Synthesis, NMR spectroscopy and conformational studies of the four anomeric methyl glycosides of the trisaccharide D-Glcp-(1→3)-[D-Glcp-(1→4)]-α-D-Glcp

Article abstract of DOI:10.1039/a707346a

The four anomeric methyl glycosides of the vicinally disubstituted trisaccharide D-Glcp-(1→3)-[D-Glcp-(1→4)]-α-D-Glcp have been synthesized using silver trifluoromethanesulfonate mediated glycosylations. The ¹H and ¹³C NMR resonances have been assigned and used for extraction of glycosylation shifts, i.e. the differences between chemical shifts for signals from the trisaccharides and those of the respective monomers, as well as those derived by addition of the glycosylation shifts for each disaccharide element. Glycosylation shifts are up to 0.5 ppm for proton and 10 ppm for carbon. Deviations from additivity are -0.2-0.1 ppm for proton and -4.5-2.3 ppm for carbon, usually confined to the atoms at the linkage positions. The conformational space spanned for the trisaccharides, and the constituent disaccharides, has been investigated by Metropolis Monte Carlo simulations using the HSEA force field. The α-linked glucosyl groups show larger conformational changes with multiple energy minima, whereas the β-linked glucosyl groups have a single energy minimum, close to that identified for the constituent disaccharide.

Full text of DOI:10.1039/a707346a

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Synthesis, NMR spectroscopy and conformational studies of the four
anomeric methyl glycosides of the trisaccharide D-Glcp-(1→3)-
[D-Glcp-(1→4)]-á-D-Glcp
Peter Söderman,a Per-Erik Janssonb and Göran Widmalm*,a
a Department of Organic Chemistry, Arrhenius Laboratory, Stockholm University,
S-106 91 Stockholm, Sweden
b Clinical Research Centre, Analytical Unit, Karolinska Institutet, Huddinge Hospital, Novum,
S-141 86 Huddinge, Sweden
The four anomeric methyl glycosides of the vicinally disubstituted trisaccharide D-Glcp-(1→3)-[D-Glcp-
(1→4)]-á-D-Glcp have been synthesized using silver trifluoromethanesulfonate mediated glycosylations.
The 1H and 13C NMR resonances have been assigned and used for extraction of glycosylation shifts, i.e.
the differences between chemical shifts for signals from the trisaccharides and those of the respective
monomers, as well as those derived by addition of the glycosylation shifts for each disaccharide element.
Glycosylation shifts are up to 0.5 ppm for proton and 10 ppm for carbon. Deviations from additivity are
؊0.2–0.1 ppm for proton and ؊4.5–2.3 ppm for carbon, usually confined to the atoms at the linkage
positions. The conformational space spanned for the trisaccharides, and the constituent disaccharides,
has been investigated by Metropolis Monte Carlo simulations using the HSEA force field. The á-linked
glucosyl groups show larger conformational changes with multiple energy minima, whereas the â-linked
glucosyl groups have a single energy minimum, close to that identified for the constituent disaccharide.
complexity of all these factors determining the conformation is
best seen through some examples.
Introduction
Complex carbohydrates play important roles in many bio-
chemical processes and the biological activity is a function of
their surface properties which are governed by their structure
and conformation, the latter arising mainly from rotation of the
glycosidic linkages. Most monosaccharides have a defined ring
shape, leaving flexibility only at the glycosidic linkage. This is
true for most monosaccharides, with exceptions that are fairly
easy to predict. The analysis of disaccharides, the simplest
models for larger oligosaccharides, may be made, inter alia,
The disaccharides can be divided into two main groups. The
difference between I and II is the position of the ring oxygen in
a
c
a
c
c
c
a
a
O
b
R1
R1
d
b
d
O
O
O
b
d
b
d
R2
R2
H
H
H
H
1
with H and 13C NMR spectroscopy and computer modelling.
I
II
NMR chemical shift assignments have been made for a large
number of disaccharides and it has been concluded that a
number of stereochemical factors influence the chemical shifts.
Thus, collections of NMR data for disaccharides containing
hexoses, deoxy-hexoses, 2-acetamido-sugars etc. can be found
in the literature.1,2 In our laboratory we have synthesized and,
under similar conditions and temperature (mostly 70 ЊC), anal-
ysed several disaccharides. These include (1→2)-,3–6 (1→3)-,3–7
(1→4)-8,9 and (1→6)-linkages.3,10
Conformational analyses of pyranosidic hexose disacchar-
ides have shown that (1→2)-, (1→3)- and (1→4)-linked disac-
charides have one main conformation. This conformation is
essentially determined by the non-bonded interactions between
atoms on linkage carbons and on neighboring atoms. In add-
ition, changes contributed by the exoanomeric effect must also
be considered. Expressed with dihedral angles φ (H1᎐C1᎐OA᎐
CA, A = aglycon) and ψ (C1᎐OA᎐CA᎐HA), values are close to
60Њ (abs) and a low value near 0Њ, respectively. Apart from the
absolute and anomeric configuration of the glycosyl group, the
equatorial substituents on the neighboring carbons in the agly-
con (R1 and R2, see below) are the most important factor. They
may be hydrogen, hydroxy groups, hydroxymethyl groups or
methyl groups. The latter three are similar in size and have
roughly the same influence on the NMR spectra. In most cases
these are either a proton and a larger group, or two larger
groups. Hydrogens in both positions are seldom encountered.
The substitution itself may also be equatorial or axial. The
the glycosyl group. If, in I, rings continue via a and c, a disac-
charide with an α--sugar substituting an axial hydroxy group
is obtained (A). For continuation via a and d, the same α--
linkage is formed but substitution is now of an equatorial
hydroxy group (B). If rings go through b and c, a β--sugar
is obtained which substitutes an axial hydroxy group (C).
Finally, if rings go through b and d, the β--sugar substitutes an
equatorial hydroxy group (D). The similarity between α- and
β- is then obvious and of course derives from the position of
the ring oxygen. For the stereochemical arrangement in II the
disaccharide formed via a and c gives an α--sugar substituting
an axial hydroxy group (E), via a and d the same but substitut-
ing an equatorial hydroxy group (F) and for combinations b
and c, and b and d, a disaccharide with a β--sugar substituting
an axial (G) and an equatorial hydroxy group (H), respectively,
is formed. The similarity here between α-- and β--substitution
also easily follows from II. For different combinations of
sugars, different inter-residue interactions arise. The effect for
three different groups of disaccharides will be given in order to
show some typical glycosylation shifts, i.e. changes in chemical
1
shift compared to those of monomers, for both 13C and H
NMR spectroscopy.
Thus, if in I, R1 is a hydrogen and R2 is a hydroxy group or
another large group an interaction between H1 and R1 is pres-
ent, a so-called γ-gauche interaction. This is manifested, inter
alia, through an NOE between those protons. Disaccharides
J. Chem. Soc., Perkin Trans. 2, 1998
639

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with this stereochemistry are for instance α--Fuc-(1→3)--Gal
where the α- can be exchanged to β- (type I) and α--Fuc-
(1→3)--Man, where the α- sugar can be exchanged to β-
(type II), and still the same interactions are present. For 13C
NMR spectra this stereochemistry brings about relatively small
glycosylation shifts for the substituted carbons C1 and CA, and
a relatively large negative glycosylation shift for the signal of
the carbon to which R1 is substituted. Significant glycosylation
shifts for this and other disaccharides are obtained with only a
few exceptions for signals from substituted (α-effect) or neigh-
boring carbons (β-effect). For the α--Fuc-(1→3)--Gal
disaccharide the values for ClЈ, C3 and C4, are ca. 3.0, 5.0
and Ϫ3.5 ppm, respectively. The remaining carbon signal (for
C2 in Gal) is shifted Ϫ1.5 ppm, a typical β-effect. The changes
for 1H NMR signals are for H1Ј, H2, H3 and H4, Ϫ0.14,
0.12, 0.04 and 0.20 ppm, respectively. The negative value is pre-
sumably a result of the proton–proton contact (H1Ј, H3/H4)
giving the NOE, a shift that however is not observed for H4.
As a comparison, for β-linked disaccharides the effects are
normally less pronounced. Thus, β--Fuc-(1→3)--Gal has for
C1Ј, C3 and C4 signals, the glycosylation shifts 4.4, 7.7 and
Ϫ2.4 ppm.
formally linear but effectively branched trisaccharides with a 2-
substituted residue in the middle, i.e. Sug-(1→2)-Sug-(1→X)-
Sug.19–21 In those oligosaccharides it could be expected that in
several cases the glycosylation shifts should differ from those
of the constituent disaccharides and from those calculated by
additivity. One of the main reasons would of course be that the
φ- and ψ-angles in the trisaccharide differ from those in the
disaccharide because of interactions between the substituting
sugars, or because the conformational freedom is limited. In
general, few large glycosylation shifts are found apart from the
expected α- and β-effects.
For NMR chemical shifts of trisaccharides a division into
groups similar to that described for the disaccharides can be
made. Thus, the substituting groups can take different anomeric
and absolute configurations. The diols can be equatorial–
equatorial or axial–equatorial. The highly unusual diaxial
substitution is not taken into consideration. Further divisions
can be made by allowing for variations at other places in the
trisaccharide. For symmetry reasons some stereochemistries
may give similar values. The trisaccharides used in previous
studies generally had one of the substituting groups linked to
an axial hydroxy group. To exemplify that some trisaccharide
elements have substantial deviations of 13C NMR data from
those of the disaccharides, data for α--Fuc-(1→3)-[α--Fuc-
(1→4)]-Gal and α--Glc-(1→3)-[α--Fuc-(1→4)]-Gal are
compared. The former is an example of how large the changes
in displacements may be. Signals from the anomeric carbon of
the Sug-(1→3)- and Sug-(1→4)-groups, and C3 and C4 in the
disubstituted residue, are 0, Ϫ2.8, Ϫ1.1 and Ϫ5.0 ppm, respect-
ively, i.e. signals appear far more upfield than expected from
comparisons with the disaccharides. If, on the other hand, the
3-O-glycosyl group is α--Glc instead the values are 0.9, Ϫ2.1,
0.5 and Ϫ2.6 ppm, thus significantly less upfield. There are
examples, however, for which signals shift almost 2 ppm in the
positive direction.
If the aglycon changes from Gal to Man in the disaccharide
from the first example to make α--Fuc-(1→3)--Man (type I)
the proton–proton contact is removed and instead H1 will
interact with a hydroxy group. Values for the same carbon
signals as above are now 8.5, 8.0 and Ϫ0.8 ppm, i.e. relatively
large and with no large negative value. The value for the C2
signal is now close to zero as compared to Ϫ1.5 ppm above. The
1
changes for H NMR signals are for H1Ј, H2, H3 and H4,
Ϫ0.02, 0.08, 0.05 and 0.18 ppm, thus the large negative effect
for H1Ј is no longer present. Values for β--Glc-(1→3)--Gal
(type II), an analogous ‘mirror image’ to β--Fuc-(1→3)--
Man, are 8, 9.9 and Ϫ0.3 ppm for signals from C1Ј, C3 and
C4, respectively, also relatively large.
The third and last group has no equatorial hydrogen and
α--Glc-(1→3)--Glc is a typical representative for this group.
Signals from C1Ј, C3 and C4, are shifted 7, 7.4 and 0.2 ppm
respectively, and this is taken as an average starting point. The
Results and discussion
1
values for H NMR glycosylation shifts are for H1Ј, H2, H3
Synthesis of trisaccharides
and H4, 0.09, 0.10, 0.14 and 0.25 ppm. Disaccharides with
groups other than hydroxy follow the general pattern but may
vary slightly.
In the synthesis of the four anomers of the trisaccharide
-Glcp-(1→3)-[-Glcp-(1→4)]-α--Glcp-OMe 1–4, three were
made by glycosylation of a suitably protected derivative of the
O-methyl glycoside, followed by selective deprotection and one
more glycosylation. The last trisaccharide 4 was synthesized
by di-glycosylation of a derivative of the O-methyl glycoside
having two hydroxy groups free. All glycosylations were medi-
ated by silver trifluoromethanesulfonate (AgOTf).22,23 Diethyl
ether was used as solvent in the formation of α-linked groups
whereas for β-linked groups dichloromethane was used. The
isolated yields in the glycosylation reactions were generally
>70%. Data for the reaction conditions used in the conversions
are given in Table 1.
For the synthesis of the first two trisaccharides, 1 and 2, both
of which have an α-(1→3)-linkage, the key intermediate was a
methyl 2-O-benzyl-4,6-O-benzylidene-α--glucopyranoside24
(9). This compound was acetylated or allylated to give methyl
2-O-benzyl-3-O-acetyl-4,6-O-benzylidene-α--glucopyranoside
(10) and methyl 2-O-benzyl-3-O-allyl-4,6-O-benzylidene-α--
glucopyranoside (11), respectively, which were subsequently
reductively opened by NaCNBH3 in hydrochloric acid–THF25
to give methyl 2,6-di-O-benzyl-3-O-acetyl-α--glucopyranoside
(12) in 82% yield (over two steps) and methyl 2,6-di-O-benzyl-3-
O-allyl-α--glucopyranoside (13) in 63% yield (over two steps),
respectively. Glycosylation of 12 with 2,3,4,6-tetra-O-benzyl-α-
-glucopyranosyl bromide26 (16), generated in situ from ethyl
2,3,4,6-tetra-O-benzyl-1-thio-β--glucopyranoside26 (15), in
diethyl ether at Ϫ30 ЊC gave the α-(1→4)-linked disaccharide
18 in 70% yield. Deprotection of 18 by Zemplen deacetylation
(sodium methoxide in methanol) gave 19 to be used as an
For the 6-linked disaccharides the situation is more complex
as an additional degree of freedom is introduced, rotation
around the C5᎐C6 linkage. The hydroxymethyl group occupies
two out of three staggered conformations, different depending
on the chirality at C4. The conformational space is likely to be
shallow and a number of conformations may be occupied.
Some preferences can however be seen, e.g. in general only one
of the H6 protons has an NOE to H1 in the glycosyl group.10
A
distinction between the disaccharides which have an axial ano-
meric substitution and those that have an equatorial can be
made and there is also a dependence on whether the two con-
stituents are  or . As stated above the effects are wholly
dependent on the stereochemistry and therefore the effects are
most similar if the sugars are α- or α-. The glycosylation
shifts are around 5.9, Ϫ1.5 and 5.2 for C1Ј, C5 and C6 in α--
Glc-(1→6)--Glc. Intermediate values are obtained for α- and
β- combinations. Glycosylation shifts of around 6.7, Ϫ1.0
and 7.8 are found for the same carbon signals for β--Glc-
(1→6)--Glc.
For larger oligosaccharides a first assumption can be made
that the conformation adopted by the disaccharide is also kept
in the oligosaccharide. It is quite clear that for most linear oligo-
saccharides this is true as data obtained for the disaccharide
elements can be transferred to the oligosaccharide. It has been
shown that additivity holds for the glycosylation shifts both for
proton and carbon.11 For vicinally branched trisaccharides data
are available for 2,3-,12,13 and 3,4-disubstituted14–18 as well as
640
J. Chem. Soc., Perkin Trans. 2, 1998

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OR1
OR1
Ph
O
O
O
O
HO
R2O
R1O
R1O
O
R2O
OR1
R1O
O
R1O
R1O
O
OMe
OMe
O
R1O
R1O
9 R1 = Bn, R2 = H
12 R1 = Bn, R2 = Ac
13 R1 = Bn, R2 = Allyl
14 R1 = Bn, R2 = H
OMe
10 R1 = Bn, R2 = Ac
R1O
R1O
11 R1 = Bn, R2 = Allyl
O
OR1
OR3
O
R3O
R3O
24 R1 = Bn
1 R1 = H
R1
R3O
OR2
R2
OR1
R2O
R2O
O
15 R1 = SEt, R2 = H, R3 = Bn
16 R1 = H, R2 = Br, R3 = Bn
17 R1 = H, R2 = Br, R3 = Bz
O
O
2O
R1O
OR
R1O
OMe
R1O
R1O
OR1
O
OR1
OR1
O
R1O
R1O
25 R1 = Bn, R2 = Bz
2 R1 = R2 = H
R1O
O
O
R2O
OR1
O
R1O
R1O
R1O
OMe
18 R1 = Bn, R2 = Ac
19 R1 = Bn, R2 = H
OR1
OR2
R1O
O
O
O
O
OR3
OR1
R3O
R3O
R1O
R2O
O
R2O
OMe
O
R2O
R2O
O
OR3
26 R1 = Bn, R2 = Bz
3 R1 = R2 = H
R1O
OMe
20 R1 = Bn, R2 = Allyl, R3 = Bz
21 R1 = Bn, R2 = H, R3 = Bz
R2O
R2O
R2O
OR2
OR2
R2O
O
Ph
O
O
O
O
O
O
OR2
O
O
O
R1O
R1O
R2O
OMe
R2O
OMe
R2O
OR2
R2O
22 R1 = Bn, R2 = Bz
2
R O
27 R1 = Bn, R2 = Bz
4 R1 = R2 = H
OR1
OR2
O
HO
O
O
26, in 79% yield. Hydrogenolysis of 26, debenzoylation and gel
filtration gave compound 3, in 89% yield.
R1O
R2O
OMe
R2O
R2O
The last trisaccharide 4, was formed by di-glycosylation of
the selectively protected acceptor methyl 2,6-di-O-benzyl-α-
-glucopyranoside30 (14) with 2.5 equiv. of 2,3,4,6-tetra-O-
benzoyl-α--glucopyranosyl bromide (17) in dichloromethane
at Ϫ30 ЊC to give 27, in 93% yield. Trisaccharide 27 was depro-
tected, as described for 26, to give 4 in 85% yield.
The order of glycosylation played an important role, in par-
ticular for trisaccharide 2, which has an α-(1→3)-linkage. For
this compound the β-(1→4)-linkage was formed first since the
reverse procedure led to a low yield of trisaccharide. Hence, the
same order of glycosylation was used for compound 1. The
sequence was reversed for 3 in which the β-(1→3)-linkage was
formed first, followed by the α-(1→4)-linkage. The last trisac-
charide, 4, could be formed in good yield by a di-glycosylation.
23 R1 = Bn, R2 = Bz
acceptor in the subsequent glycosylation. Compound 13 was
condensed with 2,3,4,6-tetra-O-benzoyl-α--glucopyranosyl
bromide27 (17) in dichloromethane at Ϫ30 ЊC to give the β-
(1→4)-linked disaccharide 20 in 74% yield. The allyl group in
compound 20 was rearranged28 to give the prop-1-enyl isomer
by reflux in ethanol using Wilkinson’s catalyst [tris(triphenyl-
phosphine)rhodium() chloride] and diisopropylethylamine
(DIPEA), followed by treatment with toluene-p-sulfonic acid to
give the deprotected 21 with a free hydroxy group. Glycosyl-
ations of the acceptors 19 and 21 with 16 as donor using the
same conditions as above gave the trisaccharides 24 and 25 in 55
and 76% yield, respectively. Removal of the benzyl groups in 24
by hydrogenolysis using palladium on carbon gave, after gel
filtration, compound 1 in 70% yield. Hydrogenolysis of 25,
followed by debenzoylation gave, after gel filtration, compound
2 in 85% yield.
Monte Carlo calculations
The conformational flexibility of trisaccharides 1–4 and of the
constituent disaccharides 5–8 was investigated by Metropolis
Monte Carlo (MMC) calculations.31,32 The global energy
minimum for each molecule was also identified by energy
minimization. Each MMC run employed 106 macro steps at 300
K. The results as averaged values of angles, angle differences
between trisaccharides and the corresponding disaccharides,
and rms deviations from average values of angles and energy
minimized conformations are given in Table 2. The global
energy conformers of 1–4 are shown in Fig. 1. The dihedral
For the synthesis of compound 3, the β-(1→3)-linkage was
formed first to give disaccharide 22.29 The 4,6-benzylidene
group of 22 was subsequently opened by reductive cleavage to
give the disaccharide 23 in 83% yield, with a free hydroxy group
at O4. The α-(1→4)-linkage was formed by glycosylation of
23 with 16 in diethyl ether at Ϫ30 ЊC to give the trisaccharide
J. Chem. Soc., Perkin Trans. 2, 1998
641

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Table 1 Data on reaction conditions, physical constants and selected NMR chemical shifts
Conversiona Solvent (cm3) T/ЊCc Purif. (solv)d,e Yield (%) m/z
Reagents (mg, mmol) treact/h
[α]578/Њ (c)g
δ
C(anomeric)
98.7
13
9→10
CH2Cl2 (25)
9 (1000, 2.69),
pyridine (0.70 cm3,
10.8), DMAP
2
rt
T–E
6:1
92
(33, 0.27)
10→12
THF (50)
3 Åb
Acetyl chloride (0.29
cm3, 4.04), 10 (900,
2.1), NaCNBH3
(1680, 26.4), HCl–
Ether
0.5
rt
T–E
4:1
89
439.2
65
(0.83)
97.4
[M ϩ Na]ϩ
9→11
DMF (25)
9 (2000, 5.39), NaH
(272, 8.08), Allyl
bromide (0.547 cm3,
6.46)
11 (1600, 3.9),
NaCNBH3 (3000,
48.3), HCl–Ether
1
rt
rt
T–E
7:1
90
70
99.3
98.2
11→13
THF (50)
3 Å
0.5
T–E
7:1–4:1
437.2
16
(1.01)
[M ϩ Na]ϩ
15→16
CH2Cl2 (10)
15 (906, 1.55), Br2
(0.087 cm3, 1.70)
12 (500, 1.20),
AgOTf (400, 1.55),
collidine (0.079 cm3,
0.59)
0
12 ϩ 16→18 Ether (30)
1
3
Ϫ30
T–E
7:1
70
90
961.7
50
(1.14)
97.6
97.5
[M ϩ Na]ϩ
18→19
15→16
MeOH
18 (720, 0.77),
rt
0
T–E
7:1
919.5
41
(0.80)
100.6
98.3
NaOMe (0.10 mol
[M ϩ Na]ϩ
dmϪ1
)
CH2Cl2 (10)
15 (316, 0.54), Br2
(0.030 cm3, 0.59)
19 (320, 0.357),
AgOTf (138, 0.54),
collidine (0.024 cm3,
0.18)
19 ϩ 16→24 Ether (30)
0.75 Ϫ30
T–E
14:1
55
1441.5
68
(0.77)
97.0
96.0
93.3
[M ϩ Na]ϩ
24→1
EtOAc–HOAc 15 (240, 0.169), H2/
20
rt
P2
70
74
517.2
117
(1.08)
Pd-C
[M Ϫ H]Ϫ
13 ϩ 17→20 CH2Cl2
17 (1030, 1.56), 13
(500, 1.2), AgOTf
(402, 1.56), collidine
(0.080 cm3, 0.6)
20 (800, 0.806),
DIPEA (0.15 cm3,
0.887), [(C6H5)3P]3
RhCl (8.2, 0.0089),
TPS f
2
3
Ϫ30
T–E
10:1
1015.5
23
(0.95)
100.6
98.5
[M ϩ Na]ϩ
20→21
EtOH (40)
90
T–E
7:1
78
975.6
22
(1.01)
100.3
98.5
[M ϩ Na]ϩ
15→16
CH2Cl2 (10)
15 (560, 1.18), Br2
(0.066 cm3, 1.29)
21 (560, 0.59),
0
21 ϩ 16→25 Ether (30)
0.5
Ϫ30
T–E
15:1
76
1497.9
30
(1.11)
98.6
97.8
95.3
AgOTf (303, 1.18),
collidine (0.039 cm3,
0.295)
[M ϩ Na]ϩ
25→2
25 (400, 0.27), H2/
rt
rt
P2
517.2
115
(0.83)
Pd-C, NaOMe (0.10
[M Ϫ H]Ϫ
mol dmϪ1
)
22→23
15→16
THF (50)
3 Å
22 (1500, 1.58),
0.25
T–E
4:1
83
975.7
101.9
98.2
NaCNBH3 (1830,
19.6), HCl–Ether
15 (380, 0.63), Br2
(0.036 cm3, 0.69)
23 (400, 0.42),
[M ϩ Na]ϩ
CH2Cl2 (10)
0
23 ϩ 16→26 Ether (30)
0.5
0.5
Ϫ30
T–E
20:1
10:1
79
89
1474.6
22
(1.53)
100.3
97.1
94.2
AgOTf (163, 0.63),
collidine (0.029 cm3,
0.21)
[M ϩ Na]ϩ
26→3
EtOAc
MeOH
26 (400, 0.27), H2/ Pd-
rt
P2
517.2
99
(1.04)
C, NaOMe (0.10 mol
[M Ϫ H]Ϫ
dmϪ1
)
14 ϩ 17→27 CH2Cl2 (20)
14 (300, 0.802), 17
(1610, 2.44), AgOTf
(628, 2.44), collidine
(0.055 cm3, 0.5)
0.75 Ϫ40/
Ϫ20
T–E
14:1
93
85
1553.6
1
100.4
98.7
97.9
4 Å
[M ϩ Na]ϩ
(1.01)
27→4
EtOAc
MeOH
27 (1.0, 0.65), H2/ Pd- 15
C, NaOMe (0.10 mol
dmϪ1
rt
P2
517.2
61
(1.46)
2
[M Ϫ H]Ϫ
)
a For coding see figures. b Molecular sieves, 3 Å. c rt = room temperature. d Solvents for chromatography separations; T = toluene, E = ethyl acetate.
e Gel permeation chromatography on Bio-Gel P-2. f Toluene-p-sulfonic acid. g Concentration (g/100 cm3).
642
J. Chem. Soc., Perkin Trans. 2, 1998

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Table 2 Conformational analysis of trisaccharides 1–4 and disaccharides 5–8 by Monte Carlo simulationsa and energy minimizations
Dihedral angles in glycosidic linkages/Њ
〈MMC〉
EM
(1→3)
φ
(1→3)
(1→4)
(1→4)
Molecule
φ
ψ
φ
ψ
ψ
φ
ψ
α--Glcp(1→3)[α--Glcp(1→4)]α--Glcp-OMe (1)
Ϫ29
42
(56)
[12]
1
(15)
[22]
27
(21)
[7]
8
Ϫ43
Ϫ33
(Ϫ10)
[11]
16
(15)
[5]
Ϫ11
(12)
[22]
6
Ϫ34
(10)
31
(54)
Ϫ41
(Ϫ9)
Ϫ31
(Ϫ3)
(10)b
(Ϫ16)
[11]
59
(7)
[9]
Ϫ24
(3)
[21]
50
[8]c
Ϫ28
(11)
[24]
55
(5)
[11]
46
(Ϫ4)
[9]
α--Glcp(1→3)[β--Glcp(1→4)]α--Glcp-OMe (2)
β--Glcp(1→3)[α--Glcp(1→4)]α--Glcp-OMe (3)
β--Glcp(1→3)[β--Glcp(1→4)]α--Glcp-OMe (4)
Ϫ32
(12)
14
(37)
61
(7)
10
(8)
55
(2)
19
(14)
Ϫ17
(15)
8
(36)
46
(Ϫ7)
5
(0)
47
(Ϫ7)
4
(2)
(2)
[6]
(Ϫ2)
[9]
(5)
[5]
α--Glcp(1→3)α--Glcp-OMe (5)
α--Glcp(1→4)α--Glcp-OMe (6)
β--Glcp(1→3)α--Glcp-OMe (7)
β--Glcp(1→4)α--Glcp-OMe (8)
Ϫ39
[12]
Ϫ14
[17]
Ϫ44
Ϫ23
Ϫ27
[11]
Ϫ23
[12]
Ϫ32
Ϫ28
50
[11]
6
[12]
53
5
52
[11]
1
[10]
54
2
a 106 Macro steps Metropolis Monte Carlo simulations at 300 K performed with a total acceptance ratio between 0.35 and 0.49. b Angle differences
between trisaccharides and corresponding disaccharide in parentheses. c Rms deviation from average angles in square brackets.
cation of conformational states can be obtained by scatter plots
in which highly populated conformational states show cluster-
ing (Fig. 2). The β-linked residues show one conformational
state at the global energy minimum region and low rms devi-
ations of the dihedral angles over the MMC simulation. One
conformational state is identified for the α-(1→3)-linkage in 1
and two states for the α-(1→4)-linkage, although only one is
major. In 2 and 3 the α-linked sugar residues have an ‘S-shaped’
conformational space. For 2 three conformational states are
readily identified whereas for 3 these are less pronounced. The
rms fluctuations for the dihedral angles in the α-linkage in 2 and
3 are the largest for any of the trisaccharides, >20Њ, for both φ
and ψ. The conformational flexibility of a molecule can be
visualized by an overlay plot as shown for 2 in Fig. 3. The three
conformational states of the α-(1→3)-linkage have been chosen
and the overlay generated from the branch point residue. The
larger flexibility for the α-linkage is contrasted to the lower
flexibility for the β-linkage.
In the study of carbohydrate conformation two approaches
have often been used, i.e. the application of sugar rings as rigid
entities and non-charged atoms (hard sphere exo-anomeric,
HSEA) or a full molecular mechanics force field with atoms
carrying partial charges in which all degrees of freedom can be
relaxed. In general, less conformational space is accessible in
the former case. In the interpretation of experimental data,
from NMR spectra for example, a choice of a force field has to
be made, which may limit the extent to which conclusions can
be drawn. These types of force fields do not account for geo-
Fig. 1 Minimum energy conformers of trisaccharides 1–4
metrical effects of lone pairs or bond polarization, two factors
angles for trisaccharides and their constituent disaccharides dif-
fer significantly for the α-linked glycosyl groups, in one case
>50Њ for the ψ dihedral angle in the (1→3)-linkage of 1. Tri-
saccharides 2 and 3, which also contain α-linkages, show large
and positive deviations. In contrast, the (1→4)-linkage in 1
shows small and negative deviations. All β-linked glycosyl
groups have small differences between trisaccharides and
disaccharides.
which may influence the outcome of the NMR chemical
shifts. By the use of ab initio methods chemical shifts can be
calculated, although not yet with the accuracy to predict the
chemical shifts of the trisaccharides in this study (vide infra).
1H NMR glycosylation shifts
The 1H NMR chemical shift data for compounds 1–4 are given
in Table 3. The spectra were assigned by 1D TOCSY experi-
ments with selective excitation at the anomeric protons. The
glycosylation shifts for the trisaccharides (obs-mono) are
obtained by subtraction of the monosaccharide chemical shift
The averaged conformation from the MMC simulations is
similar to that of the global energy minimum for 1–8. Visualiz-
ation of the conformational space sampled and the identifi-
J. Chem. Soc., Perkin Trans. 2, 1998
643

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Fig. 2 Scatter plots from MMC simulations of trisaccharides 1–4. (A) (1→3)-linkages and (B) (1→4)-linkages.
from those of the trisaccharides. A major aim of this study is
the deviation from additivity of disaccharide glycosylation
shifts (obs-calc). The deviation is the difference between actual
spectra and what would have been obtained from monosacchar-
ide chemical shifts to which are added glycosylation shifts
specific for the glycosidic linkages in question. A negative value
for the deviation indicates that the actual chemical shift is
observed at a lower numerical value and vice versa for positive
deviation. The deviations can be anticipated to derive from
interactions between the substituting residues, although it is
644
J. Chem. Soc., Perkin Trans. 2, 1998

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Table 3 Chemical shifts of the signals in the 1H NMR spectra at 70 ЊC of the trisaccharides 1–4 and the corresponding hexose and methyl
hexosides, also listing calculated values of ∆δa and of ∆∆δb
Sugar residue
H-1
H-2
H-3
H-4
H-5
H-6a
H-6b
OMe
α--Glcp(1→4)
(obs)
5.51
(0.28)
[0.15]
4.82
(0.01)
[Ϫ0.01]
5.31
3.58
(0.04)
[Ϫ0.01]
3.76
(0.20)
[0.05]
3.62
3.67
(Ϫ0.05)
[Ϫ0.02]
4.02
(0.34)
[Ϫ0.05]
3.71
3.44
(0.02)
[0.00]
3.86
(0.45)
[Ϫ0.01]
3.44
3.71
(Ϫ0.13)
[Ϫ0.04]
3.78
(0.14)
[0.01]
3.96
(0.12)
[Ϫ0.01]
3.78
(0.02)
[0.00]
3.87
(0.11)
[0.07]
3.75
3.86
(0.02)
[0.00]
3.93
(0.06)
[0.05]
3.88
(obs-mono)
(obs-calc)
(obs)
(obs-mono)
(obs-calc)
(obs)
→3,4)α--Glcp-OMe
3.43
(0.00)
α--Glcp(1→3)
(1)
(obs-mono)
(obs-calc)
(0.08)
[Ϫ0.01]
(0.08)
[0.04]
(Ϫ0.01)
[Ϫ0.05]
(0.02)
[0.00]
(Ϫ0.01)
[Ϫ0.03]
(0.04)
[0.01]
β--Glcp(1→4)
(obs)
4.56
(Ϫ0.08)
[0.04]
3.30
(0.05)
[Ϫ0.03]
3.70
3.53
(0.03)
[0.00]
3.82
3.41
(Ϫ0.01)
[Ϫ0.04]
3.95
3.49
(0.03)
[Ϫ0.01]
3.78
3.79
(0.07)
[0.05]
3.86
3.96
(0.06)
[0.03]
3.93
(obs-mono)
(obs-calc)
(obs)
→3,4)α--Glcp-OMe
4.82
3.43
(0.00)
(obs-mono)
(obs-calc)
(obs)
(0.01)
[0.00]
5.22
(0.14)
[Ϫ0.01]
3.53
(0.14)
[Ϫ0.12]
3.75
(0.54)
[0.07]
3.44
(0.14)
[0.00]
3.93
(0.10)
[0.01]
3.75
(0.06)
[0.02]
3.86
α--Glcp(1→3)
(2)
(obs-mono)
(obs-calc)
(Ϫ0.01)
[Ϫ0.10]
(Ϫ0.01)
[Ϫ0.05]
(0.03)
[Ϫ0.01]
(0.02)
[0.00]
(0.09)
[Ϫ0.04]
(Ϫ0.01)
[Ϫ0.03]
(0.02)
[Ϫ0.01]
α--Glcp(1→4)
(obs)
5.36
(0.13)
[0.00]
4.78
(Ϫ0.03)
[Ϫ0.06]
4.89
3.48
(Ϫ0.06)
[Ϫ0.11]
3.80
(0.24)
[0.00]
3.30
(0.05)
[Ϫ0.07]
3.68
(Ϫ0.04)
[Ϫ0.01]
4.19
(0.51)
[0.07]
3.51
(0.01)
[Ϫ0.03]
3.42
(0.00)
[Ϫ0.02]
3.72
(0.31)
[Ϫ0.01]
3.42
3.68
(Ϫ0.16)
[Ϫ0.07]
3.75
(0.11)
[Ϫ0.03]
3.44
3.75
(Ϫ0.01)
[Ϫ0.03]
3.86
(0.10)
[0.05]
3.76
3.83
(Ϫ0.01)
[Ϫ0.03]
3.92
(0.05)
[0.01]
3.92
(obs-mono)
(obs-calc)
(obs)
(obs-mono)
(obs-calc)
(obs)
→3,4)α--Glcp-OMe
3.42
(Ϫ0.01)
β--Glcp(1→3)
(3)
(obs-mono)
(obs-calc)
(0.25)
[0.21]
(0.00)
[Ϫ0.01]
(Ϫ0.02)
[Ϫ0.06]
(0.04)
[0.02]
(0.02)
[0.03]
β--Glcp(1→4)
(obs)
4.64
(0.00)
[0.12]
4.78
3.37
(0.12)
[0.04]
3.79
3.49
(Ϫ0.01)
[Ϫ0.04]
4.13
3.40
(Ϫ0.02)
[Ϫ0.05]
3.86
3.44
(Ϫ0.02)
[Ϫ0.06]
3.80
3.73
(0.01)
[Ϫ0.01]
3.90
3.89
(Ϫ0.01)
[Ϫ0.04]
3.90
(obs-mono)
(obs-calc)
(obs)
→3,4)α--Glcp-OMe
3.41
(Ϫ0.02)
(obs-mono)
(obs-calc)
(obs)
(Ϫ0.03)
[Ϫ0.05]
4.89
(0.23)
[Ϫ0.01]
3.35
(0.45)
[0.14]
3.51
(0.45)
[0.12]
3.40
(0.16)
[0.01]
3.45
(0.14)
[0.04]
3.72
(0.03)
[Ϫ0.04]
3.90
β--Glcp(1→3)
(4)
(obs-mono)
(obs-calc)
(0.25)
[0.21]
(0.10)
[Ϫ0.02]
(0.01)
[Ϫ0.03]
(Ϫ0.02)
[Ϫ0.03]
(Ϫ0.01)
[Ϫ0.05]
(0.00)
[Ϫ0.02]
(0.00)
[Ϫ0.02]
α--Glcp
β--Glcp
α--Glcp-OMe
5.23
4.64
4.81
3.54
3.25
3.56
3.72
3.50
3.68
3.42
3.42
3.41
3.84
3.46
3.64
3.76
3.72
3.76
3.84
3.90
3.87
3.43
a Glycosylation shifts are calculated by subtraction of the chemical shifts from those of the corresponding hexose and methyl hexoside, a positive
difference indicates a downfield shift. b ∆∆δ values for the trisaccharides, in square brackets, are calculated by adding the ∆δ values of the correspond-
ing disaccharides to the chemical shift of the hexose or methylhexoside and then subtracting the resulting value from the measured chemical shift of
the trisaccharide (for the methylhexoside residues ∆δ values from both disaccharides are added).
difficult to quantify the magnitude of these. In general, for pro-
tons an upfield chemical shift displacement is observed upon a
proton–proton interaction, whereas a downfield chemical shift
displacement occurs for proton–oxygen interactions. A problem
in the analysis is that different effects may cancel and the
change may not be apparent. Previous work has included, inter
alia, 3,4-disubstituted methyl galactosides and different 1,2-
disubstituted sugar residues. The latter are linear oligosacchar-
ides but formally vicinally disubstituted.
In compounds 1–4 glycosylation shifts are between Ϫ0.13
and 0.54 ppm. In examination of the trisaccharide chemical
shifts and those obtained by the additivity approach a signifi-
cance level of 0.1 ppm has been chosen for the present discus-
sion. Only a few signals show differences of this magnitude,
namely, H1Љ in 1 (downfield), H3 and H1Ј in 2 (upfield), H1Ј in
3 (downfield), H2Љ in 3 (upfield), and H3, H4, H1Ј and H1Љ in 4
(downfield), i.e. in all cases but one the deviations are confined
to protons at glycosidic linkages.
cussed previously,33 and may come from a changed localiz-
ation of the lone pairs of the oxygen involved in a glycosidic
linkage compared to its conformational preference in a
hydroxy group. Thus, the downfield displacement of H1Љ may
be due to interactions with O3, through increased overlap of
O3 lone pairs and H1Љ. In 2 the protons at the α-(1→3)-
linkage show upfield displacements from additivity. Changes
in positions in energy minima and their averages from the
MMC calculations lead to a conformation with φ and ψ
dihedral angles with smaller numerical values, i.e. closer to
zero degrees. This in effect leads to a shorter H1Ј–H3 distance
in 2, 2.14 Å, than in 5, 2.47 Å, as observed for the global
energy minimum structures, as well as the average distance
from the MMC simulation. The mutual upfield displacement
can thus be explained by a shorter effective proton–proton
distance. The β-(1→3)-linkage in 3 and 4 shows similarities as
observed in the MMC and small deviations compared to its
constituent disaccharide. As deduced from NMR data this
linkage exhibits conformational similarities in 3 and 4 since
H1Ј and H3 show downfield shift displacements of ca. 0.2
and 0.1 ppm, respectively, together with a deviation for C2
of ca. 1.3 ppm (vide infra) which is not at a glycosylation
position. Finally, for the β-(1→4)-linkage in 4, significant
In 1 the value of the anomeric proton resonance that shows
a deviation from additivity is not at the glycosidic α-(1→3)-
linkage that shows the largest difference in conformation
compared to the constituent disaccharide, but at the α-(1→4)-
linkage. One reason for this type of change has been dis-
J. Chem. Soc., Perkin Trans. 2, 1998
645

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the analysis of complex NMR spectra of large oligo- and poly-
saccharides knowledge of these deviations will be indispens-
able. The conformational flexibility of the four anomeric com-
binations of the 3,4-disubstituted glucopyranoside was investi-
gated by energy minimization and Metropolis Monte Carlo
simulations which in general showed larger flexibility and larger
deviations for the α-linked glucosyl groups than for the β-linked
groups. Simulation using MMC leads to rapid identification of
both oligosaccharide flexibility and possible changes in con-
formation between different oligosaccharides.
Experimental
General
Atoms in the (1→4)-linked glucosyl group are labeled by a
double prime, in the (1→3)-linked glucosyl group by a prime
and in the methyl glucoside the atoms are unprimed. Dichloro-
methane was distilled and dried over molecular sieves (4 Å)
before use in coupling reactions. Diethyl ether was dried over
sodium wire. Concentrations were performed under reduced
pressure at temperatures <50 ЊC (bath). TLC was conducted on
precoated plates (Merck Silica Gel 60 F254) and detected by UV
at 245 nm or developed by charring with 8% aqueous sulfuric
acid. Column chromatography was carried out on Matrex silica
gel 60 Å (35–70 µm, Amicon) and on Bio-Gel P-2. Optical
rotations were determined at 578 nm with a Perkin-Elmer 241
polarimeter and measured in chloroform or water. [α]578 values
Fig. 3 Overlay plot for compound 2 of the conformers from the three
low energy minima identified
deviations from additivity are also observed for the protons at
the glycosidic linkage.
Since deviations from additivity of disaccharide glyco-
sylations shifts are not larger than ca. 0.2 ppm, then it is pos-
sible to make reasonable predictions of 1H NMR trisaccharide
chemical shifts using only the disaccharide shifts.
are given in units of 103 deg cm2 gϪ1
.
Synthesis
13C NMR glycosylation shifts
General procedure for á-glycosylation. To a cooled solution
(0 ЊC) of ethyl 2,3,4,6-tetra-O-benzyl-thio-β--glucopyranoside
(1.3 equiv.) in dichloromethane, bromine (10 cm3, 1.4 equiv.)
was added. After 10 min the reaction mixture was diluted with
toluene (10 cm3) and the mixture was concentrated in vacuo and
coevaporated twice with toluene. The resulting 2,3,4,6-tetra-O-
benzyl-α--glucopyranosyl bromide as donor was dissolved in
diethyl ether (20 cm3). To the solution were added aglycon (1
equiv.), s-collidine‡ (0.7 equiv.) and molecular sieves 4 Å (1 g).
The mixture was stirred for 30 min, cooled to Ϫ30 ЊC, where-
after AgOTf (1.3 equiv.) was added and the temperature was
kept at Ϫ30 ЊC. When TLC showed complete reaction, pyridine
(0.5 cm3) was added and the reaction mixture was filtered, con-
centrated and purified from small amounts of the β-anomer by
silica gel column chromatography.
General procedure for â-glycosylation. A mixture of aglycon
(1 equiv.), 2,3,4,6-tetra-O-benzoyl-α--glucopyranosyl bromide
(1.3 equiv., except for compound 14 for which 2.5 equiv. were
used) was dissolved in dichloromethane (20 cm3). To the solu-
tion were added s-collidine (0.7 equiv.) and molecular sieves 4 Å
(1 g) and the mixture was stirred for 30 min, cooled to Ϫ30 ЊC,
whereafter AgOTf (1.3 equiv.) was added and the temperature
was kept at Ϫ30 ЊC. When TLC showed complete reaction,
pyridine (0.5 cm3) was added and the reaction mixture
was filtered, concentrated and purified by silica gel column
chromatography.
The 13C NMR chemical shift data for compounds 1–4 are given
in Table 4. The spectra were assigned by 1H, 13C HSQC and 1H,
13C HMBC experiments.† The significance level for the 13C
NMR chemical shifts was set to 1 ppm. Deviations are obtained
for signals from glycosyloxylated carbons, namely, C3 (down-
field) and C4 (upfield) in 1 and 2, C3 (upfield) in 3 and C3 and
C4 (upfield) in 4. In 3 and 4, which both have a β-(1→3)-linked
glucosyl group the C2 resonance shows a downfield chemical
shift displacement, i.e. the β-effect is quite large. Furthermore,
the signals from anomeric carbons of the β-linked glucosyl
groups in 4 have upfield chemical shift displacements.
In cases where significant changes from additivity take place
for carbon signals and deviations also occur for protons, it can
be observed that carbons with signals shifted upfield are associ-
ated with downfield displacements of the covalently linked pro-
tons and vice versa, compare for example H3/C3 in 2 and 4. For
the atoms at or next to glycosyloxylated carbons in compound 4
the direct α-effects and the adjacent β-effects add to an absolute
deviation from additivity which is larger than 14 ppm. It would
be detrimental not to include deviations of this magnitude in
a simulation of a 13C NMR spectrum of an oligo- or poly-
saccharide. For 13C NMR chemical shifts the deviations are
frequent and significant and in some cases large, ranging from
Ϫ4.5 to 2.3 ppm.
General procedure for reductive ring opening of a 4,6-
benzylidene group. NaCNBH3 (12.5 equiv.), the 4,6-benzylidene
derivative (1 equiv.) and 3 Å molecular sieves were mixed in
THF, and stirred at room temperature. After 30 min diethyl
ether saturated with hydrogen chloride was added until the
reaction mixture was acidic. After 15 (12 and 23) or 30 min (13),
triethylamine was added. The quenched reaction mixture was
filtered through Celite, concentrated and purified by silica gel
column chromatography.
Conclusions
The present study describes the synthesis of four trisaccharides
which are used to investigate the vicinal disubstitution at the 3-
and 4-positions in a glucopyranoside, the hydroxy groups of
which have a trans relationship. It complements in particular
the previous investigation of 3,4-disubstituted galactopyrano-
sides with a cis relationship. Moderate deviations from additiv-
ity occur for 1H NMR signals. Deviations for 13C NMR spectra
are large with both upfield and downfield displacements. For
General procedure for removal of protecting groups. To
remove benzoyl groups the compounds were dissolved in
† HSQC and HMBC are heteronuclear single quantum correlation and
heteronuclear multiple bond correlation spectroscopy, respectively.
‡ s-Collidene = 2,4,6-trimethylpyridine.
646
J. Chem. Soc., Perkin Trans. 2, 1998

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Table 4 Chemical shifts of the signals in the 13C NMR spectra at 70 ЊC of the trisaccharides 1–4 and the corresponding hexose and methyl
hexosides, also listing calculated values of ∆δa and of ∆∆δb
Sugar residue
C-1
99.91
(6.92)
[Ϫ0.74]
99.62
(Ϫ0.57)
[Ϫ0.48]
99.80
C-2
C-3
73.58
(Ϫ0.20)
[Ϫ0.31]
84.10
(10.00)
[2.34]
73.74
C-4
70.40
(Ϫ0.31)
[Ϫ0.04]
76.28
(5.60)
[Ϫ2.31]
70.61
C-5
73.58
(1.21)
[Ϫ0.02]
71.02
(Ϫ1.50)
[0.13]
73.44
C-6
61.49
(Ϫ0.35)
[Ϫ0.10]
61.76
(0.09)
[0.29]
61.49
OMe
α--Glcp(1→4)
(obs)
72.34
(Ϫ0.13)
[Ϫ0.38]
71.31
(Ϫ0.92)
[0.69]
72.44
(Ϫ0.03)
[Ϫ0.22]
(obs-mono)
(obs-calc)
(obs)
(obs-mono)
(obs-calc)
(obs)
→3,4)α--Glcp-OMe
55.88
(Ϫ0.05)
α--Glcp(1→3)
(1)
(obs-mono)
(obs-calc)
(6.81)
[Ϫ0.15]
(Ϫ0.04)
[Ϫ0.17]
(Ϫ0.10)
[0.05]
(1.07)
[0.63]
(Ϫ0.35)
[0.01]
β--Glcp(1→4)
(obs)
102.56
(5.72)
[Ϫ0.73]
99.94
(Ϫ0.25)
[Ϫ0.09]
100.88
(7.89)
74.36
(Ϫ0.84)
[0.23]
71.47
(Ϫ0.76)
[0.89]
72.96
(0.49)
[0.30]
76.55
(Ϫ0.21)
[Ϫ0.03]
81.75
(7.65)
[1.75]
74.36
(0.58)
[0.45]
70.61
(Ϫ0.10)
[0.10]
75.71
(5.03)
[Ϫ4.32]
70.53
(Ϫ0.18)
[Ϫ0.03]
77.30
(0.54)
[0.42]
71.47
(Ϫ1.05)
[0.54]
73.23
61.73
(Ϫ0.11)
[0.08]
(obs-mono)
(obs-calc)
(obs)
(obs-mono)
(obs-calc)
(obs)
→3,4)α--Glcp-OMe
60.55
55.91
(Ϫ0.02)
(Ϫ1.12)
[Ϫ0.40]
61.46
(Ϫ0.38)
[Ϫ0.02]
α--Glcp(1→3)
(2)
(obs-mono)
(obs-calc)
(0.86)
[0.42]
[0.93]
α--Glcp(1→4)
(obs)
100.96
(7.97)
[0.31]
99.91
(Ϫ0.28)
[0.07]
103.34
(6.50)
[Ϫ0.33]
73.01
(0.54)
[0.29]
72.77
(0.54)
[1.37]
74.63
73.98c
(0.20)
[0.09]
70.45
(Ϫ0.26)
[0.01]
76.55
(5.87)
[Ϫ0.34]
70.45
(Ϫ0.26)
[Ϫ0.13]
73.69c
(1.32)
[0.88]
71.69
(Ϫ0.83)
[0.74]
77.14
61.54
(Ϫ0.30)
[0.06]
(obs-mono)
(obs-calc)
(obs)
(obs-mono)
(obs-calc)
(obs)
→3,4)α--Glcp-OMe
80.78
61.54
55.93
(0.00)
(6.68)
[Ϫ3.21]
76.71
(Ϫ0.05)
[0.11]
(Ϫ0.13)
[Ϫ0.09]
61.76
(Ϫ0.08)
[0.12]
β--Glcp(1→3)
(3)
(obs-mono)
(obs-calc)
(Ϫ0.57)
[0.21]
(0.38)
[0.25]
β--Glcp(1→4)
(obs)
101.56
(4.72)
[Ϫ1.73]
99.86
(Ϫ0.33)
[0.09]
102.37
(5.53)
73.93
(Ϫ1.27)
[Ϫ0.20]
72.66
(0.43)
[1.30]
74.23
(Ϫ0.97)
[Ϫ0.19]
76.52
(Ϫ0.24)
[Ϫ0.06]
77.87
(3.77)
[Ϫ4.36]
76.65
70.42
(Ϫ0.29)
[Ϫ0.09]
73.85
(3.17)
[Ϫ4.48]
70.56
76.79
(0.03)
[Ϫ0.09]
71.80
(Ϫ0.72)
[0.81]
77.03
(0.27)
[0.14]
61.71c
(Ϫ0.13)
[0.06]
(obs-mono)
(obs-calc)
(obs)
(obs-mono)
(obs-calc)
(obs)
→3,4)α--Glcp-OMe
61.17
55.91
(Ϫ0.02)
(Ϫ0.50)
[0.06]
β--Glcp(1→3)
(4)
61.76c
(Ϫ0.08)
[0.12]
(obs-mono)
(obs-calc)
(Ϫ0.11)
[0.05]
(Ϫ0.15)
[Ϫ0.02]
[Ϫ1.30]
α--Glcp
β--Glcp
α--Glcp-OMe
92.99
96.84
100.19
72.47
75.20
72.23
73.78
76.76
74.10
70.71
70.71
70.68
72.37
76.76
72.52
61.84
61.84
61.67
55.93
a Glycosylation shifts, in parentheses, are calculated by subtraction of the chemical shifts from those of the corresponding hexose and methyl
hexoside, a positive difference indicates a downfield shift. b ∆∆δ values for the trisaccharides, in square brackets, are calculated by adding the ∆δ
values of the corresponding disaccharides to the chemical shift of the hexose or methylhexoside and then subtracting the resulting value from the
measured chemical shift of the trisaccharide (for the methylhexoside residues ∆δ values from both disaccharides are added). c Interchangeable pairs.
dichloromethane and 0.1 mol dmϪ3 sodium methoxide in
methanol was added. The solution was stirred until TLC
showed complete reaction, and passed through a DOWEX-
50(Hϩ) ion-exchange column, after which the solvent was
removed in vacuo. For the deprotection of the benzyl
groups the compounds were dissolved in EtOAc–EtOH and
hydrogenolysed over Pd–C (cat) at 90 psi for 5–12 h. The fully
deprotected compounds were purified by gel permeation
chromatography.
Mass spectroscopy
Fast Atom Bombardment Mass Spectroscopy (FABMS) was
performed at a resolution of 2500 on a JEOL SX-102 using
a mixture of glycerol and thioglycerol, triethanolamine or
m-nitrobenzyl alcohol as a matrix. Atmospheric Pressure
Chemical Ionization (APCI) was recorded in the positive mode
for compound 27 on a Quattro mass spectrometer (Fisons
Instruments, UK).
Computational methods
NMR spectroscopy
The GEGOP program, version 2.6, was used for all calcu-
lations.36 The bond angle τ and dihedral angles φ, ψ and ω
were defined as follows: τ = C1᎐O1᎐CX, φ = H1᎐C1᎐O1᎐CX,
ψ = C1᎐O1᎐CX᎐HX, and ω = O5᎐C5᎐C6᎐O6, where X is the
linkage position. The bond angle τ was optimized starting
from 117Њ. The O-methyl group was gauche to O5 and anti to
C2. The Metropolis Monte Carlo (MMC) simulations of 1–8
were started from low energy conformations obtained from a
grid-based conformational search of the compounds. Opti-
mized angles of the hydroxy groups were also used in the start-
ing conformations for the Monte Carlo simulations.
NMR spectra were recorded on JEOL GSX-270, Varian Unity
500 and Unity Plus 600 instruments using CDCl3 or D2O as
solvents. For solutions in D2O, spectra were recorded at 70 ЊC
and chemical shifts referred to internal TSP [sodium 3-tri-
methylsilyl[2,2,3,3-2H4]propanoate, δH = 0.00) and dioxane
(δC = 67.40). For assignments of signals of 1–4 different types
of homo- and hetero-nuclear correlation spectroscopy (COSY)
experiments were used as well as 1D TOCSY34 experiments
with different spin lock times of 30, 70, 90 and 100 ms. To
establish the glycosidic connectivities long-range proton–
carbon correlated experiments, HMBC,35 were performed using
a delay time of 50 ms.
Simulations were performed at 300 K with 106 macro steps
for each of the eight molecules. The parameters were adjusted
J. Chem. Soc., Perkin Trans. 2, 1998
647

View Article Online
14 H. Baumann, B. Erbing, P.-E. Jansson and L. Kenne, J. Chem. Soc.,
with test runs to obtain a total acceptance ratio between 35 and
49%. The maximum step length for the glycosidic angles φ and
ψ was set to 20Њ. The bond angle τ and all dihedral angles φ, ψ,
ω and those of the hydroxy and O-methyl groups, were opti-
mized. The pyranose rings were treated as rigid units residing in
the 4C1 conformation.
Perkin Trans. 1, 1989, 2145.
15 H. Baumann, B. Erbing, P.-E. Jansson and L. Kenne, J. Chem. Soc.,
Perkin Trans. 1, 1989, 2153.
16 H. Baumann, B. Erbing, P.-E. Jansson and L. Kenne, J. Chem. Soc.,
Perkin Trans. 1, 1989, 2167.
17 K. Bock, J. F.-B. Guzman and R. Norrestam, Carbohydr. Res., 1988,
179, 97.
18 E. A. Khatuntseva, A. S. Shashkov and N. E. Nifant’ev, Magn.
Reson. Chem., 1997, 35, 414.
19 A. Adeyeye, P.-E. Jansson, L. Kenne and G. Widmalm, J. Chem.
Soc., Perkin Trans. 2, 1991, 963.
20 K. Hermansson, P.-E. Jansson, L. Kenne, G. Widmalm and
F. Lindh, Carbohydr. Res., 1992, 235, 69.
21 J. Ø. Duus, N. E. Nifant’ev, A. S. Shashkov, E. A. Khatuntseva and
K. Bock, Carbohydr. Res., 1996, 288, 25.
22 F. J. Kronzer and C. Scheuerch, Carbohydr. Res., 1973, 28, 279.
23 S. Hanessian and J. Banoub, Carbohydr. Res., 1977, 53, C13.
24 P. J. Garegg, T. Iversen and S. Oscarson, Carbohydr. Res., 1976, 50,
C12.
Acknowledgements
This work was supported by grants from the Swedish Natural
Science Research Council, Magn. Bergvalls Foundation and
the Swedish Research Council for Engineering Sciences. The
Swedish NMR Centre is thanked for providing access to the
U500 NMR spectrometer.
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Paper 7/07346A
Received 13th October 1997
Accepted 18th November 1997
648
J. Chem. Soc., Perkin Trans. 2, 1998