The syntheses of 6-C-alkyl derivatives of methyl α-isomaltoside for a study of the mechanism of hydrolysis by amyloglucosidase

Article abstract of DOI:10.1139/v01-005

The epimeric (6aR)- and (6aS)-C-alkyl (methyl, ethyl and isopropyl) derivatives of methyl α-isomaltoside (1) were synthesized in order to examine the effects of introducing alkyl groups of increasing bulk on the rate of catalysis for the hydrolysis of the interunit α-glycosidic bond by the enzyme amyloglucosidase, EC 3.2.1.3, commonly termed glucoamylase (AMG). It was previously established that methyl (6aR)-C-methyl α-isomaltoside is hydrolysed about 2 times faster than methyl α-isomaltoside and about 8 times faster than its S-isomer. The kinetics for the hydrolyses of the ethyl and isopropyl analogs were also recently published. As was expected from molecular model calculations, all the R-epimers are good substrates. A rationale is presented for the catalysis based on conventional mechanistic theories that includes the assistance for the decomposition of the activated complex to products by the presence of a hydrogen bond, which connects the 4a-hydroxyl group to the tryptophane and arginine units. It is proposed that activation of the initially formed complex to the transition state is assisted by the energy released as a result of both of the displacement of perturbed water molecules of hydration at the surfaces of both the polyamphiphilic substrate and the combining site and the establishment of intermolecular hydrogen bonds, i.e., micro-thermodynamics. The dissipation of the heat to the bulk solution is impeded by a shell of aromatic amino acids that surround the combining site. Such shields are known to be located around the combining sites of lectins and carbohydrate specific antibodies and are considered necessary to prevent the disruption of the intermolecular hydrogen bonds, which are of key importance for the stability of the complex. These features together with the exquisite stereoelectronic dispositions of the reacting molecules within the combining site offer a rationalization for the catalysis at ambient temperatures and near neutral pH. The syntheses involved the addition of alkyl Grignard reagents to methyl 6-aldehydo-α-D-glucopyranoside. The addition favoured formation of the S-epimers by over 90%. Useful amounts of the active R-isomers were obtained by epimerization of the chiral centers using conventional methods. Glycosylation of the resulting alcohols under conditions for bromide-ion catalysis, provided methyl (6aS)- and (6aR)-C-alkyl-hepta-O-benzyl-α-isomaltosides. Catalytic hydrogenolysis of the benzyl groups afforded the desired disaccharides. ¹H NMR studies established the absolute configurations and provided evidence for conformational preferences.

Full text of DOI:10.1139/v01-005

238
The syntheses of 6-C-alkyl derivatives of methyl
α-isomaltoside for a study of the mechanism of
hydrolysis by amyloglucosidase
Ulrike Spohr, Nghia Le, Chang-Chun Ling, and Raymond U. Lemieux
Abstract: The epimeric (6aR)- and (6aS)-C-alkyl (methyl, ethyl and isopropyl) derivatives of methyl α-isomaltoside (1)
were synthesized in order to examine the effects of introducing alkyl groups of increasing bulk on the rate of catalysis
for the hydrolysis of the interunit α-glycosidic bond by the enzyme amyloglucosidase, EC 3.2.1.3, commonly termed
glucoamylase (AMG). It was previously established that methyl (6aR)-C-methyl α-isomaltoside is hydrolysed about 2
times faster than methyl α-isomaltoside and about 8 times faster than its S-isomer. The kinetics for the hydrolyses of
the ethyl and isopropyl analogs were also recently published. As was expected from molecular model calculations, all
the R-epimers are good substrates. A rationale is presented for the catalysis based on conventional mechanistic theories
that includes the assistance for the decomposition of the activated complex to products by the presence of a hydrogen
bond, which connects the 4a-hydroxyl group to the tryptophane and arginine units. It is proposed that activation of the
initially formed complex to the transition state is assisted by the energy released as a result of both of the displace-
ment of perturbed water molecules of hydration at the surfaces of both the polyamphiphilic substrate and the combin-
ing site and the establishment of intermolecular hydrogen bonds, i.e., micro-thermodynamics. The dissipation of the
heat to the bulk solution is impeded by a shell of aromatic amino acids that surround the combining site. Such shields
are known to be located around the combining sites of lectins and carbohydrate specific antibodies and are considered
necessary to prevent the disruption of the intermolecular hydrogen bonds, which are of key importance for the stability
of the complex. These features together with the exquisite stereoelectronic dispositions of the reacting molecules within
the combining site offer a rationalization for the catalysis at ambient temperatures and near neutral pH. The syntheses
involved the addition of alkyl Grignard reagents to methyl 6-aldehydo-α-D-glucopyranoside. The addition favoured for-
mation of the S-epimers by over 90%. Useful amounts of the active R-isomers were obtained by epimerization of the
chiral centers using conventional methods. Glycosylation of the resulting alcohols under conditions for bromide-ion
catalysis, provided methyl (6aS)- and (6aR)-C-alkyl-hepta-O-benzyl-α-isomaltosides. Catalytic hydrogenolysis of the
1
benzyl groups afforded the desired disaccharides. H NMR studies established the absolute configurations and provided
evidence for conformational preferences.
Key words: amyloglucosidase (AMG), exo-anomeric effect, 6-C-alkyl-α-D-glucopyranosides and isomaltosides,
mechanism of enzyme catalysis. 255
Résumé : On a réalisé la synthèse des dérivés (6aR) et (6aS)-C-alkyles (méthyle, éthyle et isopropyle) de l’α-
isomaltoside de méthyle (1) afin d’étudier les effets provoqués par l’introduction de groupements alkyles de plus en
plus volumineux sur la catalyse de l’hydrolyse de la liaison glycosidique α par l’enzyme amyloglucosidase, EC 3.2.1.3,
que l’on dénomme souvent glucoamylase (AMG). Il a été établi antérieurement que le (6aR)-C-méthyl-α-isomaltoside
de méthyle s’hydrolyse environ deux fois plus rapidement que l’α-isomaltoside de méthyle et environ huit fois plus ra-
pidement que son isomère S. Les mesures cinétiques pour les hydrolyses des analogues éthyle et isopropyle ont aussi
été publiées récemment. Tel qu’on pouvait s’y attendre sur la base de calculs sur des modèles moléculaires, tous les
épimères R sont de bons substrats. On propose une rationalisation de la catalyse qui est basée sur la théorie mécanis-
tique conventionnelle mais qui, pour la décomposition du complexe activé en produits, implique une aide apportée par
la présence dans le complexe d’une liaison hydrogène reliant le groupe 4a-hydroxyle aux unités de tryptophane et
d’arginine qui se trouvent loin du centre réactionnel. Il est suggéré que le passage du complexe qui se forme initiale-
ment à état de transition se produit avec l’aide de l’énergie libérée en raison à la fois du déplacement des molécules
d’eau d’hydratation perturbées sur les surfaces tant du substrat polyamphiphile que du site qui se combine ainsi ce que
celle qui résulte de la création de liaisons hydrogènes intermoléculaires, de la microthermodynamique. On indique qu’il
y a eu empêchement à la dispersion de la chaleur vers l’ensemble du milieu réactionnel causé par une sorte de coquille
Received June 16, 2000. Published on the NRC Research Press Web site on March 16, 2001.
U. Spohr1, N. Le2, C.-C. Ling, and R.U. Lemieux.3 Department of Chemistry, The University of Alberta, Edmonton, AB
T6G 2G2, Canada.
1Corresponding author (e-mail: uspohr@aol.com).
2Taken in part from a Ph.D. Thesis, University of Alberta, 1990.
3Deceased.
Can. J. Chem. 79: 238–255 (2001)
© 2001 NRC Canada
DOI: 10.1139/cjc-79-2-238

Spohr et al.
239
formée d’acides aminés aromatiques qui entoure le site où se produit la combinaison. De tels coquilles ont déjà été ob-
servées autour des sites où se produit la combinaison des lectines et des anticorps spécifiques aux hydrates de carbone
et ils sont considérés comme nécessaires pour empêcher l’interruption des liaisons hydrogènes d’importance capitale à
la stabilité du complexe. Ces caractéristiques ainsi que les dispositions stéréoélectroniques exquises des molécules qui
réagissent à l’intérieur du site où se produit la combinaison permettent de rationaliser la catalyse à la température am-
biante et à un pH pratiquement neutre. Les synthèses impliquent l’addition des réactifs de Grignard alkylés sur le 6-
aldéhydo-α-D-glucopyranoside de méthyle. L’addition conduit à la formation préférentielle (90%) des épimères S. On a
obtenu des quantités utiles des isomères actifs, R, par épimérisation des centres chiraux à l’aide de méthodes conven-
tionnelles. La glycosylation des alcools qui en résultent, dans des conditions de catalyse par l’ion bromure, a permis
d’obtenir les (6aR) et (6aS)-C-alkyl-O-benzyl-α-isomaltosides de méthyle. L’hydrogénolyse catalytique des groupes
1
benzyles fournit ensuite les disaccharides désirés. Des études de RMN du H ont permis de déterminer les configura-
tions absolues et ont permis d’obtenir des données relatives aux préférences conformationnelles.
Mots clés : amyloglucidase (AMG), effet anomère exo, α-D-glucopyranosides et isomaltosides 6-C-alkylés, mécanisme
de catalyse enzymatique.
[Traduit par la Rédaction] Spohr et al.
Nomenclature and conventions: The nomenclature follows
the general rules of organic chemistry. Thus, the introduction
of a methyl group at C-6 of methyl α-D-glucopyranoside
gives methyl (6R)-C-methyl-α-D-glucopyranoside not methyl
7-deoxy-β-L-glycero-D-gluco-heptopyranoside. For reasons
of clarity and convenience, the so-called “reducing unit” of a
disaccharide is termed the a-unit. The nonreducing unit is
the b-unit. The 6a-epimers are given the same compound
number but distinguished by their absolute configurations (R
or S). The m, e and i letters that follow the number of a
compound are to identify the alkyl substituent: methyl, ethyl
or isopropyl, respectively. The rings of glucopyranosides are
Fig. 1. The structure of methyl α-isomaltoside that displays the
pro-R and pro-S hydrogen atoms at the C-6a position. Acarbose
is a strong inhibitor (∆G = –16.3 kcal mol–1) of the hydrolysis of
1 by the enzyme AMG.
4
taken as residing in an essentially rigid C1-conformation.
The three flexible bonds that link the two glucose units at
the 6a-position of an isomaltoside, namely, C-1b-O-6a,
O-6a-C-6a and C-6a-C-5a, describe three torsion angles φ° =
O-5b/C-6a, ψ° = C-1b/C-5a, and ω° = O-6a/O-5a, respectively.
Introduction
The long known (1) industrially important enzyme,
amyloglucosidase (EC 3.2.1.3), referred to herein as AMG,
is an anomeric-centre inverting exoglycosidase that catalyzes
the release of β-D-glucopyranose from the non-reducing end
of starch and other related poly- and oligosaccharides (2, 3).
AMG catalyzes the hydrolysis of the 1 → 4 linkage of malt-
ose 30–50 times more rapidly than the 1 → 6 linkage of
isomaltose. For both substrates three hydroxyl groups were
found virtually indispensable, by means of chemical map-
ping (4, 5), for high enzyme activity. These essential
hydroxyl groups circled in Fig. 1 for 1 are located at the 4a,
4b, and 6b positions for both the β- (5) and α- (6) methyl
isomaltosides. For methyl β-maltoside these key hydroxyls
are located at positions 3a, 4b, and 6b (4). As seen in Ta-
ble 1, deoxygenation of the 4a-hydroxyl of methyl α-
isomaltoside decreases the rate of hydrolysis by about a
thousand-fold (6). It is conceivable that the enzyme recog-
nizes the conformation of these substrates that have OH-4a
of an isomaltoside (1) and OH-3a of a maltoside, which are
in similar spatial relationship to the OH-4b and OH-6b of
the nonreducing b-unit (5).
and the X-ray crystal structure of the acarbose–AMG com-
plex (7, 8). Substantially weaker interactions involve the
hydroxyl groups at the 3a, 2b, and 3b positions (6). As ex-
pected from the X-ray crystal structure of the acarbose–
AMG complex the OH-2a (see Table 1) and the aglycon at
the 1a position remain in the aqueous phase. The fact that
methyl (6aR)-C-methyl-α-isomaltoside (R-2m) (9) is hydro-
lyzed 8 times faster than the S-epimer (S-2m) suggests, as
previously noted (9), that the C-methyl group of the R-isomer
does not impede complex formation. Indeed, Frandsen et al.
(10) found that the 6a-C-methyl group of R-2m projects
into a surrounding aqueous phase to become hydropho-
bically associated to the enzyme. In the simulated complex
of isomaltoside with AMG (6), it is the pro-R hydrogen that
Chemical mapping (6) of the interaction of methyl α-
isomaltoside with AMG proved to be in total agreement with
both the kinetic data reported by Bock and co-workers (4, 5)
© 2001 NRC Canada

240
Can. J. Chem. Vol. 79, 2001
Table 1. The effect of monodeoxygenations and C-alkylation on the kinetics for the hydrolysis of methyl α-isomaltoside catalyzed by AMG.
kcat
Km
(mM)
k
cat /Km
∆∆G
Compound
(s–1)
(s–1 mM–1)
(kcal mol–1)
Methyl α-isomaltoside (1)a
0.88
0.5
0.00067
0.18
0.68
1.1
20.0
19.1
8.8
21.2
0.71
90.0
3.3
4.4 × 10–2
2.6 × 10–2
7.6 × 10–5
8.5 × 10–3
9.6 × 10–1
1.2 × 10–2
3.6 × 10–1
1.7 × 10–2
0
Methyl 2a-deoxy-α-isomaltosideb
0.33
4.02
1.04
–1.95
0.82
–1.33
0.56
Methyl 4a-deoxy-α-isomaltosideb
Methyl 2b-deoxy-α-isomaltosideb
Methyl (6aR)-C-methyl-α-isomaltosidec (R-2m)
Methyl (6aS)-C-methyl-α-isomaltosidec (S-2m)
Methyl (6aR)-C-ethyl-α-isomaltosidea (R-2e)
Methyl (6aR)-C-isopropyl-α-isomaltosidea (R-2i)
1.2
0.8
47.0
aData from ref. 10.
bData from ref. 6.
cData from ref. 9.
Table 2. Calculated values for the most energetically favorable conformers of methyl α-isomaltoside and C-6a alkylated congeners.
Torsional angles for the
1 → 6 linkages (°)
Conformera
E
∆EB-A
Compound
φ
ψ
ω
(kcal mol–1)
(kcal mol–1)
Methyl α-isomaltoside (1)
A
B
–51
–45
–169
167
–60
163
–4.2
–0.96
3.3
2.4
7.3
Methyl (6aR)-C-methyl-α-isomaltoside (R-2m)
Methyl.(6aR)-C-isopropyl-α-isomaltoside (R-2i)
A
B
–54
–45
89
167
52
163
–3.7
–1.3
A
B
–54
–45
70
167
48
163
–5.8
1.56
aConformers A are the preferred conformations established by GEGOP calculation. These were found to not be compatible with the combining site of
the acarbose–AMG complex. Consequently, the values of the φ, ψ, and ω torsion angles were changed in order to achieve a fit. An acceptable fit could
not be achieved keeping the b-unit in the chair-conformation. However, a fine fit was obtained on changing the conformation of the b-unit to the half-
chair conformer. The ∆E values refer to the differences in energy that are expected to arise from relaxation of the φ, ψ, and ω torsion angles of the B
conformers to those of the A conformers.
projects into the channel of water, which in supports the
statement that only the R-isomers are good substrates (10).
Furthermore, this data suggests that a substantial body of
bulk water extends from near the 6a-CH2 group on the side
of the complex that includes the catalytic water molecule. It
was on this basis that it had seemed likely that the (6aR)-C-
methyl group of R-2m could be replaced by bulkier groups
and still retain good substrate activity. To test this hypothe-
sis, the R- and S-2e (6a-C-ethyl) and R- and S-2i (6a-C-
isopropyl) analogs were synthesized and made available to
Frandsen et al. (10) for kinetic studies.
We considered that should these compounds (R-2e and R-
2i) prove to be good substrates for AMG that it would also
follow that all are accepted into the active site with similar φ,
ψ, and ω torsion angles for the inter-unit bonds. It was also
thought that the effect of the bulky alkyl groups on the rate
of hydrolysis could provide insight to the structure of the
transition state since the bulkier groups can be expected to
better define the conformational requirements for the inter-
unit C-5a to C-1b linkages.
Fig. 6) and are compared against reference compound
methyl α-isomaltoside (1). It is seen that the different
alkylating groups do not have a large effect on the orienta-
tion of the agluconic carbon (C-6a) (φ torsion angles range
from –51° to –54°). The conformational preferences for the
more rigid compounds (R-2m and R-2i) appear to be similar
but are significantly different as compared to the global min-
imum energy conformation of 1. This inference is supported
by the nuclear Overhauser enhancement (NOE) data pre-
sented in Table 3. As expected from the GEGOP calcula-
tions (Table 2), saturation of H-1b enhanced the signal for
H-6a almost equally for the three 6a-alkylated compounds.
The NOE data for the S-isomers are also recorded in Ta-
ble 3, but the S-isomers are not further discussed because of
their poor performance as substrates for AMG (10).
Prior to a consideration of conformers B, a brief review,
for the more salient features of the mechanism for the hydro-
lysis of an α-D-glucopyranoside by AMG, is useful. An out-
line is presented in Fig. 2.
The mechanism (Fig. 2) has been established as an acid-
base catalyzed reaction. The X-ray crystal structure of the
acarbose–AMG complex (7, 8) displays a well situated “cat-
alytic” water molecule for nucleophilic attack at the
anomeric centre of the b-unit which results in the displace-
ment of the a-unit (see Fig. 3). The nucleophilicity of this
water molecule is enhanced by the involvement of both of its
hydrogen atoms in hydrogen bonds with two oxygen atoms
Discussion
Theoretical discussion
GEGOP calculations (11) were used to estimate the pre-
ferred conformations for compounds 1, R-2m, and R-2i. The
results are presented in Table 2 as conformers A (see also
© 2001 NRC Canada

Spohr et al.
241
Table 3. NOE of methyl 6-C-alkyl-α-isomaltosides (2m, 2e, and 2i).
Compound
6a-C-Substituent
Proton saturated
Proton enhancement (%)
H-1b
H-2b
H-6aR
H-6aS
R-2m
R-2m
R-2e
R-2i
S-2m
S-2e
S-2i
CH3-
CH3-
H-1b
H-6a
H-1b
H-1b
H-1b
H-1b
H-1b
—
10.8
—
—
—
15.2
—
13.2
15.5
9.5
—
—
—
—
2.4
7.6
6.7
9.9
—
8.7
13.3a
—
CH3H2-
(CH3)2CH-
CH3-
CH3H2-
(CH3)2CH-
—
—
17.1
17.7
—
—
aSignal may contain small contributions from H-4, H-3 signals.
Fig. 2. Schematic of key features in the acid-base catalyzed hydrolysis of an α-D-glucopyranoside by the enzyme amyloglucosidase
(AMG). The structural features of 1 that are involved in the formation of the complex are not shown.
The conformation of the valienamine moiety of acarbose
(Fig. 1), as previously pointed out (6), provides an accept-
able working model for the construction of the transition
state where the C-5b, O-5b, C-1b, and C-2b atoms of the b-
unit are coplanar and the oxygen of the catalytic water mole-
cule, C-1b and the leaving O-6a atoms are colinear. Evi-
dence in support of the structure of the transition state for
inversions at anomeric centres was obtained in 1955 (14).
The X-ray structure also can serve as a model for the struc-
ture of the complex displayed in Fig. 2. Although the mole
fraction of the half-chair conformation for the b-unit of 1 is
undoubtedly very small, its formation once 1 is complexed
with AMG must be virtually spontaneous, especially since
the active site conforms for the acceptance of acarbose. The
B conformers listed in Table 2 are based on this consider-
ation. Accordingly, it is expected that formation of the half-
chair conformer will substantially weaken the endo- and
exo-anomeric effects (15) and, thereby, render the anomeric
centre more susceptible to nucleophilic attack.
(see Fig. 3), one with the carboxylate group of Glu 400 and
the other with OH-6b. The OH-6b is also hydrogen bonded
to the carboxylate of Asp 55 (7, 8). Experimental evidence
in support of this type of delocalization of electron density
in conjugated hydrogen bonds was established by Lemieux
and Pavia (12) and now termed the “cooperativity effect”
(13). These and other interactions expected to be present in
the transition state were reviewed in a recent publication (6).
The acid catalysis is promoted by proton donation from Glu
179 (Fig. 3), where the acidity of the carboxyl group of Glu
179 is expected to be enhanced by electron donation to a
proton on Glu 124 (not shown in Fig. 3). It is to be noted
(Table 1) that deoxygenation of OH-2b caused little change
in the kinetic parameters. This suggestion that OH-2b is not
involved in assisting the leaving of the a-unit must take into
consideration that 2-deoxyglycosides are much more prone
to hydrolysis when the 2-substituent is more electronegative,
such as a hydroxyl group. Also, in the case of the 2b-deoxy
compound, a water molecule may assist in relaying the
electrophilicity of the Arg 305 to the 6a-oxygen atom (6).
Certainly, a hydrogen bond between OH-2b and the leaving
group can be expected to strengthen as the C-1b—O-6a
bond is stretched to achieve the transition state.
As already mentioned, hydrogen bonding of OH-4a to Trp
178 (assisted by Arg 305) greatly enhances the rate of catalysis.
Our attempts to establish these hydrogen bonds using both
4
the a- and b-units in the C1 chair conformation proved
© 2001 NRC Canada

242
Can. J. Chem. Vol. 79, 2001
unattractive. However, when the b-unit was placed in the
half-chair conformation (see Table 2 and Figs. 3 and 6), the
same hydrogen-bond networks presented in the acarbose–
AMG complex (7, 8) are well established.
Prior to a consideration of hydrophobic forces involved in
the binding of 1 by AMG, it is of interest to examine the
thermodynamics of the binding of the H-type 2-OMe tri-
saccharide by the lectin, Ulex europaeus (UE-1) (18, 19).
The main driving force for binding is enthalpic, ∆H =
–29 kcal mol–1, which is countered by a large decrease in
entropy, T∆S = –20.5 kcal mol–1 (18). Inspection of the X-ray
crystal structure for the UE-I–H-type 2-OMe complex re-
veals, as seen in Fig. 5, that two hydrophobic interactions
are present: one involves Tyr 220 with the C-6 methylene
group of the GlcNAc unit and the other involves valine 134
with the C-6 methyl group of the α-L-Fuc unit. These inter-
actions, if truly hydrophobic in character, are obviously not
compatible with the aforesaid thermodynamic parameters
that show a decrease in entropy rather than an increase in
entropy as expected for hydrophobic association. The prox-
imity of these groups to the trisaccharide requires the dis-
placement of water. Judging from the thoroughly
polyamphiphilic surface (20) of the H-type 2-OMe molecule
(19), perturbed water molecules must have been displaced,
as in the case of Tyr 220. The interaction of Val 134 with the
C-6-methyl group of the fucosyl group may result in an in-
crease in entropy from the disordering of water molecules in
an endothermic process. However, these contributions, if
present, are strongly obscured by the large decreases in both
enthalpy and entropy that are expected to arise mainly from
the return of perturbed water molecules from both the com-
bining site and the epitope of the trisaccharide to bulk (21,
22). This process has been termed the hydraphobic effect.
The binding of 1 with AMG is similar in that along with
the establishment of numerous attractive polar interactions
one hydrophobic group comes into close contact with
disaccharide 1. As seen in Fig. 4, the only nonpolar contact
of less than 2.5 Å (omitting the interactions with the R-6a-
methyl and R-6a-isopropyl groups) is between Trp 317 and
H-2b. The presence of this large aromatic amino acid unit in
the epitope is expected to contribute to the stability of the
complex, since, along with the release of the ordered water
molecules at its surface, it is also expected to participate in
the release of perturbed water molecules from the
polyamphiphilic surface of the substrate (1). This is clearly
evident from the CPK molecular models displayed in Fig. 6.
The thermodynamic parameters for the hydrolysis of 1
catalyzed by AMG are not known. However, in view of the
extensive polyamphiphilic surface of 1 and that of the com-
plementary combining site, it is expected that complexation
involves the release of a large number of perturbed water
molecules (21). The decrease in enthalpy that is expected to
result is amplified by the formation of nine intermolecular
hydrogen bonds (see Fig. 3). In view of this large polar con-
tribution and the paucity of hydrophobic interactions, the
formation of the complex must result in a substantial (per-
haps as great as 60 kcal mol–1) decrease in enthalpy. As will
be discussed below, the liberation of this heat is expected, as
previously discussed (21), to participate in promoting the
complex to the transition state (6) (see Fig. 2).
As seen in Table 2 the φ, ψ, and ω torsion angles for the
b-unit are the same for each compound. However, large
differences in conformational energy are indicated and,
therefore, complex formation involving substrate R-2m
should be slightly favored over complexation with 1 and
much more than with substrate R-2i. As seen in Table 1, the
kinetically determined Km values are 20 (1), 0.7 (R-2m), and
47 (R-2i). The increase in complexation of AMG with R-2m
as compared to AMG with 1 can be attributed to hydropho-
bic association of the 6a-C-methyl group with both Trp 120
and Trp 52, as shown in Fig. 4. This hydrophobic associa-
tion is not present during formation of the 1–AMG complex.
Also, the conformational energy of R-2m in the complex is
expected (Table 2) to be about 0.9 kcal mol–1 less than that
for substrate 1. These considerations support an increase in
the rate of hydrolysis of R-2m (10). However, this is not the
case for R-2i. As seen in Table 1, the equilibrium for com-
plex formation (Km) for R-2i is much less favorable than it is
for R-2m (Km = 0.71 for R-2m vs. 47 for R-2i). This differ-
ence is mainly due to the large conformational strain re-
quired to form the R-2i–AMG complex (Table 2). This
resistance to complex formation is undoubtedly strongly
moderated by important hydrophobic associations of the two
methyl groups of the isopropyl group as is illustrated in
Fig. 4b. Frandsen et al. (10) employed, in part, this syntheti-
cally imposed hydrophobic association in a study of site-
specific mutagenic changes in the structure of AMG to con-
clude that “the versatility of AMG catalysis….emphasizes
the significance of hydrophobic interactions in protein-
carbohydrate complexation.” We suggest that this is not nec-
essarily the case with respect to the classical definition of
the driving force for hydrophobic associations which in-
volves the release of ordered water molecules to bulk. The
strength of hydrophobic interactions was recently observed
in the binding of N-acetyllactosamine by the lectin,
Erythrina corallodendron (16). The X-ray crystal structure
of the complex shows a strong association of the H-2, H-4,
and H-5 on the α-side of the β-D-galactose unit with the ap-
propriately positioned phenylalanine group. In this regard,
although Monte Carlo simulations (17) for the hydration
of the β-D-Gal unit of the Lewis b-OMe tetrasaccharide
suggest that the formation of a shell of disordered perturbed
water molecules extends over the hydrophobic α-side, some
ordering of the water molecules cannot be discounted. In
either event, the association of complementary surfaces
should be energetically favorable in aqueous solution
whether ordered or perturbed water molecules are released
to bulk.
As seen in Table 2, the b-unit of the B conformers are
highly strained. We suggest that the relaxation of this
conformational strain energy provides the necessary assis-
tance for the stretching of the C-1b—C-6a glycosidic bond
and thereby promotes decomposition to the products. This
structural feature is key for the catalysis of the hydrogen
bonds of OH-4a to Trp 178 and Arg 305. Indeed, as previ-
ously noted (10), the kcat values for 1, R-2m, R-2e, and R-2i
are remarkably similar.
However, Sigurskjold et al. (23) have determined that the
binding of the inhibitor acarbose by AMG is driven by both
enthalpic (∆H = –9.7 kcal mol–1) and entropic (T∆S =
6.6 kcal mol–1) values. The increase in entropy is expected
to arise from an important hydrophobic contribution. This is
© 2001 NRC Canada

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243
Fig. 3. A display of the intermolecular hydrogen bonds that are formed during the binding of 1 in the half-chair conformation and C-
6a-alkyl derivatives (R = H, Me, Et, or i-Pr) into the site occupied by the c- and d-units of acarbose.
Fig. 4. Display of the near van der Waals’ contacts of hydrophobic groups of AMG with nonpolar hydrogen atoms of 6a-alkyl deriva-
tives of 1. The hydrophobic associations of the methyl group of R-2m and the isopropyl group of R-2i are not present in the complex
formed with 1. Consequently, it appears that the only van der Waals contact with 1 involves a hydrogen of Trp 317 that is 2.4 Å away
from H-2b.
not surprising since the terminal unit of the pseudo-tetra-
saccharide (Fig. 1) is a derivative of cyclohexene, which
along with the C-6a-methyl group of the neighboring 6-
deoxy glucose the cyclohexene like unit offers a substantial
hydrophobic surface to the aqueous phase (see Fig. 1). The
ordered water molecules, along with disordered water mole-
cules hydrogen bonded to the binding site, must be displaced
during the binding process. Evidently, the increase in en-
tropy caused by the hydrophobic component dominates the
decrease in entropy expected from hydrophobic contributions.
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Can. J. Chem. Vol. 79, 2001
Fig. 5. The structures of the complex of the lectin Ulex europaeus (UE-I) and the H-type 2–OMe trisaccharide (α-L-Fuc(1→2)β-D-
Gal(1→4)β-D-GlcNAc-OMe) obtained by docking into the combining site of UE-I, which accepts (R)-2-methyl-2,4-pentanediol (L.T.J.
Delbaere and M. Vandonselaar, private communication). Structure A identifies the key intermolecular hydrogen bonds that are required
to established the formation of the complex. Structure B identifies the near van der Waals’ contacts established between Tyr 220 and
the β-D-GlcNAc and β-D-Gal units and of Val 134 with the α-L-Fuc unit.
Fig. 6. CPK models based in GEGOP calculations that display the polyamphiphilic surface of substrates 1, R-2m, and R-2i: (a) the
three preferred conformations in aqueous solution. The pro-R hydrogen of 1 is presented in yellow as are the methyl groups of R-2m
and R-2i. Introduction of the alkyl groups at position 6a results in a similar conformation which is different than that of 1; (b) CPK
models representation of the preferred conformation with the b-unit in the half-chair conformation with all R-6a substituents pointing
in the same direction. These conformers allow hydrogen bonding between OH-4a and both Trp 178 and Arg 305.
© 2001 NRC Canada

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245
Fig. 7. This graphic is of AMG looking down to the combining site that is occupied by R-2m (white–hydrogens, green–carbons, and
red–oxygen atoms). It is seen that a channel exists for water molecules to access the OH-3a region of the active site. The 6a-methyl
group, which remains in the aqueous phase, is visible just below the methoxy group. The yellow atoms belong to aromatic side chains
of amino acids that surround the active site, as shown in Fig. 8.
Fig. 8. The aromatic shield that protects the polar interactions (Fig. 2) in the complex from being disrupted by water: (a) is a close-up
(amino acids are removed for clarity); (b) is a side view of the shield of largely nonpolar aromatic groups that is expected to provide
thermal insulation of the heat of dehydration to assist achievement of the transition state. The slit in the shield through which a posi-
tion of the substrate can be viewed does not exist but is occupied by other nonaromatic amino acid residues. The apparent close-
packing is not real. However, their proximities are sufficient to provide effective protection of the polar interaction of the complex.
The heat liberated from the latter effect is large enough to
cancel the endothermicity of the hydrophobic contribution
by 9.7 kcal mol–1 and, consequently, the strong exothermi-
city of the binding of 1 by AMG is supported. Recall that
this is the source of heat required to activate the complex to
the transition state.
In 1990, Delbaere et al. (24) noted that for the Lewis
b–Griffonia simplicifolia complex many of the amino acids,
except for the diglycine bridge, near the periphery of the po-
lar receptor site have aromatic side chains. These structural
features were also observed for antibodies (25) and appear to
be necessary for the combining site of all carbohydrate-binding
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246
Can. J. Chem. Vol. 79, 2001
Scheme 1.
proteins. In general, carbohydrates are held in the cavity
mainly by attractive polar interactions in the form of
intermolecular hydrogen bonds. Hydrophobic associations
are present and important, especially in the case of antibodies
(25), but are of minor importance for the binding of oligosac-
charides by many lectins. In this regard, the driving force for
association is provided by a strong decrease in enthalpy and
a slight decrease in entropy. Clearly, binding occurs in cavi-
ties in order to avoid disrupting the association of polar
bonds with the bulk water. We suggest that the purpose of
the largely aromatic coating surrounding of the polar com-
plex is to provide a strong shield from the bulk water. Not
surprisingly, therefore, the active site of the enzyme AMG
that binds isomaltose is surrounded by amino acids that con-
tain aromatic side chains (see Fig. 7). These large hydropho-
bic groups are expected to act as a shield and thus, at high
concentrations, prevent water from seeping into the complex.
It has been proposed that lower dielectric constants in the bind-
ing in a cavity favor the formation of the polar bonds. How-
ever, this stabilization of polar bonds must also apply to the
water molecules that are displaced by the carbohydrate.
Finally, we propose that the shield of hydrophobic aro-
matic groups that surround the catalytic site of AMG (see
Fig. 8) also serves as thermal insulation to lower the rate of
the dissipation of the heat of complex formation (the hydra-
phobic effect) to bulk. This energy is expected to be the
main source of activation for the 1–AMG complex to the
transition state (Fig. 2). This concept of heat being liberated
from reacting molecules and being maintained in a suitable
submicro-environment to eventually serve as the activation
energy for another reaction may be a general feature of en-
zyme catalysis. This proposal is similar to the energy dissipa-
tion in enzyme catalysis proposed by Cramer and Freist (26).
Grignard reagents in the usual fashion to yield mixtures of
the R- and S-epimers, namely, the 6-C-methyl, 6-C-ethyl,
and 6-C-isopropyl glucosides, (4m, 4e, and 4i). In agree-
ment with Cram’s rule (29), the S-isomers were produced in
excess. The ratio of the S- to R-epimers was about 10:1 for
4m and 4e and 20:1 for 4i.
In the case of the methyl Grignard, procedures to prepare
R,S-4m, involved converting the crude product into a mix-
ture of 6-O-(3,5-dinitrobenzoyl) esters from which the S-iso-
mer crystallized and was purified by recrystallization.
Zemplen methanolysis then provided the pure S-4m. The
crude products obtained from the ethyl and isopropyl Grig-
nard reagents could be separated by column chromatography
into the pure R- and S-isomers. As will be described below
(Schemes 2 and 3) larger amounts of the R-isomers, in each
case, were obtained by inversion of the C-6 chiral centres of
the S-compounds.
Both the R- and S-epimers of 4m, 4e, and 4i were debenzyl-
ated by catalytic hydrogenolysis to obtain pure preparations
of both the R- and S-C-6-epimers, 5m, 5e, and 5i (Scheme 1).
For the configurational assignments, the S-isomers, (S-
5m, S-5e, and S-5i), were converted into the 4,6-O-
benzylidene derivatives (S-6m, S-6e, and S-6i) by reaction
with α,α-dibromo- or α,α-dichlorotoluene in pyridine at ele-
vated temperature followed by acylation. The absolute con-
figuration of compound S-6m was unequivocally established
by the NOE’s obtained by saturation of the axial C-6 methyl
group (see Table 4). The configurations of compounds S-6e
and S-6i are assigned on the basis that the J5,6 coupling con-
stants (reported in Table 4). The coupling constant of 6.0 Hz
is too small for coupling of antiperiplanar hydrogen atoms.
Furthermore, the value of J5,6 of S-6m, a compound of
known configuration, is essentially the same as those for
S-6e and S-6i, indicates that all three compounds are con-
figurationally related. It is also relevant that these three S-
compounds were the major products of the Grignard reac-
tions and, therefore, are surely configurationally related.
For the preparation of larger amounts of R-4m and R-4e
(Scheme 2), mesylated S-7m and S-7e were prepared and
reacted with sodium benzoate in N,N-dimethylformamide at
Synthesis
The syntheses of the 6-C-alkyl glucosides 4m, 4e, and 4i
(Scheme 1) started from the known methyl 6-aldehydo-α-D-
glucopyranoside (3) (27) that was prepared by oxidation of
methyl 2,3,4-tri-O-benzyl-α-D-glucopyranoside (28). Mole-
cule 3 was then reacted with the methyl, ethyl and isopropyl
© 2001 NRC Canada

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247
Scheme 2.
Scheme 3.
1
methylthiomethylene ether 11 (3:2). The borohydride reduc-
tion of 10 provided an epimeric mixture of R-4i and S-4i
(5:1).
Table 4. H NMR chemical shifts (ppm) and coupling constants
(Hz) in CDCl3 for the benzylidene derivatives S-6ma, S-6e, and S-6i.
S-6m
S-6e
S-6i
Preparation of the α-linked disaccharides R-2m, R-2e, and
R-2i is outlined in Scheme 4. Bromide-ion catalyzed
glycosylation conditions using 6-O-acetyl-2,3,4-tri-O-
benzyl-α-D-glucopyranosyl bromide (12), tetraethylammonium
bromide (32) and alcohol R-4m produced R-14m. Coupling
of tetra-O-benzyl-α-D-glucopyranosyl bromide (13) with al-
cohols R-4e and R-4i was achieved in the presence of the
cupric bromide–N,N-dimethylformamide complex (33). The
protecting groups were removed in the usual manner to yield
the desired compounds. The C-6a epimeric disaccharides
S-2m, S-2e, and S-2i were prepared by similar procedures.
H-1 (J1,2
H-2 (J2,3
H-3 (J3,4
H-4 (J4,5
H-5 (J5,6
)
)
)
)
)
4.93 (3.5)
5.15 (3.5)
5.16 (3.5)
5.21 (9.5)
6.06 (9.5)
4.21 (10.0)
4.43 (5.5)
3.88 (10.0)
5.72
4.87 (10.0) 5.19 (9.5)
5.57 (9.0) 6.04 (9.5)
3.91 (10.0) 4.15 (10.0)
4.15 (6.0)
4.50 (7.0)
5.80
4.33 (6.0)
4.25 (3.5,11.0)
5.78
H-6 (J6,CH
CHPh
)
1,2,3
CH3O
3.41
3.43
3.43
1
CH3
1.48
1.13 (7.5)
2.18, 1.79
1.15, 1.13 (6.5)
2.62
The H NMR data for the disaccharides are presented in
1
CH(1,2)
—
Table 5. The large H coupling constants for J2,3, J3,4, and
J4,5 confirm the 4C1-conformations of the pyranose rings.
The J5,6 coupling constants are ≤2.5 Hz. This suggests that a
positive synclinal (+g) relationship exists between H-5a and
H-6a, which is in agreement with the calculated ω angles for
the two conformers A and B presented in Table 2.
aNOE experiments performed by irradiation of the CH3-C6 doublet at
1.48 ppm caused enhancements of the H-6, H-4, and CHPh signals by
6.2%, 2.9%, and 4.1%, respectively.
120°C followed by saponification of the products.
Dehydromesylation of S-7e also resulted in compound 9. Its
E-configuration was assigned by the large J5,6 coupling con-
stant (14.5 Hz) (30).
The inversion of configuration at C-6 of S-4i (Scheme 3)
to obtain R-4i was achieved by reduction of ketone 10 with
sodium borohydride. Ketone 10 was prepared by oxidation
of S-4i with dimethylsulfoxide and acetic anhydride (31).
The crude product was mixture of ketone 10 and
Experimental
General methods
1
The H NMR spectra were measured at 300 MHz and
360 MHz (Bruker AM 300 and WM 360) with
tetramethylsilane as internal standard for CDCl3 solutions.
Reference standard for D2O solutions was acetone
(2.23 ppm). The 13C NMR spectra were recorded at 75 MHz
© 2001 NRC Canada

248
Can. J. Chem. Vol. 79, 2001
Scheme 4.
1
Table 5. H NMR chemical shifts (ppm) and coupling constants (Hz) for methyl 6-C-methyl, -ethyl-, and -isopropyl-α-isomaltosidesa.
(6R)-C-alkyl-isomaltosides
(6S)-C-alkyl-isomaltosides
R-2m
R-2e
R-2i
S-2m
S-2e
S-2i
a-unit
H-1 (J1,2
H-2 (J2,3
H-3 (J3,4
H-4 (J4,5
H-5 (J5,6
)
)
)
)
)
4.80 (4.0)
3.57 (9.5)
3.67 (9.0)
3.39 (10.5)
3.83 (2.5)
4.17
4.78 (3.5)
3.56 (9.5)
3.66 (9.0)
3.47 (9.5)
3.89 (1.5)
3.94 (~5)
1.71
4.79 (3.5)
3.58
4.85 (4.0)
n.a.
4.84 (3.5)
3.55 (9.5)
3.63
4.81 (3.5)
3.51
3.65
n.a.
3.63 (9.5)
3.53 (9.5)
3.90 (<1)
3.97 (5.5)
2.29
3.68
n.a.
3.57
3.92 (1.5)
3.67 (~6.5)
2.15
3.64
3.77 (<1)
3.98 (4.5, 9.5)
1.77
H-6 (J6,CH
CH(2)
)
4.28
—
—
CH3
1.29 (6.5)
—
1.02 (7.5)
—
1.025 (6.5)
1.018 (6.8)
3.43
1.28 (6.5)
—
0.94 (7.2)
—
1.04 (7.5)
1.03 (7.5)
3.51
CH3
CH3O
b-unit
3.44
3.44
3.43
3.45
H-1 (J1,2
H-2 (J2,3
H-3 (J3,4
H-4 (J4,5
H-5
)
)
)
)
5.07 (3.5)
3.55 (9.5)
3.73 (10.0)
3.41
5.12 (3.5)
3.57 (10.0)
3.74 (9.5)
3.42 (9.5)
3.82
5.20 (3.5)
3.56 (9.5)
3.74
5.09 (3.5)
3.56 (9.5)
3.69
5.12 (3.5)
3.54 (10.0)
3.70
5.17 (3.5)
3.57 (9.5)
3.77 (9.0)
3.46 (9.5)
3.74
3.41 (9.5)
3.83
3.43
3.44 (9.5)
3.73
n.a.
n.a.
H-6 (J5,6
)
3.88 (5.0)
3.76 (4.0)
3.85–3.78
n.a.
n.a.
n.a.
n.a.
3.83
H-6′ (J5,6′
)
n.a.
n.a.
n.a.
3.80
aSpectra recorded in D2O with acetone as internal reference set at 2.23 ppm at 360 MHz.
with dioxane (67.4 ppm) as reference for D2O solutions and
the CDCl3 signal (77.0 ppm) as reference in CDCl3 solu-
tions. Optical rotations were measured at room temperature
(23 ± 1°C) in a 1 dm cell on a Perkin–Elmer 241 polari-
meter. Thin-layer chromatography was performed on precoated
plates of silica gel (60-F254, E. Merck, Darmstadt) and visu-
alized by spraying with 5% sulfuric acid in ethanol followed
by heating. For column chromatography, silica gel 60 (230–
400 mesh, E. Merck, Darmstadt) and distilled solvents were
used. All solvents and reagents were purified and dried accord-
ing to standard procedures. Melting points are uncorrected.
The molecular modeling was carried out on an SGI Indigo
II. A suite of software from Molecular Simulations Inc., San
Diego, was used to build and minimize the (6R)-C-alkylated
glucopyranosyl units using AMBER forcefield. The coordi-
nates of these (6R)-C-alkylated glucopyranosyl units were
then exported to the GEGOP program4 (11) for use in grid
searchs, minimizations, and Monte-Carlo simulations.
4 R. Stuike-Prill and B. Meyer. Geometry of GlycOProteins. Version 2.6. 1992.
© 2001 NRC Canada

Spohr et al.
249
3.5 Hz, 1H, H-1), 4.05 (dq, J5,6 = 1.5 Hz, J6,CH3 = 6.5 Hz,
1H, H-6), 3.99 (dd, J3,4 = 9.0 Hz, J2,3 = 9.5 Hz, 1H, H-3),
3.62 (dd, J4,5 = 10.0 Hz, J3,4 = 9.0 Hz, 1H, H-4), 3.50 (dd,
Methyl 6-aldehydo-2,3,4-tri-O-benzyl-α-D-glucopyranoside (3)
Dried chromium trioxide (25.9 g, 259 mmol) was added to
a solution of pyridine (40.9 g, 517 mmol) in dichloromethane
(400 mL). The mixture was mechanically stirred at room
temperature for 30 min. A solution of methyl 2,3,4-tri-O-
benzyl-α-D-glucopyranoside (28) (10.0 g, 21.6 mmol) in di-
chloromethane (22 mL) was rapidly added and the resulting
mixture stirred at room temperature for 10 min. The reaction
mixture was then decanted and the black tarry precipitate
was extracted with ether several times. The combined or-
ganic solutions were filtered through a pad of silica gel-G to
provide a colorless slightly yellow oily residue which was
co-evaporated with toluene (2 × 200 mL) to remove residual
pyridine. The oily residue was dried under vacuum overnight
before use to give crude 3 (6.94 g, 70%). The relative inten-
sity of the singlet at 9.65 ppm (1H NMR) attributed to H-6
indicated an about 90% content of 3. [α]D + 8.2° (c 1.0,
chloroform), lit. (27) 13.6° (c 2.6, chloroform). IR (cm–1):
J2,3 = 9.5 Hz, J1,2 = 3.5 Hz, 1H, H-2), 3.42 (dd, J5,6
=
1.5 Hz, J4,5 = 10.0 Hz, 1H, H-5), 3.34 (s, 3H, CH3O), 1.25
(d, J6,7 = 6.5 Hz, 3H, CH3C-6). Anal. calcd. for C29H34O6:
C 72.78, H 7.16; found: C 72.94, H 6.82.
Methyl 2,3,4-tri-O-benzyl-(6S)-C-ethyl-α-D-glucopyrano-
side (S-4e) and -(6R)-C-ethyl-α-D-glucopyranoside (R- 4e)
A solution of crude methyl 6-aldehydo-2,3,4-tri-O-
benzyl-α-D-glucopyranoside (3) (1.6 g, 3.46 mmol) in
tetrahydrofuran (20 mL) was transferred slowly into a 0.5
N ethylmagnesium chloride solution in tetrahydrofuran
(21 mL, 10.1 mmol) with stirring. After 1 h, the reaction
was processed as described for S-4m. The crude material
was applied to a column of silica gel (hexane–ethyl acetate,
5:1, 4:1) to provide in the main fraction the title compound
S-4e (1.043 g, 61%) as a syrup. [α]D + 12° (c 1.4, chloro-
1
1741 (CHO). H NMR (CDCl3) δ: 9.65 (s, 1H, H-6), 7.40–
7.10 (m, 15H, 3Ph), 5.05–4.50 (m, 7H, 3CH2Ph, H-1), 4.17
1
form). H NMR (CDCl3) δ: 7.38–7.22 (m, 15H, 3Ph), 5.01–
(d, J4,5 = 10.0 Hz, 1H, H-5), 4.08 (dd, J3,4 = 9.0 Hz, J2,3
=
4.64 (3ABq, 6H, 3CH2Ph), 4.56 (d, J1,2 = 3.5 Hz, 1H, H-1),
3.99 (t, J2,3 ~ J3,4 = 9.0 Hz, 1H, H-3), 3.70 (m, H-6), 3.66
(t, J3,4 ~ J4,5 = 9.5 Hz, 1H, H-4), 3.53 (br d, J5,6 < 1 Hz, 1H,
H-5), 3.49 (dd, J1,2 = 3.5 Hz, J2,3 = 9.5 Hz, 1H, H-2), 3.35
(s, 3H, CH3O), 1.67 (m, 1H, CH2CH3), 1.50 (m, 1H,
CH2CH3), 0.95 (t, J = 7.5 Hz, 3H, CH3CH2). Anal. calcd.
for C30H36O6: C 73.15, H 7.37; found: C 73.26, H 7.32.
Continued development of the column eluted epimer
methyl 2,3,4-tri-O-benzyl-(6R)-C-ethyl-α-D-glucopyrano-
side (109 mg, 6.4%) as a crystallizing syrup. [α]D + 35° (c
9.5 Hz, 1H, H-3), 3.57 (dd, J3,4 = 9.0 Hz, J4,5 = 10.0 Hz, 1H,
H-4), 3.50 (dd, J1,2 = 3.5 Hz, J2,3 = 9.5 Hz, 1H, H-2), 3.38 (s,
3H, CH3O).
Methyl 2,3,4-tri-O-benzyl-(6S)-C-methyl-α-D-
glucopyranoside (S-4m)5
A solution of the crude aldehyde 3 (6.94 g, 15.0 mmol) in
ether (30 mL) was added dropwise to a 0.6 N methylmag-
nsesium iodide solution in ether (50 mL, 30 mmol) and the
reaction mixture was stirred at room temperature for 2 h.
The reaction was quenched by the dropwise addition of aq
sat. ammonium chloride. The mixture was diluted with wa-
ter, extracted with ether and the organic solution was dried
1
0.4, chloroform). H NMR (CDCl3) δ: 7.40–7.20 (m, 15H,
3Ph), 5.04–4.61 (3ABq, 6H, 3CH2Ph), 4.55 (d, J1,2
3.5 Hz, 1H, H-1), 4.02 (t, J2,3 ~ J3,4 = 9.0 Hz, 1H, H-3),
3.70–3.63 (m, 1H, H-5, H-6), 3.50 (dd, J1,2 = 3.5 Hz, J2,3
=
=
1
9.5 Hz, 1H, H-2), 3.49 (t, J3,4 ~ J4,5 = 9.0 Hz, 1H, H-4),
3.38 (s, 3H, CH3O), 1.54 (m, 1H, CH2CH3), 1.42 (m, 1H,
CH2CH3), 0.93 (t, J = 7.5 Hz, 3H, CH3CH2). Anal. calcd.
for C30H36O6: C 73.15, H 7.37; found: C 72.47, H 7.47.
and evaporated to provide a yellow oil (5.96 g). The H
NMR spectrum was consistent with the presence of the de-
sired compounds S-4m and R-4m in the ratio of about 10:1.
The crude mixture of S-4m and R-4m (0.59 g, 1.2 mmol)
was reacted in pyridine (10 mL) with 3,5-dinitrobenzoyl
chloride (0.51 g, 2.25 mmol) in the presence of a catalytic
amount of 4-dimethylaminopyridine (few milligrams). The
reaction mixture was stirred at room temperature until analy-
sis by TLC indicated the reaction was completed. The usual
work-up provided a yellow residue which crystallized spon-
taneously and was recrystallized from methanol. The com-
pound characterized as the 6-O-(3,5-dinitrobenzoyl)
derivative of S-4m, namely methyl 2,3,4-tri-O-benzyl-6-O-
(3,5-dinitrobenzoyl)-(6S)-C-methyl-α-D-glucopyranoside, was
obtained in 74% yield (0.612 g), mp 68–70°C. [α]D + 52.2°
(c 0.85, chloroform).
Methyl 2,3,4-tri-O-benzyl-(6S)-C-isopropyl-α-D-glucopy-
ranoside (S-4i) and -(6R)-C-isopropyl-α-D-glucopyranoside
(R-4i)
Crude aldehyde 3 (2.47 g, 5.34 mmol) in tetrahydro-
furan (30 mL) was reacted with isopropylmagnesium
chloride as described for the preparation of S-4e The
crude reaction product was purified on a column of silica
gel (hexane–ethyl acetate, 5:1 → 3:1) to provide in the
first fraction the minor epimer R-4i (73.8 mg, 2.7%) as a
slowly crystallizing syrup. [α]D + 26.4° (c 0.4, chloro-
form). 1H NMR (CDCl3) δ: 7.40–7.23 (m, 15H, 3Ph),
5.06–4.62 (3ABq, 6H, 3CH2Ph), 4.53 (d, J1,2 = 3.5 Hz,
1H, H-1), 4.03 (t, J2,3 ~ J3,4 = 9.5 Hz, 1H, H-3), 3.69 (dd,
The 6-O-(3,5-dinitrobenzoyl) derivative of S-4m (1.00 g,
1.49 mmol) was dissolved in dioxane (50 mL) followed by
the addition of 1 N sodium hydroxide solution (20 mL).
After stirring for 1 h, the mixture was diluted with dichloro-
methane and filtered over a pad of silica gel-G, the organic
solution was washed with water, dried and evaporated to
give S-4m (0.69 mg, 97%) as a solid, mp 82–84°C. [α]D +
J4,5 = 9.5 Hz, J5,6 = 6.0 Hz, 1H, H-5), 3.60 (t, J3,4 ~ J4,5
=
9.0 Hz, 1H, H-4), 3.53 (dd, J6,CH = 4.0 Hz, J5,6 = 6.0 Hz,
1H, H-6), 3.50 (dd, J1,2 = 3.6 Hz, J2,3 = 9.5 Hz, 1H, H-2),
3.40 (s, 3H, CH3O), 1.96 (m, 1H, CH(CH3)2), 0.95 (d, J =
7.0 Hz, 3H, CH(CH3)2), 0.89 (d, J = 7.0 Hz, 3H,
CH(CH3)2). Anal. calcd. for C31H38O6: C 73.49, H 7.56;
found: C 73.27, H 7.72. Continued development of the
1
26.9° (c 1.0, chloroform). H NMR (CDCl3) δ 7.40–7.20 (m,
15H, 3Ph), 5.01–4.63 (3ABq, 6H, 3CH2Ph), 4.58 (d, J1,2
=
5 Methyl 2,3,4-tri-O-benzyl-7-deoxy-β-L-glycero-D-gluco-heptopyranoside.
© 2001 NRC Canada

250
Can. J. Chem. Vol. 79, 2001
column eluted the major epimer S-4i (1.46 g, 54%) as a
syrup. [α]D + 12.1° (c 0.7, chloroform). H NMR (CDCl3)
δ: 7.40–7.23 (m, 15H, 3Ph), 5.00–4.64 (3ABq, 6H,
9.5 Hz, J5,6 < 1 Hz, 1H, H-6), 3.44 (s, 3H, CH3O), 1.92 (m,
1H, CH(CH3)2), 1.01 (d, J = 6.5 Hz, 3H, CH(CH3)2), 0.91
(d, J = 6.5 Hz, 3H, CH(CH3)2). 13C NMR (D2O) δ: 100.48
(C-1), 74.36, 74.14, 71.99, 71.13, 70.39, 56.50 (CH3O),
30.61 (CH(CH3)2), 19.84, 19.32 (CH3)2CH).
1
3CH2Ph), 4.55 (d, J1,2 = 3.5 Hz, 1H, H-1), 3.99 (tX, J2,3
=
9.5 Hz, J3,4 = 9.0 Hz, 1H, H-3), 3.75 (br d, J4,5 = 9.5 Hz,
J5,6 < 1 Hz, 1H, H-5), 3.67 (t, J3,4 ~ J4,5 = 9.0 Hz, 1H,
H-4), 3.48 (dd, J1,2 = 3.5 Hz, J2,3 = 9.5 Hz, 1H, H-2), 3.38
(s, 3H, CH3O), 3.33 (br d, J6,CH = 8.5 Hz, 1H, H-6), 1.83
(m, 1H, CH(CH3)2), 1.01 (d, J = 7.0 Hz, 3H, (CH3)2CH),
0.89 (d, J = 7.0 Hz, 3H, (CH3)2CH). Anal. calcd. for
C31H38O6: C 73.49, H 7.56; found: C 73.53, H 7.67.
Methyl (6R)-C-methyl-α-D-glucopyranoside (R-5m)
Compound R-5m was prepared from R-4m in quantitative
yield by catalytic hydrogenolysis following the general pro-
1
cedure; mp 135–136°C. [α]D + 148° (c 1.0, water). H NMR
(D2O) δ: 4.82 (d, J1,2 = 4.0 Hz, 1H, H-1), 4.20 (dq, J5,6
2.5 Hz, J6,CH3 = 6.0 Hz, 1H, H-6), 3.71 (dd, J4,5 = 10.0 Hz,
J5,6 = 2.5 Hz, 1H, H-5), 3.68 (dd, J3,4 = 9.0 Hz, J2,3
10.0 Hz, 1H, H-3), 3.58 (dd, J1,2 = 4.0 Hz, J2,3 = 10.0 Hz,
=
=
General procedure for the catalytic hydrogenolysis of
O-benzyl derivatives
1H, H-2), 3.44 (s, 3H, CH3O), 3.38 (dd, J3,4 = 9.0 Hz, J4,5
=
A mixture of the benzyl derivative (200 mg, 0.2–
0.4 mmol) and 5% palladium-on-carbon (200 mg) in metha-
nol (10–15 mL) was hydrogenated in the hydrogen stream
until completion (TLC). The catalyst was removed by filtra-
tion and the solvent evaporated. The resultant material was
applied to a column of Iatrobeads® (chloroform-methanol-
water) followed by gel filtration on a column of Sephadex
LH 20 (50% ethanol) to provide the deprotected material.
10.0 Hz, 1H, H-4), 1.25 (d, J = 6.5 Hz, 3H, CH3C-6). 13C
NMR (D2O) δ: 100.02 (C-1), 74.17, 74,12, 72.07, 71.73,
67.14, 55.76 (CH3O), 15.85 (CH3C-6). Anal. calcd. for
C8H16O6: C 46.15, H 7.75; found: C 45.99, H 7.50.
Methyl (6R)-C-ethyl-α-D-glucopyranoside (R-5e)
Compound R-5e was prepared from R-4e by catalytic
hydrogenolysis following the general procedure and
obtained as a foam after freeze-drying its aqueous solution
(87%). [α]D + 152° (c 0.4, water). 1H NMR (D2O) δ: 4.78 (d,
Methyl (6S)-C-methyl-α-D-glucopyranoside (S-5m)
Compound S-5m was prepared from S-4m in quantitative
yield by catalytic hydrogenolysis following the general pro-
cedure. The solid material could be recrystallized from 98%
J1,2 = 3.5 Hz, 1H, H-1), 3.78 (m, 1H, H-6), 3.69 (dd, J4,5
10.0 Hz, J5,6 = 1.5 Hz, 1H, H-5), 3.64 (dd, J3,4 = 8.5 Hz, J2,3
=
=
9.5 Hz, 1H, H-3), 3.54 (dd, J1,2 = 3.5 Hz, J2,3 = 9.5 Hz, 1H,
H-2), 3.43 (dd, J3,4 = 8.5 Hz, J4,5 = 10.0 Hz, 1H, H-4), 3.41
(s, 3H, CH3O), 1.64 (m, 1H, CH2CH3), 1.53 (m, 1H,
CH2CH3), 0.98 (t, J = 7.5 Hz, 3H, CH3CH2). 13C NMR
(D2O) δ: 100.00 (C-1), 74.26, 74.05, 73.39, 72.01, 71.55,
55.78 (CH3O), 23.94 (CH2CH3), 10.84 (CH3CH2).
ethanol/hexane to give needle like crystals; mp 136–137°C;
1
[α]D + 150° (c 0.2, water). H NMR (D2O) δ: 4.80 (d, J1,2
=
3.5 Hz, 1H, H-1), 4.16 (dq, J5,6 = 1.5 Hz, J6,CH3 = 6.5 Hz,
1H, H-6), 3.62 (dd, J2,3 = 10.0 Hz, J3,4 = 9.0 Hz, 1H, H-3),
3.55 (dd, J1,2 = 3.5 Hz, J2,3 = 10.0 Hz, 1H, H-2), 3.46 (dd,
J4,5 = 10.0 Hz, J3,4 = 9.0 Hz, 1H, H-4), 3.39 (s, 3H, CH3O),
3.38 (dd, J4,5 = 10.0 Hz, J5,6 = 1.5 Hz, 1H, H-5), 1.29 (d, J =
6.5 Hz, 3H, CH3C-6). 13C NMR (D2O) δ: 100.24 (C-1),
74.49, 74.41, 72.31, 70.74, 65.26, 55.89 (CH3O), 19.72
(CH3C-6). Anal. calcd. for C8H16O6: C 46.15, H 7.75; found:
C 46.04, H 7.46.
Methyl (6R)-C-isopropyl-α-D-glucopyranoside (R-5i)
Compound R-5i was prepared from R-4i by catalytic
hydrogenolysis following the general procedure and obtained
as a white solid (85%) after lyophilization of its aqueous so-
1
lution. [α]D + 123.4° (c 0.2, water). H NMR (D2O) δ: 4.78
(d, J1,2 = 3.5 Hz, 1H, H-1), 3.74 (dd, J4,5 = 9.5 Hz, J5,6
4.0 Hz, 1H, H-5), 3.67 (dd, J3,4 = 9.0 Hz, J2,3 = 9.5 Hz, 1H,
H-3), 3.58 (dd, J3,4 = 9.0 Hz, J4,5 = 9.5 Hz, 1H, H-4), 3.57
=
Methyl (6S)-C-ethyl-α-D-glucopyranoside (S-5e)
Compound S-5e was prepared from S-4e by catalytic
hydrogenolysis following the general procedure and ob-
tained as a white solid (89%) after lyophilization of the aq
(dd, J6,CH ~ 6.5 Hz, J = 40 Hz, 1H, H-6), 3.54 (dd, J1,2
=
1
3.5 Hz,J2,3 = 9.5 Hz, 1H, H-2), 3.42 (s, 3H, CH3O), 2.04
(m, 1H, CH(CH3)2), 0.96 (d, J = 6.5 Hz, 3H, CH(CH3)2),
0.95 (d, J = 6.5 Hz, 3H, CH(CH3)2). 13C NMR (D2O) δ:
100.10 (C-1), 78.63, 74.16, 72.33, 71.86, 71.49, 56.12
(CH3O), 30.11 (CH(CH3)2), 20.09, 17.96 ((CH3)2CH).
solution. [α]D + 150° (c 0.2, water). H NMR (D2O) δ: 4.80
(d, J1,2 = 3.5 Hz, 1Η, H-1), 3.85 (m, 1H, H-6), 3.64 (dd, J2,3
9.5 Hz, J3,4 = 9.0 Hz, 1H, H-3), 3.55 (dd, J1,2 = 3.5 Hz, J2,3
9.5 Hz, 1H, H-2), 3.53 – 3.50 (m, 2H, H-4, H-5), 3.41 (s,
3H, CH3O), 1.64 (m, 2H, CH2CH3), 0.95 (t, J = 7.5 Hz, 3H,
CH3CH2); 13C NMR (D2O) δ: 100.17 (C-1), 74.27, 72.60,
72.06, 70.47, 70.30, 55.87 (CH3O), 26.70 (CH3CH2), 10.67
(CH3CH2).
=
=
Methyl 2,3-di-O-acetyl-4,6-O-benzylidene-(6S)-C-methyl-
α-D-glucopyranoside (S-6m)
A stirred solution of S-5m (16 mg, 0.053 mmol) and α,α-
dibromotoluene (112 mg, 0.45 mmol) in pyridine (15 mL)
was heated at reflux for 2 h. The mixture was cooled and
then acetic anhydride (3 mL) was added. After 48 h at room
temperature, the mixture was poured into ice water followed
by extraction with chloroform, washing and drying of the or-
ganic solution and evaporation. The resultant product was
purified by thin layer chromatography on silica gel (hexane–
ethyl acetate, 3:2) to give S-6m (15 mg, 51%) as a solid, mp
Methyl (6S)-C-isopropyl-α-D-glucopyranoside (S-5i)
Compound S-5i was prepared from S-4i by catalytic
hydrogenolysis following the general procedure and ob-
tained as a white solid (95%) after lyophilization of its aq
1
solution. [α]D + 150° (c 0.3, water). H NMR (D2O) δ: 4.79
(d, J1,2 = 3.5 Hz, 1Η, H-1), 3.70 (dd, J4,5 = 9.5 Hz, J5,6
1 Hz, 1H, H-5), 3.64 (dd, J3,4 = 8.5 Hz, J2,3 = 9.5 Hz, 1H, H-
3), 3.56 (dd, J3,4 = 8.5 Hz, J4,5 = 9.5 Hz, 1H, H-4), 3.54 (dd,
<
1
J1,2 = 3.5 Hz, J2,3 = 9.5 Hz, 1H, H-2), 3.47 (dd, J6,CH
=
132–134°C. [α]D + 50.2° (c 0.3, chloroform). The H NMR
© 2001 NRC Canada

Spohr et al.
251
data are reported in Table 4. 13C NMR (CDCl3) δ: 170.48,
169.76 (2CO), 137.40, 128.92, 128.22, 126.24 (Ph), 97.59
(C-1), 94.07 (CHPh), 72.74, 71.50, 70.49, 69.66 (C-2, C-3,
C-4, C-5), 64.45 (C-6), 55.34 (CH3O), 20.79, 20.65
(2CH3CO), 11.28 (CH3C-6). Anal. calcd. for C19H24O8: C
59.99, H 6.36; found: C 59.55, H 6.06.
5.12 (br t, J6,CH = 7.5 Hz, 1H, H-6), 5.06–4.63 (3ABq, 6H,
2
3CH2Ph), 4.62 (d, J1,2 = 3.5 Hz, 1H, H-1), 4.02 (t, J2,3 ~ J3,4
=
9.5 Hz, 1H, H-3), 3.76 (br d, J4,5 = 9.5 Hz, 1H, H-5), 3.64
(t, J3,4 ~ J4,5 = 9.5 Hz, 1H, H-4), 3.55 (dd, J1,2 = 3.5 Hz,
J2,3 = 9.5 Hz, 1H, H-2), 3.38 (s, 3H, CH3O), 3.07 (s, 3H,
CH3SO2), 1.94 (m, 2H, CH2CH3), 0.99 (t, J = 7.5 Hz, 3H,
CH3CH2). Anal. calcd. for C31H38O8S: C 65.24, H 6.71; found:
C 64.86, H 7.03.
Methyl 2,3-di-O-benzoy-4,6-O-benzylidene-(6S)-C-ethyl-
α-D-glucopyranoside (S-6e)
A solution of S-5e (49 mg, 0.22 mmol) and α,α-
dichlorotoluene (100 µL, 0.78 mmol) in pyridine (7 mL) was
heated at reflux for 4 h. Then more reagent (75 µL,
0.58 mmol) was added and heating was continued for 4.5 h.
The mixture was cooled to ice bath temperature and benzoyl
chloride (100 µL, 0.86 mmol) was added. After 2 h at this
temperature, the mixture was poured into ice water, ex-
tracted with dichloromethane, followed by washing of the
organic solution with water, drying and evaporation. The re-
sultant material was purified on a column of silica gel (hex-
ane-ethyl acetate, 9:1, 0.05% triethylamine) to provide
labile, solid S-6e (56 mg, 49%) which was not analytically
Methyl 2,3,4-tri-O-benzyl-(6R)-C-methyl-α-D-glucopy-
ranoside (R-4m)
A stirring mixture of mesylate S-7m (600 mg, 1.08 mmol)
and sodium benzoate (1.0 g, 7.0 mmol) in N,N-dimethyl-
formamide (20 mL) was heated at 120° for 17 h. It was di-
luted with dichloromethane, filtered and the solution washed
with water, dried and evaporated. The resultant material was
purified by HPLC (hexane–acetone, 10:1) to give methyl 6-
O-benzoyl-2,3,4-tri-O-benzyl-(6R)-C-methyl-α-D-glucopyranoside
(R-8m) (294 mg, 47%) as a syrup. [α]D + 9.8° (c 1.04, chlo-
1
roform). H NMR (CDCl3) δ: 8.40–7.20 (m, 20H, 3Ph, Bz),
5.47 (dq, J5,6 = 2.0 Hz, J6,CH = 7.0 Hz, 1H, H-6), 5.03–4.65
(m, 6H, 3CH2Ph), 4.64 (d, J13,2 = 4.0 Hz, 1H, H-1), 4.04 (dd,
1
pure and was only characterized by H NMR spectroscopy.
1
The H NMR data are reported in Table 5.
J2,3 = 9.5 Hz, J3,4 = 9.0 Hz, 1H, H-3), 3.93 (dd, J4,5
10.0 Hz, J5,6 = 2.0 Hz, 1H, H-5), 3.52 (dd, J1,2 = 4.0 Hz,
J2,3 = 9.5 Hz, 1H, H-2), 3.42 (dd, J3,4 = 9.0 Hz, J4,5
10.0 Hz, 1H, H-4), 3.38 (s, 3H, CH3O), 1.18 (d, J = 7.0 Hz,
3H, CH3C-6). Anal. calcd. for C36H38O7: C 74.20, H 6.57;
found: C 74.33, H 6.55.
Benzoate R-8m (137 mg, 0.24 mmol) was dissolved in
dioxane (10 mL) and 1 N sodium hydroxide solution (6 mL)
was added. After stirring for 48 h at 60°C, the mixture was
diluted with dichloromethane and filtered over a pad of silica
gel-G. The dichloromethane solution was washed with wa-
ter, dried and evaporated to give solidifying R-4m (80 mg,
71%), mp 89–90°C. [α]D + 28° (c 0.4, chloroform). 1H NMR
(CDCl3) δ: 7.34–7.16 (m, 15H, 3Ph), 4.98–4.53 (m, 6H,
=
=
Methyl 2,3-di-O-benzoyl-4,6-O-benzylidene-(6S)-C-
isopropylidene-α-D-glucopyranoside (S-6i)
S-5i (37 mg, 0.16 mmol) was converted into S-6i (41 mg,
49%) as described for the preparation of S-6e. Labile S-6i
contained a minor component (13%) that could not be re-
1
moved. The H NMR data are reported in Table 4.
Methyl 2,3,4-tri-O-benzyl-6-O-methanesulfonyl-(6S)-C-
methyl-α-D-glucopyranoside (S-7m)
To a solution of S-4m (135 mg, 0.28 mmol) in pyridine
(5 mL) at 0°C was added methanesulfonyl chloride (87 mg,
0.76 mmol). After 2 h at room temperature, the mixture was
poured into ice-water, extracted with dichloromethane and
the organic solution washed with saturated aqueous sodium
bicarbonate and water, followed by drying and evaporation.
Purification of the resultant material on a column of silica
gel (toluene–acetone, 6:1) provided mesylate S-7m (104 mg,
3CH2Ph), 4.50 (d, J1,2 = 3.5 Hz, 1H, H-1), 3.95 (dd, J2,3
9.5 Hz, J3,4 = 9.0 Hz, 1H, H-3), 3.88 (m, 1H, H-6), 3.55 (dd,
J4,5 = 10.0 Hz, J5,6 = 4.5 Hz, 1H, H-5), 3.43 (dd, J1,2
3.5 Hz, J2,3 = 9.5 Hz, 1H, H-2), 3.35 (dd, J3,4 = 9.0 Hz, J4,5
10.0 Hz, 1H, H-4), 3.31 (s, 3H, CH3O), 2.49 (d, J6,OH
=
=
=
=
1
6.0 Hz, 1H, OH), 1.05 (d, J6,CH = 6.5 Hz, 3H, CH3C-6).
66%) as a syrup. [α]D + 9.3° (c 1.2, chloroform). H NMR
3
Anal. calcd. for C29H34O6: C 72.78, H 7.16; found: C 72.81,
H 7.07.
(CDCl ) δ: 7.40–7.25 (m, 15H, 3Ph), 5.32 (dq, J6,CH
=
6.5 Hz3, J6,5 = 1.0 Hz, 1H, H-6), 5.06–4.62 (m, 7H, 3CH2Ph,
H-1), 4.02 (m, 1H, H-5), 3.65–3.53 (m, 3H, H-3, H-4, H-2),
3.36 (s, 3H, CH3O), 3.04 (s, 3H, CH3SO2), 1.53 (d, J =
XX Hz, 3H, CH3C-6). 13C NMR (CDCl3) δ: 138.54–127.74
(Ph), 98.26 (C-1), 82.30, 79.71, 77.17, 73.55, 72.38 (C-2, C-
3, C-4, C-5, C-6), 75.74, 75.31, 75.00 (3CH2Ph), 55.33
(CH3O), 39.54 (CH3SO2-), 17.73 (CH3C-6). Anal. calcd. for
C30H36O8S: C 64.73, H 6.52, S 5.76; found: C 64.85, H
6.55, S 5.52.
3
Methyl 2,3,4-tri-O-benzyl-(6R)-C-ethyl-α-D-glucopy-
ranoside (R-4e)
A stirring mixture of mesylate S-7e (430 mg, 0.75 mmol)
and sodium benzoate (600 mg, 4.16 mmol) in N,N-dimethyl-
formamide (20 mL) was heated at 120° for 20 h. The solu-
tion was diluted with dichloromethane, filtered and the
solution washed with water, dried and evaporated. The resul-
tant material (methyl 6-O-benzoyl-2,3,4-tri-O-benzyl-(6R)-
C-ethyl-α-D-glucopyranoside (R-8e)) and potassium hydrox-
ide (2.5 g) in 90% ethanol (50 mL) was kept at reflux for
3 h. The mixture was neutralized with acetic acid, evapo-
rated, and the remainder taken up in dichloromethane.
Washing of the solution with water, drying and evaporation
was followed by column chromatography of the crude prod-
uct on a column of silica gel (hexane–ethyl acetate, 4:1 →
3:1). Evaporation of the first fraction provided (E)-methyl
Methyl 2,3,4-tri-O-benzyl-(6S)-C-ethyl-6-O-methanesul-
fonyl-α-D-glucopyranoside (S-7e)
S-4e (419 mg, 0.85 mmol) was reacted with methane-
sulfonyl chloride (100 µL, 1.29 mmol) as described for the
preparation of S-7m. Purification of the resultant material on
a column of silica gel (toluene–ethyl acetate, 10:1) provided
mesylate S-7e (439 mg, 90%) as a syrup. [α]D – 15.2° (c 0.4,
1
chloroform). H NMR (CDCl3) δ: 7.40–7.20 (m, 15H, 3Ph),
© 2001 NRC Canada

252
Can. J. Chem. Vol. 79, 2001
2,3,4-tri-O-benzyl-6,7,8-trideoxy-α-D-gluco-oct-6-enopyranoside
(9) (105 mg, 22%) as a crystallizing syrup. [α]D + 1.8°,
[α]365 –33.4° (c 0.5, chloroform). H NMR (CDCl3) δ: 7.40–
(22 mg, 11%) identical to the major Grignard product of al-
dehyde 3.
1
7.20 (m, 15H, 3Ph), 5.85 (m, 1H, H-7), 5.42 (ddd, J6,5
=
Methyl 6-O-(6-O-acetyl-2,3,4-tri-O-benzyl-α-D-glucopy-
ranosyl)-2,3,4-tri-O-benzyl-(6R)-C-methyl-α-D-glucopy-
ranoside (R-14m)
7.5 Hz, J6,CH = 1.5 Hz, J6,7 = 14.5 Hz, 1H, H-6), 4.98–4.59
3
(3ABq, 6H, 3CH2Ph), 4.56 (d, J1,2 = 3.5 Hz, 1H, H-1), 4.01
(m, 1H, H-5), 3.96 (t, J2,3 ~ J3,4 = 9.5 Hz, 1H, H-3), 3.51 (dd,
J1,2 = 3.5 Hz, J2,3 = 9.5 Hz, 1H, H-2), 3.37 (s, 3H, CH3O),
3.23 (t, J3,4 ~ J4,5 = 9.5 Hz, 1H, H-4), 1.73 (m, 3H,
CH3CH=CH). Anal. calcd. for C30H34O5: C 75.92, H 7.22;
found: C 76.05, H 7.31. Continued development eluted R-4e
(159 mg, 43%), identical to the minor Grignard product of
aldehyde 3.
A solution of 6-O-acetyl-2,3,4-tri-O-benzyl-α-D-glucopy-
ranosyl bromide (12) (34) (2.1 mmol) in dichloromethane
(5 mL) was added to a stirring mixture of R-4m (500 mg,
1.04 mmol), tetraethylammonium bromide (420 mg,
2.00 mmol), N,N-dimethylformamide (1 mL) and 4 Å mo-
lecular sieves (powdered, 2 g) in dichloromethane (5 mL).
The mixture was stirred under nitrogen for several days until
completion (TLC), diluted with dichloromethane and filtered.
The solution was washed with sat. aq NaHCO3 and water,
followed by drying and evaporation. The crude product was
applied to a column of silica gel (hexane–ethyl acetate, 4:1)
to provide R-14m (587 mg, 59%). [α]D + 78.2° (c 1.1,
Methyl 2,3,4-tri-O-benzyl-7,8-dideoxy-7-C-methyl-α-D-
gluco-octopyranos-6-uloside (10)
A solution of S-4i (100 mg, 0.20 mmol) in dimethyl-
sulfoxide (1.5 mL) and acetic anhydride (0.75 mL) was kept
at room temperature for 15 h. The solution was evaporated
and the residue was taken up in dichloromethane, washed
1
chloroform). H NMR (CDCl3) δ: 7.40–7.20 (m, 30H, 6Ph),
5.00 (d, J1b,2b = 3.5 Hz, 1H, H-1b), 4.67 (d, J1a,2a = 3.5 Hz,
1H, H-1a), 5.03–4.51 (m, 12H, 6CH2Ph), 4.27 (dd, J5b,6b
=
1
with water, and evaporated to dryness. The H NMR spec-
4.5 Hz, J6b,6b′ = 11.5 Hz, 1H, H-6b), 4.21 (dd, J5b,6b′ = 2.5 Hz,
J6b,6b′ = 11.5 Hz, 1H, H-6b′), 4.08–3.91 (m, 4H, H-3a,
H-3b, H-6a, H-5b), 3.78 (dd, J5a,6a = 0.5 Hz, J4a,5a = 10.0 Hz,
1H, H-5a), 3.57–3.43 (m, 3H, H-4a, H-4b, H-2a), 3.41 (dd,
J2b,3b = 9.5 Hz, J1b,2b = 3.5 Hz, 1H, H-2b), 3.29 (3, 3H,
CH3O), 1.99 (s, 3H, CH3CO), 1.16 (d, J6a,CH3 = 6.5 Hz, 3H,
CH3C-6a). Anal. calcd. for C58H64O12: C 73.09, H 6.77;
found: C 73.46, H 6.61.
trum indicated a product mixture of ketone 10 and ether 11
in the ratio 1.6:1. Column chromatography on silica gel
(toluene–ethyl acetate, 49:1) provided pure ketone 10 (55 mg,
55%) as an oil. However, a crude product mixture was used
for the subsequent reduction because of the difficult separa-
tion. [α]D + 6.4° (c 0.6, chloroform). IR (CHCl3) cm–1: 1730
1
(CO). H NMR (CDCl3) δ: 7.40–7.15 (m, 15H, 3Ph), 5.00–
4.50 (3ABq, 6H, 3CH2Ph), 4.58 (d, J1,2 = 3.5 Hz, 1H, H-1),
4.35 (d, J4,5 = 9.5 Hz, 1H, H-5), 4.02 (t, J3,4 ~ J2,3 = 9.5 Hz,
1H, H-3), 3.72 (t, J3,4 ~ J4,5 = 9.5 Hz, 1H, H-4), 3.54 (dd,
J1,2 = 3.5 Hz, J2,3 = 9.5 Hz, 1H, H-2), 3.42 (s, 3H, CH3O),
2.85 (m, 1H, CH(CH3)2), 1.12 (d, J = 6.5 Hz, 3H,
CH(CH3)2), 1.07 (d, J = 6.5 Hz, 3H, CH(CH3)2). Anal. calcd.
for C31H36O6: C 73.79, H 7.19; found: C 73.45, H 7.11.
2,3,4,6-Tetra-O-benzyl-α-D-glucopyranosyl bromide (13)
(32)
Oxalyl bromide (190 µL, ~1.8 mmol) was added to a stir-
ring solution of 2,3,4,6-tetra-O-benzyl-D-glucopyranose
(650 mg, 1.20 mmol) in dichloromethane (10 mL) and N,N-
dimethylformamide (0.5 mL, 7.0 mmol). After 20 min at
room temperature, the mixture was poured into ice water
and the organic solution was washed twice with ice-cold wa-
ter, dried and evaporated to provide 13 (quant.) as a syrup.
Methyl 2,3,4-tri-O-benzyl-(6R)-C-isopropyl-α-D-glucopy-
ranoside (R-4i)
To a solution of crude ketone 10 (0.40 mmol) in methanol
(10 mL) was added, with stirring, sodium borohydride
(65 mg, 1.72 mmol) at ice bath temperatures. After 30 min,
the solution was evaporated, and the residue was taken up in
dichloromethane, washed with water, dried, and evaporated.
The product mixture was applied to a column of silica gel
(hexane–ethyl acetate, 6:1 → 5:1 → 4:1) to provide in the
first fraction methyl 2,3,4-tri-O-benzyl-(6S)-C-isopropyl-6-
O-methylthiomethylene-α-D-glucopyranoside (11) (63 mg,
Methyl 2,3,4-tri-O-benzyl-(6R)-C-ethyl-6-O-(2,3,4,6-tetra-
O-benzyl-α-D-glucopyranosyl)-α-D-glucopyranoside (R-14e)
and -β-D-glucopyranoside
A solution of bromide 13 (1.12 mmol) in 1,2-dichloro-
ethane (4 mL) was transferred into a stirring mixture of R-4e
(159 mg, 0.32 mmol), copper(II) bromide (260 mg,
1.16 mmol), N,N-dimethylformamide (350 µL, 4.93 mmol)
in 1,2-dichloroethane (2 mL) containing powdered 4 Å mo-
lecular sieves (1 g). After 44 h, toluene (15 mL) and moist
pyridine (7.5 mL) were added. After stirring for 30 min, the
mixture was filtered and the solvent evaporated. The resultant
material was applied to a column of silica gel (hexane–ethyl
acetate, 5:1) to first provide the β-anomer of R-14e (50 mg,
1
28%). [α]D + 30° (c 0.8, chloroform). H NMR (CDCl3) δ:
7.40–7.20 (m, 15H, 3Ph), 5.06–4.58 (m, 8H, CH2S,
3CH2Ph), 4.65 (d, J1,2 = 3.5 Hz, 1H, H-1), 4.02 (dd, J2,3
=
9.5 Hz, J3,4 = 9.0 Hz, 1H, H-3), 3.83 (br d, J4,5 = 9.5 Hz, 1H,
H-5), 3.73 (br d, J6,CH = 7.0 Hz, 1H, H-6), 3.65 (dd, J3,4
9.0 Hz, J4,5 = 9.5 Hz, 1H, H-4), 3.56 (dd, J1,2 = 3.5 Hz, J2,3
=
=
1
15%) as a syrup. [α]D + 26.4° (c 0.6, chloroform). H NMR
9.5 Hz, 1H, H-2), 3.43 (s, 3H, CH3O), 2.20 (br s, 3H,
CH3S), 2.17 (m, 1H, CH(CH3)2), 1.01 (d, J = 6.5 Hz, 3H,
CH(CH3)2), 0.99 (d, J = 6.5 Hz, 3H, CH(CH3)2). Anal.
calcd. for C33H42O6S: C 69.94, H 7.47, S 5.66; found: C
69.81, H 7.65, S 5.73. Continued elution afforded R-epimer
R-4i (90 mg, 44%) identical to the minor Grignard product
of aldehyde 3. Further development eluted S-epimer S-4i
(CDCl3) δ: 7.40–7.15 (m, 35H, 7Ph), 5.01–4.51 (m, 35H,
7CH2Ph), 4.60 (d, overlapped, H-1a), 4.45 (d, J1b,2b = 7.5 Hz,
1H, H-1b), 4.00 (t, J2a,3a ~ J3a,4a = 9.5 Hz, 1H, H-3a), 3.48
(dd, J1a,2a = 3.5 Hz, J2a,3a = 9.5 Hz, 1H, H-2a), 3.40 (s, over-
lapped, CH3O), 1.63 (m, 1H, CH2CH3), 1.40 (m, 1H,
CH2CH3), 0.88 (t, J = 7.5 Hz, 3H, CH3CH2). Anal. calcd. for
C64H70O11: C 75.71, H 6.95; found: C 75.40, H 6.91.
© 2001 NRC Canada

Spohr et al.
253
Continued elution of the column provided the α-anomer R-
14e (240 mg, 73%) as a syrup. [α]D + 57° (c 1.0, chloro-
form). H NMR (CDCl3) δ: 7.40–7.10 (m, 35H, 7Ph), 5.12 (d,
(243 mg, 69%) as a syrup. [α]D + 48° (c 0.5, chloroform).
1H NMR (CDCl3) δ: 7.35–7.10 (m, 35H, 7Ph), 5.02 (d, J1b,2b
=
1
3.5 Hz, 1H, H-1b), 4.96–4.43 (m, 14H, 7CH2Ph), 4.59 (d,
J1a,2a = 3.5 Hz, 1H, H-1a), 3.98–3.96 (m, 3H, H-3b, H-5b,
H-3a), 3.84 (t, J3a,4a ~ J4a,5a = 9.0 Hz, 1H, H-4a), 3.81 (br t,
J1b,2b = 3.5 Hz, 1H, H-1b), 4.97–4.42 (m, 14H, 7CH2Ph),
4.52 (d, overlapped, J1a,2a = 3.5 Hz, 1H, H-1a), 4.01 (t, J2b,3b
~
J3b,4b = 9.5 Hz, 1H, H-3b), 3.97 (t, J2a,3a ~ J3a,4a = 9.5 Hz,
1H, H-3a), 3.93 (m, 1H, H-5b), 3.85 (br d, J4a,5a = 9.5 Hz,
J5a,6a < 1 Hz, 1H, H-5a), 3.73 (m, 3H, H-4a, H-6a, H-6b),
overlapped, J6 ,CH = 7.0 Hz, 1H, H-6a), 3.68–3.66 (m, 2H,
a
H-5a, H-4b), 3.642(dd, overlapped, J6b,6b′ = 11.0 Hz, J6b,5b
=
2.5 Hz, 1H, H-6b), 3.52–3.51 (m, 2H, H-2b, H-6b′), 3.45
(dd, J2a,3a = 9.5 Hz, J1a,2a = 3.5 Hz, 1H, H-2a), 3.37 (s, 3H,
CH3O), 1.78 (m, 2H, CH3CH2), 0.94 (t, J = 7.5 Hz, 3H,
CH3CH2). Anal. calcd. for C64H70O11: C 75.71, H 6.95;
found: C 75.52, H 7.08.
3.64 (t, J3b,4b ~ J4b,5b = 9.5 Hz, 1H, H-4b), 3.60 (dd, J6b,6b′
11.0 Hz, J6b′,5b = 1.5 Hz, 1H, H-6b′), 3.53 (dd, J1b,2b′
=
=
3.5 Hz, J2b,3b = 9.5 Hz, 1H, H-2b), 3.35 (dd, J1a,2a = 3.5 Hz,
J2a,3a = 9.5 Hz, 1H, H-2a), 3.32 (s, 3H, CH3O), 1.61 (m, 2H,
CH2CH3), 0.89 (t, J = 7.5 Hz, 3H, CH3CH2). Anal. calcd. for
C64H70O11: C 75.71, H 6.95; found: C 75.38, H 6.88.
Methyl 2,3,4-tri-O-benzyl-(6S)-C-isopropyl-6-O-(2,3,4,6-
tetra-O-benzyl-α-D-glucopyranosyl)-α-D-glucopyranoside
(S-14i)
Methyl 2,3,4-tri-O-benzyl-(6R)-C-isopropyl-6-O-(2,3,4,6-
tetra-O-benzyl-α-D-glucopyranosyl)-α-D-glucopyranoside
(R-14i)
Alcohol S-4i (160 mg, 0.32 mmol) was reacted with bro-
mide 13 (1.29 mmol) for 5 days as described for the prepa-
ration of R-14e. Column chromatography (hexane–ethyl
acetate, 6:1, 5:1) provided S-14i (207 mg, 64%) as a tough
Alcohol R-4i (150 mg, 0.30 mmol) was reacted with bro-
mide 13 (1.24 mmol) for 6 days as described for the prepa-
ration of R-14e. Column chromatography (hexane–ethyl
acetate, 8:1 → 7:1) provided R-14i (178 mg, 58%) as a thick
1
syrup. [α]D + 57.4° (c 0.4, chloroform). H NMR (CDCl3) δ:
7.35–7.10 (m, 35H, 7Ph), 5.02–4.36 (m, 16H, 7CH2Ph, H-
1a, H-1b), 4.04 (t, J2b,3b ~ J3b,4b = 9.5 Hz, 1H, H-3b), 4.00
(m, overlapped, 1H, H-5b), 3.95 (t, J2a,3a ~ J3b,4b = 9.0 Hz,
1H, H-4a), 3.90 (t, J3a,4a ~ J4a,5a = 9.0 Hz, 1H, H-4a), 3.74
1
syrup. [α]D + 56.4° (c 0.3, chloroform). H NMR (CDCl3) δ:
7.40–7.10 (m, 35H, 7Ph), 5.26 (d, J1b,2b = 3.5 Hz, 1H, H-
1b), 4.96–4.42 (m, 14H, 7CH2Ph), 4.52 (d, J1a,2a = 3.5 Hz,
1H, H-1a), 4.01 (t, J2b,3b ~ J3b,4b = 9.5 Hz, 1H, H-3b), 3.96
(t, overlapped, J2a,3a ~ J3a,4a = 9.5 Hz, 1H, H-3a), 3.74, (dd,
(br d, J5a,6a < 1 Hz, 1H, H-5a), 3.68 (t, J3a,4a ~ J4b,5b
9.0 Hz, 1H, H-4b), 3.62 (dd, J6b,6b′ = 11.0 Hz, J6b,5b
2.5 Hz, 1H, H-6b), 3.57 (dd, J6b′,5b = 1.5 Hz, J6b,6b′
=
=
=
J6b,6b′ = 11.0 Hz, J6b,5b = 3.5 Hz, 1H, H-6b), 3.61 (dd, J6b′,5b
1.5 Hz, J6b,6b′ = 11.0 Hz, overlapped,1H, H-6b′), 3.54 (dd,
J1b,2b = 3.5 Hz, J2b,3b = 9.5 Hz, 1H, H-2b), 3.51 (d, J6a,CH
=
11.0 Hz, 1H, H-6b′), 3.55 (d, J6a,CH = 6.5 Hz, 1H, H-6a),
3.51 (dd, J1b,2b = 3.5 Hz, J2b,3b = 9.5 Hz, 1H, H-2b), 3.41 (s,
3H, CH3O), 3.38 (dd, J1a,2a = 3.5 Hz, J2a,3a = 9.0 Hz, 1H, H-
2a), 2.03 (m, 1H, CH(CH3)2), 1.03 (d, 3H, J = 6.5 Hz,
CH(CH3)2), 1.00 (d, 3H, J = 6.5 Hz, CH(CH3)2). Anal.
calcd. for C65H72O11: C 75.85, H 7.05; found: C 75.67, H
7.14.
=
8.5 Hz, 1H, H-6a), 3.34 (dd, J1a,2a = 3.5 Hz, J2a,3a = 9.5 Hz,
1H, H-2a), 3.32 (s, 3H, CH3O), 2.08 (m, 1H, CH(CH3)2),
0.98 (d, J = 6.5 Hz, 3H, CH(CH3)2), 0.89 (d, J = 6.5 Hz, 3H,
CH(CH3)2). Anal. calcd. for C65H72O11: C 75.85, H 7.05;
found: C 75.78, H 7.27.
Methyl 6-O-(6-O-acetyl-2,3,4-tri-O-benzyl-α-D-
glucopyranosyl)-2,3,4-tri-O-benzyl-(6R)-C-methyl-α-D-
glucopyranoside (S-14m)
Compound S-4m (100 mg, 0.21 mmol) was reacted with
bromide 12 (0.42 mmol) as described for the preparation of
R-14m. Usual processing and chromatography provided
S-14m (101 mg, 51%) [α]D + 0.5° (c 1.06, chloroform). H
NMR (CDCl3) δ: 7.40–7.20 (m, 30H, 6Ph), 5.09 (d, J1b,2b
3.5 Hz, 1H, H-1b), 5.02–4.49 (m, 7H, 3CH2Ph, H-1a), 4.26–
4.06 (m, 3H, 2H-6b, H-1a), 4.06–3.94 (m, 3H, H-3b, H-3a,
H-5b), 3.77 (dd, J3a,4a = 9.5 Hz, J4a,5a = 9.0 Hz, 1H, H-4a),
3.56 (dq, J5a,6a = 2.0 Hz, J4a,5a = 9.0 Hz, 1H, H-5a), 3.56–
Methyl 6-O-(α-D-glucopyranosyl)-(6R)-C-methyl-α-D-
glucopyranoside (R-2m)
To a solution of compound R-14m (440 mg, 0.46 mmol)
in methanol (20 mL) was added a catalytic amount of so-
dium methoxide. After stirring overnight, it was deionized
with Amberlite IRC-50 H+. Evaporation of the methanol
provided methyl 2,3,4-tri-O-benzyl-(6R)-C-methyl-6-O-
(2,3,4-tri-O-benzyl-α-D-glucopyranosyl)-α-D-glucopyranoside
1
=
1
(347 mg, 83%) as an oil. [α]D + 65.2° (c 0.34, water). H
NMR (CDCl3) δ: 7.40–7.20 (m, 30H, 6Ph), 5.00 (d, J1b,2b
=
4.5 Hz, 1H, H-1b), 4.98–4.53 (m, 13H, 6CH2Ph, H-1a),
4.08–3.91 (m, 3H, H-3a, H-3b, H-6a), 3.81–3.67 (m, 4H,
H-5a, H-5b, H2-6b), 3.56–3.37 (m, 4H, H-4a, H-4b, H-2a,
H-2b), 3.29 (s, 3H, CH3O), 1.13 (d, J6a,CH3 = 6.5 Hz, 3H,
CH3C-6a). Anal. calcd. for C56H62O11: C 73.82, H 6.86;
found: C 73.23, H 7.23.
3.47 (m, 2H, H-2a, H-2b), 3.46 (dd, J3b,4b = 9.0 Hz, J4b,5b
=
10.0 Hz, 1H, H-4b), 3.38 (s, 3H, CH3O), 1.92 (s, 3H,
CH3CO), 1.33 (d, J6 ,CH = 6.0 Hz, 3H, CH3C-6a). Anal.
a
3
calcd. for C57H64O12: C 73.09, H 6.77; found: C 72.84, H
6.85.
The deacetylation product of R-14m, namely methyl
2,3,4-tri-O-benzyl-(6R)-C-methyl-6-O-(2,3,4-tri-O-benzyl-α-
D-glucopyranosyl)-α-D-glucopyranoside, (247 mg, 0.27 mmol)
was debenzylated following the general procedure to give
solid R-2m (80 mg, 80%), mp 231–232°C. [α]D + 228.1° (c
Methyl 2,3,4-tri-O-benzyl-(6S)-C-ethyl-6-O-(2,3,4,6-tetra-
O-benzyl-α-D-glucopyranosyl)-α-D-glucopyranoside (S-
14e)
Alcohol S-4e (170 mg, 0.35 mmol) was reacted with bro-
mide 13 (1.24 mmol) for 4 days as described for the prepa-
ration of R-14e. Column chromatography (hexane–
dichloromethane-ethyl acetate, 20:10:1–2) provided S-14e
1
0.43, water). The H NMR data are reported in Table 5. 13C
NMR (D2O) δ: 100.13 (C-1a), 97.98 (C-1b), 73.89, 73.73,
73.37, 72.98, 72.02, 71.89, 71.80, 71.48, 70.51, 56.09
© 2001 NRC Canada

254
Can. J. Chem. Vol. 79, 2001
(CH3O), 15.12 (CH3C-6a). Anal. calcd. for C14H25O11: C
45.53, H 6.82; found: C 45.18, H 6.97.
Methyl 6-O-(α-D-glucopyranosyl)-(6S)-C-isopropyl-α-D-
glucopyranoside (S-2i)
Compound S-2i was prepared from S-14i by catalytic
hydrogenolysis following the general procedure and obtained
as a white solid (98%) after lyophilization of an aqueous
Methyl (6R)-C-ethyl-6-O-(α-D-glucopyranosyl)-α-D-
glucopyranoside (R-2e)
1
solution. [α]D + 192° (c 0.2, water). The H NMR data are
Compound R-2e was prepared from R-14e by catalytic
hydrogenolysis following the general procedure and obtained
as a white solid (89%) after lyophilization of an aqueous so-
reported in Table 5. 13C NMR (D2O) δ: 100.63 (C-1a), 96.52
(C-1b), 76.45, 74.20, 73.70, 73.05, 72.67, 71.90, 70.86,
70.51, 70.33, 61.15 (C-6b), 57.43 (CH3O), 28.42
(CH(CH3)2, 19.04, 17.72 (CHCH3)2).
1
lution. [α]D + 182.5° (c 0.3, water). The H NMR data are
reported in Table 5. 13C NMR (D2O) δ: 100.36 (C-1a), 97.01
(C-1b), 78.21, 73.94, 73.73, 73.10, 72.09, 71.89, 71.28,
70.92, 70.47, 61.43 (C-6b), 56.46 (CH3O), 22.73 (CH2CH3),
11.48 (CH3CH2).
Acknowledgements
This research was supported by the Pharmaceutical Manu-
facturers Association of Canada (Prize to R.U.L.) and in part
by the Natural Sciences and Engineering Council of Canada.
We are deeply grateful to Professor D.R. Bundle for the use
of his computer graphic facilities and the collaboration of
his Research Associate, Dr. Chang-Chun Ling.
Methyl 6-O-(α-D-glucopyranosyl)-(6R)-C-isopropyl-α-D-
glucopyranoside (R-2i)
Compound R-2i was prepared from R-14i by catalytic
hydrogenolysis following the general procedure and ob-
tained as a white solid (87%) after lyophilization of an aque-
ous solution. [α]D + 190.5° (c 0.2, water). The 1H NMR data
are reported in Table 5. 13C NMR (D2O) δ: 100.23 (C-1a),
97.86 (C-1b), 82.69, 73.99, 73.64, 73.27, 72.36, 71.88,
71.73, 71.34, 70.47, 61.45 (C-6b), 56.39 (CH3O), 30.23
(CH(CH3)2), 20.75, 19.94 (CH(CH3)2).
References
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Methyl 6-O-(α-D-glucopyranosyl)-(6S)-C-methyl-α-D-
glucopyranoside (S-2m)
Compound S-14m (292 mg, 0.31 mmol) was deacetylated
as described for R-14m to provide methyl 2,3,4-tri-O-
benzyl-(6S)-C-methyl-6-O-(2,3,4-tri-O-benzyl-α-D-glucopy-
ranosyl)-α-D-glucopyranoside (269 mg, 96%) as an oil. [α]D
1
+ 47.6° (c 2.0, chloroform). H NMR (CDCl3) δ: 7.40–7.10
(m, 30H, 6Ph), 5.03 (d, J1b,2b = 3.5 Hz, 1H, H-1b), 5.01–
4.57 (m, 13H, 6CH2Ph, H-1a), 4.32 (dq, J5a,6a = 1.5 Hz,
7. A.E. Aleshin, L.M. Firsov, and R.B. Honzatko. J. Biol. Chem.
269, 15 631 (1994).
J6 ,CH = 6.5 Hz, 1H, H-6a), 4.01 (dd, J2b,3b = 9.5 Hz, J3b,4b
=
a
3
9.0 Hz, 1H, H-3b), 3.98 (dd, J2a,3a = 9.5 Hz, J3a,4a = 10.0 Hz,
1H, H-3a), 3.84–3.72 (m, 2H, H-5b, H-4a), 3.66–3.49 (m,
5H, 2H-6a, H-5a, H-4b, H-2a), 3.46 (dd, J1b,2b = 3.5 Hz,
J2b,3b = 9.5 Hz, 1H, H-2b), 3.36 (s, 3H, CH3O), 1.32 (d,
J6 ,CH = 6.5 Hz 3H, CH3C-6). Anal. calcd. for C56H62O11:
8. A.E. Aleshin, B. Stoffer, L.M. Firsov, B. Svensson, and R.B.
Honzatko. Biochemistry, 35, 8319 (1996).
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10. T.P. Frandsen, M.M. Palcic, C. Dupont, and B. Svensson.
Carbohydr. Res. 314, 127 (1998).
a
3
C 73.82, H 6.86; found: C 73.20, H 6.95.
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J. Chem. 63, 2653 (1985).
The deacetylation product of S-14m, namely methyl
2,3,4-tri-O-benzyl-(6S)-C-methyl-6-O-(2,3,4-tri-O-benzyl-α-
D-glucopyranosyl)-α-D-glucopyranoside, (126 mg, 0.138 mmol)
was debenzylated following the general procedure to give
crystalline S-2m (40 mg, 78%), mp 127–128°. [α]D + 175.3°
1
(c 0.3, water). The H NMR data are reported in Table 5.
13C NMR (D2O) δ: 100.03 (C-1a), 95.56 (C-1b), 74.49,
74.15, 73.68, 73.04, 72.25, 71.99, 70.24, 68.50, 61.17, 55.67
(CH3O), 14.76 (CH3-6a).
Methyl-(6S)-C-ethyl-6-O-(α-D-glucopyranosyl)-α-D-
glucopyranoside (S-2e)
19. U. Spohr, E. Paszkiewicz-Hnatiw, N. Morishima, and R.U.
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Compound S-2e was prepared from S-14e by catalytic
hydrogenolysis following the general procedure and obtained
as a white solid (94%) after lyophilization of an aqueous so-
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1
lution. [α]D + 198° (c 0.2, water). The H NMR data are re-
ported in Table 5. 13C NMR (D2O) δ: 100.31 (C-1a), 96.31
(C-1b), 74.77, 74.47, 73.85. 73.15, 72.37, 72.01, 70.93,
70.29, 70.14, 61.21 (C-6b), 56.38 (CH3O), 21.67 (CH2CH3),
10.02 (CH3CH2).
© 2001 NRC Canada

Spohr et al.
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© 2001 NRC Canada