Synthesis of the sugar moiety of TIME-EA4, a glycopeptide isolated from silkworm diapause eggs

Article abstract of DOI:10.1016/j.carres.2006.04.015

We describe the efficient synthesis of the tetrasaccharide, 2-O-acetyl-3,4,6-tri-O-benzyl-α-d-mannopyranosyl-(1→6)-2,4-di-O-acetyl-3-O-allyl-β-d-mannopyranosyl-(1→4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-β-d-glucopyranosyl-(1→4)-3,6-di-O-benzyl-2-deoxy-2-

Full text of DOI:10.1016/j.carres.2006.04.015

Carbohydrate Research 341 (2006) 1796–1802
Synthesis of the sugar moiety of TIME-EA4, a glycopeptide
isolated from silkworm diapause eggs
Shouji Hiro, Yoshinosuke Usuki and Hideo Iio*
Department of Material Science, Graduate School of Science, Osaka City University, Osaka 558-8585, Japan
Received 21 February 2006; received in revised form 26 March 2006; accepted 12 April 2006
Available online 15 May 2006
Abstract—We describe the efficient synthesis of the tetrasaccharide, 2-O-acetyl-3,4,6-tri-O-benzyl-a-D-mannopyranosyl-(1!6)-2,4-
di-O-acetyl-3-O-allyl-b-D-mannopyranosyl-(1!4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-b-D-glucopyranosyl-(1!4)-3,6-di-O-benz-
yl-2-deoxy-2-phthalimido-b-D-glucopyranosyl azide, which is the protected form of the sugar unit of TIME-EA4 that is isolated from
the diapausing eggs of the silkworm, Bombyx mori. The b-linked D-mannoside of the tetrasaccharide was obtained using the conven-
tional oxidation–reduction method for inversion of the configuration at the C-2 hydroxyl group of b-D-glucoside. The reduction was
effected with NaBH4 in a methanolic solution in a ratio of 98:2 in favor of the b-D-mannoside that was obtained in 87% yield.
Ó 2006 Elsevier Ltd. All rights reserved.
Keywords: TIME-EA4; N-Glycan; Glycosylation; b-D-Mannoside; Oxidation–reduction
1. Introduction
The glycan structure and the glycosylation site of EA4
were unambiguously determined to be Man-Man-Glc-
NAc-GlcNAc-Asn22 in order from the nonreducing
end (Fig. 1); this was done by using an electrospray-ion-
ization (ESI)-tandem quadrupole/orthogonal-accelera-
tion time-of-flight (Q-TOF) mass spectrometer with a
nano-HPLC system (LC–ESI-Q-TOF-MS).5 The same
sugar structure was also reported for the membrane gly-
coproteins of three insect cell lines.6 Recently, a core
pentasaccharide was additionally observed at the same
linkage site of the peptide from the Showa strain of
the silkworm.7 In this paper, we describe an efficient syn-
thesis of the sugar moiety of TIME-EA4 in the fully pro-
tected form.
Glycoproteins play an important role in biological pro-
cesses such as cellular and immunogenic recognition.
Moreover, the oligosaccharide moiety is thought to con-
tribute to the solubility and thermal stability of proteins
and to structural roles such as cell adhesion.1 The sugar
moieties are linked to the protein component through
either O-glycosidic or N-glycosylic bonds. All N-glycans
have a core pentasaccharide that links to the amide
nitrogen in the side chain of Asn in the consensus se-
quence Asn-Xaa-Ser/Thr, where Xaa can be any amino
acid except Pro and Asp.2
During the course of our studies on the synthesis of
N-glycopeptides, we began the initial studies on the
sugar moiety of TIME-EA4 (EA4), an ATPase isolated
from the diapausing eggs of the silkworm, Bombyx mori,
by Isobe and co-workers.3 EA4 is a glycoprotein con-
taining 156 amino acids and an N-linked tetrasaccha-
ride, which appears to be involved in measuring the
duration of diapause in the life cycle of the silkworm.4
OH
O
HO
HO
HO
O
OH
O
Gly21
Asn22
OH
O
NHAc
H
HO
O
O
HO
HO
N
HO
O
NHAc
Ile23 Thr24
OH
*
Figure 1. Proposed tetrasaccharide structure for the carbohydrate
Corresponding author. Fax: +81 6 6605 3152; e-mail: iio@sci.osaka-
moiety attached to Asn22 in TIME-EA4.
cu.ac.jp
0008-6215/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2006.04.015

S. Hiro et al. / Carbohydrate Research 341 (2006) 1796–1802
1797
2. Results and discussion
according to the oxidation–reduction protocol12 to ob-
tain a b-(1!4)-linked D-mannosyl derivative (Scheme
1).
The key challenges for the synthesis of N-glycans have
been the construction of b-D-mannosides, which are
especially difficult to accomplish since both neighboring
group assistance and the anomeric effect uniformly favor
the formation of a-D-mannosides during glycosylation.
Methods for the construction of b-D-mannosidic link-
ages have been reviewed,8 and several approaches,
including the intramolecular aglycon delivery9 and
direct b-D-mannoside coupling protocol,10 have been re-
ported to effectively solve this problem. Epimerizations
of b-D-glucopyranosides at the C-2 position by direct
SN2 inversion11 or oxidation–reduction of the C-2 hy-
droxyl group12 are established methods for the indirect
preparation of b-D-mannosides; additionally, a two-step
protocol for b-selective glycosylation using a glyco-ulo-
syl bromide and an acceptor, followed by manno-selec-
tive reduction of the resulting b-2-ketoglucoside, is also
established.13 In this study, we have accomplished the
synthesis of the oligosaccharide by applying the conven-
tional oxidation–reduction method of inversion of the
configuration of the b-D-glucoside to yield the b-D-man-
noside. In other words, our strategy for the synthesis of
the b-D-mannoside was to first construct the b-(1!4)-
linked D-glucosyl derivative and then invert the configu-
ration of the C-2 hydroxyl group of the D-Glc residue
For this purpose, we selected the 3-O-allyl-protected
glucosyl trichloroacetimidate 114 as a donor, which
was coupled with glucosamine acceptor 215 using tri-
methylsilyl triflate (TMSOTf)16 as a promoter to give
the b-D-gluco-configured disaccharide. Prior to the con-
version of the b-D-glucoside to the b-D-mannoside, the
2II-OH group had to be selectively deprotected. Thus,
the removal of the acetates and the regioselective ben-
zylidenation of the intermediate yielded disaccharide 3,
which was suitably functionalized for the ensuing inver-
sion sequence to the b-D-mannoside. First, compound 3
was oxidized by dimethyl sulfoxide (DMSO)–acetic
anhydride12 to afford uloside 4, and the benzylidene
group of the resulting compound was removed to yield
diol uloside 5.
The reductions of 4 and 5 were evaluated for the
inversion sequence under several conditions. The results
obtained with a series of different reagents and solvents
are shown in Table 1. The reduction of the 2-keto group
of ulosides 4 and 5 by sodium borohydride in a polar
protic solvent yielded the corresponding b-D-mannoside
as the major product with a distinctly higher stereoselec-
tivity. In entry 1, in particular, the conventional sodium
borohydride reduction of 4 in methanol stereoselectively
AcO
AcO
BnO
HO
BnO
NPhth
NH
Ph
O
a
O
O
O
BnO
O
O
OMP
+
OCCCl3
OMP
O
O
O
OAc
OH
NPhth
OBn
2
1
3
R1O
R2O
R1O
OH
O
NPhth
NPhth
b
d
O
O
BnO
O
R2O
BnO
O
OMP
OMP
O
O
O
O
OBn
OBn
6: R1, R2 = PhCH
7: R1 = R2 = H
4: R1, R2 = PhCH
5: R1 = R2 = H
c
c
Scheme 1. Reagents and conditions: (a) (i) TMSOTf, CH2Cl2, 0 °C, 5 min; (ii) NaOMe, 3:1 MeOH–CH2Cl2, rt, 18 h; (iii) benzaldehyde
dimethylacetal, CSA, MeCN, 56% (three steps from 1 and 2); (b) Ac2O, DMSO, rt, 3 h, 79%; (c) ethylene glycol, TsOHÆH2O, MeCN, rt; (d) NaBH4,
MeOH.
Table 1. Stereoselectivities of the hydride reductions of 4 and 5
Entry
Uloside
Reagent
Solvent
Time
(min)
Ratioa
manno:gluco
Total
yield (%)
1
2
3
4
5
6
7
8
4
4
4
4
5
5
5
5
NaBH4
NaBH4
NaBH4
Bu4NBH4
NaBH4
NaBH4
NaBH4
Bu4NBH4
MeOH
1:1 MeOH–CH2Cl2
CH2Cl2
THF
MeOH
1:1 MeOH–CH2Cl2
CH2Cl2
THF
1
5
10
30
1
5
10
30
98:2
89
84
82
70
82
76
88
75
87:13
55:45
52:48
92:8
89:11
10:90
5:95
a The mixture of manno- and gluco-isomers was separated by column chromatography.

1798
S. Hiro et al. / Carbohydrate Research 341 (2006) 1796–1802
provided b-D-mannoside 6, an epimer of 3, in excellent
yield. On the other hand, sodium borohydride reduction
in 1:1 dichloromethane–methanol, the solvent system
recommended for manno-selective reduction,13 resulted
in unsatisfactory stereoselectivity. The epimeric b-D-glu-
coside and b-D-mannoside were readily separated by
column chromatography to afford pure compounds.
The stereochemistry of the reduction products was con-
firmed by 1H NMR analyses of 6 and 7. Curiously,
reduction of the deprotected compound 5 by sodium
borohydride or tetrabutylammonium borohydride in
an aprotic solvent provided stereoselectivity in favor of
the b-D-glucoside (entries 7 and 8), while the benzylidene
compound 4 was reduced nonselectively under identical
conditions (entries 3 and 4). A reversal in the carbonyl
reduction selectivities of ulosides was also reported:
3,4,6-O-tribenzoylulosides are reduced by the boraneÆ
pyridine complex in tetrahydrofuran (THF) in a gluco:-
manno ratio greater than 8:1.13b The reasons for
manno:gluco selectivity are not clearly apparent at the
moment.
In order to obtain the tetrasaccharide, a mannoside
unprotected at the 6-O position was required. Therefore,
as depicted in Scheme 2, compound 6 was treated with
ethylene glycol and p-toluenesulfonic acid in acetonitrile
for 10 min to obtain triol 7 in 92% yield. Subsequently, 7
was selectively protected at the 6-position with a bulky
tert-butyldimethylsilyl group17 to provide 8 in 78%
yield. Diol 8 was acetylated with acetic anhydride and
triethylamine; this was followed by deprotection of the
silyl group with hydrogen fluoride18 to give 9 in 92%
yield in two steps. Next, having obtained 9, we pro-
ceeded to the synthesis of the protected tetrasaccharide
by using the fluoride donor 1019 and the azide acceptor
13.19 The reaction between 9 and the fluoride donor 10
in the presence of boron trifluoride etherate (BF3ÆOEt2)
proceeded smoothly to yield the trisaccharide as a single
a-anomer, the p-methoxyphenyl (MP) group of which
was oxidatively cleaved by ceric ammonium nitrate
(CAN)20 to provide 11 in 68% yield in two steps. Subse-
quently, treatment with dimethylaminosulfur trifluoride
(DAST)21 for 10 min at 0 °C afforded fluoride 12, which
was finally glycosylated with the azide acceptor 13 in the
presence of BF3ÆOEt2 to yield the desired tetrasaccharide
14 as a single b-anomer. The stereostructures of 12 and
1
14 were fully characterized by 1D and 2D H NMR
spectroscopy.
In summary, we achieved a stereocontrolled synthe-
sis of the protected tetrasaccharide 14 in TIME-EA4,
a N-glycopeptide isolated from diapausing eggs of the
silkworm, B. mori. The b-linked D-mannoside of the
tetrasaccharide was formed using an oxidation–reduc-
tion inversion sequence on the b-D-glucoside. The
reduction was effected with NaBH4 in a methanolic
solution in a ratio of 98:2 in favor of the b-D-manno-
side that was obtained in 87% yield. Compound 14 is
fully functionalized and protected for further manipu-
lation toward the synthesis of the N-glycopeptide.
The glycosylasparagine of EA4 could be synthesized
by the reaction between tetrasaccharide 14 and
Fmoc-aspartic acid tert-butyl ester in the presence of
triethylphosphine,22 which would be used as the oligo-
saccharide donor to N-acetylglucosaminyl peptide
using the transglycosylation activity of endo-b-N-acet-
ylglucosaminidase.23 This oxidation–reduction strategy
for the synthesis of the sugar moiety of the N-glycan
could be applicable to the other higher sugar deriva-
tives and is under investigation.
OAc
O
BnO
BnO
BnO
OAc
O
BnO
BnO
BnO
F
O
R1O
R2O
a
OR3
O
10
NPhth
OAc
O
7
BnO
O
NPhth
OMP
BnO
O
O
O
AcO
c
R
O
O
OBn
OBn
8: R1 = TBDMS, R2 = R3 = H
9: R1 = H, R2 = R3 = OAc
11: R = OH
12: R = F
b
d
OAc
BnO
BnO
BnO
O
BnO
HO
BnO
O
N3
NPhth
O
OBn
O
13
OAc
O
NPhth
O
BnO
O
AcO
e
N3
NPhth
O
O
BnO
OBn
14
Scheme 2. Reagents and conditions: (a) TBDMSCl, NEt3, DMF, rt, 78%; (b) (i) Ac2O, Et3N, DMAP, rt; (ii) HFÆpy, CH2Cl2, 0 °C, 91% (two steps
from 8); (c) (i) 10/BF3ÆOEt2, CH2Cl2, 10 min, 0 °C; (ii) CAN, 10:1 MeCN–H2O, 5 min, 68% (two steps from 9 and 10); (d) DAST, CH2Cl2, 0 °C, 78%;
(e) 13/BF3ÆOEt2, CH2Cl2, 10 min, 0 °C, 70%.

S. Hiro et al. / Carbohydrate Research 341 (2006) 1796–1802
1799
3. Experimental
3.1. General methods
CH2CH–CH2–), 4.81, 4.60 (1H, d, J 12.3 Hz, OCH2Ph),
4.79 (1H, d, J 11.9 Hz, OCH2Ph), 4.68 (1H, d, J 7.3 Hz,
H-1II), 4.46–4.38 (4H, m, H-2I, OCH2Ph, CH2CH–
CH2–), 4.24–4.15 (3H, m, H-3I, 4I,II), 4.05 (1H, dd, J
3.7 and 11.0 Hz, H-6aI), 3.85 (1H, dd, J 1.8 and
11.0 Hz, H-6bI), 3.70 (3H, s, OMe), 3.75–3.66 (1H, m,
H-5I), 3.56–3.42 (4H, m, H-2II, 3II, 6II), 3.27–3.19 (1H,
m, H-5II), 3.06 (1H, s, OH). 13C NMR (CDCl3,
100 MHz, selected signals): d 101.13 (C-1II), 97.73 (C-
1I). HRFABMS (positive-ion mode): Calcd for
C51H52NO13 [M+H]+, 886.3440; found, 886.3416.
1H and 13C NMR spectra were recorded, respectively, at
600 and 150 MHz (Bruker DRX-600), 400 and 100 MHz
(JEOL JNMLA400) or 300 and 75 MHz (JEOL
JNMLA300). Chemical shifts are reported in ppm rela-
tive to Me4Si and CDCl3 with CHCl3 as the internal ref-
1
erence (7.26 ppm for H NMR and 77.0 ppm for 13C
NMR). Coupling constants are reported in hertz (Hz)
and determined directly from 1H NMR spectra. Spectral
splitting patterns were designated as s (singlet), d (dou-
blet), t (triplet), m (multiplet), and br (broad). Mass
spectra were obtained on JEOL JMS-700T or JEOL
JMS-AX500 spectrometers.
3.3. p-Methoxyphenyl 3-O-allyl-4,6-O-benzylidene-b-D-
arabino-hex-2-ulopyranosyl-(1!4)-3,6-di-O-benzyl-2-
deoxy-2-phthalimido-b-D-glucopyranoside (4)
All air- and moisture-sensitive reactions were carried
out in flame-dried, argon-flushed, two-necked flasks
sealed with rubber septa, and dry solvents and reagents
were introduced with a syringe. THF and Et2O were
freshly distilled from sodium benzophenone ketyl.
CH2Cl2 was freshly distilled from P2O5. Flash column
chromatography was carried out on KANTO CHEMI-
CAL silica gel 60 N (spherical, neutral, 40–50 lm), and
precoated E. Merck silica gel plates (Art5715 Kieselgel
60F257 0.25 mm) were used for TLC analyses.
A solution of 3 (85 mg, 96 lmol) in 1:2 Ac2O–DMSO
(3.0 mL) was stirred for 3 h at room temperature. The
reaction was quenched with aq NaHCO3. The mixture
was extracted with Et2O and the organic layer was
washed with brine, dried (Na2SO4), and concentrated.
Flash chromatography of the residue gave 4 (67 mg,
20
76 lmol, 79%): ½aꢀD +10 (c 0.02, CHCl3); 1H NMR
(CDCl3, 300 MHz): d 7.68–6.68 (23H, m, arom), 6.02–
5.86 (1H, m, CH2CH–CH2–), 5.59 (1H, d, J 10.8 Hz,
H-1I), 5.45 (1H, s, benzylidene), 5.31 (1H, dd, J 1.5
and 17.2 Hz, CH2CH–CH2–), 5.21 (1H, dd, J 1.5 and
10.2 Hz, CH2CH–CH2–), 4.86, 4.49 (1H, d, J 10.0 Hz,
OCH2Ph), 4.77, 4.65 (1H, d, J 11.9 Hz, OCH2Ph), 4.59
(1H, s, H-1II), 4.46–4.34 (4H, m, H-2I, 3I, CH2CH–
CH2–), 3.84–3.57 (6H, m, H-4II, 5I, 6I,II), 3.71 (3H, s,
OMe), 3.53 (1H, d, J 10.7 Hz, H-3II), 3.25–3.18 (1H,
m, H-5II), small peaks were additionally observed due
to the ketone hydrate of compound 4.12c HRFABMS
(positive-ion mode): Calcd for C51H50NO13 [M+H]+,
884.3212; found, 884.3274.
3.2. p-Methoxyphenyl 3-O-allyl-4,6-O-benzylidene-b-D-
glucopyranosyl-(1!4)-3,6-di-O-benzyl-2-deoxy-2-phthal-
imido-b-D-glucopyranoside (3)
A mixture of acceptor 2 (0.12 g, 0.20 mmol), donor 1
˚
(0.15 g, 0.31 mmol) and 4 A molecular sieves (MS,
0.10 g) in CH2Cl2 (2.0 mL) was stirred for 1 h. TMSOTf
(10 lL, 52 lmol) was added at 0 °C. After stirring for
5 min, the reaction was quenched with aq NaHCO3.
The mixture was extracted with CH2Cl2, and the organic
layer was washed with brine and dried (Na2SO4). Evap-
oration of solvent gave the crude b-D-gluco-configured
disaccharide, which was dissolved in 3:1 MeOH–CH2Cl2
(3.0 mL) and treated with NaOMe (11 mg) overnight at
room temperature. After neutralization with an excess
of Amberlite IR-120 (H+) cation-exchange resin, filtra-
tion and evaporation of the solvent gave the crude tet-
raol. This was taken up in MeCN (1.0 mL) and
treated with benzaldehyde dimethylacetal (45 lL,
0.30 mmol) and D-camphor-10-sulfonic acid mono-
hydrate (23 mg, 0.10 mmol) for 1 h. The reaction was
quenched by the addition of Et3N, and the solvent was
evaporated to dryness. Flash chromatography of the
residue gave 3 (0.10 g, 0.11 mmol, 55%, three steps from
3.4. p-Methoxyphenyl 3-O-allyl-b-D-arabino-hex-2-ulo-
pyranosyl-(1!4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-
b-D-glucopyranoside (5)
To a solution of 4 (67 mg, 76 lmol) and ethylene glycol
(50 lL) in MeCN (1.0 mL) was added p-TsOHÆH2O
(13 mg, 68 lmol). After stirring for 30 min at room tem-
perature, the reaction was quenched with aq NaHCO3.
The mixture was diluted with Et2O, and the organic
layer was washed with brine, dried (Na2SO4), and con-
centrated. Flash chromatography of the residue gave 5
20
1
(46 mg, 57 lmol, 75%): ½aꢀD +40 (c 0.19, CHCl3); H
NMR (CDCl3, 300 MHz): d 7.68–6.67 (18H, m, arom),
6.02–5.89 (1H, m, CH2CH–CH2–), 5.59 (1H, d, J
8.4 Hz, H-1I), 5.34 (1H, dd, J 1.5 and 17.3 Hz,
CH2CH–CH2–), 5.21 (1H, dd, J 1.5 and 10.0 Hz,
CH2CH–CH2–), 5.12 (1H, s, H-1II), 4.91, 4.48 (1H, d,
J 11.9 Hz, OCH2Ph), 4.70, 4.64 (1H, d, J 11.9 Hz,
OCH2Ph), 4.59 (1H, dd, J 8.3 and 10.6 Hz, H-3I), 4.46
20
1
2 and 8): ½aꢀD +38 (c 0.06, CHCl3); H NMR (CDCl3,
400 MHz): d 7.67–6.66 (23H, m, arom), 6.02–5.89 (1H,
m, CH2CH–CH2–), 5.60 (1H, d, J 8.4 Hz, H-1I), 5.46
(1H, s, benzylidene), 5.31 (1H, dd, J 1.5 and 17.1 Hz,
CH2CH–CH2–), 5.20 (1H, dd, J 1.5 and 11.6 Hz,

1800
S. Hiro et al. / Carbohydrate Research 341 (2006) 1796–1802
20
1
(1H, dd, J 8.4 and 10.6 Hz, H-2I), 4.35–4.23 (2H, m,
CH2CH–CH2–), 4.13 (1H, dd, J 8.3 and 9.6 Hz, H-4I),
4.13–4.08 (1H, m, H-5II), 3.92–3.74 (6H, m, H-4II, 5I,
6I,II), 3.71 (3H, s, OMe), 2.06 (1H, d, J 9.7 Hz, H-3II).
0.12 mmol, 92%): ½aꢀD +12 (c 0.1, CHCl3); H NMR
(CDCl3, 300 MHz): d 7.67–6.68 (18H, m, arom), 5.92–
5.87 (1H, m, CH2CH–CH2–), 5.60 (1H, d, J 8.4 Hz,
H-1I), 5.28 (1H, dd, J 1.7 and 17.2 Hz, CH2CH–CH2–),
5.20 (1H, dd, J 1.7 and 10.4 Hz, CH2CH–CH2–), 4.88
and 4.52 (2H, d, J 12.1 Hz, OCH2Ph), 4.73 and 4.53
(2H, d, J 11.9 Hz, OCH2Ph), 4.66 (1H, s, H-1I), 4.53
(1H, d, J 11.9 Hz, OCH2Ph), 4.45–4.43 (2H, m, H-2I,
3I), 4.19–3.95 (2H, m, CH2CH–CH2–), 4.16 (1H, dd, J
9.5 and 9.5 Hz, H-4I), 3.99 (1H, d, J 2.9 Hz, H-2II),
3.85–3.62 (6H, m, H-4II, 5I, 6I,II), 3.70 (3H, s, OMe),
3.22–3.13 (1H, m, H-5II), 3.15 (1H, dd, J 2.9 and
9.7 Hz, H-3II). 13C NMR (CDCl3, 100 MHz, selected
signals): d 99.97 (C-1II), 97.67 (C-1I). HRFABMS (neg-
ative-ion mode): Calcd for C44H47NO13 [M]ꢁ, 797.3047;
found, 797.3034.
HRFABMS
(negative-ion
mode):
Calcd
for
C44H45NO13 [M]ꢁ, 795.2891; found, 795.2889.
3.5. p-Methoxyphenyl 3-O-allyl-4,6-O-benzylidene-b-D-
mannopyranosyl-(1!4)-3,6-di-O-benzyl-2-deoxy-2-
phthalimido-b-D-glucopyranoside (6)
To a stirred solution of 4 (81 mg, 91 lmol) in dry MeOH
(2.0 mL) was added NaBH4 (10 mg, 0.27 mmol). After
stirring for 1 min at 0 °C, the mixture was diluted with
CH2Cl2, and the reaction mixture was quenched by
the addition of water and aq NaHCO3. The organic
layer was separated and dried (Na2SO4), and the solvent
was evaporated. Flash chromatography of the residue
gave the b-configured D-mannoside 6 (71 mg, 80 lmol,
3.7. p-Methoxyphenyl 3-O-allyl-6-O-tert-butyldimethyl-
silyl-b-D-mannopyranosyl-(1!4)-3,6-di-O-benzyl-2-
deoxy-2-phthalimido-b-D-glucopyranoside (8)
20
87%): ½aꢀD +11 (c 0.03, CHCl3); 1H NMR (CDCl3,
400 MHz): d 7.68–6.68 (23H, m, arom), 5.96–5.86 (1H,
m, CH2CH–CH2–), 5.63 (1H, d, J 6.8 Hz, H-1I), 5.49
(1H, s, benzylidene), 5.31 (1H, dd, J 1.4 and 15.6 Hz,
CH2CH–CH2–), 5.20 (1H, dd, J 1.5 and 10.5 Hz,
CH2CH–CH2–), 4.86 and 4.49 (2H, d, J 12.0 Hz,
OCH2Ph), 4.47 and 4.53 (2H, d, J 12.9 Hz, OCH2Ph),
4.73 (1H, s, H-1II), 4.47–4.42 (2H, m, H-2I, 3I), 4.28–
3.76 (7H, m , H-5I, 6I,II, CH2CH–CH2–), 4.02 (1H, d,
J 2.0 Hz, H-2II), 3.95 (1H, dd, J 8.9 and 9.2 Hz, H-4I),
3.71 (3H, s, OMe), 3.60 (1H, dd, J 10.7 and 10.7 Hz,
H-4II), 3.43 (1H, dd, J 2.0 and 10.7 Hz, H-3II), 3.22–
3.17 (1H, m, H-5II), 2.26 (1H, s, OH). HRFABMS (neg-
ative-ion mode): Calcd for C51H51NO13 [M]ꢁ, 885.3360;
found, 885.3361.
To a solution of 7 (40 mg, 50 lmol) and Et3N (0.1 mL)
in DMF (1.0 mL) was added TBDMSCl (15 mg,
0.10 mmol). After stirring for 30 min at room tempera-
ture, the reaction was quenched with aq NaHCO3.
The mixture was extracted with Et2O, and the organic
layer was washed with brine, dried (Na2SO4), and con-
centrated. Flash chromatography of the residue gave 8
(39 mg, 43 lmol, 86%): ½aꢀD +30 (c 0.03, CHCl3); H
NMR (CDCl3, 400 MHz): d 7.67–6.71 (18H, m, arom),
5.99–5.90 (1H, m, CH2CH–CH2–), 5.62 (1H, d, J
8.3 Hz, H-1I), 5.32–5.20 (2H, m, CH2CH–CH2–), 4.86
and 4.49 (2H, d, J 12.2 Hz, OCH2Ph), 4.73 and 4.57
(2H, d, J 12.0 Hz, OCH2Ph), 4.67 (1H, s, H-1II), 4.42–
4.38 (2H, m, H-2I, 3I), 4.14–4.06 (3H, m, H-4I,
CH2CH–CH2–), 3.99 (1H, d, J 3.0 Hz, H-2II), 3.84
(3H, s, OMe), 3.86–3.70 (5H, m, H-5I, 6I, 6II), 3.65
(1H, dd, J 7.3 and 9.2 Hz, H-4II), 3.21–3.16 (1H, m,
H-5II), 3.19 (1H, dd, J 3.0 and 9.2 Hz, H-3II), 3.35
(1H, s, OH), 2.37 (1H, s, OH), 0.84 (9H, s, t-Bu), 0.04
(3H, s, Me), 0.02 (3H, s, Me). HRFABMS (negative-
ion mode): Calcd for C50H61NO13Si [M]ꢁ, 911.3912;
found, 911.3922.
20
1
3.6. p-Methoxyphenyl 3-O-allyl-b-D-mannopyranosyl-
(1!4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-b-D-gluco-
pyranoside (7)
3.6.1. From 5. To a stirred solution of 5 (30 mg,
37 lmol) in dry MeOH (1.0 mL) was added NaBH4
(41 mg, 0.11 mmol). After stirring for 1 min at 0 °C,
the mixture was diluted with CH2Cl2, and the reaction
was quenched by the addition of water and aq NaHCO3.
The organic layer was separated and dried (Na2SO4),
and the solvent was evaporated. Flash chromatography
of the residue gave 7 (23 mg, 28 lmol, 76%).
3.8. p-Methoxyphenyl 2,4-di-O-acetyl-3-O-allyl-b-D-
mannopyranosyl-(1!4)-3,6-di-O-benzyl-2-deoxy-2-
phthalimido-b-D-glucopyranoside (9)
3.6.2. From 6. To a solution of compound 6 (120 mg,
0.13 mmol) and ethylene glycol (50 lL) in MeCN
(1.0 mL) was added p-TsOHÆH2O (27 mg, 0.14 mmol).
After stirring for 30 min at room temperature, the reac-
tion was quenched with aq NaHCO3. The mixture was
extracted with Et2O, and the organic layer was washed
with brine, dried (Na2SO4), and concentrated. Flash
To a solution of 8 (20 mg, 22 lmol) in Et3N (0.5 mL)
was added Ac2O (1.0 mL), and the mixture was stirred
for 30 min at room temperature. The mixture was
poured into aq NaHCO3, then extracted with Et2O
and concentrated in vacuo. The residue was dissolved
in CH2Cl2 (2.0 mL) and treated with HFÆpyridine
(0.1 mL) for 30 min at room temperature. The reaction
chromatography of the residue gave
7 (99 mg,

S. Hiro et al. / Carbohydrate Research 341 (2006) 1796–1802
1801
was quenched with aq NaHCO3. The mixture was ex-
3.10. 2-O-Acetyl-3,4,6-tri-O-benzyl-a-D-mannopyran-
tracted with Et2O, and the organic layer was washed
successively with aq CuSO4, and aq NaHCO3 and then
concentrated. Flash chromatography of the residue
osyl-(1!6)-2,4-di-O-acetyl-3-O-allyl-b-D-mannopyran-
osyl-(1!4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-b-D-
glucopyranosyl fluoride (12)
20
gave 9 (18 mg, 20 lmol, 91%): ½aꢀD +35 (c 0.06,
CHCl3); 1H NMR (CDCl3, 400 MHz): d 7.70–6.67
(18H, m, arom), 5.84–5.72 (1H, m, CH2CH–CH2–),
5.62 (1H, d, J 8.1, H-1I), 5.35 (1H, d, J 3.1, H-2II),
5.36–5.15 (2H, m, CH2CH–CH2–), 4.93 (1H, dd, J
9.7 and 9.8 Hz, H-4II), 4.89 and 4.46 (2H, d, J
11.9 Hz, OCH2Ph), 4.79 and 4.51 (2H, d, J 11.9 Hz,
OCH2Ph), 4.67 (1H, s, H-1II), 4.39 (1H, dd, J 8.1
and 9.3 Hz, H-2I), 4.31 (1H, dd, J 8.4 and 9.3 Hz,
H-3I), 4.17 (1H, dd, J 8.4 and 9.1 Hz, H-4I), 4.04
(1H, dd, J 5.1 and 13.1 Hz, CH2CH–CH2–), 3.71
(3H, s, OMe), 3.84–3.24 (7H, m, 5I,II, 6I,II, CH2CH–
CH2–), 3.30 (1H, dd, J 3.1 and 9.7 Hz, H-3II), 2.13
(3H, s, Me), 2.07 (3H, s, Me). HRFABMS (positive-
ion mode): Calcd for C48H52NO15 [M+H]+, 882.3339;
found, 882.3346.
To a stirred mixture of 11 (49 mg, 39 lmol) in dry
CH2Cl2 (2.0 mL) was added DAST (0.10 mL,
0.95 mmol). After stirring for 10 min at 0 °C, the reac-
tion was quenched with aq NaHCO3. The mixture was
extracted with Et2O, and the organic layer was washed
with aq NaHCO3 and brine, dried (Na2SO4), and con-
centrated. Flash chromatography of the residue gave
20
12 (38 mg, 30 lmol, 77%): ½aꢀD +35 (c 0.13, CHCl3);
1H NMR (CDCl3, 600 MHz): d 7.66–6.75 (29H, m,
arom), 5.80 (1H, dd, J 53.9 and 7.4, H-1I), 5.74–5.73
(1H, m, CH2CH–CH2–), 5.34 (1H, d, J 3.0, H-2II),
5.31 (1H, dd, J 2.8 and 1.8 Hz, H-2III), 5.20 (1H, dd, J
1.5 and 17.5 Hz, CH2CH–CH2–), 5.15 (1H, dd, J 1.5
and 10.3 Hz, CH2CH–CH2–), 5.06 (1H, dd, J 9.8 and
9.9 Hz, H-4II), 4.85 (1H, d, J 1.8 Hz, H-1III), 4.69 (1H,
s, H-1II), 4.81 and 4.51 (2H, d, J 11.0 Hz, OCH2Ph),
4.79 and 4.46 (2H, d, J 13.0 Hz, OCH2Ph), 4.77 and
4.55 (2H, d, J 12.3 Hz, OCH2Ph), 4.60 and 4.45 (2H,
d, J 12.0 Hz, OCH2Ph), 4.52 and 4.34 (2H, d, J
11.0 Hz, OCH2Ph), 4.27–4.17 (3H, m, H-2I, 3I, 4I),
4.01 (1H, dd, J 5.0 and 13.1 Hz, CH2CH–CH2–), 3.90
(1H, dd, J 2.8 and 9.3 Hz, H-3III), 3.85 (1H, dd, J 9.2
and 9.3 Hz, H-4III), 3.81–3.61 (8H, m, H-5I, 5III, 6I,
6aII, 6III, CH2CH–CH2–), 3.55 (1H, dd, J 2.9 and
11.5 Hz, H-6bII), 3.42–3.39 (1H, m, H-5II), 3.24 (1H,
dd, J 3.0 and 9.9 Hz, H-3II), 2.14 (3H, s, Ac), 2.05
(3H, s, Ac), 1.99 (3H, s, Ac). 13C NMR (CDCl3,
150 MHz, selected signals): d 104.87 (JCF 235 Hz, C-
1I), 98.77 (C-1II), 97.84 (C-1III). HRFABMS (negative-
ion mode): Calcd for C70H74FNO19 [M]ꢁ, 1251.4839;
found, 1251.4843.
3.9. 2-O-Acetyl-3,4,6-tri-O-benzyl-a-D-mannopyranosyl-
(1!6)-2,4-di-O-acetyl-3-O-allyl-b-D-mannopyranosyl-
(1!4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-b-D-gluco-
pyranose (11)
A mixture of 9 (50 mg, 57 lmol), 10 (33 mg, 68 lmol)
˚
and 4 A MS (0.10 g) in CH2Cl2 (2.0 mL) was stirred
for 1 h. BF3ÆOEt2 (10 lL, 78 lmol) was added to solu-
tion at 0 °C. After stirring for 5 min, the reaction was
quenched with aq NaHCO3. The mixture was extracted
with CH2Cl2, and the organic layer was washed with
brine, dried (Na2SO4), and concentrated. The residue
was dissolved in 10:1 CH3CN–water (2.0 mL) and trea-
ted with CAN ((NH4)2Ce(NO3)6) (62 mg, 0.11 mmol)
for 30 min at room temperature. The mixture was ex-
tracted with Et2O, and the organic layer was washed
with water and brine, dried (Na2SO4), and evaporated
in vacuo. Flash chromatography of the residue gave
3.11. 2-O-Acetyl-3,4,6-tri-O-benzyl-a-D-mannopyran-
osyl-(1!6)-2,4-di-O-acetyl-3-O-allyl-b-D-mannopyran-
osyl-(1!4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-b-D-
glucopyranosyl-(1!4)-3,6-di-O-benzyl-2-deoxy-2-phthal-
imido-b-D-glucopyranosyl azide (14)
20
11 (49 mg, 39 lmol, 68%): ½aꢀD +36 (c 0.05, CHCl3);
1H NMR (CDCl3, 400 MHz): d 7.65–6.82 (29H, m,
arom), 5.75–5.71 (1H, m, CH2CH–CH2–), 5.35 (1H, d,
J 3.2 Hz, H-2II), 5.34 (1H, s, H-2III), 5.23–5.15 (3H, m,
H-1I, CH2CH–CH2–), 5.02 (1H, dd, J 9.9 and 9.8 Hz,
H-4II), 4.86 (1H, s, H-1III), 4.82 and 4.64 (2H, d, J
11.2 Hz, OCH2Ph), 4.80 and 4.53 (2H, d, J 12.2 Hz,
OCH2Ph), 4.79 and 4.43 (2H, d, J 12.0 Hz, OCH2Ph),
4.58 and 4.46 (2H, d, J 11.9 Hz, OCH2Ph), 4.50 and
4.28 (2H, d, J 10.5 Hz, OCH2Ph), 4.78 (1H, s, H-1II),
4.31 (1H, dd, J 8.4 and 9.3 Hz, H-3I), 4.15 (1H, dd, J
8.4 and 9.1 Hz, H-4I), 4.05–3.90 (3H, m, H-2I, 3III,
A mixture of acceptor 13 (6.0 mg, 12 lmol), donor 12
˚
(10 mg, 8.0 lmol) and 4 A MS (0.10 g) in CH2Cl2
(1.0 mL) was stirred for 1 h. BF3ÆOEt2 (5 lL, 39 lmol)
was added at 0 °C. After stirring for 5 min, the reaction
was quenched with aq NaHCO3. The mixture was ex-
tracted with CH2Cl2, and the organic layer was washed
with brine, dried (Na2SO4), and concentrated. Flash
chromatography of the residue gave 14 (9.8 mg,
20
CH2CH–CH2–), 3.85–3.40 (9H, m, H-4III, 5III, 6I,II,III
,
5.6 lmol, 70%): ½aꢀD +26 (c 0.01, CHCl3); 1H NMR
CH2CH–CH2–), 3.65–3.60 (1H, m, H-5I), 3.51–3.45
(1H, m, H-5II), 3.24 (1H, dd, J 9.9 and 3.2 Hz, H-3II).
(CDCl3, 600 MHz): d 7.77–6.71 (43H, m, arom), 5.79–
5.72 (1H, m, CH2CH–CH2–), 5.36 (1H, d, J 3.0 Hz,
H-2III), 5.28 (1H, d, J 3.2 Hz, H-2IV), 5.23 (1H, d, J
8.3 Hz, H-1II), 5.20 (1H, dd, J 1.5 and 17.5 Hz,
HRFABMS
(negative-ion
mode):
Calcd
for
C70H75NO20 [M]ꢁ, 1249.4882; found, 1249.4901.

1802
S. Hiro et al. / Carbohydrate Research 341 (2006) 1796–1802
5. Kurahashi, T.; Miyazaki, A.; Murakami, Y.; Suwan, S.;
Franz, T.; Isobe, M.; Tani, N.; Kai, H. Bioorg. Med.
Chem. 2002, 10, 1703–1710.
6. Kubelka, V.; Altman, F.; Kornfeld, G.; Ma¨rz, L. Arch.
Biochem. Biophys. 1994, 308, 148–157.
7. Pitchayawasin, S.; Isobe, M.; Tani, N.; Kai, T. Bioorg.
Med. Chem. Lett. 2004, 14, 2527–2531.
8. Pozsgay, V. Stereoselective Synthesis of b-Mannosides. In
Carbohydrates in Chemistry and Biology. Part 1: Chemis-
CH2CH–CH2–), 5.14 (1H, dd, J 1.5 and 9.2 Hz,
CH2CH–CH2–), 5.13 (1H, d, J 9.3 Hz, H-1I), 5.10
(1H, dd, J 9.1 and 9.9 Hz, H-4III), 4.81 (1H, s, H-1IV),
4.72 (1H, s, H-1III), 4.82 and 4.45 (2H, d, J 12.8 Hz,
OCH2Ph), 4.81 and 4.46 (2H, d, J 9.5 Hz, OCH2Ph),
4.77 and 4.42 (2H, d, J 11.1 Hz, OCH2Ph), 4.61 and
4.52 (2H, d, J 12.3 Hz, OCH2Ph), 4.59 and 4.41 (2H,
d, J 12.2 Hz, OCH2Ph), 4.51 and 4.49 (2H, d, J
11.8 Hz, OCH2Ph), 4.48 and 4.30 (2H, d, J 10.9 Hz,
OCH2Ph), 4.25–4.09 (5H, m, H-2II, 3I, 3II, 4I, 4II), 4.02
(1H, dd, J 9.3 and 10.4 Hz, H-2I), 4.04–4.00 (1H, m,
CH2CH–CH2–), 3.86 (1H, dd, J 9.4 and 3.2 Hz, H-
3IV), 3.79 (1H, dd, J 9.4 and 9.5 Hz, H-4IV), 3.26 (1H,
J 3.0 and 9.9 Hz, H-3III), 3.75–3.25 (13H, m, H-
5I,II,III,IV, H-6I,II,III,IV, CH2CH–CH2–), 2.17 (3H, s,
Ac), 2.01 (3H, s, Ac), 1.91 (3H, s, Ac). 13C NMR
(CDCl3, 150 MHz, selected signals): d 99.20 (C-1III),
97.76 (C-1IV), 96.67 (C-1II), 85.49 (C-1I). HRFABMS
(negative-ion mode): Calcd for C98H99N5O25 [M]ꢁ,
1745.6644; found, 1745.6644.
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We thank Professor Minoru Isobe, Nagoya University,
for providing helpful discussion. This work was sup-
ported in part by a Grant-in-Aid for Scientific Research
(Grant No. 11680592) and Grants-in-Aid for Priority
Area Research Program (Grant No. 12045256) of the
Ministry of Education, Science, Culture, and Sports of
Japan.
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