Synthesis and Conformational Analysis of Novel N(OCH3)-linked Disaccharide Analogues

Article abstract of DOI:10.1002/chem.200305587

N(OMe)-linked disaccharide analogues, isosteric to the corresponding natural disaccharides, have been synthesized by chemoselective assembly of unprotected natural monosaccharides with methyl 6-deoxy-6-methoxyamino-α -D-glucopyranoside in an aqueous environment. The coupling reactions were found to be chemo- and stereoselective affording β-(1→6) disaccharide mimics when using Glc and GlcNAc; in the case of Gal, the β-anomer was prevalent (β:α = 7:1). An iterative method for the synthesis of linear N(OMe) oligosaccharide analogues was demonstrated, based on the use of an unprotected monosaccharide building block in which an oxime functionality at C-6 is converted during the synthesis into the corresponding methoxyamino group. The conformational analysis of these compounds was carried out by using NMR spectroscopy, ab initio, molecular mechanics, and molecular dynamics methods. Optimized geometries and energies of fourteen conformers for each compound have been calculated at the B3LYP/631G* level. Predicted conformational equilibria were compared with the results based on NMR experiments and good agreement was found. It appears that N(OMe)-linked disaccharide analogues exhibit a slightly different conformational behavior to their parent natural disaccharides.

Full text of DOI:10.1002/chem.200305587

FULL PAPER
Synthesis and Conformational Analysis of Novel N(OCH )-linked
3
Disaccharide Analogues
[
a]
[b]
[b]
Francesco Peri,* JesÇs Jimÿnez±±arbero, VÌctor GarcÌa±Aꢀaricio,
[
c]
[a]
Igor Tvaro sœ ka, and Francesco Nicotra
Abstract: N(OMe)-linked disaccharide
analogues, isosteric to the correspond-
ing natural disaccharides, have been
synthesized by chemoselective assem-
bly of unprotected natural monosac-
charides with methyl 6-deoxy-6-me-
thoxyamino-a-d-glucopyranoside in an
aqueous environment. The coupling re-
actions were found to be chemo- and
stereoselective affording b-(1!6) dis-
accharide mimics when using Glc and
GlcNAc; in the case of Gal, the b-
anomer was prevalent (b:a=7:1). An
iterative method for the synthesis of
linear N(OMe) oligosaccharide ana-
logues was demonstrated, based on the
use of an unprotected monosaccharide
building block in which an oxime func-
tionality at C-6 is converted during the
synthesis into the corresponding me-
thoxyamino group. The conformational
analysis of these compounds was car-
ried out by using NMR spectroscopy,
ab initio, molecular mechanics, and
molecular dynamics methods. Opti-
mized geometries and energies of four-
teen conformers for each compound
have been calculated at the B3LYP/6-
31G* level. Predicted conformational
equilibria were compared with the re-
sults based on NMR experiments and
good agreement was found. It appears
that N(OMe)-linked disaccharide ana-
logues exhibit a slightly different con-
formational behavior to their parent
natural disaccharides.
Keywords: carbohydrates ¥ confor-
mation analysis ¥ glycosylation ¥
NMR spectroscopy
Introduction
Two main strategies have been explored in the construc-
tion of oligosaccharide mimics:the substitution of the O-
glycosidic bond with non-natural linkages or the replace-
ment of the entire glycosidic scaffold with a new, non-carbo-
hydrate framework bearing the required functional groups
in the same spatial orientation as the parent sugar structure.
The former type of mimics would ideally have increased re-
sistance to enzymatic hydrolysis and chemical degradation,
while conserving the global geometry of the native oligosac-
charide.
The specific molecular recognition by protein receptors of
oligosaccharides covalently attached to lipids and proteins
at cell surfaces plays a key role in a number of physiological
and pathological events such as cell adhesion, inflammation,
metastasis, and embryonic development. It is therefore not
surprising that a large effort has recently been focused on
the design of organic molecules that mimic the active con-
formation of a carbohydrate ligand, thus reproducing its bio-
logical function.
[1]
Carbon-linked oligosaccharides are an interesting class of
mimics in which a methylene group substitutes the intergly-
cosidic oxygen. The conformational properties of C-glyco-
[
a] Dr. F. Peri, Prof. F. Nicotra
Department of Biotechnology and Biosciences
University of Milano-Bicocca
[2]
sides have been debated. Since the substitution of intergly-
cosydic oxygen atom by the methylene group evidently re-
sults in a change in both the size and the electronic proper-
ties of the glycosidic linkages, the flexibility and the energy
barriers to rotation around the inter-residual linkages are
2
0126 Milano (Italy)
Fax :( +39)264483565
E-mail:francesco.peri@unimib.it
[
b] Prof. J. Jimÿnez±Barbero, Dr. V. GarcÌa±Aparicio
Departamento de Estructura y FunciÛn de ProteÌnas Centro de
Investigaciones BiolÛgicas
[3]
expected to change to some extent. Interestingly, the con-
formation of C- and O-lactoses bound respectively to
CSIC, Ramiro de Maeztu 9, 28040 Madrid (Spain)
[4]
[5]
bovine heart galectin-1 and peanut lectin were found to
be virtually identical, while different conformers of the dis-
accharide and its C-analogue are selected and recognized by
[
c] Dr. I. Tvaro sœ ka
Institute of Chemistry
Slovak Academy of Sciences
45 38 Bratislava (Slovak Republic)
8
[2a]
the galactose-binding protein ricin-B and by an Escheri-
Supporting information for this article is available on the WWW
under http://www.chemeurj.org/ or from the author, and includes rela-
[6]
chia coli b-galactosidase.
Chem. Eur. J. 2004, 10, 1433 ± 1444
DOI: 10.1002/chem.200305587
¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1433

FULL PAPER
[7]
Another class of mimics, the S-linked oligosaccharides,
Results
have several advantages over their C-linked counterparts:
they are generally easier to prepare and have an interglyco-
sidic sulfur atom that may act as a hydrogen bond acceptor
which, as in the natural substrate, could play an important
role in the binding of the ligand. These hydrolytically inert
substrate analogues have been used to investigate binding
and recognition events with glycosidases. The S-linkage
provides a high degree of flexibility between glycosyl units
and S-glycosides in solution possess more low-energy con-
formers than their O-linked parent saccharides.
Synthesis of methoxyamino disaccharide mimics:A sche-
matic representation of the synthesized disaccharide mimics
is shown in Scheme 1. The b-(1!6)-methoxyamino intergly-
[8]
Finally, oligosaccharide mimics have been prepared that
[9]
contain a nitrogen atom at the interglycosidic linkage as
[10]
[11]
well as carbopeptoids or carbonucleotoids in which the
O-glycosidic bond has been replaced by a more rigid car-
boxyamide or phosphoramidite bond.
From a methodological point of view, the synthesis of oli-
gosaccharides and their analogues is laborious and challeng-
ing, requiring extensive use of orthogonal protecting groups
and strictly anhydrous conditions in the glycosylation reac-
tion; for these reasons it is still far from routine, both in sol-
Scheme 1. Amino(methoxy) disaccharide mimics 1±6 and their parent O-
linked natural disaccharides gentiobiose (7) and allolactose (8).
[12]
[13]
ution and in the solid phase. Synthetic strategies based
on the formation of a peptide-like interglycosidic bond (car-
cosidic bond allows one to exploit the chemoselective liga-
[10]
[19]
bopeptoids) or based on the principle of chemoselective
tion procedure and afford isosteric analogues. Disacchar-
[14]
ligation provide a powerful tool for the convergent prepa-
ration of oligosaccharide mimics. In particular, sugars bear-
ing an aminooxy group at the anomeric position have been
reacted with ketones present in a second sugar for the syn-
ide 1 is an isosteric mimic of the natural disaccharide gentio-
biose (7) and 3 a mimic of allolactose (8). Compounds 1±6
have been assembled by chemoselective condensation of an
unprotected monosaccharide bearing
a methoxyamino
[15]
thesis of complex O-glycosylated peptides. It is also possi-
ble to link a peptide bearing an aminooxy function to the
anomeric center of a free aldose in a chemoselective way,
group at C-6 and unprotected natural aldoses. The mecha-
nism that we propose for the coupling reaction consists of
the formation of an intermediate oxyiminium ion by reac-
tion of the N,O-disubstituted hydroxylamine group with the
[16]
that is, without use of protecting groups. It is well known
that when using a reacting unit bearing a ™primary∫
[18]
aldehyde of the open-chain sugar (Scheme 2).
ÀOÀNH group, an open-chain
2
[17]
sugar oxime is obtained, but
it has also been observed that
the cyclic form of the sugar is
restored when a peptide with a
™secondary∫
hydroxylamino
group
used.
(RÀOÀNHÀR’)
is
[18]
By following this last
approach, if R and R’ were
sugar units, it would be possi-
ble in principle to obtain disac-
charides and, more generally,
oligosaccharide mimics by
working in water and avoiding
the use of any activation and
Scheme 2. Stereoselective formation of b-N(OMe)-linked glycosides via an intermediate oxyiminium ion.
protection strategy. These mimics, however, differ from the
Monosaccharide 13, which has a methoxyamino group at
C-6, was prepared according to the synthetic route depicted
in Scheme 3.
natural product by the fact that two atoms (ÀOÀNÀ) substi-
tute the interglycosidic oxygen atom and they are therefore
™
non-isosteric∫ analogues.
Herein we present the synthesis and conformational anal-
Methyl 2,3,4-tri-O-acetyl-a-d-glucopyranoside (9) was oxi-
dized at C-6 (Dess±Martin periodinane, CH Cl ) to afford
2
2
ysis of a novel type of disaccharide mimic in which two
monosaccharide residues are linked through a b-(1!6)-me-
thoxyamino interglycosidic linkage.
aldehyde 10 that was converted into the corresponding O-
methyloxime 11 by treatment with O-methylhydroxylamine
hydrochloride in pyridine. Zemplÿn de-O-acetylation with
sodium methylate in methanol gave compound 12 which
was reduced with NaCNBH in glacial acetic acid to afford
3
methyl 6-deoxy-6-methoxyamino-a-d-glucopyranoside (13).
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Chem. Eur. J. 2004, 10, 1433 ± 1444

Novel N(OCH )-linked Disaccharide Analogues
1433 ± 1444
3
The preference for the b anomer in the case of glucose,
galactose, and N-acetylglucosamine can be explained in
terms of stabilization of the b conformation of the oxyimini-
um intermediate (Scheme 2) through the reverse anomeric
effect that is particularly relevant when a positively charged
[20]
nitrogen atom is linked to the anomeric position.
Iterative synthesis of methoxyamino trisaccharide mimetics:
We investigated the possibility of using our chemoselective
approach iteratively for the synthesis of linear oligosacchar-
ide mimetics linked through a b-(1!6)-methoxyamino
bond. To accomplish this task, we designed the monosac-
charide 16 that has the two complementary functionalities
required for chemoselective ligation, namely, the aldehyde
and the aminomethoxy group, the latter being masked as an
O-methyloxime.
Scheme 3. a) Dess±Martin periodinane, CH
pyridine (75%); c) MeONa, MeOH (98%); d) NaCNBH
86%).
2
Cl
2
(95%); b) MeONH
2
¥HCl,
3
, glacial AcOH
(
Compound 13 was condensed with d-glucose, d-galactose,
and N-acetylglucosamine to afford disaccharide analogues 1,
The readily available compound 16 was coupled with 13
to afford the disaccharide 17 (Scheme 5). Reduction of the
3
, and 5, respectively, which were then fully acetylated
oxime group of 17 with NaCNBH gave the disaccharide 18,
3
(
Ac O, pyridine, and 4-(dimethylamino)pyridine (DMAP))
2
leading to compounds 2, 4, and 6 (Scheme 4).
Scheme 4. a) d-Glucose, DMF/AcOH (2:1) (82%); b) d-galactose, DMF/
AcOH (2:1) (75%); c) N-acetylglucosamine, DMF/AcOH (2:1) (80%);
d) Ac O, pyridine, DMAP.
2
Scheme 5. a) Dess±Martin periodinane, CH
dine (75% over two steps); c) MeONa, MeOH (98%); d) 13, DMF/
AcOH (2:1) (72%); e) NaCNBH , glacial AcOH (75%); f) 16, DMF/
O/AcOH (2:2:1) (43%); g) Ac O, pyridine, DMAP.
2 2 2
Cl ; b) MeONH ¥HCl, pyri-
The coupling reactions proceeded at room temperature in
2±24 h, and were carried out either in aqueous N,N-dime-
1
3
thylformamide (DMF)/sodium acetate buffer (pHꢀ4.5),
water/acetic acid (1:1 v/v), or acetic acid/DMF (1:2) solvent
mixtures. Reactions were never complete and after several
hours a steady state was reached with a more or less con-
stant ratio between reactants and products. Unreacted mon-
osaccharides were recovered at the end of each reaction and
recycled.
H
2
2
which has a methoxyamino group at C-6’. Chemoselective
coupling with another monosaccharide, followed by treat-
ment with Ac O, pyridine, and DMAP, afforded the perace-
2
tylated trisaccharide mimic 19, which can be reduced and
The b-disaccharide mimics 1 and 5, which contain d-glu-
cose and N-acetylglucosamine, respectively, were obtained
with complete stereoselectivity. The H NMR analysis of the
d-galactose derivative 3 revealed the presence of a small
amount of the a form (b:a=7:1, as calculated from the inte-
gration ratio of the anomeric protons). Reaction of mannose
with 13 gave a mixture of disaccharides in low yield (20%),
the NMR analysis of which showed the presence of the pyr-
anose (a+b) and furanose (a+b) forms.
submitted to further elongation.
1
Conformational analysis:The conformational study of the
disaccharide mimics with free hydroxyls and their acetylated
analogues (1±6) was performed in solution. The protocol
used is based on a combination of theoretical methods with
ab initio and molecular mechanics and dynamics calcula-
tions, supported by experimental NMR data. Similar ap-
proaches have been demonstrated to be particularly useful
Chem. Eur. J. 2004, 10, 1433 ± 1444
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¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1435

F. Peri et al.
FULL PAPER
to deduce the conformational properties of other glycomi-
named by a three-letter code according to the conformation
around the C5±C6 linkage (GG, GT, or TG) and the
number of the minimum, for example, GT1 represents the
number 1 minimum (F=À72.28, Y=À154.38) of the gt ro-
tamer (w=58.88).
[21]
metics, and also to sample the differences with respect to
the natural analogues, gentiobiose (7) and allolactose (8). It
has to be emphasized that these glycomimetics show a par-
ticular arrangement at the pseudoglycosidic linkage, since
the nitrogen atom (which mimics the natural glycosidic
oxygen) bears an additional electronegative substituent. In
this context, six different staggered arrangements at angle F
Structure and energetics of conformers:The relative ener-
gies of the 14 minima calculated at the 6-31G* level for all
methyl disaccharides in the gas phase and aqueous solution
are reported in Tables 1S±4S (see Supporting Information)
together with the values of F, Y, and w torsional angles. The
relevant proton±proton distances of these minima, calculat-
ed at the B3LYP/6-31G* level, are listed in Tables 5S±8S
(
defined as shown below) may take place, as depicted in
Scheme 6, which also considers the two possible configura-
tions at the nitrogen atom.
(
see Supporting Information). As can be seen from Ta-
bles 1S±4S, four of the 14 calculated minima belong to the
gg rotamer and each of the gt and tg rotamers contains five
minima. Inspection of the calculated structures revealed that
the counterclockwise orientation of hydroxyl groups is pre-
ferred in both monosaccharide residues. It is well known
that the cooperative effect of hydrogen bonds in these ar-
rangements favors corresponding conformations of glucopyr-
[22]
anose and galactopyranose in the gas phase. It is evident
that the preferred conformations around C1’ÀX1’ bond
(
angle F) between the parent disaccharide and its mimics
differ, Àgauche for 7 versus +gauche for 1, 3, and 5. The
lowest-energy conformers of three N(OMe)-linked ana-
logues 1, 3, and 5 lay in the low-energy domain located at
F=608, Y=1008, whereas for the parent molecule 7a the
lowest-energy minima are at F=À718, Y=À1528 (Àgauche
region) and at F=À698, Y=À908 (Àgauche region), respec-
tively. In the case of the angle Y, two preferred orientations,
namely, +gauche and trans, were detected in the low-energy
minima for all compounds.
Scheme 6. Possible rotamers around F of compounds 1±6. The different
stereochemistries of the substituents at the nitrogen atom are also depict-
ed.
Ab initio based comꢀutational analysis:
Potential energy surfaces:The available conformational
space was monitored by means of a series of two-dimension-
al (F, Y) potential energy surfaces, each one corresponding
to the one of three different starting values of w, namely
w=À608, w=608, and w=1808 (see Experimental Section).
Figure 1 represents the B3LYP/6-31G* conformational
energy maps of the gg, gt, and tg rotamers for the disacchar-
ide analogue 5 calculated as a function of the torsion angles
F and Y. Several energy minimum domains were found on
each of these maps. For example, in the case of the gg ro-
tamer (Figure 1a), four distinct low-energy regions with (F,
Y) values close to (408, 1008), (608, 3008), (2208, 808), and
The gg, gt, and tg rotamer populations calculated from the
energy values together with the available experimental data
are compared in Table 1. For 1, 5, and 7, we found that in
Table 1. Rotamer populations of compounds 1, 3, 5, and 7a estimated
from energies calculated at the B3LYP/6-31G* level.
1
3
5
7a
Exp.(7a)
GG
GT
TG
hFi
hYi
hwi
7.7
84.9
7.4
27
78
100
31.4
68.6
0.0
9.6
77.4
13.0
30
83
109
35.1
62.1
2.8
À145
À153
149
34
66
0
À72
À120
-143
À145
107
137
(
3008, 2108), respectively, were identified. It can be seen
from these three maps in Figure 1 that for the rotation
about the C1ÀO1 linkage they all have three low-energy re-
gions centered about F=408, F=2408, and F=3008. Three
low-energy regions were found in the direction of the Y
angle for the gg and gt rotamers. In the case of the tg rotam-
er (Figure 1c), the region centered about Y=608 disap-
peared and only two regions centered around Y=1508 and
Y=3008 were found in the Y direction. Geometries from
these regions were used as the initial geometry for further
optimizations of 1, 3, 5, and the parent natural disaccharide
methyl a-d-gentiobioside (7a) allowing the torsion angles F
and Y to relax also. These optimizations yielded 14 local
minima for each disaccharide analogue. The minima are
the gas phase at the 6-31G* level, the gt rotamer is the dom-
inant species and that the stability of the hydroxymethyl ro-
tamers is in the order gt@ggꢀtg with the presence of the gt
rotamer higher than 60%. This implies that the presence of
the NHAc substituent at C2’ does not significantly influence
the conformational equilibrium around the (1!6) linkage.
The conformational preferences around the C5’ÀC6’ bond of
the galactopyranose moiety in 3 displays similar behavior to
the parent disaccharide 7a and in a comparison to the Glc-
type fragments of 1 and 5 shows a smaller dominance of the
gt rotamer. In spite of differences in the rotamer popula-
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Novel N(OCH )-linked Disaccharide Analogues
1433 ± 1444
3
Figure 1. Relaxed conformational energy maps of compound 5 calculated at the B3LYP/6-31G* level as a function of the torsional angles F and Y, with
the torsional angle w in gg (a), gt (b), and tg (c) orientation. The symbols indicate local minima.
[
24]
tions, the calculated average values of the torsional angles F
and Y in the gas phase are very similar for all three
N(OMe)-linked analogues, with hFi in the range 27±638 and
hYi in the range 100±1408, respectively. The conformational
preference around the C1’ÀN linkage in 1, 3, and 5 (Fꢀ
steric interactions of the N-substituent. In our compounds
such a substituent is the electronegative OMe group. It ap-
pears that for the calculated lowest-energy conformers (Ta-
bles 1S±4S), the oxygen atom of the OMe moiety is in the
gauche orientation (structures IIIa and IIb in Scheme 6)
with respect to the ring oxygen. This suggests that two ster-
eoelectronic effects influence the orientation about the
C1’ÀN1’ bond, namely the exo-anomeric effect through the
O5’ÀC1’ÀN1ÀC6 sequence and the gauche effect through
the O5’ÀC1’ÀN1ÀO sequence. It is noteworthy that an anal-
+
608) corresponds to the gauche orientation with respect to
the C1’ÀO5’ and C1’ÀC2’ linkages and significantly differs
from the ™usual∫ conformation (FꢀÀ608) predicted by the
combination of steric and stereoelectronic interaction, the
[23]
exo-anomeric effect, for b-linked oligosaccharides. How-
ever, this preference is not entirely surprising, since the simi-
lar behavior was already observed in modeling studies on
amino derivatives with S-configuration and probably reflects
ogous conformation about F with the torsional angle of
F=55.98 was found in the crystal structure of methyl C-gen-
tiobioside. This +gauche-type conformation has also been
[25]
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F. Peri et al.
FULL PAPER
detected by NMR spectroscopy for C-lactose as the one
bound by the active site of the enzyme E. coli b-galactosi-
dase. It appears from our experimental data (see NMR
spectroscopy section below) that solvent does not influence
equilibrium in 1, 3, and 5. Therefore, we did not calculate
solvent effects for these compounds.
the MM2* force field with the GB/SA solvation model. The
corresponding results are given in the Supporting Informa-
tion. A representation of the corresponding low-energy
minima is given in Figure 2, while the key geometric fea-
tures that may be correlated with the NMR data are given
in Table 2.
[6]
Conformational preferences around the C5ÀC6 bond in
the parent disaccharide methyl gentiobioside (7a) display
similar behavior to the N(OMe)-linked analogues. The gt ro-
tamer is the preferred rotamer in the gas phase and the pop-
ulation distribution of gg, gt, and tg rotamers was calculated
to be 35:62:3. Comparison of conformational behavior cal-
culated by this B3LYP/6-31G* method for 1, 5, and 7a show
that the effect of substitution of O1’ by the N(OMe) group,
going from 7 to 1 and 5, results in a different flexibility of 1
and 5 compared to 7a and a shift of the rotamer equilibrium
towards the gt rotamer, which is the dominant rotamer in
the N(OMe)-linked analogues 1 and 5. It is evident that cal-
culated conformational equilibrium around glycosidic link-
ages C1’ÀO1’ and O1’ÀC6 in 7a differ from those found for
1
and 5. For 7a, the average values of hFi=À1458 and
hYi=À1538 were calculated. The most significant difference
is found for the orientation around C1ÀO1: hFi=Àgauche
for 7a versus hFi= ++ gauche for 1 and 5.
The conformational characteristics of gentiobiose (7) have
been the subject of several experimental and modeling stud-
[26]
ies. The crystal structure of gentiobiose
is characterized
by the following torsional angles: F=À58.58; Y=À155.28,
w=À60.88, and F=À58.38; Y=À156.38, w=À61.58, indi-
cating that the conformation about w corresponds to the gg
rotamer and the orientation about F is in the expected posi-
tion based on the exo-anomeric effect. From the calculated
minima, the GG3 minimum characterized by the torsional
angles F=À65.68; Y=À146.98 and w=À62.48 and with the
À1
energy 1.38 kcalmol above the lowest minimum GT2 is
close to the crystal structure conformation. NMR solution
data imply conformational flexibility of gentiobiose in solu-
tion reflected in a conformational equilibrium of several
conformers about F, Y, and w. The analysis of the experi-
mental information from proton±proton and carbon±proton
vicinal coupling constants and proton±proton cross-relaxa-
tion rates yielded to the presence of two major rotamers gg
and gt about the C5ÀC6 bond, characterized by w=À68.98
ꢁ
6.38 and w=79.0ꢁ3.48, with the estimated populations of
3
4ꢁ6% and 66ꢁ6%, respectively. The calculated ratio
gg:gt:tg=35:62:3 and the calculated average torsional angles
hw i=À708 and hw i=878 are in excellent agreement
GG
GT
Figure 2. Stereoviews of the global and local minima of compounds 1±5
according to MM3* calculations. A) Conformer C (gt rotamer for w
angle) for compound 1; B) conformer C (gg rotamer for w angle) for
compound 1; C) conformer B for 1; D) conformer A for 1; E) confor-
mer D for 1; F) conformer C for compound 3; G) conformer C for com-
pound 5. See Table 1 for the F and Y torsions of the different conform-
ers.
[27]
with these estimates. In another experimental study, the
ensemble average values of vicinal heteronuclear proto-
3
n±carbon coupling constants JC,H(F)=2.68ꢁ0.06 Hz and
3
JC,H(Y)=2.36ꢁ0.06 Hz were measured across the glycosi-
dic linkage for gentiobiose. These values are compatible
with the virtual conformation of gentiobiose characterized
by the torsional angles hFi=À1458 and hYi=À1538, respec-
tively. All this suggests that conformational equilibrium in 7a
is reasonably well represented by the calculated conformers.
NMR sꢀectroscoꢀy:The predictions of the ab initio and
force field calculations were checked by using NMR spec-
troscopy. The chemical shifts in D O and [D ]acetone of
2
6
Molecular mechanics and dynamics calculations:Qualitative
compounds 1±5 are listed in Table 9S in the Supporting In-
molecular mechanics calculations were performed by using
formation. The assignment of the resonances was made
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Chem. Eur. J. 2004, 10, 1433 ± 1444

Novel N(OCH )-linked Disaccharide Analogues
1433 ± 1444
3
Table 2. Expected distances estimated from the molecular mechanics (MM) and molecular dynamics simulations (MD) for the low-energy conformers
[
a]
for compounds 1±6.
Single conformers (F/Y) according to MM2* calculations and
Average distances
from MM and MD
Observed NOEs in comparison to those
estimated from the calculations according to
a full matrix relaxation approach
their relative population [%]
Confomer [F/Y] C (54/À162)
B (54/À60)
A (54/74)
1±18
D(180/À162)
<0.5
!
Pop
82±99
0±1
!
Proton pair
Distances from MM
MM
MD
NOEs
from
MM3 [%]
7.4
NOEs
from
MD [%]
7.4
Obs.
NOE
intensity
strong
medium
weak
very
weak
weak
very
weak
very
weak
very
H1’±
H1’±H6S
2.2
3.1
2.9
3.6
3.6
2.2
3.7
3.7
2.3
3.0
2.3
2.9
1.2
1.2
H2’±H6S
4.4
4.8
4.9
2.0
4.3
4.2
4.4
0.2
0.2
H1’±H5
H1’±Me
4.5
3.2/4.3
2.7
3.2
4.8
3.2/4.3
4.3
3.6
0.3
0.3
0.3
0.3
3.2/4.3
3.0/4.7
3.0
3.2/4.3
3.0/4.7
6.4
H2’±Me
H1’±H1
3.0/4.7
3.0/4.7
6.6
4.2
1.3
1.3
6.1
6.0
6.0
0.1
0.1
weak
Coupling
constants
H5/H6proS
H5/H6proR
gg [%]
Conformer
Conformer
gt
2.5
10.7
0
Exp (1)
Exp (2)
Exp (3)
Exp (4)
Exp (5)
Exp (7a)
gg
2.9
1.2
100
0
2.6
8.3
25
2.7
8.0
29
2.8
7.8
30
2.7
8.0
29
2.8
7.6
33
2.0
6.0
66
gt [%]
100
75
71
70
71
67
34
[
a] The key distances and NOEs are given in bold. Ensembled average distances together with the calculated NOEs according to a full-matrix relaxation
approach in comparison with the intensities of the observed NOEs are also given. Two figures are given for H1’±Me and H2’±Me, indicating the two pos-
sible orientations of the OMe group at the nitrogen atom. The gt conformer was always more stable than the gg one, with relative populations greater
than 9:1 in favor of gt. The ranges in populations correspond to those obtained for the different compounds. In addition, the expected J values (Hz) for
the basic conformations around angle w for 1±5, deduced by applying the generalized Karplus equation proposed by Altona to the geometries provided
by MM3 molecular mechanics calculations, are also given. The estimated populations for the different compounds are also given in comparison to those
reported for gentiobiose (7a).
through a combination of COSY, TOCSY, 1D and 2D-
NOESY/ROESY, and HSQC experiments (Figure 3 and
Supporting Information).
mental 70%, MM2 90%). The ab initio results are in good
agreement with the observations, as they predict populations
of the gt rotamer in the 70±85% interval. Since the stereo-
lectronic effects strongly affect the conformation, different
chemical surroundings in the vicinity of w should influence
The J values for the ring protons indicate that all the pyr-
4
anose rings adopt the usual C chair, independently of the
1
[31]
size of the molecule and the nature of the glycosidic linkage.
Although this is the usual situation, in some cases alterna-
tive chair conformations have been described for some C-
the conformational populations. It is therefore not unex-
pected that the MM2 modeling data do not strictly repro-
duce the distribution of rotamers around w in compounds
1±5 and that their actual relative populations differ from
[28]
glycosyl compounds. The observed values (one small and
[30a]
one large) for the C5ÀC6 lateral chains that form part of the
those reported for the natural compounds 7 and 8.
(
1!6) linkage are in agreement with an equilibrium be-
NOESY and ROESY experiments (Figure 3) were also
carried out to deduce the relevant conformational informa-
tion of 1±5 around F and Y. The intensities of the observed
NOEs compared to those estimated by the MM2* molecular
mechanics and dynamics calculations, calculated according
to a full relaxation matrix approach, are also gathered in
Table 2. The observed NOE cross peaks were indeed very
similar for all the compounds independent of the substitu-
tion and solvent used. The analysis of the conformational
behavior of 1±5 was also facilitated by the stereospecific as-
signment of the C-6 protons. In fact, for all compounds in
D O or [D ]acetone, strong NOEs were observed in all
tween the gg and a second conformer, which appeared to be
the gt one (see Supporting Information for the detailed pro-
cedure of assignment).
The coupling values are given in Table 2, together with
the deduced relative populations of conformers around the
w angle, calculated by applying the generalized Karplus
[29]
equation proposed by Haasnoot et al. to the geometries
of the basic gg and gt rotamers. The obtained data are in
agreement with a major presence (ca. 70%) of gt rotamers,
in contrast with the observations for the natural compounds,
gentiobiose and allolactose, for which a 34:66 equilibrium
2
6
[30]
has been deduced. In addition, the experimental observa-
tions indicate that the MM2* calculations slightly overesti-
mate the experimental predominance of gt rotamers (experi-
cases between H-1’ and H-6proR, that is, the same methyl-
ene that is close to its intraresidue H-4. Therefore, the pres-
ence of this strong NOE for 1±6, as shown in Table 2, pro-
Chem. Eur. J. 2004, 10, 1433 ± 1444
www.chemeurj.org
¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1439

F. Peri et al.
FULL PAPER
1
Figure 3. H NMR 1D- and 2D-NOESY and T-ROESY data for 1±5 at 500 MHz and 300 K in D
2
O (1,3) or (CD
3
)
2
CO (2, 4, 5). Key cross peaks are indi-
cated. A) 1D-NOESY spectrum obtained after inversion of H-1’ of 1. B) 1D-NOESY spectrum obtained after inversion of H-1’ of 2. C) 1D-NOESY
spectrum obtained after inversion of H-1’ of 3. D) Trace of the 2D-T-ROESY spectrum at the frequency of H-1’ of 4. E) Trace of the 2D-T-ROESY spec-
trum at the frequency of H-1’ of 5. F) 1D-NOESY spectrum obtained after inversion of H-6proR of 3. G) 2D-T-ROESY spectrum for 3.
vides evidence for the predominance of conformers with a
C-type (FÀgauche/Yanti) orientation around the glycosidic
linkages.
Nevertheless, additional cross peaks that correspond to
the presence of the other conformers are also observed. For
instance, the presence of very weak H1’±H1 in 2, weak H1’±
1440
¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
Chem. Eur. J. 2004, 10, 1433 ± 1444

Novel N(OCH )-linked Disaccharide Analogues
1433 ± 1444
3
H5 in 1±5, and medium weak H1’±H-6proS in 1±5 indicate
the additional participation of conformers A and B. Even
the minor presence (<5%) of conformers with a (F+gau-
che/Yanti) orientation (D-type) is evidenced by the presence
of a very weak, but detectable, H2’±H-6proS NOE. The ab-
sence of the H2’±H6proR NOE cross peak in all cases also
indicates the absence of the GG4, GT5, and TG4 geome-
tries. No major orientation of the OMe group at the nitro-
gen atom could be deduced, since both the H1’±Me and
H2’±Me NOEs were equally weak in all compounds and sol-
vents. Nevertheless, the magnitudes of the observed NOEs
are in agreement with a major participation (>80%) of
anti-Y conformers, with contributions around 5±10% of the
Y+ and YÀ conformers.
existing in solution may be bound by the binding site of pro-
teins without major energy conflicts.
Exꢀerimental Section
General methods:All solvents were dried over molecular sieves (Fluka)
for at least 24 h prior to use. When dry conditions were required, the re-
actions were performed under an argon atmosphere. Thin-layer chroma-
tography (TLC) was performed on silica gel 60F254 plates (Merck) with
detection with UV light when possible, or charring with a solution con-
taining concentrated H
1
4
2
SO
808C. Column flash chromatography was performed on silica gel 230±
00 mesh (Merck). The boiling range of petroleum ether used as eluent
4 2
/EtOH/H O (5:45:45) followed by heating at
in column chromatography is 40±608C. Usual workup refers to dilution
with an organic solvent, washing with water to neutrality (pH test paper),
drying with Na SO , filtration, and evaporation under reduced pressure.
The agreement between the theoretical NOEs obtained
from the full matrix relaxation analysis from the MM2* mo-
lecular mechanics and dynamics calculations and the ob-
served experimental NOEs is more than satisfactory.
2
4
1
13
Routine H and C NMR spectra were recorded at 400 MHz on a Varian
Mercury instrument with CDCl as solvent at 300 K unless otherwise
3
stated. Chemical shifts are reported in ppm downfield from TMS as an
internal standard. Aromatic signals and methyl signals of peracetylated
Therefore, after considering the J and NOE information,
it may be considered that compounds 1±5 present a confor-
mational averaging around F, Y, and w glycosidic linkages
with participation of several conformers in the conforma-
tional equilibrium with fairly small energy barriers among
them, which somehow resembles that evidenced for the nat-
urally occurring gentiobiose and allolactose, although with
different populations in equilibrium, especially around w
1
sugars are omitted in H spectra; carbonyl and methyl carbons of perace-
1
3
tylated sugars are omitted in C spectra. Mass spectra were recorded on
a MALDI2 Kompakt Kratos instrument with gentisic acid (DHB) as
matrix. Optical rotations were measured at ambient temperature (22ꢁ
2
8C) on a Perkin±Elmer 241 polarimeter.
Model and comꢀutational ꢀrocedures:The relative orientation of two
contiguous (1!6)-linked monosaccharide residues is described by the
three torsion angles F, Y, and w, where F=V(O5ÀC1ÀXÀC6); Y=
V(C1
ÀXÀC6ÀC5); w=V(XÀC6ÀC5ÀO5), in 1±6, X represents N and in
[30]
7 and O1’ in 8, respectively. Available conformational space was moni-
tored by means of a series of two-dimensional (F, Y) potential energy
surfaces, each one corresponding to one of three different starting values
of w, namely w=À608, w=608, and w=1808. These w values character-
ize the gauche±gauche (gg), gauche±trans (gt), and trans±gauche (tg) ro-
tamers around the C5ÀC6 linkage, respectively. Torsional angles F and Y
torsion angle.
Conclusion
were varied by 308 increments, within the 0±3608 range. During the
optimization, all geometrical parameters including w were optimized.
We have demonstrated that b-(1!6)N(OMe)-linked disac-
charides can be prepared by using a chemoselective ap-
proach from unprotected precursors. Yields and stereoselec-
tivity were high in the case of compounds 1, 3, and 5. This
method has also been applied to the iterative preparation of
a trisaccharide analogue in solution. The combination of
molecular mechanics calculations and NMR experiments
permits us to conclude that compounds 1±5, which have an
N(OMe) group at the pseudoglycosidic position, have con-
formational behavior similar to their natural counterparts,
gentiobiose and allolactose (7 and 8). It seems that the dif-
ference of CÀN versus CÀO distances, together with the var-
[32]
The calculations were carried out by using the Jaguar program.
Optimization of the geometry was performed by using the B3LYP
[
33]
density functional method with the 6-31G* basis set. The geometries
of all local minima identified on the maps were then fully optimized
with no constraints on the F, Y, and w torsion angles. The single-point
energy of all minima determined at the B3LYP/6-31G* level were
calculated by using the B3LYP/6-311++G** basis set. The effect of sol-
vent on conformational equilibrium has been investigated by using a
procedure implemented in the Jaguar program at the B3LYP/6-31G*
level.
1
NMR sꢀectroscoꢀy:500 MHz H NMR spectra were recorded at 300 K
2 6
in D O (1, 3) or [D ]acetone (2, 4, 5b) on a Bruker DRX 500 spectrome-
ter. Concentrations of approximately 3mm were used. Chemical shifts are
reported in ppm, using external tetramethylsilane (TMS) or 2,2-dimethyl-
2-silapentane-5-sulfonate sodium salt (DSS, 0 ppm) as reference. The
iation of bond angles (C-N-C versus C-C-C) is at the origin
of this slightly different behavior. Regarding the w angle, it
has been reported that the conformational equilibrium be-
tween gg and gt rotamers in 7 is strongly biased by solvation
2
D-TOCSY experiment (70-ms mixing time) was performed with a
data matrix of 256î2 K to digitize a spectral width of 3000 Hz. Four
scans were used per increment with a relaxation delay of 2 s. 2D-NOESY
[30]
effects. Since the stereoelectronic effects for the natural
compounds and the N(OMe)-linked analogues must obvi-
ously be rather different at this torsion angle, these proper-
ties must be at the origin of the observed conformational
differences. Indeed, the ab initio calculations performed at
the B3LYP/6-31G* level of theory reproduce the conforma-
tional behavior of 1±5 in solution reasonably well. Neverthe-
less, all the conformations sampled by the natural com-
pounds are also accessible for these glycomimetics. On the
other hand, the estimation of relatively low energy barriers
between the different conformational regions of these ana-
logues, indicate that conformers different to the major one
(
300, 400, and 500 ms) and 2D-T-ROESY experiments (300 and 400 ms)
used the standard sequences. One-dimensional-selective NOE spectra
were acquired by using the double-echo sequence proposed by Shaka
[
34]
and coworkers at five different mixing times (200, 400, 600, 800, and
000 ms). NOESY back calculations were performed as described. All
1
the theoretical NOE calculations were automatically performed by a
home-made program, which is available from the authors upon re-
[
35]
quest.
Methyl 2,3,4-tri-O-acetyl-a-d-gluco-hexodialdo-1,5-ꢀyranoside-6-O-meth-
yloxime (11):Dess±Martin periodinane (2.98 g, 7.03 mmol) was added to
a solution of 9 (1.5 g, 4.69 mmol) in dry dichloromethane (20 mL) under
an argon atmosphere. The solution was stirred for 1 h. After dilution
with dichloromethane (30 mL), a saturated solution of sodium bicarbon-
ate and sodium thiosulfate (40 mL) was added and the mixture was vigo-
Chem. Eur. J. 2004, 10, 1433 ± 1444
www.chemeurj.org
¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1441

F. Peri et al.
FULL PAPER
+
+
rously stirred for 10 min. After usual workup compound 10 was recov-
ered as a colorless oil (1.42 g, 95%) and used without further purification
in the following reaction step. O-Methylhydroxylamine hydrochloride
55.3 ppm; MALDI-MS: m/z:408.8 [ M+Na] , 424.4 [M+K] ; elemental
analysis calcd (%) for C14
44.51, H 6.88, N 3.77.
H27NO11:C 43.63, H 7.06, N 3.63; found:C
(
(
587 mg, 7.03 mmol) was added to a solution of 10 in dry pyridine
15 mL) and the mixture was stirred for 1 h. The solvent was then evapo-
Methyl 2,3,4-tri-O-acetyl-6-deoxy-6-methoxyamino-6-N-(2’,3’,4’,6’-tetra-
O-acetyl-b-d-glucoꢀyranosyl)-a-d-glucoꢀyranoside (2): [a] =+69.4 (c=
]acetone): d=5.45 (t, 1H,
D
rated and chromatography of the residue on silica gel with petroleum
ether/ethyl acetate (60:40) gave 11 (1.16 g, 75%). [a] =+33.6 (c=1 in
methanol); H NMR: d=7.23 (d, 1H, J5,6 =7.1 Hz; H-6), 5.51 (t, 1H,
2,3 =J3,4 =10.1 Hz; H-3), 5.02 (t, 1H, J3,4 =J4,5 =10.1 Hz; H-4), 4.94 (d,
H, J1,2 =3.6 Hz; H-1), 4.86 (dd, 1H, J1,2 3.6 Hz, J2,3 =10.2 Hz; H-2), 4.30
dd, 1H, 5,6 =7.1 Hz, ),
4,5 =10.1 Hz; H-5), 3.82 (s, 3H; NÀOCH
.41 ppm (s, 3H; OCH ); C NMR: d=145.7, 97.06, 71.16, 70.30, 69.62,
7.61, 62.40, 56.05 ppm; MALDI-MS: m/z:371.7 [ M+H+Na] , 387.8
1
0
.7 in chloroform); H NMR (500 MHz, [D
6
D
J
J
2,3 =J3,4 =9.8 Hz; H-3), 5.28 (t, 1H, J2’,3’ =J3’,4’ =9.3 Hz; H-3’), 5.16 (t, 1H,
1’,2’ =J2’,3’ =9.8 Hz; H-2’), 4.99 (dd, 1H, J3’,4’ =9.3 Hz , J4’,5’ =9.5 Hz; H-4’),
1
J
1
(
3
6
4
.95 (d, 1H, J1,2 =5.0 Hz; H-1), 4.89 (t, 1H, J3,4 =J4,5 =9.8 Hz; H-4), 4.84
(
dd, 1H, J1,2 =5.0 Hz, J2,3 =9.8 Hz; H-2), 4.59 (d, 1H, J1’,2’ =9.0 Hz; H-1’),
J
J
3
4
2
2
3
.26 (dd, 1H, J5’,6’b =5.1 Hz, J6’a,6’b =12.2 Hz; H-6’b), 4.12 (dd, 1H, J5’,6’a
.4 Hz, J6’a,6’b =12.2 Hz; H-6’a), 4.01 (m, 1H; H-5), 3.85 (ddd, 1H, J5’,6’a
=
=
1
3
3
+
.4 Hz, J5’,6’b =5.1, J4’,5’ =9.5 Hz; H-5’), 3.45 (s, 3H; N OCH
À
3
), 3.44 (s,
+
[
9
M+H+K] ; elemental analysis calcd (%) for C14H21NO :C 48.41, H
H; OCH
3
), 3.21 (dd, 1H, J5,6b =8.1 Hz, J6a,6b =14.6 Hz; H-6b), 3.11 ppm
6
.09, N 4.03; found:C 48.78, H 5.87, N 4.40.
13
(
dd, 1H, J5,6a =2.2 Hz, J6a,6b =14.6 Hz; H-6a); C NMR: d=96.8, 92.4,
Methyl a-d-gluco-hexodialdo-1,5-ꢀyranoside-6-O-methyloxime (12):
catalytic amount of metallic Na was added to a solution of 11 (1.0 g,
.88 mmol) in dry methanol (30 mL) under an argon atmosphere and the
mixture was stirred for 4 h. Amberlite IRA-120 H resin was then added
until a neutral pH was reached. The mixture was then filtered to remove
A
74.5, 73.9, 71.1, 70.8, 70.3, 69.0, 68.8, 68.6, 62.5, 62.0, 55.8, 53.9 ppm;
MALDI-MS: m/z:703.0 [ M+H+Na] , 718.9 [M+H+K] ; elemental
+
+
2
analysis calcd (%) for C28
50.15, H 6.78, N 2.71.
H41NO18:C 49.48, H 6.08, N 2.06; found:C
+
Methyl 6-deoxy-6-methoxyamino-6-N-(b-d-galactoꢀyranosyl)-a-d-gluco-
yranoside (3):Standard procedure for disaccharide formation was used:
Compound 13 (100 mg, 0.45 mmol) was treated with d-galactose (97 mg,
.54 mmol) in a mixture DMF/AcOH (2:1) as solvent. Column chroma-
resin and concentrated to dryness to afford pure compound 12 (623 mg,
ꢀ
1
9
6
J
8%). [a]
D
=+23.6 (c=1 in methanol); H NMR: d=7.39 (d, 1H, J5,6
=
.1 Hz; H-6), 4.77 (d, 1H, J1,2 =3.6 Hz; H-1), 4.13 (dd, 1H, J5,6 =6.1 Hz,
4,5 =9.8 Hz; H-5), 3.88 (s, 3H; NÀOCH
), 3.77 (t, 1H, J2,3 =J3,4 =9.2 Hz;
H-3), 3.55 (dd, 1H, J1,2 =3.6 Hz, J2,3 =9.2 Hz; H-2), 3.51 (t, 1H, J3,4
0
3
tography of reaction crude with AcOEt/MeOH/water (70:20:10) afforded
=
a mixture of a- and b-disaccharides (148 mg, 85%) (b/a ratio of about 7
1
3
J
4,5 =9.5 Hz; H-4), 3.43 ppm (s, 3H; OCH
3
); C NMR: d=147.96, 99.81,
1
as determined by the integration of H-1’ protons in H NMR). Further
7
3.63, 72.35, 71.78, 69.41, 62.26, 55.91 ppm; MALDI-MS: m/z:244.2
chromatographic purification of the mixture with the same eluent afford-
+
+
[
M+Na] , 259.9 [M+K] ; elemental analysis calcd (%) for C
3.44, H 6.83, N 6.33; found:C 43.58, H 6.41, N 6.05.
Methyl 6-deoxy-6-methoxyamino-a-d-glucoꢀyranoside (13):NaCNBH
154 mg, 2.44 mmol) was added to a solution of 12 (360 mg, 1.63 mmol)
8
H15NO
6
: C
1
ed pure
3
(128 mg, 75%). [a]
D
=+74.7 (c=1 in
H
2
O); H NMR
4
(
500 MHz, D
2
O): d=4.75 (d, 1H, J1,2 =3.8 Hz; H-1), 4.05 (d, 1H, J1’,2’
=
3
9.1 Hz; H-1’), 3.89 (dd, 1H, J3’,4’ =3.4 Hz , J4’,5’ =0.6 Hz; H-4’), 3.76 (dd,
1H, J3,4 =8.2 Hz, J2,3 =9.7 Hz; H-3), 3.75 (m, 1H; H-5’), 3.74 (m, 1H; H-
6’b), 3.70 (m, 1H; H-5), 3.69 (m, 1H; H-6’a), 3.68 (dd, 1H, J3’,4’ =3.4 Hz,
(
in glacial AcOH (10 mL) under an argon atmosphere. The mixture was
stirred for 1 h, and the solvent was then evaporated in vacuo. The residue
was purified by chromatography on silica gel with ethyl acetate/methanol
J
J
2’,3’ =9.4 Hz ; H-3’), 3.58 (t, 1H, J1’,2’ =J2’,3’ =9.4 Hz; H-2’), 3.52 (dd, 1H,
1,2 =3.8 Hz, 2,3 =9.7 Hz; H-2), 3.49 (dd, 1H, 5,6b =3.7 Hz,
), 3.45 (s, 3H; OCH
J
J
6a,6b
J =
3
), 3.30 (dd,
(
90:10) to give 13 (313 mg, 86%). [a]
D
=+25.5 (c=1 in methanol);
OD): d=4.88 (brs, 1H; NH), 4.66 (d, 1H, J1,2 =3.7 Hz; H-
), 3.76 (m, 1H; H-5), 3.59 (t, 1H, J2,3 =J3,4 =9.4 Hz; H-3), 3.51 (s, 3H;
14.4 Hz; H-6b), 3.48 (s, 3H; NÀOCH
3
1
H NMR (CD
1
3
1H, J3,4 =8.2 Hz, J4,5 =9.7 Hz; H-4), 3.02 ppm (dd, 1H, J5,6a =6.7 Hz,
+
+
J
6a,6b =14.4 Hz; H-6a); MALDI-MS: m/z:408.4 [ M+Na] , 424.6 [M+K]
NÀOCH
3
), 3.41 (s, 3H; OCH ), 3.39 (dd, 1H; H-6b), 3.36 (dd, 1H, J1,2 =
; elemental analysis calcd (%) for:C 43.63, H 7.06, N 3.63; found:C
43.97, H 7.45, N 3.12.
3
3
.7 Hz, J2,3 =9.4 Hz; H-2), 3.13 (dd, 1H, J3,4 =8.8 Hz, J4,5 =9.8 Hz; H-4),
1
3
2
.82 ppm (dd, 1H,
J
5,6a =8.6 Hz,
J
6a,6b =13.4 Hz; H-6a);
C NMR
Methyl 2,3,4-tri-O-acetyl-6-deoxy-6-methoxyamino-6-N-(2’,3’,4’,6’-tetra-
O-acetyl-b-d-galactoꢀyranosyl)-a-d-glucoꢀyranoside (4): [a] =+68.6
]acetone): d=5.47 (t,
H, J2,3 =J3,4 =9.8 Hz; H-3), 5.39 (dd, 1H, J3’,4’ =3.1 Hz , J4’,5’ =1.1 Hz; H-
4’), 5.29 (dd, 1H, J1’,2’ =9.5 Hz, J2’,3’ =9.9 Hz; H-2’), 5.18 (dd, 1H, J3’,4’
3.1 Hz, J2’,3’ =9.9 Hz; H-3’), 4.97 (d, 1H, J1,2 =5.0 Hz; H-1), 4.86 (dd, 1H,
1,2 =5.0 Hz, J2,3 =9.7 Hz; H-2), 4.86 (t, 1H, J3,4 =J4,5 =9.8 Hz; H-4), 4.58
(
CD
MS: m/z:246.5 [ M+Na] , 262.6 [M+K] ; elemental analysis calcd (%)
for C :C 43.04, H 7.68, N 6.27; found:C 42.68, H 7.44, N 6.90.
Standard ꢀrocedure for disaccharide formation:Reducing sugars
0.54 mmol) were added to a solution of 13 (100 mg, 0.45 mmol) in the
3
OD): d=102.3, 76.4, 75.6, 74.9, 69.8, 62.5, 56.9, 55.0 ppm; MALDI-
D
+
+
1
(
1
c=0.7 in chloroform); H NMR (500 MHz, [D
6
8
H17NO
6
=
(
appropriate solvent mixture (5 mL), and the mixture was stirred for 24 h.
The solvents were then evaporated in vacuo and chromatography of the
residue on silica gel gave pure disaccharides. To obtain peracetylated dis-
J
(d, 1H, J1’,2’ =9.5 Hz; H-1’), 4.19 (m, 1H; H-6’b), 4.15 (m, 1H; H-6’a),
4.02 (m, 1H; H-5), 3.85 (dt, 1H, J4’,5’ =1.1 Hz, J5’,6’a =J5’,6’b =7.0 Hz; H-5’),
accharides, compounds 1, 3, and 5 (100 mg) were dissolved in Ac
dine (1:2, 5 mL) and a catalytic amount of N,N-dimethylaminopyridine
was added. The mixture was stirred 4 h and then diluted with methanol
2
O/pyri-
3.49 (s, 3H; OCH
3
), 3.48 (s, 3H; NÀOCH
3
), 3.19 (dd, 1H, J5,6b =8.3 Hz,
J6a,6b =14.8 Hz; H-6b), 3.10 ppm (dd, 1H, J5,6a =2.2 Hz, J6a,6b =14.8 Hz; H-
1
3
6a); C NMR: d=96.8, 92.8, 72.6, 72.5, 71.1, 70.8, 70.3, 69.1, 67.4, 66.3,
62.0, 61.5, 55.8, 54.0 ppm; MALDI-MS: m/z:702.8 [ M+Na] , 718.2
[M+K] ; elemental analysis calcd (%) for C28
2.06; found:C 48.85, H 6.33, N 1.91.
+
to destroy the excess of Ac
2
O, solvents were evaporated in vacuo and
+
chromatography of the residue on silica gel afforded peracetylated disac-
charides.
H
41NO18 C 49.48, H 6.08, N
Methyl 6-deoxy-6-methoxyamino-6-N-(b-d-glucoꢀyranosyl)-a-d-glucoꢀyr-
anoside (1):Standard procedure for disaccharide formation was used:
Compound 13 (100 mg, 0.45 mmol) was treated with d-glucose (97 mg,
Methyl 6-deoxy-6-methoxyamino-6-N-(2-acetamido-2-deoxy-b-d-gluco-
ꢀyranosyl)-a-d-glucoꢀyranoside (5):Standard procedure for disaccharide
formation was used:Compound 13 (100 mg, 0.45 mmol) was reacted with
d-N-acetylglucosamine (120 mg, 0.54 mmol) in a mixture DMF/AcOH/
water (2:2:1). Column chromatography of reaction crude with AcOEt/
0
.54 mmol) in a mixture DMF/0.1m aqueous sodium acetate buffer
pH 4.5 (1:1) as solvent. Column chromatography of the reaction crude
with AcOEt/MeOH/water (70:20:10) afforded 1 (140 mg, 82%). [a]
D
=
=
=
MeOH/water (70:20:10) afforded 5 (184 mg, 80%). [a]
in H O); H NMR (D
2 2
D
=+64.8 (c=0.5
O): d=4.62 (d, 1H, J1,2 =3.7 Hz; H-1), 4.09 (d,
1H, J1’,2’ =9.3 Hz; H-1’), 3.71 (brt, 1H, J1’,2’ =J2’,3’ =9.3 Hz; H-2’), 3.70 (m,
1
1
+
63.6 (c=1.5 in H
2
O); H NMR (500 MHz, D
2
O): d=4.78 (d, 1H, J1,2
4
5
.1 Hz; H-1), 4.15 (d, 1H, J1’,2’ =9.3 Hz; H-1’), 3.89 (dd, 1H, J5’,6’b
.1 Hz, J6’a,6’b =12.4 Hz; H-6’b), 3.81 (dd, 1H, J2,3 =9.7 Hz, J3,4 =8.9 Hz;
1H; H-6’a), 3.61 (m, 1H; H-5), 3.54 (dd, 1H, J5’,6’b =4.8 Hz, J6’a,6’b
12.7 Hz; H-6’b), 3.46 (t, 1H, J2,3 =J3,4 =8.9 Hz; H-3), 3.34 (dd, 1H, J1,2
=
H-3), 3.72 (dd, 1H, J5’,6’a =2.6 Hz, J6’a,6’b =12.4 Hz; H-6’a), 3.65 (m, 1H;
H-5), 3.55 (dd, 1H, J1,2 =4.1 Hz, J2,3 =9.7 Hz; H-2), 3.55 (t, 1H, J2’,3’
=
=
3.7 Hz, J2,3 =8.9 Hz; H-2), 3.36 (m, 1H; H-6b), 3.35±3.21 (m, 3H; H-3’,
H-4’, H-5’), 3.35 (s, 3H; NÀOCH
), 3.26 (s, 3H; OCH ), 3.21 (t, 1H,
3,4 =J4,5 =9.0 Hz; H-4), 2.82 ppm (dd, 1H, J5,6a =8.4 Hz, J6a,6b =14.6 Hz;
J
J
3
1
3
9
3’,4’ =9.3 Hz; H-3’), 3.51 (dd, 1H, J6a,6b =14.7 Hz; H-6b), 3.50 (t, 1H,
1’,2’ =J2’,3’ =9.3 Hz; H-2’), 3.49 (s, 3H; NÀOCH
), 3.46 (s, 3H; OCH ),
.40 (ddd, 1H, J5’,6’a =2.6 Hz, J5’,6’b =5.1 Hz, J4’,5’ =9.7 Hz; H-5’), 3.37 (dd,
H, J3’,4’ =9.3 Hz, J4’,5’ =9.7 Hz; H-4’), 3.31 (t, 1H, J3,4 =J4,5 9.6 Hz; H-4),
.01 ppm (dd, 1H, J5,6a 8.2 Hz, J6a,6b 14.7 Hz; H-6a); C NMR: d=99.8,
3.6, 77.6, 77.3, 72.9, 71.9, 71.2, 70.06, 70.05, 69.6, 62.6, 61.1, 56.4,
3
3
3
3
J
1
3
H-6a); C NMR (D
2
O): d=173.8,?99.5, 92.2, 77.7, 75.8, 73.1, 72.1, 71.3,
+
70.4, 69.8, 61.7, 61.1, 55.9, 52.8 ppm; MALDI-MS: m/z:449.7 [ M+Na] ,
1
3
+
30 2
465.6 [M+K] ; elemental analysis calcd (%) for C16H N O11:C 45.07, H
7.09, N 6.57; found:C 46.21, H 6.98, N 7.07.
1442
¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
Chem. Eur. J. 2004, 10, 1433 ± 1444

Novel N(OCH )-linked Disaccharide Analogues
1433 ± 1444
3
Methyl 2,3,4-tri-O-acetyl-6-deoxy-6-methoxyamino-6-N-(3’,4’,6’-tetra-O-
was stirred for 12 h, after which the solvents were evaporated in vacuo.
The solid residue was then dissolved in acetic anhydride/pyridine (1:2,
3 mL). The mixture was stirred for 2 h and then concentrated. Chroma-
acetyl-2-acetamido-2-deoxy-b-d-glucoꢀyranosyl)-a-d-glucoꢀyranoside (6):
1
[
a]
D
=+68.4 (c=0.5 in chloroform); H NMR (500 MHz, [D
6
]acetone):
d=5.47 (t, 1H, J2,3 =J3,4 =9.9 Hz; H-3), 5.25 (dd, 1H, J2’,3’ =9.0 Hz, J3’,4’
=
tography of the residue with petroleum ether/AcOEt (4:3) afforded pure
1
9
1
.3 Hz; H-3’), 4.96 (dd, 1H, J3’,4’ =9.3 Hz , J4’,5’ =9.6 Hz; H-4’), 4.92 (d,
H, J1,2 =5.1 Hz; H-1), 4.83 (dd, 1H, J1,2 =5.1 Hz, J2,3 =9.9 Hz; H-2), 4.81
19 (41 mg, 47%). [a]
D
=+78.8 (c=0.6 in chloroform); H NMR: d=7.16
(d, 1H, J5’’,6’’ =7.1 Hz; H-6’’), 5.09 (m, 1H; H-5), 5.01 (t, 1H, J1’,2’ =J2’,3’
=
(
t, 1H, J3,4 =J4,5 =9.9 Hz; H-4), 4.53 (d, 1H, J1’,2’ =9.0 Hz; H-1’), 4.25 (m,
9.3 Hz; H-2’), 5.40±4.80 (m, 9H; H-2’’, H-3’’, H-4’’, H-3’, H-4’, H-1, H-2,
H-3, H-4), 4.22 (d, 1H, J1’’,2’’ =9.3 Hz; H-1’’), 4.20 (d, 1H, J1’,2’ =9.3 Hz;
1
1
3
H; H-6’b), 4.16 (dd, 1H, J1’,2’ =9.0 Hz, J2’,3’ =10.1 Hz; H-2’), 4.11 (m,
H; H-6’a), 4.02 (m, 1H; H-5), 3.78 (m, 1H; H-5’), 3.52 (s, 3H; OCH ),
), 3.20 (m, 1H; H-6b), 3.14 ppm (m, 1H; H-6a);
H-1’), 4.01 (m, 2H; H-5’, H-5’’), 3.76 (s, 3H; NOCH
NOCH ), 3.38 (s, 3H; NOCH ), 3.35 (s, 3H; OCH ), 3.20 (dd, 1H, J5,6b
2.5 Hz, J6a,6b =14.6 Hz; H-6b), 2.92 (dd, 1H, J5,6b =8.6 Hz, J6a,6b =14.6 Hz;
H-6a), 2.82 (m, 1H; H-6’a), 2.72 ppm (dd, 1H, J5,6’b =7.3 Hz, J6’a,6’b
3
), 3.41 (s, 3H;
3
.49 (s, 3H; NÀOCH
3
3
3
=
3
1
3
C NMR: d=96.98, 94.22, 75.07, 74.11, 71.19, 70.52, 70.46, 68.73, 68.16,
+
6
2.75, 56.03, 51.27, 23.69 ppm; MALDI-MS: m/z:701.4 [ M+Na] , 717.6
=
1
3
+
[
M+K] ; elemental analysis calcd (%) for C28
H
42
N
2
O
17:C 49.56, H 6.24,
14.1 Hz, H-6’b); C NMR: d=145.4, 96.85, 96.79, 92.6, 74.1, 73.5, 71.2,
7
5
1.1, 70.8, 70.5, 70.06, 70.00, 69.6, 69.3, 69.1, 67.4, 63.3, 62.3, 60.7, 60.6,
N 4.13; found:C 50.65, H 6.43, N 4.86.
+
+
6.06, 56.03 ppm; MALDI-MS: m/z:1005.4 [ M+Na] , 1020.8 [M+K]
;
2
,3,4-Tri-O-acetyl a-d-gluco-hexodialdo-1,5-ꢀyranose-6-O-methyloxime
15):Compound 15 was obtained from 14 (100 mg, 0.29 mmol) through
Dess±Martin oxidation in dry CH Cl followed by reaction with O-
elemental analysis calcd (%) for C40
found:C 49.22, H 6.61, N 5.28.
59 3
H N O25:C 48.93, H 6.06, N 4.28;
(
2
2
methyl hydroxylamine hydrochloride in pyridine as described for the syn-
thesis of compound 11. Intermediate aldehyde at C-6 was not purified
and final column chromatography with ethyl acetate/petroleum ether
Acknowledgement
(
D
1:1) afforded pure 15 (81 mg, 75%). [a] =+64.8 (c=0.5 in chloroform);
1
H NMR d=7.07 (d, 1H, J5,6 =6.8 Hz, H-6), 6.25 (d, 1H, J1,2 =3.0 Hz;
We thank the EU for funding (HPRN-CT-2002±00251). J.J.-B. thanks the
Ministry of Science and Technology of Spain (grant BQU2000-C1501).
I.T. thanks the Scientific Grant Agency of the Ministry of Education of
Slovak Republic and the Slovak Academy of Sciences (grant VEGA-2/
H-1), 5.45 (t, 1H, J2,3 =J3,4 =9.9 Hz; H-3), 4.86 (t, 1H, J3,4 =J4,5 =9.6 Hz;
H-4), 4.86 (m, 1H; H-2), 4.38 (m, 1H; H-5), 3.76 ppm (s, 3H; NÀOCH
);
C NMR: d=141.7, 90.8, 72.2, 71.0, 68.8, 66.6, 63.5, 54.9 ppm; MALDI-
3
1
3
+
+
MS: m/z:398.0 [ M+Na] , 414.0 [M+K] ; elemental analysis calcd (%)
3
077/23).
for C 48.00, H 5.64, N 3.73; found:C 49.11, H 5.17, N 4.32.
a- and b-d-gluco-hexodialdo-1,5-ꢀyranose-6-O-methyloxime (16):Deace-
tylation of 15 (100 mg, 0.27 mmol) with MeONa in MeOH was carried
out according to the procedure described for the preparation of 12. Com-
pound 16 was recovered as a 1:1 mixture of a and b anomers (55 mg,
[
1] a) Y. C. Lee, R. T. J. Lee, Biomol. Eng. 1997, 3, 221±237; b) Glyco-
sciences: Status and perspectives (Eds.:H.-J. Gabius, S. Gabius),
Chapman & Hall, London, 1997.
+
+
9
8%). MALDI-MS: m/z:230.4 [ M+Na] , 246.8 [M+K] ; elemental
analysis calcd (%) for C :C 40.58, H 6.32, N 6.76; found:C
0.32, H 5.98, N 6.09.
[2] a) J.-F. Espinosa, F. J. CaÊada, J. L. Asensio, M. MartÏn-Pastor, H.
Dietrich, M. MartÏn-Lomas, R. R. Schmidt, J. Jimÿnez-Barbero, J.
Am. Chem. Soc. 1996, 118, 10862±10871; b) A. Wei, K. M. Boy, Y.
Kishi, J. Am. Chem. Soc. 1995, 117, 9432±9436.
[3] a) W. Levy, D. Chang, Chemistry of C-glycosides, Elsevier, Cam-
bridge, 1995; b) R. U. Lemieux, S. Koto, D. Voisin, ACS Symp. Ser.
1979, 87, 17±29; c) G. R. J. Thatcher, The Anomeric Effect and As-
sociated Stereoelectronic Effects, American Chemical Society, Wash-
ington DC, 1993; d) I. Tvaroska, T. Bleha, Adv. Carbohydr. Chem.
Biochem. 1989, 47, 45±103; e) A. J. Kirby, The Anomeric Effect and
Related Stereoelectronic Effects at Oxygen, Springer, Heidelberg,
Germany, 1983.
7
H13NO
6
4
Methyl 6-deoxy-6-methoxyamino-6-N-(b-d-gluco-hexodialdo-1,5-ꢀyrano-
side-6-O-methyloxime)-a-d-glucoꢀyranoside (17):Compound 16 (45 mg,
0
.22 mmol) was added to a solution of 13 (50 mg, 0.22 mmol) in DMF/
AcOH (2:1, 3 mL) and the mixture was stirred 6 h. Solvents were evapo-
rated in vacuo and column chromatography of the residue with AcOEt/
MeOH/water (60:40:1) afforded pure 17 (74 mg, 82%). [a]
D
=+25.5 (c=
OD): d=7.28 (d, 1H, J6’,5’ =7.2 Hz; H-
’), 4.65 (d, 1H, J1,2 =3.7 Hz; H-1), 4.16 (d, 1H, J1’,2’ =9.0 Hz; H-1’), 3.84
), 3.75 (m, 1H; H-5), 3.74 (m, 1H; H-5’), 3.62 (s, 3H;
), 3.61 (t, 1H, J2,3 =J3,4 =8.9 Hz; H-3), 3.52 (t, 1H, J1’,2’ =J2’,3’
3
.0 Hz; H-2’), 3.44 (s, 3H; OCH ), 3.44±3.36 (m, 4H; H-6a, H-3’, H-2, H-
1
0
.5 in methanol); H NMR (CD
3
6
(
s, 3H; NÀOCH
3
NÀOCH
=
[
4] J. L. Asensio, J. F. Espinosa, H. Dietrich, F. J. CaÊada, R. R.
Schmidt, M. MartÏn-Lomas, S. Andrÿ, H.-J. Gabius, J. Jimÿnez-Bar-
bero, J. Am. Chem. Soc. 1999, 121, 8995±9000.
3
9
4
’), 3.19 (dd, 1H, J3,4 =8.9 Hz, J4,5 =9.6 Hz; H-4), 3.05 ppm (dd, 1H,
1
3
[5] R. Ravishankar, A. Surolia, L. Vijayan, Y. Kishi, J. Am. Chem. Soc.
998, 120, 11297±11303.
[6] J. F. Espinosa, E. Montero, A. Vian, J. L. GarcÏa, J. L. Dietrich,
R. R. Schmidt, M. Martin-Lomas, A. Imberty, F. J. CaÊada, J. Jimÿ-
nez-Barbero, J. Am. Chem. Soc. 1998, 120, 1309±1318.
J
5,6a =7.3 Hz, J6a,6b =14.3 Hz; H-6b); C NMR (CD
3
OD): d=147.6, 100.4,
1
9
4.3, 77.7, 75.7, 73.7, 73.0, 72.3, 71.9, 70.4, 70.3, 61.3, 61.0, 55.4, 55.3 ppm;
+
+
MALDI-MS: m/z:435.8 [ M+Na] , 451.4 [M+K] ; elemental analysis
calcd (%) for C15 11:C 43.69, H 6.84, N 6.79; found:C 44.12, H
.11, N 6.77.
Methyl 6-deoxy-6-methoxyamino-6-N-(6-deoxy-6-methoxyamino-b-d-glu-
coꢀyranosyl)-a-d-glucoꢀyranoside (18):NaCNBH (15 mg, 0.24 mmol)
was added to a solution of 17 (50 mg, 0.12 mmol) in glacial AcOH
5 mL), and the mixture was stirred for 40 min. Solvents were evaporated
in vacuo and column chromatography of the residue with AcOEt/MeOH/
water (70:30:1) afforded pure 18 (26 mg, 53%). [a] =+31.2 (c=0.5 in
OD): d=4.64 (d, 1H, J1,2 =3.8 Hz; H-1), 3.91 (m,
H), 3.80 (m, 1H), 3.76 (m, 1H; H-5), 3.61 (m, 1H), 3.60 (t, 1H, J2,3
3,4 =9.0 Hz; H-3), 3.57 (s, 3H; NÀOCH ), 3.52 (s, 3H; NÀOCH
), 3.45 (s,
), 3.39 (dd, 1H, J1,2 =3.8 Hz, J2,3 =9.7 Hz; H-2), 3.23 (m, 2H;
28 2
H N O
6
[
7] a) B. D. Johnston, B. M. Pinto, J. Org. Chem. 2000, 65, 4607±4617;
b) G. Hummel, O. Hindsgaul, Angew. Chem. 1999, 111, 1900-1902;
Angew. Chem. Int. Ed. 1999, 38, 1782±1784.
3
[
[
8] H. Driguez, ChemBioChem 2001, 2, 311±318 and references there-
in.
9] J. S. Andrews, T. Weimar, T. P. Frandsen, B. Svensson, B. M. Pinto,
J. Am. Chem. Soc. 1995, 117, 10799±11804, and references therein.
(
D
1
2 3
H O); H NMR (CD
[
[
[
10] F. Schweizer, Angew. Chem. 2002, 114, 240-264; Angew. Chem. Int.
2
=
Ed. 2002, 41, 230±253, and references therein.
J
3
3
11] K. C. Nicolaou, H. Flˆrke, M. G. Egan, T. Barth, V. A. Estevez, Tet-
rahedron Lett. 1995, 36, 1775±1778.
12] K. H. Jung, M. M¸ller, R. R. Schmidt, Chem. Rev. 2000, 100, 4423±
3
H; OCH
3
H-6’a, H-5’), 3.19 (dd, 1H, J3,4 =9.0 Hz, J4,5 =9.7 Hz; H-4), 3.00 (dd, 1H,
5,6’b =5.2 Hz, J6’a,6’b =13.2 Hz; H-6’b), 2.90 (dd, 1H, J5,6a =7.1 Hz, J6a,6b
3.0 Hz; H-6a), 2.84 ppm (dd, 1H, J5,6a =8.0 Hz, J6a,6b =13.2 Hz; H-6a);
J
=
4
442.
1
[
[
13] P. H. Seeberger, W.-C. Haase, Chem. Rev. 2000, 100, 4349±4393.
14] L. A. Marcaurelle, C. R. Bertozzi, J. Am. Chem. Soc. 2001, 123,
1
3
C NMR (CD
3
OD): d=100.3, 74.8, 73.7, 74.0, 72.3, 70.9, 70.7, 69.4, 67.8,
+
6
[
2.3, 60.2, 60.0, 55.5, 55.2 ppm; MALDI-MS: m/z:437.2 [ M+Na] , 453.7
M+K] ; elemental analysis calcd (%) for C15H N O11:C 43.47, H 7.30,
30 2
1
587±1595.
+
[
15] E. C. Rodriguez, K. A. Winans, D. S. King, C. R. Bertozzi, J. Am.
Chem. Soc. 1997, 119, 9905±9906.
N 6.76; found:C 43.02, H 6.98, N 6.17.
Trisaccharide 19:Compound 16 (27 mg, 0.13 mmol) was added to a solu-
tion of 18 (36 mg, 0.087 mmol) in DMF/AcOH (1:1, 2 mL). The mixture
[16] S. E. Cervigni, P. Dumy, M. Mutter, Angew. Chem. 1996, 108, 1325±
1328; Angew. Chem. Int. Ed. 1996, 35, 1230±1232.
Chem. Eur. J. 2004, 10, 1433 ± 1444
www.chemeurj.org
¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1443

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