 MHz, CDCl3): δ 0.70ꢀ
1.00 (m, TIPS, 21H), 1.95 (s, Ac, 21H), 3.29 (dd, J = 10.4, 7.2 Hz, 6a-
127.47, 127.50, 127.6, 127.7, 127.77, 127.83, 127.95, 127.98, 128.1, 128.27,
128.33, 128.36, 128.40, 129.3, 120.8, 130.1, 132.7, 133.2, 137.4, 138.1,
138.29, 138.34, 138.6, 138.7, 139.1, 150.8, 155.1, 165.2, 165.7, 170.3.
MALDI-TOF MS: [M + Na]+ calcd for C103H116O23Si1Na1, 1771.76,
found 1772.12; ESI-TOF MS: [M + Na]+ calcd for C103H116O23Si1Na1,
Man, 1H), 3.38 (dd, J = 9.2, 2.8 Hz, 3-HMan, 1H), 3.44 (ddd, J = 9.0, 7.2,
1771.76, found 1771.91; HRMS ESI-TOF: [M + Na]+ calcd for C103H116
O23Si1Na1, 1771.7574, found 1771.7557.
-
H
1.2 Hz, 5-HMan, 1H), 3.67 (dd, J = 10.4, 1.2 Hz, 6b-HMan, 1H), 3.77 (t, J =
9.2 Hz, 4-HMan, 1H), 3.87 (dd, J = 13.2, 2.4 Hz, 5a-HXyl, 1H), 4.02 (dd,
J = 5.2, 2.4 Hz, 4-HXyl, 1H), 4.11 (d, J = 10.0 Hz, CH2Ph, 1H), 4.02 (dd,
J = 13.2, 2.4 Hz, 5b-HXyl, 1H), 4.30 (d, J = 12.0 Hz, CH2Ph, 1H), 4.75 (d,
4-Methoxyphenyl β-D-Mannopyranosyl-(1f4)-β-D-xylopyranosyl-
(1f4)-β-D-mannopyranosyl-(1f4)-α-D-xylopyranoside (36). To a
solution of protected 33 (15.1 mg, 8.63 μmol) in dry THF (1 mL)
was added TBAF (86 μL, 86 μmol) at room temperature. After the mix-
ture stirred for 18 h at the same temperature, the solvent was evaporated
in vacuo to give 4-methoxyphenyl 2-O-acetyl-3,6-di-O-benzyl-β-D-man-
nopyranosyl-(1f4)-2,3-di-O-benzoyl-β-D-xylopyranosyl-(1f4)-2,3,
6-tri-O-benzyl-β-D-mannopyranosyl-(1f4)-2,3-di-O-benzyl-α-D-xylopyra-
noside (34) (MALDI-TOF MS: [M + Na]+ calcd for C94H96O23Na1,
1615.62, found 1615.57; ESI-TOF MS: [M + Na]+ calcd for C94H96-
O23Na1, 1615.62, found 1615.70; HRMS ESI-TOF: [M + Na]+ calcd for
C94H96O23Na1, 1615.6240, found 1615.6224.). To the crude mixture in
MeOH (2.0 mL) was added 1 M MeOH solution of NaOMe (39 μL)
and stirred for 24 h at room temperature. After stirring for 4 h at room
temperature, the reaction was quenched by Amberlyst 15H+ resin,
filtered, and concentrated in vacuo. Then the residue was purified by
PTLC (hexane/ethyl acetate = 3:1) to give 4-methoxyphenyl 3,6-di-O-
benzyl-β-D-mannopyranosyl-(1f4)-D-xylopyranosyl-(1f4)-2,3,6-tri-O-
benzyl-β-D-mannopyranosyl-(1f4)-2,3-di-O-benzyl-α-D-xylopyranoside
(35) (MALDI-TOF MS: [M + Na]+ calcd for C78H86O20Na1, 1365.56,
found 1365.92; ESI-TOF MS: [M + Na]+ calcd for C78H86O20Na1,
1365.56, found 1365.64; HRMS ESI-TOF: [M + Na]+ calcd for
C78H86O20Na1, 1365.5610, found 1365.5586). Hydrogenolysis of the
resulting residue 35 was carried out in the presence of Pd(OH)2 (15.0
mg) in MeOHꢀH2O (2:1, 3 mL) for 24 h at room temperature. The
mixture was filtered through Celite under Ar atmosphere, and the filtrate
was concentrated in vacuo to give the title compound 36 (4.3 mg, 70% in
three steps). 36: 1H NMR (400 MHz, D2O): δ 3.31 (dd, J = 9.2, 7.8 Hz,
2-H Xyl2, 1H), 3.32ꢀ3.40 (m, 5-HMan2, 5-HXyl2, 2H), 3.48ꢀ3.63
(m, 5-HMan1, 3-HMan2, 4-HMan2, 3-HXyl2, 5-HXyl1, 5H), 3.66ꢀ3.76
J = 10.0 Hz, CH2Ph, 1H), 4.80 (s, 1-HMan, 1H), 5.18ꢀ5.23 (m, 2-HXyl
,
1H), 5.58 (d, J = 56.4 Hz, 1-HXyl, 1H), 5.60ꢀ5.66 (m, 3-HXyl, 1H), 5.67
(d, J = 2.8 Hz, 2-HMan, 1H), 7.00ꢀ8.12 (m, Ar, 20H); 13C NMR (100
MHz, CDCl3): δ 12.9, 18.0, 18.1, 20.8, 59.7 (C5Xyl), 66.7 (C2XylC1Xyl
ꢀ
F), 67.1 (C2XylC1XylꢀF), 67.3 (C3Xyl), 67.6 (C2Man), 68.4 (C4Man),
69.7 (C6Man), 70.8 (C4Xyl, Bn), 73.3 (Bn), 77.2 (C5Man), 81.1 (C3Man),
97.4 (C1Man), 103.9 (C1XylꢀF), 106.2 (C1XylꢀF), 127.4, 127.5, 127.6,
127.8, 128.0, 128.1, 128.4, 128.5, 129.0, 129.3, 129.9, 130.4, 129.9, 130.4,
133.5, 137.4, 138.1, 164.8, 165.2, 170.4; MALDI-TOF MS: [M + Na]+
calcd for C50H61F1O12Si1Na1, 923.38, found 923.72; ESI-TOF MS:
[M + Na]+ calcd for C50H61F1O12Si1Na1, 923.38, found 923.36; HRMS
ESI-TOF: [M + Na]+ calcd for C50H61F1O12Si1Na1, 923.3814, found
923.3827.
4-Methoxyphenyl 2-O-Acetyl-3,6-di-O-benzyl-4-O-triisopropylsilyl-
β-D-mannopyranosyl-(1f4)-2,3-di-O-benzoyl-β-D-xylopyranosyl-(1f4)-
2,3,6-tri-O-benzyl-β-D-mannopyranosyl-(1f4)-2,3-di-O-benzyl-α-D-xylo-
pyranoside (33). A mixture of AgClO4 (14.4 mg, 69.2 μmol), Cp2HfCl2
(13.8 mg, 34.6 μmol), and dried powdered MS 4A (250 mg) in dry
benzene (1.0 mL) was stirred for 30 min at room temperature under Ar
atmosphere. To the mixture was added through a cannula a solution of
32 (16.0 mg, 17.3 μmol) and 30 (11.7 mg, 13.5 μmol) in dry benzene
(1.0 mL + 0.5 mL for rinsing) at the same temperature, and the mixture
was stirred for 3 h at the same temperature under Ar atmosphere. The
reaction mixture was diluted with ethyl acetate, quenched with sat.
NaHCO3 aq, and filtered through Celite. The filtrate was extracted with
ethyl acetate, and the combined organic layers were washed with brine.
The washed organic layer was dried over Na2SO4 and evaporated in
vacuo. The crude product was purified by gel filtration chromatography
(Biobeads, SX-3, ethyl acetate/toluene, 1/1) followed by PTLC
(m, 3-HMan1, 4-HMan1, 6-HMan2, 2-HXyl1, 4H), 3.77ꢀ3.87 (m, 6-HMan1
5-HXyl1, 4-HXyl2, 3H), 3.79 (s, OMe, 3H), 3.88ꢀ4.02 (m, 2-HMan1
,
,
(hexane/ethyl acetate = 3/1) to give 33 (20.7 mg, 88%). 33: [α]25
6-HMan1, 2-HMan2, 6-HMan2, 3-HXyl1, 4-HXyl1, 6H), 4.10 (dd, J = 11.6, 5.2
D
7.4° (c 1.0, CHCl3); 1H NMR (400 MHz, CDCl3): δ 0.80ꢀ0.97
(m, TIPS, 21H), 1.95 (s, Ac, 3H), 3.16 (ddd, J = 9.6, 4.0, 1.6 Hz,
5-HMan1, 1H), 3.22 (dd, J = 12.0, 10.4 Hz, 5-HXyl2, 1H), 3.28ꢀ3.34
(m, 6-HMan2, 5-HMan2, 2H), 3.36 (dd, J = 9.2, 3.2 Hz, 3-HMan1, 1H), 3.39
Hz, 5-HXyl2, 1H), 4.44 (d, J = 7.8 Hz, 1-HXyl2, 1H), 4.74 (s, 1-HMan2
,
1H), 4.77 (s, 1-HMan1, 1H), 5.49 (d, J = 3.2 Hz, 1-HXyl1, 1H), 6.95ꢀ7.11
(m, Ar, 4H); 13C NMR (100 MHz, D2O): δ 56.6 (OMe), 60.4 (C5Xyl1),
61.2 (C6Man1), 61.9 (C6Man2), 63.8 (C5Xyl2), 67.6 (C4Man2), 71.3
(C2Man1), 71.6 (C2Man2), 71.8 (C2Xyl1), 72.2 (C4Xyl1), 72.4 (C3Man1),
(dd, J = 9.2, 2.4 Hz, 3-HMan2, 1H), 3.50ꢀ3.77 (m, 4-HMan2, 6-HMan2
,
2-HMan1, 6-H2Man1, 2-HXyl1, 5-H2Xyl1, 8H), 3.78 (s, OMe, 3H), 3.91
73.7 (C2Xyl2, C3Man2), 74.7 (C3Xyl2), 76.0 (C5Man1), 77.0 (C3Xyl1
,
(ddd, J = 10.0, 9.2, 6.4 Hz, 4-HXyl1, 1H), 3.98ꢀ4.04 (m, 5-HXyl2, 3-HXyl1
,
C4Xyl2), 77.2 (C4Man1, C5Man2), 98.6 (C1Xyl1, JC,H = 175.5 Hz), 98.9
2H), 4.13ꢀ4.20 (m, 4-HXyl2, CH2Ph, 2H), 4.24 (d, J = 12.0 Hz, CH2Ph,
1H), 4.28 (t, J = 9.6 Hz, 4-HMan1, 1H), 4.37 (d, J = 11.2 Hz, CH2Ph, 1H),
4.38 (s, 1-HMan1, 1H), 4.47 (d, J = 11.2 Hz, CH2Ph, 1H), 4.51 (d, J =
12.4 Hz, CH2Ph, 1H), 4.58 (s, 1-HMan2, 1H), 4.61 (d, J = 12.4 Hz,
(C1Man1, JC,H = 165.0 Hz), 99.2 (C1Man2, JC,H = 165.5Hz), 104.2 (C1Xyl2
,
JC,H = 174.5 Hz), 116.0, 119.6, 151.0, 155.5; MALDI-TOF MS:
[M + Na]+ calcd for C29H44O20Na1, 735.23, found 735.31; ESI-TOF
MS: [M + Na]+ calcd for C29H44O20Na1, 735.23, found 735.28; HRMS
ESI-TOF: [M + Na]+ calcd for C29H44O20Na1, 735.2324, found
735.2335. 1H NMR (400 MHz, D2O, pH 7.5 phosphate buffer at 40 °C): δ
CH2Ph, 1H), 4.64 (d, J = 12.0 Hz, CH2Ph, 1H), 4.71ꢀ4.77 (m, 1-HXyl2
,
CH2Ph, 4H), 4.80 (d, J = 12.4 Hz, CH2Ph, 1H), 4.85 (s, CH2Ph, 2H),
5.06 (d, J = 11.6 Hz, CH2Ph, 1H), 5.22 (d, J = 3.6 Hz, 1-HXyl1, 1H), 5.30
(dd, J = 9.2, 6.4 Hz, 2-HXyl2, 1H), 5.42 (dd, J = 9.6, 9.6 Hz, 3-HXyl2, 1H),
5.55 (d, J = 3.2 Hz, 2-HMan2, 1H), 6.80ꢀ7.98 (m, Ar, 49H); 13C NMR
(100 MHz, CDCl3): δ 12.9, 17.9, 18.1, 20.5, 55.6, 59.7 (C6Man1), 62.8
(C5Xyl2), 66.8 (C2Man2), 68.2 (C5Xyl1), 68.3 (C4Man2), 69.7 (C6Man2),
70.6 (Bn), 72.1 (C4Xyl2), 72.47 (C2Xyl2), 72.54 (C3Xyl2), 72.9 (C4Man1),
73.3 (Bn), 73.4 (Bn), 73.5 (Bn), 74.1 (Bn), 75.1 (Bn), 75.3 (C2Man1),
75.5 (Bn), 75.9 (C5Man1), 76.0 (C4Xyl1), 77.2 (C5Man2), 78.8 (C2Xyl1),
79.9 (C3Man1), 80.1 (C3Xyl1), 80.6 (C3Man2), 95.9 (C1Man2, JC,H = 157.4
Hz), 96.7 (C1Xyl1, JC,H = 176.4 Hz), 99.5 (C1Man1, JC,H = 155.5 Hz),
100.8 (C1Xyl2, JC,H = 168.8 Hz), 114.5, 118.3, 127.1, 127.2, 127.4,
3.51 (dd, J = 9.2, 8.0 Hz, 2-H Xyl2, 1H), 3.52ꢀ3.60 (m, 5-HMan2, 5-HXyl2
,
2H), 3.48ꢀ3.63 (m, 5-HMan1, 3-HMan2, 4-HMan2, 3-HXyl2, 4H),
3.86ꢀ3.96 (m, 3-HMan1, 4-HMan1, 6-HMan2, 2-HXyl1, 5-HXyl1, 5H), 3.87ꢀ
4.07 (m, 6-HMan1, 5-HXyl1, 4-HXyl2, 3H), 3.99 (s, OMe, 3H), 4.08ꢀ4.21
(m, 2-HMan1, 6-HMan1, 2-HMan2, 6-HMan2, 3-HXyl1, 4-HXyl1, 6H), 4.29
(dd, J = 12.0, 5.2 Hz, 5-HXyl2, 1H), 4.60 (d, J = 8.0 Hz, 1-HXyl2, 1H), 4.94
(s, 1-HMan2, 1H), 4.97 (s, 1-HMan1, 1H), 5.49 (d, J = 4.0 Hz, 1-HXyl1
,
1H), 7.14ꢀ7.32 (m, Ar, 4H); 13C NMR (100 MHz, D2O, pH 7.5
phosphate buffer at 40 °C, processed using native scale): δ 56.2 (OMe),
59.8 (C5Xyl1), 60.6 (C6Man1), 61.3 (C6Man2), 63.2 (C5Xyl2), 67.0 (C4Man2),
70.7 (C2Man1), 71.0 (C2Man2), 71.2 (C2Xyl1), 71.6 (C4Xyl1), 71.7 (C3Man1),
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ARTICLE
73.1 (C2Xyl2, C3Man2), 74.1 (C3Xyl2), 75.4 (C5Man1), 76.5 (C3Xyl1, C4Xyl2),
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76.5 (C4Man1, C5Man2), 98.0 (C1Xyl1, JC,H = 175.5 Hz), 98.4 (C1Man1, JC,H
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115.5, 119.1, 150.4, 155.0 (also see Table 2).
Molecular Modeling Experiment. The modelings were performed on
MacroModel, ver. 8.1,34a through a conformational search program. Con-
formational profiles were generated by 1000 step Monte Carlo (MCMM)34b
searches with Amber*39 force fields in water using the GB/SA continuum
solvation model,40 and then reminimized by multiple minimization program
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structure did not reach a gradient to <0.05 kJ/Å mol by C-search.
3
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Supporting Information. Experimental procedures and
b
results of antifreeze activity of 36; results of molecular modeling
of (ManXyl)10 and (ManXyl)2 in H2O calculated by MacroMo-
del, ver 8.1; H and 13C NMR spectra of 1, 2, 5, 8ꢀ12, 14,
1
16ꢀ20, 22ꢀ24, 30, 32, 33, 36. This material is available free of
charge via the Internet at http://pubs.acs.org.
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’ AUTHOR INFORMATION
Corresponding Author
aishiwa@riken.jp; yukito@riken.jp
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(24) When acetonide 24 was used as an acceptor for IAD, cleavage of
acetonide has been observed to give a complex mixture of products.
When 17 and 19 were used as donor for IAD, β-mannosides were
obtained in 34% and 40% in two steps, respectively.
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been applied, although methyl 4,6-O-cyclohexylidene-2-O-(4-meth-
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’ ACKNOWLEDGMENT
We thank Prof. Jun-ichi Tamura, Dr. Masakazu Hachisu and
Mr. Hidekazu Kano for their valuable advises and Ms. Akemi
Takahashi and Ms. Satoko Shirahata for their kind technical
assistance. This work was partly supported by a Grant-in-Aid for
Scientific Research from the Ministry of Education, Culture,
Sports, Science, and Technology (No. 22510243), and Fund for
Collaborative Research (2010) in RIKEN.
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(36) The structure of cellotetraose in H2O calculated by Macro-
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’ NOTE ADDED AFTER ASAP PUBLICATION
The toc/abstract graphic was replaced and this paper reposted
November 30, 2011.
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