Synthesis of sulfated phenyl 2-acetamido-2-deoxy-D-galactopyranosides. 4-O-Sulfated phenyl 2-acetamido-2-deoxy-β-D-galactopyranoside is a competitive acceptor that decreases sulfation of chondroitin sulfate by N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase

Article abstract of DOI:10.1016/j.carres.2005.06.010

We have previously cloned N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST), which transfers sulfate from 3′-phosphoadenosine 5′-phosphosulfate (PAPS) to the C-6 hydroxyl group of the GalNAc 4-sulfate residue of chondroitin sulfate A and forms chondroitin sulfate E containing GlcA-GalNAc(4,6-SO₄) repeating units. To investigate the function of chondroitin sulfate E, the development of specific inhibitors of GalNAc4S-6ST is important. Because GalNAc4S-6ST requires a sulfate group attached to the C-4 hydroxyl group of the GalNAc residue as the acceptor, the sulfated GalNAc residue is expected to interact with GalNAc4S-6ST and affect its activity. In this study, we synthesized phenyl α- or -β-2-acetamido-2-deoxy-β-D-galactopyranosides containing a sulfate group at the C-3, C-4, or C-6 hydroxyl groups and examined their inhibitory activity against recombinant GalNAc4S-6ST. We found that phenyl β-GalNAc(4SO₄) inhibits GalNAc4S-6ST competitively and also serves as an acceptor. The sulfated product derived from phenyl β-GalNAc(4SO₄) was identical to phenyl β-GalNAc(4,6-SO ₄). These observations indicate that derivatives of β-D-GalNAc(4SO₄) are possible specific inhibitors of GalNAc4S-6ST.

Full text of DOI:10.1016/j.carres.2005.06.010

Carbohydrate
RESEARCH
Carbohydrate Research 340 (2005) 1983–1996
Synthesis of sulfated phenyl
2-acetamido-2-deoxy-D-galactopyranosides. 4-O-Sulfated phenyl
2-acetamido-2-deoxy-b-D-galactopyranoside is a competitive
acceptor that decreases sulfation of chondroitin sulfate by
N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase
Toshihiko Sawada,a, Sonoko Fujii,a Hirofumi Nakano,a Shiori Ohtake,a,b
Koji Kimatab and Osami Habuchia,*
aDepartment of Chemistry, Aichi University of Education, Igaya-cho, Kariya, Aichi 448-8542, Japan
bInstitute for Molecular Science of Medicine, Aichi Medical University, Nagakute, Aichi 480-1195, Japan
Received 12 March 2005; received in revised form 14 May 2005; accepted 8 June 2005
Available online 15 July 2005
Abstract—We have previously cloned N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST), which transfers sul-
fate from 30-phosphoadenosine 50-phosphosulfate (PAPS) to the C-6 hydroxyl group of the GalNAc 4-sulfate residue of chondroitin
sulfate A and forms chondroitin sulfate E containing GlcA-GalNAc(4,6-SO4) repeating units. To investigate the function of chon-
droitin sulfate E, the development of specific inhibitors of GalNAc4S-6ST is important. Because GalNAc4S-6ST requires a sulfate
group attached to the C-4 hydroxyl group of the GalNAc residue as the acceptor, the sulfated GalNAc residue is expected to interact
with GalNAc4S-6ST and affect its activity. In this study, we synthesized phenyl a- or -b-2-acetamido-2-deoxy-b-D-galactopyrano-
sides containing a sulfate group at the C-3, C-4, or C-6 hydroxyl groups and examined their inhibitory activity against recombinant
GalNAc4S-6ST. We found that phenyl b-GalNAc(4SO4) inhibits GalNAc4S-6ST competitively and also serves as an acceptor. The
sulfated product derived from phenyl b-GalNAc(4SO4) was identical to phenyl b-GalNAc(4,6-SO4). These observations indicate
that derivatives of b-D-GalNAc(4SO4) are possible specific inhibitors of GalNAc4S-6ST.
ꢀ 2005 Elsevier Ltd. All rights reserved.
Keywords: GalNAc4S-6ST; Chondroitin sulfate; Sulfotransferase; Inhibitor; Sulfated 2-acetamido-2-deoxy-b-D-galactopyranose; Synthesis
1. Introduction
bearing sulfate moieties on various hydroxyl groups of
the sugar residues. CS-E is an isomer in which the C-4
and C-6 hydroxyl groups of the GalNAc residue are
sulfated (GlcA-GalNAc(4,6-SO4)). CS-E is present in
Chondroitin sulfate is a glycosaminoglycan composed of
b-D-GlcA-(1!3)-b-D-GalNAc-(1!4) repeating units
Abbreviations: CS-E, chondroitin sulfate E; CS-A, chondroitin sulfate A; GalNAc4S-6ST, N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase;
C4ST, chondroitin 4-sulfotransferase; C6ST, chondroitin 6-sulfotransferase; GalNAc(4SO4), 2-acetamido-2-deoxy-4-O-sulfonato-D-galactopyra-
nose; GalNAc(6SO4), 2-acetamido-2-deoxy-6-O-sulfonato-D-galactopyranose; GalNAc(4,6-SO4), 2-acetamido-2-deoxy-4,6-di-O-sulfonato-D-galac-
topyranose; GlcA, D-glucuronic acid; PAPS, 30-phosphoadenosine 50-phosphosulfate; DDi-0S, 2-acetamido-2-deoxy-3-O-(b-D-gluco-4-
enepyranosyluronic acid)-D-galactopyranose; DDi-6S, 2-acetamido-2-deoxy-3-O-(4-deoxy-b-L-threo-hex-4-enopyranosyluronic acid)-6-O-sulfonato-
D-galactopyranose; DDi-4S, 2-acetamido-2-deoxy-3-O-(4-deoxy-b-L-threo-hex-4-enopyranosyluronic acid)-4-O-sulfonato-D-galactopyranose; DDi-
diSD, 2-acetamido-2-deoxy-3-O-(4-deoxy-b-L-threo-hex-4-enopyranosyluronic acid)-6-O-sulfonato-D-galactopyranose; DDi-diSE, 2-acetamido-2-
deoxy-3-O-(4-deoxy-b-L-threo-hex-4-enopyranosyluronic acid)-4,6-di-O-sulfonato-D-galactopyranose.
*
Corresponding author. Tel.: +81 (0)566 26 2642; fax: +81 (0)566 26 2649; e-mail: ohabuchi@auecc.aichi-edu.ac.jp
Present address: Department of Applied Bio-organic Chemistry, Gifu University, Gifu 501-1193, Japan.
0008-6215/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2005.06.010

1984
T. Sawada et al. / Carbohydrate Research 340 (2005) 1983–1996
various cells such as mast cells,1–4 polymorphonuclear
granulocytes,5,6 monocytes,7–10 and macrophages11 and
is thought to be involved in the immunological response
of mast cells,12 the binding of platelet factor 4 to neutro-
phils,6 the regulation of procoagulant activity,10 and the
binding of lipoprotein lipase to macrophages.11 CS-E
was reported to inhibit the binding of versican to L-
selectin or chemokines, and to bind L-selectin or chemo-
kines directly.13,14 CS-E, as well as a tetrasaccharide
containing GlcA-GalNAc(4,6-SO4) units, was reported
to promote neurite outgrowth.15,16 The presence of non-
reducing terminal GalNAc(4,6-SO4) residues has been
reported in chondroitin sulfate attached to aggrecan17–20
and thrombomodulin.21 Proportions of the nonreducing
terminal GalNAc(4,6-SO4) residue of aggrecan was
found to be decreased in human osteoarthritis.22
GalNAc4S-6ST transfers sulfate to the C-6 hydroxyl
group of GalNAc(4SO4) residues of chondroitin sul-
fate.23,24 We identified human GalNAc4S-6ST cDNA
on the basis of the amino acid sequences of purified
squid GalNAc4S-6ST.25 Recombinant human Gal-
NAc4S-6ST transfers sulfate to the C-6 hydroxyl group
of GalNAc(4SO4) residues located at the nonreducing
terminal and internal repeating disaccharide region.25
We found that a unique nonreducing terminal structure
is present in CS-A, and GalNAc4S-6ST should be in-
volved in the production of the nonreducing terminal
structure.26 To obtain information about the function
of GalNAc(4,6-SO4) residues contained in CS-E or lo-
cated at the nonreducing terminal of CS, it is important
to regulate the activity of GalNAc4S-6ST. The enzyme
requires a sulfate group attached to the 4-hydroxyl
group of the GalNAc residue as the acceptor,25 suggest-
ing that the sulfated GalNAc residue interacts with
GalNAc4S-6ST and affects its enzymatic activity. In this
study, we synthesized the a- or b-phenyl glycoside
derivatives of 2-acetamido-2-deoxy-D-galactopyranose,
which were sulfated at the C-3, C-4, or C-6 hydroxyl
groups and examined their inhibitory activity against
recombinant GalNAc4S-6ST. We found that phenyl
b-GalNAc(4SO4) inhibited GalNAc4S-6ST activity
competitively.
ous aglycons from a readily available starting material.
For the synthesis of GalNAc(6SO4) derivatives, the
highly reactive primary 6-hydroxyl group of GalNAc
was sulfated without protection.
Phenyl a-GalNAc(4SO4) (7) was synthesized as de-
picted in Scheme 1. The reaction of fully acetylated
galactosamine 1 with phenol in the presence of ZnCl2
and activated CaSO4 gave phenyl glycoside 2 with a-
selectivity. Triol 4 was obtained from 2 after reaction
with sodium methoxide in methanol in good yield.
Galactopyranoside 5 was synthesized by the selective
3,6-di-O-pivaloylation27 of 4. Target 7 was then ob-
tained by treatment of 5 with sulfur trioxide–pyridine
complex in pyridine, followed by de-O-pivaloylation
using sodium methoxide in methanol.
As a starting material for synthesis of phenyl b-Gal-
NAc(4SO4) 15, thioglycoside 8 was prepared from D-glu-
cosamine hydrochloride by OgawaÕs method (Scheme
2).28 To invert the 4-position of GlcNPhth, 8 was sub-
jected to mesylation followed by SN2 reaction by potas-
sium acetate in the presence of 18-crown-6 ether.
Thioglycoside 10 thus obtained was converted to galac-
tosyl fluoride 11 in good yield by treating with DAST
and NBS in CH2Cl2. b-Selective glycosylation of 11 with
phenol was carried out by activation of glycosyl fluoride
with the Cp2HfCl2–AgClO4 promoter system. The
phthalimido group of 12 was converted to an acetamido
group, and then the acetyl group at the 4-position of
the GalNAc residue was removed to give compound
13. The 4-sulfated target compound 15 was obtained
by sulfation with sulfur trioxide followed by removal of
the benzyl groups with Pd–C catalyzed hydrogenation.
The GalNAc(6SO4) derivatives 16 and 18 were pre-
pared from the corresponding triol 4 and 17, respec-
tively, by sulfation with SO3–pyr (Scheme 1).
The GalNAc(3SO4) derivatives 21 and 24 were pre-
pared from triol 4 and 17, respectively, by protection
of the 4- and 6-hydroxyl groups as benzylidene acetals
19 and 22, followed by sulfation and removal of benzyl-
idene group (Scheme 1).
Phenyl b-GalNAc(4,6-SO4) 28 was synthesized from
benzylidene acetal 22 (Scheme 3), by first pivaloylation
of the 3-hydroxyl group to give 25, which was then sub-
jected to removal of benzylidene group, sulfation, and
deprotection to give phenyl b-GalNAc(4,6-SO4) 28.
2. Results and discussion
2.1. Synthesis
1
In the H NMR spectrum of the products, the signal
for the hydrogens attached to the carbon bearing the
O-sulfonate group were shifted downfield relative to
the corresponding signals in 4 or 17. For example,
the H-4 signal in the spectrum for phenyl b-Gal-
NAc(4SO4) 15 was shifted by 0.8 ppm relative to the
corresponding signal in the spectrum of 17. Similarly,
the downfield shift of the H-3 signal for the a-Gal-
NAc(3SO4) derivative 21 by 0.65 ppm relative to the
corresponding signal for 4 indicates that 21 has O-sul-
fate group at C-3.
Strategies with minimal synthetic steps from D-galactos-
amine were designed for the synthesis of the Gal-
NAc(4SO4) and GalNAc(3SO4) derivatives, in which
nonsulfated positions of the GalNAc residues were
selectively protected. For the synthesis of the b-Gal-
NAc(4SO4) derivative, D-glucosamine was used as the
starting material. This synthetic route is applicable for
the synthesis of b-GalNAc(4SO4) derivatives with vari-

T. Sawada et al. / Carbohydrate Research 340 (2005) 1983–1996
1985
OH
OPiv
OPiv
OH
HO
HO
HO
NaO3SO
PivO
O3SO
HO
NaOMe
MeOH
O
O
O
O
PivCl, pyr, CH2Cl2
SO3-pyr
pyr
PivO
AcHN
AcHN
82%
AcHN
AcHN
29%
OPh
95%
OPh
OPh
OPh
7
5
6 71%
4
NaOMe
OAc
OAc
OAc
O
AcO
AcO
AcO
AcO
AcO
O
O
ZnCl2
+
AcO
OAc
OPh
PhOH, ClCH2CH2Cl
AcHN
AcHN
AcHN
OPh
43%
CaSO4, ∆
1
1.8%
3
2
NaOMe
MeOH
OH
OSO3
O
HO
HO
HO
SO3-pyr
pyr
O
OPh
OPh
HO
AcHN
AcHN
α
β
4
17
α
β
95%
88%
16
18
6%
9%
Ph
Ph
OMe
H
OMe
OH
OH
H2, Pd-C
SO3-pyr
pyr
O
O
O
O
O
O3SO
OPh
EtOH-H2O
DMF, CSA, DMF
O
O
NHAc
HO
NaO3SO
OPh
OPh
NHAc
NHAc
α 61%
β 51%
α 82%
α 26%
β 54%
19
22
21
24
20
β 85%
23
Scheme 1. Synthetic route to 7, 16, 18, 21, and 24 from 1.
2.2. Inhibition of GalNAc4S-6ST
noglycan was digested with chondroitinase ACII and
the degradation products were separated by SAX-
HPLC. As shown in Figure 3, the proportion of the
radioactivity recovered in DDi-diSE, which was derived
from the internal repeating disaccharide units, and the
radioactivity recovered in GalNAc(4,6-SO4) and b-
GalNAc(4,6-SO4)-(1!4)-b-GlcA(2SO4)-(1!3)-GalNAc-
(6SO4), which were derived from the nonreducing termi-
nal, was not altered in the presence of the inhibitor. This
indicates that sulfation of the 6-hydroxyl group of non-
reducing terminal GalNAc(4SO4) residue and internal
GalNAc(4SO4) residues was inhibited equally by phenyl
b-GalNAc(4SO4) (15). GalNAc4S-6ST was found to re-
quire the presence of sulfate moiety attached to the C-4
hydroxyl group of GalNAc residue; therefore, phenyl b-
GalNAc(4SO4) (15) may be recognized by the acceptor
binding domain of GalNAc4S-6ST. If this is the case,
phenyl b-GalNAc(4SO4) (15) may act as a competitive
inhibitor. To clarify the mode of inhibition by phenyl
b-GalNAc(4SO4) (15), the effect of the concentration
of the acceptor and the inhibitor was determined. As
Compounds 7, 15, 16, 18, 21, and 24 were tested as
inhibitors for GalNAc4S-6ST activity. Dose dependent
inhibition of GalNAc4S-6ST was observed for com-
pound 15 (phenyl b-GalNAc(4SO4)); in the presence of
5 mM of the inhibitor, the GalNAc4S-6ST activity de-
creased to 24% of the control (Fig. 1). Compound 16
(phenyl a-GalNAc(6SO4)) was shown to inhibit rather
weakly. GalNAc4S-6ST is thus thought to recognize
not only the position of sulfate group on the GalNAc
residue but also configuration of the anomeric carbon
of the sugar residue. Neither C4ST nor C6ST was inhib-
ited by compound 15 (Fig. 2); indicating that the
observed inhibitory activity of 15 is specific to Gal-
NAc4S-6ST. GalNAc4S-6ST transfers sulfate to the C-
6 hydroxyl group of GalNAc(4SO4) residues located at
the nonreducing terminal and in the internal repeating
units.25 We examined whether the degree of inhibition
by phenyl b-GalNAc(4SO4) (15) depends on the location
of GalNAc(4SO4) residue. Thus, 35S-labeled glycosami-

1986
T. Sawada et al. / Carbohydrate Research 340 (2005) 1983–1996
OBn
OBn
OBn
AcOK,
AcO
BnO
18-crown-6
MsCl
O
O
O
HO
BnO
MsO
BnO
SPh
SPh
NPhth
88%
SPh
NPhth
10 70%
DMF, ∆
pyr, CH2Cl2
NPhth
8
9
DAST, NBS
CH2Cl2,
-15~25 °C
OBn
OBn
AcO
BnO
HO
OBn
1. NH2NH2, EtOH, ∆
2. Ac2O, pyr
AcO
BnO
O
PhOH, Cp2HfCl2, AgClO4
O
O
OPh
BnO
OPh
NHAc
38%
F
3. NaOMe, MeOH
MS4A, CH2Cl2, -78 ~ -50 °C
NPhth
12 68%
NPhth
13
65%
11
SO3-pyr
pyr
OH
OBn
O3SO
HO
15
NaO3SO
BnO
H2, Pd-C
O
O
OPh
NHAc
47%
OPh
NHAc
93%
EtOH-H2O
14
Scheme 2. Synthetic route to 15 from 8.
Ph
Ph
HO
OH
H2, Pd-C
EtOH-H2O
PivCl
pyr
O
O
O
O
PivO
O
OPh
NHAc
O
O
HO
PivO
OPh
NHAc
OPh
NHAc
66%
22
25
26 64%
SO3-pyr
pyr
O3SO
HO
NaO3SO
PivO
OSO3
OSO3Na
O
NaOMe
MeOH
O
OPh
OPh
NHAc
NHAc
27
100% (2 steps)
28
Scheme 3. Synthetic route to 28 from 22.
shown in Figure 4, it is evident that phenyl b-Gal-
NAc(4SO4) (15) inhibited GalNAc4S-6ST competi-
tively; the Km for CS-A was 0.038 mM and the Ki for
phenyl b-GalNAc(4SO4) (15) was 0.98 mM. When phe-
nyl b-GalNAc(4SO4) (15) was added to the reaction
mixture instead of CS-A, a sulfated product was de-
tected by paper electrophoresis (Fig. 5B). This material
behaved identically with phenyl b-GalNAc(4,6-SO4)
(28) in the SAX-HPLC (Fig. 6), indicating that sulfate
was transferred to the C-6 hydroxyl group of the Gal-
NAc(4SO4) residue of phenyl b-GalNAc(4SO4) (15).
These observations indicate that phenyl b-Gal-
NAc(4SO4) served not only as a competitive inhibitor,
but also as an acceptor for GalNAc4S-6ST. Phenyl b-
GalNAc(4SO4) (15) is thus a competitive acceptor that
decreases sulfation of chondroitin sulfate by N-acetylga-
lactosamine 4-sulfate 6-O-sulfotransferase. Unlike phe-
nyl b-GalNAc(4SO4) (15), phenyl b-GalNAc(4,6-SO4)

T. Sawada et al. / Carbohydrate Research 340 (2005) 1983–1996
1987
150.0
100.0
50.0
0.0
3000
34
5
6
7
A
8
400
200
0
2000
1
2
1000
0
10
20
30
40
50
3000
2000
1000
0
B
Figure 1. Inhibition of GalNAc4S-6ST activity by various sulfated
phenyl GalNAc derivatives. The sulfotransferase reaction was carried
out as described in Section 3.3. Sulfated phenyl GalNAc derivatives
were added to the reaction mixtures at a final concentration of 1.0 mM
(open bar), 2.5 mM (hatched bar), or 5.0 mM (closed bar). Values are
averages of three determinations and the standard deviation is
indicated by perpendicular lines.
10
20
30
40
50
Retention time (min)
Figure 3. Effect of phenyl b-GalNAc(4SO4) (15) on the sulfation of
internal and nonreducing terminal GalNAc(4SO4) residues. The
sulfotransferase reaction with GalNAc4S-6ST was carried out as
described in Section 3.3 in the absence (A) or presence (B) of 5 mM
phenyl b-GalNAc(4SO4) (15). The 35S-labeled glycosaminoglycans
were digested with chondroitinase ACII, and subjected to SAX-HPLC.
The arrows indicate the elution position of GalNAc(6SO4) (arrow 1);
GalNAc(4SO4) (arrow 2); DDi-6S (arrow 3); DDi-4S (arrow 4);
GalNAc(4,6-SO4) (arrow 5); DDi-diSD (arrow 6); DDi-diSE (arrow
7); and b-GalNAc(4,6-SO4)-(1!4)-b-GlcA(2SO4)-(1!3)-GalNAc-
(6SO4) (arrow 8).
150.0
100.0
50.0
0.0
0.8
[ I ] = 5.0 mM
0.6
C4ST
C6ST
[ I ] = 2.5 mM
0.4
Figure 2. Effect of phenyl b-GalNAc(4SO4) (15) on the activities of
C4ST and C6ST. The sulfotransferase reaction was carried out as
described in Section 3.3. Phenyl b-GalNAc(4SO4) (15) was added to
the reaction mixtures at a final concentration of 1.0 mM (open bar),
2.5 mM (hatched bar), or 5.0 mM (closed bar). Values are averages
of three determinations and the standard deviation is indicated by
perpendicular lines.
0.2
[ I ] = 0 mM
0
0.0
1.0
2.0
3.0
(1/[S]) x 10-4 (M-1
)
Figure 4. Competitive inhibition of GalNAc4S-6ST by phenyl b-
GalNAc(4SO4) (15). The GalNAc4S-6ST activity was determined at
the varying concentration of CS-A and phenyl b-GalNAc(4SO4) (15).
Data are expressed as a Lineweaver–Burk plot.
(28) did not inhibit GalNAc4S-6ST activity at all under
the same conditions (data not shown), suggesting that
the product of the enzymatic reaction with GalNAc4S-
6ST may not have detectable affinity for the active site
of the enzyme.
In this letter, we found that phenyl b-GalNAc(4SO4)
(15) inhibited sulfation of CS-A by GalNAc4S-6ST in
vitro. Membrane permeable glycosides were found to
act as inhibitors of glycosyl transferases in the living
cells.29,30 Therefore, it is possible that b-GalNAc(4SO4)
derivatives containing various hydrophobic might be
able to inhibit CS-E synthesis in living cells.
3. Experimental
3.1. General methods
Structures of synthetic compounds were confirmed by
1H NMR, 13C NMR, and two-dimensional NMR
1
(COSY, HMQC, HMBC). H and 13C NMR spectra

1988
T. Sawada et al. / Carbohydrate Research 340 (2005) 1983–1996
2.0
1.5
1.0
0.5
ionization mass spectra (ESI) were obtained on a
LCQ (Thermoquest) or
spectrometer.
A
SO
4
a
LCT (Micromass)
3.2. Preparation of the recombinant human GalNAc4S-
6ST, C4ST, and C6ST
Recombinant GalNAc4S-6ST (EC 2.8.2.-), C4ST (EC
2.8.2.5), and C6ST (EC 2.8.2.17) were expressed as fu-
sion proteins with FLAG peptide in COS-7 cells, and
were affinity purified as described previously.25,31
0
0
10
20
30
2.0
B
1.5
1.0
0.5
0
3.3. Assay of sulfotransferase activity
Sulfotransferase activities of GalNAc4S-6ST, C4ST,
and C6ST were assayed by the methods described previ-
ously.25,32,33 The standard assay mixture for GalNAc4S-
6ST contained, in a final volume of 50 lL, 2.5 lmol of
imidazole–HCl, pH 6.8, 0.5 lmol of CaCl2, 1 lmol of re-
duced glutathione, 25 nmol (as D-galactosamine) of CS-
A, 50 pmol of [35S]PAPS (about 5.0 · 105 cpm), the
inhibitors and the enzyme. To determine the activity to-
ward phenyl b-GalNAc(4SO4) (15), CS-A was replaced
with 25 nmol of compound 15. The standard reaction
mixture for C4ST or C6ST contained 2.5 lmol of imid-
azole–HCl, pH 6.8, 1.25 lg of protamine chloride,
0.1 lmol dithiothreitol, 25 nmol (as D-galactosamine)
chondroitin, 50 pmol [35S]PAPS (about 5.0 · 105 cpm),
and enzyme in a final volume of 50 lL. The reaction
mixtures were incubated at 37 ꢁC for 20 min and the
reaction was stopped by immersing the reaction tubes
in a boiling waterbath for 1 min. After the reaction
was stopped, 35S-labeled glycosaminoglycans were iso-
lated by the precipitation with ethanol followed by gel
chromatography with a Fast Desalting Column as de-
scribed previously,33 and the radioactivity was deter-
mined. When phenyl b-GalNAc(4SO4) (15) was used
as the acceptor, the reaction was stopped by the addition
of 30 lL 0.25 M HCl and the mixture was then incu-
bated for an additional 60 min at 37 ꢁC to hydrolyze
the remaining [35S]PAPS. Under these hydrolysis condi-
tions, sulfated products formed in the assay were com-
pletely stable. Aliquots of the reaction mixtures were
applied to paper electrophoresis in buffer B as described
below, and the 35S-labeled products were separated from
35SO4.
0
10
20
30
Segment No.
Figure 5. Sulfation of phenyl b-GalNAc(4SO4) (15) by GalNAc4S-
6ST. The sulfotransferase reaction was carried out as described in
Section 3.3 using phenyl b-GalNAc(4SO4) as the acceptor instead of
CS-A. The reaction mixtures without acceptor (A) or with acceptor (B)
were subjected to the paper electrophoresis in buffer B after degrada-
tion of [35S]PAPS by mild acid hydrolysis. The fractions indicated by a
bar in (B) were used for further analysis.
Figure 6. HPLC separation of the reaction product derived from
phenyl b-GalNAc(4SO4) (15) after the reaction with GalNAc4S-6ST.
(A) Elution pattern of phenyl b-GalNAc(4,6-SO4) (28). (B) Elution
pattern of the reaction product derived from phenyl b-GalNAc(4SO4)
(15) after the reaction with GalNAc4S-6ST. The standards were the
same as those described under the legend for Figure 3. Arrow a in (A)
indicates the elution position of phenyl b-GalNAc(4SO4) (15).
3.4. Superdex 30 chromatography, SAX-HPLC, and
paper electrophoresis
A Superdex 30 16/60 column was equilibrated with
0.2 M NH4HCO3, and run at a flow rate of 2 mL/min.
One-milliliter fractions were collected. SAX-HPLC was
carried out using a Whatman Partisil-10 SAX column
(4.6 mm · 25 cm) equilibrated with 5 mM KH2PO4.
The column was developed with 5 mM KH2PO4 for
were recorded with a JEOL LA-400 spectrometer
operating at 399.65 and 100.40 MHz, respectively.
Chemical shifts were referenced to TMS. Electrospray

T. Sawada et al. / Carbohydrate Research 340 (2005) 1983–1996
1989
10 min followed by a linear gradient from 5 to 500 mM
KH2PO4. Fractions (0.5 mL) were collected at a flow
rate of 1 mL/min and a column temperature of 40 ꢁC.
Disaccharide and monosaccharide standards for SAX-
HPLC were obtained from commercial sources. The
b-GalNAc(4,6-SO4)-(1!4)-b-GlcA(2SO4)-(1!3)-Gal-
NAc(6SO4) standard was prepared as described.26 Paper
electrophoresis was carried out on Toyo No. 1 paper
(20 cm · 60 cm) in buffer A (30 mM NH4HCO3) or on
Whatman No. 3 paper (2.5 cm · 57 cm) in buffer B
(50 mM ammonium acetate, pH 5.0) at 30 V/cm for
40 min.
157.07 (s, arom. C), 170.25 (s, 2-COCH3), 170.39 (s,
COCH3), 170.43 (s, COCH3), and 170.45 (s, COCH3).
3.7. Phenyl 2-acetamido-2-deoxy-a-D-galactopyranoside
(4)
A solution of 2 (827 mg, 1.95 mmol) in dry CH3OH
(30 mL) was treated with 1 M methanolic NaOCH3
(1.95 mL) for 40 min at room temperature. The mixture
was neutralized with Amberlite IRC-50 H+ resin, and
concentrated under reduced pressure to give
4
(552 mg, 95%) as colorless crystals. 1H NMR (CD3OD):
d 1.99 (s, 3H, NHCOCH3), 3.67 (dd, 1H, J6a,6b 11.2,
J5,6a 6.1 Hz, H-6a), 3.71 (dd, 1H, J5,6b 6.1 Hz, H-6b),
3.94 (t, 1H, H-5), 3.98 (d, 1H, J3,4 3.2 Hz, H-4), 4.00
(dd, 1H, J2,3 10.4 Hz, H-3), 4.41 (dd, 1H, J1,2 3.6 Hz,
H-2), 5.49 (d, 1H, H-1), 6.97–7.01 (m, 1H, arom. CH),
7.09–7.12 (m, 2H, arom. CH), 7.24–7.29 (m, 2H, arom.
CH); 13C NMR (CD3OD): d 22.57 (q, CH3), 51.59 (d, C-
2), 62.47 (t, C-6), 69.43 (d, C-3), 70.12 (d, C-4), 73.20 (d,
C-5), 98.37 (d, C-1), 118.26 (d, arom. CH), 123.55 (d,
arom. CH), 130.48 (d, arom. CH), 158.73 (s, arom. C),
174.10 (s, NHCOCH3).
3.5. Phenyl 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-a-D-
galactopyranoside (2)
A suspension of 1 (1.556 g, 4.00 mmol), phenol (1.88 g,
20.0 mmol), ZnCl2 (2.75 g, 20.2 mmol), and anhydrous
CaSO4 (3.05 g) in 1,2-dichloroethane (25 mL) was
heated at reflux for 18 h with stirring. After cooling,
the reaction was quenched with a satd aq NaHCO3
(40 mL). The mixture was filtered through Celite,
washed with satd aq NaHCO3 and a satd aq NaCl suc-
cessively, dried (Na2SO4), and concentrated under
reduced pressure. The residue was dissolved in toluene
and separated by silica gel chromatography eluting with
35% EtOAc–hexane to give 2 (0.731 g, 43%) as a color-
less solid, further elution with 40% EtOAc–hexane to
gave 3 (31 mg, 1.8%) as a colorless solid. 1H NMR
(CDCl3): d 1.90 (s, 3H, COCH3), 1.95 (s, 3H, COCH3),
2.02 (s, 3H, COCH3), 2.16 (s, 3H, COCH3), 4.01 (dd,
1H, J6a,6b 12.2, J5,6a 6.5 Hz, H-6a), 4.10 (dd, 1H, J5,6b
6.5 Hz, H-6b), 4.27 (t, 1H, H-5), 4.74 (ddd, 1H, J2,3
11.3, J2,NH 10.2, J1,2 3.6 Hz, H-2), 5.38 (dd, 1H, J3,4
3.4 Hz, H-3), 5.42 (d, 1H, H-4), 5.59 (d, 1H, H-1),
5.73 (br, 1H, NH), 7.02–7.07 (m, 3H, arom. CH), and
7.27–7.32 (m, 2H, arom. CH); 13C NMR (CDCl3): d
20.53 (q), 20.72 (q), 20.78 (q), 23.30 (q, 2-Ac), 47.91
(d, C-2), 61.64 (t, C-6), 67.22 (d, C-4), 67.56 (d, C-5),
68.22 (d, C-3), 96.31 (d, C-1), 116.57 (d, arom.
CH), 123.10 (d, arom. CH), 129.71 (d, arom. CH),
155.96 (s, arom. C), 170.11 (s, 2-COCH3), 170.30 (s,
COCH3 · 2), and 171.06 (s, 3-COCH3).
3.8. Phenyl 2-acetamido-2-deoxy-3,6-di-O-pivaloyl-a-D-
galactopyranoside (5)
Pivaloyl chloride (0.125 mL, 1.01 mmol) was added to a
solution of 4 (103 mg, 0.346 mmol) in dry pyridine
(1.05 mL) and CH2Cl2 (1.55 mL) at 0 ꢁC, and the mix-
ture was stirred for 15 h at 0 ꢁC. Because the reaction
was not complete, more pivaloyl chloride (0.100 mL,
0.812 mmol) was added to the mixture, and the mixture
was stirred for 7 h at room temperature. The mixture
was then diluted with CHCl3 (50 mL), washed with a
satd aq NaHCO3 and a satd aq NaCl, dried (Na2SO4),
and concentrated under reduced pressure. The residue
was separated by silica gel chromatography eluting with
30% EtOAc–hexane to give 5 (132 mg, 82%) as a color-
less oil. 1H NMR (CDCl3): d 1.04 (s, 9H, C-6-
OCOC(CH3)3), 1.22 (s, 9H, C-3-OCOC(CH3)3), 1.92
(s, 3H, NHCOCH3), 4.04 (br, 1H, H-4), 4.07–4.13 (m,
1H, H-5), 4.17 (dd, 1H, J6a,6b 11.3, J5,6a 7.4 Hz, H-6a),
4.33 (dd, 1H, J5,6b 4.3 Hz, H-6b), 4.86 (ddd, 1H, J2,3
11.2, J2,NH 9.8, J1,2 3.6 Hz, H-2), 5.32 (dd, 1H, J3,4
2.9 Hz, H-3), 5.56 (d, 1H, H-1), 5.76 (d, 1H, NH),
7.00–7.06 (m, 3H, arom. H), and 7.25–7.29 (m, 2H,
arom. H); 13C NMR (CDCl3): d 23.24 (q, NHCOCH3),
26.98 (q, C-6-OCOC(CH3)3), 27.07 (q, C-3-
OCOC(CH3)3), 38.62 (s, C-6-OCOC(CH3)3), 39.09 (s,
C-3-OCOC(CH3)3), 47.38 (d, C-2), 63.25 (t, C-6),
67.68 (d, C-4), 69.28 (d, C-5), 70.29 (d, C-3), 96.33 (d,
C-1), 116.44 (d, arom. CH), 122.83 (d, arom. CH),
129.67 (d, arom. CH), 156.09 (s, arom. C), 169.92 (s,
NHCOCH3), 178.33 (s, C-6-OCOC(CH3)3), and 178.60
(s, C-3-OCOC(CH3)3).
3.6. Phenyl 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-b-D-
galactopyranoside (3)
1H NMR (CDCl3): d 1.93 (s, 3H, COCH3), 2.01 (s, 3H,
COCH3), 2.03 (s, 3H, COCH3), 2.15 (s, 3H, COCH3),
4.06–4.26 (m, 4H), 5.27 (d, 1H, J1,2 8.5 Hz, H-1), 5.37–
5.41 (m, 2H), 5.59 (br, 1H, NH), 6.98–7.05 (m, 3H,
arom. H), and 7.26 (t, 2H, arom. H); 13C NMR
(CDCl3): d 20.65 (q), 20.67 (q), 20.69 (q), 23.47 (q, 2-
Ac), 51.73 (d, C-2), 61.52 (t, C-6), 66.63 (d, C-4), 69.60
(d, C-3), 70.93 (d, C-5), 99.19 (d, C-1), 116.88 (d, arom.
CH), 123.11 (d, arom. CH), 129.52 (d, arom. CH),

1990
T. Sawada et al. / Carbohydrate Research 340 (2005) 1983–1996
3.9. Phenyl 2-acetamido-2-deoxy-3,6-di-O-pivaloyl-4-O-
sulfonato-a-D-galactopyranoside sodium salt (6)
1.07 mmol) in dry CH2Cl2 (1.80 mL) and dry pyridine
(1.5 mL). The mixture was stirred for 11 h at 40 ꢁC
under dry nitrogen, and diluted with CHCl3 (100 mL).
The solution was washed with satd aq NaHCO3
(20 mL · 3), water (20 mL · 2), and brine (20 mL · 2).
The organic layer was dried over Na2SO4 and concen-
trated. Purification was carried out by silica gel column
chromatography eluting with 20% EtOAc–hexane to
give 9 (0.62 g, 88%) as colorless needles. 1H NMR
(CDCl3): d 2.93 (s, 3H, CH3), 3.77 (dd, 1H, J6a,6b 11.0,
J5,6a 5.4 Hz, H-6a), 3.86 (ddd, 1H, J4,5 9.9, J5,6b
2.2 Hz, H-5), 3.92 (dd, 1H, H-6b), 4.33 (t, 1H, J1,2
9.9 Hz, H-2), 4.36 (d, 1H, Jgem 11.7 Hz, C-3-
OCH(H)Ph), 4.52 (t, 1H, J2,3 9.9 Hz, H-3), 4.60 (s,
2H, C-6-OCH2Ph), 4.71 (t, 1H, J3,4 9.9 Hz, H-4), 4.74
(d, 1H, C-3-OCH(H)Ph), 5.50 (d, 1H, H-1), 6.83–6.87
(m, 1H, arom. H), 6.91–6.95 (m, 2H, arom. H), 7.00–
7.02 (m, 2H, arom. H), 7.12–7.23 (m, 3H, arom. H),
7.27–7.37 (m, 7H, arom. H), 7.65–7.78 (m, 4H, arom.
H); 13C NMR (CDCl3): d 38.57 (q, CH3), 54.81 (d, C-
2), 68.85 (t, C-6), 73.58 (t, C-6-OCH2Ph), 75.04 (t,
C-3-OCH2Ph), 77.83 (d, C-5), 78.84 (d, C-3), 78.90 (d,
C-4), 83.43 (d, C-1), 123.42 (d, arom. CH), 123.63
(d, arom. CH), 127.61 (d, arom. CH), 127.75 (d, arom.
CH), 128.00 (d, arom. CH), 128.18 (d, arom. CH),
128.32 (d, arom. CH), 128.85 (d, arom. CH), 131.33
(s, arom. C), 131.41 (s, arom. C), 131.59 (s, arom. C),
132.56 (d, arom. CH), 133.98 (d, arom. CH), 134.08
(d, arom. CH), 136.95 (s, arom. C), 137.96 (s, arom.
C), 166.84 (s, C@O), 168.17 (s, C@O).
A mixture of 5 (110.3 mg, 0.237 mmol) and sulfur triox-
ide–pyridine complex (76.7 mg, 0.482 mmol) in dry pyr-
idine (5 mL) was stirred for 6 h at 40 ꢁC. CH3OH
(1.5 mL) was then added, and the mixture was passed
through the column of Dowex 50W X8 Na+, and concen-
trated under reduced pressure. The residue was dissolved
in 20% CH3OH–CHCl3 and separated by silica gel chro-
matography eluting with 30% CH3OH–CHCl3 to give 6
(95.8 mg, 71%) as colorless crystals. 1H NMR (CD3OD):
d 0.98 (s, 9H, C-6-OCOC(CH3)3), 1.22 (s, 9H, C-3-
OCOC(CH3)3), 1.93 (s, 3H, NHCOCH3), 4.23–4.29 (m,
2H, H-5, H-6a), 4.40–4.47 (m, 1H, H-6b), 4.79 (dd, 1H,
J2,3 11.5, J1,2 3.7 Hz, H-2), 4.93 (d, 1H, J3,4 3.2 Hz, H-
4), 5.20 (dd, 1H, H-3), 5.58 (d, 1H, H-1), 6.99–7.02 (m,
1H, arom. H), 7.11–7.15 (m, 2H, arom. H), 7.25–7.30
(m, 2H, arom. H); 13C NMR (CD3OD): d 22.47 (q,
NHCOCH3), 27.42 (q, C-6-OCOC(CH3)3), 27.67 (q, C-
3-OCOC(CH3)3), 39.60 (s, C-6-OCOC(CH3)3), 39.99 (s,
C-3-OCOC(CH3)3), 48.01 (d, C-2), 65.97 (t, C-6), 70.23
(d, C-3), 70.56 (d, C-5), 73.90 (d, C-4), 97.60 (d, C-1),
117.91 (d, arom. CH), 123.68 (d, arom. CH), 130.61 (d,
arom. CH), 158.13 (s, arom. C), 173.37 (s, NHCOCH3),
179.81 (s, C-6-OCOC(CH3)3), 180.00 (s, C-3-
OCOC(CH3)3).
3.10. Phenyl 2-acetamido-2-deoxy-4-O-sulfonato-a-D-
galactopyranoside (7)
A solution of 6 (26.4 mg, 0.0465 mmol) in dry CH3OH
(1 mL) was treated with 1 M methanolic NaOCH3
(0.10 mL) for 47 h at room temperature. The mixture
was neutralized with Amberlite IRC-50 H+ resin and
concentrated under reduced pressure. The residue was
purified by paper electrophoresis in buffer A, and gel fil-
tration with Superdex 30 afforded 7 (5.3 mg, 29% as
3.12. Phenyl 4-O-acetyl-3,6-di-O-benzyl-2-deoxy-2-
phthalimido-1-thio-b-D-galactopyranoside (10)
Thioglycoside 9 (102.1 mg, 0.155 mmol) was added to a
solution of potassium acetate (150 mg, 1.53 mmol) and
a
catalytic amount of 18-crown-6 in dry DMF
(10 mL). The mixture was stirred at 140 ꢁC for 14 h
under dry nitrogen, and diluted with CHCl3 (80 mL).
The solution was washed with water (15 mL · 3) and
the aqueous layer was extracted with CHCl3
(20 mL · 2). The combined organic layer was dried over
Na2SO4 and concentrated in vacuo. The residue was
separated by silica gel column chromatography eluting
with 30% EtOAc–hexane to give 10 (67.1 mg, 70%) as
1
ammonium salt) as colorless crystals. H NMR (D2O):
d 1.90 (s, 3H, NHCOCH3), 3.57–3.67 (m, 2H, H-6),
4.07–4.10 (m, 1H, H-5), 4.15 (dd, 1H, J2,3 11.2, J3,4
2.9 Hz, H-3), 4.23 (dd, 1H, J1,2 3.5 Hz, H-2), 4.65–4.70
(m, 1H, H-4), 5.53 (d, 1H, H-1), 6.98–7.03 (m, 3H,
arom. H), 7.24–7.38 (m, 2H, arom. H); 13C NMR
(D2O): d 24.72 (q, NHCOCH3), 52.98 (d, C-2), 63.87
(t, C-6), 69.55 (d, C-3), 74.02 (d, C-5), 79.43 (d, C-4),
98.84 (d, C-1), 119.98 (d, arom. CH), 126.05 (d, arom.
CH), 132.73 (d, arom. CH), 158.92 (s, arom. C),
177.58 (s, NHCOCH3); ESI-MS (C14H18NO9Sꢀ) m/z
376 (Mꢀ).
1
a pale brown syrup. H NMR (CDCl3): d 2.11 (s, 3H,
OCOCH3), 3.56 (dd, 1H, J6a,6b 9.6, J5,6a 6.5 Hz, H-
6a), 3.64 (dd, 1H, J5,6b 6.5 Hz, H-6b), 3.95 (t, 1H, H-
5), 4.23 (d, 1H, Jgem 12.3 Hz, C-3-OCH(H)Ph), 4.29
(dd, 1H, J2,3 10.6, J3,4 3.2 Hz, H-3), 4.45 (d, 1H, Jgem
12.0 Hz, C-6-OCH(H)Ph), 4.49 (t, 1H, J1,2 10.6 Hz, H-
2), 4.56 (m, 2H, C-3-OCH(H)Ph + C-6-OCH(H)Ph),
5.55 (d, 1H, H-1), 6.87–6.94 (m, 4H, arom. H), 6.97–
7.03 (m, 1H, arom. H), 7.15–7.19 (m, 3H, arom. H),
7.28–7.38 (m, 7H, arom. H), 7.61–7.74 (m, 2H, arom.
H); 13C NMR (CDCl3): d 20.88 (q, C-4-OCOCH3),
3.11. Phenyl 3,6-di-O-benzyl-2-deoxy-4-O-methanesulfo-
nyl-2-phthalimido-1-thio-b-D-glucopyranoside (9)
Methanesulfonyl chloride (0.30 mL, 3.88 mmol) was
added to a solution of thioglycoside 828 (0.62 g,

T. Sawada et al. / Carbohydrate Research 340 (2005) 1983–1996
1991
˚
51.56 (d, C-2), 66.00 (d, C-4), 68.16 (t, C-6), 70.97 (t, C-
3-OCH2Ph), 73.56 (d, C-3), 73.62 (t, C-6-OCH2Ph),
76.24 (d, C-5), 84.22 (d, C-1), 123.25 (d, arom. CH),
123.52 (d, arom. CH), 127.61 (d, arom. CH), 127.65
(d, arom. CH), 127.83 (d, arom. CH), 127.98 (d, arom.
CH), 128.05 (d, arom. CH), 128.12 (d, arom. CH),
128.44 (d, arom. CH), 128.78 (d, arom. CH), 131.59
(s, arom. C), 131.68 (s, arom. C), 132.06 (d, arom.
CH), 132.67 (s, arom. C), 133.84 (d, arom. CH),
134.01 (d, arom. CH), 137.26 (s, arom. C), 137.67 (s,
arom. C), 167.38 (s, C@O), 167.92 (s, C@O), 170.42
(s, C-4-OCOCH3).
4 A (85.5 mg) in dry CH2Cl2 (3 mL) was stirred for
15 min at room temperature under dry nitrogen.
Hafnocene dichloride (74.1 mg, 0.195 mmol) and silver
perchlorate (78.1 mg, 0.377 mmol) were added to the
mixture at ꢀ78 ꢁC. The mixture was stirred for 1 h at
this temperature, then stirred for 23 h at ꢀ50 ꢁC, and
diluted with CHCl3 (50 mL). The mixture was washed
with satd aq NaHCO3 (10 mL · 3), water (10 mL · 3),
and brine (10 mL · 2). The organic layer was dried
over Na2SO4 and concentrated. Purification was
carried out by silica gel column chromatography
eluting with 10% EtOAc–hexane to give 12 (77.3 mg,
1
68%) as a pale brown oil. H NMR (CDCl3): d 2.15
3.13. 4-O-Acetyl-3,6-di-O-benzyl-2-deoxy-2-phthalimido-
b-D-galactopyranosyl fluoride (11)
(s, 3H, C-4-OCOCH3), 3.62 (dd, 1H, J6a,6b 9.5, J5,6a
6.5 Hz, H-6a), 3.65 (dd, 1H, J5,6b 6.5 Hz, H-6b), 4.06
(t, 1H, H-5), 4.39 (dd, 1H, J2,3 11.0, J3,4 3.6 Hz, H-
3), 4.66 (dd, 1H, J1,2 8.5 Hz, H-2), 5.70 (d, 1H, H-4),
5.77 (d, 1H, H-1), 6.79–7.05 (m, 7H, arom. H),
7.12–7.35 (m, 8H, arom. H), and 7.58–7.86 (m, 4H,
arom. H); 13C NMR (CDCl3): d 20.93 (q, COCH3),
52.73 (d, C-2), 65.76 (d), 68.03 (t, C-6), 71.18 (t,
OCH2), 72.76 (d), 72.82 (d), 73.68 (t, OCH2), 96.52
(d, C-1), 116.83 (d, arom. CH), 122.77 (d, arom.
CH), 123.24 (d, arom. CH), 123.48 (d, arom. CH),
127.64 (d, arom. CH), 127.84 (d, arom. CH), 127.96
(d, arom. CH), 128.04 (d, arom. CH), 128.16 (d, arom.
CH), 128.45 (d, arom. CH), 129.34 (d, arom. CH),
131.57 (s, arom. C), 133.83 (d, arom. CH), 134.01 (d,
arom. CH), 137.29 (s, arom. C), 137.62 (s, arom. C),
156.66 (s, arom. C), 167.59 (s, C@O), 168.20 (s,
C@O), and 170.46 (s, C-4-OCOCH3).
N-Bromosuccinimide (77.9 mg, 0.438 mmol) and dieth-
ylaminosulfur trifluoride (0.120 mL, 0.910 mmol) were
added to a solution of 10 (179.8 mg, 0.2883 mmol) in
dry CH2Cl2 (4.3 mL) at ꢀ15 ꢁC, and the mixture was
stirred for 1 h under dry nitrogen. The reaction mixture
was then gradually warmed to 0 ꢁC and stirred for 4.5 h.
The mixture was diluted with CHCl3 (100 mL), and
washed with satd aq NaHCO3 (30 mL · 3), water
(30 mL · 2), and brine (30 mL · 2). The organic layer
was dried over Na2SO4 and concentrated. Purification
was carried out by silica gel column chromatography
eluting with 20% EtOAc–hexane to give fluoride 11
(100.7 mg, 65%) as colorless powder. 1H NMR (CDCl3):
d 2.14 (s, 3H, COCH3), 3.59 (dd, 1H, J6a,6b 9.5, J5,6a
6.8 Hz, H-6a), 3.66 (dd, 1H, J5,6b 5.9 Hz, H-6b), 3.98–
4.02 (m, 1H, H-5), 4.23 (d, 1H, Jgem 12.4 Hz, C-3-
OCH(H)Ph), 4.32 (dd, 1H, J2,3 11.2, J3,4 3.0 Hz, H-3),
4.46 (ddd, 1H, JF,2 12.7, J1,2 8.0 Hz, H-2), 4.48 (d, 1H,
Jgem 11.8 Hz, C-6-OCH(H)Ph), 4.58 (d, 1H, C-3-
OCH(H)Ph), 4.60 (d, 1H, C-6-OCH(H)Ph), 5.65 (t,
J4,5 3.0 Hz, H-4), 5.83 (dd, 1H, JF,1 53.4 Hz, H-1),
6.89–6.95 (m, 4H, arom. H), 7.00–7.04 (m, 1H, arom.
H), 7.27–7.38 (m, 5H, arom. H), 7.72–7.75 (m, 4H,
arom. H); 13C NMR (CDCl3): d 20.83 (q, C-4-
OCOCH3), 52.91 (d, JF,2 21.5 Hz, C-2), 65.24 (d, C-4),
67.51 (t, C-6), 71.24 (t, C-3-OCH2Ph), 72.16 (d, JF,3
9.9 Hz, C-3), 72.48 (d, JF,5 5.7 Hz, C-5), 73.73 (t, C-6-
OCH2Ph), 104.93 (d, JF,1 214.2 Hz, C-1), 123.45 (d,
arom. CH), 127.73 (d, arom. CH), 127.98 (d, arom.
CH), 128.05 (d, arom. CH), 128.08 (d, arom. CH),
128.19 (d, arom. CH), 128.52 (d, arom. CH), 131.55
(s, arom. C), 134.08 (d, arom. CH), 137.07 (s, arom.
C), 137.35 (s, arom. C), 167.50 (s, C@O), 170.25 (s, C-
4-OCOCH3).
3.15. Phenyl 2-acetamido-4-O-acetyl-3,6-di-O-benzyl-2-
deoxy-b-D-galactopyranoside (13)
A solution of 12 (105.4 mg, 0.1735 mmol) and hydra-
zine monohydrate (0.085 mL) in ethanol (5 mL) was
heated at reflux for 2 h. After cooling, the mixture
was concentrated under reduced pressure. The residue
was treated with dry pyridine (4 mL) and Ac2O
(2 mL). The mixture was stirred for 15 h at room tem-
perature, diluted with CHCl3 (100 mL), and washed
with water (20 mL · 2), 3% HCl aq (20 mL · 3), satd
aq NaHCO3 (20 mL · 3), water (20 mL · 2), and brine
(20 mL · 2). The organic layer was dried over Na2SO4
and concentrated under reduced pressure. A solution
of the residue in dry CH3OH (5 mL) was treated with
1 M methanolic NaOCH3 (0.90 mL) for 4 h at room
temperature. The mixture was neutralized with Amber-
lite IRC-50 H+ resin and concentrated under reduced
pressure. Purification was carried out by silica gel col-
umn chromatography eluting with 50% EtOAc–hexane
to give 13 (31.4 mg, 38%) as colorless crystals. 1H
NMR (CDCl3): d 1.89 (s, 3H, OCOCH3), 3.61–3.68
(m, 1H, H-2), 3.73–3.84 (m, 3H, H-6+H-3), 4.12 (t,
3.14. Phenyl 4-O-acetyl-3,6-di-O-benzyl-2-deoxy-2-
phthalimido-b-D-galactopyranoside (12)
A mixture of 11 (99.1 mg, 0.186 mmol), phenol
(106.0 mg, 1.126 mmol), and activated molecular sieves

1992
T. Sawada et al. / Carbohydrate Research 340 (2005) 1983–1996
1H, J3,4 3.0 Hz, H-4), 4.33 (dd, 1H, J2,3 10.6 Hz, H-3),
4.52–4.55 (m, 1H, C-3-OCH(H)Ph), 4.55 (s, 2H, C-6-
OCH2Ph), 4.70 (d, 1H, Jgem 11.7 Hz, C-3-OCH(H)Ph),
5.54 (d, 1H, J1,2 8.3 Hz, H-1), 5.61 (d, 1H, J2,NH
7.1 Hz, NH), 6.97–7.01 (m, 3H, arom. H), and 7.20–
7.37 (m, 12H, arom. H); 13C NMR (CDCl3): d 23.69
(q, OCOCH3), 54.41 (d, C-2), 65.88 (d, C-4), 69.28 (t,
C-6), 71.88 (t, C-3-OCH2Ph), 73.45 (d, C-5), 73.68 (C-
6-OCH2Ph), 76.10 (d, C-3), 97.81 (d, C-1), 117.10 (d,
arom. CH), 122.66 (d, arom. CH), 127.71 (d, arom.
CH), 128.10 (d, arom. CH), 128.18 (d, arom. CH),
128.40 (d, arom. CH), 128.61 (d, arom. CH), 129.40
(d, arom. CH), 137.61 (s, arom. C), 137.95 (s, arom.
C), 157.25 (s, arom. C), and 170.81 (s, C@O).
and Superdex 30 chromatography to give 15 (5.2 mg,
47% as ammonium salt) as colorless crystals. 1H
NMR (D2O): d 1.89 (s, 3H, NHCOCH3), 3.66–3.75
(m, 2H, H-6), 3.83–3.88 (m, 2H, H-3, H-5), 4.04 (dd,
1H, J2,3 10.9, J1,2 8.4 Hz, H-2), 4.65–4.70 (m, 1H, H-
4), 5.02 (d, 1H, H-1), 6.95–7.03 (m, 3H, arom. H),
7.23–7.27 (m, 2H, arom. H); 13C NMR (D2O): d
24.99 (q, NHCOCH3), 55.59 (d, C-2), 63.65 (t, C-6),
72.61 (d, C-3), 77.57 (d, C-5), 78.33 (d, C-4), 102.79
(d, C-1), 119.59 (d, arom. CH), 126.34 (d, arom.
CH), 132.80 (d, arom. CH), 159.57 (s, arom. C),
177.92 (s, NHCOCH3); ESI-MS (C14H18NO9Sꢀ) m/z
376 (Mꢀ).
3.18. Phenyl 2-acetamido-2-deoxy-6-O-sulfonato-a-D-
galactopyranoside (16)
3.16. Phenyl 2-acetamido-3,6-di-O-benzyl-2-deoxy-4-O-
sulfonato-b-D-galactopyranoside sodium salt (14)
A mixture of 4 (50.8 mg, 0.171 mmol) and sulfur triox-
ide–pyridine complex (56.8 mg, 0.357 mmol) in dry pyr-
idine (2 mL) was stirred for 6 h at 40 ꢁC. CH3OH (2 mL)
was then added, and the mixture was passed through the
column of Dowex 50W X8 Na+, and concentrated under
reduced pressure. The residue was dissolved in water
and purified successively by gel filtration with Superdex
30, paper electrophoresis in buffer A, SAX-HPLC and
Superdex 30 chromatography to give the target com-
pound 16 (4.0 mg, 6% as ammonium salt) as colorless
Sulfur trioxide–pyridine complex (66.3 mg, 0.417 mmol)
was added to a solution of 13 (21.6 mg, 0.0452 mmol) in
dry pyridine (5 mL), and the mixture was stirred for 18 h
at room temperature. CH3OH (5 mL) was added to the
reaction mixture, and the resulting solution was sub-
jected to Dowex 50W X8 Na+ resin. The effluent was
concentrated under reduced pressure. The residue was
dissolved in 20% CH3OH–CHCl3, and separated by sil-
ica gel column chromatography eluting with 30%
CH3OH–CHCl3 to give 14 (24.4 mg, 93%) as colorless
1
crystals. H NMR (D2O): d 1.92 (s, 3H, NHCOCH3),
crystals. 1H NMR (CD3OD):
d
1.85 (s, 3H,
3.97–4.10 (m, 4H), 4.23–4.27 (m, 2H), 5.44 (d, J1,2
3.7 Hz, H-1), 7.00–7.04 (m, 3H, arom. H), 7.24–7.28
(m, 2H, arom. H); 13C NMR (CD3OD): d 22.57
(q, NHCOCH3), 51.55 (d, C-2), 67.34 (t, C-6), 69.08
(d, C-3), 69.58 (d, C-4), 71.04 (d, C-5), 98.77 (d,
C-1), 118.59 (d, arom. CH), 123.73 (d, arom. CH),
130.53 (d, arom. CH), 158.84 (s, arom. C), and
174.04 (s, NHCOCH3); ESI-MS (C14H18NO9Sꢀ) m/z
376 (Mꢀ).
NHCOCH3), 3.86–3.91 (m, 3H, H-3, H-5, H-6a), 3.97–
4.00 (m, 1H, H-6b), 4.04–4.09 (m, 1H, H-2), 4.42
(d, 1H, Jgem 11.5 Hz, C-3-OCH(H)Ph), 4.54 (d, 1H,
Jgem 11.3 Hz, C-6-OCH(H)Ph), 4.58 (d, 1H, C-6-
OCH(H)Ph), 4.95 (d, 1H, C-3-OCH(H)Ph), 4.96 (d,
1H, J3,4 3.4 Hz, H-4), 5.20 (d, 1H, J1,2 8.3 Hz, H-1),
6.94–7.03 (m, 3H, arom. H), 7.16–7.40 (m, 12H, arom.
H); 13C NMR (CD3OD): d 23.00 (q, CH3), 53.91 (d,
C-2), 71.87 (t, C-6), 72.26 (t, C-3-OCH2Ph), 73.01 (d,
C-4), 74.41 (t, C-6-OCH2Ph), 75.56 (d, C-5), 77.96
(d, C-3), 100.51 (d, C-1), 117.94 (d, arom. CH), 123.42
(d, arom. CH), 128.46 (d, arom. CH), 128.50 (d, arom.
CH), 128.89 (d, arom. CH), 129.17 (d, arom. CH),
129.26 (d, arom. CH), 129.31 (d, arom. CH), 130.39
(d, arom. CH), 139.94 (s, arom. C), 140.04 (s, arom.
C), 159.17 (s, arom. C), and 173.57 (s, C@O).
3.19. Phenyl 2-acetamido-2-deoxy-b-D-
galactopyranoside (17)
A solution of 3 (226.6 mg, 0.535 mmol) in dry CH3OH
(8 mL) was treated with 1 M methanolic NaOCH3
(0.54 mL) for 1 h at room temperature. The mixture
was neutralized with Amberlite IRC-50 H+ resin, and
concentrated under reduced pressure to give 17
(139.8 mg, 88%) as colorless crystals. 1H NMR
(CD3OD): d 1.97 (s, 3H, NHCOCH3), 3.65 (t, 1H, J5,6a
6.1 Hz, H-5), 3.71 (dd, 1H, J2,3 10.5, J3,4 2.7 Hz, H-3),
3.76 (dd, 1H, J6a,6b 11.5 Hz, H-6a), 3.80 (dd, 1H, J5,6b
6.1 Hz, H-6b), 3.90 (d, 1H, H-4), 4.17 (dd, 1H, J1,2
8.5 Hz, H-2), 5.00 (d, 1H, H-1), 6.96–7.03 (m, 3H, arom.
H), and 7.23–7.89 (m, 2H, arom. H); 13C NMR
(CD3OD): d 22.98 (q, CH3), 54.32 (d, C-2), 62.44 (t,
3.17. Phenyl 2-acetamido-2-deoxy-4-O-sulfonato-b-D-
galactopyranoside (15)
To a solution of 14 (16.2 mg, 0.0279 mmol) in 95% eth-
anol–water (2.0 mL) was added 10% Pd–C 30.4 mg and
the mixture was stirred for 21 h at 40 ꢁC under hydro-
gen. The reaction mixture was filtered through Celite
and concentrated under reduced pressure. The residue
was dissolved in water, and purified by SAX-HPLC

T. Sawada et al. / Carbohydrate Research 340 (2005) 1983–1996
1993
C-6), 69.59 (d, C-4), 72.98 (d, C-3), 77.06 (d, C-5), 101.28
(d, C-1), 117.75 (d, arom. CH), 123.45 (d, arom. CH),
130.43 (d, arom. CH), and 159.25 (s, arom. C), 174.23
(s, C@O).
117.83 (d, arom. CH), 123.58 (d, arom. CH), 127.63
(d, arom. CH), 129.05 (d, arom. CH), 129.94 (d, arom.
CH), 130.60 (d, arom. CH), 139.69 (s, arom. C),
158.45 (s, arom. C), and 174.14 (s, C@O).
3.20. Phenyl 2-acetamido-2-deoxy-6-O-sulfonato-b-D-
galactopyranoside (18)
3.22. Phenyl 2-acetamido-4,6-O-benzylidene-2-deoxy-3-
O-sulfonato-a-D-galactopyranoside sodium salt (20)
A mixture of 17 (40.4 mg, 0.136 mmol) and sulfur triox-
ide–pyridine complex (44.5 mg, 0.278 mmol) in dry pyri-
dine (2 mL) was stirred for 6 h at 40 ꢁC. CH3OH (1.5 mL)
was then added, and the mixture was passed through the
column of Dowex 50W X8 Na+, and concentrated under
reduced pressure. The residue was dissolved in water, and
purified by paper electrophoresis in buffer A, SAX-HPLC
and Superdex 30 chromatography to give the target com-
pound 18 (5.0 mg, 9% ammonium salt) as colorless crys-
Sulfur trioxide–pyridine complex (134.3 mg, 0.844 mmol)
was added to a solution of 19 (30.4 mg, 0.0789 mmol) in
dry pyridine (2 mL), and the mixture was stirred for
20.5 h at 50 ꢁC. CH3OH (2 mL) was added to the reac-
tion mixture, and the resulting solution was subjected
to Dowex 50W X8 Na+ resin. The effluent was concen-
trated under reduced pressure. The residue was dissolved
in 20% CH3OH–CHCl3, and separated by silica gel col-
umn chromatography eluting with 30% CH3OH–CHCl3
to give 20 (31.5 mg, 82%) as colorless crystals. 1H NMR
(CD3OD): d 1.98 (s, 3H, NHCOCH3), 3.89 (s, 1H, H-5),
4.09 (dd, 1H, J6a,6b 12.6, J5,6a 1.6 Hz, H-6a), 4.13 (dd,
1H, J5,6b 1.6 Hz, H-6b), 4.64 (dd, 1H, J2,3 11.5, J1,2
3.2 Hz, H-2), 4.71 (d, 1H, J3,4 3.1 Hz, H-4), 4.94 (dd,
1H, H-3), 5.66 (s, 1H, CH), 5.78 (d, 1H, H-1), 7.00–
7.04 (m, 1H, arom. H), 7.10–7.13 (m, 2H, arom. H),
7.28–7.37 (m, 5H, arom. H), 7.52–7.55 (m, 2H, arom.
H); 13C NMR (CD3OD): d 22.72 (q, CH3), 50.09 (d, C-
2), 65.33 (d, C-5), 70.19 (t, C-6), 73.57 (d, C-3), 75.53
(d, C-4), 98.23 (d, C-1), 102.04 (d, CH), 117.85 (d, arom.
CH), 123.69 (d, arom. CH), 127.54 (d, arom. CH), 128.98
(d, arom. CH), 129.86 (d, arom. CH), 130.64 (d, arom.
CH), 139.63 (s, arom. C), 158.31 (s, arom. C), 173.97
(s, NHCOCH3).
1
tals. H NMR (D2O): d 1.88 (s, 3H, NHCOCH3), 3.70–
3.73 (m, 1H, H-3), 3.93 (s, 1H, H-4), 3.97–4.15 (m, 4H,
H-2, H-5, H-6), 4.95 (d, 1H, J1,2 8.3 Hz, H-1), 6.94–7.03
(m, 3H, arom. H), and 7.22–7.83 (m, 2H, arom. H); 13
C
NMR (CD3OD): d 22.97 (q, NHCOCH3), 54.10 (d,
C-2), 67.56 (t, C-6), 69.17 (d, C-4), 72.90 (d, C-3), 74.56
(d, C-5), 101.37 (d, C-1), 117.80 (d, arom. CH), 123.46
(d, arom. CH), 130.47 (d, arom. CH), 159.25 (s, arom.
C), and 174.16 (s, NHCOCH3); ESI-MS (C14H18NO9Sꢀ)
m/z 376 (Mꢀ).
3.21. Phenyl 2-acetamido-4,6-O-benzylidene-2-deoxy-a-
D-galactopyranoside (19)
A mixture of benzaldehyde dimethyl acetal (0.13 mL,
0.866 mmol), compound 4 (100.4 mg, 0.338 mmol),
and D-camphor-10-sulfonic acid (34.9 mg, 0.150 mmol)
was stirred at 40 ꢁC for 24 h under reduced pressure.
Benzaldehyde dimethyl acetal (0.20 mL, 1.33 mmol)
and D-camphor-10-sulfonic acid (68.5 mg, 0.295 mmol)
was added to the mixture, and then it was stirred at
40 ꢁC for 17 h under reduced pressure. The mixture
was diluted with CHCl3 (50 mL), and washed with satd
aq NaHCO3 (20 mL · 3), water (20 mL · 2), and brine
(20 mL · 2). The organic layer was dried over Na2SO4,
and concentrated under reduced pressure. Purification
was carried out by silica gel column chromatography
eluting with EtOAc to give 19 (79.6 mg, 61%) as color-
less needles. 1H NMR (CD3OD): d 1.99 (s, 3H,
NHCOCH3), 3.85 (s, 1H, H-5), 4.11 (s, 2H, H-6), 4.19
(dd, 1H, J2,3 11.2, J3,4 3.4 Hz, H-3), 4.34 (d, 1H, H-4),
4.52 (dd, 1H, J1,2 3.4 Hz, H-2), 5.65 (d, 1H, H-1),
6.99–7.03 (m, 1H, arom. H), 7.09–7.12 (m, 2H, arom.
H), 7.26–7.39 (m, 5H, arom. H), and 7.55–7.58 (m,
2H, arom. H); 13C NMR (CD3OD): d 22.56 (q, CH3),
51.62 (d, C-2), 65.29 (d, C-5), 67.90 (d, C-3), 70.27 (t,
C-6), 77.17 (d, C-4), 98.38 (d, C-2), 102.33 (d, CH),
3.23. Phenyl 2-acetamido-2-deoxy-3-O-sulfonato-a-D-
galactopyranoside (21)
To a solution of 20 (18.5 mg, 0.0380 mmol) in 95% eth-
anol–water (2.0 mL) was added 10% Pd–C (21.2 mg)
and the mixture was stirred for 9 h at 40 ꢁC under
hydrogen. The reaction mixture was filtered through
Celite and concentrated under reduced pressure. The
residue was dissolved in water, and purified by
SAX-HPLC and Superdex 30 chromatography to
give the target compound 21 (3.9 mg, 26%, ammonium
salt) as colorless crystals. 1H NMR (D2O): d 1.91
(s, 3H, NHCOCH3), 3.60 (d, 2H, J5,6 6.2 Hz, H-6),
4.01 (t, 1H, H-5), 4.26 (d, J3,4 3.2 Hz, H-4), 4.43 (dd,
1H, J2,3 10.7, J1,2 4.0 Hz, H-2), 4.65 (dd, 1H, H-3),
5.55 (d, 1H, H-1), 6.99–7.06 (m, 3H, arom. H),
7.25–7.28 (m, 2H, arom. H); 13C NMR (D2O): d 24.82
(q, NHCOCH3), 50.65 (d, C-2), 63.76 (t, C-6), 69.75
(d, C-4), 74.41 (d, C-5), 78.50 (d, C-5), 99.09 (d,
C-1), 120.03 (d, arom. CH), 126.06 (d, arom.
CH), 132.72 (d, arom. CH), 158.75 (s, arom. C),

1994
T. Sawada et al. / Carbohydrate Research 340 (2005) 1983–1996
177.61 (s, NHCOCH3); ESI-MS (C14H18NO9Sꢀ) m/z
376 (Mꢀ).
CH), 128.94 (d, arom. CH), 129.82 (d, arom. CH),
130.47 (d, arom. CH), 139.64 (s, arom. C), 159.10 (s,
arom. C), and 174.07 (s, C@O).
3.24. Phenyl 2-acetamido-4,6-O-benzylidene-2-deoxy-b-
D-galactopyranoside (22)
3.26. Phenyl 2-acetamido-2-deoxy-3-O-sulfonato-b-D-
galactopyranoside (24)
A mixture of benzaldehyde dimethyl acetal (0.15 mL,
0.999 mmol), compound 17 (50.5 mg, 0.170 mmol),
and D-camphor-10-sulfonic acid (42.3 mg, 0.182 mmol)
was stirred at 40 ꢁC for 22.5 h under reduced pressure.
Additional benzaldehyde dimethyl acetal (0.10 mL,
0.666 mmol) and D-camphor-10-sulfonic acid (40.3 mg,
0.173 mmol) was added to the mixture, and then it
was stirred at 40 ꢁC for 9 h under reduced pressure.
The mixture was diluted with CHCl3 (50 mL), and
washed with satd aq NaHCO3 (20 mL · 3), water
(20 mL · 2), and brine (20 mL · 2). The organic layer
was dried over Na2SO4 and concentrated under reduced
pressure. Purification was carried out by silica gel col-
umn chromatography eluting with EtOAc to give 22
(33.7 mg, 51%) as colorless needles. 1H NMR (CD3OD):
d 1.97 (s, 3H, NHCOCH3), 3.73 (q, 1H, J 1.4 Hz, H-5),
3.90 (dd, 1H, J2,3 11.1, J3,4 3.5 Hz, H-3), 4.18 (dd, 1H,
J6a,6b 12.3, J5,6a 1.4 Hz, H-6a), 4.24 (dd, 1H, J5,6b
1.4 Hz, H-6b), 4.24–4.28 (m, 2H, H-3, H-4), 5.15 (d,
1H, J1,2 8.5 Hz, H-1), 5.67 (s, 1H, CH), 6.98–7.06 (m,
3H, arom. CH), 7.25–7.30 (m, 2H, arom. CH), 7.33–
7.38 (m, 3H, arom. CH), and 7.56–7.59 (m, 2H, arom.
CH); 13C NMR (CD3OD): d 22.98 (CH3), 54.16 (C-2),
68.19, 70.22, 71.49, 78.31, 100.96, 102.52, 117.88,
123.61, 127.73, 129.03, 129.95, 130.49, 139.64, 159.10,
and 174.23 (s, NHCOCH3).
To a solution of 23 (25.4 mg, 0.0521 mmol) in 95% eth-
anol–water (2.0 mL) was added 10% Pd–C (36.2 mg)
and the mixture was stirred for 35 h at 40 ꢁC under
hydrogen. The reaction mixture was filtered through
Celite and concentrated under reduced pressure. The
residue was dissolved in water, and purified by SAX-
HPLC and Superdex 30 chromatography to give the tar-
get compound 21 (11.2 mg, 54% as ammonium salt) as
colorless crystals. 1H NMR (D2O): d 1.87 (s, 3H,
NHCOCH3), 3.68–3.70 (m, 2H, H-6), 3.76–3.79 (m,
1H, H-5), 4.18–4.20 (m, 2H, H-2, H-4), 4.37–4.40 (m,
1H, H-3), 5.11 (d, 1H, J1,2 9.5 Hz, b-H-1), 6.96–7.03
(m, 3H, arom. H), and 7.24–7.28 (m, 2H, arom. H);
13C NMR (D2O): d 25.07 (q, NHCOCH3), 53.48 (d,
C-2), 63.56 (t, C-6), 69.03 (d, C-4), 77.95 (d, C-5),
80.37 (d, C-3), 102.55 (d, C-1), 119.65 (d, arom. CH),
126.37 (d, arom. CH), 132.84 (d, arom. CH), 159.61
(s, arom. C), and 177.85 (s, NHCOCH3); ESI-MS
(C14H18NO9Sꢀ) m/z 376 (Mꢀ).
3.27. Phenyl 2-acetamido-4,6-O-benzilidene-2-deoxy-3-
O-pivaloyl-b-D-galactopyranoside (25)
Pivaloyl chloride (0.050 mL, 0.406 mmol) was added to
a solution of 22 (15.9 mg, 0.0413 mmol) in dry pyridine
(0.5 mL) and CH2Cl2 (0.5 mL), and the mixture was stir-
red for 15 h at room temperature. The mixture was
diluted with CHCl3 (60 mL), washed with satd aq
NaHCO3 (20 mL · 3), water (20 mL · 2), and brine
(20 mL · 2), dried over Na2SO4, and concentrated
under reduced pressure. The residue was dissolved in
toluene, and separated by silica gel chromatography
eluting with 30% EtOAc–hexane to give 25 (12.8 mg,
3.25. Phenyl 2-acetamido-4,6-O-benzylidene-2-deoxy-3-
O-sulfonato-b-D-galactopyranoside sodium salt (23)
Sulfur trioxide–pyridine complex (98.7 mg, 0.620 mmol)
was added to a solution of 22 (23.7 mg, 0.0615 mmol) in
dry pyridine (2 mL), and the mixture was stirred for 13 h
at 40 ꢁC. CH3OH (2 mL) was added to the reaction mix-
ture, and the resulting solution was subjected to Dowex
50W X8 Na+ resin. The effluent was concentrated under
reduced pressure. The residue was separated by silica gel
column chromatography eluting with 30% CH3OH–
CHCl3 to give 23 (25.4 mg, 85%) as colorless crystals.
1H NMR (CD3OD): d 1.96 (s, 3H, NHCOCH3), 3.77
(t, 1H, J 1.7 Hz, H-5), 4.18 (dd, 1H, J6a,6b 12.6, J5,6a
1.7 Hz, H-6a), 4.23 (dd, 1H, J5,6b 1.7 Hz, H-6b), 4.41
(dd, 1H, J2,3 10.9, J1,2 8.4 Hz, H-2), 4.62 (dd, 1H, J3,4
3.4 Hz, H-3), 4.65 (d, 1H, H-4), 5.29 (d, 1H, H-1),
5.66 (s, 1H, CH), 6.98–7.06 (m, 3H, arom. CH), 7.25–
7.37 (m, 5H, arom. CH), and 7.54–7.57 (m, 2H, arom.
CH); 13C NMR (CD3OD): d 23.11 (q, CH3), 51.99 (d,
C-2), 68.04 (d, C-5), 70.19 (t, C-6), 75.16 (d, C-4),
76.32 (d, C-3), 101.08 (d, C-1), 102.30 (d, CH), 117.91
(d, arom. CH), 123.60 (d, arom. CH), 127.68 (d, arom.
1
66%) as colorless crystals. H NMR (CDCl3): d 1.19
(s, 9H, C(CH3)3), 1.91 (s, 3H, NHCOCH3), 3.68 (s,
1H, H-5), 4.07 (dd, 1H, J6a,6b 12.4, J5,6a 1.7 Hz, H-6a),
4.22 (dt, 1H, J2,3 11.3, J1,2 8.2 Hz, H-2), 4.35 (dd, 1H,
J5,6b 1.2 Hz, H-6b), 4.42 (d, 1H, J3,4 3.4 Hz, H-4), 5.47
(dd, 1H, J2,3 11.3 Hz, H-3), 5.49 (d, 1H, H-1), 5.50 (d,
1H, JNH,2 8.2 Hz, N-H), 5.54 (s, 1H, CH), 6.99–7.03
(m, 3H, arom. H), 7.23–7.36 (m, 5H, arom. H), and
7.48–7.50 (m, 2H, arom. H); 13C NMR (CDCl3): d
23.44 (q, CH3), 27.01 (q, C(CH3)3), 39.02 (s, C(CH3)3),
51.93 (d, C-2), 66.54 (d), 69.07 (t, C-6), 69.88 (d),
72.94 (d), 98.67 (d), 100.50 (d), 117.30 (d, arom. CH),
122.85 (d, arom. CH), 126.06 (d, arom. CH), 128.07
(d, arom. CH), 128.81 (d, arom. CH), 129.41 (d, arom.
CH), 137.65 (s, arom. C), 157.29 (s, arom. C), 170.22
(s, C@O), and 178.23 (s, C@O).

T. Sawada et al. / Carbohydrate Research 340 (2005) 1983–1996
1995
3.28. Phenyl 2-acetamido-2-deoxy-3-O-pivaloyl-b-D-
galactopyranoside (26)
10178102 from the Ministry of Education, Science,
Sports and Culture of Japan, by MEXT KAKENHI
(14082206 and 16-4208), and by a special research fund
from Seikagaku Corporation.
To a solution of 25 (10.6 mg, 0.0226 mmol) in 95% eth-
anol–water (2.0 mL) was added 10% Pd–C (19.5 mg),
and the mixture was stirred for 2 days at 40 ꢁC under
hydrogen. The reaction mixture was filtered through
Celite, and concentrated under reduced pressure. The
residue was purified by silica gel chromatography elut-
ing with EtOAc to give 26 (5.5 mg, 64%) as colorless
crystals. 1H NMR (CD3OD): d 1.21 (s, 9H, C-3-
OCOC(CH3)3), 1.90 (s, 3H, NHCOCH3), 3.71–3.82
(m, 3H, H-5, H-6), 4.05 (d, 1H, J3,4 3.2 Hz, H-4), 4.51
(dd, 1H, J2,3 11.2, J1,2 8.5 Hz, H-2), 4.94 (dd, 1H, H-
3), 5.10 (d, 1H, H-1), 6.97–7.04 (m, 3H, arom. H),
7.24–7.29 (m, 2H, arom. H); 13C NMR (CD3OD): d
22.85 (q, NHCOCH3), 27.49 (q, C-3-OCOC(CH3)3),
39.95 (s, C-3-OCOC(CH3)3), 51.47 (d, C-2), 62.16 (t,
C-6), 67.12 (d, C-4), 74.62 (d, C-3), 76.98 (d, C-5),
101.19 (d, C-1), 117.79 (d, arom. CH), 123.61 (d, arom.
CH), 130.49 (d, arom. CH), 159.14 (s, arom. C), 173.43
(s, NHCOCH3), 179.52 (s, C-3-OCOC(CH3)3).
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Acknowledgements
This work was supported by a Grant-in-Aid for Scien-
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