Synthesis of 4'-O-acetyl-maltose and alpha-D-galactopyranosyl-(1-->4)-D-glucopyranose for biochemical studies of amylose biosynthesis.

Article abstract of DOI:10.1016/S0008-6215(00)00306-2

The chemical synthesis of the title compounds as maltose analogs, in which the non-reducing end is modified by acetylation of the 4'-OH group or by reversing its configuration, is reported. For synthesis of the 4'-O-acetylated analog, beta-maltose was converted into its per-O-benzylated-4',6'-O-benzylidene derivative followed by removal of the benzylidene acetal function and selective silylation at C-6'. Acetylation at C-4' of the obtained silylated compound followed by removal of the benzyl ether protecting groups and subsequent desilylation afforded the desired analog. The other maltose analog was synthesized via the glycosidation reaction between the glycosyl donor, O-(2,3,4,6-tetra-O-benzyl-alpha/beta-D-galactopyranosyl)trichloroacetimidate and the glycosyl acceptor, phenyl 2,3,6-tri-O-benzyl-1-thio-beta-D-glucopyranoside followed by removal of the phenylthio group and debenzylation to provide the desired analog.

Full text of DOI:10.1016/S0008-6215(00)00306-2

www.elsevier.nl/locate/carres
Carbohydrate Research 330 (2001) 309–318
Synthesis of 4%-O-acetyl-maltose and
a-D
-galactopyranosyl-(1“4)-
D-glucopyranose for
biochemical studies of amylose biosynthesis
Mohammed Saddik Motawia,a,c,* Carl Erik Olsen,b,c Kay Denyer,d Alison M. Smith,d
Birger Lindberg Møllera,c
aPlant Biochemistry Laboratory, Department of Plant Biology, The Royal Veterinary and Agricultural Uni6ersity,
40 Thor6aldsens6ej, DK-1871 Frederiksberg C, Copenhagen, Denmark
bDepartment of Chemistry, The Royal Veterinary and Agricultural Uni6ersity, 40 Thor6aldsens6ej,
DK-1871 Frederiksberg C, Copenhagen, Denmark
cCenter of Molecular Plant Physiology (PlaCe), The Royal Veterinary and Agricultural Uni6ersity,
40 Thor6aldsens6ej, DK-1871 Frederiksberg C, Copenhagen, Denmark
dJohn Innes Centre, Norwich Research Park, Norfolk NR4 7UH, UK
Received 18 June 2000; received in revised form 27 October 2000; accepted 21 November 2000
Abstract
The chemical synthesis of the title compounds as maltose analogs, in which the non-reducing end is modified by
acetylation of the 4%-OH group or by reversing its configuration, is reported. For synthesis of the 4%-O-acetylated
analog, b-maltose was converted into its per-O-benzylated-4%,6%-O-benzylidene derivative followed by removal of the
benzylidene acetal function and selective silylation at C-6%. Acetylation at C-4% of the obtained silylated compound
followed by removal of the benzyl ether protecting groups and subsequent desilylation afforded the desired analog.
The other maltose analog was synthesized via the glycosidation reaction between the glycosyl donor, O-(2,3,4,6-tetra-
O-benzyl-a/b-
D
-galactopyranosyl)trichloroacetimidate and the glycosyl acceptor, phenyl 2,3,6-tri-O-benzyl-1-thio-b-
D
-glucopyranoside followed by removal of the phenylthio group and debenzylation to provide the desired analog.
© 2001 Elsevier Science Ltd. All rights reserved.
Keywords: Maltose derivatives; Phenyl thioglycosides; Glycosyl trichloroacetamidate; Glycosidation; Hydrogenolysis; a-D-Galac-
topyranosyl-(1“4)-D-glucopyranose
chains bound at their reducing end to the
main chain via a-(1“6) linkages. Amylo-
pectin is a much larger molecule built of clus-
1. Introduction
Starch consists of two
amylose and amylopectin. Amylose is an es-
sentially linear molecule composed of a back-
bone of a-(1“4)-linked
decorated with a few short a-(1“4) glucan
D
-glucose polymers,
ters of a-(1“4)-linked
D-glucose chains, each
of which are bound at their reducing end via
an a-(1“6) linkage to another glucan chain
resulting in an uneven distribution of the a-
(1“6) linkages along the molecule and in the
formation of intermittent crystalline and
D
-glucose residues
* Corresponding author. Tel.: +45-35-283369; fax: +45-
35-283333.
E-mail address: mosm@kvl.dk (M.S. Motawia).
amorphous regions with a unit length of 90
1–3
,
A.
The formation of all glucosidic link-
0008-6215/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0008-6215(00)00306-2

310
M.S. Motawia et al. / Carbohydrate Research 330 (2001) 309–318
promote amylose synthesis may reflect their
involvement as primers for initiation of new
amylose molecules or a function as effectors–
activators of GBSSI. Discrimination between
these two possibilities would require the
availability of malto-oligosaccharides specifi-
cally blocked or altered at their non-reducing
end to prevent their functioning as primers
while supposedly retaining their function as
effectors–activators. In the present study, we
report the synthesis of two such compounds,
ages in amylose and amylopectin may be ac-
counted for by the action of multiple isoforms
of starch synthases (SS) and starch branching
enzymes (SBE).3–9
Studies using mutants have demonstrated
that one particular isoform of starch synthase,
granule-bound starch synthase I (GBSS I), is
responsible for amylose synthesis.9 In the ab-
sence of GBSS I, other soluble and granule-
bound isoforms of starch synthases are unable
to synthesize amylose. Accordingly, these iso-
forms are assigned a role in amylopectin syn-
thesis. Amylose biosynthesis takes place in the
polysaccharide matrix and it is experimentally
possible to obtain amylose synthesis in vitro
using purified granules.3,10 Two models have
been proposed to explain the mechanism of
amylose synthesis.11 The first model is based
on studies in pea and involves small-chain
malto-oligosaccharides.12 The second model is
based on studies in Chlamydomonas and pro-
poses that external a-glucans of amylopectin
serve as primers for amylose synthesis. In this
model, primer extension is terminated by
cleavage to produce amylose.13 No evidence
for the operation of this mechanism was
found in starch granules from developing
pea.14 The ability of malto-oligosaccharides to
namely 4-O-acetyl-a-
4)- -glucopyranose (9) and a-
osyl-(1“4)- -glucopyranose (26).
D
-glucopyranosyl-(1“
D
D
-galactopyran-
D
2. Results and discussion
For the synthesis of compound 9 from malt-
ose, the following strategy for introducing spe-
cifically, an acetyl group at the 4%-OH of the
non-reducing end, was devised. The strategy is
also applicable to other (1“4)-linked
oligomers of hexopyranoses. Starting with the
free carbohydrate, a 4,6-O-benzylidene acetal
is formed at the non-reducing end. Then all
remaining free OH groups are protected by
benzylation. Removal of the 4,6-O-benzyli-
dene acetal now exposes the C-6 hydroxyl for
selective protection with sterically demanding
groups like trityl15 or diphenyl-tert-butyl-
silyl16, providing a specific access to the 4-OH
position.
Based on this strategy, the synthesis of the
target molecule 9 from b-maltose monohy-
drate (1) is outlined in Scheme 1. b-Maltose
monohydrate (1) was treated with a,a-
dimethoxytoluene in N,N-dimethylformamide
in the presence of p-toluenesulfonic acid
monohydrate17 to give 4-O-(4,6-O-benzyli-
dene-a-
D
-glucopyranosyl)- -glucopyranose
D
(2).17 Benzylation of 2 with benzyl bromide in
DMF–NaH afforded the corresponding O-
benzylated-4,6-O-benzylidene derivative 3 as
an anomeric mixture with an a/b ratio of 2:5
in 88% yield after chromatographic purifica-
tion. Removal of the benzylidene functional
group from the anomeric mixture 3 with (3:2
v/v) acetic acid–water gave benzyl 2,3,6-O-
benzyl-4-O-(2,3-di-O-benzyl-a-
D
-glucopyra-
Scheme 1. Synthesis of 4%-O-acetyl maltose (8).
nosyl)- -glucopyranoside (4) in 85% yield
D

M.S. Motawia et al. / Carbohydrate Research 330 (2001) 309–318
311
1
Fig. 1. H NMR spectra at 400.13 MHz of compounds 8 and 9 in CD3OD.
of the expected formation of 9. This is in
contradiction with a previous report by Nico-
laou et al.18 where desilylation using exactly
the same reaction conditions and a similar
functional group pattern was reported not to
result in acetyl migration. However, attempted
desilylation of 7 using H3PO4 in 50% aq
MeOH furnished the formation of the desired
target molecule 9 in quantitative yield. Under
these desilylation reaction conditions, only 8–
10% acetyl migrations occurred as revealed by
after chromatographic purification. From the
anomeric mixture 4 pure b anomer could be
isolated as a crystalline product by crystalliza-
tion from diethyl ether. The 6%-OH group of 4
was then regioselectively tert-butyldiphenylsi-
lylated by treatment with tert-butylchloro-
diphenylsilane in DMF–imidazole at room
temperature to afford compound 5 in quanti-
tative yield after chromatographic purifica-
tion. The 4%-OH group of 5 was readily
acetylated with acetic anhydride in dry pyri-
dine resulting in the corresponding 4%-O-acetyl
derivative 6 in quantitative yield. Hydrogenol-
ysis of 6 to remove the benzyl ether protecting
groups was carried out under atmospheric
pressure of hydrogen over 10% palladium on
carbon with (2:1 v/v) EtOH–EtOAc as the
solvent and produced compound 7 in 65%
yield after chromatographic purification. Fi-
nally, removal of the tert-butyldiphenylsilyl
protecting group from C-6% of 7 was achieved
using tetrabutylammonium fluoride in THF.16
As expected, desilylation of 7 using TBAF in
THF resulted in complete removal of the TB-
DPS group. However, a simultaneous migra-
tion of the acetyl group from C-6 toC-6% was
observed probably due to the presence of the
fluoride ion, resulting in 8 in 95% yield instead
1
the H NMR spectrum of compounds 8 and 9
1
shown in Fig. 1. Extensive H and 13C NMR
chemical shift assignments for 8 and 9 listed in
Table 1 were done using 2D spectra (COXY,
TOCSY, HSQC and HMBC).
1
In the H NMR spectrum of 8 (Fig. 1(a)), a
one-proton doublet of doublets assignable to
H-6%b (a,b anomeric mixture; J6%a,6%b=11.8 and
J5%,6%b=2.1 Hz) observed at l 4.38 ppm and a
one-proton multiplet (two doublet of dou-
blets) assignable to H-6%a (a anomer; J6%a,6%b
11.7 and J5%,6%a=1.2 Hz) and H-6%a (b anomer;
6%a,6%b=11.8 and J5%,6%a=1.3 Hz) observed at l
=
J
4.18 and 4.15 ppm, respectively. The appear-
ance of these signals at lower magnetic fields
dictates that the acetyl group is located at C-6%
after its migration from C-4%. In contrast, the

312
M.S. Motawia et al. / Carbohydrate Research 330 (2001) 309–318
1H NMR spectrum of 9 (Fig. 1(b)) reveals
that the H-4% proton (a,b anomeric mixture)
resonates at l 4.73 ppm as a doublet of dou-
blets (appearing as a triplet). Integration of
acetyl group have remained at C-4% whereas
only 7% have migrated. An unambiguous de-
termination of the location of the acetyl group
in analogs 8 and 9 was confirmed from long
1
range H–13C COSY [1heteronuclear multiple
1
the relevant H-signals shows that 93% of the
Table 1
1H and 13C NMR data (400.13 MHz) for analogs 8 and 9 in CD3OD
Chemical shifts l (ppm)
8
9
(J in Hz)
a anomer
b anomer
a anomer
b anomer
H-1
5.11 d
(3.8)
93.8
3.42 dd
(9.5)
73.4
3.90 t
(9.4)
74.7
4.50 d
(7.6)
98.2
3.18 dd
(9.5)
75.8
3.60 t
(9.4)
77.9
5.10 d
(3.7)
93.8
3.41 dd
(9.8)
73.5
3.92 t
(9.5)
74.6
3.55 t
4.49 d
(7.8)
98.2
3.18 dd
(9.2)
75.8
3.62 t
(9.5)
77.8
3.55 t
(J1,2
C-1
H-2
(J2,3
C-2
H-3
(J3,4
C-3
H-4
(J4,5
C-4
)
)
)
)
3.47 t
(9.5)
3.47 t
(9.5)
(9.5)
81.6
(9.5)
81.8
a
a
82.3
82.5
H-5 C-5
3.36–3.50 m
76.7
3.36–3.50 m
76.8
3.46–3.57 m
71.6
3.37–3.42 m
76.6
H-6a
(J6a,6b) (J5,6a,
H-6b
3.91 dd
(11.9) (1.8)
3.76 dd
(5.2)
3.80–3.88 m
3.77 dd
(11.9) (2.1)
3.80–3.88 m
3.77 dd
(11.9) (2.1)
3.80–3.88 m
)
3.80–3.88 m
62.3
(J5,6b
)
C-6
H-1%
62.5
5.11 d
62.2
5.21 d
(3.7)
102.5
3.52 dd
(9.5)
62.4
5.22 d
(3.7)
102.5
3.52 dd
(9.5)
b
b
5.12 d
(J1%,2%
C-1%
H-2%
(J2%,3%
C-2%
H-3%
(J3%,4%
C-3%
H-4%
(J4%,5%
C-4%
H-5%
C-5%
)
)
)
)
(3.8)
103.1
3.44–3.48 m
(4.1)
103.1
3.44–3.48 m
74.1
3.61 t
74.4
3.63 t
74.1
74.2
3.80–3.88 m
c
c
3.76 t
(9.5)
(9.1)
75.1
(9.1)
75.0
c
c
d
73.5
72.7 d
4.73 t
(9.5)
72.6
3.80–3.88 m
72.9
3.25 t
(9.5)
71.7
3.80–3.88 m
72.2
3.25 t
(9.5)
71.4
3.80–3.88 m
72.2
4.73 t
(9.5)
72.6
3.80–3.88 m
72.9
H-6%a
(J6%a,6%b
4.38 dd
(12)
4.38 dd
(11.9)
3.58 dd
(12)
3.58 dd
(12)
)
(J5%,6%a,
)
(1.9)
(1.7)
(2.5)
(2.5)
H-6%b
(J5%,6%b
4.17 dd
(2.2)
4.16 dd
(2.3)
3.49 dd
(6.4)
3.49 dd
(6.4)
)
C-6%
65.2
65.2
62.4
62.4
CH3CO
2.08
2.08
2.09
2.08
20.8, 172.9
20.8, 172.9
20.9, 172.2
20.9, 172.2
a Assignments may be reversed.
b Assignments may be reversed.
c Assignments may be reversed.
d Assignments may be reversed.

M.S. Motawia et al. / Carbohydrate Research 330 (2001) 309–318
313
Scheme 2. Synthesis of a-D-galactopyranosyl-(1“4)-glucopyranose (25).
sium carbonate as
a
catalyst in dry
bond connectivity (HMBC)] spectra of 8 and
9. Thus, the HMBC spectrum of 8 showed
cross-peaks (4.38/172.2 and 4.18/172.9) which
connect the carbonyl carbon of the acetyl
group to the H-6% indicating that the acetyl
group is located at C-6%. Correspondingly, the
HMBC spectrum of 9 showed a cross-peak
(4.73/172.2) which connects the carbonyl car-
bon of the acetyl group to H-4% indicating that
the acetyl group is located at C-4%-position.
The target compound 9 was obtained in 96%
yield after chromatographic purification.
dichloromethane to obtain the glycosyl donor
16. The potential of O-glycosyl-trichloroace-
timidates as strong glycosylation donors under
mild acidic catalytic conditions has previously
been documented.25 We have previously
reported25 synthesis of the glycosyl acceptor
23 in six steps starting from
D-glucose (17).
The glycosidation reaction between 16 and 23
was performed in dry diethyl ether using
trimethylsilyl triflate as a catalyst and pro-
vided the desired disaccharide derivative 24 in
79% yield after chromatographic purification.
Minor amounts of the b anomer were re-
moved during the chromatographic step. The
replacement of the phenylthio group of 24
with a free OH group was achieved using the
N-bromosuccinimide method22 and provided
2,3,6-tri-O-benzyl -4-O- (2,3,4,6-tetra-O-ben-
The route for the second target molecule 26
is summarized in Scheme 2.
D
-Galactose (10)
was acetylated19 to produce 11 which upon
treatment with phenylthiotrimethylsilane in
the presence of trimethylsilyl trifluoro-
methanesulfonate as catalyst afforded 12
which was subsequently deacetylated to
phenyl 1-thio-b-
D
-galactopyranoside (13) as
zyl
a-D-galactopyranosyl)- -glucopyranose
D
previously described.20 Standard benzylation
of 13 using benzyl bromide in DMF–NaH
afforded the known21 per-O-benzylated thio-
glycoside (14) in 72% yield. By treating the
thioglycoside 14 with N-bromosuccinimide–
acetone–water22 for 5 min at room tempera-
ture, the phenylthio group was replaced with a
(25) in quantitative yield after chromato-
graphic purification. Hydrogenolysis of 25 for
removal the benzyl ether protecting groups
was performed under hydrogen in the pres-
ence of 10% palladium on carbon, and pro-
duced the target compound 26 in 80% yield
after chromatographic purification.
free OH group to afford 2,3,4,6-O-benzyl-
D
-
Biosynthetic experiments were carried out
using specific malto-oligosaccharides as well
as the two chemically synthesized maltose
analogs 9 and 26 in which the non-reducing
end was modified to exclude their function as
galactopyranose21,23 (15) in 99% yield. Com-
pound
15
was
converted
into
its
trichloroacetimidate24 by the action of
trichloroacetonitrile with anhydrous potas-

314
M.S. Motawia et al. / Carbohydrate Research 330 (2001) 309–318
4.0 Hz, H-1%, b anomer), 5.24 (d, 0.4 H, J1,2
3.8 Hz, H-1a), 4.64 (d, 0.6 H, J1,2 8.2 H,
primers in amylose synthesis. De novo synthe-
sis of amylose was obtained upon incubation
of GBSS I bound to starch granules in the
presence of malto-oligosaccharides. Neither of
the maltose analogs 9 and 26 were able to
support amylose synthesis.14 This demon-
strates that the ability of malto-oligosacchar-
ides to stimulate amylose synthesis reflects
that GBSSI use these malto-oligosaccharides
as direct primers for amylose synthesis and
not as effectors–activators of GBSS I.14
13
H-1b); C NMR (D2O): l 137.0, 130.7, 129.6,
129.6, 127.1, 127.1 (Ph), 102.6 (2 C, C-1%, a
and b anomers), 101.3 (PhCH, a anomer),
101.2 (PhCH, b anomer), 96.6 (C-1b), 92.8
(C-1a), 81.0, 77.9, 75.2, 74.1, 73.2, 72.2, 70.6,
64.1 (C-2,3,4,5,2%,3%,4%,5% a anomer), 81.0, 77.8,
77.0, 74.8, 73.1, 70.9, 70.9, 64.1 (C-
2,3,4,5,2%,3%,4%,5% b anomer), 68.8 (2 C, C-6%, a
and b anomers), 61.4 (C-6, b anomer), 61.3
(C-6, a anomer).
Benzyl 2,3,6-tri-O-benzyl-4-O-(4,6-O-benz-
ylidene-2,3-di-O-benzyl-h-
D
-glucopyranosyl)-
3. Experimental
D-glucopyranoside (3).—A solution of 2 (3 g,
7 mmol) in dry DMF (100 mL) was stirred
with NaH (3.4 g, 60% dispersion in mineral
oil) for 2 h at rt and the mixture cooled to
0 °C. Benzyl bromide (10.5 mL) was added
dropwise to the mixture and stirring was con-
tinued overnight at rt. The mixture was cooled
to 0 °C and excess NaH decomposed by drop-
wise addition of MeOH. The solution was
concentrated in vacuo and diluted with EtOAc
(250 mL) and water (100 mL). The organic
phase was separated, washed with water (4×
50 mL) and brine (50 mL). After drying, the
solvent was evaporated and the residue chro-
matographed on silica gel (210 g) with (7:3
v/v) n-pentane–Et2O as eluent to give 3 (6 g,
colorless syrup, 88%); [h]D +9.1° (c 0.55;
CHCl3); 1H NMR (CDCl3):l 7.52–7.14 (m, 35
H, ArH), 5.75 (d, 0.3 H, J1%,2% 3.8 Hz, H-1%, a
anomer), 5.70 (d, 0.7 H, J1%,2% 3.9 Hz, H-1%, b
anomer), 5.53 (s, 0.7 H, PhCH, b anomer),
5.52 (s, 0.3 H, PhCH, a anomer), 5.08–4.44
(m, 13 H, PhCH2 and anomeric protons),
4.20–3.47 (m, 12 H, skeleton protons); 13C
NMR (CDCl3): l 102.3 (C-1b), 101.1 (2 C,
PhCH, a and b anomers), 97.2 (2 C, C-1%, a
and b anomers), 95.0 (C-1a), 84.9, 82.3, 82.3,
78.7, 78.7, 74.3, 71.7, 63.2 (C-2,3,4,5,2%,3%,4%,5%
b anomer), 82.2, 82.2, 82.0, 80.2, 78.8, 71.9,
69.6, 63.2 (C-2,3,4,5,2%,3%,4%,5% a anomer), 75.2,
75.2, 74.6, 73.6, 73.4, 70.9 (6×PhCH2, b
anomer), 74.1, 73.8, 73.8, 73.3, 72.8, 72.8 (6×
PhCH2, a anomer), 69.1 (C-6, a anomer), 68.9
(C-6, b anomer), 68.8 (C-6%, b anomer), 68.6
(C-6%, a anomer); FAB+MS: m/z 993 [M+
Na]+.
General methods.—Melting points were de-
termined using a Mettler FP81 MBC Cell
connected to a Mettler FP80 central processor
unit. Optical rotations were measured at 219
2 °C with an optical activity Ltd AA-1000
polarimeter.
All reactions were monitored by TLC on
aluminum sheets coated with silica Gel 60F254
(0.2 mm thickness, E. Merck, Darmstadt,
Germany) and the components present were
detected by charring with 10% H2SO4 in
MeOH. Column chromatography was carried
out using Silica Gel 60 (particle size 0.040–
0.063 mm, 230–400 mesh ASTM, E. Merck).
Solvent extracts were dried with anhyd
MgSO4 unless otherwise specified. Microanal-
ysis was performed at DB Lab Danish Bio-
protein A/S, Stenhuggervej 22, PO Box 829,
DK-5230 Odense M, Denmark.
The 1H and 13C NMR spectra were
recorded on a Bruker AC250P spectrometer at
250 and 63 MHz, respectively. In water, diox-
ane was used as an internal reference [lH
(dioxane)=3.75; lC (dioxane)=67.4]. In
other solvents, lH values are relative to inter-
nal Me4Si and lC values are referenced to the
solvent [lC (CDCl3)=77.0; lC (Me2SO-d6)=
39.4].
4-O-(4,6-O-benzylidene-h- -glucopyranosyl)-
D
D
-glucopyranose (2).—Compound 2 was syn-
thesized as reported previously;17 [h]D +79.5°
(c 1.06; D2O), lit. [h]D +81.1° (c 1.40; D2O)14;
1H NMR (D2O): l 7.58–7.47 (m, 5 H, ArH),
5.72 (s, 1 H, benzylic-H), 5.45 (d, 0.4 H, J1%,2%
4.0 Hz, H-1%, a anomer), 5.44 (d, 0.6 H, J1%,2%

M.S. Motawia et al. / Carbohydrate Research 330 (2001) 309–318
315
0.6 H, J1%,2% 3.6 Hz, H-1% b anomer), 5.08–4.40
(m, 13 H, PhCH2 and anomeric protons),
4.14–3.36 (m, 12 H, skeleton protons), 1.05,
1.03 (2s, 9 H, SiC(CH3)3; 13C NMR (CDCl3):
b anomer: l 102.2 (C-1), 96.1 (C-1%), 84.8,
82.0, 81.1, 79.1, 77.2, 72.6, 71.9, 71.1 (C-
2,3,4,5,2%,3%,4%,5%), 75.3, 75.3, 74.6, 73.3, 72.8,
70.1 (6×PhCH2), 69.3 (C-6), 63.9 (C-6%), 26.9
[(SiC(CH3)3], 19.3 [(SiC(CH3)3]; a anomer: l
96.1 (C-1%), 94.9 (C-1), 81.9, 81.0, 80.0, 79.3,
72.5, 71.9, 71.0 (C-2,3,4,5,2%,3%,4%,5%), 74.6,
74.1, 73.7, 73.2, 72.9, 72.9 (6×PhCH2), 69.1
(C-6), 63.7 (C-6%), 26.9 [(SiC(CH3)3], 19.3
[(SiC(CH3)3]; FAB+MS: m/z 1143 [M+Na]+.
Benzyl 2,3,6-tri-O-benzyl-4-O-(4-O-acetyl-
2,3-di-O-benzyl-6-O-tert-butyldiphenylsilyl-h-
Benzyl
benzyl-h-
2,3,6-tri-O-benzyl-4-O-(2,3-di-O-
-glucopyranosyl)- -glucopyranoside
D
D
(4).—A stirred solution of 3 (5.8 g, 5.97
mmol) in AcOH (60 mL) was heated to 95 °C,
water (40 mL) was added dropwise, and the
stirring was continued for 30 min at 95 °C.
The mixture was cooled, evaporated in vacuo
and the residue dissolved in EtOAc (200 mL)
and water (100 mL). The organic phase was
separated, washed with 1 M aq NaOH (3×50
mL), water (4×100 mL) and brine (50 mL).
After drying, the solvent was evaporated and
the residue chromatographed on silica gel (210
g) using a gradient of EtOAc in n-pentane (20,
30, and 40%) as eluent to obtain 4 (4.6 g,
colorless syrup, 85%); [h]D +22.5° (c 0.69;
1
CHCl3); H NMR (CDCl3): l 7.44–7.16 (m,
D
-glucopyranosyl)- -glucopyranoside (6).—To
D
30 H, ArH), 5.72 (d, 0.4 H, J1%,2% 3.6 Hz, H-1%
a anomer), 5.67 (d, 0.6 H, J1%,2% 3.6 Hz, H-1% b
anomer), 5.11–4.44 (m, 13 H, PhCH2 and
anomeric protons), 4.12–3.36 (m, 12 H, skele-
a stirred solution of 5 (2.24 g, 2.0 mmol) in
dry pyridine (30 mL) was added Ac2O (2.0
mL) at rt and stirring was continued
overnight. The solvents were removed in
vacuo and the last trace solvents were re-
moved by repeated co-evaporation with tolu-
ene. The residue was dissolved in EtOAc (100
mL) and washed with satd aq NaHCO3 (3×
25 mL), water (3×25 mL), and brine (25
mL). After drying, the solvent was evaporated
and the residue chromatographed on silica gel
(210 g) with (4:1, v/v) n-pentane–Et2O to
obtain 6 (2.2 g, colorless syrup, 95%); [h]D
+35.8° (c 1.1; CHCl3); 1H NMR data
(CDCl3): l 7.68–7.15 (m, 40 H, ArH), 5.60 (d,
0.7 H, J1%,2% 3.6 Hz, H-1% b anomer), 5.59 (d,
0.3 H, J1%,2% 3.6 Hz, H-1% a anomer), 5.11–4.42
(m, 13 H, PhCH2 and anomeric protons),
4.18–3.43 (m, 12 H, skeleton protons), 1.82,
1.81 (2s, 3 H, COCH3), 1.03, 1.02 [(2s, 9 H,
SiC(CH3)3]; 13C NMR (CDCl3): b anomer: l
169.3 (COCH3), 102.2 (C-1), 96.1 (C-1%), 84.7,
82.0, 79.5, 79.1, 74.7, 73.4, 71.2, 68.9 (C-
2,3,4,5,2%,3%,4%,5%), 75.0, 75.0, 74.6, 73.3, 73.1,
70.9 (6×PhCH2), 69.6 (C-6), 62.6 (C-6%), 26.8
[SiC(CH3)3], 20.8 (COCH3), 19.2 [SiC(CH3)3];
a anomer: l 169.3 (COCH3), 96.3 (C-1%), 95.0
(C-1), 81.8, 79.8, 79.6, 79.0, 73.6, 73.2, 71.1,
70.0 (C-2,3,4,5,2%,3%,4%,5%), 74.3, 73.8, 73.8,
73.2, 73.1, 72.8 (6×PhCH2), 69.4 (C-6), 62.6
(C-6%), 26.8 [SiC(CH3)3], 20.8 (COCH3),
19.2 [SiC(CH3)3]; FAB+MS: m/z 1185 [M+
Na]+.
13
ton protons); C NMR (CDCl3): b anomer: l
102.3 (C-1), 96.5 (C-1%), 84.8, 82.2, 81.2, 78.9,
74.6, 72.6, 71.4, 70.6 (C-2,3,4,5,2%,3%,4%,5%),
75.2, 75,2, 74.6, 73.3, 73.0, 70.9 (6×PhCH2),
69.3 (C-6), 62.3 (C-6%); a anomer: l 96.5 (C-
1%), 95.0 (C-1), 82.0, 81.1, 80.2, 79.0, 71.5,
71.4, 70.5, 68.9 (C-2,3,4,5,2%,3%,4%,5%), 74.2,
73.8, 73.8, 73.5, 73.2, 72.8 (6×PhCH2), 69.1
(C-6), 62.2 (C-6%).
Benzyl
benzyl-6-O-tert-butyldiphenylsilyl-h-
pyranosyl)- -glucopyranoside (5).—A stirred
2,3,6-tri-O-benzyl-4-O-(2,3-di-O-
D
-gluco-
D
solution of 4 (4.2 g, 4.76 mmol) in dry DMF
(100 mL) and imidazol (1.3 g, 19.1 mmol) was
cooled to 0 °C and tert-butylchloro-diphenyl-
silane (2.5 mL, 9.55 mmol) was added drop-
wise during a period of 5 min. The cooling
bath was removed and the mixture was stirred
for 1 h at rt. The mixture was diluted with
EtOAc (300 mL) and water (100 mL). The
organic phase was washed successively with
water (3×100 mL), satd aq NaHCO3 (3×50
mL), water (3×100 mL), and brine (50 mL).
After drying, the solvent was evaporated and
the residue chromatographed on silica gel (210
g) with (7:3, v/v) n-pentane–Et2O to afford 5
(5.12 g, colorless syrup, 96%, a/b ratio:2:3);
[h]D +23.6° (c 0.84; CHCl3); 1H NMR
(CDCl3): l 7.68–7.19 (m, 40 H, ArH), 5.66 (d,
0.4 H, J1%,2% 3.6 Hz, H-1% a anomer), 5.63 (d,

316
M.S. Motawia et al. / Carbohydrate Research 330 (2001) 309–318
monitored by complete disappearance of the
4-O-(4-O-Acetyl-6-O-tert-butyldiphenylsilyl-
-glucopyranosyl)- -glucopyranose (7).—A
h-D
D
starting material. After neutralization with
NaHCO3, the mixture was taken to dryness on
a rotavapor at rt. The residue was chro-
matographed on silica gel (15 g) with (4:1, v/v)
EtOAc–MeOH to obtain pure 9 (91 mg, white
powder, 96%); ESIMS positive mode: m/z 407
[M+Na]+.
solution of 6 (1.8 g) in (1:2, v/v) EtOAc–
EtOH (60 mL) was hydrogenated in the pres-
ence of 10% Pd–C (0.5 g) at normal pressure
for 3 days at rt. The catalyst was removed by
filtration through a Celite pad and a silica gel
layer, and thoroughly washed with EtOH
(6×30 mL). The combined filtrates were
evaporated in vacuo and the residue chro-
matographed on silica gel (50 g) with (4:1 v/v)
CH2Cl2 –MeOH as eluent to obtain 7 (0.63,
Phenyl 2,3,4,6-tetra-O-benzyl-1-thio-h- -
D
galactopyranoside (14).—Compound 1319
(3.70 g, 13.59 mmol) was benzylated as re-
ported previously20 to obtain 14 (6.21 g, 72%);
mp 88–89 °C (Et2O–n-pentane), lit.20 mp 88–
89 °C; [h]D +1.3° (c 0.50; CHCl3), lit.20 [h]D
white powder, 65%); mp 98–99 °C; [h]D
+
1
73.9° (c 0.38; CH3OH); H NMR (CD3OD): l
7.78–7.30 (m, 10 H, ArH), 5.24 (d, 0.5 H, J1%,2%
3.8 Hz, H-1% b anomer), 5.22 (d, 0.5 H, J1%,2%
3.8 Hz, H-1% a anomer), 5.11 (d, 0.5 H, J1,2 3.8
Hz, H-1 a anomer), 4.99 (t, 0.5 H, J3%,4% 9.6,
J4%,5% 10 Hz, H-4%, a anomer), 4.96 (t, 0.5 H,
J3%,4%=9.4, J4%,5%=10.2 Hz, H-4%, b anomer),
4.49 (d, 0.5 H, J1,2 7.9 Hz, H-1 b anomer),
3.98–3.17 (m, 11 H, skeleton protons), 1.99
(2s, 3 H, CH3CO), 1.03 (s, 9 H, [SiC(CH3)3]);
13C NMR (CD3OD): l 171.8, 171.7 (2×
COCH3 a and b anomers), 102.8 (C-1% b
anomer), 102.7 (C-1% a anomer), 98.1 (C-1 b
anomer), 93.8 (C-1 a anomer), 82.0, 81.6,
77.7, 76.8, 75.9, 74.5, 74.3, 74.1, 73.5, 73.2,
73.1, 72.9, 72.8, 72.1, 72.1, 71.8 (C-
2,3,4,5,2%,3%,4%,5% a and b anomers), 64.1 (C-6,
b anomer), 64.0 (C-6, a anomer), 62.6 (C-6% b
anomer), 62.4 (C-6%, a anomer), 27.1 ([SiC-
(CH3)3]), 21.0 (COCH3), 20.0 ([SiC(CH3)3]).
Anal. Calcd for C30H42O12Si·H2O: C, 56.9; H,
6.9. Found: C, 56.6; H, 7.1.
1
+1.0° (c 1.0; CHCl3); H NMR (CDCl3): l
7.58–7.16 (m, 25 H, ArH), 4.96, 4.59 (2d, 2 H,
J 11.5 Hz, PhCH2), 4.79, 4.73 (2d, 2 H, J 11.3
Hz, PhCH2), 4.71 (s, 2 H, PhCH2), 4.64 (d, 1
H, J1,2 9.6 Hz, H-1b), 4.47, 4.41 (2d, 2 H, J
10.5 Hz, PhCH2), 3.98–3.57 (m, 12 H, skele-
ton protons); 13C NMR (CDCl3): l 138.7–
127.0 (C arom.), 87.7 (C-1), 84.2, 77.3, 77.3,
73.6 (C-2,3,4,5), 75.6, 74.4, 73.5, 72.7 (4×
PhCH2), 68.7 (C-6).
2,3,4,6 - tetra - O - Benzyl - - galactopyranose
D
(15).—N-Bromosuccinimide (1 g, 6.74 mmol)
was added to a stirred solution of 14 (2 g, 3.16
mmol) in (9:1 v/v) acetone–water (20 mL) and
stirred for 5 min at rt, at which time all
thioglycoside had disappeared. Solid NaHCO3
(5 g) was added and the solvents evaporated
in vacuo. The residue was dissolved in EtOAc
(150 mL) and water (50 mL) and the organic
phase was separated and successively washed
with satd aq NaHCO3 (3×50 mL), water
(3×50 mL), and brine (25 mL). After drying,
the solvent was evaporated in vacuo and the
residue chromatographed on silica gel (60 g)
using (9:1 v/v) n-pentane–EtOAc as the initial
eluent to remove all impurities and (7:3 v/v)
n-pentane–EtOAc to elute 15 (1.7 g, colorless
syrup, quantitative) as an anomeric mixture
(a/b ratio:5:2 as determined by 13C NMR;
[h]D +11.5° (c 0.8; CHCl3); 1H NMR
(CDCl3):l 7.39–7.22 (m, 20 H, ArH), 5.26 (d,
0.7 H, J1,2 3.5 Hz, H-1 a anomer), 4.95–4.35
(m, 8.3 H, PhCH2 and H-1b), 4.18–3.42 (m, 6
H, skeleton protons); 13C NMR (CDCl3): b
anomer: l 97.7 (C-1), 82.1, 80.7, 76.5, 73.6
(C-2,3,4,5), 75.0, 74.5, 73.5, 73.5 (4×PhCH2),
4-O-(6-O-Acetyl-h-
D
-glucopyranosyl)- -
D
glucopyranose (8).—Tetrabutylammonium
fluoride (0.5 mL, 1.1 M in THF) was added to
a stirred solution of 7 (0.24 g) in THF (5 mL)
at rt and stirring was continued for 2 h after
which the solvent was evaporated and the
residue chromatographed on silica gel (26 g)
with (4:1, v/v) EtOAc–MeOH to give 8 (0.14
g, white powder, 95%); [h]D +102.9° (c 0.28;
CH3OH); FAB+MS: m/z 407 [M+Na]+.
4-O-(4-O-Acetyl-h-
D
-glucopyranosyl)- -
D
glucopyranose (9).—A clear solution of 7 (154
mg) in 50% aq MeOH (10 mL) was treated
with H3PO4 (291.3 mL, 2.68 M) at rt and the
1
mixture was let stand for 40 h. H NMR
experiments revealed complete desilylation as

M.S. Motawia et al. / Carbohydrate Research 330 (2001) 309–318
317
15 to give 25 (0.73 g, colorless syrup, 95%)
after chromatographic purification on silica
gel (60 g) using (3:2 v/v) Et2O–n-pentane as
eluent; [h]D +32.5° (c 0.88; CHCl3); 1H NMR
(CDCl3): l 7.35–7.14 (m, 35 H, ArH), 5.69 (d,
0.7 H, J1%,2% 3.6 Hz, H-1% a anomer), 5.68 (d,
0.3 H, J1%,2% 3.6 Hz, H-1% b anomer), 5.18 (d,
0.7 H, J1,2 3.5 Hz, H-1 a anomer), 4.89–4.27
(m, 14.3 H, PhCH2 and H-1b), 4.19–3.35 (m,
68.9 (C-6); a anomer: l 91.8 (C-1), 78.8, 76.5,
74.7, 69.4 (C-2,3,4,5), 74.6, 73.4, 73.4, 72.9
(4×PhCH2), 69.0 (C-6).
O-(2,3,4,6-tetra-O-Benzyl-h,i-
D
-galactopy-
ranosyl)trichloroacetimidate24
(16).—Com-
pound 16 was prepared from 15 (1.54 g)
according to the previously published
procedure.24
Phenyl
tetra-O-benzyl-h-
i- -glucopyranoside (24).—A mixture of the
crude product 16 (1.64 g, 2.39 mmol) and
2,3,6-tri-O-benzyl-4-O-(2,3,4,6-
13
12 H, skeleton protons); C NMR (CDCl3): a
D
-galactopyranosyl)-1-thio-
anomer: l 97.6 (C-1%), 90.7 (C-1), 81.3, 80.0,
79.0, 75.6, 74.7, 74.1, 69.9, 69.7 (C-
2,3,4,5,2%,3%,4%,5%), 74.7, 73.7, 73.4, 73.1, 73.1,
72.9, 72.7 (7×PhCH2), 69.5, 68.7 (C-6,6%); b
anomer: l 97.4 (C-1), 97.2 (C-1%), 84.4, 83.1,
77.2, 75.4, 74.6, 74.2, 73.9, 70.1 (C-
2,3,4,5,2%,3%,4%,5%), 74.7, 74.5, 73.5, 73.5, 73.4,
73.2, 72.9 (7×PhCH2), 69.7, 68.8 (C-6,6%);
FAB+MS: m/z 995 [M+Na]+.
D
phenyl
2,3,6-tri-O-benzyl-1-thio-b- -gluco-
D
pyranoside26 (0.8 g, 1.47 mmol) was co-evapo-
rated with dry CH2Cl2 (3×25 mL) and dried
for 1 h at 70 °C in vacuo. The residue was
dissolved in dry (2:1 v/v) Et2O–CH2Cl2 (75
,
mL) and stirred with 4 A molecular sieves (3
g, activated powder) for 1 h at rt under Ar.
The stirred mixture was then cooled to
−50 °C and trimethylsilyl trifluoromethane-
sulfonate (100 mL, 0.55 mmol) was added in
one portion, and stirring was continued for
1.5 h during which the temperature was grad-
ually raised to rt. Sodium hydrogen carbonate
(2 g) was added and the reaction mixture was
stirred for additional 20 min and filtered
through a pad of sea sand on the top of a
silica gel layer. The filtrate was evaporated,
EtOAc (150 mL) was added to the residue and
the organic phase was successively washed
with satd aq NaHCO3 (2×25 mL), water
(3×25 mL), and brine (25 mL). After drying,
the solvent was evaporated and the residue
was chromatographed on silica gel (180 g)
using (4:1 v/v) n-pentane–Et2O as eluent to
give pure 24 (1.24 g, gum, 79%); [h]D +30.7°
h-
D
-Galactopyranosyl-(1“4)- -glucopyra-
D
nose (26).—Compound 25 (0.5 g, 0.51 mmol)
was debenzylated as described for compound
7
and the crude product was chro-
matographed on silica gel (28 g) using (4:1
v/v) EtOAc–MeOH as eluent to give pure 26
1
(0.14 g, white powder, 80%); H NMR (D2O):
l 5.41 (d, 0.6 H, J1%,2% 3.6 Hz, H-1% b anomer),
5.40 (d, 0.4 H, J1%,2% 3.6 Hz, H-1% a anomer),
5.23 (d, 0.4 H, J1,2 3.8 Hz, H-1 a anomer),
4.65 (d, 0.6 H, J1,2 7.8 Hz, H-1 b anomer),
3.99–3.24 (m, 12 H, skeleton protons); 13C
NMR (D2O): a anomer: l 100.8 (C-1%), 92.7
(C-1), 77.8, 74.0, 72.6, 72.1, 70.8, 70.2, 70.0,
69.5 (C-2,3,4,5,2%,3%,4%,5%), 62.0, 61.4 (C-6,6%); b
anomer: l 100.7 (C-1%), 96.6 (C-1), 77.8, 77.0,
75.4, 74.8, 72.6, 70.2, 70.0, 69.4 (C-
2,3,4,5,2%,3%,4%,5%), 62.0, 61.6 (C-6,6%); FAB+
MS: m/z 365 [M+Na]+.
1
(c 0.85; CHCl3); H NMR (CDCl3): l 7.58–
7.12 (m, 40 H, Ar H), 5.70 (d, 1 H, J1%,2% 3.7
Hz, H-1%), 4.93–4.25 (m, 13 H, PhCH2 and
H-1), 4.13–3.41 (m, 12 H, skeleton protons);
13C NMR (CDCl3): l 97.7 (C-1%), 87.2 (C-1),
86.7, 81.0, 79.1, 78.6, 75.4, 74.6, 72.8, 69.9
(C-2,3,4,5,2%,3%,4%,5%), 75.2, 74.7, 74.2, 73.9,
73.4, 73.1, 72.6 (7×PhCH2), 69.9, 68.6 (C-
6,6%); FAB+MS: m/z 1087 [M+Na]+.
Acknowledgements
This work was financially supported by the
EU FAIR programme contract CT95-0568,
by the Danish National Research Foundation.
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D
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D
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