A stereocontrolled construction of 2-azido-2-deoxy-1,2-cis-α- galactosidic linkages utilizing 2-azido-4,6-O-benzylidene-2- deoxygalactopyranosyl diphenyl phosphates: Stereoselective synthesis of mucin core 5 and core 7 structures

Article abstract of DOI:10.1016/j.tet.2012.04.059

TMSOTf-promoted glycosidation of 2-azido-4,6-O-benzylidene-2- deoxygalactosyl diphenyl phosphates with fluorenylmethoxycarbonyl (Fmoc)-protected serine and threonine derivatives in THF/Et₂O (1:1) gave glycosyl amino acids in high yields and with excellent levels of α-selectivity (α/β=94:6-95:5). The synthetic utility of the present glycosidation method was demonstrated by a stereoselective synthesis of mucin-type glycopeptide core 5 and core 7 building blocks, which are suitable for Fmoc-based solid-phase synthesis of O-glycopeptides.

Full text of DOI:10.1016/j.tet.2012.04.059

Tetrahedron 68 (2012) 5005e5017
Contents lists available at SciVerse ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
A stereocontrolled construction of 2-azido-2-deoxy-1,2-cis-a-galactosidic linkages
utilizing 2-azido-4,6-O-benzylidene-2-deoxygalactopyranosyl diphenyl
phosphates: stereoselective synthesis of mucin core 5 and core 7 structures
Kosuke Kakita, Toshifumi Tsuda, Noritoshi Suzuki, Seiichi Nakamura, Hisanori Nambu,
*
Shunichi Hashimoto
Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
TMSOTf-promoted glycosidation of 2-azido-4,6-O-benzylidene-2-deoxygalactosyl diphenyl phosphates
with fluorenylmethoxycarbonyl (Fmoc)-protected serine and threonine derivatives in THF/Et2O (1:1)
Received 24 March 2012
Received in revised form 12 April 2012
Accepted 14 April 2012
gave glycosyl amino acids in high yields and with excellent levels of
a
-selectivity (
a
/
b
¼94:6e95:5). The
synthetic utility of the present glycosidation method was demonstrated by a stereoselective synthesis of
mucin-type glycopeptide core 5 and core 7 building blocks, which are suitable for Fmoc-based solid-
phase synthesis of O-glycopeptides.
Available online 21 April 2012
Keywords:
Ó 2012 Elsevier Ltd. All rights reserved.
Carbohydrates
Glycosylation
TN-antigen
Mucin
Phosphates
1. Introduction
reported approaches toward the construction of this type of
linkage rely on the methodology introduced by Paulsen in 1978,7 in
2-Acetamido-2-deoxy-
D
-glycopyranosides are the structural
which the nonparticipating azido group is selected as a latent
amino functionality at C-2. 2-Azido-2-deoxygalactosyl halides,8
trichloroacetimidates,8h,9e11 and thioethers12 are the most com-
monly used glycosyl donors to prepare this type of linkage.13e15
Although the stereochemical outcome of the glycosidation with
units in glycoproteins, one of the most important classes of natu-
rally occurring oligosaccharides and glycoconjugates.1 Mucin-type
glycoproteins, the oligosaccharide moiety of which is O-glycosidi-
cally linked to
L-serine and
L-threonine through 2-acetamido-2-
deoxy-
a
-
D
-galactopyranose (GalNAc), have attracted much atten-
these donors often resulted in moderate to good
a
-selectivity,5f
-glyco-
tion due to their major roles in numerous biological aspects.2 The
glycoproteins can serve as a source of antigens to the immune
system. Since a variety of tumor-associated antigens are O-linked
glycans,3 synthetic mucin-type O-linked glycopeptides have been
pursued as anti-tumor vaccines.4 Eight core structures of mucin-
several examples of highly stereocontrolled 1,2-cis-
a
sidation reactions were reported by the Mukaiyama,14 Dani-
shefsky,8h Polt,8i Field,8j and Boons12h groups. In contrast, there are
only a few reports on glycosidations using C-2 functionalities ca-
pable of neighboring group participation, such as NHAc16 and
NHTroc.17 Kiso and co-workers reported that reactions of 4,6-O-di-
tert-butylsilylene-protected GalNTroc donors with serine and
type glycopeptides have been identified to date. The GalNAca-1-
O-Ser/Thr structure, commonly referred to as the TN-antigen, forms
the biosynthetic foundation for a diverse array of core structures
generated by glycosylation at the C-3 and/or C-6 hydroxy groups of
GalNAc (Fig. 1).
threonine derivatives produced exclusively
a-glycosides in
high yields despite the presence of a participating NHTroc group at
C-2.5g,17b,c Conceptionally different approaches were developed by
the Schmidt18 and Gin19 groups. Schmidt and co-workers reported
a Michael-type addition of serine and threonine derivatives to 2-
The formation of the 1,2-cis-a-glycosidic linkage between Gal-
NAc and serine or threonine has garnered much attention and
has been extensively reviewed in the literature.5,6 The majority of
nitrogalactal to give the corresponding 2-deoxy-2-nitro-
tosides.18 Despite the variety of methods available, stereoselective
synthesis of GalNAc -1-O-Ser/Thr structures continues to be
a-galac-
a
a challenge because reports on high-yielding and highly stereo-
* Corresponding author. Tel.: þ81 11 706 3236; fax: þ81 11 706 4981; e-mail
address: hsmt@pharm.hokudai.ac.jp (S. Hashimoto).
selective construction of this type of linkage are limited.8h,12h,14,17
0040-4020/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2012.04.059

5006
K. Kakita et al. / Tetrahedron 68 (2012) 5005e5017
OH
O
Ph
HO
HO
Ph
HO
HO
HO
O
NHFmoc
CO2Bn
OH
O
OH
O
HO
O
O
O
O
AcHN
OH
AcHN
O
O
O
glycosidation
OP(OPh)2
O
O
O
Ser/Thr
Ser/Thr
Ser/Thr
RO
RO
TN-antigen
NHFmoc
CO2Bn
Core 1
N3
N3
R = Ac, chloroacetyl
O
OH
O
cassette
HO
HO
O
HO
R = chloroacetyl
R = Ac
OH
O
OH
O
AcHN
HO
HO
1. removal of
4,6-O-benzylidene
group
1. removal of
HO
HO
HO
O
OH
O
3-O-chloroacetyl
group
AcHN
AcHN
O
O
2. glycosylation
2. glycosylation
OH
AcHN
O
Ph
Core 3
Ser/Thr
OAc
O
Core 2
AcO
AcO
O
Ph
O
OH
O
O
OH
HO
HO
O
O
HO
HO
N3
AcO
O
O
OH
OH
O
O
HO
AcO
O
N3
AcHN
OH
O
O
O
AcHN
HO
NHFmoc
CO2Bn
O
N3
NHFmoc
CO2Bn
AcHN
O
O
HO
HO
N3
O
O
Ser/Thr
O
Core 5
AcHN
Core 4
AcHN
O
Ser/Thr
mucin core 5 building block
mucin core 7 building block
OH
O
OH
O
HO
HO
Scheme 1. Synthesis of mucin core 5 and core 7 building blocks by the cassette
method.
HO
HO
OH
O
HO
HO
O
AcHN
HO
AcHN
HO
OH
OH
O
O
Table 1
HO
O
O
AcHN
Screening of promoters in the glycosidation of 3-O-acetyl-2-azido-4,6-O-benzyli-
dene-2-deoxy-a-D-galactosyl diphenyl phosphate (1aa) with serine derivative 2
O
HO
HO
Core 6
AcHN
O
AcHN
O
Ser/Thr
O
Ser/Thr
Ser/Thr
Core 7
Core 8
Fig. 1. Structures of mucin-type glycopeptide cores.
For the past two decades, we have been engaged in the de-
velopment of novel stereocontrolled glycosidation reactions capi-
talizing on phosphorus-containing leaving groups.20,21 For
Entry
Promoter
TMSOTf
TMSClO4
TMSNTf2
BF3$OEt2
HClO4
t (h)
Yield (%)
a/
ba
stereoselective construction of 1,2-cis-a
-glycosidic linkages,22 we
1
2
0.1
0.1
0.1
10
83
83
59
46
63
71:29
73:27
55:45
69:31
67:33
b
recently reported that catalytic stereoselective glycosidations of
glycosyl diphenyl phosphates using a commercially available HClO4
solution (0.1 M solution in dioxane) in dioxane/Et2O (1:1) gave
3
4c
5
0.5
glycosides in good yields and with good to high a-selectivities (up
a
The ratio was determined by HPLC (column, ZorbaxÒ Sil, 4.6ꢁ250 mm; eluent,
to
a
/
b
¼92:8).23 Herein, we report a high-yielding and highly ster-
hexane/AcOEt 3:2; flow rate, 1.0 mL/min).
eoselective construction of 2-azido-2-deoxy-1,2-cis-a-galactosidic
b
Prepared from TMSCl and AgClO4.
The reaction was performed at 23 ꢀC.
c
linkages by TMSOTf-promoted glycosidation of 2-azido-4,6-O-
benzylidene-2-deoxygalactosyl diphenyl phosphates with fluo-
renylmethoxycarbonyl (Fmoc)-protected serine and threonine de-
rivatives in THF/Et2O (1:1) and its application to the synthesis of
mucin core 5 and core 7 building blocks employing the cassette
approach developed by the groups of Meldal and Paulsen24 and
Danishefsky’s group25 as outlined in Scheme 1.26
TMSNTf2-promoted glycosidation provided modest yield and poor
a
-selectivity (59%,
required a significantly longer time (10 h) to reach completion
even at room temperature and provided 3a in 46% yield with an
a
/b¼55:45, entry 3). The reaction with BF3$OEt2
a
/
b
ratio of 69:31 (entry 4). Although HClO4 was the optimal pro-
moter in our previous work,23 the use of a commercially available
0.1 M solution of anhydrous HClO4 in dioxane gave moderate yield
2. Results and discussion
and
a-selectivity due to partial hydrolysis of the benzylidene
acetal functionality (63%,
AgClO4 is necessary to prepare TMSClO4, our efforts focused on the
use of conveniently handled TMSOTf.
a
/
b
¼67:33, entry 5). Since explosive
2.1. Glycosidation of 3-O-acetyl-2-azido-4,6-O-benzylidene-2-
deoxy-D-galactosyl diphenyl phosphates 1a with serine
derivative 2
Using TMSOTf as a promoter, we next studied the effects of
solvents on stereoselectivity (Table 2). Switching the solvent from
At the outset of this work, the glycosidation of 3-O-acetyl-2-
azido-4,6-O-benzylidene-2-deoxy- -galactopyranosyl diphenyl
was explored using Fmoc-protected serine de-
Et2O to CH2Cl2 or toluene slightly increased
a
-selectivities at the
-se-
a-D
27
expense of product yields (entries 2 and 3). Good yield and
a
phosphate (1aa)
lectivity were obtained in dioxane, although the high melting point
of dioxane precluded a direct comparison with those obtained with
the foregoing solvents at 0 ꢀC (entry 4). Since the use of THF im-
rivative 2 (1.1 equiv) as an acceptor alcohol. The reaction using
ꢀ
TMSOTf (1.1 equiv) as a promoter and 5-A molecular sieves (MS) in
Et2O proceeded at 0 ꢀC to completion within 0.1 h, giving glycoside
proved
a
-selectivity (
a
/
b
¼86:14, entry 5), we then explored
3a in 83% yield (Table 1, entry 1). The a/b ratio of 3a was determined
to be 71:29 by HPLC (ZorbaxÒ Sil column). The use of a 0.5 M so-
lution of TMSClO4 in toluene, prepared from TMSCl and AgClO4,28
a mixed solvent system of THF and Et2O. Gratifyingly, the reaction
in THF/Et2O (1:1) gave 3a in high yield and a-selectivity (85%, a/
b
¼89:11, entry 6).29 An examination of the temperature profile
resulted in a similar level of
a
-selectivity (
a
/b
¼73:27, entry 2).

K. Kakita et al. / Tetrahedron 68 (2012) 5005e5017
5007
Table 2
(
a
/
b
¼68:32) in 92% yield. Although phosphorylation of 6 with
diphenyl chlorophosphate using DMAP35 provided diphenyl
phosphate 1b in only 15% yield due to the formation of several by-
Effects of solvents, temperature and anomeric composition of the donor on
selectivity
a
products, the Tanabe protocol using N-methylimidazole and trie-
thylamine36 afforded phosphates 1b
yield.
a
and 1b
b
(
a/
b¼70:30) in 86%
Entry
Donor
Solvent
T (ꢀC)
t (h)
Yield (%)
a/
ba
1
2
3
4
5
6
7
8
1a
1a
1a
1a
1a
1a
1a
1a
1a
1a
1a
1a
1a
a
a
a
a
a
a
a
a
a
a
a
a
b
Et2O
CH2Cl2
Toluene
Dioxane
0
0
0
23
0
0.1
0.1
0.1
0.1
0.1
0.1
0.5
1.5
10
83
73
64
80
76
85
91
93
93
63
91
95
94
71:29
75:25
77:23
80:20
86:14
89:11
91:9
95:5
95:5
94:6
94:6
THF
THF/Et2O (1:1)
THF/Et2O (1:1)
THF/Et2O (1:1)
THF/Et2O (1:1)
THF/Et2O (2:1)
THF/Et2O (1:2)
THF/Et2O (1:5)
THF/Et2O (1:1)
0
ꢂ20
ꢂ40
ꢂ60
ꢂ40
ꢂ40
ꢂ40
ꢂ40
9
10
11
12
13
1.5
2
2
Scheme 2. Preparation of 3-O-chloroacetyl-protected galactosyl phosphate 1b.
93:7
94:6
1.5
Under optimized reaction conditions, the TMSOTf-promoted
a
The ratio was determined by HPLC (column, ZorbaxÒ Sil, 4.6ꢁ250 mm; eluent,
glycosidation of diphenyl phosphates 1aa and 1ba with serine
hexane/AcOEt 3:2; flow rate, 1.0 mL/min).
and threonine derivatives 2 and 7 was examined (Table 3, Fig. 2).
The reaction of 3-O-CA-protected galactosyl phosphate 1ba with
alcohol 2 (1.1 equiv) at ꢂ40 ꢀC gave glycoside 3b in a similar high
demonstrated that lowering the reaction temperature to ꢂ20 or
yield and
a
-selectivity as those obtained with 1a
a
(92%,
a/b
¼95:5,
ꢂ40 ꢀC increased
a-selectivity (entries 7 and 8). Although the a-
entry 2). When threonine derivative 7 was used as an acceptor,
1.5 equiv of 7 was required to provide good yield because of the
selectivity and yield obtained at ꢂ60 ꢀC were the same as those at
ꢂ40 ꢀC (entries 8 vs 9), a much longer time (10 h) was necessary to
complete the reaction. We next investigated the effect of the ratio
of a mixed solvent system at ꢂ40 ꢀC. Increasing the ratio of THF to
Et2O (2:1) resulted in lower yield due to the formation of un-
identified glycosyl polytetrahydrofurans (entry 10),30 while in-
creasing the ratio of Et2O to THF (THF/Et2O¼1:2e1:5) had little
attenuated reactivity of the acceptor. Coupling of 1a
a with 7
afforded glycoside 10a in 84% yield with an
3). The use of less reactive glycosyl donor 1b
same stereoselectivity as that found with 1a
decrease in product yield (69%,
a
a
a
/
b
ratio of 94:6 (entry
exhibited nearly the
but led to a marked
a
/b
¼93:7, entry 4) due to the
competitive formation of glycosyl polytetrahydrofurans. When the
impact on product yield and
we selected THF/Et2O (1:1) as a solvent system for further experi-
ments.31 The use of
-phosphate 1a gave virtually the same
product yield and -selectivity as those obtained with -phosphate
1a (entries 8 vs 13), because thermodynamically less-stable
phosphates anomerize to the corresponding
presence of acids.27,32 Therefore, stereoselective preparation of
or
parison of the
2-deoxy-
a-selectivity (entries 11 and 12). Thus,
reaction was conducted at ꢂ60 ꢀC for 48 h, higher yield and
a-se-
lectivity were obtained (80%,
a
/
b
¼95:5, entry 5). Since the
a
- and b-
b
b
anomers in all cases (3a, 3b, 10a and 10b) are readily separated by
column chromatography on silica gel, the present protocol provides
easy access to appropriately protected TN-antigen building blocks.
a
a
a
b
-
-
a
-phosphates in the
The glycoside 10ba is a key intermediate for the synthesis of anti-
a
freeze glycoproteins reported by Chen.13b,37
b-phosphates is not a requirement for this method. From com-
a/b
ratios obtained with 2-azido-3,4,6-tri-O-benzyl-
Table 3
a
-
D
-galactosyl diphenyl phosphate and 3,4,6-tri-O-acetyl-
protected diphenyl phosphate 19 (vide infra),33 the presence of
TMSOTf-promoted glycosidation of 2-azido-4,6-O-benzylidene-2-deoxy-
D-galacto-
syl diphenyl phosphates 1 with acceptor alcohols 2, 7e9
a 4,6-O-benzylidene acetal group proved to be crucial for high
levels of a-selectivity.
2.2. TMSOTf-promoted glycosidation of diphenyl phosphates
1 with acceptor alcohols 2, 7e9
As mentioned above, the core structures of mucin-type glyco-
peptides contain additional glycosyl residues at position 3 and/or
position 6 to form complex O-glycans (Fig. 1). Therefore, regiose-
Entry
Donor
R0OH
T (ꢀC)
t (h)
Glycoside
Yield (%)
lective deprotection of 3a
the 4,6-O-benzylidene acetal group in 3a
a
was explored. Although hydrolysis of
could be achieved (vide
Equiv
a/
ba
a
1
2
3
4
5
6
7
1a
1b
1a
a
a
a
2
2
7
7
7
8
9
1.1
1.1
1.5
1.5
1.5
1.1
1.1
ꢂ40
ꢂ40
ꢂ40
ꢂ40
ꢂ60
ꢂ60
ꢂ60
1.5
6
4
10
48
1.5
5
3a
93
92
84
69
80
92
76
95:5
95:5
94:6
93:7
95:5
82:18
94:6
infra), attempts at deprotection of the 3-O-acetyl group with vari-
ous bases were unsuccessful because of the instability of the Fmoc
group under these conditions. Since removal of the chloroacetyl
(CA) group in glycosides under mild conditions could be easily
achieved,13b,34 we prepared 3-O-CA-protected galactosyl diphenyl
phosphates 1b (Scheme 2). Treatment of the known alcohol 49a
with chloroacetyl chloride and pyridine followed by removal of
the TBS-protecting group with TBAF gave 3-O-CA-protected lactol 6
3b
10a
10b
10b
11
1b
1b
a
a
1a
1a
a
a
12
a
The ratio was determined by HPLC (column, ZorbaxÒ Sil, 4.6ꢁ250 mm; eluent,
hexane/AcOEt 2:1e3:2; flow rate, 1.0 mL/min).

5008
K. Kakita et al. / Tetrahedron 68 (2012) 5005e5017
OBn
O
OH
O
NHFmoc
CO2Bn
HO
BnO
BnO
HO
BnO
BnO
OMe
9
BnO
OMe
Ph
7
8
Ph
O
O
O
O
O
O
AcO
NHFmoc
CO2Bn
R1O
N3
O
O
N3
O
BnO
BnO
R2
BnO
11
OMe
Ph
O
3b: R1 = CA, R2 = H
10a: R1 = Ac, R2 = Me
10b: R1 = CA, R2 = Me
O
O
OBn
O
AcO
N3
O
BnO
BnO
OMe
12
Fig. 2. Acceptor alcohols and products in Table 3.
To further evaluate this glycosidation method, we next ex-
amined the reaction of diphenyl phosphate 1a with 1.1 equiv of
glycoside alcohols 8 and 9 (entries 6 and 7). According to the
general trend of -selectivity in this system, high -selectivity
was observed in the reaction with less-reactive 4-O-unprotected
glycoside 9 (
¼94:6, entry 7), whereas the primary alcohol 8
led to moderate -selectivity (
¼82:18, entry 6).
a
a
a
Scheme 3. Synthesis of mucin core 5 building block.
a/b
a
a/b
3aa
with 80% AcOH gave 4,6-O-unprotected glycoside 17 in 80%
yield. TMSOTf-promoted coupling of 1aa with diol 17 (1.3 equiv) in
2.3. Synthesis of mucin core 5 and core 7 building blocks
THF/Et2O (1:1) at ꢂ78 ꢀC resulted in 89% yield of disaccharides 18
with less satisfactory
a
-selectivity (
a
/
b
¼86:14). In an effort to en-
With appropriately protected TN-antigen derivatives in hand,
we set out to synthesize mucin-type glycopeptide core 5 and core 7
building blocks. O-Glycan with core 5 was found in sialylated form
of a human rectal adenocarcinoma glycoprotein38 and in meconium
glycoproteins.39 Oligosaccharide with core 7 was detected in bovine
submaxillary-gland mucin.40
hance the
be achieved by our previously reported method.23 HClO4-catalyzed
glycosidation of 3,4,6-tri-O-acetyl-2-azido-2-deoxygalactosyl
diphenyl phosphate (19) (
¼31:69) with 17 (1.1 equiv) in di-
oxane/Et2O (1:1) at 0 ꢀC greatly improved the stereoselectivity,
affording disaccharides 20 and 20 in 95% yield with an ratio
of 94:6. Protection of the C4 hydroxy group in 20 with Ac2O and
a-selectivity, we were pleased to find that this goal could
a/b
a
b
a/b
In 1995, Paulsen and co-workers reported the synthesis of core 5
and core 7 building blocks employing the KoenigseKnorr method,
in which glycosidations of disaccharide donors with a threonine
a
pyridine followed by reductive acetylation gave acetamide 21 in
86% yield. Finally, hydrogenolysis of 21 completed the synthesis of
core 7 building block 22. The present synthetic routes to these
building blocks offer distinct advantages in overall yield over other
methods.18c,41,42
acceptor gave exclusively
a
-glycosides in 73e74% yields.41 This
protocol depends on conventional labile glycosyl bromides that
must be prepared just prior to glycosidation, thus clearly dimin-
ishing synthetic flexibility. Koganty and co-workers accomplished
the synthesis of a core 5 building block, although coupling of 2-
acetamido-4,6-O-benzylidene-2-deoxygalactosyl trichloroacetimi-
date with 3-O-unprotected TN-antigen derivatives in THF resulted
in poor yields (30e35%).42 Recently, Schmidt and co-workers
reported the synthesis of a variety of mucin core building blocks,
including core 5 and core 7 building blocks, by capitalizing on the
Michael-type addition described above.18c
2.4. Mechanistic aspects
The beneficial effect of ethereal solvents,44,45 such as Et2O,46
dioxane,47 and THF,8h,16a,25b,48 on 1,2-cis-
a-glycosidations has
been well documented.49 We previously demonstrated that HClO4-
catalyzed glycosidation with per-O-benzyl-protected glucosyl
diphenyl phosphate in dioxane/Et2O (1:1) would proceed via a ki-
The chloroacetylated
a-glycoside 3ba was deblocked with
thiourea and sodium bicarbonate43 to give 3-O-unprotected
glycoside alcohol 13 in 88% yield (Scheme 3). The present pro-
tocol was found to be applicable to the acceptor alcohol 13,
netically favored
separated ion pair, leading to the preferential formation of
coside.23 Under similar conditions, the use of THF resulted in poor
-selectivity. In the TMSOTf-promoted glycosidation of 2-azido-
4,6-O-benzylidene-2-deoxy- -galactosyl diphenyl phosphate
1a , the use of Et2O or dioxane exhibited lower -selectivity than
a
-face attack of an acceptor alcohol on a solvent-
a
-gly-
providing disaccharides 14
a
and 14
b
in 94% yield with an
a
/
a
b
ratio of 91:9. After chromatographic separation of the anomers,
a-D
transformation of azide 14
a
to acetamide 15 with Zn in THF/
a
a
Ac2O/AcOH (3:2:1) followed by hydrogenolysis of the benzyl
ester over 10% Pd/C in EtOH furnished core 5 building block 16 in
71% yield. The carboxylic acid 16 could be a key intermediate for
Fmoc-based solid-phase synthesis of mucin-type O-linked
glycopeptides.
that found with THF (Table 2, entries 1 and 4 vs 5). Consequently,
the glycosidation mechanism is assumed to be different from the
previous one. Wulff and co-workers reported that an excess of THF
can compete with an acceptor alcohol to give mainly a highly re-
active
alcohol to afford the
we propose the reaction pathway for the present glycosidation in
b
-tetrahydrofuranium ion, which, in turn, reacts with the
We next explored the synthesis of mucin core 7 building block
from 3aa (Scheme 4). Removal of the benzylidene acetal group in
a
-glycoside.48b On the basis of Wulff’s work,

K. Kakita et al. / Tetrahedron 68 (2012) 5005e5017
5009
selectivity in a mixed solvent system, such as THF/Et2O (1:1) is
unclear at present, it is clear that the use of Et2O as a co-solvent
suppresses the formation of glycosyl polytetrahydrofurans from
oxonium ions 25a and 25b.
Scheme 5. Plausible mechanism of the TMSOTf-promoted glycosidation using
2-azido-4,6-O-benzylidene-2-deoxygalactosyl diphenyl phosphates in THF/Et2O.
3. Conclusion
TMSOTf-promoted glycosidation of 2-azido-4,6-O-benzylidene-
2-deoxygalactosyl diphenyl phosphates with Fmoc-protected ser-
ine and threonine derivatives in THF/Et2O (1:1) gave glycosyl amino
Scheme 4. Synthesis of mucin core 7 building block.
acids in high yields and with excellent
a-selectivities (a/
b
¼94:6e95:5), regardless of the anomeric composition of the do-
THF/Et2O as shown in Scheme 5. Diphenyl phosphate
1 is
nor. Comparative studies with 3,4,6-tri-O-benzyl- and 3,4,6-tri-O-
acetyl-protected diphenyl phosphates under the same conditions
demonstrated that the presence of a 4,6-O-benzylidene acetal
activated by silylation on the phosphoryl oxygen atom, resulting in
cleavage of the phosphate group to provide glycosyl triflate 24,
along with trimethylsilyl diphenyl phosphate. Intermediate 24 is
rapidly trapped by THF to form an anomeric mixture of tetrahy-
group is crucial for high levels of
a-selectivity. Regioselective
deprotections of the obtained 3-O-chloroacetyl- and 3-O-acetyl-
4,6-O-benzylidene-protected TN-antigen derivatives provided 3-
and 4,6-O-unprotected glycosides, respectively, which were suit-
able acceptor alcohols to construct core structures of mucin-type
glycopeptides. Using the 3-O-unprotected acceptor alcohol, we
achieved the synthesis of core 5 building block employing the
present glycosidation method. In the synthesis of core 7 building
block, HClO4-catalyzed glycosidation of 3,4,6-tri-O-acetyl-2-azido-
2-deoxygalactosyl diphenyl phosphate with the 4,6-O-unprotected
glycosyl amino acid afforded the corresponding disaccharides in
drofuranium ions 25
of THF than those of Et2O and dioxane.50 In this step, the equilib-
rium between the -oxonium ion 25 and the -oxonium ion 25
would heavily lie to 25
a and 25b because of the higher donor ability
b
b
a
a
b
, which occupies an equatorial position due
to the steric hindrance.51 Although actual glycosyl tetrahy-
drofuranium salts have yet to be observed, there have been some
examples of the formation of THF ring-opening products from
glycosyl tetrahydrofuranium ion intermediates.52 The
28a arises from the a
a
-glycoside
-axial attack of the acceptor alcohol on the
molecule-oxocarbenium ion complex 26 derived from the -oxo-
nium ion 25b 53
along with generation of TfOH, while the -gly-
coside 28 results from the SN2-like displacement by the acceptor
b
b
95% yield with an a/b ratio of 94:6. Thus, we demonstrated efficient
,
routes to mucin core 5 and core 7 building blocks that are useful in
Fmoc-based solid-phase synthesis of O-linked glycopeptides. Fur-
ther application of this methodology to the synthesis of biologically
active mucin-type glycopeptides is currently in progress.
b
alcohol at the anomeric carbon of the a-oxonium ion 25a via an
‘exploded’ transition state 27.54 Owing to the kinetic anomeric ef-
fect55 and the steric hindrance of the 4,6-O-benzylidene acetal
group,56 the
nium ion 25
placement of 25
a
b
-axial attack of the acceptor alcohol on the
might have lower activation energy than the dis-
via a loose association of the acceptor alcohol
with the anomeric center. The proportion of 1,2-cis- -galactosides
b-oxo-
4. Experimental section
4.1. General
a
a
decreased with highly reactive alcohols, such as the sterically less
demanding 6-O-unprotected glycoside alcohols 8 and 17, compared
to less reactive ones. The nucleophilic attack of highly reactive al-
€
Melting points were measured on a Buchi 535 digital melting
point apparatus and were uncorrected. Optical rotations were
measured on a JASCO P-1030 digital polarimeter with a sodium lamp
(589 nm). Infrared (IR) spectra were recorded on a JASCO FT/IR-5300
cohols might competitively occur on 25
a and 26, resulting in the
decrease of -selectivity. Although the reason for the enhanced a-
a

5010
K. Kakita et al. / Tetrahedron 68 (2012) 5005e5017
spectrometer and absorbance bands are reported in wavenumber
(cmꢂ1). Proton nuclear magnetic resonance (1H NMR) spectra were
recorded on JEOL JNM-AL400 (400 MHz), JNM-ECX400P (400 MHz),
JNM-ECS400 (400 MHz) or JNM-ECA500 (500 MHz) spectrometers
with tetramethylsilane (dH 0.00), CHCl3 (dH 7.26), benzene (dH 7.16)
or methanol (dH 3.31) as an internal standard. Coupling constants (J)
are reported in hertz (Hz). Abbreviations of multiplicity are as fol-
lows: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br,
broad. Data are presented as follows: chemical shift, multiplicity,
coupling constants, integration and assignment. Carbon nuclear
magnetic resonance (13C NMR) spectra were recorded on JEOL JNM-
ECX400P (100 MHz) or JNM-ECA500 (126 MHz) spectrometers with
128.2,129.2,137.4,166.9; ESI-HRMS m/z calcd for C21H30N3O6ClNaSi
(MþNa)þ 506.1490, found 506.1478.
4.2.2. 2-Azido-4,6-O-benzylidene-3-O-chloroacetyl-2-deoxy-D-gal-
actopyranose (6). Tetrabutylammonium fluoride in THF (1.0 M,
10.0 mL, 10.0 mmol) was added to a stirred solution of 5 (3.73 g,
7.71 mmol) in THF (10 mL) and AcOH (0.90 mL) at 0 ꢀC. After
stirring for 2 h, saturated aqueous NaHCO3 (12 mL) was added, and
the whole was extracted with AcOEt (200 mL). The organic layer
was successively washed with saturated aqueous NaHCO3 (50 mL)
and brine (2ꢁ50 mL), and dried over anhydrous Na2SO4. Filtration
and evaporation in vacuo furnished the crude product (3.60 g),
which was purified by column chromatography (silica gel 90 g, 4:1
CDCl3 (dC 77.0) or CD3OD (dC 49.0) as an internal standard. Phos-
phorus nuclear magnetic resonance (31P NMR) spectra were recor-
ded on JEOL JNM-ECX400P (160 MHz) or JNM-ECA500 (202 MHz)
hexane/AcOEt) to give lactol 6 (2.65 g, 93%,
a
/
b
¼68:32) as a white
amorphous solid. The anomeric a/b ratio of 6 was determined by
spectrometers with H3PO4
(dP 0.00) as an external standard. Elec-
1H NMR: Rf 0.37 (2:1 hexane/AcOEt); ½a 2D3
þ170.1 (c 1.00, CHCl3)
ꢃ
trospray ionization (ESI) mass spectra were obtained on a Thermo
Scientific Exactive spectrometer. Fast atom bombardment (FAB)
mass spectra were obtained on a JEOL JMS-700TZ spectrometer by
the Center for Instrumental Analysis, Hokkaido University.
(
a
/
b
¼68:32); IR (KBr) 3450, 2952, 2918, 2871, 2116, 1760, 1408,
1313, 1167, 996, 749, 700 cmꢂ1
(br s, 0.3H,
-OH), 3.56 (ddd, J¼1.1, 1.7, 1.7 Hz, 0.3H,
-H-2), 4.03 (ddd, J¼1.1, 1.7, 1.7 Hz, 0.7H,
-H-2), 4.07 (dd, J¼1.7,
-H-6a), 4.09 (dd, J¼1.7, 12.6 Hz, 0.7H, -H-6a), 4.17
-ClCH2), 4.26 (dd, J¼1.7, 12.6 Hz,
-H-6b), 4.34 (dd, J¼1.7, 12.3 Hz, 0.3H, -H-6b), 4.39 (dd,
J¼1.1, 3.4 Hz, 0.3H, -H-4), 4.53 (dd, J¼1.1, 3.4 Hz, 0.7H, -H-4),
4.69 (d, J¼8.0 Hz, 0.3H, -H-1), 4.80 (dd, J¼3.4, 10.9 Hz, 0.3H, -H-
3), 5.41 (dd, J¼3.4, 10.9 Hz, 0.7H, -H-3), 5.51 (d, J¼3.4 Hz, 0.7H,
H-1), 5.537 (s, 0.3H, -CHPh), 5.544 (s, 0.7H, -CHPh), 7.37e7.42
(m, 3H, Ar), 7.48e7.51 (m, 2H, Ar); 13C NMR (100 MHz, CDCl3)
40.7 (ab), 57.7 ( ), 61.4 ( ), 62.1 ( ), 66.2 ( ), 68.8 ( ), 69.0 ( ),
71.4 ( ), 72.2 ( ), 73.0 ( ), 73.7 ( ), 92.4 ( -C-1), 96.1 ( -C-1), 100.6
-CHPh), 100.7 ( -CHPh), 126.0, 128.19, 128.23, 129.1, 129.2, 137.0,
;
1H NMR (500 MHz, CDCl3)
d 3.03
b
b
-H-5), 3.89
(dd, J¼8.0, 10.9 Hz, 0.3H,
b
Column chromatography was carried out on Kanto silica gel
a
-H-5), 4.06 (dd, J¼3.4, 10.9 Hz, 0.7H,
a
60 N (40e50
mm or 63e210
mm) or Wakogel C-200 (75e150
mm).
12.3 Hz, 0.3H,
(s, 1.4H,
0.7H,
b
a
Analytical and preparative thin layer chromatography (TLC) was
carried out on 0.25-mm Merck Kieselgel 60 F254 plates. Visualiza-
tion was accomplished with ultraviolet light and anisaldehyde or
phosphomolybdic acid stain, followed by heating. Analytical high-
performance liquid chromatography (HPLC) was performed on
a-ClCH2), 4.18 (s, 0.6H, b
a
b
b
a
b
b
a
a-
a JASCO PU-980 and UV-970 (detector,
(tR) and peak ratios were determined with a JASCO-Borwin. Hexane
was HPLC grade, and filtered and degassed prior to use.
l
¼254 nm). Retention times
b
a
d
a
b
a
b
b
a
Reagents and solvents were purified by standard means or used
as received unless otherwise noted. Dehydrated CH2Cl2, Et2O, THF
(stabilizer free) and toluene were purchased from Kanto Chemical
Co., Inc. Dioxane was distilled from sodium metal/benzophenone
ketyl prior to use. A 0.1 M solution of anhydrous HClO4 in dioxane
a
b
a
b
a
b
(a
b
137.2, 166.97
(
b), 167.02
(a); ESI-HRMS m/z calcd for
C15H16N3O6ClNa (MþNa)þ 392.0625, found 392.0621.
ꢀ
was purchased from Kishida Chemical Co., Ltd. 5-A molecular sieves
4.2.3. 2-Azido-4,6-O-benzylidene-3-O-chloroacetyl-2-deoxy-a-D-
was finely ground in mortar and heated in vacuo at 200 ꢀC for 12 h.
All reactions were conducted under an argon atmosphere. 3-O-
galactopyranosyl diphenyl phosphate (1b
ophosphate (0.33 mL, 1.60 mmol) was added to a stirred solution of
lactol (492 mg, 1.33 mmol), N-methylimidazole (0.12 mL,
1.60 mmol) and Et3N (0.22 mL, 1.60 mmol) in toluene (10 mL) at
0
ꢀC. After stirring for 5 min, the reaction was quenched with
a). Diphenyl chlor-
Acetyl-2-azido-2-deoxy-4,6-O-benzylidene-
diphenyl phosphate (1a)27 and 3,4,6-tri-O-acetyl-2-azido-2-deoxy-
-galactopyranosyl diphenyl phosphate (19)23 were prepared
D-galactopyranosyl
6
D
according to literature procedures.
crushed ice, followed by stirring for 10 min. The mixture was
poured into a two-layer mixture of AcOEt (15 mL) and saturated
aqueous NaHCO3 (15 mL), and the whole mixture was extracted
with AcOEt (60 mL). The organic extract was successively washed
with saturated aqueous NaHCO3 (20 mL) and brine (20 mL), and
dried over anhydrous Na2SO4. Filtration and evaporation in vacuo
furnished the crude product (1.20 g), which was purified by column
chromatography (WakogelÒ 20 g, 15:1 toluene/AcOEt) to give
4.2. Preparation of glycosyl donor 1b
4.2.1. tert-Butyldimethylsilyl 2-azido-4,6-O-benzylidene-3-O-chlor-
oacetyl-2-deoxy-b-D-galactopyranoside (5). Chloroacetyl chloride
(0.23 mL, 2.83 mmol) was added to a stirred solution of 49a (1.05 g,
2.58 mmol) and pyridine (1.4 mL, 17.2 mmol) in CH2Cl2 (8.0 mL) at
ꢂ20 ꢀC. After stirring for 15 min, the reaction was quenched with
crushed ice followed by stirring for 15 min. The mixture was
extracted with AcOEt (60 mL). The organic layer was washed with
HCl (3ꢁ20 mL), water (20 mL) and brine (2ꢁ20 mL), and dried over
anhydrous Na2SO4. Filtration and evaporation in vacuo furnished
the crude product, which was purified by column chromatography
(silica gel 40 g, 4:1 hexane/AcOEt) to give 5 (1.24 g, 99%) as a white
diphenyl phosphate 1b
a
(482 mg, 60%) and 1b
b
(206 mg, 26%) as
a white amorphous solid. Data for
a
-anomer 1b
a
: Rf 0.54 (3:1 tol-
uene/AcOEt); ½a D20
ꢃ
þ149.9 (c 1.00, CHCl3); IR (KBr) 3067, 2918, 2116,
1765, 1590, 1489, 1290, 1188, 958, 759 cmꢂ1
;
1H NMR (500 MHz,
CDCl3)
d
3.71 (d, J¼1.7 Hz, 1H, H-5), 3.88 (dd, J¼1.7, 12.6 Hz, 1H, H-
6a), 3.99 (dd, J¼1.7, 12.6 Hz, 1H, H-6b), 4.17 (s, 2H, ClCH2), 4.24 (ddd,
J¼3.2, 10.9, 3.2 (JHeP) Hz, 1H, H-2), 4.48 (dd, J¼1.7, 3.4 Hz, 1H, H-4),
5.26 (dd, J¼3.4, 10.9 Hz, 1H, H-3), 5.49 (s, 1H, CHPh), 6.12 (dd, J¼3.2,
6.0 (JHeP) Hz, 1H, H-1), 7.21e7.46 (m, 15H, Ar); 13C NMR (100 MHz,
amorphous solid: Rf 0.68 (2:1 hexane/AcOEt); ½a D24
þ31.6 (c 1.00,
ꢃ
CHCl3); IR (CHCl3) 2955, 2931, 2859, 2117, 1762, 1406, 1312, 1171,
698 cmꢂ1
;
1H NMR (500 MHz, CDCl3)
d
0.18 (s, 3H, SiCH3), 0.20 (s,
CDCl3)
d
40.5, 57.1 (d, JCeP¼8.6 Hz, C-2), 64.2, 68.3, 71.4, 72.2, 97.5 (d,
3H, SiCH3), 0.95 (s, 9H, SiC(CH3)3), 3.47 (dt, J¼1.4, 1.1 Hz, 1H, H-5),
3.83 (dd, J¼7.4, 10.9 Hz, 1H, H-2), 4.06 (dd, J¼1.1, 12.3 Hz, 1H, H-6a),
4.15 (s, 1H, ClCH2), 4.16 (s, 1H, ClCH2), 4.28 (dd, J¼1.1, 12.3 Hz, 1H, H-
6b), 4.34 (dd, J¼1.4, 3.4 Hz,1H, H-4), 4.64 (d, J¼7.4 Hz, 1H, H-1), 4.73
(dd, J¼3.4, 10.9 Hz, 1H, H-3), 5.50 (s, 1H, CHPh), 7.37e7.41 (m, 3H,
JCeP¼5.7 Hz, C-1), 100.6 (CHPh), 120.0 (d, JCeP¼4.8 Hz), 120.2 (d,
JCeP¼4.8 Hz), 125.6, 125.7, 126.0, 128.2, 129.3, 129.77, 129,81, 136.9,
150.17 (d, JCeP¼3.9 Hz), 150.24 (d, JCeP¼4.8 Hz), 166.8; 31P NMR
(160 MHz, CDCl3)
d
ꢂ12.9; FAB-HRMS m/z calcd for C27H26N3O9ClP
(MþH)þ 602.1095, found 602.1094. Data for
b-anomer 1bb: Rf 0.36
Ar), 7.49e7.51 (m, 2H, Ar); 13C NMR (100 MHz, CDCl3)
d
18.0, 25.6,
(3:1 toluene/AcOEt); ½a D23
þ80.7 (c 1.00, CHCl3); IR (KBr) 3067,
ꢃ
40.7, 62.4, 66.1, 69.0, 72.3, 73.4, 97.3 (C-1), 100.9 (CHPh), 126.2,
2920, 2118, 1763, 1590, 1489, 1406, 1187, 957, 766 cmꢂ1 1H NMR
;

K. Kakita et al. / Tetrahedron 68 (2012) 5005e5017
5011
(500 MHz, CDCl3)
d
3.61 (d, J¼1.7 Hz, 1H, H-5), 3.99 (dd, J¼1.7,
(d, J¼7.6 Hz, 2H, Ar); 13C NMR (126 MHz, CDCl3)
d
20.9, 47.0, 54.3,
12.6 Hz, 1H, H-6a), 4.03 (dd, J¼8.0, 10.9 Hz, 1H, H-2), 4.14 (s, 2H,
ClCH2), 4.20 (dd, J¼1.7, 12.6 Hz, 1H, H-6b), 4.40 (d, J¼3.4 Hz, 1H, H-
4), 4.80 (dd, J¼3.4, 10.9 Hz, 1H, H-3), 5.26 (dd, J¼6.3 (JHeP), 8.0 Hz,
1H, H-1), 5.51 (s, 1H, CHPh), 7.26e7.53 (m, 15H, Ar); 13C NMR
60.2, 66.3, 67.3, 67.6, 68.7, 69.7, 72.0, 72.4, 100.8 (CHPh), 102.4 (C-1),
119.9, 125.17, 125.24, 126.2, 127.0, 127.7, 128.1, 128.2, 128.3, 128.5,
128.6, 128.9, 129.0, 129.1, 135.2, 137.5, 141.20, 141.24, 143.7, 143.9,
156.0, 169.5, 170.4; FAB-HRMS m/z calcd for C40H39N4O10 (MþH)þ
735.2667, found 735.2659.
(100 MHz, CDCl3)
d
40.5, 60.4 (d, JCeP¼10.5 Hz, C-2), 66.8, 68.3, 71.7,
73.8 (d, JCeP¼1.9 Hz, C-3), 98.1 (d, JCeP¼4.8 Hz, C-1), 100.8 (CHPh),
120.0 (d, JCeP¼4.8 Hz), 120.1 (d, JCeP¼4.8 Hz), 125.6, 126.2, 128.3,
129.3, 129.6, 129.8, 137.2, 150.1 (d, JCeP¼7.6 Hz), 150.3 (d,
4.3.2. N-(9-Fluorenylmethoxycarbonyl)-O-(3-O-acetyl-2-azido-4,6-
O-benzylidene-2-deoxy-D-galactopyranosyl)-L-threonine benzyl ester
JCeP¼7.6 Hz), 166.7; 31P NMR (160 MHz, CDCl3)
d
ꢂ13.1; FAB-HRMS
(10a). The glycosidation was performed according to the typical
m/z calcd for C27H25N3O9ClPNa (MþNa)þ 624.0915, found 624.0915.
4.3. Glycosidation
procedure (1:1 THF/Et2O 1.0 mL, ꢂ40 ꢀC, 4 h) employing diphenyl
phosphate 1a
a (56.7 mg, 0.10 mmol), alcohol 7 (69.4 mg,
0.15 mmol), TMSOTf (1.0 M in CH2Cl2, 0.11 mL, 0.11 mmol), and
ꢀ
pulverized 5-A MS (50 mg). An anomeric mixture of glycoside 10a
4.3.1. Typical procedure for glycosidation of 3-O-acetyl-2-azido-4,6-O-
(62.9 mg, 84%,
a
/
b
¼94:6) was obtained as a white amorphous solid
benzylidene-2-deoxy-
fluorenylmethoxycarbonyl)-O-(3-O-acetyl-2-azido-4,6-O-benzyli-
dene-2-deoxy- -galactopyranosyl)- -serine benzyl ester (3a). TMSOTf
in CH2Cl2 (1.0 M, 0.11 mL, 0.11 mmol) was added to a stirred solution
of diphenyl phosphate 1a (56.7 mg, 0.10 mmol), alcohol 2 (46.0 mg,
a-D-galactopyranosyl diphenyl phosphate: N-(9-
from the crude product (105.6 mg) after column chromatography
(silica gel 4 g, 20:1 CH2Cl2/AcOEt). The anomeric ratio of 10a was
determined by HPLC analysis [eluent, 2:1 hexane/AcOEt; flow rate,
D
L
1.0 mL minꢂ1; detection, 254 nm; tR
(
a
-anomer)¼10.2 min, tR
(b-
a
anomer)¼20.0 min]. The
a- and b-glycosides were separated by
ꢀ
0.11 mmol) and pulverized 5-A MS (50 mg) in THF/Et2O (1:1, 1 mL)
at e40 ꢀC. After stirring for 1.5 h, the reaction was quenched with
saturated aqueous NaHCO3 (0.1 mL), and the mixture was filtrated
through a Celite pad. The filtrate was poured into a two-layer
mixture of AcOEt (3 mL) and saturated aqueous NaHCO3 (6 mL),
and the whole was extracted with AcOEt (30 mL). The organic ex-
tract was successively washed with saturated aqueous NaHCO3
(10 mL) and brine (10 mL), and dried over anhydrous Na2SO4. Fil-
tration and evaporation in vacuo furnished the crude product
(90.8 mg), from which an anomeric mixture of glycosides 3a
flash chromatography with 25:1 CH2Cl2/AcOEt. Data for a-anomer
a 22
10a
a
: Rf 0.43 (20:1 CH2Cl2/AcOEt); ½ ꢃD þ115.8 (c 1.01, CHCl3); IR
(KBr) 3427, 3358, 3065, 2980, 2112, 1755, 1730, 1514, 1452, 1375,
1240, 1043 cmꢂ1; 1H NMR (500 MHz, CDCl3)
d
1.34 (d, J¼6.2 Hz, 3H,
Thr-CH3), 2.18 (s, 3H, CH3CO), 3.72 (br s, 1H, H-5), 3.94 (dd, J¼3.4,
11.1 Hz, 1H, H-2), 4.00 (br d, J¼12.4 Hz, 1H, H-6a), 4.23 (br d,
J¼12.4 Hz,1H, H-6b), 4.28 (dd, J¼6.7, 7.8 Hz,1H, Fmoc-CH), 4.38 (dd,
J¼7.8, 10.2 Hz, 1H, Fmoc-CH), 4.46e4.52 (m, 4H, H-4, Thr-
a-CH, Thr-
b
-CH, Fmoc-CH), 5.00 (d, J¼3.4 Hz, 1H, H-1), 5.23 (d, J¼12.2 Hz, 1H,
OCHPh), 5.265 (dd, J¼3.3, 11.1 Hz, 1H, H-3), 5.271 (d, J¼12.2 Hz, 1H,
OCHPh), 5.51 (s, 1H, CHPh), 5.87 (d, J¼8.2 Hz, 1H, NH), 7.32e7.43 (m,
12H, Ar), 7.52e7.54 (m, 2H, Ar), 7.66 (d, J¼7.4 Hz, 2H, Ar), 7.78 (d,
(68.3 mg, 93%,
a
/
b
¼95:5) was obtained as a white amorphous solid
after column chromatography (silica gel 10 g, 15:1 toluene/AcOEt).
The anomeric ratio of the product was determined by HPLC analysis
[column, ZorbaxÒ Sil, 4.6ꢁ250 mm; eluent, 3:2 hexane/AcOEt; flow
J¼7.5 Hz, 2H, Ar); 13C NMR (126 MHz, CDCl3)
d 18.5, 20.7, 46.9, 57.4,
58.5, 62.7, 67.2, 67.5, 68.7, 69.4, 73.0, 75.9, 98.7 (C-1), 100.5 (CHPh),
119.8, 125.01, 125.04, 125.9, 126.91, 126.93, 127.5, 128.0, 128.3, 128.4,
128.5, 128.9, 134.8, 137.3, 141.06, 141.07, 143.6, 143.7, 156.6, 169.9,
170.2; FAB-HRMS m/z calcd for C41H41N4O10 (MþH)þ 749.2822,
rate, 1.0 mL/min; tR (
a
-anomer)¼6.4 min, tR ( -anomer)¼14.6 min].
b
The - and b-glycosides were separated by flash column chroma-
a
tography with 20:1 toluene/AcOEt. Data for
a
-anomer 3a
a: Rf 0.59
(3:1 toluene/AcOEt); ½a D18
ꢃ
þ122.5 (c 1.00, CHCl3); IR (KBr) 3356,
found 749.2814. Data for b-anomer 10ab: Rf 0.29 (20:1 CH2Cl2/
3036, 2947, 2112, 1743, 1728, 1514, 1452, 1336, 1240, 1145,
AcOEt); ½a 2D3
ꢃ
þ9.30 (c 0.52, CHCl3); IR (KBr) 3429, 3065, 2937, 2116,
1043 cmꢂ1; 1H NMR (500 MHz, CDCl3)
s, 1H, H-5), 3.86 (d, J¼11.3 Hz, 1H, H-6a), 3.89 (dd, J¼3.2, 10.9 Hz, 1H,
H-2), 4.01 (dd, J¼2.5,11.1 Hz,1H, Ser- -CH), 4.17 (d, J¼11.3 Hz,1H, H-
6b), 4.19 (dd, J¼3.6, 11.1 Hz, 1H, Ser- -CH), 4.24 (t, J¼7.5 Hz, 1H,
Fmoc-CH), 4.35 (dd, J¼7.5, 10.4 Hz, 1H, Fmoc-CH), 4.37 (d, J¼2.7 Hz,
d
2.17 (s, 3H, CH3CO), 3.58 (br
1748, 1728, 1512, 1450, 1371, 1232, 1055 cmꢂ1 1H NMR (500 MHz,
;
CDCl3)
d
1.36 (d, J¼6.2 Hz, 3H, Thr-CH3), 2.15 (s, 3H, CH3CO), 3.08 (br
b
s, 1H, H-5), 3.85 (dd, J¼8.1, 10.9 Hz, 1H, H-2), 3.90 (br d, J¼12.3 Hz,
1H, H-6a), 4.16 (br d, J¼12.3 Hz, 1H, H-6b), 4.21 (d, J¼3.5 Hz, 1H, H-
4), 4.23 (dd, J¼6.4, 7.5 Hz, 1H, Fmoc-CH), 4.31e4.39 (m, 3H, H-1,
b
1H, H-4), 4.43 (dd, J¼7.5, 10.4 Hz, 1H, Fmoc-CH), 4.60 (m, 1H, Ser-
a
-
Fmoc-CH ꢁ2), 4.52 (dd, J¼1.4, 9.6 Hz, 1H, Thr-
a
-CH), 4.60 (dd, J¼3.5,
CH), 4.96 (d, J¼3.2 Hz, 1H, H-1), 5.19 (dd, J¼2.7, 10.9 Hz, 1H, H-3),
5.22 (d, J¼12.2 Hz, 1H, OCHPh), 5.26 (d, J¼12.2 Hz, 1H, OCHPh), 5.47
(s, 1H, CHPh), 5.97 (d, J¼8.6 Hz, 1H, NH), 7.33e7.39 (m, 12H, Ar),
7.46e7.48 (m, 2H, Ar), 7.61 (m, 2H, Ar), 7.77 (d, J¼7.7 Hz, 2H, Ar); 13C
10.9 Hz, 1H, H-3), 4.66 (dq, J¼1.4, 6.2 Hz, 1H, Thr-
b
-CH), 5.18 (d,
J¼12.7 Hz, 1H, OCHPh), 5.21 (d, J¼12.7 Hz, 1H, OCHPh), 5.46 (s, 1H,
CHPh), 5.81 (d, J¼9.6 Hz, 1H, NH), 7.22e7.38 (m, 12H, Ar), 7.48e7.50
(m, 2H, Ar), 7.61 (m, 2H, Ar), 7.74 (d, J¼7.6 Hz, 2H, Ar); 13C NMR
NMR (126 MHz, CDCl3)
d
20.8, 46.9, 54.5, 57.0, 62.8, 67.2, 67.6, 68.7,
(126 MHz, CDCl3) d 17.4, 20.9, 47.1, 58.6, 60.2, 66.1, 67.5, 68.6, 71.7,
69.0, 69.8, 73.0, 99.6 (C-1), 100.4 (CHPh), 119.9, 124.9, 125.0, 125.9,
126.96, 126.98, 127.6, 128.0, 128.3, 128.4, 128.5, 128.9, 134.9, 137.3,
141.1, 143.5, 143.6, 155.8, 169.5, 170.2; FAB-HRMS m/z calcd for
72.5, 74.9, 100.1, 100.9 (C-1, CHPh), 119.9, 125.3, 126.3, 127.1, 127.60,
127.62, 128.2, 128.4, 128.6, 129.1, 135.5, 137.6, 141.20, 141.24, 143.8,
144.0, 156.8, 170.1, 170.4; FAB-HRMS m/z calcd for C41H41N4O10
(MþH)þ 749.2822, found 749.2822.
C40H39N4O10 (MþH)þ 735.2667, found 735.2667. Data for
b-anomer
a 19
3a
b
: Rf 0.36 (3:1 toluene/AcOEt); ½ ꢃD þ29.7 (c 0.50, CHCl3); IR (KBr)
3356, 3036, 2947, 2110, 1743, 1730, 1514, 1450, 1336, 1240, 1145,
1043 cmꢂ1; 1H NMR (500 MHz, CDCl3)
2.17 (s, 3H, CH3CO), 3.37 (s,
1H, H-5), 3.90 (dd, J¼8.0, 10.6 Hz, 1H, H-2), 3.93 (dd, J¼2.7, 10.3 Hz,
1H, Ser-
4.3.3. N-(9-Fluorenylmethoxycarbonyl)-O-(2-azido-4,6-O-benzyli-
dene-3-O-chloroacetyl-2-deoxy-D-galactopyranosyl)-L-serine benzyl
d
ester (3b). The glycosidation was performed according to the typ-
b
-CH), 4.02 (dd, J¼1.7, 12.6 Hz, 1H, H-6a), 4.23 (t, J¼7.7 Hz,
ical procedure (1:1 THF/Et2O 6.0 mL, ꢂ40 ꢀC, 6 h) employing
1H, Fmoc-CH), 4.29 (dd, J¼1.7, 12.6 Hz, 1H, H-6b), 4.32 (d, J¼3.4 Hz,
1H, H-4), 4.35 (dd, J¼7.7, 11.7 Hz, 1H, Fmoc-CH), 4.36 (d, J¼8.0 Hz,
1H, H-1), 4.39 (dd, J¼7.7, 11.7 Hz, 1H, Fmoc-CH), 4.50 (dd, J¼2.7,
diphenyl phosphate 1ba (296 mg, 0.49 mmol), alcohol 2 (225 mg,
0.54 mmol), TMSOTf (1.0 M in CH2Cl2, 0.54 mL, 0.54 mmol) and
ꢀ
pulverized 5-A MS (500 mg). An anomeric mixture of glycoside 3b
10.3 Hz, 1H, Ser-
b
-CH), 4.64 (dt, J¼8.6, 2.7 Hz, 1H, Ser-
a
-CH), 4.69
(344 mg, 92%,
a
/
b
¼95:5) was obtained as a white amorphous solid
(dd, J¼3.4, 10.6 Hz, 1H, H-3), 5.22 (d, J¼12.2 Hz, 1H, OCHPh), 5.26 (d,
J¼12.2 Hz,1H, OCHPh), 5.52 (s, 1H, CHPh), 5.94 (d, J¼8.6 Hz,1H, NH),
7.20e7.39 (m, 12H, Ar), 7.49e7.51 (m, 2H, Ar), 7.60 (m, 2H, Ar), 7.75
from the crude product (613 mg) after column chromatography
(silica gel 18 g, 10:1/6:1 toluene/AcOEt). The anomeric ratio of 3b
was determined by HPLC analysis [eluent, 2:1 hexane/AcOEt; flow

5012
K. Kakita et al. / Tetrahedron 68 (2012) 5005e5017
rate, 1.0 mL/min; detection, 254 nm; tR
(
a
-anomer)¼9.2 min, tR
(
b
-
137.2, 141.22, 141.24, 143.7, 143.8, 156.7, 169.9, 170.0. Data for b-
anomer)¼23.9 min]. The
a
- and
b
-glycosides were separated by
-anomer
þ121.1 (c 1.00, CHCl3); IR
anomer 10b
b
: Rf 0.27 (3:1 toluene/AcOEt); ½a D22
þ11.0 (c 0.74,
ꢃ
flash chromatography with 15:1 toluene/AcOEt. Data for
a
CHCl3); IR (KBr) 3429, 3066, 2932, 2116, 1751, 1726, 1513, 1451,
3b
a
: Rf 0.70 (3:1 toluene/AcOEt); ½a D21
ꢃ
1369, 1055 cmꢂ1; 1H NMR (500 MHz, CDCl3)
d
1.37 (d, J¼6.3 Hz, 3H,
(KBr) 3425, 3250, 3066, 2951, 2112, 1744, 1721, 1451, 1338, 1267,
Thr-CH3), 3.02 (br s, 1H, H-5), 3.87 (dd, J¼8.0, 10.9 Hz, 1H, H-2), 3.90
(dd, J¼1.1, 12.6 Hz, 1H, H-6a), 4.16 (s, 1H, ClCH2), 4.166 (dd, J¼1.1,
12.6 Hz, 1H, H-6b), 4.167 (s,1H, ClCH2), 4.230 (t, J¼7.4 Hz, 1H, Fmoc-
CH), 4.234 (d, J¼3.4 Hz, 1H, H-4), 4.33 (dd, J¼7.4, 10.3 Hz, 1H, Fmoc-
CH), 4.34 (d, J¼8.0 Hz, 1H, H-1), 4.38 (dd, J¼7.4, 10.3 Hz, 1H, Fmoc-
1080 cmꢂ1; 1H NMR (500 MHz, CDCl3)
d, J¼12.6 Hz, 1H, H-6a), 3.90 (dd, J¼3.4, 11.5 Hz, 1H, H-2), 4.02 (dd,
J¼3.4, 10.9 Hz, 1H, Ser- -CH), 4.17 (s, 2H, ClCH2), 4.18 (br d,
J¼12.6 Hz, 1H, H-6b), 4.20 (dd, J¼3.4, 10.9 Hz, 1H, Ser- -CH), 4.24 (t,
J¼7.4 Hz, 1H, Fmoc-CH), 4.33e4.39 (m, 2H, Fmoc-CH, H-4), 4.44 (dd,
-CH),
d 3.55 (br s,1H, H-5), 3.85 (br
b
b
CH), 4.52 (dd, J¼1.7, 9.7 Hz, 1H, Thr-
1H, H-3), 4.67 (m, 1H, Thr-
a
-CH), 4.62 (dd, J¼3.4, 10.9 Hz,
J¼7.4, 10.9 Hz, 1H, Fmoc-CH), 4.61 (dt, J¼8.0, 3.4 Hz, 1H, Ser-
a
b
-CH), 5.18 (d, J¼12.6 Hz, 1H, OCHPh),
4.97 (d, J¼3.4 Hz, 1H, H-1), 5.216 (dd, J¼3.4, 11.5 Hz, 1H, H-3), 5.220
(d, J¼12.0 Hz, 1H, OCHPh), 5.26 (d, J¼12.0 Hz, 1H, OCHPh), 5.47 (s,
1H, CHPh), 5.94 (d, J¼8.0 Hz, 1H, NH), 7.31e7.78 (m, 18H, Ar); 13C
5.22 (d, J¼12.6 Hz, 1H, OCHPh), 5.48 (s, 1H, CHPh), 5.81 (d, J¼9.7 Hz,
1H, NH), 7.18e7.39 (m, 12H, Ar), 7.47e7.49 (m, 2H, Ar), 7.60 (dd,
J¼3.4, 7.4 Hz, 2H, Ar), 7.75 (d, J¼8.5 Hz, 2H, Ar); 13C NMR (100 MHz,
NMR (100 MHz, CDCl3)
d
40.6, 46.9, 54.5, 57.0, 62.8, 67.3, 67.8, 68.7,
CDCl3) d 17.4, 40.7, 47.0, 58.4, 59.9, 65.9, 67.47, 67.50, 68.5, 72.1, 73.2,
70.0, 71.0, 72.7, 99.6 (C-1), 100.5 (CHPh), 120.0, 125.0, 125.1, 126.0,
127.06, 127.08, 127.7, 128.1, 128.5, 128.6, 129.1, 134.9, 137.1, 141.19,
141.21, 143.6, 143.7, 155.9, 166.8, 169.6; FAB-HRMS m/z calcd for
75.9, 100.0 (C-1), 100.9 (CHPh), 119.9, 125.3, 126.2, 127.1, 127.62,
127.63, 128.2, 128.5, 128.6, 129.2, 135.5, 137.3, 141.19, 141.22, 143.7,
143.9, 156.9, 167.0, 170.0.
C40H38N4O10Cl (MþH)þ 769.2276, found 769.2278. Data for
b-
anomer 3b
b
: Rf 0.54 (3:1 toluene/AcOEt); ½a D24
ꢃ
þ33.6 (c 1.00,
4.4. Synthesis of mucin core 5 building block
CHCl3); IR (KBr) 3327, 2949, 2112, 1750, 1732, 1538, 1453, 1283,
1056 cmꢂ1; 1H NMR (500 MHz, CDCl3)
3.91 (dd, J¼8.3, 10.6 Hz, 1H, H-2), 3.95 (dd, J¼2.9, 10.0 Hz, 1H, Ser-
d
3.36 (d, J¼1.4 Hz, 1H, H-5),
4.4.1. N-(9-Fluorenylmethoxycarbonyl)-O-(2-azido-4,6-O-benzylidene-
b
-
2-deoxy-
(0.66 g, 8.7 mmol) and NaHCO3 (0.73 g, 8.7 mmol) was added to
a solution of glycoside 3b (0.67 g, 0.87 mmol) in CH2Cl2/MeOH
D-galactopyranosyl)-L
-serine benzyl ester (13).18c Thiourea
CH), 4.03 (dd, J¼1.4, 12.5 Hz, 1H, H-6a), 4.177 (s, 1H, ClCH2), 4.182 (s,
1H, ClCH2), 4.23 (t, J¼8.3 Hz, 1H, Fmoc-CH), 4.30 (dd, J¼1.4, 12.5 Hz,
1H, H-6b), 4.34 (dd, J¼8.3, 10.6 Hz, 1H, Fmoc-CH), 4.35 (d, J¼3.7 Hz,
1H, H-4), 4.37 (d, J¼8.3 Hz, 1H, H-1), 4.40 (dd, J¼8.3, 10.6 Hz, 1H,
a
(1:1, 10 mL) at room temperature. After stirring at room temper-
ature for 8 h, saturated aqueous NaHCO3 (60 mL) was added, and
the whole was extracted with AcOEt (200 mL). The organic layer
was successively washed with saturated aqueous NaHCO3 (50 mL)
and brine (2ꢁ50 mL), and dried over anhydrous Na2SO4. Filtration
and evaporation in vacuo furnished the crude product (0.88 g),
which was purified by column chromatography (silica gel 25 g,10:1
toluene/AcOEt) to give alcohol 13 (0.55 g, 88%) as a white solid: Rf
Fmoc-CH), 4.50 (dd, J¼2.9, 10.0 Hz, 1H, Ser-
b
-CH), 4.64 (dt, J¼8.6,
2.9 Hz, 1H, Ser-
a
-CH), 4.73 (dd, J¼3.7, 10.6 Hz, 1H, H-3), 5.21 (d,
J¼12.6 Hz, 1H, OCHPh), 5.26 (d, J¼12.6 Hz, 1H, OCHPh), 5.52 (s, 1H,
CHPh), 5.91 (d, J¼8.6 Hz, 1H, NH), 7.27e7.77 (m, 18H, Ar); 13C NMR
(100 MHz, CDCl3)
d 40.6, 46.9, 54.1, 60.0, 66.1, 67.3, 67.5, 68.5, 69.7,
72.1, 73.4, 100.7 (CHPh), 102.2 (C-1), 120.0, 125.1, 125.2, 126.1, 127.0,
127.6, 128.1, 128.2, 128.3, 128.5, 129.1, 135.1, 137.2, 141.1, 141.2, 143.6,
143.8, 155.9, 166.8, 169.4; FAB-HRMS m/z calcd for C40H38N4O10Cl
(MþH)þ 769.2276, found 769.2277.
0.50 (2:1 hexane/AcOEt); mp 86.0e87.0 ꢀC; ½a D20
þ80.3 (c 1.42,
ꢃ
CHCl3); IR (KBr) 3500, 3303, 3065, 2914, 2100, 1750, 1725, 1693,
1549, 1063, 739 cmꢂ1; 1H NMR (500 MHz, CDCl3)
3.51 (dd, J¼3.4, 10.6 Hz, 1H, H-2), 3.58 (s, 1H, H-5), 3.89 (d,
J¼12.2 Hz, 1H, H-6a), 3.97e4.03 (m, 2H, H-3, Ser- -CH), 4.15e4.19
(m, 3H, H-4, H-6b, Ser-
-CH), 4.23 (t, J¼7.0 Hz, 1H, Fmoc-CH), 4.34
(dd, J¼7.0, 10.4 Hz, 1H, Fmoc-CH), 4.46 (dd, J¼7.0, 10.4 Hz, 1H,
d 2.38 (s, 1H, OH),
4.3.4. N-(9-Fluorenylmethoxycarbonyl)-O-(2-azido-4,6-O-benzyli-
b
dene-3-O-chloroacetyl-2-deoxy-
D
-galactopyranosyl)-
L
-threonine
b
benzyl ester (10b).13b The glycosidation was performed according
to the typical procedure (1:1 THF/Et2O 1.0 mL, ꢂ60 ꢀC, 48 h)
Fmoc-CH), 4.60 (dt, J¼8.2, 3.1 Hz, 1H, Ser- -CH), 4.91 (d, J¼3.2 Hz,
a
employing diphenyl phosphate 1b
a
(60.8 mg, 0.10 mmol), alcohol
1H, H-1), 5.23 (s, 1H, OCHPh), 5.25 (s, 1H, OCHPh), 5.52 (s, 1H,
CHPh), 5.90 (d, J¼8.2 Hz, 1H, NH), 7.31e7.43 (m, 12H, Ar), 7.46e7.49
(m, 2H, Ar), 7.60 (dd, J¼4.3, 7.7 Hz, 2H, Ar), 7.78 (d, J¼7.7 Hz, 2H, Ar);
7 (65.4 mg, 0.15 mmol), TMSOTf (1.0 M in CH2Cl2, 0.11 mL,
ꢀ
0.11 mmol) and pulverized 5-A MS (100 mg). An anomeric mixture
of glycoside 10b (61.4 mg, 80%,
a
/
b
¼95:5) was obtained as a white
13C NMR (100 MHz, CDCl3)
d 47.0, 54.6, 60.4, 63.2, 67.0, 67.8, 68.9,
amorphous solid from the crude product (105.6 mg) after column
chromatography (silica gel 4 g, 10:1 toluene/AcOEt). The anomeric
ratio of 10b was determined by HPLC analysis [eluent, 2:1 hexane/
69.8, 75.2, 100.0 (C-1), 101.1 (CHPh), 120.0, 125.0, 125.1, 126.1,
127.07, 127.10, 127.8, 128.3, 128.62, 128.64, 129.3, 135.0, 137.2, 141.3,
143.6, 143.7, 155.9, 169.7.
AcOEt; flow rate, 1.0 mL minꢂ1; detection, 254 nm; tR (
a
-anomer)¼
6.3 min, tR
(
b
-anomer)¼11.1 min]. The
a
- and
b
-glycosides were
4.4.2. O-(3-O-Acetyl-2-azido-4,6-O-benzylidene-2-deoxy-
actopyranosyl)-(1/3)-(2-azido-4,6-O-benzylidene-2-deoxy-
actopyranosyl)-N-(9-fluorenylmethoxycarbonyl)- -serine benzyl ester
D
-gal-
separated by flash chromatography with 15:1 toluene/AcOEt. Data
a-D
-gal-
for
a
-anomer 10b
a
: Rf 0.64 (3:1 toluene/AcOEt); ½a D22
ꢃ
þ109.1 (c
L
0.54, CHCl3); IR (KBr) 3369, 3065, 3036, 2926, 2112, 1749, 1727,
(14). The glycosidation was performed according to the typical
1515, 1451, 1405, 1041 cmꢂ1 1H NMR (500 MHz, CDCl3)
; d 1.31 (d,
procedure (1:1 THF/Et2O 1.0 mL, ꢂ60 ꢀC, 6 h) employing diphenyl
J¼6.9 Hz, 3H, Thr-CH3), 3.71 (d, J¼1.1 Hz, 1H, H-5), 3.92 (dd, J¼3.4,
10.9 Hz, 1H, H-2), 4.03 (dd, J¼1.1, 12.6 Hz, 1H, H-6a), 4.16 (s, 2H,
ClCH2), 4.24 (dd, J¼1.1, 12.6 Hz, 1H, H-6b), 4.26 (t, J¼7.4 Hz, 1H,
Fmoc-CH), 4.35 (dd, J¼7.4, 10.9 Hz, 1H, Fmoc-CH), 4.45 (dd, J¼7.4,
phosphate 1aa (56.7 mg, 0.10 mmol), alcohol 13 (76.0 mg,
0.11 mmol), TMSOTf (1.0 M in CH2Cl2, 0.11 mL, 0.11 mmol) and
ꢀ
pulverized 5-A MS (100 mg). An anomeric mixture of disaccharide
14 (95.0 mg, 94%,
a
/
b
¼91:9) was obtained as a white amorphous
10.9 Hz, 1H, Fmoc-CH), 4.46e4.50 (m, 3H, H-4, Thr-
a
-CH, Thr-
b
-
solid from the crude product (110 mg) after flash column chro-
matography (silica gel 10 g, 20:1 toluene/AcOEt). The anomeric
ratio of 14 was determined by HPLC analysis [eluent, 5:1 hexane/i-
CH), 4.98 (d, J¼3.4 Hz, 1H, H-1), 5.22 (s, 1H, OCHPh), 5.23 (s, 1H,
OCHPh), 5.23e5.26 (m, 1H, H-3), 5.52 (s, 1H, CHPh), 5.75 (d,
J¼9.7 Hz, 1H, NH), 7.30e7.41 (m, 12H, Ar), 7.47e7.49 (m, 2H, Ar),
7.62 (d, J¼7.5 Hz, 2H, Ar), 7.77 (d, J¼7.5 Hz, 2H, Ar); 13C NMR
PrOH; flow rate, 1.0 mL/min; detection, 254 nm; tR
(
a
-anomer)¼
6.5 min, tR - and -glycosides were
(
b
-anomer)¼15.0 min]. The
a
b
(100 MHz, CDCl3)
d
18.6, 40.6, 47.1, 57.6, 58.6, 62.7, 67.4, 67.7, 68.9,
separated by flash chromatography with 15:1 toluene/AcOEt. Data
71.6, 72.7, 76.3, 98.7 (C-1), 100.7 (CHPh), 119.9, 125.1, 125.2, 125.3,
126.0, 127.1, 127.7, 128.2, 128.5, 128.6, 128.7, 129.0, 129.2, 134.9,
for
a
-anomer 14
a
: Rf 0.59 (3:1 toluene/AcOEt); ½a D20
þ184.1 (c 1.00,
ꢃ
CHCl3); IR (KBr) 3326, 2914, 2112, 1745, 1728, 1452, 1242,

K. Kakita et al. / Tetrahedron 68 (2012) 5005e5017
5013
1046 cmꢂ1; 1H NMR (500 MHz, CDCl3)
d
2.15 (s, 3H, CH3CO), 3.48 (s,
Fmoc-CH), 4.60 (br s, 1H, Ser-
a
-CH), 4.68 (ddd, J¼2.9, 10.3, 10.6 Hz,
1H, H-50), 3.79 (dd, J¼3.4, 10.9 Hz, 1H, H-20), 3.89 (d, J¼12.6 Hz, 1H,
1H, H-20), 4.80 (ddd, J¼4.0, 9.7, 11.5 Hz, 1H, H-2), 4.81 (d, J¼2.9 Hz,
1H, H-10), 4.97 (dd, J¼3.4, 11.5 Hz, 1H, H-3), 5.19 (d, J¼12.6 Hz, 1H,
OCHPh), 5.23 (d, J¼12.6 Hz, 1H, OCHPh), 5.26 (d, J¼4.0 Hz, 1H, H-1),
5.48 (s, 1H, CHPh), 5.52 (s, 1H, CHPh), 5.81 (d, J¼8.6 Hz, 1H,
NHFmoc), 5.83 (d, J¼10.3 Hz, 1H, NHAc0), 6.25 (d, J¼9.7 Hz, 1H,
NHAc), 7.31e7.59 (m, 21H, Ar), 7.77 (d, J¼7.7 Hz, 2H, Ar); 13C NMR
H-60a), 3.97 (dd, J¼3.4, 11.5 Hz, 1H, H-2), 3.98 (s, 1H, H-5), 3.99 (dd,
J¼2.7, 8.0 Hz, 1H, Ser- -CH), 4.07 (dd, J¼1.1, 12.9 Hz, 1H, H-6a), 4.10
b
(dd, J¼2.6, 10.9 Hz, 1H, H-30), 4.20 (d, J¼12.6 Hz, 1H, H-60b), 4.21
(dd, J¼2.7, 8.0 Hz, 1H, Ser- -CH), 4.23 (t, J¼7.2 Hz, 1H, Fmoc-CH),
b
4.27 (d, J¼2.6 Hz, 1H, H-40), 4.32 (dd, J¼1.1, 12.9 Hz, 1H, H-6b), 4.35
(dd, J¼7.2, 10.6 Hz, 1H, Fmoc-CH), 4.46 (dd, J¼7.2, 10.6 Hz, 1H,
Fmoc-CH), 4.54 (d, J¼2.9 Hz, 1H, H-4), 4.59 (dt, J¼8.0, 2.7 Hz, 1H,
(100 MHz, CDCl3)
d 21.0, 22.7, 23.3, 46.4, 47.0, 47.7, 54.6, 63.0, 63.3,
67.1, 67.8, 69.1, 69.2, 69.3, 71.0, 71.3, 73.3, 94.2 (C-10), 99.8 (C-1),
100.7 (CHPh), 100.9 (CHPh), 120.1, 124.76, 124.81, 126.0, 126.3, 127.1,
127.8, 127.9, 128.2, 128.3, 128.4, 128.8, 129.0, 129.2, 134.5,
137.0, 137.3, 141.3, 143.4, 143.5, 156.0, 170.1, 170.4, 170.7, 171.2; FAB-
HRMS m/z calcd for C57H59N3O16Na (MþNa)þ 1064.3793, found
1064.3781.
Ser-
a
-CH), 4.94 (d, J¼3.4 Hz, 1H, H-10), 5.21 (d, J¼12.0 Hz, 1H,
OCHPh), 5.23 (d, J¼12.0 Hz, 1H, OCHPh), 5.32 (d, J¼3.4 Hz, 1H, H-1),
5.39 (d, J¼2.9, 11.5 Hz, 1H, H-3), 5.54 (s, 1H, CHPh), 5.55 (s, 1H,
CHPh), 5.93 (d, J¼8.0 Hz, 1H, NH), 7.32e7.42 (m, 15H, Ar), 7.50e7.54
(m, 4H, Ar), 7.60 (d, J¼7.5 Hz, 2H, Ar), 7.77 (d, J¼7.5 Hz, 2H, Ar); 13C
NMR (100 MHz, CDCl3) d 20.9, 47.0, 54.6, 56.5, 58.2, 63.1, 63.2, 67.2,
67.7, 68.8, 69.0, 69.1, 70.1, 71.1, 71.4, 73.3, 95.3 (C-10), 99.9 (C-1),
100.7 (CHPh ꢁ2), 120.0, 124.94, 125.03, 125.2, 125.9, 126.1, 127.06,
127.09, 127.8, 128.1, 128.16, 128.18, 128.5, 128.7, 128.9, 129.0, 129.1,
135.0, 137.2, 137.4, 141.1, 143.57, 143.64, 155.9, 169.7, 170.3; FAB-
HRMS m/z calcd for C53H52N7O14 (MþH)þ 1010.3572, found
4.4.4. O-(2-Acetamido-3-O-acetyl-4,6-O-benzylidene-2-deoxy-
galactopyranosyl)-(1/3)-(2-acetamido-4,6-O-benzylidene-2-deoxy-
-galactopyranosyl)-N-(9-fluorenylmethoxycarbonyl)- -serine
a-D-
a
-
D
L
(16). 10% Pd/C (30.0 mg) was added to a solution of glycoside 15
(30.1 mg, 0.029 mmol) in EtOH (1.0 mL) under an argon atmo-
sphere, and the mixture was vigorously stirred under 1 atm of
hydrogen for 15 min. The catalyst was filtered through a Celite
pad, and the filtrate was evaporated in vacuo. Purification of the
crude product (28.2 mg) by column chromatography (silica gel,
1.20 g, 19:1:0.1 CHCl3/MeOH/AcOH) afforded carboxylic acid 16
(22.6 mg, 82%) as a white solid: Rf 0.21 (4:1 CHCl3/MeOH); mp
1010.3569. Data for
b-anomer 14b: Rf 0.36 (3:1 toluene/AcOEt);
a 24
½ ꢃD þ95.6 (c 0.85, CHCl3); IR (KBr) 3424, 2904, 2116, 1746, 1728,
1509, 1452, 1235, 1048 cmꢂ1 1H NMR (500 MHz, CDCl3)
; d 2.16 (s,
3H, CH3CO), 3.41 (s, 1H, H-5), 3.59 (s, 1H, H-50), 3.87 (d, J¼12.6 Hz,
1H, H-60a), 3.90 (dd, J¼3.4, 10.9 Hz, 1H, H-2), 3.97 (dd, J¼7.4,
10.9 Hz, 1H, H-20), 4.00 (d, J¼12.9 Hz, 1H, H-6a), 4.04 (dd, J¼2.9,
8.6 Hz, 1H, Ser-
J¼2.9, 8.6 Hz, 1H, Ser-
b
-CH), 4.06 (dd, J¼3.2, 10.9 Hz, 1H, H-3), 4.16 (dd,
180.0e181.0 ꢀC (decomp.); ½a 2D3
ꢃ
þ163.3 (c 0.48, CHCl3); IR (KBr)
b
-CH), 4.17 (d, J¼12.6 Hz, 1H, H-60b), 4.24 (t,
3407, 2924, 1721, 1662, 1523, 1243, 1050, 759, 743 cmꢂ1 1H NMR
;
J¼7.2 Hz, 1H, Fmoc-CH), 4.26 (d, J¼12.9 Hz, 1H, H-6b), 4.31 (d,
J¼3.7 Hz, 1H, H-40), 4.35 (dd, J¼7.2, 10.6 Hz, 1H, Fmoc-CH), 4.41 (d,
J¼3.2 Hz, 1H, H-4), 4.46 (dd, J¼7.2, 10.6 Hz, 1H, Fmoc-CH), 4.616 (m,
(500 MHz, CD3OD) d 1.45 (s, 3H, CH3CO), 1.98 (s, 3H, CH3CO), 2.02
(s, 3H, CH3CO), 3.72 (s, 1H, H-50), 3.79 (s, 1H, H-5), 3.93e3.97 (m,
2H, Ser-
b
-CH ꢁ2), 4.00 (dd, J¼3.2, 11.2 Hz, 1H, H-30), 4.06 (d,
1H, Ser-
a
-CH), 4.618 (d, J¼7.4 Hz, 1H, H-10), 4.71 (dd, J¼3.7, 10.9 Hz,
J¼12.6 Hz, 1H, H-60a), 4.12 (d, J¼12.6 Hz, 1H, H-60b), 4.13 (d,
J¼12.0 Hz, 1H, H-6a), 4.21 (d, J¼12.0 Hz, 1H, H-6b), 4.24 (t,
1H, H-30), 4.98 (d, J¼3.4 Hz, 1H, H-1), 5.24 (s, 2H, OCHPh), 5.50 (s,
1H, CHPh), 5.54 (s, 1H, CHPh), 5.90 (d, J¼8.6 Hz, 1H, NH), 7.32e7.43
(m, 15H, Ar), 7.48e7.53 (m, 4H, Ar), 7.59e7.61 (m, 2H, Ar), 7.78 (d,
J¼6.6 Hz, 1H, Fmoc-CH), 4.37 (m, 1H, Ser- -CH), 4.43 (dd, J¼6.6,
a
11.5 Hz, 1H, Fmoc-CH), 4.45 (m, 2H, H-4, H-40), 4.49 (dd, J¼6.6,
11.5 Hz, 1H, Fmoc-CH), 4.59 (ddd, J¼4.0, 7.8, 11.2 Hz, 1H, H-20), 4.62
(ddd, J¼3.4, 8.9, 11.5 Hz, 1H, H-2), 4.92 (d, J¼4.0 Hz, 1H, H-10), 5.03
(dd, J¼3.4, 11.5 Hz, 1H, H-3), 5.22 (d, J¼3.4 Hz, 1H, H-1), 5.59 (s,
1H, CHPh), 5.60 (s, 1H, CHPh), 7.31e7.82 (m, 18H, Ar); 13C NMR
J¼7.5 Hz, 2H, Ar); 13C NMR (100 MHz, CDCl3)
d 20.9, 47.0, 54.6, 59.1,
60.2, 63.5, 66.2, 67.3, 67.8, 68.85, 68.90, 69.7, 72.2, 72.3, 74.1, 75.6,
100.0 (C-1), 100.5 (CHPh), 100.9 (CHPh), 102.7 (C-10), 120.1, 125.0,
125.1,126.1,126.2, 127.1,127.2,127.8, 128.1, 128.3, 128.4, 128.6, 128.7,
128.8,129.2, 134.9, 137.5, 141.3, 143.5, 143.7, 155.9, 169.7, 170.5; FAB-
HRMS m/z calcd for C53H51N7O14Na (MþNa)þ 1032.3392, found
1032.3385.
(100 MHz, CD3OD)
d 20.7, 23.0, 23.8, 56.5, 58.3, 64.3, 64.4, 67.8,
70.0, 70.3, 70.4, 71.1, 71.8, 74.6, 94.4 (C-10), 100.6 (C-1), 101.9
(CHPh), 102.1 (CHPh), 121.0, 126.06, 126.13, 127.5, 127.8, 128.3,
128.9, 129.1, 129.2, 130.0, 130.1, 139.5, 139.6, 142.6, 145.2, 145.3,
158.4, 172.2, 173.46, 173.54, 174.0; ESI-HRMS m/z calcd for
C50H52N3O16 (MꢂH)ꢂ 950.3348, found 950.3355.
4.4.3. O-(2-Acetamido-3-O-acetyl-4,6-O-benzylidene-2-deoxy-
galactopyranosyl)-(1/3)-(2-acetamido-4,6-O-benzylidene-2-deoxy-
-galactopyranosyl)-N-(9-fluorenylmethoxycarbonyl)- -serine
benzyl ester (15). Zn powder (40 mg) was added to a solution of
glycoside 14 (46.0 mg, 0.046 mmol) in THF/Ac2O/AcOH (3:2:1,
a-D-
a
-
D
L
4.5. Synthesis of mucin core 7 building block
a
1 mL) at room temperature. After stirring for 5 min, the mixture
was filtered through a Celite pad. The filtrate was poured into
a two-layer mixture of AcOEt (3 mL) and saturated aqueous
NaHCO3 (4 mL), and the whole mixture was extracted with AcOEt
(12 mL). The organic extract was successively washed with satu-
rated aqueous NaHCO3 (2ꢁ4 mL) and brine (4 mL), and dried over
anhydrous Na2SO4. The filtrate was concentrated and purified by
column chromatography (silica gel 2 g, 1:4 hexane/AcOEt) to give
acetamide 15 (41.0 mg, 86%) as a white solid: Rf 0.20 (1:4 hexane/
4.5.1. N-(9-Fluorenylmethoxycarbonyl)-O-(3-O-acetyl-2-azido-2-
deoxy-a-D-galactopyranosyl)-L-serine benzyl ester (17). Compound
3aa (515 mg, 0.70 mmol) was dissolved in 80% AcOH (14 mL)
and the solution was stirred for 8 h at 50 ꢀC. The mixture was
poured into a two-layer mixture of AcOEt (15 mL) and saturated
aqueous NaHCO3 (20 mL), and the whole was extracted with
AcOEt (150 mL). The organic layer was washed with saturated
aqueous NaHCO3 (35 mL) and brine (2ꢁ35 mL), and dried over
anhydrous Na2SO4. Filtration and evaporation in vacuo furnished
the pale yellow residue (490 mg), which was purified by column
chromatography (silica gel 15 g, 1:2 hexane/AcOEt) to give diol
17 (360 mg, 80%) as a white amorphous solid. Rf 0.17 (1:2 hex-
AcOEt); ½a 2D5
ꢃ
þ141.6 (c 1.01, CHCl3); mp 138.0e139.0 ꢀC; IR (KBr)
3414, 3065, 2923, 1740, 1727, 1667, 1522, 1244, 1050, 743 cmꢂ1 1H
;
NMR (500 MHz, CDCl3)
d 1.60 (s, 3H, CH3CO), 2.00 (s, 6H, CH3CO),
2.04 (s, 3H, CH3CO), 3.51 (s, 1H, H-50), 3.71 (s, 1H, H-5), 3.78 (dd,
ane/AcOEt); ½a 2D0
ꢃ
þ83.9 (c 1.01, CHCl3); IR (KBr) 3430, 3066,
1H NMR
2.20 (s, 3H, CH3CO), 2.89 (br s, 1H, OH), 3.68
(dd, 1H, J¼3.4, 10.9 Hz, H-2), 3.65e3.78 (m, 4H, OH, H-5, H-6a, H-
6b), 3.94 (dd, J¼2.6, 11.2 Hz, 1H, Ser- -CH), 4.13 (d, J¼2.9 Hz, 1H,
H-4), 4.23 (t, J¼6.9 Hz, 1H, Fmoc-CH), 4.26 (dd, J¼2.6, 11.2 Hz, 1H,
J¼2.0, 10.6 Hz, 1H, H-30), 3.89e3.98 (m, 2H, Ser-
b-CH ꢁ2), 3.93 (d,
2948, 2112, 1742, 1725, 1524, 1451, 1247, 1044 cmꢂ1
;
J¼13.2 Hz, 1H, H-60a), 4.05 (d, J¼11.5 Hz, 1H, H-6a), 4.20 (d,
J¼13.2 Hz, 1H, H-60b), 4.23 (t, J¼7.2 Hz, 1H, Fmoc-CH), 4.27 (d,
J¼11.5 Hz,1H, H-6b), 4.31 (br s,1H, H-40), 4.33 (d, J¼3.4 Hz,1H, H-4),
4.41 (dd, J¼7.2, 10.6 Hz, 1H, Fmoc-CH), 4.52 (dd, J¼7.2, 10.6 Hz, 1H,
(500 MHz, CDCl3) d
b

5014
K. Kakita et al. / Tetrahedron 68 (2012) 5005e5017
Ser-
2.6 Hz, 1H, Ser-
b
-CH), 4.44 (d, J¼6.9 Hz, 2H, Fmoc-CH ꢁ2), 4.58 (dt, J¼8.0,
125.13, 125.15, 126.1, 126.98, 127.04, 127.6, 128.2, 128.4, 128.5, 128.6,
a
-CH), 4.83 (d, J¼3.4 Hz, 1H, H-1), 5.12 (dd, J¼2.9,
129.2, 135.0, 137.3, 141.2, 143.9, 155.9, 169.6, 169.8, 170.5; FAB-
HRMS m/z calcd for C48H49N7O15Na (MþNa)þ 986.3184, found
986.3182.
10.9 Hz, 1H, H-3), 5.22 (d, J¼12.0 Hz, 1H, OCHPh), 5.23 (d,
J¼12.0 Hz, 1H, OCHPh), 6.10 (d, J¼8.0 Hz, 1H, NH), 7.31e7.42 (m,
9H, Ar), 7.62 (d, J¼7.4 Hz, 2H, Ar), 7.77 (d, J¼7.4 Hz, 2H, Ar); 13C
NMR (100 MHz, CDCl3)
d
20.9, 47.0, 54.6, 57.1, 62.9, 67.1, 67.8,
4.5.3. O-(3,4,6-Tri-O-acetyl-2-azido-2-deoxy-
(1/6)-(3-O-acetyl-2-azido-2-deoxy- -galactopyranosyl)-N-(9-
fluorenylmethoxycarbonyl)- -serine benzyl ester (20). HClO4 in di-
D-galactopyranosyl)-
68.6, 69.7, 70.2, 70.3, 99.8 (C-1), 120.0, 125.1, 127.1, 127.7, 128.4,
128.59, 128.63, 134.9, 141.21, 141.23, 143.6, 143.7, 156.0, 169.7,
170.1; FAB-HRMS m/z calcd for C33H35N4O10 (MþH)þ 647.2353,
found 647.2351.
a-D
L
oxane (0.1 M, 0.20 mL, 0.02 mmol) was added to a stirred solution
of diphenyl phosphate 19 (56.3 mg, 0.10 mmol), diol 17 (71.1 mg,
ꢀ
0.11 mmol) and pulverized 5-A MS (100 mg) in dioxane/Et2O (1:1,
1.0 mL) at 0 ꢀC. After stirring at this temperature for 10 h, the re-
action was quenched with saturated aqueous NaHCO3 (0.1 mL), and
the mixture was filtrated through a Celite pad. The filtrate was
poured into a two-layer mixture of AcOEt (3 mL) and NaHCO3
(6 mL), and the whole was extracted with AcOEt (30 mL). The or-
ganic layer was successively washed with saturated aqueous
NaHCO3 (10 mL) and brine (10 mL), and dried over anhydrous
Na2SO4. Filtration and evaporation in vacuo furnished the crude
product (121.5 mg), from which an anomeric mixture of di-
4.5.2. O-(3-O-Acetyl-2-azido-4,6-O-benzylidene-2-deoxy-
actopyranosyl)-(1/6)-(3-O-acetyl-2-azido-2-deoxy- -galactopyr-
anosyl)-N-(9-fluorenylmethoxycarbonyl)- -serine benzyl ester
D-gal-
a-D
L
(18). The glycosidation was performed according to the typical
procedure (1:1 THF/Et2O 1.0 mL, ꢂ78 ꢀC, 48 h) employing diphenyl
phosphate 1aa (56.7 mg, 0.10 mmol), diol 17 (80.8 mg, 0.13 mmol),
ꢀ
TMSOTf (1.0 M in CH2Cl2, 0.11 mL, 0.11 mmol) and pulverized 5-A
MS (100 mg). An anomeric mixture of disaccharide 18 (85.8 mg,
89%,
a
/
b
¼86:14) was obtained as a white amorphous solid from the
saccharide 20 (91.2 mg, 95%,
a
/
b
¼94:6) was obtained as a white
crude product (120 mg) after flash column chromatography (silica
gel 12 g, 4:1/2:1 toluene/AcOEt). The anomeric ratio of 18 was
determined by HPLC analysis [eluent, 3:1 hexane/i-PrOH; flow rate,
amorphous solid after flash column chromatography (silica gel 10 g,
10:1 CH2Cl2/acetone). The anomeric ratio of 20 was determined by
HPLC analysis [eluent, 2:1 hexane/THF; flow rate, 1.0 mL/min; de-
1.5 mL/min; detection, 254 nm; tR
anomer)¼6.6 min]. The - and -glycosides were separated by
-anomer
þ143.0 (c 1.00, CHCl3); IR
(
a
-anomer)¼3.5 min, tR
(b-
tection, 254 nm; tR
(
a
-anomer)¼15.3 min, tR
(
b
-anomer)¼
a
b
19.9 min]. The - and
a
b glycosides were separated by flash column
flash chromatography with 1:2 hexane/AcOEt. Data for
a
chromatography with 20:1 CH2Cl2/acetone. Data for
a
-anomer 20a:
18
a
: Rf 0.54 (1:2 hexane/AcOEt); ½a D20
ꢃ
Rf 0.35 (10:1 CH2Cl2/acetone); ½a D20
þ103.8 (c 1.00, CHCl3); IR (KBr)
ꢃ
(KBr) 3434, 2938, 2111, 1742, 1726, 1517, 1452, 1374, 1236,
3424, 3066, 2945, 2112, 1749, 1727, 1520, 1451, 1230, 1050,
1036 cmꢂ1; 1H NMR (500 MHz, CDCl3)
d 2.13 (s, 3H, CH3CO), 2.21 (s,
742 cmꢂ1; 1H NMR (500 MHz, CDCl3)
d 1.99 (s, 3H, CH3CO), 2.03 (s,
3H, CH3CO), 2.81 (d, J¼2.3 Hz, 1H, OH), 3.70 (br s, 1H, H-50), 3.72
(dd, J¼3.4, 11.5 Hz, 1H, H-2), 3.76 (dd, J¼7.2 12.3 Hz, 1H, H-6a),
3.85e3.89 (m, 2H, H-5, H-6b), 3.94 (br d, J¼12.6 Hz, 1H, H-60a), 4.01
3H, CH3CO), 2.10 (s, 3H, CH3CO), 2.20 (s, 3H, CH3CO), 2.66 (d,
J¼3.4 Hz, 1H, OH), 3.70 (dd, J¼4.0, 10.9 Hz, 1H, H-2), 3.74 (dd, J¼2.3,
8.0 Hz, 1H, H-6a), 3.76 (dd, J¼3.4, 10.9 Hz, 1H, H-20), 3.83e3.88 (m,
(dd, J¼3.4, 10.9 Hz, 1H, Ser-
b
-CH), 4.04 (dd, J¼3.4, 10.9 Hz, 1H, H-20),
2H, H-5, H-6b), 3.98e4.08 (m, 3H, Ser-b
-CH, H-50, H-60a), 4.13e4.20
4.17 (br d, J¼12.6 Hz, 1H, H-60b), 4.15e4.18 (m, 2H, H-4, Ser-
b-CH),
(m, 2H, Ser-
b
-CH, H-60b), 4.14 (m, 1H, H-4), 4.25 (t, J¼7.7 Hz, 1H,
4.24 (dd, J¼7.4, 10.3 Hz, 1H, Fmoc-CH), 4.34 (dd, J¼7.4, 10.3 Hz, 1H,
Fmoc-CH), 4.33 (dd, J¼7.7, 10.6 Hz, 1H, Fmoc-CH), 4.41 (dd, J¼7.7,
Fmoc-CH), 4.41 (m, 2H, H-40, Fmoc-CH), 4.63 (dt, J¼8.0, 3.4 Hz, 1H,
10.6 Hz,1H, Fmoc-CH), 4.65 (dt, J¼8.6, 3.4 Hz,1H, Ser-
a-CH), 4.90 (d,
Ser-
a
-CH), 4.88 (d, J¼3.4 Hz, 1H, H-1), 5.11 (d, J¼3.4 Hz, 1H, H-10),
J¼4.0 Hz, 1H, H-1), 5.05 (d, J¼3.4 Hz, 1H, H-10), 5.19 (dd, J¼4.0,
10.9 Hz, 1H, H-3), 5.22 (d, J¼12.5 Hz, 1H, OCHPh), 5.27 (dd, J¼2.9,
10.9 Hz, 1H, H-30), 5.28 (d, J¼12.5 Hz, 1H, OCHPh), 5.41 (d, J¼2.9 Hz,
1H, H-40), 5.99 (d, J¼8.6 Hz, 1H, NH), 7.26e7.42 (m, 9H, Ar),
7.61e7.63 (m, 2H, Ar), 7.77 (d, J¼7.5 Hz, 2H, Ar); 13C NMR (100 MHz,
5.188 (dd, J¼2.6, 10.9 Hz, 1H, H-30), 5.190 (dd, J¼3.4, 11.5 Hz, 1H, H-
3), 5.22 (d, J¼12.6 Hz, 1H, OCHPh), 5.28 (d, J¼12.6 Hz, 1H, OCHPh),
5.48 (s, 1H, CHPh), 5.97 (d, J¼8.0 Hz, 1H, NH), 7.26e7.42 (m, 12H,
Ar), 7.46 (dd, J¼2.9, 6.9 Hz, 2H, Ar), 7.61 (dd, J¼4.0, 7.5 Hz, 2H, Ar),
7.75 (d, J¼7.5 Hz, 2H, Ar); 13C NMR (100 MHz, CDCl3)
d 20.88, 20.90,
CDCl3)
d 20.5, 20.6, 20.9, 21.0, 46.9, 54.3, 57.1, 57.6, 60.4, 61.4, 66.7,
46.9, 54.4, 57.1, 62.6, 66.8, 67.4, 67.67, 67.70, 68.8, 68.9, 69.5, 69.8,
70.2, 73.1, 98.2 (C-10), 99.5 (C-1), 100.6 (CHPh), 119.9, 125.1, 125.2,
126.0, 127.0, 127.1, 127.7, 128.1, 128.3, 128.4, 128.5, 129.0, 135.0,
137.3, 141.2, 143.6, 143.8, 155.9, 169.7, 169.8, 170.4; FAB-HRMS m/z
calcd for C48H50N7O15 (MþH)þ 964.3365, found 964.3365. Data for
67.3, 67.4, 67.7, 68.56, 68.61, 69.5, 70.2, 97.6 (C-10), 99.3 (C-1), 119.9,
125.1, 125.2, 127.1, 127.7, 128.4, 128.5, 128.6, 135.1, 141.15, 141.17,
143.7, 143.8, 155.9, 169.70, 169.72, 169.8, 169.9, 170.4; FAB-HRMS m/
z calcd for C45H50N7O17 (MþH)þ 960.3263, found 960.3260. Data
for
b
-anomer 20
b
: Rf 0.30 (10:1 CH2Cl2/acetone); ½a D24
þ34.8 (c
ꢃ
b
-anomer 18
b
: Rf 0.27 (1:2 hexane/AcOEt); ½a D24
þ65.2 (c 1.01,
ꢃ
0.40, CHCl3); IR (KBr) 3370, 2944, 2115, 1750, 1736, 1523, 1236,
CHCl3); IR (KBr) 3430, 2948, 2117, 1747, 1726, 1508, 1451, 1371, 1236,
1039 cmꢂ1; 1H NMR (500 MHz, CDCl3)
d 2.037 (s, 3H, CH3CO), 2.042
1052 cmꢂ1; 1H NMR (500 MHz, CDCl3)
d 2.14 (s, 3H, CH3CO), 2.16 (s,
(s, 3H, CH3CO), 2.08 (s, 3H, CH3CO), 2.20 (s, 3H, CH3CO), 2.49 (d,
J¼3.4 Hz, 1H, OH), 3.67 (dd, J¼7.7, 10.6 Hz, 1H, H-20), 3.68 (dd, J¼4.0,
10.9 Hz, 1H, H-2), 3.80 (dd, J¼6.0, 6.6 Hz, 1H, H-50), 3.84 (dd, J¼6.0,
10.0 Hz, 1H, H-60a), 3.96ꢂ4.02 (m, 3H, H-5, H-6a, H-6b), 4.05 (dd,
3H, CH3CO), 2.65 (br s, 1H, OH), 3.46 (br s, 1H, H-50), 3.67 (dd, J¼3.4,
10.9 Hz, 1H, H-2), 3.81 (dd, J¼6.0, 10.0 Hz, 1H, H-5), 3.97 (dd, J¼8.0,
10.9 Hz, 1H, H-20), 3.98 (t, J¼6.0 Hz, 1H, H-6a), 4.02 (br d, J¼12.0 Hz,
1H, H-60a), 4.03e4.08 (m, 3H, H-6b, Ser-
b
-CH ꢁ2), 4.15 (t, J¼6.9 Hz,
J¼6.6, 10.0 Hz, 1H, H-60b), 4.08e4.09 (m, 2H, Ser-
-CH ꢁ2),
b
1H, Fmoc-CH), 4.20 (m, 1H, H-4), 4.25 (d, J¼12.0 Hz, 1H, H-60b), 4.33
(d, J¼3.4 Hz, 1H, H-40), 4.35 (dd, J¼7.4, 10.9 Hz, 1H, Fmoc-CH), 4.390
(dd, J¼7.4, 10.9 Hz, 1H, Fmoc-CH), 4.394 (d, J¼8.0 Hz, 1H, H-10), 4.58
4.21e4.26 (m, 2H, H-4, Fmoc-CH), 4.33 (dd, J¼7.2, 10.6 Hz, 1H,
Fmoc-CH), 4.34 (d, J¼7.7 Hz, 1H, H-10), 4.44 (dd, J¼7.2, 10.6 Hz, 1H,
Fmoc-CH), 4.62 (dt, J¼8.4, 3.2 Hz, 1H, Ser- -CH), 4.76 (dd, J¼3.4,
a
(dt, J¼8.4, 3.2 Hz, 1H, Ser-
a
-CH), 4.72 (dd, J¼3.4, 10.9 Hz, 1H, H-30),
10.9 Hz, 1H, H-30), 4.86 (d, J¼3.4 Hz, 1H, H-1), 5.22 (d, J¼12.4 Hz, 1H,
OCHPh), 5.23 (dd, J¼3.2, 10.9 Hz, 1H, H-3), 5.27 (d, J¼12.5 Hz, 1H,
OCHPh), 5.30 (d, J¼3.4 Hz, 1H, H-40), 5.86 (d, J¼8.4 Hz, 1H, NH),
7.26e7.42 (m, 9H, Ar), 7.62 (d, J¼7.5 Hz, 2H, Ar), 7.77 (d, J¼7.5 Hz, 2H,
4.88 (d, J¼3.4 Hz, 1H, H-1), 5.19 (d, J¼12.0 Hz, 1H, OCHPh), 5.21 (dd,
J¼3.4, 10.9 Hz, 1H, H-3), 5.25 (d, J¼12.0 Hz, 1H, OCHPh), 5.50 (s, 1H,
CHPh), 5.80 (d, J¼8.4 Hz, 1H, NH), 7.30e7.40 (m, 12H, Ar), 7.47 (dd,
J¼2.9, 6.9 Hz, 2H, Ar), 7.54 (d, J¼7.4 Hz, 1H, Ar), 7.63 (d, J¼7.4 Hz, 1H,
Ar), 7.75 (dd, J¼2.9, 7.7 Hz, 2H, Ar); 13C NMR (100 MHz, CDCl3)
Ar); 13C NMR (100 MHz, CDCl3)
d 20.5, 20.58, 20.64, 21.0, 47.0, 54.4,
57.2, 60.5, 61.4, 66.2, 67.1, 67.4, 67.8, 68.9, 69.1, 70.2, 70.9, 71.1, 99.1
(C-1), 102.4 (C-10), 120.0, 125.17, 125.24, 127.1, 127.7, 128.3, 128.5,
128.6, 128.7, 135.0, 141.2, 143.7, 143.9, 155.9, 169.6, 169.8, 169.9,
d
20.9, 21.0, 46.9, 54.4, 57.2, 60.1, 66.4, 67.2, 67.3, 67.7, 68.7, 68.8,
69.1, 70.0, 72.2, 72.4, 99.1 (C-1), 100.8 (CHPh), 101.7 (C-10), 119.9,

K. Kakita et al. / Tetrahedron 68 (2012) 5005e5017
5015
170.0, 170.4; FAB-HRMS m/z calcd for C45H49N7O17Na (MþNa)þ
9.5 Hz,1H, H-6b), 4.34 (t, J¼6.9 Hz,1H, Fmoc-CH), 4.35e4.39 (m, 2H,
H-5, H-50), 4.41 (dd, J¼6.9,10.3 Hz,1H, Fmoc-CH), 4.49e4.55 (m, 3H,
982.3083, found 982.3084.
H-2, H-20, Ser-
a-CH), 4.57 (dd, J¼6.9, 10.3 Hz, 1H, Fmoc-CH), 4.88 (d,
4.5.4. O-(2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-
anosyl)-(1/6)-(2-acetamido-3,4-di-O-acetyl-2-deoxy-
actopyranosyl)-N-(9-fluorenylmethoxycarbonyl)- -serine benzyl ester
(21). Pyridine (0.039 mL, 0.48 mmol) was added to a solution of
disaccharide 20 (46.0 mg, 0.048 mmol) in CH2Cl2 (1 mL) at room
a
-
D
-galactopyr-
J¼3.4 Hz, 1H, H-1), 5.00 (d, J¼4.0 Hz, 1H, H-10), 5.24 (dd, J¼2.9,
11.5 Hz, 2H, H-3, H-30), 5.42 (d, J¼2.9 Hz, 1H, H-4), 5.55 (d, J¼2.9 Hz,
1H, H-40), 7.39e7.43 (m, 2H, Ar), 7.47 (t, J¼7.4 Hz, 2H, Ar), 7.78 (t,
J¼6.6 Hz, 2H, Ar), 7.88 (d, J¼7.4 Hz, 2H, Ar); 13C NMR (100 MHz,
a-D
-gal-
L
a
CD3OD) d 20.5, 20.57, 20.60, 20.66, 20.68, 22.6, 22.7, 55.9, 63.0, 67.2,
temperature followed by addition of acetic anhydride (0.009 mL,
0.10 mmol). After stirring at room temperature for 30 min, the
reaction was quenched with crushed ice, followed by stirring for
15 min. The mixture was extracted with AcOEt (15 mL). The or-
ganic layer was washed with 10% HCl (3ꢁ4 mL) and brine (4 mL),
and dried over anhydrous Na2SO4. Filtration and evaporation in
vacuo furnished the crude product (55.0 mg). The crude mixture
thus obtained was suspended in THF/Ac2O/AcOH (3:2:1, 1 mL), and
Zn powder (50 mg) was added. After stirring at room temperature
for 5 min, the mixture was filtered through a Celite pad. The fil-
trate was poured into a two-layer mixture of AcOEt (3 mL) and
saturated aqueous NaHCO3 (4 mL), and the whole mixture was
extracted with AcOEt (12 mL). The organic extract was succes-
sively washed with saturated aqueous NaHCO3 (2ꢁ4 mL) and
brine (4 mL), and dried over anhydrous Na2SO4. The filtrate was
concentrated and purified by column chromatography (silica gel
2 g, 40:1 CHCl3/MeOH) to give acetamide 21 (40.0 mg, 81%) as
68.0, 68.1, 68.7, 69.0, 69.7, 70.8, 99.0 (C-10), 100.5 (C-1), 121.0, 126.2,
126.3, 126.4, 128.2, 128.3, 128.9, 129.2, 129.9, 142.6, 145.2, 145.2,
158.3, 171.9, 172.0, 172.08, 172.10, 173.4, 173.6, 173.8.
Acknowledgements
This research was supported, in part, by a Grant-in-Aid for Sci-
entific Research on Innovative Areas ‘Organic Synthesis Based on
Reaction Integration’ (No. 2105) from the Ministry of Education,
Culture, Sports, Science and Technology, Japan. We thank Ms.
S. Oka, and M. Kiuchi of the Center for Instrumental Analysis at
Hokkaido University for technical assistance in the MS and ele-
mental analyses.
References and notes
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1770e1791.
a colorless film: Rf 0.65 (9:1 CHCl3/MeOH); ½a D22
þ93.8 (c 0.93,
ꢃ
CHCl3); IR (KBr) 3372, 3066, 2955, 1748, 1676, 1529, 1373, 1242,
1048 cmꢂ1 1H NMR (500 MHz, CDCl3)
; d 1.94 (s, 3H, CH3CO), 1.96
(s, 6H, CH3CO), 2.01 (s, 3H, CH3CO), 2.03 (s, 3H, CH3CO), 2.16 (s, 6H,
CH3CO), 3.19 (dd, J¼2.7, 8.6 Hz, 1H, H-5), 3.71e3.78 (m, 2H, H-6a,
H-6b), 3.902 (dd, J¼2.3 Hz, 1H, H-50), 3.904 (dd, J¼7.7, 10.4 Hz, 1H,
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Ser-
b
-CH), 3.99 (dd, J¼7.7, 10.4 Hz, 1H, Ser-
b-CH), 4.16e4.25 (m,
3H, Fmoc-CH, H-60a, H-60b), 4.34 (dd, J¼7.0, 10.6 Hz, 1H, Fmoc-CH),
4.49 (dd, J¼7.0, 10.6 Hz, 1H, Fmoc-CH), 4.55 (ddd, J¼3.6, 9.5,
11.3 Hz, 1H, H-2), 4.66 (ddd, J¼3.6, 10.0, 10.8 Hz, 1H, H-20), 4.76 (m,
1H, Ser-
a
-CH), 4.81 (d, J¼3.6 Hz, 1H, H-10), 4.82 (d, J¼3.6 Hz, 1H, H-
1), 4.93 (dd, J¼3.0, 11.3 Hz, 1H, H-3), 5.12 (d, J¼3.0 Hz, 1H, H-4),
5.18 (d, J¼11.8 Hz, 1H, OCHPh), 5.27 (dd, J¼2.8, 10.8 Hz, 1H, H-30),
5.30 (d, J¼11.8 Hz, 1H, OCHPh), 5.37 (d, J¼2.8 Hz, 1H, H-40), 5.60 (d,
J¼9.5 Hz, 1H, NHAc), 6.48 (d, J¼9.1 Hz, 1H, NHFmoc), 6.59 (d,
J¼10.0 Hz, 1H, NHAc0), 7.28e7.43 (m, 9H, Ar), 7.63 (d, J¼7.2 Hz, 2H,
6. The chemical synthesis of TN-antigen structure was first described by Kaifu and
Osawa, in which glycosidation of 3,4,6-tri-O-acetyl-2-deoxy-2-(2,4-
dinitroanilino)-
a
-D
-galactopyranosyl bromide with N-tosyl-L-serine methyl
ester provided the corresponding
a
- and -galactosides in 53% and 11% yields,
b
respectively Kaifu, R.; Osawa, T. Carbohydr. Res. 1977, 58, 235e239.
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Ar), 7.77 (d, J¼7.7 Hz, 2H, Ar); 13C NMR (100 MHz, CDCl3)
d 20.5,
20.7, 20.8, 23.0, 23.3, 29.6, 47.0, 47.2, 47.8, 53.7, 60.4, 61.2, 65.7,
66.8, 67.3, 67.7, 67.8, 68.0, 68.26, 68.29, 69.1, 97.4 (C-10), 98.3 (C-1),
120.0, 125.0, 125.1, 127.0, 127.1, 127.79, 127.81, 128.5, 128.8, 128.9,
134.6, 141.2, 141.3, 143.6, 155.8, 170.1, 170.30, 170.34, 170.5, 170.7,
171.2, 171.5; FAB-HRMS m/z calcd for C51H60N3O20 (MþH)þ
1034.3770, found 1034.3750.
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anosyl)-(1/6)-(2-acetamido-3,4-di-O-acetyl-2-deoxy-
actopyranosyl)-N-(9-fluorenylmethoxycarbonyl)- -serine (22). 10%
a
-
D
-galactopyr-
a-D
-gal-
L
9. (a) Grundler, G.; Schmidt, R. R. Liebigs Ann. Chem. 1984, 1826e1847; (b) Toyo-
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Pd/C (30.0 mg) was added to a solution of glycoside 21 (32.0 mg,
0.031 mmol) in EtOH (1.0 mL) under an argon atmosphere, and the
mixture was vigorously stirred under 1 atm of hydrogen for 15 min.
The catalyst was filtered through a Celite pad, and the filtrate was
evaporated in vacuo. Purification of the crude product (29.5 mg) by
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€
2000, 8, 1017e1025; (g) Gotze, S.; Fitzner, R.; Kunz, H. Synlett 2009,
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0.23 (4:1 CHCl3/MeOH); mp 146.0e147.2 ꢀC; ½a D22
þ89.4 (c 0.90,
ꢃ
ꢂ
a glycosyl donor, see: Adinolfi, M.; Iadonisi, A.; Ravida, A.; Valerio, S. Tetrahe-
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CHCl3); IR (KBr) 3372, 2933, 1748, 1664, 1533, 1373, 1242, 1048, 762,
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742 cmꢂ1; 1H NMR (500 MHz, CD3OD)
d 1.95 (s, 3H, CH3CO), 1.98 (s,
3H, CH3CO), 2.00 (s, 3H, CH3CO), 2.01 (s, 3H, CH3CO), 2.02 (s, 3H,
CH3CO), 2.16 (s, 3H, CH3CO), 2.22 (s, 3H, CH3CO), 3.46 (dd, J¼5.2,
9.5 Hz, 1H, H-60a), 3.86 (dd, J¼7.7, 9.5 Hz, 1H, H-60b), 3.93 (dd, J¼6.9,
10.9 Hz, 1H, H-6a), 4.02e4.10 (m, 2H, Ser-
b
-CH ꢁ2), 4.14 (dd, J¼6.0,

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a
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D
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/
b
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with C1-O-hemiacetal nucleophiles to form
a
-O-glycosyl serine conjugates in
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a
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a
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¼94:6) as

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