Synthesis of core-class 2 O-linked glycopeptides: a benzyl-protected tetrasaccharyl serine and its derivative carrying a hydrophobic cholestanyl group.

Article abstract of DOI:10.1271/bbb.66.1904

A core-class 2 tetrasaccharide-linked serine was synthesized in a convergent manner. The coupling reaction of disaccharide glycosyl donor 3 and acceptor 4 stereoselectively afforded tetrasaccharide 15, which was converted to glycosyl fluoride 20. Glycosyl

Full text of DOI:10.1271/bbb.66.1904

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Synthesis of Core-class 2 O-Linked Glycopeptides:
a Benzyl-protected Tetrasaccharyl Serine and its
Derivative Carrying a Hydrophobic Cholestanyl Group
Jun WATABEa, Latika SINGHb, Yuko NAKAHARAb, Yukishige ITOb, Hironobu HOJOa &
Yoshiaki NAKAHARAab
a Institute of Glycotechnology, Department of Applied Biochemistry, Tokai University
Kitakaname 1117, Hiratsuka-shi, Kanagawa 259-1292, Japan
b The Institute of Physical and Chemical Research (RIKEN) Hirosawa 2-1, Wako-shi,
Saitama 351-0198, Japan CREST, Japan Science and Technology Corporation (JST)
Published online: 22 May 2014.
To cite this article: Jun WATABE, Latika SINGH, Yuko NAKAHARA, Yukishige ITO, Hironobu HOJO & Yoshiaki NAKAHARA
(2002) Synthesis of Core-class 2 O-Linked Glycopeptides: a Benzyl-protected Tetrasaccharyl Serine and its Derivative
Carrying a Hydrophobic Cholestanyl Group, Bioscience, Biotechnology, and Biochemistry, 66:9, 1904-1914, DOI: 10.1271/
bbb.66.1904
To link to this article: http://dx.doi.org/10.1271/bbb.66.1904
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Biosci. Biotechnol. Biochem., 66 (9), 1904–1914, 2002
Synthesis of Core-class 2
O-Linked Glycopeptides: a Benzyl-protected
Tetrasaccharyl Serine and its Derivative Carrying a Hydrophobic
Cholestanyl Group
2
Jun WATABE,1 Latika SINGH,2 Yuko NAKAHARA,2 Yukishige ITO
,
1,2,
†
Hironobu HOJO,1 and Yoshiaki NAKAHARA
1Institute of Glycotechnology, Department of Applied Biochemistry, Tokai University, Kitakaname 1117,
Hiratsuka-shi, Kanagawa 259–1292, Japan
2The Institute of Physical and Chemical Research (RIKEN), Hirosawa 2-1, Wako-shi, Saitama 351-0198, Japan
CREST, Japan Science and Technology Corporation (JST)
Received March 20, 2002; Accepted April 25, 2002
A core-class 2 tetrasaccharide-linked serine was syn-
thesized in a convergent manner. The coupling reaction
of disaccharide glycosyl donor 3 and acceptor 4
stereoselectively aŠorded tetrasaccharide 15, which was
converted to glycosyl ‰uoride 20. Glycosylation of
order to establish e‹cient synthetic routes to
homogeneous samples which can be used to obtain
more insight into carbohydrated-mediated biological
reactions. We have recently reported the syntheses of
several Ser Thr-linked
O-glycan derivatives of this
W
Fmoc serine allyl ester 5 with 20 produced
a
- and
b
-
class by using stereoselective glycosylation reac-
tions.4–6) Other workers have also disclosed a number
of synthetic works on the related oligosaccha-
rides.7–13) We describe in this report a novel access to
core 2 tetrasaccharide-linked serine 1 which is suita-
bly protected as a potent building block for the syn-
thesis of glycopeptides. Conversion of the tetrasac-
charide into compound 2 carrying a hydrophobic
cholestanyl group, which is used in the immunologi-
cal assay on the plastic micro plates, is also investi-
gated.
glycosides in 40 and 33 yields, respectively.
%
%
a-
Isomer 21 was converted into 1, a useful building block
for the solid-phase synthesis of glycopeptides. On the
other hand, 21 was
N-deprotected and condensed with
hydrophobic cholestanol through a succinyl spacer. The
same compound was alternatively synthesized by
coupling 20 and 28. Functional group manipulation
and hydrogenation aŠorded core 2 tetrasaccharide-
cholestanol conjugate 2.
Key words:
O
-glycopeptide; core-class 2; cholestanol;
glycosylation; serine
Results and Discussion
It has been shown that the
N
- and
O
-linked
On the basis of the benzyl-protection strategy for
glycopeptide synthesis conducted in this laborato-
ry,5,6,14,15) three building blocks, disaccharides 3 and
4, and amino acid derivative 5, were designed to
perform convergent synthesis of target molecule 1.
To synthesize 2, additional building block 6 was
designed. The synthetic plan involves (1) coupling 3
oligosaccharides of glycoprotein play important roles
in a variety of biological phenomena such as cell-cell
and receptor-ligand interaction, viral infection, and
cancer metastasis.1) The
O-linked core 2 structures
have been found not only in the common mucins of
normal human milk2) but also in the oligosaccharides
derived from T-lymphocytic leukaemia cells.3) In
such biological events as development, diŠerentia-
tion, and oncogenesis, the core 2 glycans often ap-
pear as the altered glycoform. We are interested in
the biological signiˆcance of such oligosaccharides
and 4 to give a
b
-glycoside of high stereoselectivity
-phthaloyl group in 3, (2)
with the help of the 2-
N
condensation of 5 with the resulting tetrasaccharide
by the glycosyl ‰uoride method, and (3) conjugation
with 6 by acylation of the amino group of the serine
residue. A conceptually similar approach to a
and have studied the syntheses of
O-glycopeptides in
†
To whom correspondence should be addressed. Fax: +81-463-50-2075; E-mail: yonak
＠
keyaki.cc.u-tokai.ac.jp
Abbreviations: AgOTf, silver tri‰uoromethanesulfonate; DTBMP, 2,6-di-tert-butyl-4-methylpyridine; MS 4A, molecular sieves 4A;
TMSOTf, trimethylsilyl tri‰uoromethanesulfonate; DBU, 1,8-diazabicyclo[5.4.0]undec-7-ene; TFA, tri‰uoroacetic acid; GalN3, 2-azido-2-
deoxy-
D
-galactopyranose; TBAF, tetra-
n
-butylammonium ‰uoride; DAST, (diethylamino)sulfur tri‰uoride; EDC
・
HCl, 1-(3-
-glucopyra-
D-galactopyranose; MALDI-TOF MS, matrix-assisted laser desorption ionization time of ‰ight mass
dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; HOBt, 1-hydroxybenzotriazole; GlcNAc, 2-acetamido-2-deoxy-
D
nose; GalNAc, 2-acetamido-2-deoxy-
spectrometry

Synthesis of Core 2
O
-Glycopeptides
1905
Fig. 1. Structures of the Target Core-class 2 Tetrasaccharides and the Building Blocks.
tetrasaccharide-linked serine, employing glycosyla-
tion of the -benzyloxycarbonylserine benzyl ester
cetal 19 was then converted to a mixture of
a
- and
b
-
‰uorides 20 (a b 3 4) by treating with DAST17,18) in
＝
N
W
W
with an acetyl-protected tetrasaccharide glycosyl
THF. The Cp2Zr(ClO4)2-promoted glycosylation19) of
Fmoc serine allyl ester 5 with tetrasaccharyl donor 20
in CH2Cl2 was poorly stereoselective to aŠord 21 and
donor, has already been reported.8)
The synthesis of lactosamine block 3 commenced
by coupling tetra-
O
-acetyl-
a
-
D
-galactopyranosyl
22 in 40 and 33
z
z
yields, respectively, although
bromide 7 and known
N
-phthaloylated glucosamine
highly a-selective glycosylation of the serine and
derivative 816) by using AgOTf, DTBMP and MS 4A
in CH2Cl2. The reaction gave disaccharide 9 and a
stereoisomeric mixture of orthoesters 10. The latter
was readily converted to 9 by treating with TMSOTf.
Compound 9 was deacetylated and then benzylated
threonine derivatives had been achieved with the
related disaccharyl glycosyl ‰uoride in the previous
studies.14,20,21) Other glycosylation methods or
promoters were not tested. Separated
was then heated with thiourea in DMF to remove the
chloroacetyl group in an 85 yield. Reduction of the
a-glycoside 21
to give 12 in an 82
z
yield. Desilylation of 12 was fol-
z
lowed by reacting resulting hemiacetal 13 with
azide group of 23 with zinc and AcOH was followed
by acetylation to furnish 24. Compound 24 was
deallylated with Pd(PPh3)4 and dimedone to produce
1, a useful building block for the synthesis of
glycopeptides carrying a core 2 tetrasaccharide.
Using intermediary tetrasaccharide 23, conversion
to 2 was next investigated. Removal of the Fmoc
CCl3CN in the presence of DBU to furnish
trichloroacetimidate 3 in a 94
z
yield. On the other
hand, known disaccharide 1414) was treated with 80
z
aq. TFA in CH2Cl2 to exclusively give 4. The
coupling reaction of 3 and 4 was promoted by cata-
lytic TMSOTf (0.2 equiv.) in CH2Cl2 at „40
9
C. The
reaction stereoselectively produced desired tetrasac-
group was carried out by treating with 20
z
piperi-
charide 15 in an 86
z
yield. The structure of 15 was
dine in CH2Cl2 to give 25 in a 91z yield. Resulting
1
fully assigned by H- and 13C-NMR spectroscopy. It
amine 25 was coupled with 6, which had been pre-
is noteworthy that the glycosylation reaction was less
pared from cholestanol and succinic anhydride, in
＝
・
stereoselective (b a 3 1) when the 2-azide sugar
the presense of EDC HCl and HOBt. However, the
W
W
had been employed as a precursor of the GlcNAc
residue in our previous study.5,6) The phthaloyl group
was then removed by reacting with ethylenediamine,
and liberated amine 16 was acetylated in MeOH to
reaction was very sluggish, and desired coupling
product 26 was produced in only a small quantity,
even at an elevated temperature. We then altered the
synthetic route to this compound. Condensation of
cholestanyl hemisuccinate 6 with the serine allyl ester
led to 28, whose glycosylation with ‰uoride 20 was
then attempted. The reaction was smoothly promot-
ed by Sn(OTf)2 but with no stereoselectivity to aŠord
aŠord 17 in a 91
z overall yield. The 4-hydroxyl
group in the GalN3 residue was protected as a chlo-
roacetate, before the anomeric silyl group was split in
the presence of TBAF and AcOH. Resulting hemia-

1906
J. WATABE et al.
Scheme 1. Synthetic Routes to Building Blocks 3 and 4.
Scheme 2. Synthesis of the Tetrasaccharide-serine Framework.
＝
a mixture of
a
- and
b
-glycosides (a b 1 1) in a 65
z
the oligosaccharide portion was deprotected by cata-
lytic hydrogenation to secure target compound 2.
The structure of the synthesized product was as-
signed by NMR and MALDI-TOF MS data.
In conclusion, the tetrasaccharide framework of
core 2-type glycans was synthesized in a benzyl-
protected form. The GlcNAc-b-(1ª6)-GalNAc link-
W
W
yield.
a
-Isomer 29 was readily coverted into com-
yield by selective cleavage of the
pound 26 in an 80
z
chloroacetyl group with thiourea. Compound 26 was
then treated with zinc and AcOH to reduce the azide
group, and subsequent acetylation with Ac2O in
MeOH CH2Cl2 aŠorded 31 in a 55
z
yield. Finally,
W

Synthesis of Core 2
O
-Glycopeptides
1907
age was stereoselectively achieved by 2-
N
-phthaloyl-
assisted glycosidation of donor disaccharide 3 and
acceptor disaccharide 4. The resulting tetrasaccha-
ride was condensed with 5 by the glycosyl ‰uoride
method. The glycosylation reaction unexpectedly dis-
played poor stereoselectivity. The
a-glycoside was
transformed into 1, a building block suitably protect-
ed for glycopeptide synthesis. The tetrasaccharide
carrying a hydrophobic cholestanyl attachment was
also synthesized by coupling 20 and 28, a lack of
stereoselectivity again being observed. These results
are of crucial importance in designing a new synthetic
strategy to the core-class 2 oligosaccharides required
for use in the solid-phase assembly of glycopeptides.
An immunological study with deprotected glycosyl
cholestanol 2 will be reported elsewhere.
Experimental
Optical rotation values were determined with a
Jasco DIP-370 polarimeter for solutions in CHCl3 at
Scheme 3. Synthesis of 1 and
Tetrasaccharide-cholestanol Conjugate.
N
-Acylation Approach to the
20+2
9C, unless noted otherwise. Column chro-
matography was performed on Silica Gel-60 (E.
Scheme 4. Glycosylation of the Serine-cholestanol Conjugate and Synthesis of 2.

1908
J. WATABE et al.
Merck 70–230 mesh or 230–400 mesh), and TLC was
ched by stirring with excess solid NaHCO3. The mix-
ture was ˆltered through Celite and the ˆltrate was
concentrated in vacuo to give a crude product, which
was puriˆed by column chromatography as just de-
1
performed on Silica Gel 60 F254 (E. Merck). H- and
13C-NMR spectra were recorded 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 a solution in CDCl3.
TOF mass spectra were recorded with a PerSeptive
scribed to aŠord 9 (423 mg, 83
z
).
tert-Butyldiphenylsilyl 2,3,4,6-tetra-O-benzyl-
-galactopyranosyl-(1 4)-3,6-di-O-benzyl-2-deoxy-
2-phthalimido- -glucopyranoside (12). To a stirred
b-
Voyager-DE PRO spectrometer (
a
-cyano-4-hydroxy-
D
ª
cinnamic acid or 2,5-dihydroxybenzoic acid).
b
-
D
solution of 9 (1.02 g, 0.96 mmol) in anhydrous
tert-Butyldiphenylsilyl 2,3,4,6-tetra-O-acetyl-
b
-
D
-
MeOH (10 ml) was added 0.2 N NaOMe MeOH
W
galactopyranosyl-(1 4)-3,6-di-O-benzyl-2-deoxy-2-
ª
(0.96 ml, 0.19 mmol). The mixture was stirred at
room temperature for 2 h and then neutralized with
Amberlist 15. After the resin had been ˆltered oŠ, the
methanolic solution was concentrated in vacuo to
give 11 (0.86 g, quant.) which was used for the next
reaction without puriˆcation. To an ice-cooled
mixture of 11 (1.95 g, 2.2 mmol), Bu4NI (0.65 g,
1.8 mmol), and benzyl bromide (2.08 ml, 17.5 mmol)
phthalimido-b- -glucopyranoside (9). A mixture of 7
D
(0.85 g, 2.0 mmol), 8 (1.0 g, 1.4 mmol), DTBMP
(0.83 g, 4.0 mmol), and dried MS 4A powder (2 g) in
anhydrous CH2Cl2 (20 ml) was stirred under Ar at
room temperature for 30 min, and then at „20
1 h. AgOTf (0.69 g, 2.7 mmol) was added to the mix-
ture, which was stirred for 1 h at „20 C before being
9C for
9
quenched with NaHCO3 and ice. The mixture was
stirred for 1 h at room temperature and then ˆltered
through Celite. The combined ˆltrate and washings
were successively washed with aq. NaHCO3, water
and brine, dried over Na2SO4, and concentrated in
vacuo. The crude product was chromatographed on
silica gel with hexane-EtOAc (2.5:1) to give more mo-
in anhydrous DMF (40 ml) was added 60
z
NaH
C-
(1.05 g, 26.3 mmol). The mixture was stirred at 0
9
room temperature for 4 h. The mixture was then
cooled on an ice bath, and the reaction was quenched
by carefully adding AcOH. The mixture was concen-
trated under vacuum, the resulting residue being ex-
tracted with EtOAc. The extract was successively
washed with water and brine, dried over Na2SO4, and
concentrated in vacuo. The crude product was chro-
matographed on silica gel with hexane-EtOAc
bile orthoesters 10 (0.29 g, 20
(1.02 g, 70 ).
z) and disaccharide 9
z
Compound
9
: Rf 0.29 (3:2 hexane-EtOAc). [
(c 1.0). 1H-NMR
: 7.78–6.85 (24H, m, Ar),
2.4 Hz, Gal-H4), 5.19 (1H, d,
a
]
D
+14.1
9
d
(95:5–80:20) to give 12 (2.30 g, 84
hexane-EtOAc). [ +13.7
(c 1.0). 1H-NMR
7.74–6.70 (44H, m, Ar), 5.10 (1H, d,
GlcN-H1), 4.38 (1H, d, 7.6 Hz, Gal-H1), 4.26
z
). Rf 0.30 (4:1
＝
5.28 (1H, d,
J
a
]
D
9
d:
＝
＝
＝
J 8.1 Hz,
J
7.8 Hz, GlcN–H1), 4.90 (1H, dd,
J
3.4,
8.3 Hz, Gal-H1),
7.8, 10.7 Hz, GlcN-H2), 4.20 (1H,
10.3 Hz, Gal-H3), 4.68 (1H, d,
J
＝
J
＝
＝
＝
＝
J
4.28 (1H, dd,
dd,
J
(1H, brd,
J
8.1 Hz, GlcN-H2), 4.12 (1H, dd,
9.6 Hz,
2.6 Hz, Gal-H4), 3.07
(1H, m, GlcN-H5), 0.81 (9H, s, t-Bu). 13C-NMR
102.7 (Gal-C1), 98.1 (GlcN-C1). Anal. Calcd. for
C78H81O12NSi: C, 74.79; H, 6.52; N, 1.12 . Found:
C, 74.52; H, 6.35; N, 1.04
J
＝
8.3, 10.7 Hz, GlcN-H3), 4.10 (1H, m, GlcN-
8.6, 10.7 Hz, GlcN-H3), 3.98 (1H, brt, J
＝
＝
GlcN-H4), 3.81 (1H, brd, J
H4), 3.97 (2H, m, 2 Gal-H6), 3.71 (1H, m, Gal-H5),
＝
3.65 (1H, brd,
J
10.0 Hz, GlcN–H6), 3.17 (1H, m,
d:
GlcN-H5), 2.05, 2.02, 1.97, 1.96 (12H, 4s, 4 Ac),
0.89 (9H, s, t-Bu). 13C-NMR
d
: 100.2 (Gal-C1), 93.4
z
(GlcN-C1). Anal. Calcd. for C58H67O17NSi
・
H2O: C,
z
.
64.61; H, 6.26; N, 1.30
N, 1.19
z
. Found: C, 64.70; H, 5.93;
z.
2,3,4,6-Tetra-O-benzyl-
b
- -galactopyranosyl-
D
1
Compound 10: Rf 0.36 (3:2 hexane-EtOAc). H-
(1
ª
4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido- -
D
＝
NMR
d
: 7.65–6.77 (24H, m, Ar), 5.83 (1H, d,
J
4.4
glucopyranose (13). To a mixture of 12 (4.68 g,
Hz, Gal-H1), 5.45 (1H, t,
J
＝
3.2 Hz, Gal-H4), 5.18
3.7 mmol) and AcOH (3.2 ml, 56 mmol) in freshly
＝
＝
(1H, d,
6.1 Hz, Gal-H3), 4.47 (1H, dd,
H2), 4.31 (1H, dt, 3.4, 6.9 Hz, Gal-H5), 3.85
8.3, 9.7 Hz, GlcN–H4), 3.67 (1H, dd,
J
7.8 Hz, GlcN-H1), 4.96 (1H, dd,
J
2.9,
distilled THF (100 ml) was added 1M TBAF THF
W
＝
J
4.4, 6.1 Hz, Gal-
(18.7 ml, 18.7 mmol). The mixture was stirred at
room temperature for 24 h before being concentrated
in vacuo. The residue was dissolved in EtOAc, suc-
cessively washed with water, aq. NaHCO3, and brine,
dried over Na2SO4, and concentrated in vacuo. The
crude product was chromatographed on silica gel
J
＝
＝
J
(1H, dd,
＝
＝
2.2,
J
4.4, 11.0 Hz, GlcN-H6), 3.57 (1H, dd,
J
11.0 Hz, GlcN-H6), 3.21 (1H, m, GlcN-H5), 2.09,
2.05, and 1.93 (9H, 3s, 3 Ac), 1.70 (3H, s, CH3), 0.89
(9H, s, t-Bu).
with hexane-EtOAc (1:1) to aŠord 13 (4.20 g, 91
z
).
Synthesis of
(511 mg, 0.48 mmol) and MS 4A (1.2 g) in anhydrous
CH2Cl2 (12 ml) was added TMSOTf (14 l, 78 mol)
at „10 C. Completion of the reaction after 20 min
was ascertained by TLC, and the reaction was quen-
9 from 10. To a stirred mixture of 10
Rf 0.33 (3:2 hexane-EtOAc). Anal. Calcd. for
・
C62H61O12N 0.5H2O: C, 72.92; H, 6.12; N, 1.37
z.
m
m
Found: C, 72.76; H, 6.11; N, 1.33
z
.
9
2,3,4,6-Tetra-O-benzyl-
b
-D-galactopyranosyl-

Synthesis of Core 2
O
-Glycopeptides
1909
＝
(1
ª
4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-
b
-
D
-
NMR
(1H, d,
GalN-H1), 4.05 (1H, d,
d
: 5.00 (1H, d,
J
7.9 Hz, GlcN-H1), 4.31
＝
＝
7.9 Hz,
glucopyranosyl trichloroacetimidate (
3
). To a mix-
J
7.6 Hz, Gal-H1), 4.12 (1H, d,
J
＝
7.6 Hz, Gal-H1), 3.81
ture of 13 (366 mg, 0.36 mmol) and CCl3CN
J
＝
＝
2.6 Hz,
(0.36 ml, 3.6 mmol) in anhydrous CH2Cl2 (5 ml) was
(1H, d,
J
3.0 Hz, Gal-H4), 3.65 (1H, d,
J
added DBU (11
m
l, 72
m
mol) at „10
9
C. The mixture
Gal-H4), 3.44 (1H, m, GlcN-H4), 0.89 (9H, s, t-Bu).
＝
was stirred for 2 h and then concentrated in vacuo at
20 C. The residue was chromatographed on silica gel
with hexane-EtOAc (2:1) to give 3 (382 mg, 92 ). Rf
0.58 (3:2 hexane-EtOAc). [ +39.9 (c 1.0). H-
NMR : 8.45 (1H, s, N ), 7.62–6.72 (34H, m, Ar),
6.33 (1H, d, 8.8 Hz, GlcN–H1), 4.40 (1H, br,
13C-NMR
d
: 103.7 (1JC-H 161.3 Hz, Gal-C1), 103.0
1
1
＝
＝
9
(
JC-H 160.4 Hz, Gal-C1), 98.3 ( JC-H 167.1 Hz,
GlcN-C1), 96.8 (1JC-H 164.6 Hz, GalN-C1). Anal
.
＝
z
1
a
]
D
9
Calcd. for C118H122O21N4Si 0.5H2O: C, 71.97; H,
6.30; N, 2.84 . Found: C, 71.88; H, 6.22; N,
2.87
・
d
H
z
＝
J
z.
＝
8.3 Hz, Gal-H1), 4.32 (1H,
Glc-H2), 4.39 (1H, d,
J
＝
m, GlcN-H3), 4.11 (1H, brt,
J
9.0 Hz, GlcN-H4),
: 102.7
tert-Butyldiphenylsilyl 2,3,4,6-tetra-O-benzyl-
galactopyranosyl-(1 4)-2-amino-3,6-di-O-benzyl-2-
deoxy- -glucopyranosyl-(1 6)-[2,3,4,6-tetra-O-
benzyl- -galactopyranosyl-(1 3)]-2-azido-2-
deoxy- -galactopyranoside (16). A mixture of 15
(206 mg, 0.11 mmol) and ethylenediamine (1 ml) in
n-BuOH (8 ml) was stirred overnight at 90 C. After
b-D-
3.81 (1H, d,
J
2.7 Hz, Gal-H4). 13C-NMR
d
ª
＝
(Gal-C1), 94.1 (GlcN-C1), 90.4 (CCl3). Anal. Calcd.
b
-
D
ª
・
for C64H61O12N2Cl3 H2O: C, 65.43; H, 5.41; N,
b
-
D
ª
2.38 . Found: C, 65.23; H, 5.23; N, 2.32
z
z.
b
-
D
tert-Butyldiphenylsilyl O-(2,3,4,6-tetra-O-benzyl-
-galactopyranosyl)-(1 4)-2-azido-2-deoxy-
galactopyranoside ( ). A solution of 14 (5.13 g,
4.9 mmol) in CH2Cl2 (20 ml) was stirred with 50
aq. TFA (30 ml) at 0 C-room temperature for 3 h.
9
b
-
D
ª
b
-
D
-
cooling, the mixture was concentrated in vacuo, and
remaining n-BuOH was removed by evaporating with
toluene. The residue was chromatographed on silica
gel with CHCl3-MeOH (98:2) to give 16 (184 mg,
4
z
9
The mixture was neutralized by carefully adding of
solid NaHCO3 and extracted with EtOAc. The ex-
tract was successively washed with sat. NaHCO3 and
96
z
). Rf 0.27 (7:3 toluene-EtOAc). [
a
]
D
+6.7
8.0 Hz, Gal-H1),
7.7 Hz, Gal-H1), 4.32 (1H,
9(c
1
1.0). H-NMR
4.35 (1H, d,
d: 4.45 (1H, d, J
＝
＝
J
＝
7.8 Hz, GlcN–H1), 2.71
brine, dried over Na2SO4, and concentrated in vacuo
The residue was chromatographed on silica gel with
hexane-EtOAc (9:1–3:2) to give 4 (4.23 g, 90 ). Rf
.
GalN–H1), 4.05 (1H, d, J
(1H, m, GlcN–H2), 1.11 (9H, s, t-Bu). 13C-NMR d:
z
104.0 (Gal-C1), 103.6 (GlcN-C1), 102.7 (Gal-C1),
1
0.28 (3:2 hexane-EtOAc). [
a
]
D
+25.0
9
(c 1.0). H-
96.9 (GalN-C1). Anal. Calcd. for C110H120O19N4Si: C,
NMR
d
: 7.70–7.13 (30H, m, Ar), 4.42 (1H, d,
72.19; H, 6.61; N, 3.06
N, 3.08
z
. Found: C, 72.19; H, 6.65;
＝
＝
7.8 Hz, Gal-
J
7.8 Hz, GalN-H1), 4.37 (1H, d,
J
z.
H1), 3.80 (1H, m, Gal-H2), 3.74–3.66 (3H, m, Gal-
H4, GalN-H4, GalN-H2), 3.58 (1H, m, GalN-H6),
3.47–3.42 (3H, m, Gal-H3, Gal-H5, Gal-H6),
3.30–3.24 (3H, m, GalN-H3, Gal-H6, GalN-H6),
2.98 (1H, m, GalN-H5), 1.04 (9H, s, t-Bu). 13C-NMR
tert-Butyldiphenylsilyl 2,3,4,6-tetra-O-benzyl-
galactopyranosyl-(1 4)-2-acetamido-3,6-di-O-ben-
zyl-2-deoxy- -glucopyranosyl-(1 6)-[2,3,4,6-
tetra-O-benzyl- -galactopyranosyl-(1 3)]-2-
azido-2-deoxy- -galactopyranoside (17). To a solu-
tion of 16 (150 mg, 0.08 mmol) in MeOH (5 ml) was
added Ac2O (1 ml) at 0 C. The mixture was stirred
b
-
D
-
ª
b
-
D
ª
b
-
D
ª
d
: 104.0 (Gal-C1), 97.2 (GalN-C1). Anal. Calcd. for
C56H63O10N3Si: C, 69.61; H, 6.58; N, 4.35 . Found:
C, 69.34; H, 6.56; N, 4.45
b
-
D
z
z
.
9
for 2 h before its concentration in vacuo. The
remaining Ac2O was coevaporated with toluene. The
crude product was chromatographed on silica gel
with hexane-EtOAc (3:1–1:1) to give 17 (145 mg,
tert-Butyldiphenylsilyl 2,3,4,6-tetra-O-benzyl-
galactopyranosyl-(1 4)-3,6-di-O-benzyl-2-deoxy-2-
phthalimido- -glucopyranosyl-(1 6)-[2,3,4,6-
tetra-O-benzyl- -galactopyranosyl-(1 3)]-2-
azido-2-deoxy- -galactopyranoside 15). To
b
-
D
-
ª
b
-
D
ª
b
-
D
ª
95
z
). Rf 0.41 (7:3 toluene-EtOAc). [
9
a]D+5.8 (c
＝
b
-
D
(
a
1.0). 1H-NMR
4.45 (1H, d,
d
: 4.47 (1H, d,
J
7.3 Hz, GlcN–H1),
＝
stirred mixture of 3 (350 mg, 0.30 mmol), 4 (244 mg,
J
7.6 Hz, Gal-H1), 4.34 (1H, d,
7.6 Hz,
GalN–H1), 1.58 3H, s, Ac), 1.08 (9H, s, t-Bu). 13C-
NMR : 103.9 (Gal-C1), 102.9 (Gal-C1), 100.0
(GlcN-C1), 96.7 (GalN-C1). Anal
0.25 mmol) and dried MS 4A (300 mg) in anhydrous
J 7.6 Hz, Gal-H1), 4.32 (1H, d, J
＝ ＝
CH2Cl2 (7 ml) was added TMSOTf (11
m
l,
0.06 mmol) at „40
9
C. After stirring for 30 min, the
d
reaction was quenched by adding solid NaHCO3 and
a few pieces of ice. The mixture was then diluted with
EtOAc and ˆltered through Celite. The ˆltrate was
successively washed with water and brine, dried over
Na2SO4, and concentrated in vacuo. The crude
product was chromatographed on silica gel with tol-
.
Calcd. for
・
C
112H122O20N4Si H2O: C, 71.16; H, 6.61; N, 2.96
Found: C, 71.28; H, 6.62; N, 2.91
z.
z
.
tert-Butyldiphenylsilyl 2,3,4,6-tetra-O-benzyl-
galactopyranosyl-(1
zyl-2-deoxy- -glucopyranosyl-(1
tetra-O-benzyl- -galactopyranosyl-(1
b
-
D
-
ª
4)-2-acetamido-3,6-di-O-ben-
uene-EtOAc (98:2–92:8) to aŠord 15 (426 mg, 86
z).
b
-
D
ª
6)-[2,3,4,6-
3)]-2-
1
Rf 0.29 (9:1 toluene-EtOAc), [
a
]D
+6.9
9
(c 1.0). H-
b
-
D
ª

1910
J. WATABE et al.
＝
azido-4-O-chloroacetyl-2-deoxy-
b
-
D
-galactopyrano-
52.4 Hz, GalN-H1), 5.70 (1H, d,
J
7.3 Hz, N
3.2 Hz, GalN-H4), 3.89 (2H, brs,
H2CO-), 1.83 (3H, s, Ac). Anal. Calcd. for
H ),
＝
J
side (18). To an ice-cooled mixture of 17 (51 mg,
5.50 (1H, brd,
ClC
C98H104O20N4ClF H2O: C, 68.02; H, 6.17; N, 3.24;
Cl, 2.05; F, 1.10 . Found: C, 67.88; H, 6.19; N,
3.31; Cl, 1.74; F, 1.12
Compound 20 : Rf 0.16 (7:3 toluene-EtOAc). [
+12.0 (c 1.0). H-NMR
NH), 5.30 (1H, brs, GalN-H4), 5.01 (1H, dd,
51.8 Hz, GalN-H1), 4.06 and 4.03 (2H, 2d,
28
(2 ml) was added chloroacetic anhydride (7 mg,
41 mol). The mixture was stirred at 0 C for 3 h and
m
mol) and pyridine (250
m
l) in anhydrous CH2Cl2
・
z
m
9
concentrated with toluene in vacuo. The product was
puriˆed by gel-permeation chromatography on Bio-
beads S X3 with toluene-EtOAc (1:1) to aŠord 18
z.
b
a
]
D
1
＝
7.5 Hz,
9
d
: 5.63(1H, d,
J
＝
7.1,
(52 mg, quantitative). Rf 0.78 (7:3 toluene-EtOAc).
J
1
[
a
]
D
+2.5
Hz, N
(1H, d,
Hz, Gal-H1), 4.35 (1H, d,
9
(c 1.0). H-NMR
d
: 5.26 (1H, d,
J 7.3
＝
＝
＝
H
), 5.16 (1H, d,
J
3.2 Hz, GalN-H4), 4.63
J
15.5 Hz, ClC
Calcd. for C98H104O20N4ClF: C, 68.74; H, 6.12; N,
3.27; Cl, 2.07; F, 1.11 . Found: C, 68.52; H, 6.12;
N, 3.23; Cl, 1.70; F, 0.97
H2CO-), 1.82 (3H, s, Ac). Anal.
＝
＝
J
7.3 Hz, GlcN-H1), 4.44 (1H, d,
J
7.3
7.8 Hz, GalN-H1),
7.8 Hz, Gal-H1), 3.99 and 3.94 (2H,
15.4 Hz, ClC 2CO-), 2.99 (1H, m, GlcN-
H2), 1.63(3H, s, Ac), 1.09 (9H, s, t-Bu). 13C-NMR
103.0 (Gal-C1), 102.8 (Gal-C1), 98.7 (GlcN-C1),
96.9 (GalN-C1), 57.0 (- H2Cl). Anal. Calcd. for
114H123O21N4SiCl 2H2O: C, 68.99; H, 6.45; N, 2.82;
Cl, 1.79 . Found: C, 68.80; H, 6.56; N, 2.77; Cl,
1.82
＝
J
z
＝
4.31 (1H, d,
2d,
J
z.
J
＝
H
d
:
N-(9-Fluorenylmethoxycarbonyl)-O-
tetra-O-benzyl- -galactopyranosyl-(1
acetamido-3,6-di-O-benzyl-2-deoxy- -glucopyra-
nosyl-(1 6)-[2,3,4,6-tetra-O-benzyl-
topyranosyl-(1 3)]-2-azido-4-O-chloroacetyl-2-
deoxy- - and -galactopyranosyl -serine allyl es-
ter (21) and (22). A mixture of Cp2ZrCl2 (51 mg,
175 mol), AgClO4 (73 mg, 350 mol), and dried MS
s
2,3,4,6-
b
-
D
ª
4)-2-
C
b
-
D
・
D
C
ª
b- -galac-
z
ª
z
.
a
b
-
D
t-L
2,3,4,6-tetra-O-benzyl-
4)-2-acetamido-3,6-di-O-benzyl-2-deoxy-
glucopyranosyl-(1 6)-[2,3,4,6-tetra-O-benzyl-
galactopyranosyl-(1
2-deoxy- -galactopyranose
mixture of 18 (52 mg, 27 mol) and AcOH (24
410 mol) in distilled THF (2 ml) was added 1M
TBAF THF (137 l, 137 mol). The mixture was
b
-
D
-galactopyranosyl-
m
m
(1
ª
b-
b-
D
-
-
4A (840 mg) in anhydrous CH2Cl2 (2 ml) was stirred
under Ar for 1 h at room temperature and then
ª
D
ª
3)]-2-azido-4-O-chloroacetyl-
19). To stirred
l,
cooled at „15
stirred mixture was added a mixture of 20
88 mol) and 5 (64 mg, 175 mmol) in anhydrous
CH2Cl2 (5.3 ml). The mixture was stirred overnight
at „15 C-room temperature before the reaction was
quenched by adding pyridine (210 l). The mixture
9
C on an ice-MeOH bath. To the
b
-
D
(
a
b
(150 mg,
m
m
m
m
m
m
9
W
stirred at room temperature overnight and concen-
trated in vacuo. The residue was extracted with
EtOAc, successively washed with water and brine,
dried over Na2SO4, and concentrated in vacuo. The
crude product was chromatographed on Bio-beads S
X3 with toluene-EtOAc (1:1) to give 19 (41 mg,
m
was diluted with ether and ˆltered through Celite.
The combined ˆltrate and ethereal washings were
successively washed with water and brine, dried over
Na2SO4, and concentrated in vacuo. The crude
product was chromatographed on Bio-beads S X3
with toluene-EtOAc (1:1) and then on silica gel with
90
Calcd. for C98H105O21N4Cl: C, 68.82; H, 6.19; N,
3.28; Cl, 2.07 . Found: C, 68.71; H, 6.18; N, 3.29;
Cl, 2.10
z). Rf 0.32 and 0.21 (7:3 toluene-EtOAc). Anal.
hexane-EtOAc (1:1) to give 21 (72 mg, 39.9
z
) and 22
z
(60 mg, 33.3z).
z
.
Compound 21: Rf 0.38 (1:1 hexane-EtOAc). [
a
]
D
1
＝
+34.9
9
(c 1.0). H-NMR
d
: 6.28 (1H, d,
J
7.0 Hz,
D
-galactopyranosyl-
N
H
), 6.10 (1H, d,
-CH
5.30 (1H, brd,
J
＝
7.3 Hz, N
H
), 5.88 (1H, m,
2,3,4,6-tetra-O-benzyl-
4)-2-acetamido-3,6-di-O-benzyl-2-deoxy-
glucopyranosyl-(1 6)-[2,3,4,6-tetra-O-benzyl-
galactopyranosyl-(1
2-deoxy- - and
To a stirred solution of 19 (500 mg, 0.29 mmol) in
anhydrous CH2Cl2 (21 ml) was added DAST (108 l,
0.41 mmol) at 0 C. The mixture was stirred for
b
-
(1
ª
b-
b-
D
D
-
-
CH2), 5.40 (1H, brd, J 2.7 Hz, GalN-H4),
＝ ＝
＝
＝
CH
ª
J
17.1 Hz, -CH
2), 5.21 (1H,
ª
3)]-2-azido-4-O-chloroacetyl-
-galactopyranosyl ‰uoride (20).
brd,
ClC
J 10.3 Hz, -CH CH2), 4.03 (2H, brs,
＝ ＝
a
b
-
D
H
＝
2CO-), 1.84 (3H, s, Ac). 13C-NMR
d
: 103.9
1
1
＝
(
JCH 160.8 Hz, Gal-C1), 102.9 ( JCH 162.5 Hz,
m
Gal-C1), 99.6
(
1JCH
＝
169.2 Hz, GlcN-C1), 98.2
＝
JCH 170.0 Hz, GalN-C1). Anal. Calcd. for
1
9
(
・
30 min before the reaction was quenched with MeOH
(0.3 ml). The mixture was diluted with CHCl3, suc-
cessively washed with water and brine, dried over
Na2SO4, and concentrated in vacuo. The crude
product was chromatographed on silica gel with
C
119H124O25N5Cl H2O: C, 68.79; H, 6.11; N, 3.37
z.
Found: C, 68.88; H, 6.15; N, 3.01
z
. MALDI-TOF
MS: m z 2080.82 (M+Na, calcd. for C119H124O25-
W
N5Cl
Compound 22: Rf 0.13 (1:1 hexane-EtOAc). [
+12.1 5.99 (1H, brd,
(c 1.0). 1H-NMR
7.6 Hz, N ), 5.99 (1H, brd,
5.89 (1H, m, -C
-CH 2), 5.29 (1H, brd,
・
Na: 2080.82).
a
]
D
toluene-EtOAc (7:3) to give
29 ) and ‰uoride 20 (208 mg, 42
Compound 20 : Rf 0.39 (7:3 toluene-EtOAc). [
+29.9 (c 1.0). H-NMR
a
‰uoride 20
a
(145 mg,
9
d:
＝
＝
8.0 Hz, N
z
b
b
z
).
J
H
J
H
),
17.1 Hz,
＝
J 2.9 Hz, GalN-H4),
a
a
]
D
H
＝ ＝
CH2), 5.33 (1h, brd, J
1
d
: 5.72 (1H, dd,
J
＝
2.2,
＝
C
H
9

Synthesis of Core 2
O
-Glycopeptides
1911
＝
＝
CH
5.22 (1H, brd,
J
10.2 Hz, -CH
2), 4.03 (2H,
acetamido-3,6-di-O-benzyl-2-deoxy-
b
-
D
-glu-
brs, ClC
H
2CO-), 1.82 (3H, s, Ac). 13C-NMR
d
: 103.5
copyranosyl-(1
ª
6)-[2,3,4,6-tetra-O-benzyl-
b
-
D
-
-
1
1
＝
＝
(
JCH 160.9 Hz, Gal-C1), 103.0 ( JCH 161.7 Hz,
galactopyranosyl-(1
ª
3)]-2-acetamido-2-deoxy-
).
a-
D
Gal-C1), 102.3 (1JCH 160.9 Hz, GalN-C1), 99.5
galactopyranosyl
t
- -serine (
L
1
＝
(
1JCH
＝
167.5 Hz, GlcN-C1). Anal
.
Calcd. for
119H124O25N5Cl 2H2O: C, 68.20; H, 6.16; N, 3.34
Found: C, 68.16; H, 6.14; N, 3.03 . MALDI-TOF
A mixture of 24 (21 mg, 11
m
mol), Pd(PPh3)4
l,
・
C
z.
(3 mg, 2.6 mol) and N-methylaniline (26 m
m
z
0.24 mmol) in DMSO-CH2Cl2 (1:1, 2 ml) was stirred
for 19 h. The mixture was diluted with CHCl3, suc-
cessively washed with 0.1 N HCl, water, and brine,
dried over Na2SO4, and concentrated in vacuo. The
residue was chromatographed on Bio-beads S X3
with EtOAc and then on silica gel with CHCl3-MeOH
MS: m z 2081.20 (M+Na, calcd. for C119H124O25-
N5Cl Na: 2080.82).
W
・
N-(9-Fluorenylmethoxycarbonyl)-O-s2,3,4,6-
tetra-O-benzyl-
b
-
D
-galactopyranosyl-(1
ª
4)-2-
-glu-
acetamido-3,6-di-O-benzyl-2-deoxy-
copyranosyl-(1
galactopyranosyl-(1
topyranosyl -serine allyl ester (23). A stirred
mixture of 21 (50 mg, 24 mol) and thiourea (11 mg,
145 mol) in anhydrous DMF (1.5 ml) was heated at
70 C under Ar for 1 day. After cooling, the mixture
b
-
D
(9:1, containing 0.1
74
0.1
(2H, brd,
z
AcOH) to aŠord 1 (15 mg,
ª
6)-[2,3,4,6-tetra-O-benzyl-
b
-
D
-
z). Rf 0.38 (9:1 CHCl3-MeOH, containing
1
ª
3)]-2-azido-2-deoxy- -galac-
a
-
D
z
AcOH). [
a
]
D
9
+35.6 (c 0.3). H-NMR d: 7.72
＝
＝
t
-
L
J
7.3 Hz, Fmoc-Ar), 7.58 (2H, brd,
J
m
6.9 Hz, Fmoc-Ar), 1.89 (3H, s, Ac), 1.53 (3H, s, Ac).
13C-NMR
: 106.7, 103.1, 102.8, 100.7. Anal. Calcd.
for C117H123O25N3 H2O: C, 70.46; H, 6.37; N, 2.13
. Found: C, 70.31; H, 6.35; N, 2.06 . MALDI-
m
9
d
・
was diluted with EtOAc-ether (1:1), successively
washed with water and brine, dried over Na2SO4, and
concentrated in vacuo. The crude product was chro-
matographed on Bio-beads S X3 with toluene-EtOAc
(1:1) and then on silica gel with hexane-EtOAc (1:1)
z
z
TOF MS: m z 1980.54 (M+Na, calcd. for
W
・
117H123O25N3 Na: 1980.83).
C
O-
4)-2-acetamido-3,6-di-O-benzyl-2-deoxy-
6)-[2,3,4,6-tetra-O-benzyl-b
s
2,3,4,6-Tetra-O-benzyl-
b
-
D
-galactopyranosyl-
to give 23 (41 mg, 85
EtOAc). [ +25.9
7.6 Hz, N
5.89 (1H, m, -C
z
). Rf 0.33 (1:1 hexane-
(1
ª
b
-
-
D
-
-
1
a
]
D
9
(c 0.5). H-NMR
d
: 6.21 (1H,
7.1 Hz, N ),
CH2), 5.31 (1H, brd,
glucopyranosyl-(1
ª
D
＝
＝
D
d,
J
H
), 6.09 (1H, d,
J
H
galactopyranosyl-(1
topyranosyl -serine allyl ester (25). Compound 23
(69 mg, 35 mol) was stirred in 20 piperidine-
ª3)]-2-azido-2-deoxy-a- -galac-
H
＝
J
＝
t-L
＝
＝
10.8 Hz,
17.3 Hz, -CH
-CH 2), 1.84 (3H, s, Ac). Anal. Calcd. for
117H123O24N5 H2O: C, 70.22; H, 6.30; N, 3.50
Found: C, 70.17; H, 6.27; N, 3.40
C
H
2), 5.22 (1H, brd,
J
m
z
＝
C
H
CH2Cl2 (1.5 ml) at room temperature for 1 h. The
mixture was concentrated twice with toluene (10 ml)
in vacuo. The residue was chromatographed on Bio-
・
C
z
.
z.
beads S X1 in toluene to give 25 (55 mg, 91
z
). Rf
N-(9-Fluorenylmethoxycarbonyl)-O-
tetra-O-benzyl- -galactopyranosyl-(1
acetamido-3,6-di-O-benzyl-2-deoxy-
nosyl-(1 6)-[2,3,4,6-tetra-O-benzyl-
topyranosyl-(1 3)]-2-acetamido-2-deoxy-
topyranosyl
solution of 23 (45 mg, 23
were added powdered Zn (181 mg) and AcOH
(52 l). The mixture was stirred for 1 h. before being
s
2,3,4,6-
0.63 (9:1 CHCl3-EtOH). [
a
]
D
+27.8
9
H
(c 0.5). 1H-
), 5.88 (1H,
b
-
D
ª
4)-2-
NMR
m, -C
d
: 6.10 (1H, brd,
J
＝
8.1 Hz, N
＝
＝
＝
b
-
D
-glucopyra-
H
CH2), 5.30 (1H, brd,
J
17.3 Hz, -CH
D
-galac-
-galac-
C
H
2), 5.21 (1H, brd,
J
＝
10.5 Hz, -CH
＝
CH
2), 1.84
ª
b
-
ª
a
-
D
(3H, s, Ac). 13C-NMR
d: 104.1(Gal-C1), 103.0 (Gal-
t
-
L
-serine allyl ester (24). To a stirred
C1), 100.4 (GalN-C1), 99.0 (GlcN-C1). Anal. Calcd.
for C102H113O22N5 H2O: C, 68.86; H, 6.52; N,
3.94 . Found: C, 68.96; H, 6.67; N, 3.79
m
mol) in CH2Cl2 (1 ml)
・
z
z
.
m
ˆltered through Celite. The ˆltrate was concentrated
with toluene, and the residue was stirred with Ac2O
Succinic acid mono-3
stirred mixture of
b
-cholestanyl ester (
6). To
a
3b
-cholestanol (200 mg,
(32
m
l, 340
m
mol) in MeOH-CH2Cl2 (3:1, 2 ml) for
0.52 mmol) and succinic anhydride (103 mg,
1.03 mmol) in pyridine-CH2Cl2 (2:1, 1.5 ml) was
added Et3N (290 ml, 2.6 mmol). After stirring for 6
days, the mixture was concentrated with toluene in
vacuo. The residue was chromatographed on silica
gel with toluene-EtOAc (1:1) to give 6 (251 mg,
1 h. The mixture was concentrated with toluene, and
the residue was extracted with CHCl3. The extract
was successively washed with sat. NaHCO3 and
brine, dried over Na2SO4, and concentrated in vacuo
.
The crude product was puriˆed by preparative thin-
layer chromatography on silica gel with toluene-
quantitative). Rf 0.53 (1:1 toluene-EtOAc). [
a
]
D
1
EtOAc (1:2) to give 24 (21 mg, 46
z), which was only
+12.6
9
(c 1.0, 1:1 CHCl3-MeOH). H-NMR
d
: 4.71
characterized by mass spectrometry and used for the
next reaction. Rf 0.28 (1:2 toluene-EtOAc).
(1H, m, chol. H-4), 2.68–2.58 (4H, m, -COCH2
-
＝
C
H
2CO-), 0.99 (3H, d,
J
6.6 Hz, C
H
3), 0.863 (3H,
＝
＝
6.6 Hz, C
MALDI-TOF MS: m z 2020.79 (M+Na, calcd. for
d,
J
6.6 Hz, C
H
H
3), 0.858 (3H, d,
3), 0.64 (3H, s, C
J
H
H3),
W
・
C119H127O25N3 Na: 2020.86).
0.81 (3H, s, C
3). Anal. Calcd.
. Found:
・
N-(9-Fluorenylmethoxycarbonyl)-O-
s
2,3,4,6-
for C31H52O4 0.2H2O: C, 75.63; H, 10.73
z
tetra-O-benzyl-
b
-
D
-galactopyranosyl-(1
ª
4)-2-
z
C, 75.74; H, 10.85 .

1912
J. WATABE et al.
N-[3-(3
b
-Cholestanyloxycarbonyl)propionyl]-O-
allyl ester (29 and 30). A mixture of Sn(OTf)2 (24 mg,
58 mol) and dried MS 4A (300 mg) in anhydrous
CH2Cl2 (1 ml) was stirred under Ar at room tempera-
ture for 1 h and then cooled at „15 C. To the stirred
mixture was added a mixture of 20 (50 mg, 29 mmol)
and 28 (27 mg, 44 mol) in anhydrous CH2Cl2 (3 ml).
The mixture was stirred for 2.5 h at „15 C-room
temperature, before the reaction was quenched with
pyridine (100 l). The mixture was diluted with ether,
successively washed with water and brine, dried over
Na2SO4, and concentrated in vacuo. The crude
product was chromatographed on Bio-beads S X1
with toluene-EtOAc (1:1) to aŠord the coupling
s
(1
2,3,4,6-tetra-O-benzyl-
b
-
D
-galactopyranosyl-
m
ª
4)-2-acetamido-3,6-di-O-benzyl-2-deoxy-
6)-[2,3,4,6-tetra-O-benzyl-
3)]-2-azido-2-deoxy-
b
-
-
D
D
-
-
glucopyranosyl-(1
ª
ª
b
9
galactopyranosyl-(1
topyranosyl
6 (31 mg, 63
(13 mg, 94 mol), EDC HCl (18 mg, 94
Et3N (9 ml, 62 mol) in anhydrous DMF (1.5 ml) was
stirred at 45 C for 24 h. After DMF had been evapo-
a
-
D
-galac-
t
-
L
-serine allyl ester (26). A mixture of
mol), 25 (53 mg, 30 mol), HOBt
mol), and
m
m
m
9
・
m
m
m
m
9
rated under reduced pressure, the resulting mixture
was extracted with ether-EtOAc (1:1). The extract
was successively washed with water and brine, dried
over Na2SO4, and concentrated in vacuo. The crude
product was chromatographed on Bio-beads S X1 in
EtOAc-toluene (1:1) to aŠord 26 (19 mg, 29
0.77 (9:1 CHCl3-EtOH). [
product as a mixture of
a- and b-glycosides (124 mg,
65 a b 1 1). The isomers were separated by
z
,
＝
W
W
z
). Rf
chromatography on silica gel with toluene-EtOAc
(1:1).
a
]
D
+39.6
9
H
(c 0.5). 1H-
), 6.51 (1H,
＝
＝
NMR
d
: 6.95 (1H, brd,
J
7.3 Hz, N
), 5.82 (1H, m, -C
17.1 Hz, -CH
Compound 29: Rf 0.49 (1:1 toluene-EtOAc). [
+36.6 6.58 (1H, brd,
(c 1.0). 1H-NMR
7.1 Hz, N ), 5.88 (1H, m, -C CH2), 5.47
3.4 Hz, GalN3 H-4), 5.30 (1H, brd,
a
]
D
＝
brd,
J
7.6 Hz, N
H
＝
H
CH2),
9
d:
5.22 (1H, brd,
J
＝
C
H
2), 5.15 (1H,
2), 2.60–2.43 (4H, m,
2CO-), 1.80 (3H, s, Ac), 0.82–0.78 (9H,
J
＝
H
H
＝
＝
＝
＝
＝
J
＝
10.5 Hz,
brd,
-COC
m, C
Calcd. for C133H163O25N5 2H2O: C, 70.44; H, 7.42
Found: C, 70.54; H, 7.38
J
10.5 Hz, -CH
2C
3), 0.70 (3H, s, C
C
H
(1H, brd,
J
＝
H
H
17.3 Hz, -CH
C
H
2), 5.22 (1H, brd,
2), 4.03 (2H, s, ClC 2CO-), 2.60–2.45
(4H, m, -COC 2C 2CO-), 1.87 (3H, s, Ac), 0.89–
0.86 (9H, m, C 3), 0.78 (3H, s, C 3), 0.63 (3H, s,
3). 13C-NMR
: 103.9 (Gal-C1), 102.8 (Gal-C1),
99.6 (GalN-C1), 98.1 (GlcN-C1). Anal. Calcd. for
C135H164O26N5Cl H2O: C, 69.70; H, 7.19; N, 3.01
J
＝
H
H
・
3), 0.55 (3H, s, C
H
3). Anal
.
.
-CH
C
H
H
z
H
H
H
z
.
H
C
H
d
N-[3-(3
b
-Cholestanyloxycarbonyl)propionyl]-
L
-
serine allyl ester (28). A mixture of 6 (100 mg,
・
z
.
・
205
(118 mg, 614
stirred for 30 min and then added to a stirred solution
of 27 (30 mg, 205 mol) in anhydrous DMF. After
stirring for 1 h more, the mixture was stirred over-
night with Et3N (86 l, 614 mol). DMF was evapo-
m
mol), HOBt (83 mg, 614
m
mol), and EDC HCl
Found: C, 69.54; H, 7.04; N, 2.77z.
m
mol) in anhydrous DMF (1 ml) was
Compound 30: Rf 0.20 (1:1 toluene-EtOAc). [
a
]
D
1
＝
+13.9
9
(c 1.0). H-NMR
d
: 6.86 (1H, d,
J
7.1 Hz,
), 5.85 (1H, m,
17.3 Hz, -CH
＝
m
N
H
), 6.22 (1H, brd,
J
7.3 Hz, N
H
-C
H
＝
CH2), 5.29 (1H, brd,
J
＝
＝
＝
m
m
C
H
2), 5.25 (1H, brd,
(1H, brd, 10.5 Hz, -CH
m, -COC 2CO-), 1.87 (3H, s, Ac), 0.88 (3H, d,
J
3.1 Hz, GalN3 H-4), 5.19
rated under reduced pressure, and the residue was
extracted with ether-EtOAc (1:1). The extract was
successively washed with water and brine, dried over
Na2SO4, and concentrated in vacuo. The crude
product was chromatographed on silica gel with
J
＝
＝
CH
2), 2.65–2.46 (4H,
H
2C
6.6 Hz, C
(3H, s, C 3), 0.63 (3H, s, C
(Gal-C1), 102.9 (Gal-C1), 102.1 (GalN-C1), 99.5
(GlcN-C1). Anal. Calcd. for C135H164O26N5Cl H2O:
C, 69.70; H, 7.19; N, 3.01 . Found: C, 69.48; H,
7.14; N, 2.78
H
＝
＝
J
H
3), 0.86 (6H, d,
J
H
6.6 Hz, C 3), 0.79
H
H
3). 13C-NMR
d: 103.6
toluene-EtOAc (1:1) to give 28 (61 mg, 48
(1:1 toluene-EtOAc). [ +22.8
7.3 Hz, N
CH2), 5.35 (1H, brd, 17.1 Hz, -CH
z
). Rf 0.32
・
1
a
]
D
9
(c 0.5 ). H-NMR
z
＝
＝
d
: 6.57 (1H, d,
J
H
), 5.90 (1H, m, -C
2), 5.28
2), 4.72–4.65 (4H,
CH2), 4.00 (2H, m,
H), 2.77–2.50 (4H, m, -COC 2C 2CO-), 0.89
3), 0.862 (3H, d,
6.6 Hz, C 3), 0.81 (3H, s,
3). Anal Calcd. for
H
z.
J
＝
＝
C
H
＝
＝
CH
(1H, brd,
m, chol. H-4, Ser-
Ser-
(3H, d,
H
H
J
10.4 Hz, -CH
Conversion of 29 to 26
mol) and thiourea (11 mg, 145
drous DMF (1 ml) was heated overnight at 70
9
.
A mixture of 29 (54 mg,
mol) in anhy-
C.
a
H, -C 2CH
H
＝
23
m
m
b
H
H
＝
＝
6.6 Hz,
J
6.6 Hz, C
3), 0.858 (3H, d,
3), 0.64 (3H, s,
H
J
DMF was evaporated under reduced pressure, and
the residue was extracted with ether-EtOAc (1:1).
The extract was successively washed with water and
brine, dried over Na2SO4, and concentrated in vacuo.
The product was puriˆed by gel permeation column
chromatography on Bio-beads S X1 in EtOAc-
toluene (1:1) to give 26 (42 mg, 80 ), which was
z
identical with the sample prepared by coupling 6 and
25.
C
C
J
＝
H
C
H
.
・
C37H61O6N 0.2H2O: C, 71.74; H, 9.99; N, 2.26
Found: C, 71.67; H, 10.09; N, 2.28
z.
z
.
N-[3-(3
2,3,4,6-tetra-O-benzyl-
(1 4)-2-acetamido-3,6-di-O-benzyl-2-deoxy-
glucopyranosyl-(1 6)-[2,3,4,6-tetra-O-benzyl-
galactopyranosyl-(1
chloroacetyl- - and
b
-Cholestanyloxycarbonyl)propionyl]-O-
s
b
-
D
-galactopyranosyl-
ª
b
-
-
D
-
-
ª
b
D
ª
3)]-2-azido-2-deoxy-4-O-
-galactopyranosyl -serine
N-[3-(3
b
-Cholestanyloxycarbonyl)propionyl]-O-
-galactopyranosyl-
a
b
-
D
t
-
L
s
2,3,4,6-tetra-O-benzyl-
b
-
D

Synthesis of Core 2
O
-Glycopeptides
1913
(1
glucopyranosyl-(1
galactopyranosyl-(1
galactopyranosyl
of 26 (52 mg, 24
AcOH (54 l) in CH2Cl2 (1.5 ml) was stirred for 2 h.
The solid material was ˆltered oŠ through Celite, and
the ˆltrate was concentrated with toluene. The
residue was stirred with Ac2O (33 ml, 353 mmol) in
MeOH-CH2Cl2 (3:1, 2 ml) for 0.5 h. The mixture was
concentrated with toluene, and the residue was
extracted with CHCl3. The extract was successively
washed with sat. NaHCO3 and brine, dried over
Na2SO4, and concentrated in vacuo. The crude
product was puriˆed by gel permeation chro-
matography on Sephadex LH-20 with CHCl3-MeOH
ª
4)-2-acetamido-3,6-di-O-benzyl-2-deoxy-
6)-[2,3,4,6-tetra-O-benzyl-
3)]-2-acetamido-2-deoxy-
b
-
-
-
D
D
D
-
-
-
References
ª
b
1) Reviews. In ``Essentials of Glycobiology,'' eds.
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ª
a
t
-
L
-serine allyl ester (31). A mixture
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mol) powdered Zn (400 mg) and
m
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(1:1) to give 31 (29 mg, 55
z). Rf 0.49 (1:3 toluene-
1
EtOAc). [
a
]
D
+29.3
9
(c 0.3). H-NMR
d
: 6.13 (1H,
＝
CH2), 5.60
＝
d,
J
8.1 Hz, N
H
), 5.85 (1H, m, -CH
(1H, brd,
J
＝
7.3 Hz,
NH ), 5.28 (1H, brd,
＝
＝
＝
J
17.3 Hz, -CH
C
H
2), 5.20 (1H, brd,
2), 2.65–2.45 (4H, m, -COC
1.83 (3H, s, Ac), 1.56 (3H, s, Ac), 0.89–0.85 (9H, m,
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Calcd. for C135H167O26N3Cl: C, 72.14; H, 7.49; N,
1.87 . Found: C, 71.77; H, 7.47; N, 1.85
J
9.8 Hz,
＝
-CH
C
H
H2CH2CO-),
C
H
H
H
.
z
z
.
N-[3-(3
b
-Cholestanyloxycarbonyl)propionyl]-O-
-galactopyranosyl-(1 4)-2-acetamido-2-deoxy-
-glucopyranosyl-(1
s
b
-
D
ª
7) Kinzy, W., and Schmidt, R. R., Application of the
trichloroacetimidate method to the synthesis of
a
-
D
ª
6)-[
b
a
-
D
-galactopyranosyl-
(1
ª
3)]-2-acetamido-2-deoxy-
-
D
-galactopyranosylt-
glycopeptides of the mucin type containing a
Gal -(1 3)- -Gal NAc unit. Carbohydr. Res., 166,
265–276 (1987).
b- -
D
L
-serine propyl ester (
22 mol) was hydrogenated with a catalytic amount
of 10 Pd-C in MeOH-AcOH (1:1, 3 ml) for 10
2). Compound 31 (49 mg,
p
ª
D
p
m
8) Paulsen, H., Rauwald, W., and Weichert, U.,
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catalyst was ˆltered oŠ through Celite. The catalyst
was then washed with CHCl3-MeOH (1:1), and the
combined ˆltrate and washings were concentrated in
vacuo. The crude product was puriˆed by gel perme-
ation on Sephadex LH-20 with CHCl3-MeOH (1:1) to
O-
N
give 2 (12 mg, 42
(4H, m, -COC
1.98 (3H, s, Ac), 0.97 (3H, t,
z
H
). 1H-NMR
2C 2CO-), 2.02 (3H, s, Ac),
7.5 Hz, -OCH2CH2-
9
d (CD3OD, 50 C) 2.61
H
J
＝
a
＝
C
H
3), 0.92 (3H, d,
J
6.1 Hz, C
H
3), 0.87 (6H, d,
3), 0.69 (3H, s,
blocks and some unexpected reactions. Tetrahedron
Lett., 38, 45–48 (1997).
J
＝
6.6 Hz, C 3), 0.85 (3H, s, C
H
H
11) Mathieux, N., Paulsen, H., Meldal, M., and Bock,
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CH3). MALDI-TOF MS: m z 1370.28 (M+Na,
W
・
calcd. for C65H109O26N3 Na: 1370.72).
Acknowledgments
This work was supported by the CREST program
of Japan Science and Technology Corporation, in
part by grant-aid for scientiˆc research from the
Ministry of Education, Science, Sports and Culture
of Japan, and by grant-aid for high-technology
research from Tokai University. We thank Dr. T.
Chihara and his staŠ for elemental analyses, and Ms.
A. Takahashi for technical assistance.
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derivatives of
a
-
D
-Neup5Ac-(2
ª
3)-
b
-
D
-Galp-(1
ª3)-
[
a
-
D
-Neup5Ac-(2
ª
6)]- -GalNAc-(1ª
a
-
D
3)-L-Ser
W
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