Novel features of acceptor recognition by β-(1 ← 4)- galactosyltransferase

Article abstract of DOI:10.1016/S0008-6215(97)10093-3

In order to understand how β-(1→4)-galactosyltransferase recognizes its glycosyl acceptor, substrate specificities were investigated using synthetic 2-acetamido-2-deoxy-D-glucopyranose (N-acetylglucosamine) derivatives in which the 1-, 2-, 3-, 4-, and 6-p

Full text of DOI:10.1016/S0008-6215(97)10093-3

Carbohydrate Research 306 (1998) 361±378
Novel features of acceptor recognition by
ꢀ-(1!4)-galactosyltransferase
Yasuhiro Kajiharaa, Hisashi Kodamab, Tsuyoshi Endoa,
Hironobu Hashimotoa*
aDepartment of Life Science, Faculty of Bioscience and Biotechnology,
Tokyo Institute of Technology, Nagatsuta, Midori-ku Yokohama 227, Japan
bLife Science Research Laboratory, Japan Tobacco Inc., 6-2 Umegaoka,
Midori-ku Yokohama 227, Japan
Received 17 June 1997; accepted 3 November 1997
Abstract
In order to understand how ꢀ-(1!4)-galactosyltransferase recognizes its glycosyl acceptor,
substrate speci®cities were investigated using synthetic 2-acetamido-2-deoxy-d-glucopyranose
(N-acetylglucosamine) derivatives in which the 1-, 2-, 3-, 4-, and 6-positions were systematically
substituted. The hydroxyl groups at the 3-, 4-, and 6-positions were substituted by ¯uoride, thiol
or hydrogen. For modi®cation of the 2- position, the acetamido group was converted to ethyla-
mino-, N-methylacetamido- and acetyloxy groups. For the anomeric position, several sugar resi-
dues were introduced as the aglycon of N-acetylglucosaminide. Galactose transfer assay using
synthetic N-acetylglucosamine derivatives indicated that both the acetamido group and the
4-hydroxyl group were essential for binding of N-acetylglucosamine toward the ꢀ-(1!4)-galacto-
syltransferase. The assay also showed that the N-acetylglucosamine having a large substitution at
the 6-position can be recognized as an acceptor. It is suggested that in this case the bulky sub-
stitutent is positioned away from the catalytic site or out of enzyme. Since the 2-acetamido and the
4-hydroxyl group are essential for recognition, the side composed of the 2, 3, and 4-positions may
face the acceptor-binding site. # 1998 Elsevier Science Ltd. All rights reserved
Keywords: ꢀ-(1!4)-galactosyltransferase; Acceptor supeci®city; Galactosyl transfer; N-acetylglucosamine
1. Introduction
2-acetamido-2-deoxy-d-glucopyranose (N-acetyl-
glucosamine). Since the GalTase is available in
large quantities bovine milk, it is useful as synthetic
catalyst for enzymatic or chemoenzymatic synth-
eses of galactosyl oligosaccharides [1]. This
enzyme has the following unique characteristics.
When GalTase forms a complex with lactal-
bumin, the substrate speci®city changes from N-
Bovine ꢀ-(1!4)-galactosyltransferase (GalTase,
EC 2.4.1.90) catalyzes the transfer of a galactose
residue from UDP-galactose to the 4-position of
* Corresponding author.
0008-6215/98/$19.00 # 1998 Elsevier Science Ltd. All rights reserved
PII S0008-6215(97)10093-3

362
Y. Kajihara et al./Carbohydrate Research 306 (1998) 361±378
acetylglucosamine to glucose [2,3]. The GalTase is
also the most extensively studied enzyme among
many kinds of glycosyltransferases. Cloning of
GalTase was performed by Narimatsu et al. [4]
in 1986, and investigation of the binding site of
UDP-galactose was reported by Brew et al. [5] in
1990. In addition, in order to investigate substrate
speci®city and to design inhibitors, modi®ed
N-acetylglucosamines [6±8], as well as UDP-mod-
i®ed-galactoses [1,9], have been synthesized. Since
modi®cation at the 4-position of N-acet-
ylglucosamine interferes with the binding to the
GalTase [6,7,10], an acceptor analogue±inhibitor
has not been found. Hindsgaul and co-workers
reported inhibitory activities of several deox-
ygenated acceptor±analogue inhibitors toward
eight kinds of glycosyltransferases and concluded
that the glycosyltransferases were divided into two
groups [11]. In the ®rst group the hydroxyl group
to be glycosylated in glycosyl acceptor is essential
for the binding to the enzyme. In contrast, the
enzyme belonging to the second group does not
require the hydroxyl group for binding. GalTase
belongs to the former group. The 4-hydroxyl
group, especially the hydrogen of the 4-hydroxyl
group of N-acetylglucosamine, is deduced to form
a hydrogen bond to the GalTase. However, recog-
nition by the GalTase of N-acetylglucosamine has
not been investigated in detail. In order to under-
stand how the GalTase recognizes the acceptor
moiety, we studied substrate speci®cities system-
atically [10] by use of modi®ed N-acet-
ylglucosamines of which hydroxyl groups are
substituted by ¯uoride, thiol or hydrogen. For
both the ¯uoro and thiol functions, the nature of
the hydrogen-bond formation is expected to be
biased to a hydrogen-accepting and a hydrogen-
donating role, respectively. In addition, deoxy-
genation is expected to remove both the hydrogen-
accepting and -donating roles of the hydroxyl
group. We report here the behaviors of a series of
synthetic N-acetylglucosamine derivatives toward
GalTase.
In order to check for speci®city toward the
anomeric position, we prepared N-acet-
ylglucosaminyl disaccharides whose aglycon moi-
eties were varied (14±17). A disaccharide, ꢀ-d-
GlcNAc-ꢀ-(1!3)-Gal-(1!O)Me 14, was synthesized
[12] by glycosylation of galactoside 19 with 20 [13]
and by subsequent deprotection (Scheme 1). The
disaccharide 15 was also synthesized in moderate
yield in the same manner as in the preparation of
14. Other disaccharides, 16 [14] and 17 [15] were
synthesized according to the reported method.
Deoxygenation at the 6-position of 26 was per-
formed by iodination and subsequent radical
reduction. The 3-deoxygenated derivative 8 was
derived from benzylidene acetal derivative 28
(Scheme 2) [16]. Thiocarbonylimidazolylation at
the 3-position of 28 and subsequent radical reduc-
tion and deprotection a€orded the 3-deoxygenated
derivative 8. The 4-deoxygenated glycosyl donor 33
was synthesized by radical reduction of the corre-
sponding iodo derivative 32. Glycosylation of 23
with deoxygenated donor 33 in the presence of
MeOTf and subsequent deprotection a€orded dis-
accharide 5.
Synthetic routes for glycosyl donors having
¯uoro or thiol groups are shown in Scheme 3.
Fluorination at the 6-position of 35 and the 3-
position of 39 were performed with diethylamino-
sulfur tri¯uoride (DAST) [17]. The 6-thioacetyl
derivative 38 was derived from tosyl derivative 37
by the treatment of potassium thioacetate.
Synthesis of the disaccharides having a ¯uorine
atom or a thiol group are shown in Scheme 4.
Glycosylation with the ¯uorinated 36 or 41 or
2. Results
Synthesis of N-acetylglucosamine analogues.ÐIn
order to investigate substrate speci®city, we plan-
ned to synthesize several N-acetylglucosamine
derivatives modi®ed at the 1, 2, 3, 4, and 6-posi-
tions as shown in 1±17.

Y. Kajihara et al./Carbohydrate Research 306 (1998) 361±378
363
ꢀ
.
Scheme 1. (a) H2, Pd(OH)2, MeOH; (b) MeOTf, MS 4A, CH2Cl2, 21: 92%, 24: 80%; (c) (1) H2NNH2 H2O, EtOH, 90 C, (2)
Ac2O, pyridine, DMAP, 22: 90% , 25: 83%; (d) NaOMe, MeOH, 14: 90%, 15: 81%.
thioacetylated donor 38 in the presence of MeOTf
and subsequent deprotection a€orded desired di-
saccharides 2, 9, and 3, respectively. S-deacetylation
of 47 was carried out in the presence of dl-dithio-
threitol in order to avoid disul®de formation.
However, the disul®de 4 was also derived from the
thiol 3 by spontaneous oxidation [18] in water, and
it was then used in the GalTase assay.
The 4-¯uoro 6 and 4-thiol derivatives 7 were
synthesized from methyl 2-azido-2-deoxy-ꢀ-d-
galactopyranoside 48 [19] that was derived from d-
galactal by the azidonitration method (Scheme 5).
In order to introduce a ¯uorine atom at the 4-
position, the 3- and 6-positions of 48 were selec-
tively acetylated and then treated with DAST to
a€ord the 4-deoxy-4-¯uoro compound 50. After
reduction of the azido group by hydrogen sul®de,
N-acetylation, followed by O-deacetylation, a€or-
ded 6. Formation of the tri¯ate at the 4-position of
49 and subsequent nucleophilic substitution with
potassium thioacetate a€orded the 4-thioacetyl
derivative 52. N-Acetylation and O-deacetylation
was performed in the same manner as for the pre-
paration of 6.
The two kinds of 2-modi®ed derivatives were
synthesized from the known derivative 54 (Scheme
6) [20]. In order to remove the carbonyl group
from the acetamido group, compound 54 was
reduced with LiAlH4 to a€ord the 2-ethylamino
derivative 10. N-Methylated derivative 11 was
obtained by the treatment of 54 with sodium
hydride and methyl iodide, and subsequent O-
debenzylation. In this case, the desired derivative
11 was obtained as an E/Z mixture. The 2-O-acetyl
derivative 12 [21] was prepared from the 4,6-O-
benzylidene derivative 57 by acid hydrolysis.
Enzyme assays.ÐEnzyme assays were performed
by the reported method of Babad and Hassid
[22]. Radioactive galactose was transferred from
UDP-[U-14C]-galactose to synthetic N-acetyl-
glucosamine derivatives by the action of the
GalTase, and then the 14C-labelled 2-acetamido-2-
deoxy-lactoside (N-acetyllactosaminide) was quan-
titated by liquid scintillation counting. The kinetic

364
Y. Kajihara et al./Carbohydrate Research 306 (1998) 361±378
parameters of N-acetylglucosamine derivatives
obtained for GalTase reactions are summarized
in Table 1.
In the case of the 6-modi®ed analogues, the
assay indicated that deoxy- 1, ¯uoro- 2 and thiol-
derivatives 3 were good acceptors. However, in the
case of disul®de derivatives 4, the Vmax value was
far lower than that of native disaccharide 15.
The 4-modi®ed N-acetylglucosamine derivatives,
deoxy- 5, ¯uoro- 6, and thiol-derivatives 7 were
assayed. However, the assay indicated that these
derivatives were neither acceptors nor inhibitors.
In addition, even with a 10-fold higher concentra-
tion compared to the Km value of N-acet-
ylglucosamine, derivatives 5, 6, and 7 did not
inhibit the reaction in which N-acetyllactosamine
was formed by the action of UDP-galactose, N-
acetylglucosamine, and GalTase. Therefore, this
suggests that both hydrogen and oxygen in the 4-
hydroxyl group are essential for the binding of N-
acetylglucosamine to GalTase.
In order to investigate recognition at the 3-posi-
tion of N-acetylglucosamine, 3-deoxygenated 8 and
the 3-deoxy-3-¯uoro-derivative 9 were used. In the
case of the 3-deoxy derivative 8, the Km value was
elevated by 2.8 fold and the Vmax value was
decreased to 34% compared to those of N-acet-
ylglucosamine 13. On the other hand, in the case of
the 3-deoxy-3-¯uoro derivative 9, no transfer
Scheme 2. (a) (1) Nal, DME, (2) Bu3SnH, AlBN, toluene,
77% (2 steps); (b) NaOMe, MeOH, 60%; (c) (1) Thiocarbo-
nyldiimidazole, DMAP, CH2Cl2; (2) Bu3SnH, AlBN, toluene,
85% (2 steps); (d) (1) 60% AcOH, (2) Ac2O, pyridine, (3)
NaOMe, MeOH, 77% (3 steps); (e) (1) BzCl, pyridine, (2)
60% AcOH, (3) PivCl, pyridine, 84% (3 steps); (f) (1) Tf2O,
pyridine, CH2Cl2, (2) NaI, DME, 99% (2 steps); (g) Bu3SnH,
AlBN, toluene, 87%; (h) (1) MeOTf, MS4A, CH2Cl2, (2)
activity was observed due to a decrease of Vmax
.
However, the anity was changed only slightly
(Ki=4.2 mM). These results suggested that mod-
i®cation at the 3-position a€ected the reaction
velocity rather than the anity.
.
H2NNH2 H2O, EtOH, (3) Ac2O, pyridine, 66% (3 steps); (i)
NaOMe, MeOH, 88%.
It is known that 2-deoxy-2-propionamido-d-glu-
cose is a good acceptor for GalTase [23]. However,
GalTase can not recognize 2-ethylamino- 10, 2-N-
methylacetamido- 11 and 2-O-acetyl-d-glucopyr-
anosides 12. In addition, even with a 10-fold higher
concentration compared to the Km value of N-
acetylglucosamine, these derivatives did not act as
inhibitors. These results suggested that the whole
acetamido group is essential for binding of N-acet-
ylglucosamine to the GalTase.
In order to investigate the recognition of the
reducing end, or aglycon, of N-acetylglucosamine
by GalTase, four disaccharides, 14, 15, 16, and 17,
having di€erent aglycons and linkages, were used
in the GalTase assay. Since their Km values were
similar, we conclude that GalTase does not recog-
nize the aglycon sugar moiety and linkage to a sig-
ni®cant degree.
Scheme 3. (a) (1) DAST, CH2Cl2, (2) Ac2O, pyridine, 38% (2
steps); (b) (1) TsCl, puridine, (2) Ac2O, pyridine, 92% (2
steps); (c) KSAc, DMF, 82%; (d) (1) Swern oxidation, (2)
NaBH4, EtOH±H2O, 96% (2 steps), gluco:allo=1:4, (e)
DAST, CH2Cl2, 50%, (f) (1) 60% AcOH, (2) Ac2O, pyridine,
63% (2 steps.

Y. Kajihara et al./Carbohydrate Research 306 (1998) 361±378
365
.
Scheme 4. (a) MeOTf, MS4A, CH2Cl2, 43: 44%, 46:75%; (b) (1) H2NNH2 O, EtOH, (2) Ac2O, pyridine, DMAP, 44: 44% (3
steps), 45: 56% (2 steps), 47: 76%; (c) NaOMe, MeOH, 2: 82%, 9: 71%; (d) NH4OH, dl-dithiothreitol, MeOH, 56%; (e) H2O
(56 mM). 37 ꢀC, 67%.
Scheme 5. (a) Ac2O, pyridine, CH2Cl2, 66%; (b) DAST,
CH2Cl2, 48%; (c) (1) H2S, pyridine, triethylamine, H2O, (2)
Scheme 6. (a) LiAlH4, Et2O, re¯ux 76%; (b) Pd/C, H2,
MeOH, 10: 87%, 11: 85%; (c) NaH, Mel, DMF, 99%; (d)
60% AcOH, 72%.
Ac2O, pyridine, 51: 98%, 53: 84%; (d) NaOMe, MeOH, 91%;
(e) (1) Tf2O, pyridine, CH2Cl2, (2) KSAc, DMF, 85% (2
steps); (g) NH4OH, dl-dithiothreitol, MeOH, 62%.

366
Y. Kajihara et al./Carbohydrate Research 306 (1998) 361±378
Table 1
Kinetics parameters of GlcNAc-OR derivatives for the
galactosylation with bovine ꢀ-1,4GalTase
Compd
Km (nM)
Vmax (relative
1
2
3
4
0.5
1.0
1.2
0.2
55
94
33
1.7
5
6
7
no binding
no binding
no binding
8
9
4.2
2.7a
34
<1
10
11
12
13
14
15
16
17
no binding
no binding
no binding
Fig. 1. Schematic representation of the binding of methyl 2-
acetamido-2 deoxy-ꢀ-d-glucopyranoside to GalTase.
1.5
1.4
0.7
1.0
2.8
100
94
66
108
100
to the ꢀ-anomeric position of 3-acetamido-3-
deoxy-d-glucopyranose [24,25]. This unusual
transfer also suggests that the GalTase recognized
the W-like orientation as a recognition marker.
The assay shows that the disul®de disaccharide 4
is an acceptor (Km=0.2 mM). Hindsgaul and co-
workers have also reported the same tendency for
the N-acetylglucosamine having a large substitu-
tion at the 6-position such as a sialic acid methyl
ester or fucose to act as galactosyl acceptor [7].
Therefore, the 6-position of N-acetylglucosamine is
not of importance for its binding to the GalTase. It
is reasonable to assume that in this case the bulky
substitutent is positioned away from the catalytic
site or outside of the enzyme altogether.
Considering the assay results described above,
the binding mode of N-acetylglucosamine to Gal-
Tase can be deduced. Since the 2-acetamido and
the 4-hydroxyl groups are essential for recognition,
the side that is formed by the 2-, 3-, 4-positions may
face the acceptor-binding site. The 6-position, as
well as the aglycon at the reducing end, may be
positioned outside of the acceptor-binding site.
These interrelationships are shown in Fig. 1.
aKi value.
3. Discussion
The GalTase assay using the synthetic N-acet-
ylglucosamine derivatives suggests that modi®ca-
tion at both the 2- and 4-position severely
interfered the binding of the N-acetylglucosamine
to the GalTase. Therefore, GalTase recognized
both the acetamido and the 4-hydroxyl groups. In
other words, a W-like orientation of the acetamido
group and the 4-hydroxyl group is characteristic
enough to distinguish N-acetylglucosamine from
other amino sugars. Since there is no other sugar
that has the same W-like orientation among the
sugar residues of the glycoconjugates, it may be
reasonable that GalTase recognizes the W-like
orientation in N-acetylglucosamine as a crucial
marker, (see structures below).
4. Conclusions
In order to investigate the substrate speci®city of
the ꢀ-(1!4)-galactosyltransferase in detail, several
N-acetylglucosamine analogues, in which the 1-, 2-,
3-, 4-, and 6-positions were individually chemically
modi®ed, were synthesized and subjected to
enzyme assay. The enzyme assay indicated that the
ꢀ-(1!4)-galactosyltransferase recognizes the W-
like orientation of the 2-acetamido and 4-hydroxyl
Nishida et al. reported that the GalTase cata-
lyzes the transfer of a galactose from UDP-galactose

Y. Kajihara et al./Carbohydrate Research 306 (1998) 361±378
367
groups. Since there is no report of an X-ray crystal
structure of the GalTase, derivatization of sub-
strates and their enzyme assays may serve as the
best methods to obtain clues for the binding of N-
acetylglucosamine to the acceptor-binding site of
the enzyme. The results obtained here, together
with previous ones concerning substrate speci®city
toward the UDP-galactose [26], enabled us to
design and synthesize a potent tricomponent Gal-
Tase-inhibitor composed of UDP, galactose, and
N-acetylglucosamine [27].
excess benzyl bromide by the addition of the
NaOMe (excess), the mixture was concentrated in
vacuo. Then saturated aq KF solution 100 mL
was added to the residue, which was then stirred
at room temperature. The mixture was then diluted
with EtOAc, and the organic phase was washed
with water and then dried with MgSO4. After con-
centration of the mixture, the residue was dissolved
in acetic anhydride and pyridine (1:1, 10 mL).
After stirring for 0.5 h at room temperature, the
mixture was concentrated in vacuo. Puri®cation
of this residue by the ¯ash column chromato-
graphy (3:2 hexane±ethyl acetate) a€orded 18
(7.4 g, 70%) : [ꢁ]d23+40.66 ꢀ(c 0.9, CHCl3); mp 120±
5. Experimental section
122 ꢀC; H NMR: ꢂ 7.4±7.18 (m, 5 H, Ph), 5.52 (d,
1
General.Ð1H NMR spectra were recorded with
JEOL EX-270 or Bruker AM 500 instruments at
298 K. The chemical shifts are presented in ppm
and referenced to tetramethylsilane in CDCl3, or
sodium 3-(trimethylsilyl)propionate in D2O as the
internal or external standard, respectively. 13C
NMR spectra were recorded with a JEOL EX-270
at 298 K. The chemical shifts are expressed in ppm
and referenced to tetramethylsilane in CDCl3, or
dioxane (67.4 ppm) in D2O as the internal or
external standard, respectively. Optical rotations
were measured with a JASCO DIP-4. High-resolu-
tion mass spectra were recorded on a JMS-SX102A
or Shimazu/Kratos concept-IIH under FAB con-
ditions. All reactions were monitored by TLC
(Silica Gel 60-F254, E. Merck) by charring after
spraying with 5% H2SO4 in methanol. Wako-Gel
C-300 was used for ¯ash column chromatography.
Bovine ꢀ-(1!4)-galactosyltransferase (EC 2.4.1.90)
and calf intestinal alkaline phosphatase (EC
3.1.3.1) were purchased from Sigma Chemical Co.
and Boehringer Mannheim, respectively. UDP-ꢁ-
[U-14C]-d-galactose were purchased from NEN
Research Products.
1 H, J4,3 2.6 Hz, H-4), 5.12 (dd, 1 H, J2,3 9.9, J2,1
7.9 Hz, H-2), 4.71, 4.39 (d, 1 H, J 12.1 Hz, benzyl
CH2), 4.30 (d, 1 H, H-1), 4.19 (d, 2 H, J6,5 6.6 Hz,
H-6), 3.83 (dd, 1 H, H-5), 3.55 (dd, 1 H, H-3), 3.48
(s, 3 H, OMe), 2.15, 2.08, 2.03 (s, 3 H, Ac). Anal.
Calcd for C20H26O9: C, 58.53; H, 6.39. Found C,
58.35; H, 6.53.
Methyl 2,4,6-tri-O-acetyl-b-d-galactopyranoside
(19). To a solution of 18 (3.4 g, 8.27 mmol) in
MeOH (70 mL) was added Pd(OH)2 (600 mg, 10%/
C), and the mixture was stirred under a hydrogen
atmosphere for 12 h. (Caution. Extreme ®re
hazard!) After stirring for 12 h, the mixture was
passed through a Celite pad, and the ®ltrate was
concentrated in vacuo. Puri®cation of the residue
by the ¯ash column chromatography (1:2 hexane±
ethyl acetate) a€orded 19 (2.5 g, 95%): [ꢁ]d23
40.0 ꢀ (c 0.4, CHCl3); mp 98±100 ꢀC; H NMR: ꢂ
1
5.33 (dd, 1 H, J4,3 1.0, J4,3 3.6 Hz, H-4), 4.96 (dd, 1
H, J2,3 9.9, J2,1 7.9 Hz, H-2), 4.37 (d, 1 H, H-1),
4.17 (d, 2 H, J6,5 6.6 Hz, H-6), 3.88±3.82 (m, 2 H,
H-3, 5), 3.52 (s, 3 H, OMe), 2.41 (d, 1 H, JOH,3
6.3 Hz, OH), 2.18, 2.14, 2.07 (s, 3 H, Ac). Anal.
Calcd for C13H20O9: C, 48.75; H, 6.29. Found C,
49.17; H, 6.63.
Methyl
2,4,6-tri-O-acetyl-3-O-benzyl-b-d-gal-
actopyranoside (18). To a solution of methyl ꢀ-d-
galactopyranoside [28] (5.0 g, 25.7 mmol) in dry
MeOH (100 mL) was added di-n-butyltin oxide
(6.42 g, 25.7 mmol), and the mixture was stirred
under re¯ux for 2 h. The mixture was allowed to
cool to room temperature, and it was concentrated
in vacuo. The residue was dried by coevaporating
with pyridine (twice). To a solution of this residue
in dry benzene (200 mL) was added n-butylammo-
nium iodide (11.4 g, 30.93 mmol) and benzyl bro-
mide (6 mL, 51.5 mmol), and the mixture was
stirred under re¯ux for 2 h. After quenching the
Methyl 3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-
b-d-glucopyranosyl-(1!3)-2,4,6-tri-O-acetyl-b-d-
galactopyranoside (21). To a solution of acceptor
19 (47 mg, 147 ꢃmol), donor 20 (50 mg, 98 ꢃmol) in
CH2Cl2 (2.5 mL) was added MS 4A (250 mg). After
stirring for 1 h at room temperature, MeOTf
(55 ꢃL, 490 ꢃmol) was added at 0 ꢀC, and the mix-
ture was stirred at room temperature for 18 h.
After addition of triethylamine (136 ꢃL), the mix-
ture was passed through a Celite pad and con-
centrated in vacuo. Puri®cation of the residue by
the ¯ash column chromatography (1:1 hexane±

368
Y. Kajihara et al./Carbohydrate Research 306 (1998) 361±378
ethyl acetate) a€orded 21 (66 mg, 92%): [ꢁ]d25
+7.0 ꢀ (c 0.9, CH2Cl2); 1H NMR: ꢂ 7.86±7.72
(1 g). After stirring for 1 h at room temperature,
MeOTf (199 ꢃL, 1.76 mmol) was added at 0 ꢀC,
and the mixture was stirred at room temperature
for 3 h. After addition of triethylamine (490 ꢃL),
the mixture was passed through a Celite pad and
concentrated in vacuo. Puri®cation of the residue
by ¯ash column chromatography (1:2 hexane±
ethyl acetate) a€orded 24 (220 mg, 80%): [ꢁ]d23
+65.2 ꢀ (c 2.6, CH2Cl2); 1H NMR: ꢂ 7.87±7.31 (m,
0
0
0
0
(m, 4 H, Phth), 5.76 (dd, 1 H, J3 ,4 8.9, J3 ,2
0
10.9 Hz, H-30), 5.43 (d, 1 H, J1 ,2 8.3 Hz, H-10),
5.38 (dd, 1 H, J4,5 1.0, J4,3 4.6 Hz, H-4), 5.19
(dd, 1 H, J4 ,5 9.9 Hz, H-40), 4.91 (dd, 1 H, J2,1
0
0
0
0
0
7.9, J2,3 9.9 Hz, H-2), 4.40 (dd, 1 H, J6a ,5 2.6,
J6 a,6 b 12.2 Hz, H-60a), 3.38 (s, 3 H, OMe), 2.15,
2.10, 2.07, 2.03, 1.84, 1.77 (each s, each 3 H,
Ac). Anal. Calcd for C33H39NO18: C, 53.73; H,
5.33; N,1.90. Found C, 53.70; H, 5.69; N,1.78.
Methyl (3,4,6-tri-O-acetyl-2-acetamido-2-deoxy-
b-d-glucopyranosyl)-(1!3)-2,4,6-tri-O-acetyl-b-d-
galactopyranoside (22). To a solution of 21 (46 mg,
62.4 mmol) in EtOH (0.5 mL) was added
0
0
0
0
0
0
4 H, Phth), 5.78 (dd, 1 H, J3 ,4 9.2, J3 ,2 10.6 Hz,
H-30), 5.41 (d, 1cH, J1 ,2 8.6 Hz, H-10), 5.34 (dd,
0
0
0
0
1H, J3,2=J3,4 9.6 Hz, H-3), 5.18 (dt, 1 H, J4 ,5
9.6 Hz, H-40), 4.76 (bt, 1 H, J4,5 9.9 Hz, H-4), 4.68
(dd, 1 H, J2,1 3.3 Hz, H-2), 4.46 (d, 1 H, H-1), 3.49
(dd, 1 H, J6b,5 7.3, J6b,6a 10.6 Hz, H-6b), 3.02 (s, 3
H, OMe), 2.13, 2.04, 2.01, 1.93, 1.92, 1.87 (each s,
each 3 H, Ac). Anal. Calcd for C33H39NO18: C,
53.73; H, 5.32; N, 1.90. Found C, 53.91; H, 5.08;
N, 1.85.
.
H2NNH2 H2O (0.5 mL), and the mixture stirred at
90 ꢀC. After 24 h the mixture was concentrated in
vacuo. The residue was dissolved in Ac2O
(0.25 mL) and pyridine (0.25 mL) and stirred for
12 h. After concentration of the mixture in vacuo,
puri®cation of the residue by ¯ash column chro-
Methyl (3,4,6-tri-O-acetyl-2-acetamido-2-deoxy-
b-d-glucopyranosyl)-(1!_6)-2,3,4-tri-O-acetyl-a-
d-glucopyranoside (25). To a solution of 24
(100 mg, 1.28 mmol) in EtOH (1.0 mL) was added
matography (ethyl acetate) a€orded 22 (36 mg,
90%): [ꢁ]2d3 +22.95 (c 2.9, CH2Cl2); H NMR: ꢂ
ꢀ
1
.
0
5.66 (d, 1 H, JNH,2 7.9 Hz, NH), 5.52 (t, 1 H,
0
H2NNH2 H2O (1.0 mL), and the mixture was stir-
J3 ,4 =J3 ,2 9.2 Hz, H-30), 5.38 (d, 1 H, J4,3 3.3 Hz,
red at 90 ꢀC. After 24 h, the mixture was con-
centrated in vacuo. The residue was dissolved in
acetic anhydride (1.0 mL) and pyridine (1.0 mL)
and stirred for 12 h. After concentration of the
mixture in vacuo, puri®cation of the residue by
¯ash column chromatography (1:2 hexane±ethyl
acetate) a€orded 25 (73 mg, 83%): [a]2d3 +7.9 ꢀ (c
0
0
0
H-4), 5.15±5.00 (m, 3 H, H-2, 10, 40), 4.32 (dd, 1 H,
J6 a,5 2.6, J6 a,6 b 12.2 Hz, H-60a), 4.31 (d, 1 H, J1 ,2
0
0
0
0
0
0
7.9 Hz, H-10), 3.70±3.66 (m, 1 H, H-50), 3.39 (s, 3
H, OMe), 2.12 (s, 3 H, Ac), 2.11 (s, 6 H, AcÂ2),
2.08 (s, 3 H, Ac), 2.02 (s, 6 H, AcÂ2), 1.91 (s, 3 H,
Ac). Anal. Calcd for C27H39NO17: C, 49.92; H,
6.05; N, 2.16. Found C, 49.69; H, 5.94; N, 2.34.
Methyl (2-acetamido-2-deoxy-b-d-glucopyrano-
syl)-(1!3)-b-d-galactopyranoside (14). To a solu-
tion of 22 (95 mg, 147 ꢃmol) in dry MeOH (2 mL)
was added NaOMe (8 mg, 147 ꢃmol), and the
mixture was stirred at room temperature. After
20 h the mixture was neutralized by the addition of
the resin (Dowex-50WÂ8, H+) and passed through
a Celite pad. The ®ltrate was concentrated in
vacuo. Puri®cation of the residue over a gel-per-
meation column (Shephadex G-15, water) a€orded
14 [11] (52mg, 90%): 1H NMR (D2O, HOD=4.81):
ꢀ
4.3, CH2Cl2); mp 220±223 C; 1H NMR: ꢂ 5.97 (d,
0
1 H, JNH,2 8.6 Hz, NH), 5.48 (t, 1 H, J3,2=J3,4
9.2 Hz, H-3), 5.21 (t, 1 H, J3 ,2 =J3 ,4 9.2 Hz, H-30),
5.14±5.05 (m, 2 H, H-4, 40), 4.94 (d, 1 H, J1,2
3.6 Hz, H-1), 4.85 (dd, 1 H, J2,3 10.2 Hz, H-2), 4.50
0
0
0
0
(d, 1 H, J1 ,2 8.6 Hz, H-10), 4.24 (dd, 1 H, J6 a,5 4.6,
0
0
0
0
J6 a,6 b 12.2 Hz, H-60a), 3.39 (s, 3 H, OMe), 2.08,
2.07, 2.06, 2.04, 2.02, 2.00, 1.97 (s, 3 H, Ac). Anal.
Calcd for C27H39NO17: C, 49.92; H, 6.05; N, 2.16.
Found C, 49.97; H, 5.62; N, 2.05.
0
0
Methyl (2-acetamido-2-deoxy-b-d-glucopyrano-
ꢂ 4.70 (d, 1 H, J1 ,2 8.6 Hz, H-10), 4.31(d, 1 H, J1,2
8.6 Hz, H-1), 4.14 (d, 1 H, J4,3 3.1 Hz, H-4), 3.90 (bd,
syl)-(1!6)-a-d-glucopyranoside (15). To
a
0
0
solution of 25 (52 mg, 80 ꢃmol) in dry MeOH
(1 mL) was added NaOMe (2 mg, 40 ꢃmol), and
the mixture was stirred at room temperature. After
20 h, the mixture was neutralized by the addition of
the resin (Dowex-50WÂ8, H+) and then passed
through a Celite pad. The ®ltrate was concentrated
in vacuo. Puri®cation of the residue over a gel-
permeation column (Shephadex G-15, water)
J6 a,6 b 12.2 Hz, H-60a), 3.57 (s, 3 H, OMe), 2.04 (s, 3
H, Ac).
0
0
Methyl (3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-
b-d-glucopyranosyl)-(1!6)-2,3,4,-tri-O-acetyl-a-
d-glucopyranoside (24). To a solution of acceptor
23 [29] (170 mg, 530 ꢃmol), donor 20 (180 mg,
354 ꢃmol) in CH2Cl2 (3 mL) was added MS 4A

Y. Kajihara et al./Carbohydrate Research 306 (1998) 361±378
369
a€orded 15 (26 mg, 81%): [ꢁ]2d3 +51.7 ꢀ (c 0.7,
H2O); H NMR (D2O, HOD=4.81): ꢂ 4.56 (d, 1
(ethyl acetate) a€orded 27 (98 mg, 77%): [ꢁ]d23
38.0 ꢀ (c 0.3, DMSO); H NMR: ꢂ 5.44 (d, 1 H,
1
1
H, J1 ,2 8.5 Hz, H-10), 3.69 (dd, 1 H, J3,4=J3,2
9.2 Hz, H-3), 3.57 (dd, 1 H, J2,1 3.6 Hz, H-2), 3.44
(s, 3 H, OMe), 2.08 (s, 3 H, Ac); m/z Calcd for
C15H27NO11Na: 420.1482 (M + Na); Found
420.1502.
JNH,2 8.3 Hz, NH), 5.19 (dd, 1 H, J3,2 10.6, J3,4
1
0
0
9.2 Hz, H-3), 4.82 (t, H, J4,5 9.6 Hz, H-4), 4.50 (d,
1 H, J1,2 8.6 Hz, H-1), 3.89 (dt, 1 H, H-2), 3.59±
3.51 (m, 3 H, H-5, 6a, 6b), 3.49 (s, 3 H, OMe),
2.04, 2.03, 1.95 (s, 3 H, Ac), 1.25 (d, 3 H, J6,5
6.3 Hz, H-6). Anal. Calcd for C13H21NO7: C,
51.48; H, 6.98; N, 4.62. Found C,51.90; H, 6.80;
N, 4.38.
Methyl 3,4-di-O-acetyl-2-acetamido-2-deoxy-6-
O-p-toluenesulfonyl-b-d-glucopyranoside (26). To a
solution of methyl 2-acetamido-2-deoxy-b-d-glu-
copyranoside (13) [30] (400 mg, 1.74 mmol) in pyr-
idine (5 mL) was added p-toluenesulfonyl chloride
(398 mg, 2.08 mmol) at 0 ꢀC, and the mixture was
stirred for 2 h at 0 ꢀC. The mixture was diluted with
ethyl acetate and washed with aq NaHCO3. The
organic phase was dried with MgSO4 and con-
centrated in vacuo. The residue was dissolved in
acetic anhydride and pyridine (0.5 mL:0.5 mL),
and the mixture was stirred at room temperature.
Concentration of this mixture gave crystalline 26
that was recrystallized from ethanol (390 mg,
48%): [ꢁ]2d3 +19.7 ꢀ (c 0.6, CH2Cl2); mp 192 ꢀC
(dec); 1H NMR: ꢂ 7.78, 7.35 (d, each 2 H, J 8.6 Hz,
Ar), 5.42 (d, 1 H, JNH,2 9.2 Hz, NH), 5.22 (dd, 1 H,
J3,4 9.2, J3,2 10.6 Hz, H-3), 4.90 (bdd, 1 H, J4,5
9.9 Hz, H-4), 4.51 (d, 1 H, J1,2=8.3 Hz, H-1), 4.13
(dd, 1 H, J6a,5 3.3, J6a,6b 10.9 Hz, H-6a), 4.07 (dd, 1
H, J6b,5 5.6 Hz, H-6b), 3.85±3.71 (m, 2 H, H-2, 5),
3.43 (s, 3 H, OMe), 2.46 (s, 3 H, Ts-Me), 2.01, 2.00,
1.94 (s, 3 H, Ac). Anal. Calcd for C20H27NO10S: C,
50.73; H, 5.75; N, 2.96. Found C,50.26; H, 5.72; N,
3.14.
Methyl 2-acetamido-2,6-dideoxy-b-d-glucopyr-
anoside (1). To a solution of methyl 3,4-di-O-
acetyl-2-acetamido-2,6-dideoxy-b-d-glucopyrano-
side (27) (41 mg, 0.135 mmol) in dry MeOH
(0.5 mL) was added a catalytic amount of NaOMe,
and the mixture was stirred at room temperature.
After 5 h, the mixture was neutralized by the addi-
tion of the resin (Dowex-50WÂ8, H+) and ®ltered.
Puri®cation of the concentrated ®ltrate over a gel-
permeation column (Shephadex G-15, water)
a€orded 1 (18 mg, 60%): [ꢁ]2d3 46.3 ꢀ (c 2.0,
1
H2O); H NMR (D2O, HOD=4.81): ꢂ 4.21 (d, 1
H, J1,2 8.6 Hz, H-1), 3.47 (dd, 1 H, J2,3 10.2 Hz, H-
2), 3.30±3.23 (m, 2 H, H-3, 5), 3.27 (s, 3 H, OMe),
2.99 (t, 1 H, J4,3 9.2 Hz, H-4), 1.82 (s, 3 H, Ac),
1.11 (d, 3 H, J6,5 6.3 Hz, H-6); m/z Calcd for
C9H17NO5Na: 242.1005 (M + Na); Found
242.1032.
Methyl 2-acetamido-4,6-O-benzylidene-2,3-di-
deoxy-b-d-glucopyranoside (29). To a solution of
methyl 2-acetamido-4,6-O-benzylidene-2-deoxy-b-
d-glucopyranoside (28) [16] (250 mg, 771 ꢃmol)
and 4-dimethylaminopyridine (47 mg, 385 ꢃmol) in
CH2Cl2 (12 mL) was added 1,1-thiocarbonyldiimi-
dazole (165 mg, 925 ꢃmol), and the mixture was
stirred under re¯ux for 1 h. The mixture was
allowed to cool to room temperature, and then it
was diluted with CHCl3. The organic phase was
washed with brine and then dried with MgSO4.
After concentration of the organic phase in vacuo,
a solution of this residue in toluene (30 mL) was
treated with AIBN (10 mg) and tri-n-butyltin
hydride (415 ꢃL, 1.54 mmol) and stirred under
re¯ux. After 2 h, the mixture was allowed to cool to
room temperature and n-hexane (20 mL) was
added. Crystalline 29 was isolated by ®ltration
(203 mg, 85%): [ꢁ]2d5 86.4ꢀ (c 0.5, DMSO); mp
270 ꢀC (dec); 1H NMR (DMSO-d6): ꢂ 7.94 (d, 1 H,
JNH,2 8.6 Hz, NH), 7.43±7.33 (m, 5 H, Ph), 5.59 (s,
1 H, PhCH), 4.37 (d, 1 H, J1,2 8.3 Hz, H-1), 4.20
(dd, 1 H, J6a,5 5.0, J6a,6b 10.2 Hz, H-6a), 3.73 (t, 1
H, J6b,5 10.2 Hz, H-6b), 2.13 (ddd, 1 H, J3e,4=J3e,2
Methyl 3,4-di-O-acetyl-2-acetamido-2,6-dideoxy-
b-d-glucopyranoside (27). To a solution of methyl
3,4-di-O-acetyl-2-acetamido-2-deoxy-6-O-p-toluene-
sulfonyl-b-d-glucopyranoside
(26)
(200 mg,
0.422 mmol) in dimethoxyethane (3 mL) was added
NaI (126 mg, 0.846 mmol), and the mixture was
stirred at 80 ꢀC. After 4 h the mixture was diluted
with ethyl acetate and washed with brine (Â2). The
organic phase was dried and then concentrated in
vacuo. After drying this residue by vacuum pump,
a solution of this residue in dry toluene (20 mL)
was treated with AIBN (10 mg) and tri-n-butyltin
hydride (227 ꢃL, 0.846 mmol), and the mixture was
stirred under re¯ux. After 2 h the mixture was
concentrated in vacuo and ethyl acetate and satur-
ated aq KF (5 mL each) were added to the residue,
and the mixture was stirred for 1 h. The organic
phase was washed with brine (Â3), dried with
MgSO4 and concentrated in vacuo. Puri®cation of
this residue by ¯ash column chromatography

370
Y. Kajihara et al./Carbohydrate Research 306 (1998) 361±378
4.6, J3e,3a 7.6 Hz, H-3e), 1.82 (s, 3 H, Ac), 1.58
(ddd, 1 H, J3a,4=J3a,2 11.9 Hz, H-3a). Anal. Calcd
for C16H21NO5: C, 62.53; H, 6.89; N, 4.56. Found
C, 62.12; H, 6.66; N, 4.77.
NMR: d 8.11±7.33(m, 9 H, Ar), 5.93 (dd, 1 H, J3,2
10.2, J3,4 8.6 Hz, H-3), 5.54 (d, 1 H, J1,2 10.6 Hz, H-
1), 4.50 (t, 1 H, H-2), 4.50 (dd, 1 H, J6a,5 2.6, J6a,6b
12.2 Hz, H-6a), 4.43 (dd, 1 H, J6b,5 4.3 Hz, H-6b),
3.88±3.83 (m, 1 H, H-5), 3.75 (t, 1 H, J4,5 8.6 Hz,
H-4), 2.78±2.61 (m, 2 H, SCH2), 1.25 (m, 12 H, t-
Bu, SCH2CH3). Anal. Calcd for C28H31NO8S: C,
62.09; H, 5.77; N, 2.59. Found C, 62.38; H, 5.35;
N, 2.53.
Methyl 2-acetamido-2,3-dideoxy-b-d-glucopy-
ranoside (8). The 3-deoxy derivative 29 (101 mg,
328 ꢃmol) was dissolved in 60% acetic acid and
stirred at 100 ꢀC. After 6 h the mixture was con-
centrated in vacuo. In order to purify the crude
product, this residue was dissolved in acetic anhy-
dride and pyridine (0.25 mL:0.25 mL). After 1 h,
the mixture was concentrated in vacuo, and pur-
i®cation of this residue by ¯ash column chromato-
graphy (3:97 MeOH±ethyl acetate) a€orded per
acetylated 8. To a solution of the acetate derivative
in dry MeOH (1 mL) was added NaOMe (catalytic
amount), and the mixture was stirred at room
temperature. The mixture was neutralized by the
addition of the resin (Dowex-50WÂ8, H+) and
passed through a Celite pad. Puri®cation of the
concentrated ®ltrate over a gel-permeation column
(Shephadex G-15, water) a€orded 8 (56 mg, 77%):
Ethyl 3-O-benzoyl-2,4-dideoxy-4-iodo-2-phthal-
imido-6-O-pivaloyl-1-thio-b-d-galactopyranoside
(32). To a solution of pyridine (1.5 g, 19.2 mmol) in
CH2Cl2 (50 mL) was added tri¯uoromethanesufonic
anhydride (2.7 g, 9.6 mol) at 0 ꢀC. After 15 min,
thioglycoside 31 (2.6 g, 4.8 mmol) in CH2Cl2 (5 mL)
was added, and the mixture was stirred at room
temperature for 30 min. The mixture was diluted
with CHCl3 and washed with aq 1% HCl, water
and NaHCO3. The organic phase was dried with
MgSO4 and concentrated in vacuo. The residue
was used directly for the next step without pur-
i®cation. The residue was dissolved in dry dime-
thoxyethane (50 mL) and treated with NaI (3.5 g,
24 mol). After stirring at room temperature for
12 h, the mixture was diluted with ethyl acetate and
washed with brine. The organic phase was dried
with MgSO4 and concentrated in vacuo. Puri®ca-
tion of the residue by ¯ash column chromato-
graphy (3:2 hexane±ethyl acetate) a€orded 32
[ꢁ]2d3
34.8 ꢀ (c 1.2, H2O); 1H NMR (D2O,
HOD=4.81): d 4.46 (d, 1 H, J1,2 8.6 Hz, H-1), 3.95
(dd, 1 H, J6a,5 2.3, J6a,6b 12.2 Hz, H-6a), 3.74 (dd, 1
H, J6b,5 6.3, H-6b), 3.54 (s, 3 H, OMe), 3.52±3.46
(m, 1 H, H-5), 2.32 (ddd, 1 H, J3e,4=J3e,2 4.9, J3e,3a
12.5 Hz, H-3e), 2.02 (s, 3 H, Ac), 1.58 (ddd, 1 H,
J3a,4=J3a,2 12.2 Hz, H-3a); m/z Calcd for
C9H17NO5Na: 242.1005 (M + Na); Found
242.1003.
(2.55 g, 99%): [ꢁ]2d3 +63.8 ꢀ (c 4.4, CH2Cl2); H
1
NMR: d 7.97±7.36 (m, 9 H, Ar), 5.54 (d, 1 H, J1.2
10.6 Hz, H-1), 5.35 (dd, 1 H, J3,2 10.6, J3,4 4.3 Hz,
H-3), 4.97 (dd, 1 H, J4,5 1.0 Hz, H-4), 4.91 (t, 1 H,
H-2), 4.39 (dd, 1 H, J6a,5 6.6, J6a,6b 11.6 Hz, H-6a),
4.14 (dd, 1 H, J6b,5 5.9 Hz, H-6b), 3.48 (m, 1 H, H-
5), 2.82±2.68 (m, 2 H, SCH2), 1.23 (m, 12 H, t-Bu,
SCH2CH3). Anal. Calcd for C28H30INO7S: C,
51.56; H, 4.60; N, 2.15. Found C, 51.29; H, 4.54;
N, 2.01.
Ethyl 3-O-benzoyl-2,4-dideoxy-2-phthalimido-6-
O-pivaloyl-1-thio-b-d-glucopyranoside (33). To a
solution of 32 (740 mg, 1.38 mmol) in toluene
(13 mL) was added AIBN (10 mg) and tri-n-butyl-
tin hydride (0.74 mL, 2.80 mmol), and the mixture
was stirred under re¯ux. After 2.5 h the mixture
was allowed to cool to room temperature, and the
mixture was diluted with a solution of ether and
saturated aq KF (50 mL:50 mL). The mixture was
stirred for 2.5 h, and then the organic phase was
washed with brine (Â3) and dried with MgSO4.
After concentration in vacuo, puri®cation of the
residue by ¯ash column chromatography
Ethyl 3-O-benzoyl-2-deoxy-2-phthalimido-6-O-
pivaloyl-1-thio-b-d-glucopyranoside (31). To a solu-
tion of ethyl 4,6-O-benzylidene-2-deoxy-2-phthali-
mido-1-thio-b-d-glucopyranoside 30 [13] (7.3 g,
16.5 mmol) in pyridine (5 mL) was added benzoyl
chloride (2.5 g, 18.2 mmol), and the mixture was
stirred at room temperature for 30 min. The mix-
ture was diluted with CHCl3 and washed with 1%
HCl solution and then water. After drying with
MgSO4, the organic phase was concentrated in
vacuo. The residue was dissolved in 60% acetic
acid, and the mixture was stirred at 100 ꢀC. After
1 h, the mixture was concentrated in vacuo. To a
solution of this residue in pyridine (20 mL) was
slowly added pivaloyl chloride (2.19 g, 18.1 mmol)
at 0 ꢀC, and the mixture was stirred for 2 h. The
mixture was then diluted with ethyl acetate and
washed with aq NaHCO3, and concentrated. Pur-
i®cation of these residue by ¯ash column chroma-
tography (3:2 hexane±ethyl acetate) a€orded 31
(7.55 g, 84%): [ꢁ]2d3 +45.9 ꢀ (c 2.2, CH2Cl2); H
1

Y. Kajihara et al./Carbohydrate Research 306 (1998) 361±378
371
(hexane! 3:2 hexane±ethyl acetate) a€orded 33
temperature. After 1 h, MeOH (2 mL) was added at
10 ꢀC, and the mixture was concentrated in
vacuo. Acetic anhydride and pyridine were added
to the residue with stirring at room temperature.
After concentration of the mixture, puri®cation of
the residue by ¯ash column chromatography (3:1
hexane±ethyl acetate) a€orded 36 (145 mg, 38%):
(492 mg, 87%): [ꢁ]2d3 +59.9 ꢀ (c 6.0, EtOH); H
1
NMR: ꢂ 7.90±7.33 (m, 9 H, Ar), 5.92 (ddd, 1 H,
J3,2=J3,4a 10.6, J3,4e 5.3 Hz, H-3), 5.53 (d, 1 H, J1,2
10.2 Hz, H-1), 4.45 (t, 1 H, H-2), 4.27±4.04 (m, 3
H, H-5, 6, 60), 2.80±2.60 (m, 2 H, SCH2), 2.47 (dd,
1 H, J4e,4a 11.6 Hz, H-4e), 1.77 (dt, 1 H, J4a,5
11.6 Hz, H-4a), 1.40±1.17 (m, 12 H, t-Bu,
SCH2CH3). Anal. Calcd for C28H31NO7S: C,
63.98; H, 5.95; N, 2.67. Found C, 64.32; H, 6.10;
N, 2.56.
[ꢁ]2d3 +43.0 ꢀ (c 0.4, CHCl3); mp 127±129 ꢀC; H
1
NMR: ꢂ 7.88±7.33 (m, 4 H, Phth), 5.86 (dd, 1 H,
J3,2=J3,4 9.9 Hz, H-3), 5.50 (d, 1 H, J1,2 10.6 Hz,
H-1), 5.14 (bdd, 1 H, J4,5 10.2 Hz, H-4), 4.52 (dd, 2
H, J6,5 4.3, J6,F 47.18 Hz, H-6), 4.39 (bt, 1 H, H-2),
3.99±3.85 (m, 1 H, H-5), 2.76±2.61 (m, 2 H, SCH2),
2.06, 1.87 (s, 3 H, Ac), 1.22 (t, 3 H, J 7.6 Hz,
SCH2CH3). Anal. Calcd for C20H22FNO7S: C,
54.67; H, 5.01; N, 3.18. Found C, 54.55; H, 5.06;
N, 3.14.
Methyl (3,6-di-O-acetyl-2-acetamido-2,4-dideoxy-
b-d-glucopyranosyl)-(1!6)-2,3,4-tri-O-acetyl-a-d-
glucopyranoside (34). The preparation of di-
saccharide 34 with donor 33 (111 mg, 211 ꢃmol)
and acceptor 23 (130 mg, 406 ꢃmol) was performed
with the same manner in the preparation of 22.
Puri®cation of this disaccharide by ¯ash column
chromatography (ethyl acetate) a€orded 34
(105 mg, 66% 2 steps): [ꢁ]2d3 +50.0 ꢀ (c 1.9,
Ethyl 3,4-di-O-acetyl-2-deoxy-2-phthalimido-6-O-
p-toluenesulfonyl-1-thio-b-d-glucopyranoside (37).
To a solution of ethyl 2-deoxy-2-phthalimido-1-
thio-b-d-glucopyranoside (35) (4.26 g, 12.1 mmol)
in pyridine (30 mL) was added p-toluenesulfonyl
chloride (2.76 g, 14.5 mmol) at 0 ꢀC. After 1 h the
mixture was poured into aq NaHCO3 and extrac-
ted with CHCl3. The organic phase was washed
with 1% aq HCl and water. After drying with
MgSO4, the mixture was concentrated in vacuo.
The residue was dissolved in acetic anhydride and
pyridine and stirred for 2 h. After concentration of
the mixture in vacuo, puri®cation of the con-
centrated mixture by ¯ash column chromato-
graphy (3:1 hexane±ethyl acetate) a€orded 37
ꢀ
CH2Cl2); mp 172±174 C; 1H NMR: ꢂ 5.93 (d, 1 H,
JNH,2 8.6 Hz, NH), 5.48 (dd, 1 H, J3,2=J3,4 9.6 Hz,
H-3), 5.12 (dd, 1 H, J4,5 9.9 Hz, H-4), 5.92 (ddd, 1
H, J3 ,2 =J3 ,4 a 11.2, J3 ,4 e 5.0 Hz, H-30), 4.94 (d, 1
H, J1,2 3.6 Hz, H-1), 4.86 (dd, 1 H, J2,3 9.9 Hz, H-
0
0
0
0
0
0
2), 4.32 (d, 1 H, J1 ,2 8.3 Hz, H-10), 4.22±4.05 (m, 3
H, H-60a, 60b, 6a), 3.93±3.83 (m, 2 H, H-20, 5),
3.76±3.70 (m,1 H, H-50), 3.39 (s, 3 H, OMe), 3.36
(dd, 1 H, J6b,5 4.0, J6b,6a 11.6 Hz, H-6b), 2.08 (s, 3
H, Ac), 2.06 (s, 6 H, AcÂ2), 2.06, 1.99 (s, 3 H, Ac),
0
0
0
0
0
0
0
0
1.63 (ddd, 1 H, J4 a,5 =J4 a,3 =J4 a,4 e 11.9 Hz, H-
40a). Anal. Calcd for C25H37NO15: C, 50.76; H,
6.30; N, 2.37. Found C, 50.48; H, 6.20; N, 2.32.
Methyl (2-acetamido-2,4-dideoxy-b-d-glucopyr-
anosyl)-(1!6)-a-d-glucopyranoside (5). O-Deace-
tylation was performed in the same manner as for
the preparation of 14. Puri®cation with the gel-
permeation column (Sephadex G-15, water) a€or-
(6.57 g, 92%): [ꢁ]2d3 +46.7 ꢀ (c 2.4, CH2Cl2); H
1
NMR: ꢂ 7.86±7.75 (m, 6 H, Ar), 7.36 (d, 2 H, J
7.9 Hz, Ar), 5.79 (dd, 1 H, J3,4 9.2, J3,2 10.2 Hz, H-
3), 5.42 (d, 1 H, J1,2 10.6 Hz, H-1), 5.02 (dd, 1 H,
J4,5 10.2 Hz, H-4), 4.31 (dd, 1 H, H-2), 4.18 (dd, 1
H, J6a,5 3.3, J6a,6b 11.2 Hz, H-6a), 4.10 (dd, 1 H,
J6b,5 3.3 Hz, H-6b), 3.95±3.88 (m, 1 H, H-5), 2.69±
2.54 (m, 2 H, SCH2), 2.46 (s, 3 H, Ts), 2.00, 1.84 (s,
3 H, Ac), 1.18 (t, 3 H, J 7.3 Hz, SCH2CH3). Anal.
Calcd for C27H29NO10S2: C, 54.81; H, 4.94; N,
2.37. Found C, 54.94; H, 4.57; N, 2.25.
Ethyl 3,4-di-O-acetyl-2-deoxy-2-phthalimido-6-
thioacetyl-1-thio-b-d-glucopyranoside (38). To a
solution of 37 (5.57 g, 9.45 mmol) in N,N-dime-
thylformamide (30 mL) was added potassium
thioacetate (5.39 g, 47.2 mmol), and the mixture
was stirred at 80 ꢀC under an argon atmosphere.
After 3 h the mixture was concentrated in vacuo.
Puri®cation of the residue by ¯ash column chro-
matography (2:1 hexane±ethyl acetate) a€orded 38
ded 5 (88%): [ꢁ]2d5 +35.6 ꢀ (c 1.0, H2O); H NMR
1
0
0
(D2O, HOD=4.81): ꢂ 4.47 (d, 1 H, J1 ,2 12.9 Hz,
H-10), 3.43 (s, 3 H, OMe), 2.08 (s, 3 H, Ac), 2.06
(ddd, 1 H, J4 e,5 =J4 a,3 4.6, J4 a,4 e 11.5 Hz, H-40e),
0
0
0
0
0
0
1.48 (ddd, 1 H, J4 a,5 =J4 a,3 11.5 Hz, H-40a); m/z
Calcd for C15H27NO10Na: 404.1533; (M + Na);
Found 404.1539.
0
0
0
0
Ethyl
3,4-di-O-acetyl-2,6-dideoxy-6-¯uoro-2-
phthalimido-1-thio-b-d-glucopyranoside (36). To a
solution of ethyl 2-deoxy-2-phthalimido-1-thio-b-
d-glucopyranoside (35) [13] (310 mg, 0.87 mmol) in
CH2Cl2 (6.4 mL) was added diethylaminosulfur
tri¯uoride (DAST, 634 ꢃL, 5.20 mmol) at 40 ꢀC,
and the mixture was allowed to warm to room

372
Y. Kajihara et al./Carbohydrate Research 306 (1998) 361±378
(4.08 g, 82%): [ꢁ]2d3 +47.2 ꢀ (c 0.5, CHCl3); mp
concentrated mixture by ¯ash column chromato-
graphy (3:1 hexane±ethyl acetate) a€orded 40
(431 mg, 50%): [ꢁ]2d3 +7.1 ꢀ (c 2.2, CHCl3); mp
148±150 ꢀC; H NMR: ꢂ 7.92±7.67 (m, 4 H, Phth),
1
5.80 (dd, 1 H, J3,2 9.0, J3,4 10.11 Hz, H-3), 5.45 (d,
1 H, J1,2 10.6 Hz, H-1), 5.06 (dd, 1 H, J4,5 9.9 Hz,
H-4), 4.36 (dd, 1 H, H-2), 3.82 (ddd, 1 H, J5,6a 4.0,
J5,6b 8.0 Hz, H-5), 3.35 (dd, 1 H, J6a,6b 16.0 Hz, H-
6a), 3.07 (dd, 1 H, H-6b), 2.81±2.56 (m, 2 H,
SCH2), 2.36, 2.09, 1.85 (s, 3 H, Ac), 1.23 (t, 3H, J
7.6 Hz, SCH2CH3). Anal. Calcd for C22H25NO8S2:
C, 53.32; H, 5.09; N, 2.83. Found C, 53.25; H,
5.09; N, 2.85.
149±151 ꢀC; H NMR: ꢂ 7.98±7.20 (m, 9 H, Ar),
1
5.59 (s, 1 H, PhCH), 5.50 (ddd, 1 H, J3,2=J3,4 8.6,
J3,F 44.4 Hz, H-3), 5.40 (d, 1 H, J1,2 10.6 Hz, H-1),
2.68 (q, 2 H, J 7.3 Hz, SCH2), 1.17 (t, 3 H,
SCH2CH3). Anal. Calcd for C23H22FNO5S: C,
62.29; H, 5.00; N, 3.16. Found C, 61.82; H, 4.95;
N, 3.27.
Ethyl
4,6-di-O-acetyl-2,3-dideoxy-3-¯uoro-2-
Ethyl 4,6-O-benzylidene-2-deoxy-2-phthalimido-
1-thio-b-d-allopyranoside (39). To a cold ( 78 ꢀC)
stirred solution of oxalyl chloride (11.5 g,
90.7 mmol) in CH2Cl2 (300 mL) was slowly added
DMSO (8.84 g, 113 mmol). After the mixture was
stirred at 78 ꢀC for 10 min, ethyl 4,6-O-benzyli-
dene-2-deoxy-2-phthalimido-1-thio-b-d-glucopyrano-
side 30 (10.0 g, 22.7 mmol) in CH2Cl2 (50 mL)
was added, and the mixture was stirred at that
temperature for 20 min. Triethylamine (ca. 25 mL)
was slowly added, and then the mixture was
allowed to warm to room temperature. The mix-
ture was diluted with CHCl3 and washed with aq
NaHCO3. After drying with MgSO4, the mixture
was concentrated in vacuo. The residue was dis-
solved in ethyl acetate and H2O (300 mL, 3:1), and
to this mixture was added NaBH4 (4.2 g) at 0 ꢀC.
After 30 min the mixture was diluted with ethyl
acetate and washed with water. After drying with
MgSO4, the organic phase was concentrated in
vacuo. Puri®cation of the residue by ¯ash column
chromatography (3:1 hexane±ethyl acetate) a€or-
ded a diastereomeric mixture (9.6 g, 96%; glu-
co:allo=1:4). The allopyranoside 39 was
recrystallized from hexane±ethanol: [ꢁ]2d3 80.5 ꢀ (c
1.3, CHCl3); mp 160±162 ꢀC; 1H NMR: ꢂ 7.90±7.33
(m, 9 H, Ar), 6.02 (d, 1 H, J1,2 10.2 Hz, H-1), 5.62
(s, 1 H, PhCH), 4.47±4.41 (m, 3 H, H-2, 3, 4), 4.27
(ddd, 1 H, J 5.3, 9.6 Hz, H-5), 3.82 (bt, 2 H, H-6a,
6b), 3.14 (s, 1 H, OH), (dd, 1 H, H-6b), 2.78±2.65
(m, 2 H, SCH2), 1.24 (t, 3 H, J 7.6 Hz, SCH2CH3).
Anal. Calcd for C23H23NO6S: C, 62.57; H, 5.25; N,
3.17. Found C, 62.54; H, 5.21; N, 3.15.
phthalimido-1-thio-b-d-glucopyranoside (41). The
¯uoro sugar 40 (2.5 g, 5.63 mmol) was dissolved in
70% acetic acid (50 mL) and the mixture was stir-
red at 60 ꢀC. After 3 h the mixture was con-
centrated in vacuo, and the residue was treated
with acetic anhydride and pyridine (10 mL:10 mL)
for 30 min. Puri®cation of the concentrated mix-
ture by ¯ash column chromatography (3:1 hexane±
ethyl acetate) a€orded 41 (1.57 g, 63%): [ꢁ]d23
+22.9 ꢀ (c 2.5, CHCl3); H NMR: ꢂ 7.88±7.76 (m,
1
4 H, Phth), 5.48±5.24 (m, 2 H, H-3,4), 5.33 (d, 1 H,
J1,2 10.9 Hz, H-1), 4.51 (ddd, 1 H, J2,3=J2,F 9.9 Hz,
H-2), 4.32 (dd, 1 H, J6a,5 4.9, J6a,6b 12.5 Hz, H-6a),
4.20 (bd, 1 H, H-6b), 3.85 (m, 1 H, H-5), 2.77±2.62
(m, 2 H, SCH2), 2.12 (s, 6 H, Ac), 1.22 (t, 3 H, J
7.6 Hz, SCH2CH3). 13C NMR: ꢂ 170.51, 169.15,
167.92, 166.86, 89.18 (d, J3,F 190.4 Hz, C-3), 80.73
(d, J1,F 6.1 Hz, C-1), 75.20 (d, J5,F 7.4 Hz, C-5),
68.92 (d, J4,F 18.3 Hz, C-4), 61.98 (C-6), 53.69 (d,
J2,F 18.3 Hz, C-2), 24.10 (SCH2), 20.60, 20.52 (each
Ac), 14.68 (SCCH3). Anal. Calcd for C20H22
FNO7S: C, 54.67; H, 5.01; N, 3.18. Found C,
54.64; H, 5.17; N, 3.10.
Methyl(3,4-di-O-acetyl-2-acetamido-2,6-dideoxy-
6-¯uoro-b-d-glucopyranosyl)-(1!3)-2,4,6-tri-O-
acetyl-b-d-galactopyranoside (44). Preparation of
disaccharide 42 with donor 36 (200 mg, 420 ꢃmol)
and acceptor 19 (270 mg, 850 ꢃmol) was performed
in the same manner as in the preparation of 21.
Puri®cation of crude disaccharide by ¯ash column
chromatography (1:5 hexane±ethyl acetate) a€or-
ded disaccharide 42 (176 mg). This disaccharide
.
was dissolved in EtOH:H2NNH2 H2O (1.4 mL:
Ethyl 4,6-O-benzylidene-2,3-dideoxy-3-¯uoro-2-
phthalimido-1-thio-b-d-glucopyranoside (40). To a
solution of allopyranoside 39 (850 mg, 1.93 mmol)
in CH2Cl2 (20 mL) was added DAST (932 mg,
5.78 mmol) at 40 ꢀC. The mixture was allowed to
slowly warm to room temperature and stirred for
2 h. The mixture was again cooled to 10 ꢀC, and
MeOH (6 mL) was added. Puri®cation of the
1.4 mL) and the mixture was stirred at 90 ꢀC. After
24 h the mixture was concentrated in vacuo. The
residue was treated with acetic anhydride (0.5 mL),
pyridine (0.5 mL) and DMAP at room tempera-
ture. After 6 h the mixture was concentrated in
vacuo and puri®cation of this residue by ¯ash col-
umn chromatography (1:5 hexane±ethyl acetate)
a€orded 44 (113 mg, 44%): [ꢁ]2d3 +43.0 ꢀ (c 2.6,

Y. Kajihara et al./Carbohydrate Research 306 (1998) 361±378
373
1
0
0
CH2Cl2); H NMR: ꢂ 5.72 (d, 1 H, JNH,2 7.6 Hz,
123.56, 123.36, 101.69 (C-1), 97.58 (d, J1 ,F 11.0 Hz,
0
NH), 5.53 (dd, 1 H, J3 ,2 10.6, J 9.6 Hz, H-30), 5.41
(bd, 1 H, J4,3 3.3 Hz, H-4), 5.13 (dd, 1 H, J2,3 9.9,
C-10), 88.12 (d, J3 ,F 190.4 Hz, C-30), 75.96 (C-3),
0
0
30,40
71.12 (d, J5 ,F 4.9 Hz, C-50), 71.09, 70.39, 68.97 (d,
0
J2,1 8.3 Hz, H-2), 5.09 (d, 1 H, J1 ,2 8.2 Hz, H-10),
J4 ,F 18.3 Hz, C-40), 68.89, 62.14, 60.99, 56.51
0
0
0
4.92 (bdd, 1 H, J4 ,5 9.9 Hz, H-40), 4.45 (dd, 2 H,
(OMe), 54.90 (d, J2 ,F 19.0 Hz, C-20), 20.81, 20.74,
0
0
0
J6 ,5 3.3, J6 ,F 46.5 Hz, H-60), 4.31 (d, 1 H, J1,2
7.9 Hz, H-1), 4.17±4.08 (m, 2 H, H-6a.b), 3.49 (s, 3
H, OMe), 3.37 (dt, 1 H, H-20), 2.13, 2.11, 2.07,
2.03, 2.02, 1.92 (s, 3H, Ac); 13C NMR: ꢂ 170.60,
170.44, 170.10, 169.75, 169.7, 169.56 (each carbon-
20.70, 20.61, 20.52 (each Me). Anal. Calcd for
C31H34FNO16: C, 53.53; H, 4.93; N, 2.01. Found
C, 53.81; H, 5.35; N, 2.06.
0
0
0
Methyl (4,6-di-O-acetyl-2-acetamido-2,3-dideoxy-
3-¯uoro-b-d-glucopyranosyl)-(1!3)-2,4,6-tri-O-
acetyl-b-d-galactopyranoside (45). N-Dephthalimi-
dation and acetylation of 43 (100 mg, 0.14 mmol)
by the same manner in the preparation of 44
a€orded crude disaccharide. Puri®cation of this
crude disaccharide by ¯ash column chromato-
graphy (ethyl acetate) a€orded 45 (49 mg, 56%):
yl), 101.94 (C-1), 99.68 (C-10), 81.57 (d, J6 ,F
0
174.6 Hz, C-60), 76.26, 72.66 (d, J5 ,F 20.8 Hz, C-50),
0
71.19 (d, J3 ,F 4.9 Hz, C-30), 70.78, 69.20, 68.59 (d,
0
J4 ,F 7.3, C-40), 62.16 (C-6), 56.84 (OMe), 55.99
0
(C-20), 23.29, 21.10, 20.77, 20.74, 20.66, 20.63
(each Ac-Me); Anal. Calcd for C25H36FNO15: C,
49.26; H, 5.95; N, 2.30. Found C, 49.35; H, 5.84;
N, 2.91.
[ꢁ]2d3 8.0 ꢀ (c 0.3, CHCl3); mp 189±191 ꢀC; H
1
0
NMR: ꢂ 5.88 (d, 1 H, JNH,2 6.6 Hz, NH), 5.38 (d, 1
0 0
H, J4,3 3.3 Hz, H-4), 5.31 (d, 1 H, J1 ,2 7.9 Hz, H-
Methyl (2-acetamido-2,6-dideoxy-6-¯uoro-b-d-
glucopyranosyl)-(1!3)-b-d-galactopyranoside (2).
O-Deacetylation of 44 (162 mg, 266 ꢃmol) by the
10), 4.36 (bd, 1 H, J6 a,6 b 12.2 Hz, H-60a), 4.31 (d, 1
H, J1,2 7.9 Hz, H-1), 3.65±3.61 (m, 1 H, H-50), 3.50
(s, 3 H, OMe), 2.99±2.91 (m, 1 H, H-20), 2.12 (s, 3
H, Ac), 2.11(s, 6 H, AcÂ2), 2.10, 2.08, 1.99 (each s,
each 3 H, Ac); 13C NMR: ꢂ 171.16, 170.74, 170.64,
169.77, 169.70, 169.43 (each carbonyl), 102.03 (C-
0
0
same manner in the preparation of 14 a€orded 2
(87 mg, 82%): [ꢁ]2d3 8.0 (c 0.4, H2O); H NMR
ꢀ
1
0
0
(D2O, HOD=4.81): d 4.76 (d, 1 H, J1 ,2 8.6 Hz, H-
0
10), 4.73 (bd, 2 H, J6 ,F 47.1 Hz, H-60), 4.12 (bd, 1 H,
0
0
J4,3 2.9 Hz, H-4), 3.57 (s, 3 H, OMe), 2.04 (s, 3 H,
Ac); 13C NMR (TPS=0.00 ppm): ꢂ 177.69, 106.60
1), 98.34 (d, J1 ,F 11.0 Hz, C-10), 88.70 (d, J3 ,F
185.6 Hz, C-30), 76.57, 71.21, 70.67 (d, J5 ,F 9.8 Hz,
0
(C-1), 105.45 (C-10), 84.79 (d, J6 ,F 167.9 Hz, C-60),
C-50), 70.48 (C-2), 69.43 (C-4), 69.14 (d, J4 ,F
0
0
0
84.84 (C-3), 77.44 (C-5), 76.96 (d, J5 ,F 17.7 Hz, C-
0
18.3 Hz, C-40), 62.03 (C-6), 61.17 (C-60), 58.40 (d,
50), 76.09, 72.54, 71.31 (d, J4 ,F 6.23 Hz, C-40), 71.13
J2 ,F 17.1 Hz, C-20), 56.87 (OMe), 23.58, 21.04,
0
(C-4), 63.63 (C-6), 59.90 (OMe), 58.37 (C-20), 24.88
(Me); m/z Calcd for C15H26FNO10Na: 422.1439 (M
+ Na); Found 422.1438.
20.84, 20.74 (Ac-Me). Anal. Calcd for C25H36
FNO15: C, 49.26; H, 5.95; N, 2.30. Found C, 49.03;
H, 6.02; N, 2.31.
Methyl (4,6-di-O-acetyl-2,3-dideoxy-3-¯uoro-2-
phthalimido-b-d-glucopyranosyl)-(1!3)-2,4,6-tri-
O-acetyl-b-d-galactopyranoside (43). Glycosylation
of 19 (434 mg, 1.36 mmol) with donor 41 (400 mg,
0.905 mmol) in the same manner in the preparation
of 21 a€orded crude disaccharide. Puri®cation of
crude disaccharide by ¯ash column chromato-
graphy (1:1 hexane±ethyl acetate) a€orded 43
(280 mg, 44%): [ꢁ]2d3 1.4 ꢀ (c 2.0, EtOH); 1H
NMR: ꢂ 7.87±7.74 (m, 4 H, Phth), 5.38 (bd, 1 H,
Methyl (2-acetamido-2,3-dideoxy-3-¯uoro-b-d-
glucopyranosyl)-(1!3)-b-d-galactopyranoside (9).
O-Deacetylation of 45 (84 mg, 138 ꢃmol) in the
same manner in the preparation of 2 a€orded 9
(39 mg, 71%): [ꢁ]2d3 17.5 (c 0.8, H2O); H NMR
ꢀ
1
1
0
0
(D2O, DSS=0.00 ppm): ꢂ 4.76 (d, H, J1 ,2 7.9 Hz,
H-10), 4.50 (ddd, 1 H, J3 ,2 8.6, 9.8, J3 ,F 52.2 Hz,
H-30), 4.29 (d, 1 H, J1,2 7.9 Hz, H-1), 4.12 (bd, 1 H,
J4,3 3.4 Hz, H-4), 3.55 (s, 3 H, OMe), 2.03 (s, 3 H,
Ac); m/z Calcd for C15H26FNO10Na: 422.1439 (M
+ Na); Found 422.1470.
0
0
0
0
0
0
0
0
J4,3 3.6 Hz, H-4), 5.24 (dt, 1 H, J3 ,4 =J3 ,2 8.9, J3 ,F
0
48.2 Hz, H-30), 5.25 (d, 1 H, J1 ,2 8.6 Hz, H-1 ), 4.92
(dd, 1 H, J2,1 7.9, J2,3 9.9 Hz, H-2), 4.41 (bd, 1 H,
Methyl (3,4-di-O-acetyl-2-deoxy-2-phthalimido-
6-thioacetyl-b-d-glucopyranosyl)-(1!6)-2,3,4-tri-O-
acetyl-a-d-glucopyranoside (46). Glycosylation of
acceptor 23 (170 mg, 0.53 mmol) with donor 38
(185 mg, 0.35 mmol) in the same manner as in the
preparation of 42 a€orded crude disaccharide.
Puri®cation of this disaccharide by ¯ash column
chromatography (1:2 hexane±ethyl acetate) a€orded
0
0
J6 a,6 b 12.2 Hz, H-60a), 4.36 4.24 (m, 1 H, H-20),
4.21 (d, 1 H, H-1), 4.19±4.04 (m, 3 H, H-60b, 6a,
6b), 3.84±3.70 (m, 3 H, H-3. 5. 50), 3.38 (s, 3 H,
OMe), 2.16, 2.12, 2.11, 2.07, 1.78 (each s, each 3 H,
Ac); 13C NMR: ꢂ 170.83, 170.64, 170.10, 169.92,
169.14, 169.96 (each carbonyl), 134.36, 131.55,
0
0

374
Y. Kajihara et al./Carbohydrate Research 306 (1998) 361±378
46 (211mg, 75%): [ꢁ]2d3 +67.7 ꢀ (c 4.2, CH2Cl2); 1H
NMR: ꢂ 7.85±7.72 (m, 4 H, Phth), 5.74 (dd, 1 H,
Methyl
(2-acetamido-2-deoxy-6-thio-b-d-gluco-
pyranosyl)-(1!6)-a-d-glucopyranoside-60 -yl di-
sul®de (4). The disaccharide 3 (6 mg, 14 ꢃmol) was
dissolved in H2O (250 mL), and this solution was
stirred at 37 ꢀC. After 48 h the mixture was con-
centrated in vacuo. Puri®cation of this residue
over a gel-permeation column (Sephadex G-15,
H2O) a€orded disul®de tetrasaccharide 4 (4 mg,
67%): [ꢁ]2d3 +128.8 ꢀ (c 0.2, H2O); 1H NMR
J3 ,4 8.9, J3 ,2 10.6 Hz, H-30), 5.36 (d, 1 H, J1 ,2
8.6 Hz, H-10), 5.35 (dd, 1 H, J3,2=J3,4 9.9 Hz, H-3),
0
0
0
0
0
0
5.05 (dt, 1 H, J4 ,5 9.9 Hz, H-40), 4.76 (dd, 1 H, J4,5
10.2 Hz, H-4), 4.70 (dd, 1 H, J2,1 3.3Hz, H-2), 4.53
(d, 1 H, H-1), 4.29 (dd, 1 H, H-20), 3.90±3.77 (m, 3
H, H-5, 50, 6a), 3.48 (dd, 1 H, J6b,5 7.3, J6b,6a
0
0
0
0
0
0
10.9 Hz, H-6b), 3.30 (dd, 1 H, J6 a,5 3.0, J6 a,6 b
14.2 Hz, H-60a), 3.12 (dd, 1 H, J6 b,5 14.2 Hz, H-
60a), 3.06 (s, 3 H, OMe), 2.37, 2.09, 2.01, 1.93, 1.92,
1.85 (each s, each 3 H, Ac). Anal. Calcd for
C33H39NO17S: C, 52.59; H, 5.22; N,1.86. Found C,
53.05; H, 4.70; N, 1.64.
(HOD=4.81): ꢂ 4.53 (d, 1 H, J1 ,2 8.2 Hz, H-10),
3.64 (dd, 1 H, J2 ,3 10.2 Hz, H-20), 3.64 (dd, 1 H,
0
0
0
0
0
0
0
0
J4 ,3 =J4 ,5 9.7 Hz, H-4 ), 3.52 (dd, 1 H, J2,1 3.6, J2,3
0
0
0
10.0 Hz, H-2), 3.38 (s, 3 H, OMe), 3.35±3.29 (m,1
H, H-60a), 2.93 (dd, 1 H, J6 b,5 8.4, J6 b,6 a 13.6 Hz,
H-60b), 2.03 (s, 3 H, Ac). m/z Calcd for
C30H52N2O20S2Na: 847.2453 (M + Na); Found
847.2472.
0
0
0
0
Methyl (3,4-di-O-acetyl-2-acetamido-2-deoxy-6-
thioacetyl-b-d-glucopyranosyl)-(1!6)-2,3,4-tri-O-
acetyl-a-d-glucopyranoside (47). N-Dephthalation
and acetylation of 46 (230 mg, 0.288 mmol) in the
same manner as in the preparation of 44 a€orded
crude disaccharide. Puri®cation of this di-
saccharide by ¯ash column chromatography (1:3
hexane±ethyl acetate) a€orded 47 (156 mg, 76%):
[ꢁ]d23 +56.6 ꢀ (c 2.2, CH2Cl2); 1H NMR: ꢂ 5.91 (d, 1
Methyl 3,6-di-O-acetyl-2-azido-2-deoxy-b-d-gal-
actopyranoside (49). To a solution of methyl 2-
azido-2-deoxy-b-d-galactopyranoside (48) [19]
(341 mg, 1.56 mmol) and pyridine (541 mg) in
CH2Cl2 (10 mL) stirring at 0 ꢀC was added acetic
anhydride (349 mg, 3.43 mmol) in CH2Cl2 (7 mL)
over 15 min. After 1 h the mixture was allowed to
warm to room temperature and stirred for 3 h. The
reaction was quenched by addition of MeOH
(1 mL), and the mixture was concentrated in vacuo.
Puri®cation of the residue by ¯ash column chro-
matography (1:1 hexane±ethyl acetate) a€orded 49
(310 mg, 66%): [ꢁ]2d3 +1.4 ꢀ (c 1.1, CH2Cl2); mp
145±147 ꢀC; 1H NMR: ꢂ 4.72 (dd, 1 H, J3,4 3.0, J3,2
10.6 Hz, H-3), 4.36 (dd, 1 H, J6a,5 6.6, J6a,6b
11.2 Hz, H-6a), 4.27 (dd, 1 H, J6b,5 5.9 Hz, H-6b),
4.37 (d, 1 H, J1,2 7.9 Hz, H-1), 3.98 (dd, 1 H, J4,OH
5.3 Hz, H-4), 3.73 (dd, 1 H, H-2), 3.68 (bt, 1 H, H-
5), 3.59 (s, 3 H, OMe), 2.33 (d, 1 H, 4-OH), 2.18,
2.09 (s, 3 H, Ac). Anal. Calcd for C11H17N3O7: C,
43.57; H, 5.65; N, 13.86. Found C, 43.40; H, 5.71;
N, 13.42.
0
H, JNH,2 8.6 Hz, NH), 5.48 (dd, 1 H, J3,2=J3,4
0
0
0
0
9.6 Hz, H-3), 5.17 (dd, 1 H, J3 ,2 =J3 ,4 9.6 Hz, H-
30), 5.10 (dd, 1 H, J4,5 9.6 Hz, H-4), 4.95 (dd, 1 H,
J4 ,5 9.6 Hz, H-40), 4.95 (d, 1 H, J1,2 4.0 Hz, H-1),
0
0
0
0
4.86 (dd, 1 H, H-2), 4.43 (d, 1 H, J1 ,2 8.3 Hz, H-
10), 4.08±3.87 (m, 3 H, H-20, 5, 6a), 3.57 (t, 1 H, H-
50), 3.40 (s, 3 H, OMe), 3.38 (dd, 1 H, J6b,5 3.6,
0
J6b,6a 12.2 Hz, H-6b), 3.27 (dd, 1 H, J6 a,5 3.0,
J6 a,6 b 14.5 Hz, H-60a), 3.00 (dd, 1 H, J6 b,5 7.3 Hz,
H-60b), 2.34, 2.08 (each s, each 3 H, Ac), 2.07 (s, 6
H, AcÂ2), 2.03, 2.00, 1.97 (s, 3 H, Ac). Anal. Calcd
for C27H39NO16S: C, 48.72; H, 5.91; N, 2.10.
Found C, 48.51; H, 5.85; N, 2.06.
0
0
0
0
Methyl (2-acetamido-2-deoxy-6-thio-b-d-gluco
pyranosyl) - (1!6) - a - d - glucopyranoside (3).
To a solution of disaccharide 47 (95 mg, 133 ꢃmol)
and dl-dithiothreitol (204 mg, 1.33 mol) in degas-
sed MeOH (0.5 mL) was added NH4OH (28%,
3 mL), and the mixture was stirred at room tem-
perature. After 12 h the mixture was concentrated
in vacuo at 35 ꢀC. Puri®cation of residue over a gel-
permeation column (Sephadex G-15, H2O) a€or-
Methyl
3,6-di-O-acetyl-2-azido-2,4-dideoxy-4-
¯uoro-b-d-glucopyranoside (50). To a solution of
methyl 3,6-di-O-acetyl-2-azido-2-deoxy-b-d-galac-
topyranoside (49) (80 mg, 0.27 mmol) in CH2Cl2
(2 mL) was added diethylaminosulfur tri¯uoride
(97 ꢃL, 0.80 mmol) at 40 ꢀC under argon atmo-
sphere. After 4 h the mixture was allowed to warm
ded 3 (31 mg, 56%): [ꢁ]2d3 +37.9 ꢀ (c 2.9, H2O); H
1
0
0
NMR: (D2O, HOD=4.81): ꢂ 4.58 (d, 1 H, J1 ,2
to
10 ꢀC, and excess diethylaminosulfur tri-
8.2 Hz, H-10), 3.44 (s, 3H, OMe), 3.06 (bd, 1 H,
¯uoride was quenched by the addition of MeOH
(0.3 mL). The mixture was directly concentrated in
vacuo. Puri®cation of the residue by ¯ash column
chromatography (1:3 hexane±ethyl acetate) a€or-
ded 50 (38 mg, 48%): [ꢁ]2d5 32.7 ꢀ (c 3.3, CH2Cl2);
J6 a,6 b 13.5 Hz, H-60a), 2.80 (dd, 1 H, J6 b,5 6.9 Hz,
H-60b), 2.08 (s, 3H, Ac); negative-ion FAB: m/z
Calcd for C15H26NO10S: 412.1278 (M - H); Found
412.1286.
0
0
0
0

Y. Kajihara et al./Carbohydrate Research 306 (1998) 361±378
375
1H NMR: ꢂ 5.13 (ddd, 1 H, J3,2 10.2, J3,4 8.9, J3,F
13.9 Hz, H-3), 4.42 (ddd, 1 H, J6a,5=J6a,F 2.0,
J6a,6b 12.2 Hz, H-6a), 4.41 (ddd, 1 H, J4,5 8.9, J4,F
50.8 Hz, H-4), 4.33 (d, 1 H, J1,2 7.9 Hz, H-1), 4.26
(ddd, 1 H, J6b,5 5.0, J6b,F 1.7 Hz, H-6b), 3.76±3.65
(m, 1 H, H-5), 3.59 (s, 3 H, OMe), 3.42 (bdd, 1 H,
J2,F 0.5 Hz, H-2), 2.17, 2.11 (s, 3 H, Ac); 13C NMR:
ꢂ 170.75, 169.83, 103.16 (C-1), 87.15 (d, J4,F
188.0 Hz, C-4), 72.11 (d, J3,F 19.5 Hz, C-3), 71.46
(d, J5,F 23.2 Hz, C-5), 63.78 (d, J2,F 7.3 Hz, C-2),
62.23 (C-6), 57.77 (OMe), 21.13, 20.99 (each
Ac).Anal. Calcd for C11H16FN3O6: C, 43.28;
H, 5.28; N, 13.77. Found C, 43.71; H, 5.35; N,
13.69.
Methyl 3,6-di-O-acetyl-2-azido-2-deoxy-4-thio-
acetyl-b-d-glucopyranoside (52). To a solution of
pyridine (158 mg, 1.99 mmol) in CH2Cl2 (5 mL)
stirring at 0 ꢀC was added tri¯uoromethanesulfonic
anhydride (167 ꢃL, 1.0 mmol). After 10 min 49
(150 mg, 0.50 mmol in CH2Cl2 (5 mL) was added to
this mixture at 0 ꢀC. After 30 min the mixture was
diluted with CHCl3 and washed with 1% aq HCl,
H2O and aq NaHCO3. The organic phase was
dried with MgSO4 and was concentrated in vacuo.
The residue was further dried by use of a vacuum
pomp and then dissolved in N,N-dimethylform-
amide (2 mL). To this solution was added potassium
thioacetate (280 mg, 2.49 mmol) at 0 ꢀC. After
being stirred for 30 min, the mixture was directly
applied to ¯ash column chromatography (3:1
Methyl 3,6-di-O-acetyl-2-acetamido-2,4-dideoxy-
4-¯uoro-b-d-glucopyranoside (51). To a solution
of methyl 3,6-di-O-acetyl-2-azido-2,4-dideoxy-4-
¯uoro-b-d-glucopyranoside (50) (25 mg, 83 ꢃmol)
in triethylamine±pyridine±H2O (0.1 mL:0.5 mL:
0.25 mL) was introduced H2S gas, and the mixture
was stirred at room temperature. After 30 min the
mixture was concentrated in vacuo. The residue
was treated with acetic anhydride and pyridine.
After being stirred for 30 min at room temperature,
the mixture was concentrated in vacuo. Puri®ca-
tion of the residue by ¯ash column chromato-
graphy (ethyl acetate) a€orded 51 (26 mg, 98%):
hexane±ethyl acetate) to give 52 (151 mg, 85%):
[ꢁ]2d5 16.4 (c 0.6, CH2Cl2); H NMR: ꢂ 5.04 (dd,
ꢀ
1
1 H, J3,4 9.9, J3,2 10.9 Hz, H-3), 4.37 (dd, 1 H,
J6a,5 5.3, J6a,6b 12.2 Hz, H-6a), 4.28 (d, 1 H, J1,2
8.3 Hz, H-1), 4.22 (dd, 1 H, J6b,5 2.0 Hz, H-6b),
3.79 (m, 1 H, H-5), 3.61(dd, 1 H, J4,5 10.9 Hz, H-
4), 3.59 (s, 3 H, OMe), 3.45 (dd, 1 H, H-2), 2.33,
2.10, 2.09 (s, 3 H, Ac). Anal. Calcd for
C13H19N3O7S: C, 43.21; H, 5.30; N, 11.63. Found
C, 43.66; H, 5.33; N, 12.22.
Methyl 3,6-di-O-acetyl-2-acetamido-2-deoxy-4-
thioacetyl-b-d-glucopyranoside (53). Reduction of
azido 52 (218 mg, 0.61 mmol) and acetamidation in
the same manner as in the preparation of 51 a€or-
ded crude 53. Puri®cation of this crude product by
¯ash column chromatography (9:1 ethyl acetate±
MeOH) a€orded 53 (190 mg, 84%): [ꢁ]2d5 18.5 ꢀ
(c 4.0, CH2Cl2); mp 149±151 ꢀC;1H NMR: ꢂ 5.73
(d, 1 H, JNH,2 8.9 Hz, NH), 5.25 (dd, 1 H, J3,4=J3,2
10.6 Hz, H-3), 4.53 (d, 1 H, J1,2 8.6 Hz, H-1), 4.37
(dd, 1 H, J6a,5 4.6, J6a,6b 11.9 Hz, H-6a), 4.26 (dd, 1
H, J6b,5 2.3 Hz, H-6b), 3.97±3.83 (m, 2 H, H-2, 5),
3.65(dd, 1 H, J4,5 10.6 Hz, H-4), 3.49 (s, 3 H,
OMe), 2.33, 2.09, 2.04, 1.95 (s, 3 H, Ac). Anal.
Calcd for C15H23NO8S: C, 47.74; H, 6.14; N, 3.71.
Found C, 47.88; H, 5.62; N, 3.77.
[ꢁ]2d5 32.9 ꢀ (c 1.73, CH2Cl2); mp 180±182 ꢀC; H
1
NMR: ꢂ 5.80 (d, 1 H, JNH,2 9.2 Hz, NH), 5.29
(ddd, 1 H, J3,2 9.2, J3,4 9.2, J3,F 14.5 Hz, H-3), 4.50
(ddd, 1 H, J4,5 9.2, J4,F 50.8 Hz, H-4), 4.49 (d, 1 H,
J1,2 8.2 Hz, H-1), 4.25 (dd, 1 H, J6b,5 4.3, J6b,6a
12.0 Hz, H-6b), 3.97 (ddd, 1 H, H-2), 3.76±3.71 (m,
1 H, H-5), 3.49 (s, 3 H, OMe), 2.11 (s, 6 H, AcÂ2),
1.97 (s, 3 H, Ac); 13C NMR: ꢂ 1701.01, 170.64,
170.35 (each carbonyl), 101.85 (C-1), 86.87 (d, J4,F
186.8 Hz, C-4), 72.46 (d, J3,F 18.3 Hz, C-3), 71.25
(d, J5,F 24.4 Hz, C-5), 62.14 (C-6), 56.87 (OMe),
53.93 (d, J2,F 6.1 Hz, C-2), 23.32 (NAc), 20.77
(AcÂ2). Anal. Calcd for C13H20FNO7: C, 48.60;
H, 6.30; N, 4.36. Found C, 48.60; H, 6.42; N,
4.34.
Methyl 2-acetamido-2,4-dideoxy-4-¯uoro-b-d-
glucopyranoside (6). O-Deacetylation of 51 (63 mg,
0.20 mmol) in the same manner as in the prepara-
tion of 14 a€orded 6 (42 mg, 91%): [ꢁ]2d3 22.7 ꢀ (c
2.5, H2O); 1H NMR (D2O, HOD=4.81): ꢂ 4.54 (d,
Methyl 2 - acetamido - 2 - deoxy - 4 - thio - b - d -
glucopyranoside (7). 4-Thioacetyl derivative 53
(20 mg, 54 ꢃmol) and dl-dithiothreitol (83 mg,
0.54 mmol) were dissolved in MeOH±NH4OH
(0.5 mL:3 mL). After stirring for 12 h at room
temperature, the mixture was concentrated in
vacuo. Puri®cation of the residue by ¯ash column
chromatography (7:3 ethyl acetate±MeOH) and
1
H, J1,2=8.6 Hz, H-1), 4.42 (bdt,
1
H,
J4,3=J4,5=8.9, J4,F=51.5 Hz, H-4), 3.55 (s, 3 H,
OMe), 2.08 (s,3 H, Ac); m/z Calcd for
C9H16FNO5Na: 260.0911 (M + Na); Found
260.0925.
subsequent
(Sephadex G-15, H2O) a€orded 7 (8 mg, 62%):
gel-permeation
chromatography

376
Y. Kajihara et al./Carbohydrate Research 306 (1998) 361±378
60.2 ꢀ (c 0.7, H2O); 1H NMR (D2O,
[ꢁ]2d3
room temperature. After 30 min, MeI (222 mg,
1.58 mmol) was added to this mixture, and the
mixture was stirred for another 30 min. The mix-
ture was diluted with ethyl acetate and washed
with brine (twice). The organic phase was dried
with MgSO4 and concentrated in vacuo. Puri®ca-
tion of the crude product by ¯ash column chro-
matography (3:2 hexane±ethyl acetate) a€orded 56
(406 mg, 99%): [ꢁ]2d5 25.3 ꢀ (c 2.0, CHCl3); 1H
NMR (CDCl3): ꢂ 7.35ꢁ7.20 (m, 15 H, Ph), 4.35 (d,
1 H, J1,2 7.3 Hz, H-1) 3.48 (s, 3 H, OMe), 2.72 (s, 3
H, NMe), 2.16 (s, 3 H, Ac); Anal. Calcd for
C31H37NO6: C, 71.65; H, 7.17; N, 2.69. Found C,
71.16; H, 7.10; N, 2.61.
HOD=4.81): ꢂ 4.47 (d, 1 H, J1,2 8.6 Hz, H-1),
4.10 (dd, 1 H, J6a,5 2.0, J6a,6b 12.5 Hz, H-6a), 3.91
(dd, 1 H, J6b,5 5.3, H-6b), 3.68 (dd, 1 H, J2,3
9.9 Hz, H-2), 3.59±3.50 (m, 1 H, H-5), 3.54 (s, 3
H, OMe), 3.47 (dd, 1 H, J3,4 9.9 Hz, H-3), 2.77
(dd, 1 H, J4,5 9.9 Hz, H-4), 2.07 (s, 3 H, Ac). m/z
Calcd for C9H18NO5S: 252.0906 (M + H); Found
252.0911.
Methyl 2-aminoethyl-3,4,6-tri-O-benzyl-2-deoxy-
b-d-glucopyranoside (55). To a solution of methyl
2-acetamido-3,4,6-tri-O-benzyl-2-deoxy-b-d-gluco-
pyranoside (54) [20] (300 mg, 0.59 mmol) in THF
(3 mL) was added LiAlH4 (113 mg, 2.97 mmol),
and the mixture was stirred under re¯ux. After 2
days, the mixture was poured into water and
extracted with ethyl acetate. The organic phase was
dried with MgSO4 and concentrated in vacuo.
Puri®cation of the crude product by ¯ash column
chromatography (ethyl acetate) a€orded 55
(203 mg, 70%): [ꢁ]2d5 16.6 ꢀ (c 0.7, CHCl3); 1H NMR
(300 MHz): ꢂ 7.35ꢁ7.19 (m, 15 H, Ph), 4,95 (d, 1
H, J 11.5 Hz, benzyl CH2), 4.79 (d, 1 H, J 10.8 Hz,
benzyl CH2), 4.70ꢁ4.54 (m, 4 H, benzyl CH2), 4.19
(d, 1 H, J1,2 7.9 Hz, H-1), 3.66 (s, 3 H, OMe),
2.88ꢁ2.86, 2.69ꢁ2.63 (each m, each 1 H, CH2),
2.57 (dd, 1 H, J2,3 10.0 Hz, H-2), 0.97 (t, 1 H, J
7.2 Hz, CH3); Anal. Calcd for C30H37NO5: C,
73.29; H, 7.58; N, 2.85. Found C, 73.08; H, 7.82;
N, 2.83.
Methyl 2-methylacetamido-2-deoxy-b-d-glucopyr-
anoside (11). O-Debenzylation of 56 (400 mg,
0.77 mmol) in the same manner as in the prepara-
tion of 10 a€orded 11 as an E, Z mixture (ca. 1:1,
1
163 mg, 85%): H NMR (D2O, HOD=4.81, 323
K): ꢂ 4.99 (d, 0.5 H, J1,2 8.0 Hz, H-1), 4.22ꢁ4.18
(m, 1 H, H-6a), 4.04ꢁ4.00 (m, 1 H, H-6b), 3.77 (s,
3 H, OMe), 3.30, 3.14 (each s, each 1.5 H, Me),
2.43 (s, 3 H, Ac); m/z Calcd for C10H19NO6Na:
272.1110 (M + Na); Found 272.1124.
Methyl 2-O-acetyl-4,6-O-benzylidene-b-d-gluco-
pyranoside (57). To a solution of methyl 4,6-O-
benzylidene-b-d-glucopyranoside [21] (140 mg,
0.49 mmol) in CH2Cl2 (14 mL) were added acetic
anhydride (60 mg, 0.59 mmol), pyridine (58 mg,
0.74 mmol) and DMAP (40 mg) in CH2Cl2 (1 mL)
at 0 ꢀC. After stirring 1 h at that temperature,
MeOH (2 mL) was added and the mixture was
concentrated in vacuo. Puri®cation of the crude
product by ¯ash column chromatography (1:1
hexane±ethyl acetate) a€orded 2-O-acetyl deriva-
tive 57 [21] (46 mg, 29%), 3-O-acetyl derivative
Methyl 2-aminoethyl-2-deoxy-b-d-glucopyrano-
side (10). To a solution of tri-O-benzyl derivative
55 (200 mg, 0.41 mmol) and Pd/C (cat) in MeOH
(2 mL) (Caution: Extreme ®re hazard!) was intro-
duced hydrogen gas. After stirring for 5 h at room
temperature, the mixture was ®ltered and the ®l-
trate was concentrated in vacuo. Puri®cation of the
crude product over a gel-permeation column
(Sephadex G-15, H2O) a€orded 10 (78 mg, 87%):
(51 mg) and 2,3-di-O-acetyl derivative (23 mg)
[ꢁ]2d5 69.6 (c 1.2, CHCl3); H NMR (CDCl3): ꢂ
ꢀ
1
7.51ꢁ7.25 (m, 15 H, Ph), 5.53 (s, 1 H, CH), 4.92
(dd, J2,1 7.9, J2,3 9.2 Hz, H-2), 4.42 (d, 1 H, H-1)
4.36 (dd, 1 H, J6a,5 5.0, J6a,6b 10.5 Hz, H-6a), 3.86
(ddd, 1 H, J3,OH 3.6, J3,4=J3,2 9.2 Hz, H-3), 3.79
(dd, 1 H, J6b,5 10.2 Hz, H-6b), 3.57 (dd, J4,5 9.3 Hz,
H-4), 3.50 (s, 3 H, OMe), 2.74 (d, 1 H, OH), 2.13
(s, 3 H, Ac); m/z Calcd for C16H20O7Na: 347.1107
(M + Na); Found 347.1112.
Methyl 2-O-acetyl-b-d-glucopyranoside (12).
2-O-Acetyl derivative 57 (24 mg, 74 ꢃmol) was dis-
solved in 60% acetic acid (2.0 mL). The mixture
was stirred 1 h at 100 ꢀC. After concentration in
vacuo, the crude mixture was puri®ed on a gel-
permeation column (Sephadex G-15, H2O) to
[ꢁ]2d5
23.9 ꢀ (c 0.4, H2O); 1H NMR (D2O,
HOD=4.81): ꢂ 4.45 (d, 1 H, J1,2 8.3 Hz, H-1), 3.92
(dd, 1 H, J6a,5 1.9, J6a,6b 12.2 Hz, H-6a), 3.72 (dd, 1
H, J6b,5 5.5 Hz, H-6b), 3.56 (s, 3 H, OMe),
3.00ꢁ2.74 (m, 1 H, CH2), 2.53 (dd, 1 H, J2,3
8.3 Hz, H-2), 1.11 (t, 3 H, J 7.2 Hz, CH3); m/z
Calcd for C9H19NO5Na: 244.1161 (M + Na);
Found 244.1174.
Methyl 2-methylacetamido-3,4,6-tri-O-benzyl-2-
deoxy-b-d-glucopyranoside (56). To a solution of
tri-O-benzyl derivative 54 (400 mg, 0.79 mmol) in
DMF (2 mL) was added NaH (60% in oil, 63 mg,
1.58 mmol), and the mixture was stirred at

Y. Kajihara et al./Carbohydrate Research 306 (1998) 361±378
377
1
a€ord 12 [20] (13 mg, 72%): H NMR (300 MHz,
References
D2O, HOD=4.81): ꢂ 4.68 (dd, J2,1=J2,3=9.4 Hz,
H-2), 4.57 (d, 1 H, H-1), 3.97 (dd, 1 H, J6a,5 1.6,
J6a,6b 12.6 Hz, H-6a), 3.522 (s, 3 H, OMe),
3.49ꢁ3.47 (m, 2 H, H-4,5), 2.16 (s, 3 H, Ac); m/z
Calcd for C9H16O7Na: 259.0794 (M + Na); Found
259.0821.
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Enzyme assay of synthetic N-acetylglucosaminide
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(1!4)-galactosyltransferase toward synthetic N-
acetylglucosaminide analogues was examined
under the following conditions. An assay solution
containing UDP-[U-14C]-galactose (9.92 GBq/
mmol, 90 pmol), the various concentrations of N-
acetylglucosaminide analogue, bovine serum albu-
min (0.1 ꢃg) MnCl2 (10 mM) and b,-(1!4)-galacto-
syltransferase in TRIS bu€er (20 mM, pH 7.5, total
volume 30 ꢃL) was incubated 37 ꢀC. The reaction
was followed up to 15% consumption of UDP-
galactose. The 14C-labeled N-acetyllactosaminide
analogue was isolated by passage of the mixture
diluted with 1 mL of water through a Pasteur pip-
ette column of Dowex- 1Â8 (Cl ), and further
eluted by 1 mL of water. The 14C-labeled N-acet-
yllactosaminide analogue was measured by scintil-
lation counting. The assay was performed in
duplicate, and Km and Vmax were determined using
Lineweaver±Burk plot, and the results are sum-
marized in Table 1.
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