Synthesis of an unnatural N-glycan-linked dolichyl pyrophosphate precursor

Article abstract of DOI:10.1271/bbb.67.1761

An unnatural α-D-mannopyranose-linked chitobiosyl dolichyl pyrophosphate, a stereoisomer of the N-glycan biosynthesis intermediate, was synthesized. The protected trisaccharide, α-D-Man-(1→4)-β-D- GlcNAc-(1→4)-D-GlcNAc, carrying a 4-methylbenzoyl group wa

Full text of DOI:10.1271/bbb.67.1761

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Synthesis of an Unnatural N-Glycan-linked Dolichyl
Pyrophosphate Precursor
Yuko NAKAHARAa, Tomoharu NONAKAa, Hironobu HOJOa & Yoshiaki NAKAHARAa
a Institute of Glycotechnology, Department of Applied Biochemistry, Tokai
UniversityKitakaname 1117, Hiratsuka, Kanagawa 259-1292, Japan
Published online: 22 May 2014.
To cite this article: Yuko NAKAHARA, Tomoharu NONAKA, Hironobu HOJO & Yoshiaki NAKAHARA (2003) Synthesis of
an Unnatural N-Glycan-linked Dolichyl Pyrophosphate Precursor, Bioscience, Biotechnology, and Biochemistry, 67:8,
1761-1766, DOI: 10.1271/bbb.67.1761
To link to this article: http://dx.doi.org/10.1271/bbb.67.1761
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Biosci. Biotechnol. Biochem., 67 (8), 1761–1766, 2003
Synthesis of an Unnatural
N
-Glycan-linked Dolichyl Pyrophosphate Precursor
†
Yuko NAKAHARA, Tomoharu NONAKA, Hironobu HOJO, and Yoshiaki NAKAHARA
Institute of Glycotechnology, Department of Applied Biochemistry, Tokai University, Kitakaname 1117,
Hiratsuka, Kanagawa 259-1292, Japan
Received March 12, 2003; Accepted April 24, 2003
An unnatural
dolichyl pyrophosphate, a stereoisomer of the
biosynthesis intermediate, was synthesized. The protect-
ed trisaccharide, -Man-(1 4)- -GlcNAc-(1 4)-
GlcNAc, carrying a 4-methylbenzoyl group was pre-
pared for the convenience of a TLC analysis. 1-
a
-
D
-mannopyranose-linked chitobiosyl
As part of our ongoing project designed to eluci-
date the nature of the functional importance of the
N
-glycan
N
- and
natural trisaccharide-linked dolichyl pyrophosphate,
a potent probe for substrate-speciˆcity studies on
O
-glycan structures in glycoproteins,7) an un-
a
-
D
ª
b
-
D
ª
D
-
N
-
O
-
glycan biosynthesis-associated enzymes such as
mannosyl transferase and OT, was synthesized. In
Phosphorylation, condensation with dolichyl phos-
phate, and subsequent deprotection aŠorded the title
compound.
this paper, we describe synthesis of
-GlcNAc-(1 4)- -GlcNAc-PP-Dol 1, a man-
nopyranoside-linkage stereoisomer of native glycan
-Man-(1 4)- -GlcNAc-]. Synthesis of the
related unnatural pentasaccharides linked to the
a
-D
-Man-(1ª4)-
b
-
D
ª
a
-
D
Key words: synthesis; glycosylation;
N
-glycan biosyn-
[
b
-
D
ª
b
-
D
thesis; unnatural -glycan; dolichyl
pyrophosphate precursor
N
N
-
acetylasparagine amide has recently been reported by
this group.8)
Although it has been recognized that
N
-glycans of
Coupling reaction of known glycosyl donor 28) and
acceptor 38) was promoted with Cp2ZrCl2–AgClO4
9)
glycoprotein serve as recognition signals in a variety
of biological phenomena such as cell-cell adhesion,
receptor-ligand interaction, and cancer metastasis,
the relationship between the glycan structure and
glycopeptide function has not been well elucidated.1)
Transfer `en bloc' of Glc3Man9GlcNAc2 oligosaccha-
ride from the dolichyl pyrophosphate (Dol-PP)
precursor to asparagine residues on nascent proteins
to give trisaccharide 4 in an 85
z yield. Ir-catalyzed
isomerization of the allyl group10) and subsequent
hydrolysis with mercuric salt aŠorded 5 (92 ), which
was converted to corresponding acetamide derivative
6 (88 ) by dephthaloylation and acetylation. The
z
z
3,6-hydroxyl groups on the mannose residue were
protected with a 4-methylbenzoyl (MBz) group to
aŠord 7 (91 ), with the expectation that the presence
z
is a conserved biological process for the
N
-glycosyla-
tion of proteins in eukaryotic cells.2) Subsequent
processing in the endoplasmic reticulum and glycosyl
of this UV-absorbing group would facilitate a
TLC analysis of the protected glycosyl dolichyl
pyrophosphate. The characteristic methyl proton
signal of MBz, which appears apart from the huge
signals of dolichols, would also provide an easy
structural assignment of the compound by 1H-NMR.
The silyl group was removed, and resulting hemia-
transfer in the Golgi apparatus produce
N-glycans of
diverse structure. It is intriguing that the shorter
oligosaccharides can also be transferred to protein
acceptors by oligosaccharyl transferase (OT) owing
to the broad substrate speciˆcity of the enzyme.3,4)
Tai and Imperiali have recently reported a radiolabel-
ling experiment aiming to clarify the minimal struc-
tural requirement for glycosyl donors in a yeast OT
system by using chitobiosyl donor analogs with
substitution at the C-2 acetamide sites.5)
cetal 8 was acetylated (9: 81
z in two steps) before
hydrogenolytic cleavage of the benzyl ether, since an
attempted conversion of the benzylated sugar into
the acetylated one by hydrogenation of 7 and subse-
quent acetylation resulted in a complex mixture
arising from hydrogenation of the TBDPS group and
scission of the silyl ether linkage in part. Hydrogeno-
On the other hand, enzyme deˆciency in the early
process of
N-glycan biosynthesis is known to be
responsible for some severe genetic diseases.6) More
detailed studies on the speciˆcity of the related
enzymes as well as on the disordered mechanism of
gene expression are required to establish a precise
diagnosis and suitable therapeutic approach.
lyzed product 10 was acetylated to give 11 (85
z
in two steps), which was treated with hydrazine
acetate11) to selectively split the anomeric acetyl group
(85
z). Resulting hemiacetal 12 was phosphorylated
with tetrabenzyl pyrophosphate in the presence of
†
To whom correspondence should be addressed. Fax:
81-463-50-2075; E-mail: yonak keyaki.cc.u-tokai.ac.jp
＋ ＠
Abbreviations: TBDPS, tert-butyldiphenylsilyl; TLC, thin-layer chromatography; LDA, lithium diisopropylamide

1762
Y. NAKAHARA et al.
Fig. 1. Reagents and Conditions.
－
(a) Cp2ZrCl2, AgClO4, CH2Cl2, 10
9C, 2 h; (b) 1. Ir-complex, THF; 2. HgCl2, HgO, aq. acetone, 5 h; (c) 1. (CH2NH2)2, BuOH,
100 C, 48 h; 2. Ac2O, MeOH, 24 h; (d) MBzCl, pyridine, 5 h; (e) TBAF, AcOH, THF, 16 h; (f ) Ac2O, pyridine, 4 h; (g) H2, Pd–C,
9
－
AcOH, 42 h; (h)) Ac2O, pyridine, 48 h; (i) hydrazine acetate, DMF, 30 min; (j) LDA, [(BnO)2P(O)]2O, 789C, 1.5 h; (k) H2, Pd–C,
MeOH, 3 h; (l) 1. (C3H3N2)2CO, DMF; 2. 15, CH2Cl2, 48 h.
LDA according to the literature3) to aŠord 13 in an
85 yield. Cleavage of the benzyl phosphate by
with a Jeol AL400 spectrometer [1H at 400 MHz and
13C at 100 MHz. Chemical shifts are expressed in
ppm downˆeld from the signal for internal Me4Si for
solutions in CDCl3. MALDI-TOF mass spectra were
obtained with a Bruker AUTOFLEX-T spectrome-
ter, 2,5-dihydroxybenzoic acid being used as a
matrix. HPLC was performed with Mightysil RP-18
z
hydrogenation exclusively gave key intermediate 14.
Compound 14 was activated with carbonyldiimida-
zole and reacted with dolichyl phosphate 1512) which
had been prepared from semi-synthesized dolichol.13)
The coupling reaction was readily monitored by TLC
and completed in 48 h. Pyrophosphate 16 was
×
×
column (4.6 150 mm for analysis and 10 250 mm
obtained in an 85
z
yield after puriˆcation by gel-
for preparation; Kanto Chemical Co.).
permeation chromatography and subsequent chro-
matography on silica gel. The structure of 16 was
tert-Butyldiphenylsilyl 3,6-di-O-allyl-2,4-di-O-ben-
1
proved by H- and 13C-NMR as well as by MALDI
zyl-
deoxy-2-phthalimido-
d i - O - b e n z yl -2 - d e o xy - 2 -p h t h a l i m i d o -
glucopyranoside ( ). A mixture of Cp2ZrCl2 (0.72 g,
a
-
D
-mannopyranosyl-(1
ª
4)-3,6-di-O-benzyl-2-
TOF-MS studies. Finally, the acetyl and 4-methyl-
benzoyl protecting groups on the trisaccharide
were removed by a treatment with NaOMe in
MeOH–CH2Cl2 to produce the target compound 1.
Successful deprotection was clearly demonstrated by
the characteristic mass spectrum. A biological study
of this synthesized unnatural precursor will be
reported elsewhere.
b
-
D
-glucopyranosyl-(1 4)-3,6-
ª
b
- D -
4
2.46 mmol), AgClO4 (1.02 g, 4.92 mmol), and dried
MS 4A (5 g) in anhydrous CH2Cl2 (20 ml) was stirred
under Ar for 1 h at room temperature and then
cooled to
－
10 C in an ice-MeOH bath. To the
9
stirred mixture was added a solution of 3 (1.07 g,
1.47 mmol) in CH2Cl2 (5 ml). The mixture was stirred
for 1.5 h before adding a solution of 2 (1.12 g,
1.23 mmol) in CH2Cl2 (5 ml). The reaction mixture
was stirred for 2 h, and the reaction was quenched by
adding sat. NaHCO3. The resulting mixture was
ˆltered through Celite, and the ˆltrate was extracted
with CHCl3. The extract was successively washed
with water and brine, dried over Na2SO4, and
concentrated in vacuo. The crude product was chro-
Experimental
Optical rotation data were determined with a Jasco
DIP-370 polarimeter for solutions in CHCl3, unless
otherwise noted. Column chromatography was
performed on PSQ 100B silica gel (Fuji Silysia). TLC
and HPTLC were performed on 60 F254 silica gel (E.
Merck). 1H- and 13C-NMR spectra were recorded

Synthesis of an
N
-Glycan Precursor Analog
1763
＝
matographed on silica gel with toluene-EtOAc (10:1)
to give 4 (1.70 g, 85 ). f 0.51 (6:1 toluene-EtOAc).
1
a
), 4.47 (1H, d,
J
7.8 Hz, H-1
b
), 1.79 (3H, s, Ac),
z
R
1.59 (3H, s, Ac), 1.04 (9H, s, t-Bu). 13C-NMR
d: 99.4
1
＋
＝
[
a
]
39.5
CH–), 5.28–5.21 (4H, m, H-1
5.14–5.09 (2H, m, CH2 CH–), 5.02 (1H, d,
8.0 Hz, H-1 : 99.9
), 0.82 (9H, s, t-Bu). 13C-NMR
(C-1c), 96.6 (C-1 ), 93.1 (C-1 ). Anal. Calcd. for
C98H100O18N2Si: C, 72.57; H, 6.21; N, 1.73
Found: C, 72.47; H, 6.38; N, 1.62
9
(c 1). H-NMR
d
: 5.29 (2H, m, CH2
(C-1
C80H92O16N2Si 0.5H2O: C, 69.90; H, 6.89; N,
2.04 . Found: C, 69.90; H, 6.72; N, 2.28
b), 98.4 (C-1c), 95.5 (C-1a). Anal. Calcd. for
D
＝
, H-1c, CH2 CH–),
a
＝
J
＝
z
z
.
b
d
a
b
tert-Butyldiphenylsilyl 2,4-di-O-benzyl-3,6-di-O-
(4-methylbenzoyl)- -mannopyranosyl-(1 4)-2-
ac e t a m i d o - 3 , 6 -d i - O - b e n z y l - 2 - d e o x y -
glucopyranosyl-(1 4)-2-acetamido-3,6-di-O-benzyl-
2-deoxy- -glucopyranoside ( ). A mixture of 6
z
.
a
-
D
ª
b
z.
D
- -
ª
tert-Butyldiphenylsilyl 2,4-di-O-benzyl-
nopyranosyl-(1 4)-3,6-di-O-benzyl-2-deoxy-2-
phthalimido- -glucopyranosyl-(1 4)-3,6-di-O-
benzyl-2-deoxy-2-phthalimido- -glucopyranoside
). red suspension of the Ir complex
[Ir(COD)(PMePh2)]2PF6, 30 mg, 36 mol in fresh-
a
-
D
-man-
b
-D
7
ª
(1.52 g, 1.11 mmol) and 4-methylbenzoyl chloride
(0.36 ml, 2.67 mmol) in CH3CN-pyridine (1:4, 30 ml)
was stirred for 5 h before being concentrated in
vacuo. The residue was extracted with EtOAc,
successively washed with sat. NaHCO3, water and
b
-
D
ª
b
-
D
(
5
A
s
m
t
ly distilled THF (5 ml) was stirred in an atmosphere
of H2 at room temperature for 30 min to give a color-
less solution of the activated catalyst, and then the
atmosphere was replaced with Ar. To the solution
was added a carefully degassed solution of 4 (1.10 g,
0.68 mmol) in dry THF (10 ml). After stirring for
brine, dried (Na2SO4), and concentrated in vacuo
The crude product was chromatographed on silica gel
with toluene-EtOAc (7:3) to give 7 (1.62 g, 91 ). Rf
0.41 (3:2 toluene-EtOAc). [ 3.0
(c 1). 1H-NMR
), 5.57 (1H, dd,
.
z
a
]
－
H
9
D
＝
＝
d
: 5.61 (1H, d,
J
9.0 Hz, N
J
＝
2.9, 9.3 Hz, H-3
c
), 5.24 (1H, d,
J
2.0 Hz, H-1
c
),
＝
8.8 Hz, NH), 4.66 (1H, d, J
＝
3 h, the reaction mixture was concentrated in vacuo
.
5.04 (1H, d,
6.4 Hz, H-1 ), 4.44 (1H, dd,
4.42 (1H, d, 7.6 Hz, H-1
11.5 Hz, H-6 ), 2.43 (3H, s, C
s, C 3C6H4–), 1.80 (3H, s, Ac), 1.59 (3H, s, Ac),
1.04 (9H, s, t-Bu). 13C-NMR
: 99.7 (C-1 ), 99.4 (C-
), 95.6 (C-1 ). Anal. Calcd. for C96H104O18N2Si: C,
71.98; H, 6.54; N, 1.75 . Found: C, 71.71; H, 6.51;
N, 1.73
J
＝
The residue was dissolved in 90 aq. acetone (10 ml)
z
a
J
4.0, 11.5 Hz, H-6
), 4.38 (1H, dd, 2.0,
3C6H4–), 2.35 (3H,
c),
and stirred with HgCl2 (770 mg, 1.63 mmol) and
HgO (60 mg, 0.28 mmol) at room temperature for
5 h. The mixture was concentrated in vacuo to
remove the acetone, the residue was extracted with
J
＝
b
J
＝
c
?
H
H
d
b
CHCl3, and the extract was washed with 10
z
aq. KI
1
c
a
and brine, dried (Na2SO4), and concentrated in va-
cuo. The crude product was puriˆed by column chro-
matography on silica gel with toluene-EtOAc (5:1) to
z
z
.
give 5 (0.96 g, 92
z
). Rf 0.36 (3:1 toluene-EtOAc).
2,4-Di-O-benzyl-3,6-di-O-(4-methylbenzoyl)-
mannopyranosyl-(1 4)-2-acetamido-3,6-di-O-ben-
zyl-2-deoxy- -glucopyranosyl-(1 4)-2-acetamido-
1-O-acetyl-3,6-di-O-benzyl-2-deoxy-
glucopyranose ( ). To a mixture of 7 (300 mg,
a
-
D
-
1
＋
[
a
]
37.8
5.26 (1H, d,
8.1 Hz, H-1
), 0.84 (9H, s, t-Bu). 13C-NMR
(C-1 ), 96.5 (C-1 ), 93.2(C-1 ). Anal. Calcd. for
9
(c 1). H-NMR
d
: 5.32 (1H, brs, H-1
), 5.04 (1H, d,
: 98.9
c
),
ª
D
J
＝
8.3 Hz, H-1
a
J
＝
b
-
D
ª
b
d
a
,
b
-D -
c
a
b
9
C92H92O18N2Si 0.5H2O: C, 71.25; H, 6.04; N,
0.19 mmol) and AcOH (0.22 ml, 3.73 mmol) in
freshly distilled THF (3 ml) was added 1M tetrabutyl-
ammonium ‰uoride-THF (0.76 ml, 0.76 mmol). The
mixture was stirred at room temperature for 16 h and
then concentrated in vacuo. The residue was extract-
ed with EtOAc, successively washed with sat.
NaHCO3, water and brine, dried (Na2SO4), and
concentrated in vacuo. The crude product was
chromatographed on Bio-beads S X1 with CHCl3 to
give 8 (230 mg), which was dissolved in pyridine
(2 ml) and stirred with Ac2O (0.5 ml) for 4 h. The
mixture was concentrated in vacuo, and solution of
the residue in CHCl3 was washed with sat. NaHCO3,
water, and brine, dried (Na2SO4), and concentrated
in vacuo. The product was puriˆed by gel permeation
chromatography on Bio-beads S X3 with EtOAc and
then by silica gel chromatography with toluene-
1.81
z. Found: C, 71.15; H, 6.01; N, 1.93
z.
tert-Butyldiphenylsilyl 2,4-di-O-benzyl-
nopyranosyl-(1
deoxy- -glucopyranosyl-(1
di-O-benzyl-2-deoxy-
a
-
D
-man-
ª
4)-2-acetamido-3,6-di-O-benzyl-2-
4)-2-acetamido-3,6-
).
b
-
D
ª
b
-
D
-glucopyranoside
(
6
A
mixture of 5 (968 mg, 0.63 mmol) and ethylenedia-
mine (0.84 ml, 12.6 mmol) in n-BuOH (20 ml) was
heated at 1009C for 48 h and concentrated in vacuo.
The residue was dissolved in MeOH (5 ml), stirred
with Ac2O (2.4 ml) at 0 C-room temperature for
9
24 h, and then concentrated in vacuo. The residue
was dissolved in CHCl3, successively washed with sat.
NaHCO3, water and brine, dried (Na2SO4), and
concentrated in vacuo. The crude product was
chromatographed on silica gel with toluene-EtOAc
(1:1) to give 6 (752 mg, 88
z
). Rf 0.44 (2:1 toluene-
EtOAc (1:1) to aŠord 9 (215 mg, 81
z
). Rf 0.60 and
(c 1). 1H-NMR
d
: 5.62 (1H, d,
J
0.67 (9:1 CHCl3–MeOH). 1H-NMR (
a
-acetate)
d
:
－
EtOAc). [
a
]
1.7
9
D
＝
＝
＝
＝
9.0 Hz, N
(1H, d,
H
), 5.23 (1H, d,
J
1.5 Hz, H-1
c
), 5.15
6.16 (1H, d,
9.3 Hz, H-3
J
3.4 Hz, H-1
), 5.22 (1H, d,
a
), 5.62 (1H, dd,
J
2.9,
J
＝
8.5 Hz, N
H
), 4.67 (1H, d,
J
＝
6.4 Hz, H-
c
J
＝
2.0 Hz, H-1
c
), 2.44

1764
Y. NAKAHARA et al.
(3H, s, C
H
3C6 H4–), 2.35 (3H, s, C
H
3C6H4–), 2.07
101.7 (C-1
b
), 99.4 (C-1
c
), 91.5 [C-1
a(
a
-OH)].
1
＋
＋
(3H, s, Ac), 1.75 (3H, s, Ac), 1.72 (3H, s, Ac). H-
MALDI TOF-MS: m z 1097.56 (M Na) ; calcd for
W
＝
NMR (
5.58 (1H, dd,
2.0 Hz, H-1 ), 2.43 (3H, s, C
3C6H4–), 2.00 (3H, s, Ac), 1.99 (3H, s, Ac), 1.80
(3H, s, Ac). Anal. Calcd. for C82H88O19N2 0.5H2O:
C, 69.62; H, 6.34; N, 1.98 . Found: C, 69.55; H,
6.34; N, 1.98
b
-acetate)
d
: 5.60 (1H, d,
J
6.4 Hz, H-1
a
),
C50H62O24N2Na, 1097.36.
＝
＝
c), 5.29 (1H, d, J
J
3.0, 9.5 Hz, H-3
3C6H4–), 2.37 (3H, s,
c
H
2,4-Di-O-acetyl-3,6-di-O-(4-methylbenzoyl)-
mannopyranosyl-(1 4)-2-acetamido-3,6-di-O-
acetyl-2-deoxy- -glucopyranosyl-(1 4)-2-
a c e t a m i d o - 3 , 6 - d i - O - a c e t y l - 2 - d e o x y - - -
a
-D-
C
H
ª
D
b
-
ª
z
a
D
z.
glucopyranosyl dibenzyl phosphate (13). To a stirred
solution of 12 (75 mg, 0.07 mmol) in anhydrous THF
2,4-Di-O-acetyl-3,6-di-O-(4-methylbenzoyl)-
mannopyranosyl-(1 4)-2-acetamido-3,6-di-O-
acetyl-2-deoxy- -glucopyranosyl-(1 4)-2-
acetamido-1,3,6-tri-O-acetyl-2-deoxy-
glucopyranose 11). Compound
0.04 mmol) was hydrogenated with 10
a
-
D
-
(3 ml) was added 0.76M LDA-hexane THF (0.2 ml,
W
－
ª
D
0.15 mmol) at 78
9
C under Ar. The mixture was
b
-
ª
stirred for 30 min. A
solution of tetrabenzyl
a
,
b
-
D
-
pyrophosphate (45 mg, 0.08 mmol) in anhydrous
THF (1 ml) was added, and the mixture was stirred
for 1.5 h before being concentrated in vacuo. The
residue was dissolved in EtOAc, successively washed
with sat. NaHCO3, water and brine, dried (Na2SO4),
and concentrated in vacuo. The crude product was
puriˆed by gel permeation chromatography on Bio-
(
9
(56 mg,
Pd–C
z
(50 mg) in AcOH (5 ml) for 72 h. The catalyst was
ˆltered oŠ through Celite, and the ˆltrate was
concentrated in vacuo. The residue was dissolved in
pyridine (2.5 ml), stirred with Ac2O (0.3 ml) for 48 h
at room temperature, and concentrated in vacuo
.
beads S X3 with EtOAc to give 13 (79 mg, 85
0.60 (9:1 CHCl3–MeOH). [
z
). Rf
37.39
(c 1). 1H-
＋
The residue was extracted with EtOAc, successively
washed with sat. NaHCO3, water and brine, dried
(Na2SO4), and concentrated in vacuo. The crude
product was chromatographed on silica gel with
a
]
D
＝
8.0 Hz, Ar), 7.83 (2H, d, J
NMR
d
: 7.98 (2H, d,
J
＝
8.0 Hz, Ar), 7.39–7.33 (10H, m, Ar), 7.30–7.23
(4H, m, Ar), 6.17 (1H, d,
J
＝
9.7 Hz, N
H
), 5.74 (1H,
＝
＝
hexane-acetone (1:2) to give 11 (38 mg, 85
z
).
-acetate)
), 5.09 (1H, d,
), 2.41 (3H, s, C 3C6H4–), 2.40 (3H, s,
3C6H4–), 2.19 (3H, s, Ac), 2.17 (3H, s, Ac),
R
f 0.66
: 6.10
2.2 Hz,
d,
5.63 (1H, dd,
3.1, 9.9 Hz, H-3
H-1 , H-2 , 2 PhC
12.7 Hz, H-6 ), 4.44–4.15 (7H, m, H-2
, H-5 , H-6
), 3.93 (1H, m, H-5
), 3.71 (1H, t,
), 2.41 (3H, s, C
3C6H4–), 2.11 (3H, s, Ac), 2.09 (6H, s, Ac), 2.05
(3H, s, Ac), 1.98 (3H, s, Ac), 1.95 (3H, s, Ac), 1.93
(3H, s, Ac), 1.70 (3H, s, Ac). 13C-NMR
: 101.6 (C-
), 99.3 (C-1 ), 96.0 (d, JCP 5.8 Hz, C-1 ). Anal.
Calcd. for C64H75 O27N2P: C, 57.57; H, 5.66; N,
2.10 . Found: C, 57.94; H, 5.62; N, 2.08
J
9.3 Hz, N
H
), 5.68 (1H, t,
J
9.9 Hz, H-4
c
),
1
＝
(14:1 CHCl3–MeOH). H-NMR (
(1H, d, 3.7 Hz, H-1
H-1
a
d
J
3.1, 5.8 Hz, H-1
), 5.15–5.03 (8H, m, H-3
2–), 4.55 (1H, dd,
, H-6a
a
), 5.45 (1H, dd,
J
J
＝
a
J
＝
＝
c
a
, H-3
＝
J
b
,
2.7,
, H-
c
H
c
c
H
C
H
c
b
a
2.12 (3H, s Ac), 2.09 (3H, s, Ac), 2.05 (3H, s, Ac),
2.01 (3H, s, Ac), 1.96 (3H, s, Ac), 1.95 (3H, s, Ac),
1
6
b
, H-6
b
, H-6
b
?
c
c?), 4.06–3.97 (2H, m, H-
＝
J
a
?
, H-2
a
), 3.87 (1H, t,
9.6 Hz, H-4 ), 3.60
H3C6H4–), 2.40 (3H, s,
1.93 (3H, s, Ac). 13C-NMR (
a
-acetate)
d
: 101.9
9.2 Hz, H-4
b
b
J
＝
a
(C-1
b
), 99.3 (C-1
c
), 90.5 (C-1
a
). Anal. Calcd. for
(1H, m, H-5
CH
C52H64O25N2 1.5H2O: C, 54.59; H, 5.90; N, 2.45
z
.
Found: C, 54.17; H, 5.62; N, 2.38
z
. MALDI
＋
＋
TOF-MS: m z 1138.90 (M Na) ; calcd. for
d
W
C52H64O25N2Na, 1139.37.
1
b
c
＝
a
2,4-Di-O-acetyl-3,6-di-O-(4-methylbenzoyl)-
mannopyranosyl-(1 4)-2-acetamido-3,6-di-O-
acetyl-2-deoxy- -glucopyranosyl-(1 4)-2-
a c e t a m i d o - 3 , 6 - d i - O - a c e t y l - 2 - d e o x y -
a
-
D
-
z
z
.
ª
D
b
-
ª
2,4-Di-O-acetyl-3,6-di-O-(4-methylbenzoyl)-
mannopyranosyl-(1 4)-2-acetamido-3,6-di-O-
acetyl-2-deoxy- -glucopyranosyl-(1 4)-2-
a c e t a m i d o - 3 , 6 - d i - O - a c e t y l - 2 - d e o x y -
glucopyranosyl phosphate 14). Compound 13
(145 mg, 0.11 mmol) was hydrogenated with 10
Pd–C (27 mg) in MeOH (10 ml) for 3 h. The catalyst
was ˆtered oŠ, and the ˆltrate was concentrated with
MeOH (5 ml) and pyridine (1 ml) in vacuo. The resid-
ual oil was dissolved in MeOH (5 ml) and stirred with
a
D
- -
b
-
D
-
ª
D
glucopyranose (12). To a solution of 11 (483 mg,
0.43 mmol) in anhydrous DMF (20 ml) was added
crystalline hydrazine acetate (171 mg, 1.86 mmol).
The mixture was stirred for 30 min at room tempera-
ture. The mixture was diluted with EtOAc, succes-
sively washed with sat. NaHCO3, water and brine,
dried (Na2SO4), and concentrated in vacuo. The
crude product was chromatographed on silica gel
b
-
ª
a
- D -
(
z
with CHCl3–MeOH (39:1) to give 12 (394 mg, 85
z
d
).
tri-
n-butylamine (80 ml, 2.6 eq). To the mixture was
1
Rf 0.30 and 0.40 (9:1 CHCl3–MeOH). H-NMR
:
added distilled water (1.2 ml). The excess tri-n-
＝
5.19 [1H, brt,
J
4.0 Hz, H-1a(
2.0 Hz, H-1
(3H, s, C
Ac), 2.08 (3H, s Ac), 2.05 (6H, s, Ac), 1.97 (3H, s,
Ac), 1.96 (3H, s, Ac), 1.94 (3H, s, Ac). 13C-NMR
a
-OH)], 5.06 (1H,
butylamine was extracted three times with hexane
(3 ml). The resulting aq. MeOH solution was concen-
trated in vacuo. The residual water in the product
was co-evaporated with toluene in vacuo to give 14 as
＝
brd,
J
c), 2.41 (3H, s, CH3C6H4–), 2.40
H
3C6H4–), 2.16 (3H, s, Ac), 2.12 (3H, s,
d
:
z
a tri-n-butylammonium salt (132 mg, 89 ), which

Synthesis of an
N
-Glycan Precursor Analog
1765
was used for the next reaction without further puriˆ-
cation.
Acknowledgments
P1-[2,4-Di-O-acetyl-3,6-di-O-(4-methylbenzoyl)-
a
-
This work was supported by grant-aid for scientiˆc
research from the Ministry of Education, Culture,
Sports, Science, and Technology of Japan. We thank
Kuraray Co., Ltd. and Dr. T. Takigawa for
generously presenting the synthesized dolichol, and
Dr. Chihara and his staŠ (Riken) for elemental
analyses. We also thank Tokai University for grant-
aid for high-technology research.
D
-mannopyranosyl-(1
ª
4)-2-acetamido-3,6-di-O-
acetyl-2-deoxy-
b
-
D
-glucopyranosyl-(1ª4)-2-
a c e t a m id o - 3 , 6 - d i- O - a c e t y l - 2 - d e o x y -
a
D
- -
glucopyranosyl]-P2-dolichyl pyrophosphate (16).
mixture of the tri- -butylammonium salt of 14
mol) and 1,1 -carbonyldiimidazole
mol) in anhydrous DMF (2 ml) was
A
n
(66 mg, 49
(60 mg, 370
m
m
?
stirred at room temperature for 4 h. The excess
reagent was then decomposed by stirring with MeOH
(25 ml) for 30 min. The mixture was diluted with
References
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Vliegenthart, J. F. G., and Schachter, H., Elsevier,
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MeOH and extracted with hexane. The methanolic
layer was concentrated in vacuo. The residue was dis-
solved in anhydrous CH2Cl2 (1 ml) and stirred with a
solution of dolichyl phosphate tri-n-butylammonium
salt 153) (150 mg, 2 equiv. based on C90-dolichol) in
anhydrous CH2Cl2 (1 ml) at room temperature for
48 h. The solvent was evaporated in vacuo, and the
residue was chromatographed on Bio-beads S X3
with toluene-EtOH (9:1). The product was further
puriˆed by column chromatography on silica gel with
CHCl3–MeOH–H2O (80:20:1) to aŠord 16 (106 mg,
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Nakahara, Y., Ito, Y., and Nakahara, Y., Synthesis
85
CHCl3–MeOH–H2O). H-NMR
8.0 Hz, Ar), 7.82 (2H, d, 8.0 Hz, Ar), 7.27–7.20
(4H, m, Ar), 5.67 (1H, brt, c),
z
based on C90-dolichol). Rf 0.66 (70:30:3
1
＝
: 7.97 (2H, d, J
d
J
＝
＝
J
9.3 Hz, H-4
), 5.12 (ca. 34H, br),
3C6H4–), 2.38 (3H, s, C 3C6H4–),
5.45 (1H, brd,
2.40 (3H, s, C
J 6.6 Hz, H-4c
＝
H
H
2.04 (ca. 130H, br), 1.67 (ca. 78H, s), 0.85 [3H, m,
–CH(C : 99.4 (C-1 ), 99.2 (C-1 ),
3)–]. 13C-NMR
100.8 (C-1 ), 143.8 and 144.2 (MBz C-4), 162.5,
H
d
a
c
b
165.2, 166.0, 169.5, 169.6, 170.7, 170.9, 171.4,
171.5, and 171.7 (CO). MALDI TOF-MS Found:
m z (M－) 2393.93, 2461.98, 2530.04, 2598.10,
W
2666.01. Calcd.: 2393.38 (C135H202N2O30P2), 2461.44
(C140 H210N2O30P2),
2597.57
(C155 H234N2O135P2).
2529.51
(C145H218N2O30P2),
(C150 H226N2O135P2),
2665.63
P1-[
deoxy-
a
-
D
-Mannopyranosyl-(1
-glucopyranosyl-(1
ª
4)-2-acetamido-2-
of
N-linked pentasaccharides with isomeric glycosidic
b
-
D
ª
4)-2-acetamido-2-
linkage. Glycoconj. J., 17, 361–375 (2000).
d e o x y -
a
-
- g l u c o p y r a n o s y l ] - P 2 - d o l i c h y l
D
9) Matsumoto, T., Maeta, H., Suzuki, K., and
Tsuchihashi, G., New glycosidation reaction 1. Com-
binational use of Cp2ZrCl2–AgClO4 for activation of
pyrophosphate (
1
). To a stirred solution of 16
(28 mg) in anhydrous CH2Cl2 (1 ml) was added 0.2
M
glycosyl ‰uorides and application to highly
glycosidation of -mycinose. Tetrahedron Lett., 29,
3567–3570 (1988).
b-selective
NaOMe MeOH (1 ml). The mixture was stirred for
W
D
6 h at room temperature and neutralized with excess
Dowex 8A (pyridine form). The resin was ˆltered oŠ,
and the ˆltrate was concentrated in vacuo to give 1
10) Oltvoort, J. J., van Boeckel, C. A. A., de Koning, J.
H., and van Boom, J. H., Use of cationic iridium
(18 mg, 80
z based on the C90-dolichyl congener). Rf
complex
1,5-cyclooctadiene-bis[methyldiphenyl-
0.19 (60:35:6 CHCl3–MeOH–H2O). MALDI TOF-
phosphine]-iridium hexa‰uorophosphate in carbohy-
drate chemistry: Smooth isomerization of allyl ethers
to 1-propenyl ethers. Synthesis, 305–308 (1981).
MS Found: m z (M－) 1905.67, 1973.75, 2041.84,
W
2109.92, 2178.01. Calcd: 1905.23 (C107H178N2O22P2),
1973.30
(C117 H194N2O22P2),
2177.49 (C127H210N2O22 P2).
(C112H186N2O22P2),
2041.36
11) Exco‹er, G., Gagnaire, D., and Utille, J.-P.,
Coupure se
á
lective par l'hydrazine des groupements
res de residus glycosyles acetyles.
2109.42
(C122H202N2O22P2),
acetyles anome
á
à
á
á
á

1766
Y. NAKAHARA et al.
Carbohydr. Res., 39, 368–373 (1975).
Ninagawa, Y., Nishida, T., Mizuno, M., and
Tanaka, Y., Synthesis of mammalian dolichols from
plant polyprenols. Tetrahedron Lett., 24, 5103–5106
(1983).
12) Danilov, L. L., and Chojnacki, T., A simple proce-
dure for preparing dolichyl monophosphate by the
use of POCl3. FEBS Lett., 131, 310–312 (1981).
13) Suzuki, S., Mori, F., Takigawa, T., Ibata, K.,