Chemical synthesis of 6'''-α-maltotriosyl-maltohexaose as substrate for enzymes in starch biosynthesis and degradation

Article abstract of DOI:10.1016/S0008-6215(99)00131-7

A branched nonasaccharide 6'''-α-maltotriosyl-maltohexaose was synthesised in 40 steps from D-glucose and maltose. Phenyl O-(2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl)-(1→4)-O-(2,3,6-tri-O-benzyl-α-D-glucopyranosyl)-(1→4)-2,3-di-O-benzyl-1-thio-β-D-glucop

Full text of DOI:10.1016/S0008-6215(99)00131-7

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Carbohydrate Research 320 (1999) 19–30
Chemical synthesis of 6§-a-maltotriosyl-maltohexaose as
substrate for enzymes in starch biosynthesis and
degradation
Iben Damager a,c, Carl Erik Olsen b,c, Birger Lindberg Møller a,c,
Mohammed Saddik Motawia a,c,
*
a Plant Biochemistry Laboratory, Department of Plant Biology, The Royal Veterinary and Agricultural Uni6ersity,
40 Thor6aldsens6ej, DK-1871 Frederiksberg C, Copenhagen, Denmark
b Department of Chemistry, The Royal Veterinary and Agricultural Uni6ersity, 40 Thor6aldsens6ej,
DK-1871 Frederiksberg C, Copenhagen, Denmark
c Center for Molecular Plant Physiology (PlaCe), The Royal Veterinary and Agricultural Uni6ersity,
40 Thor6aldsens6ej, DK-1871 Frederiksberg C, Copenhagen, Denmark
Received 25 January 1999; accepted 22 May 1999
Abstract
A branched nonasaccharide 6§-a-maltotriosyl-maltohexaose was synthesised in 40 steps from
maltose. Phenyl O-(2,3,4,6-tetra-O-benzyl-a- -glucopyranosyl)-(1“4)-O-(2,3,6-tri-O-benzyl-a- -glucopyranosyl)-
(1“4)-2,3-di-O-benzyl-1-thio-b- -glucopyranoside and O-(2,3,4,6-tetra-O-benzyl-a- -glucopyranosyl)-(1“4)-O-
(2,3,6-tri-O-benzyl-a- -glucopyranosyl trichloroacetimidate were
D-glucose and
D
D
D
D
D
-glucopyranosyl)-(1“4)-2,3,6-tri-O-benzyl-a,b-
D
coupled by a general condensation reaction to form the per-O-benzylated branched hexasaccharide phenyl thiogly-
coside. The phenylthio group of this compound was converted into a trichloroacetimidate, which was coupled with
phenyl O-(2,3,6-tri-O-benzyl-a-
D
-glucopyranosyl)-(1“4)-O-(2,3,6-tri-O-benzyl-a-
D
-glucopyranosyl)-(1“4)-2,3,6-tri-
O-benzyl-1-thio-b- -glucopyranoside to afford the per-O-benzylated branched nonasaccharide phenyl thioglycoside.
D
Replacement of the phenylthio group with a free OH-group followed by hydrogenolysis gave the desired product.
The synthons reported for this synthesis constitute a versatile tool for the chemical synthesis of other complex
carbohydrates. © 1999 Elsevier Science Ltd. All rights reserved.
Keywords: Carbohydrates; Branched oligosaccharide; Nonasaccharide; 6§-a-Maltotriosyl-maltohexaose
lose, which is a predominantly linear a-(1“
1. Introduction
4)-glucan, and of amylopectin, which is a
higher-molecular-mass a-(1“4)-glucan with
frequent a-(1“6)-glucan branch points. Al-
though studies within biochemistry, molecular
biology and genetics have resulted in isolation,
cloning and heterologous expression of a large
number of enzymes in starch metabolism, the
precise nature and sequential action of the
enzymatic reactions involved in starch biosyn-
thesis [1–3] and starch degradation [4,5] have
Starch can be distinguished from glycogen
by the presence of highly ordered and densely
packed glucan chains permitting packaging of
the starch as large insoluble granules in the
plastids of plants. Starch is composed of amy-
* Corresponding author. Tel.: +45-3-528-3353; fax: +45-
3-528-3333.
E-mail address: mosm@kvl.dk (M. Saddik Motawia)
0008-6215/99/$ - see front matter © 1999 Elsevier Science Ltd. All rights reserved.
PII: S0008-6215(99)00131-7

20
I. Damager et al. / Carbohydrate Research 320 (1999) 19–30
based on a limited number of synthons. In this
context, we have previously reported the syn-
thesis of a protected branched tetrasaccharide
[6] and its use in the synthesis of 6%-a-maltosyl-
maltotriose [7]. Here, we report the synthesis
of 6§-a-maltotriosyl-maltohexaose.
proven difficult to elucidate. First, starch de-
position and mobilisation involves the com-
plex interplay of a large number of enzymes.
Secondly, most of these enzymes occur as
multiple isoforms. Thirdly, the structure of the
a-(1“4)/a-(1“6)-glucan molecules that serve
as substrates for these enzymes are poorly
defined. Attempts to resolve the function of
the individual enzymes and isoforms by pro-
duction of plants in which expression of the
particular gene is blocked using anti-sense
technology have been greatly hampered by the
simultaneous presence of a multitude of en-
zymes or isoforms that exhibit partly overlap-
ping catalytic activities. The bottle neck in
defining the exact substrate specificity and cat-
alytic actions of the different enzymes and
differences in the properties of specific iso-
forms thus remains the availability of struc-
turally well-defined a-(1“4)/a-(1“6)-glucan
substrates. Accordingly, we have embarked on
a research programme to chemically synthe-
sise a range of complex oligosaccharides to be
used as substrates or primers for enzymes in
starch biosynthesis and as substrates for
starch-degrading enzymes. The approach is
2. Results and discussion
The strategy was to synthesise the nonasac-
charide 6§-a-maltotriosyl-maltohexaose (26)
from three trisaccharide building blocks (15,
18 and 20), which were meant to be retrosyn-
thesised from four building blocks – two
monosaccharide glycosyl donors (5 and 9) and
two disaccharide glycosyl acceptors (11 and
12) (Scheme 1). Chemical synthesis of 20 from
9 and 12 was accomplished. However,
difficulties encountered in separating interme-
diary products as well as 20 from reactants
and products formed prompted us to synthe-
sise 20 from the monosaccharide glycosyl ac-
ceptor 6, which is an intermediate in the
synthesis of 9, and the disaccharide 13
(Scheme 1).
Scheme 1. Retrosynthesis of the nonasaccharide 6§-a-maltotriosyl-maltohexaose (26) (Scheme 4) via three trisaccharide building
blocks synthesised from derivatives of glucose and maltose.

I. Damager et al. / Carbohydrate Research 320 (1999) 19–30
21
Scheme 2. Synthesis of the monosaccharide building blocks.
routes [20,21]. The glycosyl trichloroacetimi-
date derivative 9 (to be used as glycosyl
donor) was obtained in 98% yield by treating
8 with trichloroacetonitrile in the presence of
anhydrous potassium carbonate.
Synthesis of the disaccharide building blocks
(Scheme 1).—The three disaccharides 11, 12
and 13 were synthesised from maltose (10) (to
be published elsewhere).
Synthesis of the monosaccharide building
blocks (Scheme 2).—The synthesis of the de-
sired glycosyl donors 5 and 9 and of the
glycosyl acceptor 6 from
D
-glucose 1 is shown
in Scheme 2. -Glucose 1 was converted into
D
phenyl 1-thio-b- -glucopyranoside 2 in two
D
steps [7]. Benzylation of 2 with NaH and
benzyl bromide gave the known phenyl
2,3,4,6-tetra-O-benzyl-1-thio-b- -glucopyra-
D
noside (3) in 60% yield [8–10]. By treating the
thioglycoside 3 with N-bromosuccinimide in
aqueous acetone [11], the phenylthio group
was replaced with a free OH-group affording
an anomeric mixture of 2,3,4,6-tetra-O-ben-
Synthesis of the trisaccharide building blocks
(Scheme 3).—All three trisaccharide building
blocks were synthesised by coupling the ap-
propriate monosaccharide derivative with the
corresponding disaccharide derivative. All
zyl-a,b- -glycopyranose (4) in 92% yield after
D
chromatographic purification. Based on mea-
surements of optical rotation and melting
point, 4 has previously been reported as the a
anomer [12–16]. In the present study, 4 was
identified as an a,b mixture by NMR spec-
troscopy. Reaction of 4 with trichloroacetoni-
trile in the presence of anhydrous potassium
carbonate afforded the desired glycosyl donor
O-(2,3,4,6-tetra-O-benzyl-a,b- -glucopyra-
D
nosyl) trichloroacetimidate (5), which has
been described previously [17–19], in quanti-
tative yield. In four known steps, 2 was con-
verted into the glycosyl acceptor phenyl
2,3,6-tri-O-benzyl-1-thio-b- -glucopyranoside
D
(6) [7], which was acetylated using acetic an-
hydride in pyridine to give phenyl 4-O-acetyl-
2,3,6-tri-O-benzyl-1-thio-b- -glucopyranoside
D
(7) in 98% yield. Treatment of 7 with N-bro-
mosuccinimide in aqueous acetone to replace
the phenylthio group with a free OH-group
gave 4-O-acetyl-2,3,6-tri-O-benzyl-a,b- -glu-
D
copyranose (8) in 91% yield [11]. Compound 8
has been prepared previously by different
Scheme 3. Synthesis of the three trisaccharide building blocks.

22
I. Damager et al. / Carbohydrate Research 320 (1999) 19–30
the desired building block 18 in quantitative
yield. Coupling of the glycosyl donor 9 and
the glycosyl acceptor 12 in diethyl ether, using
trimethylsilyl triflate as catalyst, produced a
mixture of 19 and 12. Attempts to separate
the product 19 from the reactant 12 by chro-
matography were unsuccessful. Subsequent
deacetylation of this mixture with sodium
methoxide in methanol produced a mixture of
20 and 12, which could not be separated ei-
ther. To overcome these problems, 19 was
synthesised from the monosaccharide glycosyl
acceptor 6 and the disaccharide glycosyl
donor 13 in 55% yield after chromatographic
purification. Exclusive introduction of an a-
linkage by the coupling reaction was verified
by H and C NMR spectroscopy. In the H
NMR spectrum, a doublet at l 5.50 with J1,2
3.7 Hz and a doublet at l 5.58 with J1,2 3.7 Hz
reflect the two a-linkages. In the 13C NMR
spectrum a signal at 87.3 ppm reflects C-1 and
two signals at 96.7 and 96.8 ppm reflect the
a-linkage configuration of C-1% and C-1¦.
Deacetylation of 19 with sodium methoxide in
methanol gave the desired building block 20 in
quantitative yield after chromatographic
purification.
synthetic schemes are based on the possibility
of quantitative removal of the phenylthio
group protecting the anomeric centre by treat-
ment with N-bromosuccinimide in aqueous
acetone [11]. Diethyl ether favouring the for-
mation of 1,2-cis-glycosides [22,23] was the
choice of solvent in all glycosidation reactions.
Coupling the glycosyl donor 5 with the glyco-
syl acceptor 11 in dry diethyl ether, using
trimethylsilyl triflate as catalyst, provided the
4-phenylbenzoyl derivative 14 in 48% yield
after chromatographic purification. The cou-
pling reaction resulted in the expected forma-
tion of an a-linkage as verified from the
diagnostic signal for C-1¦ at 97.2 ppm in the
13C NMR spectrum. The introduction of a
b-linkage would have given rise to a signal
with a value higher than 102 ppm [24,25]. The
signal at 86.9 ppm reflects C-1 and the signal
at 101.3 ppm reflects C-1% as also seen in 11
(unpublished data). In the 1H NMR spectrum,
the presence of a doublet at l 5.61 with J1,2
3.5 Hz reflects the newly formed a-linkage.
The two other anomeric protons are over-
lapped by the methylene protons of the benzyl
groups. The desired phenyl thioglycoside
building block 15, having a free OH-6 at the
1
13
1
Synthesis of the hexasaccharides (Scheme
4).—The branched hexasaccharide 21 was
synthesised from the glycosyl acceptor 15 and
the glycosyl donor 18 in 64% yield using dry
diethyl ether and trimethylsilyl triflate as cata-
lyst. The coupling reaction resulted in the
specific introduction of an a-linkage as ver-
ified by 13C NMR spectroscopy. A signal at
87.2 ppm reflects C-1 and five signals at 96.7,
96.8, 97.0, 97.3, 100.3 reflect the five internal
reducing terminal -glucopyranoside unit, was
D
obtained in 80% yield after chromatographic
purification by treating 14 with sodium
methoxide in methanol. The phenyl thiogly-
coside 16 was obtained in 80% yield after
chromatographic purification by coupling the
glycosyl donor 5 and the glycosyl acceptor 12
in diethyl ether with trimethylsilyl triflate as
catalyst. Less than 3% of formed b isomer as
well as other impurities were removed by
1
anomeric carbons. In the H NMR spectrum,
1
chromatography. In the H NMR spectrum, a
only three anomeric protons are identified.
The signals of the remaining anomeric protons
are overlapped with signals from the
methylene protons of the benzyl groups.
Treatment of the thioglycoside 21 with N-bro-
mosuccinimide in aqueous acetone afforded
22 in 87% yield after chromatographic purifi-
cation. Reaction of 22 with trichloroacetoni-
trile in the presence of anhydrous potassium
carbonate provided the desired building block
23 in quantitative yield.
doublet at l 5.50 with J1,2 3.6 Hz and a
doublet at l 5.62 with J1,2 3.6 Hz reflect the
two a-linkages. In the 13C NMR spectrum a
signal at 87.2 ppm reflects C-1 and two signals
at 96.8 and 96.9 ppm reflect C-1% and C-1¦a-
linkages. By treatment of the thioglycoside 16
with N-bromosuccinimide in aqueous acetone
[11], the phenylthio group was replaced with a
free OH-group to give 17 [26,27] in 91% yield
after chromatographic purification. Reaction
of 17 with trichloroacetonitrile in the presence
of anhydrous potassium carbonate afforded
Synthesis of the nonasaccharides (Scheme
4).—The trisaccharide 20 (glycosyl acceptor)

I. Damager et al. / Carbohydrate Research 320 (1999) 19–30
23
Scheme 4. Synthesis of hexasaccharides and nonasaccharides.
and the hexasaccharide 23 (glycosyl donor)
were coupled in diethyl ether and trimethylsi-
lyl triflate to afford the protected branched
nonasaccharide thioglycoside 24, in 56% yield,
after chromatographic purification. The cou-
pling resulted in formation of the expected
mosuccinimide in aqueous acetone to afford
25 in 86% yield. 6§-a-Maltotriosyl-malto-
hexaose (26) was obtained in 80% yield after
chromatographic purification by hydrogenoly-
sis of the benzyl ether protecting groups with
10% PdꢀC under hydrogen (Fig. 1).
Conclusions.—The availability of the syn-
thons used for chemical synthesis of 26 com-
bined with those previously used for chemical
synthesis of 6%-a-maltosyl-maltotriose [7] of-
fers excellent possibilities for chemical synthe-
sis of an infinite number of other complex
carbohydrates, which may be either linear
oligosaccharides with a defined degree of poly-
merisation, or branched oligosaccharides en-
compassing one or more branch points with
defined position and spacing. The use of N-
bromosuccinimide for removal of the thio-
phenyl group at the anomeric position to
generate a reducing sugar with the original
1
a-linkage as verified by 13C and H NMR
spectroscopy. In the 13C NMR spectrum, a
signal at 87.2 ppm reflects the C-1 and eight
signals at 95.9, 96.1, 96.6, 96.7, 96.8, 97.0,
97.4, 98.7 reflect the eight internal anomeric
1
carbon atoms. In the H NMR spectrum, four
doublets at l 5.18 with J1,2 3.6 Hz, l 5.32 with
J1,2 3.3 Hz, l 5.50 with J1,2 3.5 Hz, l 5.54 with
J1,2 3.5 Hz and a multiplet containing four
protons reflect the eight internal anomeric
protons. The H-1b is overlapped by the signals
from the methylene protons of the benzyl
groups. The phenylthio group in 24 was re-
placed with a free OH-group by using N-bro-

24
I. Damager et al. / Carbohydrate Research 320 (1999) 19–30
3. Experimental
protecting groups still present [11] constitutes
a key reaction in all synthetic schemes. The
outlined strategy for chemical synthesis ren-
ders it possible to chemically synthesise de-
sired partial structures of the starch polymers,
amylopectin and amylose and to test these as
substrates for starch-synthesising or -degrad-
ing enzymes.
General methods.—Melting points were de-
termined using a Mettler FP81 MBC Cell
connected to a Mettler FP80 Central Proces-
sor unit. Optical rotations were measured at
2791 °C with an Optical Activity Ltd AA-
1000 Polarimeter. All reactions were moni-
1
Fig. 1. (a) MALDI spectrum of 26; (b) 500 MHz H NMR spectrum of 26 in D2O; (c) 126 MHz 13C NMR spectrum of 26 in
D2O.

I. Damager et al. / Carbohydrate Research 320 (1999) 19–30
25
tored by thin layer chromatography (TLC) on
aluminium sheets coated with Silica Gel
60F254 (0.2 mm thickness, E. Merck, Darm-
stadt, Germany) and the components were
visualised by charring with 10% H2SO4 in
MeOH. Column chromatography was carried
out using Silica Gel (particle size 0.040–0.063
mm, 230–400 mesh ASTM, E. Merck) and
stepwise elution with the solvent mixtures,
known from TLC chromatography, to permit
separation of the individual components. Sol-
vent extracts were dried with anhyd MgSO4
2,3,4,6-Tetra-O-benzyl-h,i-
D
-glucopyranose
(4).—Acetone–water (9:1 v/v, 276 mL) was
added to a soln of 3 (17.00 g, 26.86 mmol) in
acetone (138 mL). NBS (9.56 g, 53.72 mmol)
was added to the soln in one portion and after
5 min the reaction was quenched by adding
solid NaHCO3 (20 g). The mixture was stirred
for 10 min, evaporated in vacuo at rt and then
diluted with EtOAc (300 mL). The organic
phase was washed with water until pH neu-
tral, then with brine, dried and evaporated to
dryness. The residue was chromatographed on
Silica Gel (220 g) with CH2Cl2–MeOH (19:1
v/v) as eluent to give 4 as white solid (13.29 g,
92%) as an a,b mixture (2:1): mp 151–152 °C,
lit. 151–152 °C [16]; [h]D 919.3° (c 0.99,
1
unless otherwise specified. The H and 13C
NMR spectra were recorded on a Bruker
AC250P spectrometer or a Bruker AM500
spectrometer. In D2O, dioxane was used as
1
internal
reference
(lH(dioxane)93.75;
CHCl3), lit.921.7° (c 2.19, CHCl3) [16]; H
lC(dioxane)967.4). In CDCl3, the lH-values
are relative to internal Me4Si and the lC-val-
ues are referenced to the solvent (lC(CD-
Cl3)977.0). MALDI spectra were recorded
on a Tofspec E spectrometer (Micromass).
NMR (CDCl3): l 3.36–4.07 (m, 6 H, H-2,
H-3, H-4, H-5, 2 H-6), 4.44–4.97 (m, 8.3 H, 4
CH2Ph and H-1b), 5.22 (d, 0.7 H, J1,2 3.5 Hz,
H-1a), 7.10–7.36 (m, 20 H, H-arom, 4 PhCH2;
13C NMR (CDCl3): For the a anomer: l 68.6
(C-6), 73.1, 73.4, 74.9, 75.6 (4 CH2Ph), 70.2,
77.7, 80.0, 81.7 (C-2, C-3, C-4, C-5), 91.2
(C-1), 127.6–138.7 (C-arom, PhCH2). For the
b anomer: l 68.9 (C-6), 74.6, 75.6, 74.6, 75.6
(4 CH2Ph), 74.7, 77.8, 83.1, 84.5 (C-2, C-3,
C-4, C-5), 97.4 (C-1), 127.6–138.7 (C-arom,
PhCH2). Anal. Calcd for C34H36O6: C, 75.53;
H, 6.71. Found: C, 75.33; H, 6.85.
Phenyl 2,3,4,6-tetra-O-benzyl-1-thio-i- -
D
glucopyranoside (3).—A soln of 2 (12.26 g,
45.02 mmol) in dry DMF (200 mL) was
stirred with NaH (14.53 g, 60% in mineral oil)
for 3 h at room temperature (rt). The mixture
was cooled to 0 °C and benzyl bromide (43.2
mL) was added dropwise and stirring was
continued at rt for 45 h. After cooling to 0 °C
and addition of MeOH to decompose excess
NaH, the soln was concd in vacuo and diluted
with EtOAc (150 mL) and water (50 mL). The
organic phase was washed with water (4×25
mL) and brine, dried and evaporated. Com-
pound 3 was obtained by crystallisation from
EtOH as white needles (17.03 g, 60%): mp
93 °C, lit. 92–93 °C [8]; [h]D 90.01° (c 0.31,
O-(2,3,4,6-Tetra-O-benzyl-h,i- -glucopyra-
D
nosyl) trichloroacetimidate (5).—A soln of 4
(12.50 g, 23.12 mmol) in dry CH2Cl2 (150 mL)
was stirred vigorously with CCl3CN (30 mL)
and K2CO3 (15 g) for 23 h at rt under N2. The
reaction mixture was filtered through a sea
sand pad and a layer of Silica Gel. The filtrate
was evaporated to dryness to give 5 as colour-
less syrup (15.79 g, quantitative) as an a,b
mixture (1:10), which was used directly for the
1
CHCl3), lit. [h]D 91° (CHCl3) [8]; H NMR
(CDCl3): l 3.47–3.54 (m, 2 H, H-2 and H-5),
3.61–3.75 (m, 3 H, H-3, H-4 and H-6b), 3.79
(dd, 1 H, J6a,6b 10.9 Hz, J6a,5 2.0 Hz, H-6a),
4.67 (d, 1 H, J1,2 9.8 Hz, H-1), 4.50–4.92 (m,
8 H, 4 CH2Ph), 7.17–7.60 (m, 25 H, H-arom,
SPh and PhCH2); 13C NMR (CDCl3): l 69.0
(C-6), 73.4, 75.0, 75.4, 75.8 (4 CH2Ph), 77.8,
79.1, 80.8 (C-2, C-4, C-5), 86.7 (C-3), 87.4
(C-1), 127.4–138.4 (C-arom, SPh and
PhCH2). Anal. Calcd for C40H40O5S: C, 75.92;
H, 6.37; S, 5.07. Found: C, 75.77; H, 6.66; S,
5.52.
1
next step without further purification. The H
NMR spectral data were identical to the liter-
ature data [17–19]; 13C NMR (CDCl3): For
the a anomer: l 68.0 (C-6), 72.8, 73.4, 75.1,
75.3 (4 CH2Ph), 73.1, 76.8, 79.4, 81.4 (C-2,
C-3, C-4, C-5), 91.3 (CCl3), 94.4 (C-1), 127.6–
138.4 (C-arom, PhCH2), 161.3 (CꢁNH). For
the b anomer: l 68.2 (C-6), 73.3, 74.8, 74.9,
75.6 (4 CH2Ph), 75.9, 77.3, 80.9, 84.6 (C-2,
C-3, C-4, C-5), 91.0 (CCl3), 98.3 (C-1), 127.6–
138.4 (C-arom, PhCH2), 161.1 (CꢁNH).

26
I. Damager et al. / Carbohydrate Research 320 (1999) 19–30
H-3, H-4, H-5, 2 H-6), 4.41–5.23 (m, 6 H,
Phenyl
thio-i-
4-O-acetyl-2,3,6-tri-O-benzyl-1-
-glucopyranoside (7).—Acetic anhy-
CH2Ph), 5.85 (d, 0.3 H, J1,2 7.6 Hz, H-1b),
6.50 (d, 0.7, J1,2 3.5 Hz, H-1a), 7.17–7.36 (m,
15 H, H-arom, PhCH2), 8.63, 8.75 (2s, 1 H,
NH, a and b anomer); 13C NMR (CDCl3):
For the a anomer: l 20.6 (COCH3), 68.3
(C-6), 72.8, 73.3, 74.8 (3 CH2Ph), 69.5, 71.5,
78.1, 78.8 (C-2, C-3, C-4, C-5), 91.0 (CCl3),
93.9 (C-1), 127.4–138.4 (C-arom, PhCH2),
160.8 (CꢁNH). For the b anomer: l 20.6
(COCH3), 68.8 (C-6), 73.2, 74.9, 75.0 (3
CH2Ph), 70.2, 74.2, 80.4, 81.4 (C-2, C-3, C-4,
C-5), 90.6 (CCl3), 97.8 (C-1), 127.6–138.4 (C-
arom, PhCH2), 160.9 (CꢁNH). Anal. Calcd
for C29H32O7: C, 70.71; H, 6.55. Found: C,
70.55; H, 6.90.
D
dride (50 mL) was added to a soln of 6 (5.13
g, 9.45 mmol) in dry pyridine (100 mL) and
stirred for 24 h at rt. The reaction mixture was
concd, coevaporated with toluene (3×50 mL)
and then dissolved in EtOAc (250 mL). The
organic phase was successively washed with
satd aq NaHCO3, water until pH neutral, then
with brine and dried before evaporation to
dryness. Compound 7 was obtained by crys-
tallisation from EtOH as white fibres (5.43 g,
98%): mp 81–84 °C; [h]D-21.1° (c 0.39,
1
CHCl3); H NMR (CDCl3): l 1.84 (s, 3 H,
COCH3), 3.50–3.70 (m, 5 H, H-2, 2 H-6, H-3,
H-5), 4.50 (s, 2 H, CH2Ph), 4.63, 4.81 (2 d, 2
H, J 11.4 Hz, CH2Ph), 4.69 (d, 1 H, J1,2 9.5
Hz, H-1), 4.70, 4.88 (2 d, 2 H, J 10.4 Hz,
CH2Ph), 5.00 (m, 1 H, H-4), 7.20–7.59 (m, 20
H, H-arom, PhCH2 and SPh); 13C NMR
(CDCl3): l 20.8 (COCH3), 69.7 (C-6), 73.5,
75.4, 75.5 (3 CH2Ph), 70.8 (C-4), 77.6 (C-5),
80.6 (C-2), 84.0 (C-3), 87.5 (C-1), 127.5–138.1
(C-arom, SPh and PhCH2), 169.7 (CꢁO).
Anal. Calcd for C35H36O6S: C, 71.89; H, 6.21;
S, 5.48. Found: C, 71.34; H, 6.30; S, 5.55.
Phenyl O-(2,3,4,6-tetra-O-benzyl-h-
copyranosyl)-(1“4)-O-(2,3,6-tri-O-benzyl-h-
-glucopyranosyl)-(1“4)-2,3-di-O-benzyl-6-
O - (4 - phenylbenzoyl) - 1 - thio - i - - glucopyr-
D-glu-
D
D
anoside (14).—A soln of 5 (6.40 g, 9.34 mmol)
and 11 (6.60 g, 6.20 mmol) in dry diethyl ether
(200 mL) was stirred for 1 h at rt under Ar in
,
the presence of 4 A molecular sieves (2.5 g,
activated powder). After cooling to −20 °C
and
addition
of
trimethylsilyl
trifl-
4-O-Acetyl-2,3,6-tri-O-benzyl-h,i- -gly-
D
uoromethanesulfonate (290 mL, 1.57 mmol),
stirring was continued for 1.5 h; during this
period the temperature was raised to rt. After
addition of solid NaHCO3 (9 g), the reaction
mixture was stirred for 10 min, diluted with
EtOAc (150 mL) and filtered through a sea
sand pad and a layer of Silica Gel. The filtrate
was successively washed with satd aq
NaHCO3, water until pH neutral, brine, dried
and evaporated to dryness. The residue was
chromatographed on Silica Gel (210 g) with
diethyl ether–n-pentane (1:4 and 3:7 v/v) as
eluent to give 14 as gum (4.68 g, 48%): [h]D 9
copyranose (8).—The procedure was the same
as described for 4 using acetone–water (9:1
v/v, 50 mL), 7 (3.00 g, 5.13 mmol) in acetone
(25 mL) and NBS (1.83 g, 10.26 mmol). Reac-
tion time: 3.5 min. The residue was chro-
matographed on Silica Gel (220 g) with
EtOAc–n-pentane (3:7 v/v) as eluent to give 8
as white fibers (2.30 g, 91%) as an a,b mixture
(2:5): mp 115–116 °C, lit. 112–113° [21];
[h]D99.8° (c 0.43, CHCl3), lit.910.9° (c 1.0,
CHCl3) [21]. The NMR data were identical to
the literature data [20,21]. Anal. Calcd for
C29H32O7: C, 70.71; H, 6.55. Found: C, 70.55;
H, 6.90.
1
29.7° (c 0.34, CHCl3); H NMR (CDCl3): l
5.61 (d, 1 H, J1,2 3.6 Hz, H-1, internal
anomeric). Two anomeric protons were over-
lapped by the methylene protons of the benzyl
4-O-Acetyl-2,3,6-tri-O-benzyl-h,i- -glu-
D
copyranosyl trichloroacetimidate (9).—The
procedure was the same as described for 5
using 8 (2.97 g, 6.03 mmol) in dry CH2Cl2 (75
mL), CCl3CN (9 mL) and K2CO3 (4.5 g) to
give 9 as colourless syrup (3.78 g, 98%) as an
a,b mixture (2:1), which was used directly for
13
groups; C NMR (CDCl3): l 64.0 (C-6), 68.1,
68.6 (C-6%, C-6¦), 73.3, 73.4, 73.5, 74.5, 74.9,
74.9, 74.9, 75.0, 75.5 (9 CH2Ph), 71.1, 71.5,
72.9, 76.3, 77.6, 78.6, 79.3, 79.4, 80.0, 82.0,
82.1, 82.7 (C-2, C-3, C-4, C-5, C-2%, C-3%, C-4%,
C-5%, C-2¦, C-3¦, C-4¦, C-5¦), 86.9 (C-1), 97.3
(C-1¦), 101.3 (C-1%), 126.4–145.7 (C-arom,
SPh, PhCH2 and C6H4Ph), 165.8 (CꢁO).
1
the next step without further purification: H
NMR (CDCl3): l 1.80, 1.84 (2s, 3 H, COCH3,
b and a anomer), 3.43–4.09 (m, 6 H, H-2,

I. Damager et al. / Carbohydrate Research 320 (1999) 19–30
27
Phenyl O-(2,3,4,6-tetra-O-benzyl-h-
copyranosyl)-(1“4)-O-(2,3,6-tri-O-benzyl-h-
-glucopyranosyl)-(1“4)-2,3-di-O-benzyl-1-
thio-i- -glucopyranoside (15).—NaOMe (4.4
D
-glu-
81.5, 82.0 (C-2, C-4, C-5, C-2%, C-3%, C-4%,
C-5%, C-2¦, C-3¦, C-4¦, C-5¦), 86.5 (C-3), 87.2
(C-1), 96.8, 96.9 (C-1%, C-1¦), 126.5–138.8 (C-
arom, SPh and PhCH2).
D
D
mL, 30% in MeOH) was added to a soln of 14
(2.41 g, 1.52 mmol) in MeOH–EtOAc (30
mL, 1:1 v/v) at 0 °C followed by stirring at rt
for 32 h. Acetic acid was added to obtain
neutral pH. The reaction mixture was concd
to dryness and the residue was dissolved in
CHCl3 (50 mL) and successively washed with
1 M aq NaOH (25 mL), water until pH neu-
tral, brine, dried and concd. The residue was
chromatographed on Silica Gel (60 g) with
diethyl ether–n-pentane (1:4 and 3:7 v/v) as
eluent to give 15 as colourless syrup (1.72 g,
O-(2,3,4,6-Tetra-O-benzyl-h-
nosyl)-(1“4)-O-(2,3,6-tri-O-benzyl-h-
pyranosyl)-(1“4)-2,3,6-tri-O-benzyl-h,i-
D
-glucopyra-
D
-gluco-
D-
glycopyranose (17).—The same procedure as
described for 4 using acetone–water (9:1 v/v,
60 mL), 16 (4.62 g, 3.08 mmol) in acetone (30
mL) and NBS (1.10 g, 6.16 mmol). Reaction
time: 5 min. The residue was chromato-
graphed on Silica Gel (95 g) with EtOAc–n-
pentane (1:4 and 100:0 v/v) as eluent to give
17 as colourless syrup (3.94 g, 91%) as an a,b
mixture (2:1): [h]D 948° (c 0.1, CHCl3). The
1H NMR spectral data were identical to the
1
80%): [h]D932.3° (c 0.23, CHCl3); H NMR
13
(CDCl3): l 4.96 (d, 1 H, J1,2 3.6 Hz, H-1,
internal anomeric), 5.58 (d, 1 H, J1,2 3.6 Hz,
H-1, internal anomeric). H-1b was overlapped
by the signals from the methylene protons of
literature data [26]; C NMR (CDCl3): l 90.8
(C-1a), 97.3 (C-1b), 96.5, 96.7 (C-1%, C-1¦),
126.5–138.9 (C-arom, PhCH2) [26].
O-(2,3,4,6-Tetra-O-benzyl-h-
D
-glucopyra-
-gluco-
pyranosyl)-(1“4)-2,3,6-tri-O-benzyl-h,i-
13
the benzyl groups; C NMR (CDCl3): l 61.9
nosyl)-(1“4)-O-(2,3,6-tri-O-benzyl-h-
D
(C-6), 68.2, 69.0 (C-6%, C-6¦), 73.3, 73.4, 73.5,
73.5, 74.4, 74.6, 75.0, 75.0, 75.5 (9 CH2Ph),
71.0, 71.3, 73.3, 77.7, 78.5, 78.7, 79.2, 79.7,
79.9, 81.4, 81.9, 81.9 (C-2, C-3, C-4, C-5, C-2%,
C-3%, C-4%, C-5%, C-2¦, C-3¦, C-4¦, C-5¦), 87.0
(C-1), 97.3 (C-1¦), 100.6 (C-1%), 126.3–138.6
(C-arom, SPh and PhCH2).
D-
glucopyranosyl trichloroacetimidate (18).—The
same procedure as described for 5 using 17
(3.15 g, 2.24 mmol) in dry CH2Cl2 (60 mL),
CCl3CN (6 mL) and K2CO3 (3 g) to give 18 as
colourless syrup (3.47 g, quantitative) as an
a,b mixture (1:2), which was used directly for
Phenyl O-(2,3,4,6-tetra-O-benzyl-h-
copyranosyl)-(1“4)-O-(2,3,6-tri-O-benzyl-h-
-glucopyranosyl)-(1“4)-2,3,6-tri-O-benzyl-
1-thio-i- -glucopyranoside (16).—The proce-
D-glu-
1
the next step without further purification: H
NMR (CDCl3): for the a anomer: l 5.58 (d,
0.3 H, J1,2 3.6 Hz, H-1, internal anomeric),
5.68 (d, 0.3 H, J1,2 3.6 Hz, H-1, internal
anomeric), 6.54 (d, 0.3 H, J1,2 3.4 Hz, H-1),
8.59 (s, 0.3 H, NH). For the b anomer: l 5.51
(d, 0.7 H, J1,2 3.6 Hz, H-1, internal anomeric),
5.64 (d, 0.7 H, J1,2 3.6 Hz, H-1, internal
anomeric), 5.91 (d, 0.7 H, J1,2 7.0 Hz, H-1),
8.69 (s, 0.7 H, NH); 13C NMR (CDCl3): for
the a anomer: l 91.3 (CCl3), 94.1 (C-1), 96.5,
96.7 (C-1%, C-1¦), 161.4 (CꢁNH), 126.5–138.8
(C-arom, PhCH2). For the b anomer: l 91.0
(CCl3), 96.6, 96.9 (C-1%, C-1¦), 98.1 (C-1),
D
D
dure was the same as described for 14 using 5
(2.75 g, 4.01 mmol) and 12 (2.60 g, 2.67
,
mmol) in dry diethyl ether (200 mL), 4 A
molecular sieves (2.5 g, activated powder) and
trimethylsilyl trifluoromethanesulfonate (123
mL, 0.66 mmol). The residue was chro-
matographed on Silica Gel (175 g) with di-
ethyl ether–n-pentane (1:4 and 3:7 v/v) as
eluent to give 16 (3.19 g, 80%, syrup): [h]D9
1
47° (c 0.1, CHCl3); H NMR (CDCl3): l 5.50
(d, 1 H, J1,2 3.6 Hz, H-1, internal anomeric),
5.62 (d, 1 H, J1,2 3.6 Hz, H-1, internal
anomeric). H-1b was overlapped by the signals
from the methylene protons of the benzyl
161.0
PhCH2).
(CꢁNH),
126.5–138.8
(C-arom,
Phenyl O-(4-O-acetyl-2,3,6-tri-O-benzyl-h-
- glucopyranosyl) - (1“4) - O - (2,3,6 - tri - O -
D
13
groups; C NMR (CDCl3): l 68.2, 69.0, 69.1
benzyl-h-
D
-glucopyranosyl)-(1“4)-2,3,6-tri-
-glucopyranoside (19).
(C-6, C-6%, C-6¦), 72.9, 73.1, 73.2, 73.2, 73.4,
74.0, 74.4, 74.9, 75.2, 75.4 (10 CH2Ph), 70.9,
70.9, 71.0, 73.7, 77.6, 78.8, 79.3, 79.5, 80.8,
O-benzyl-1-thio-i-
D
—The same procedure as described for 14
using 6 (1.21 g, 2.23 mmol) and 13

28
I. Damager et al. / Carbohydrate Research 320 (1999) 19–30
(2.00 g, 1.87 mmol) in dry diethyl ether (100
ethyl ether–n-pentane (3:7 and 1:1 v/v) as
eluent to give 21 as white foam (3.09 g, 64%):
[h]D 964.7° (c 0.18, CHCl3); 1H NMR
(CDCl3): l 5.55 (d, 1 H, J1,2 3.5 Hz, H-1,
internal anomeric), 5.65 (d, 2 H, J1,2 3.6 Hz, 2
H-1, internal anomeric). The rest of the
anomeric protons were overlapped with sig-
nals from the methylene protons of the benzyl
,
mL), 4 A molecular sieves (1.5 g, activated
powder) and a soln of trimethylsilyl trifl-
uoromethanesulfonate (67 mL, 0.37 mmol) in
dry diethyl ether (10 mL), which was added
dropwise. The residue was chromatographed
on Silica Gel (120 g) with diethyl ether–n-
pentane (1:9, 1:4 and 3:7 v/v) as eluent to give
19 as colourless syrup (1.5 g, 55%): [h]D9
13
groups; C NMR (CDCl3): l 87.2 (C-1), 96.7,
1
40.7° (c 0.36, CHCl3); H NMR (CDCl3): l
96.8, 97.0, 97.3, 100.3 (5 internal anomeric C),
125.5–138.9 (C-arom, SPh and PhCH2).
5.50 (d, 1 H, J1,2 3.7 Hz, H-1, internal
anomeric), 5.58 (d, 1 H, J1,2 3.7 Hz, H-1,
internal anomeric). H-1b was overlapped by
the signals from the methylene protons of the
benzyl groups; 13C NMR (CDCl3): l 20.8
(COCH3), 87.3 (C-1), 96.7, 96.8, (C-1%, C-1¦),
126.5–138.7 (C-arom, SPh and PhCH2), 169.4
(CꢁO).
O-(2,3,4,6-Tetra-O-benzyl-h-
osyl)-(1“4)-O-(2,3,6-tri-O-benzyl-h-
pyranosyl)-(1“4)-O-(2,3,6-tri-O-benzyl-h-
glucopyranosyl)-(1“6) - O - [(2,3,4,6 - tetra - O-
benzyl-h- -glucopyranosyl)-(1“4)-O-(2,3,6-
tri-O-benzyl-h- -glucopyranosyl)-(1“4)]-2,3-
di-O-benzyl-h,i- -glycopyranose (22).—The
D
-glucopyran-
D
-gluco-
D
-
D
D
D
Phenyl O-(2,3,6-tri-O-benzyl-h-
ranosyl)-(1“4)-O-(2,3,6-tri-O-benzyl-h-
glucopyranosyl)-(1“4)-2,3,6-tri-O-benzyl-1-
thio-i- -glucopyranoside (20).—NaOMe (0.5
D
-glucopy-
same procedure as described for 4 using ace-
tone–water (9:1 v/v, 36 mL), 21 (2.77 g, 0.99
mmol) in acetone (18 mL) and NBS (705 mg,
3.96 mmol). Reaction time: 6.5 min. The
residue was chromatographed on Silica Gel
(95 g) with EtOAc–n-pentane (1:4 and 3:7
v/v) as eluent to give 22 as colourless syrup
(2.17 g, 87%) as an a,b mixture (1:2): [h]D 9
D-
D
mL, 30% in MeOH) was added to a soln of 19
(1.15 g, 0.80 mmol) in MeOH (100 mL) at
0 °C followed by stirring at rt for 32 h. The
reaction mixture was worked up as described
for 15. The residue was chromatographed on
Silica Gel (50 g) with diethyl ether–n-pentane
(3:7 v/v) as eluent to give 20 as colourless
syrup in quantitative yield (1.12 g): [h]D9
1
71.4° (c 0.1, CHCl3); H NMR (CDCl3): l
5.15 (d, 0.3 H, J1,2 3.6 Hz, H-1a), 5.60–5.69
(m, 3 H, 3 H-1, internal anomeric). The rest of
the anomeric protons were overlapped with
signals from the methylene protons of the
benzyl groups; 13C NMR (CDCl3): For the a
anomer: l 91.1 (C-1), 95.5, 96.0, 96.3, 96.8,
100.0 (5 internal anomeric C), 126.3–139.0
(C-arom, PhCH2). For the b anomer: l 97.2
(C-1), 96.8, 96.9, 97.0, 97.2, 100.4 (5 internal
anomeric C), 126.3–139.0 (C-arom, PhCH2).
1
38.4° (c 0.11, CHCl3); H NMR (CDCl3): l
5.50 (d, 1 H, J1,2 3.4 Hz, H-1, internal
anomeric), 5.63 (d, 1 H, J1,2 3.6 Hz, H-1,
internal anomeric). H-1b was overlapped by
the signals from the methylene protons of the
benzyl groups; 13C NMR (CDCl3): l 87.3
(C-1), 96.7, 96.8, (C-1%, C-1¦), 126.5–138.7
(C-arom, SPh and PhCH2).
O-(2,3,4,6-Tetra-O-benzyl-h-
osyl)-(1“4)-O-(2,3,6-tri-O-benzyl-h-
pyranosyl)-(1“4)-O-(2,3,6-tri-O-benzyl-h-
glucopyranosyl) - (1“6) - O - [(2,3,4,6 - tetra-O-
benzyl-h- -glucopyranosyl)-(1“4)-O-(2,3,6-
tri-O-benzyl-h- -glucopyranosyl)-(1“4)]-2,3-
di-O-benzyl-h,i- -trichloroacetimidate (23).—
D
-glucopyran-
Phenyl O-(2,3,4,6-tetra-O-benzyl-h-
copyranosyl)-(1“4)-O-(2,3,6-tri-O-benzyl-h-
-glucopyranosyl)-(1“4)-O-(2,3,6-tri-O-benzyl-
h- -glucopyranosyl)-(1“6)-O-[(2,3,4,6-tetra-
O-benzyl-h- -glucopyranosyl)-(1“4)-O-(2,3,
6-tri-O-benzyl-h- -glucopyranosyl)-(1“4)]-
2,3-di-O-benzyl-1-thio-i- -glucopyranoside
D-glu-
D
-gluco-
D
-
D
D
D
D
D
D
D
D
The procedure was the same as described for 5
using 22 (1.19 g 0.44 mmol) in dry CH2Cl2 (20
mL), CCl3CN (2.25 mL) and K2CO3 (1.13 g)
to give 23 as colourless syrup (1.25 g, quanti-
tative) as an a,b mixture (1:1), which was used
directly for the next step without further
(21).—The same procedure as described for
14 using 15 (2.44 g, 1.73 mmol) and 18 (3.23 g,
,
2.08 mmol) in dry diethyl ether (200 mL), 4 A
molecular sieves (2.5 g, activated powder) and
trimethylsilyl trifluoromethanesulfonate (80
mL, 0.43 mmol). The residue was chro-
matographed on Silica Gel (180 g) with di-
1
purification. H NMR (CDCl3): l 5.60–5.75
(m, 5 H, 5 H-1, internal anomeric), 5.73 (d,

I. Damager et al. / Carbohydrate Research 320 (1999) 19–30
29
glycopyranose (25).—The procedure was the
same as described for 4 using acetone–water
(9:1 v/v, 16 mL), 24 (857 mg, 0.209 mmol) in
acetone (8 mL) and NBS (149 mg, 0.837
mmol). Reaction time: 10 min. The residue
was chromatographed on Silica Gel (45 g)
with diethyl ether–n-pentane (1:4, 1:1 and 4:1
v/v) as eluent to give 25 as white solid (720
mg, 86%) as an a,b mixture (4:3): mp 88–
0.5 H, J1,2 7.9 Hz, , H-1b), 6.40 (d, 0.5 H, J1,2
3.6 Hz, H-1a), 8.79 (s, 0.5 H, NH, a anomer),
8.82 (s, 0.5 H, NH, b anomer); 13C NMR
(CDCl3): l 90.8 (CCl3, b anomer), 91.2 (CCl3,
a anomer), 93.7 (C-1a), 97.6 (C-1b), 95.5, 96.1,
96.5, 96.6, 96.7, 96.8, 97.1, 97.3, 100.0, 100.2
(internal anomeric C), 160.3 (CꢁNH, b
anomer), 161.0 (CꢁNH, a anomer).
Phenyl O-(2,3,4,6-tetra-O-benzyl-h-
copyranosyl)-(1“4)-O-(2,3,6-tri-O-benzyl-h-
- glucopyranosyl) - (1“4) - O - (2,3,6 - tri - O-
benzyl-h- -glucopyranosyl)-(1“6)-O-[(2,3,4,6-
tetra-O-benzyl-h- -glucopyranosyl)-(1“4)-O-
(2,3,6 - tri - O - benzyl - h - - glucopyranosyl)-
(1“4)]-O-(2,3-di-O-benzyl-h- -glucopyran-
osyl)-(1“4)-O-(2,3,6-tri-O-benzyl-h- -gluco-
pyranosyl)-(1“4)-O-(2,3,6-tri-O-benzyl-h-
glucopyranosyl)-(1“4)-2,3,6-tri-O-benzyl-1-
thio-i- -glucopyranoside (24).—The proce-
D-glu-
1
90 °C; [h]D 972.9° (c 0.21, CHCl3); H NMR
D
(CDCl3): l 5.18 (d, 1 H, J1,2 3.0 Hz, H-1,
internal anomeric), 5.22 (bs, 0.6 H, H-1a), 5.31
(d, 1 H, J1,2 3.3 Hz, H-1, internal anomeric),
5.52 (d, 1 H, J1,2 3.7 Hz, H-1, internal
anomeric), 5.58 (d, 1 H, J1,2 3.3 Hz, H-1,
internal anomeric), 5.66–5.73 (m, 4 H, 4 H-1,
internal anomeric). H-1b was overlapped by
the signals from the methylene protons of the
benzyl groups; 13C NMR (CDCl3): l 90.7
(C-1a), 97.2 (C-1b), 95.8, 95.8, 96.5, 96.6, 96.6,
96.6, 97.3, 98.6 (8 internal anomeric C),
126.2–138.8 (C-arom, PhCH2). Anal. Calcd
for C250H260O46: C, 75.05; H, 6.55. Found: C,
74.81; H, 6.80.
D
D
D
D
D
D-
D
dure was the same as described for 14 using 20
(570 mg, 0.405 mmol) and 23 (1.07 g, 0.376
,
mmol) in dry diethyl ether (80 mL), 4 A
molecular sieves (1 g, activated powder) and
trimethylsilyl trifluoromethanesulfonate (17.4
mL, 0.094 mmol). The residue was chro-
matographed on Silica Gel (55 g) with diethyl
ether–n-pentane (2:3 v/v) as eluent to give 24
as white foam (867 mg, 56%): mp 96–99 °C;
[h]D971.6° (c 0.22, CHCl3); 1H NMR
(CDCl3): l 5.18 (d, 1 H, J1,2 3.6 Hz, H-1,
internal anomeric), 5.32 (d, 1 H, J1,2 3.3 Hz,
H-1, internal anomeric), 5.50 (d, 1 H, J1,2 3.5
Hz, H-1, internal anomeric), 5.54 (d, 1 H, J1,2
3.5 Hz H-1, internal anomeric), 5.62–5.73
(m, 4 H, 4 H-1, internal anomeric). H-1b
was overlapped by the signals from the
methylene protons of the benzyl groups; 13C
NMR (CDCl3): l 87.2 (C-1), 95.9, 96.1, 96.6,
96.7, 96.8, 97.0, 97.4, 98.7 (8 internal
anomeric C), 125.5–138.9 (C-arom, SPh and
PhCH2).
6§-h-Maltotriosyl-maltohexaose (26).—To
a soln of 25 (720 mg, 0.18 mmol) in THF–
MeOH–H2O (130 mL, 6:6:1 v/v) was added
10% PdꢀC (500 mg) and the reaction mixture
was stirred under hydrogen (1 atm) for 30 h at
rt. The catalyst was removed by filtration on a
layer of Silica Gel and the filtrate was concd
to dryness. The residue was chromatographed
on Silica Gel (60 g) with MeOH–H2O (7:3
v/v) as eluent and 26 was collected as a white
powder from EtOH (212 mg, 80%) as an a,b
mixture (ꢀ1:2, estimated from 13C NMR):
1
[h]D 9166° (c 0.09, H2O); H NMR (D2O):
l 4.65 (d, 0.6 H, J1,2 8.0 Hz, H-1b), 4.96 (1 H,
J1,2 3.7 Hz, H-1, branch point), 5.22 (d, 0.4 H,
J1,2 3.7 Hz, H-1a), 5.31 (1 H, J1,2 3.7 Hz, H-1,
internal anomeric), 5.33 (1 H, J1,2 3.7 Hz, H-1,
internal anomeric), 5.35 (1 H, J1,2 3.7 Hz, H-1,
internal anomeric), 5.37–5.40 (4 H, 4 H-1,
internal anomeric); 13C NMR (D2O): l 92.7
(C-1a), 96.6 (C-1b), 99.3 (C-1, branch point),
100.2, 100.3, 100.5, 100.6, 100.7, 100.8,
100.9 (internal anomeric C). MALDI; Calcd
for C54H92O46+Na: 1500.3. Found 1500.5.
Calcd for C54H92O46 +K: 1516.4. Found
1516.6.
O-(2,3,4,6-Tetra-O-benzyl-h-
osyl)-(1“4)-O-(2,3,6-tri-O-benzyl-h-
pyranosyl)-(1“4)-O-(2,3,6-tri-O-benzyl-h-
glucopyranosyl)-(1“6) - O - [(2,3,4,6 - tetra - O-
benzyl-h- -glucopyranosyl)-(1“4)-O-(2,3,6-
tri-O-benzyl-h- -glucopyranosyl)-(1“4)]-O-
(2,3-di-O-benzyl-h- -glucopyranosyl)-(1“4)-
O-(2,3,6-tri-O-benzyl-h- -glucopyranosyl)-
(1“4) - O - (2,3,6 - tri - O - benzyl - h - - gluco-
pyranosyl)-(1“4)-2,3,6-tri-O-benzyl-h,i-
D
-glucopyran-
D
-gluco-
D-
D
D
D
D
D
D-

30
I. Damager et al. / Carbohydrate Research 320 (1999) 19–30
[11] M.S. Motawia, J. Marcussen, B.L. Møller, J. Carbohydr.
Acknowledgements
Chem., 14 (9) (1995) 1279–1294.
[12] M.E. Tate, C.T. Bishop, Can. J. Chem., 41 (1963) 1801–
1806.
[13] O.T. Schmidt, T. Auer, H. Schmadel, Chem. Ber., 93
(1960) 556–557.
[14] F. Weygand, H. Ziemann, Liebigs Ann. Chem., 657
(1962) 179–198.
[15] J. Gigg, R. Gigg, J. Chem. Soc. C, (1966) 82–86.
[16] T.D. Perrine, C.P.J. Glaudemans, R.K. Ness, J. Kyle,
H.G. Fletcher Jr., J. Org. Chem., 32 (1967) 664–669.
[17] R.R. Schmidt, M. Stumpp, Liebigs Ann. Chem., (1983)
1249–1256.
This work was financially supported by the
EU FAIR programme contract CT95-0568,
by the Danish Directorate for Development
(non-food program), and by the Danish Na-
tional Research Foundation.
References
[18] H. Rathore, T. Hashimoto, K. Igarashi, H. Nukaya,
D.S. Fullerton, Tetrahedron, 41 (1985) 5427–5438.
[19] R.R. Schmidt, J. Michel, Tetrahedron Lett., 25 (1984)
821–824.
[1] S. Ball, H. Guan, M. James, A. Myers, P. Keeling, G.
Mouille, A. Bule´on, P. Colonna, J. Preiss, Cell, 86 (1996)
349–352.
[20] S. Koto, N. Morishima, K. Takenaka, K. Kanemitsu, N.
Shimoura, M. Kase, S. Kojiro, T. Nakamura, T.
Kawase, S. Zen, Bull. Chem. Soc. Jpn., 62 (1989) 3549–
3566.
[21] A. Ernst, A. Vasella, Hel6. Chim. Acta, 79 (1996) 1279–
1294.
[22] S. Hashimoto, M. Hayashi, R. Noyori, Tetrahedron
Lett., 25 (1984) 1379–1382.
[23] B. Wegmann, R.R. Schmidt, J. Carbohydr. Chem., 6
(1987) 357–375.
[2] K. Denyer, B. Clarke, C. Hylton, H. Tatge, A.M. Smith,
Plant J., 10 (6) (1996) 1135–1143.
[3] C. Martin, A.M. Smith, Plant Cell, 7 (1995) 971–985.
[4] M. Steup, in J. Preiss (Ed.), The Biochemistry of Plants,
Vol. 14, Academic Press, London, 1988, pp. 255–296.
[5] W.J. Whelan, Methods Carbohydr. Chem., 4 (1963) 252–
260.
[6] M.S. Motawia, C.E. Olsen, B.L. Møller, J. Marcussen,
Carbohydr. Res., 252 (1994) 69–84.
[7] M.S. Motawia, C.E. Olsen, K. Enevoldsen, J. Mar-
cussen, B.L. Møller, Carbohydr. Res., 277 (1995) 109–
123.
[24] K. Bock, C. Pedersen, Ad6. Carbohydr. Chem. Biochem.,
41 (1983) 27–66.
[25] K. Bock, C. Pedersen, H. Pedersen, Ad6. Carbohydr.
Chem. Biochem., 42 (1984) 193–225.
[26] H.P. Wessel, B. Mayer, G. Englert, Carbohydr. Res., 242
(1993) 141–151.
[8] R.J. Ferrier, R.W. Hay, N. Vethaviyasar, Carbohydr.
Res., 27 (1973) 55–61.
[9] R.J. Ferrier, R.H. Furneaux, Carbohydr. Res., 52 (1976)
63–68.
[10] A. Liptak, I. Jodal, J. Harangi, P. Nanasi, Acta Chim.
Hung., 113 (4) (1983) 415–422.
[27] K. Takeo, K. Mine, T. Kuge, Carbohydr. Res., 48 (1976)
197–208.
.