The activation of fibroblast growth factors by heparin: Synthesis and structural study of rationally modified heparin-like oligosaccharides

Article abstract of DOI:10.1139/v02-023

Heparin-like hexasaccharide 3 and octasaccharide 4 have been synthesized using a convergent block strategy and their solution conformations have been determined by NMR spectroscopy. Both oligosaccharides contain the basic structural motif of the regular region of heparin but have been constructed as to display negatively charged sulfate groups only on one side of their solution helical structures. This charge distribution along the saccharide chain has been designed to get insight into the proposed mechanism for fibroblast growth factors (FGFs) activation that involves heparin-induced FGF dimerization.

Full text of DOI:10.1139/v02-023

917
The activation of fibroblast growth factors by
heparin: Synthesis and structural study of
rationally modified heparin-like oligosaccharides
Rafael Ojeda, Jesús Angulo, Pedro M. Nieto, and Manuel Martín-Lomas
Abstract: Heparin-like hexasaccharide 3 and octasaccharide 4 have been synthesized using a convergent block strategy
and their solution conformations have been determined by NMR spectroscopy. Both oligosaccharides contain the basic
structural motif of the regular region of heparin but have been constructed as to display negatively charged sulfate
groups only on one side of their solution helical structures. This charge distribution along the saccharide chain has
been designed to get insight into the proposed mechanism for fibroblast growth factors (FGFs) activation that involves
heparin-induced FGF dimerization.
Key words: heparin oligosaccharides, synthesis design, conformational analysis, FGF activation.
Résumé : On a réalisé la synthèse d’un hexasaccharide (3) et d’un octasaccharide (4) apparenté à l’héparine en faisant
appel à une stratégie de synthèse convergente par blocs et on a déterminé leurs conformations en solution à l’aide de la
spectroscopie RMN. Les deux oligosaccharides comportent le motif structurel de base de la région régulière de
l’héparine, mais leurs structures ont été organisées d’une façon telle qu’elles ne comportent des groupes sulfates
chargés négativement que sur un seul côté de leurs structures hélicoïdales en solution. Cette distribution des charges
ainsi que la chaîne du saccharide ont été choisies afin d’avoir un aperçu du mécanisme proposé pour l’activation des
facteurs de croissance du fibroblaste (FCF) qui implique une dimérisation des FCF induite par l’héparine.
Mots clés : oligosaccharides de l’héparine, modèle de synthèse, analyse conformationnelle, activation des FCF.
[Traduit par la Rédaction] Ojeda et al. 936
Introduction
oligomerization and that these oligomers would interact with
FGFRs in the presence of divalent cations giving rise to
FGFR dimerization, thus inducing the cellular response (13).
According to sedimentation equilibrium experiments, the
formation of FGF-2 dimers in the presence of heparin hav-
ing a trans (with two FGF molecules on each side of the
polysaccharide chain) or a cis (with two FGF molecules on
the same side of the polysaccharide chain) configuration has
been proposed (14). The existence of both types of dimeric
structures has been confirmed by NMR spectroscopy and
light scattering (15). The trans dimer has also been observed
for the FGF-1-heparin complex using X-ray crystallography
(7).Whether this type of mechanism, which was originally
proposed for FGF-2 (14), would be generally applicable to
all the members of the FGF family is still a matter of discus-
sion as the biologically active configuration of the heparin-
FGF and the heparin-FGF–FGFR complexes are still open
questions (16, 17). The establishment of the molecular basis
of the entire biological process and the understanding of the
The fibroblast growth factors (FGFs) constitute a family
of signaling polypeptides that intervene in a variety of bio-
logical processes by stimulating key cellular functions after
binding to specific receptor tyrosine kinases at the cell sur-
face (FGFRs) (1). The biological activity of FGFs is tightly
regulated by heparin or heparan sulfate glycosaminoglycans
(GAGs) (2, 3). Binding of these GAGs to FGFs protects
FGFs from degradation (4), however, the precise structural
requirements for the GAG chains to regulate the biological
activity of the FGFs are not yet well understood at the mo-
lecular level. A number of crystal structures of FGF com-
plexes with heparin fragments (5–7), and of FGF-2 (basic
FGF) and FGF-1 (acidic FGF) bound to variants of FGFR
(8–11) have been determined and several hypotheses on the
structure–activity relationship of these complexes have been
expressed (12). It is widely accepted that heparin and (or)
heparan sulfate would bind to FGFs to cause FGF
Received 7 August 2001. Published on the NRC Research Press Web site at http://canjchem.nrc.ca on 19 July 2002.
Dedicated to the memory of Ray Lemieux whose outstanding vision of chemistry and strong personality deeply influenced the early
days of the Carbohydrate Group in Seville. We consider ourselves privileged to have been connected to him during the last years
of his fruitful life.
R. Ojeda, J. Angulo, P.M. Nieto, and M. Martín-Lomas.1 Grupo de Carbohidratos. Instituto de Investigaciones Químicas, CSIC,
Américo Vespucio s/n, Isla de la Cartuja, 41092 Seville, Spain.
1Corresponding author (e-mail: manuel.martin-lomas@iiq.cartuja.csic.es).
Can. J. Chem. 80: 917–936 (2002)
DOI: 10.1139/V02-023
© 2002 NRC Canada

918
Can. J. Chem. Vol. 80, 2002
different functions of the FGF family at the molecular level
remain, therefore, a difficult problem of chemical and bio-
medical interest.
heparin-like oligosaccharides to investigate the activation of
FGF-1 (21). Both compounds 1 and 2 induced the mitogenic
activity of FGF-1 but, as may be expected (31–35), the acti-
vating effect of octasaccharide 2 reached a maximum at ap-
proximately the same concentration than natural low
molecular weight heparin while considerably higher concen-
trations of 1 were needed to reach an equivalent maximal ac-
tivation (21). Most interestingly, sedimentation equilibrium
analysis results showed that the profile of FGF-1 solutions
in the presence of activating concentrations of 2 corre-
sponded to monomeric species thus suggesting that heparin-
induced dimerization of FGF-1 in the culture medium sur-
rounding the cell may not be an absolute requirement for
FGF-1-induced mitogenesis signaling (21).
The above result prompted us to apply our synthetic strat-
egy to the preparation of hexasaccharide 3 and octa-
saccharide 4 and to investigate their behavior in FGF-1
activation. We reasoned that, assuming that 3 and 4 would
present a solution conformation similar to that shown by 1
and 2, these structures (3 and 4) would display negatively
charged sulfate groups only on one side of their right-handed
helical structures (Fig. 1). Therefore, if the interaction with
the polypeptide is primarily electrostatic in nature, the abil-
ity of 3 and 4 to form trans dimers with FGF-1would be
greatly decreased and, because of their relatively reduced
size, both of them would hardly be involved in the formation
of cis dimers.
One of the difficulties of these investigations resides in
the inherent heterogeneity of the GAGs. Heparin and
heparan sulfate are primarily constituted by disaccharide re-
peating units of L-iduronic acid and D-glucosamine linked by
α1 → 4 glycosidic linkages and typically containing sulfate
groups at position 2 of the L-iduronic acid units and posi-
tions 2 and 6 of the D-glucosamine units. But these polysac-
charides show significant heterogeneity in terms of sulfation
pattern, size, and even carbohydrate sequence (18–20). For
this reason it is highly desirable to develop effective synthe-
ses of these heparin-like oligosaccharide chains with defined
size, sequence, and charge distribution to be used in the in-
teraction studies. In this context, we recently reported a syn-
thetic strategy the effectiveness of which was illustrated by
the preparation of hexasaccharide 1 and octasaccharide 2
(21). It has to be mentioned that these oligosaccharides con-
tain L-iduronic acid residues at the reducing end, a sequence
which is not available either by enzymatic or chemical deg-
radation of heparin, and that oligosaccharides with the alter-
native sequence (22–24) and di- and tetrasaccharide
sequences containing the above structural motif (25) have
previously been synthesized. The overall structures of 1 and
2 in solution (21) resulted in stable right hand helixes with
four residues per turn as those reported for natural heparin
fragments (26–30). Therefore, 1 and 2 were used as chemi-
cally well-defined structural models of naturally occurring
In this paper, we report the synthesis of 3 and 4 and pres-
ent data that indicate that, as expected, their overall solution
OSO3
O
OSO3
O2C
OH
O
O
O
OSO3
O2C
OH
O
O
O
O2C
OH
O
HO
O
O
NH
HO
HO
O
SO3
NH
SO3
HO
O3SO
O
NH
SO3
O3SO
O3SO
E
n
n =1
n = 2
1
2
F
D
C
B
A
OH
OSO3
O
O2C
OH
O
3
O
O
O2C
OH
OH
O
O
O
O2C
OH
O
HO
O
O
NH
SO3
HO
O
HO
NHAc
HO
O3SO
O
NH
SO3
HO
O3SO
F
E
D
C
B
A
OH
OSO3
O
O
O2C
OH
O
O
OH
O2C
OH
O
O
OSO3
O
O
O2C
OH
O
HO
O
O
NH
SO3
O2C
4
OH
HO
O
O
NHAc
HO
HO
HO
O
O
NH
OSO3
O
HO
SO3
NHAc
OSO3
HO
H
G
F
E
D
C
B
A
© 2002 NRC Canada

Ojeda et al.
919
Fig. 1. Crystallographic structure of an FGF-1 cis-dimer (protein data bank accession code 1axm) (7) and schematic representation of
hexasaccharides 1 and 3.
6
NH
2
6
G
1
G
G
I
G
G
I
I
I
I
6
NH
2
NH
2
G
I
3
6
2
NH
NH
2
G
I
Glucosamine
Iduronic acid
Sulfate group
structures are right-handed helixes with four residues per
turn. These compounds showed an unexpected biological be-
havior that will be discussed in detail elsewhere.
Scheme 2 summarizes the synthesis of the disaccharide
building blocks. The nonreducing-end building block 6 was
prepared from 9 as already reported (21). This disaccharide
(6) also provided the reducing-end building block 8 after
glycosylation with isopropanol to give 12 (21) followed by
regioselective reductive opening of the benzylidene acetal
(36). The alternative building block 14 was also prepared
from 12 that was transformed into diol 13 (21) and
regioselectively acetylated (4% of 13 was recovered). The
inner region building block 7 was also prepared from 9 that
was transformed into 17 either by selective reduction of the
azido group with thioacetic acid (37) to give 15 followed by
conventional acetylation or by using the reverse sequence
9 → 16 → 17. This latter route was more convenient as
acetylation of 15 took place sluggishly most likely as a con-
sequence of hydrogen bonding between the hydroxyl and the
acetamido groups.
Glycosylation of low-reactive acceptor 8 with donor 7 af-
forded tetrasaccharide 19 in 50% yield and unreacted accep-
tor (46%) (Scheme 3). Compound 19 was deacetylated to
give 20 which was benzylated under neutral conditions (38)
to yield 21. Hydrolysis of the benzylidene acetal (39) gave
diol 22 that was regioselectively benzoylated (40) to afford
tetrasaccharide acceptor 23.
Results and discussion
Synthesis
We have reported the synthesis of hexasaccharide 1 and
octasaccharide 2 using a convergent n + 2 block approach
from key disaccharide structures, that may operate as gly-
cosyl donors or as glycosyl acceptors, having protective
group patterns that permit formation of the required
stereochemistry of the glycosidic linkages and to locate the
sulfate groups at the desired positions at a later stage (21).
This approach has now proven to be of wide applicability
allowing the synthesis of oligosaccharide sequences with
different size and sulfation patterns as the target hexa-
saccharides 3 and 4. As indicated in Scheme 1 the synthesis
of 3 was envisaged from the fully protected hexasaccharide
5 that could be prepared from disaccharide building blocks
6, 7, and 8. These building blocks were obtained from a
common disaccharide (9) that is readily available from the
2-azido-2-deoxy-D-glucopyranosyl trichloroacetimidate 10
and the iduronic acid glycosyl acceptor 11 (21).
© 2002 NRC Canada

920
Can. J. Chem. Vol. 80, 2002
Scheme 1.
OH
OSO3Na
O
O
NaO2C
OH
O
O
OH
NaO2C
OH
O
O
O
NaO2C
OH
O
HO
O
O
NH
SO3Na
HO
O
HO
HO
NHAc
NaO3SO
O
NH
SO3Na
HO
3
NaO3SO
OBn
O
OBz
O
MeO2C
OBn
O
O
MeO2C
O
OBn
O
O
BnO
O
O
MeO2C
OBn
O
O
N3
Ph
BnO
O
O
NHAc
BnO
PivO
O
N3
BnO
5
PivO
OBn
NH
O
NH
O
O
O
MeO2C
O
OBn
O
O
MeO2C
MeO2C
O
OBn
OBn
O
O
Ph
Ph
O
HO
BnO
O
CCl3
O
O
CCl3
O
BnO
BnO
NHAc
O
N3
N3
AcO
7
PivO
PivO
8
6
O
O
MeO2C
OBn
Ph
O
O
BnO
O
N3
OTDS
HO
9
OBn
O
O
Ph
NH
O
OTDS
MeO2C
OH
O
BnO
O
CCl3
N3
OH
10
11
In spite of the moderate yield of the glycosylation reac-
tion, this synthetic route to 23 was more convenient than that
using acceptor building block 14 since the deactivating ef-
fect of the acetyl group in 14 further complicated the cou-
pling with 7 to give 24 with formation of orthoester and
considerable elimination (25% of 2-acetoxyglycal). Com-
pound 24 could be conventionally deacetylated to give 25
that was benzylated to yield 21 (52%).
The coupling of 23 with donor 6 afforded hexasaccharide
5 in 65% yield. The methoxycarbonyl and acyl groups in 5
were removed by treatment with lithium hydroperoxide and
then hydroalcoholic potasium hydroxide (41, 42) and the re-
sulting partially protected hexasaccharide 26 was sulfonated
and isolated as the sodium salt 27. Hydrogenolytic cleavage
of the benzylidene acetal and benzyl groups and simulta-
neous reduction of the azido groups in 27 followed by selec-
tive N-sulfonation yielded compound 3 that was purified
using a protocol partially based on the reported purification
of synthetic oligosaccharides of the irregular region of hepa-
rin (42).
building block (34) was prepared as indicated in Scheme 4
from the iduronic acid derivative 11 (21, 43) and
trichloroacetimidate 29 that was obtained from 28 (21, 44).
Coupling of 29 with 11 gave disaccharide 30 that was
transformed into trichloroacetimidate 34 following the se-
quence 30 → 31 → 32 → 33 → 34 as indicated in Scheme 4.
Building block acceptor 35 was prepared by regioselective
reductive opening of the benzylidene acetal group in 5
(Scheme 5). As expected (21), the glycosylation reaction be-
tween 34 with 35 took place in only moderate yield (40%).
However, no attempt was made to optimize this reaction
since a reasonable amount of octasaccharide 36 was isolated
that allowed the subsequent transformations leading to suffi-
cient 4 for structural and biological investigation. The rele-
vance of 4 for these further studies, which was not yet
known at this stage, would dictate whether working out some
changes in this synthetic strategy may be worthwhile in the
near future. The introduction of a needed permanent protect-
ing group at position 2 of the G unit was satisfactorily car-
ried out by selective deacylation using catalytic sodium
methoxide in methanol to obtain 37 (80%) followed by
benzylation under neutral conditions (38) to afford 38. The
carboxymethyl and O-acyl groups were then removed by
treatment with lithium hydroperoxide then alcoholic potasium
The elongation of the oligosaccharide chain to obtain an
octasaccharide (4) was performed by adding
a new
disaccharide building block (34) to the nonreducing end of
the previously constructed hexasaccharide structure 5. This
© 2002 NRC Canada

Ojeda et al.
921
Scheme 2. Reagents and conditions: (a) NaBH3CN, THF, HCl–Et2O, 92%; (b) EtSH, PTSA, CH2Cl2, 90%; (c) Ac2O, Py, CH2Cl2,
–10°C, 85%; (d) AcSH, 73–77%; (e) Ac2O, Py, 93–77%; (f) (HF)n·Py, THF, –15°C, 78%; (g) K2CO3, Cl3CCN, CH2Cl2, 94%.
9
d,c or c,d
See ref. 21
6
O
O
MeO2C
O
OBn
O
Ph
O
OR1
BnO
X
See ref. 21
R2O
15
16
R =TDS, R =H, X=NHAc
1 2
O
O
MeO2C
OBn
O
O
Ph
O
R =TDS, R =Ac, X=N
3
BnO
1
2
O
N3
12
PivO
b and c
17
18
7
R =TDS, R =Ac, X=NHAc
1 2
f
a or
R =H, R =Ac, X=NHAc
1
2
g
R =C(NH)CCl , R =Ac, X=NHAc
1
3
2
OR2
O
8
R =H, R =Bn
1 2
MeO2C
OBn
O
O
R1O
BnO
O
13 R =R =H
1 2
N3
PivO
14 R =H, R =Ac
1
2
hydroxide to give 39 that was O-sulfonated to yield 40.
Hydrogenolytic cleavage of the benzyl groups with concom-
itant reduction of the azido groups of 40 followed by N-sul-
fonation gave finally octasaccharide 4 that was purified as 3.
in the C and G units and the further modifications in the
neighboring glucosamine residues, absence of 6-O-sulfate
group in the B and F units and of N-sulfate group in the D
and H units, do not seem to qualitatively modify the
conformational behavior of these iduronate rings with re-
spect to heparin. This result is also in agreement with previ-
ous findings that similar structural changes in natural
heparin fragments do not influence the inner iduronate ring
conformational equilibrium (48).
With regard to the conformation around the glycosidic
linkages, the NOEs pattern observed for hexasaccharide 3
was compared to that previously obtained for the parent
hexasaccharide 1 (see supplementary data).2 There were no
indications of conformational changes as no major differ-
ences were observed and only small intensity variations,
which could be attributed to differences in correlation times,
were detected. The possibility that the changes in charge dis-
tribution in 3 with respect to 1 may partially drive the
glycosidic angles towards an anti-ψ arrangement could,
therefore, be discarded. Furthermore, the exclusive NOEs
that should be expected for such anti-ψ situations (H1′-H5
for the IdoA–GlcN linkages and H5′-H3 for the GlcN–IdoA
ones) could not be observed in NOESY experiments with
hexasaccharide 3.
Structural study
The 1H and 13C NMR spectra of 3 and 4 were fully
assigned using conventional 1D and 2D spectroscopy (Table 1).
These assignments were carried out by identification of the
spin systems of the residues and further connection based on
inter-residue NOE. The substitution pattern in 3 and 4 led to
a better signal dispersion than that observed in the spectra of
hexasaccharide 1 and octasaccharide 2. The chemical shift
values were in agreement with those expected according to
reported data for chemicaly modified heparin (45, 46). Three
bond coupling constants were obtained from 2D DQF-
COSY(47). As expected, the D-glucosaminyl residues
4
showed a single C1 conformation and the ω rotamer of the
6-O-sulfated units appeared as in 1 (21) as the gg conformer.
The observed values for the coupling constants of the
iduronate residues could be accounted for by the existence
1
2
of a conformational equilibrium between C4 and So forms
as reported for heparin (Table 2), also confirmed by the H2-
H5 NOE (30). Therefore, the lack of the 2-O-sulfate group
2 Supplementary material may be purchased from the Depository of Unpublished Data, Document Delivery, CISTI, National Research Coun-
cil Canada, Ottawa, ON K1A 0S2, Canada (http://www.nrc.ca/cisti/irm/unpub_e.shtml for information on ordering electronically).
© 2002 NRC Canada

922
Can. J. Chem. Vol. 80, 2002
Scheme 3. Reagents and conditions: (a) TMSOTf, CH2Cl2, 50%; (b) MeONa, MeOH, 96%; (c) Ag2O, BnBr, DMF, 88%; (d) EtSH,
PTSA, CH2Cl2, 83%; (e) BzCN, Et3N, CH3CN, –40°C, 88%; ( f ) TMSOTf, CH2Cl2, 65%; (g) (i) H2O2, LiOH aq; (ii) KOH aq,
MeOH, 98%; (h) SO3·Py, Py, 72%; (i) Pd/C, H2, MeOH–H2O (9:1); (j) SO3·Py, H2O, pH = 9.5, 75% from 27.
OR
1
OR
O
MeO C
2
4
OBn
O
O
O
MeO C
2
OBn
O
O
BnO
a
O
3
N
7
+
8 or 14
R O
3
BnO
O
NHAc
PivO
R O
2
from 8
from 14
R =R =Ac, R , R =PhCH
24
25
21
1
2
3
4
19
20
21
R =Bn, R =Ac, R , R =PhCH
1 2 3 4
b
c
b
c
R =R =H, R , R =PhCH
1
2
3
4
R =Bn, R =H, R , R =PhCH
1
2
3
4
R =R =Bn, R , R =PhCH
1
2
3
4
d
e
22
23
R =R =Bn, R =R =H
1 2 3 4
R =R =Bn, R =H, R =Bz
1
2
3
4
6
f
OR
2
OR
O
3
RO C
2
OR
O
2
O
O
OR
5
RO C
2
OR
2
O
O
RO C
2
OR
O
2
O
R O
2
O
O
Y
R O
4
R O
O
2
X
R O
2
O
Y
R O
1
R O
2
R O
1
5
R=Me, R =Piv, R =Bn, R =Bz, R ,R =PhCH, Y=N , X=NHAc
1 2 3 4 5 3
g
26 R= R =R =H, R =Bn, R ,R =PhCH, Y=N , X=NHAc
1
3
2
4
5
3
h
27 R=Na, R =R =SO Na, R =Bn, R ,R =PhCH, Y=N , X=NHAc
1
3
3
2
4
5
3
i, j
3
Scheme 4. Reagents and conditions: (a) KOH, MeOH; (b) BzCl, Py, 96% from 28; (c) BnNH2, Et2O, 96%; (d) DBU, Cl3CCN,
CH2Cl2, 87%; (e) TMSOTf, CH2Cl2, 0°C, 88%; (f) Ac2O, Py, 43% from 29; (g) AcSH, 68%; (h) (FN)n·Py, THF, –15°C, 81%; (i)
K2CO3, Cl3CCN, CH2Cl2, 71%.
OBz
O
OBz
O
MeO2C
O
OBn
O
NH
e
BnO
BnO
BnO
BnO
11
+
X
O
CCl3
OR1
N3
R2O
29
30
31
32
R1=TDS, R2=H, X=N3
R1=TDS, R2=Ac, X=N3
R1=TDS, R2=Ac, X=NHAc
f
a, b, c, d
g
OAc
h
i
O
33
34
R1=H, R2=Ac, X=NHAc
BnO
BnO
OAc
N3
R1=C(NH)CCl3, R2=Ac, X=NHAc
28
The above qualitative NMR study indicated that the syn-
thetic compounds 3 and 4 present the main structural features
of natural heparin oligosaccharides (46). The structural defi-
nition of 3 was further improved with a semiquantitative
analysis of the NOE data. Heparin fragments larger than five
monosaccharide units usually show an anisotropic hydrody-
namic behavior with a shape close to a prolate ellipsoid.
This feature can be determined through the analysis of σ NOE
and (or) the apparent correlation times for the fixed
glucosamine interprotonic vectors H1-H2 and H2-H4. As the
H1-H2 vector is almost parallel and the H2-H4 almost per-
pendicular to the main axis of inertia and both distances are
© 2002 NRC Canada

Ojeda et al.
923
Scheme 5. Reagents and conditions: (a) NaBH3CN, THF, HCl–Et2O, 66%; (b) TMSOTf, CH2Cl2, 40%; (c) MeONa, MeOH–CH2Cl2,
80%; (d) Ag2O, BnBr, DMF, 72%; (e) (i) H2O2, LiOH aq; (ii) KOH aq, MeOH, 94%; (h) SO3·Py, Py, 64%; (i) Pd/C, H2, MeOH–H2O
(9:1); (j) SO3·Py, H2O, pH = 9.5, 66% from 40.
OR2
OBz
O
MeO2C
OBn
O
O
O
OBn
MeO2C
O
OBn
O
O
BnO
O
MeO2C
OBn
O
O
BnO
O
N3
a
HO
BnO
5
NHAc
O
N3
PivO
BnO
PivO
34
35
b
OR2
OR3
O
RO2C
O
OR2
O
O
O
OR2
RO2C
O
OR2
O
O
OR3
O
RO2C
O
OR2
O
O
O
R2O
Y
RO2C
O
OR2
O
O
R2O
R2O
X
R2O
R2O
Y
R1O
X
R2O
R1O
R4O
36 R=Me, R =Piv, R =Bn, R =Bz, R =Ac, Y=N , X=NHAc
1
2
3
4
3
c
37 R=Me, R =Piv, R =Bn, R =Bz, R =H, Y=N , X=NHAc
1
2
3
4
3
d
e
38 R=Me, R =Piv, R ,R =Bn, R =Bz, R =Ac, Y=N , X=NHAc
1
2
4
3
4
3
39 R= R =R =H, R ,R =Bn, Y=N , X=NHAc
1
3
2
4
3
f
40 R=Na, R =R =SO Na, R ,R =Bn, Y=N , X=NHAc
1
3
3
2
4
3
g,h
4
Table 1. Proton and carbon chemical shifts for hexasaccharide 3.
A
B
C
D
E
F
H-1
H-2
H-3
H-4
H-5
H-6/H-6′
C-1
C-2
C-3
C-4
C-5
5.22
4.16
4.19
4.00
4.50
5.32
3.23
3.64
3.67
3.83
3.84/3.78
99.7
60.8
72.1
79.7
73.6
62.5
4.89
3.66
3.85
4.05
4.74
5.15
3.95
3.74
3.76
3.99
4.37/4.23
97.1
74.1
72.4
78.4
72.0
69.0
5.18
4.31
4.19
4.07
4.77
5.38
3.18
3.61
3.42
3.80
3.85/3.76
99.4
60.7
73.8
72.6
74.4
63.0
99.8
78.6
71.0
78.6
70.8
104.2
72.5
72.2
77.5
72.6
101.9
78.5
71.7
78.4
71.9
C-6
nearly equal, the observed variation of σ NOE can be attrib-
uted to a difference in parallel and perpendicular correlation
times. The σ NOE values for 3 were calculated from the NOE
growing rate, assuming the isolated spin pairs approxima-
tion, by integration of the NOE cross-peaks at several mix-
ing times (200–600 ms) (49). The apparent correlation times
for glucosamine vectors H1-H2 and H2-H4, calculated from
the σ NOE ratio at 400 and 500 MHz provided a clear indication
of this anisotropic tumbling (Table 3) (50). This behavior,
which also was previously observed for 1 (21), prevented
the accurate calculation of distances from NOE data and a
complex full-motion model, which accounts both for internal
motions and anisotropy, has to be considered. However,
because of the above-mentioned parallel and perpendicular
orientation of the H1-H2 and H2-H4 vectors with respect to
the main axis of inertia, the known glucosamine H1-H2 and
H2-H4 distances can be used as extreme reference values for
the estimation of distances from interglycosidic NOEs. The
upper and the lower distance limits may be given by
considering the parallel and perpendicular correlation times
derived from H1-H2 and H2-H4 σ NOE, respectively. The dis-
tances thus obtained were in agreement with those expected
for a syn-Φ disposition of the glycosidic linkages (Table 4)
as that reported for heparin and related oligosaccharides.
© 2002 NRC Canada

924
Table 2. JHH observed for iduronate residues of 3.
Can. J. Chem. Vol. 80, 2002
3
IdoA (A)
IdoA (C)
IdoA (E)
1C4 (teor.)
2SO (teor.)
4C1 (teor.)
3J1, 2
3J2, 3
3J3, 4
3J4, 5
3.0
4.9
5.4
2.9
3.5
6.6
4.5
3.3
3.0
5.8
4.0
2.8
2.9
4.0
4.0
0.8
5.6
10.1
6.0
8.0
10.1
9.8
4.8
3.9
Table 3. σ NOE (500) and σ NOE (400) (s–1) and apparent correla-
tion times (ns) for internal glucosamine NOEs of 3.
Fig. 2. Relaxed structures of hexasaccharides 1 and 3.
GlcN (B)
GlcN (D)
GlcN (F)
H1-H2
H2-H4
σ NOE (500)
σ NOE (400)
τc
σ NOE (500)
σ NOE (400)
τc
0.16
0.14
1.04
0.05
0.03
0.60
0.20
0.19
1.37
0.09
0.08
1.22
0.13
0.12
1.10
0.02
0.01
0.55
The small differences observed for the H1′-H3:H1′-H4 ratio
of the C–D linkage in comparison with those of the A–B and
E–F linkages can be attributed to changes of the local flexi-
bility as a consequence of the different substitution pattern.
Finally, a model of hexasaccharide 3 was constructed on
the basis of a previous model constructed for 1. A
minimization including TIP3P water and sodium counterions
was performed. The system was preequilibrated by several
cycles of molecular dynamics with frozen solute coordinates.
After minimization of the overall system, the relaxed struc-
ture showed the typical helical symmetry of heparin with the
five sulfate groups located on one face. The backbone of this
structure can be superimposed on the backbone of structure
1 (Fig. 2) This model is in agreement with the key inter-
glycosidic distances calculated from NOE data (Table 4).
Although more detailed structural studies are presently
underway, the results presented here clearly indicate that the
key three-dimensional elements present in the structure of
heparin, helical symmetry and iduronate ring conformational
equilibrium, are adequatelly modeled by compounds 3 and 4.
The induction of the mitogenic activity of FGF-1 by these
synthetic compounds (3 and 4) in comparison with that pro-
moted by compounds 1 and 2 is now under investigation.
Preliminary results indicate that hexasaccharide 3 shows the
maximum activating effect. On the other hand, sedimenta-
tion equilibrium analysis also shows, in this case, that the
profile of FGF-1 solutions in the presence of activating con-
centrations of 3 and 4 correspond to monomeric species.
These results and further studies on the structure–reactivity
relationship will be reported in due time.
performed using Sephadex LH-20 and G-25 (Pharmacia).
Ionic exchange chromatography was carried out using
Dowex 50WX4 resin (Fluka). Thin-layer chromatography
was performed on precoated plates of silica gel (60-F254, E.
Merck, Darmstadt) and visualized with H2SO4–EtOH (1:9,
v/v) or anisaldehyde followed by heating. For column chro-
matography, silica gel 60 (0.2–0.5 mm, 0.2–0.063 mm, or
0.04–0.015 mm, E. Merck, Darmstadt) and distilled solvents
were used. All solvents and reagents were purified and dried
according to standard procedures.
NMR measurements
1D and 2D experiments were recorded in D2O at 298 K
on Bruker DRX-500 and DRX-400 instruments using ace-
tone as the external reference in a 6 mM sample. pH* was
adjusted to 7.0. DQF-COSY (51), TOCSY (52), and HMQC
(53) experiments were recorded using standard z-pulsed field
gradient enhanced or selected pulse sequences when possi-
ble. Phase-sensitive experiments were performed in all cases
using the TPPI (time proportional phase increment) method
(54). Data were transformed into phase-sensitive mode after
weighting with shifted- square sine-bells functions.
Experimental
NOESY (55) experiments were carried out at 500 and
400 MHz with mixing times of 250, 300, 325, 350, 400,
500, 550, and 600 ms. The volumes of the cross and
diagonal peaks were integrated using standard Bruker soft-
ware. The normalized cross-peak’s volume variation as a
function of the mixing time indicated good linearity of the
NOE build up curves for the range of mixing times consid-
ered. Cross-relaxation rates (σ) were calculated from these
General methods
The H NMR spectra were measured at 500 MHz (Bruker
1
DRX-500) with tetramethylsilane or the residual signal of
the solvent as the internal standard. The 13C NMR spectra
were recorded at 125 MHz. Optical rotations were measured
at room temperature in a 1 dm cell on a PerkinElmer 341
polarimeter. Elemental analyses were carried out in a Leco
CHNS 932 analyzer. Permeation gel chromatography was
© 2002 NRC Canada

Ojeda et al.
925
Table 4. Experimental and theoretical interglycosidic distances of hexasaccharide 3.
Residue
Distance
Minimun
Maximum
Average
Model distance
A
B
H2—H5
3.26
2.22
2.42
2.62
2.26
2.42
2.22
2.37
2.1
2.72
2.29
2.16
2.85
1.82
1.92
3.81
2.6
3.54
2.41
2.62
2.79
2.45
2.62
2.41
2.53
2.24
2.90
2.45
2.3
3.96
2.39
2.33
3.78
2.35
2.31
3.90
2.57
2.22
4.02
2.45
2.42
3.84
2.51
2.21
H1—H4(A)
H1—H3(A)
H2—H5
H1—H4(B)
H1—H6(B)
H1—H6′(B)
H1—H4(C)
H1—H3(C)
H2—H5
H1—H4(D)
H1—H6(D)
H1—H6′(D)
H1—H4(E)
H1—H3(E)
2.83
2.97
2.64
2.83
2.6
2.69
2.38
3.08
2.61
2.45
3.24
2.44
2.57
C
D
E
3.04
2.13
2.24
F
curves assuming the isolated spin pairs approximation at
short mixing times by extrapolation to zero time. The appar-
ent correlation times were calculated using the spectral den-
sity function of an isotropic rigid molecule, from the ratio σ
(500)/σ (400) (56).
Methyl (isopropyl 4-O-(2-azido-3-O-benzyl-2-deoxy-α-D-
glucopyranosyl)-3-O-benzyl-2-O-pivaloyl-α-L-idopyran-
osyl) uronate (13)
To a solution of 12 (800 mg, 1.02 mmol) in dry CH2Cl2
(9 mL), EtSH (374 µL, 5.06 mmol), and catalytic PTSA
were added. After stirring for 3 h under an argon atmo-
sphere, the reaction was neutralized with saturated NaHCO3
solution, diluted with CH2Cl2 (200 mL), and washed with
H2O (200 mL). The organic layer was dried (MgSO4) and
concentrated to dryness. The mixture was purified by flash
chromatography (hexane–EtOAc, 1:1) to yield 13 (639 mg,
90%). TLC: 0.14 (hexane–EtOAc, 2:1). [α]2D3 –6.6° (c 0.6,
Molecular modeling
Molecular modeling was performed using AMBER force
field (57) (parameter set “parm91”) as integrated in the
AMBER 5.0 program (58), modified for carbohydrate mol-
ecules by the GLYCAM_93 parameter set (59). Specific
parameters for sulfates and sulfamate groups were also in-
cluded, as described by Huige and Altona (60). The initial
structure of the hexasaccharide 3 was built using the φ/ψ
values found for a minimized structure of the regular hepa-
rin-like synthetic hexasaccharide 1 (21). Minimization was
carried out using periodic boundary conditions with TIP3P
water molecules. Box dimensions were 53 × 50 × 50 Å,
large enough to allow for the faces of the box to extend
12 Å beyond the sugar in each direction, reducing in this
way the possibility of border effects that could take place if
a reorientation of the solute happened during the calcula-
tion. It involved the introduction of 3933 water molecules
and eight sodium counterions. To enable the cations to
solvate appropriately, they were initially located randomly
far enough from the solute. The initial water and
counterions configuration was subjected to 500 cycles of
energy minimization, with the conformation of the sugar
frozen. Following this step, a 50 ps volume constant MD
simulation was performed, in which only the water mole-
cules were allowed to move. After this preequilibration of
the solvent, the next step consisted of 500 cycles of energy
minimization of the water molecules, keeping constant the
Cartesian coordinates of the solute and counterions. To
reach the equilibrium value for the density of the system,
the water molecules were subjected to 50 ps pressure con-
stant MD simulation with both the solute and the
counterions frozen. Finally, the solvent equilibration proce-
dure involved 500 cycles of steepest descent energy
minimization of the water molecules. At this point, the en-
ergetic minimum of 3 was obtained using 1500 cycles of
conjugated gradient minimization for the whole system.
1
CHCl3). H NMR (500 MHz, CDCl3) δ: 7.37–7.24 (m, 10H,
2 Ph), 5.18 (d, 1H, H-1, J1,2 = 4.5 Hz), 5.05 (d, 1H, H-1′,
J1,2 = 3.6 Hz), 4.93 (m, 1H, H-2), 4.89 (d, 1H, 1 CH2Ph,
J
gem = 11.2 Hz), 4.79−4.69 (m, 4H, 3 CH2Ph, H-5), 4.15 (m,
1H, H-4), 3.97–3.91 (m, 2H, H-3, CH(CH3)2), 3.80–3.55
(m, 5 H, H-3′, H-4′, H-5′, H-6′a, H-6′b), 3.75 (s, 3H,
COOCH3), 3.17 (t, 1H, H-2′, J2,1 ≈ J2,3 = 10.7 Hz), 1.22 (s,
9H, C(CH3)3), 1.19–1.15 (2d, 6H, CH(CH3)2, J = 6.2 Hz).
Methyl (isopropyl 4-O-(6-O-acetyl-2-azido-3-O-benzyl-2-
deoxy-α-D-glucopyranosyl)-3-O-benzyl-2-O-pivaloyl-β-L-
idopyranosyl) uronate (14)
To a solution of 13 (540 mg, 0.77 mmol) in dry CH2Cl2
(12 mL) at –10°C, Py (63 µL, 0.78 mmol) and acetic anhy-
dride (74 µL, 0.78 mmol) were added. After stirring for
24 h, MeOH (1 mL) was added, and was concentrated in
vacuo. The crude was purified by flash chromatography
(hexane–EtOAc, 2:1) to yield 14 (488 mg, 85%) as well as
nonreacting starting material 13 (22 mg, 4%). TLC: 0.33
(hexane–EtOAc, 2:1). [α]2D3 –10.1° (c 1.0, CHCl3). MALDI-
TOF (m/z): 766 (M + Na+), 782 (M + K+). 1H NMR
(500 MHz, CDCl3) δ: 7.37–7.23 (m, 10H, 2 Ph), 5.18 (d,
1H, H-1, J1,2 = 4.4 Hz), 5.04 (d, 1H, H-1′, J1,2 = 3.4 Hz),
4.92 (m, 1H, H-2), 4.85 (m, 2H, 2 CH2Ph), 4.78 (d, 1H,
1 CH2Ph, Jgem = 11.3 Hz), 4.74 (d, 1H, H-5, J5,4 = 4.0 Hz),
4.69 (d, 1H, 1 CH2Ph CH2Ph, Jgem = 11.2 Hz), 4.54 (dd, 1H,
H-6′a, J6a,6b = 12.5 Hz, J6a,5 = 3.5 Hz), 4.13 (m, 2H, H-4,
H-6′b), 3.93 (m, 2H, H-3, CH(CH3)2), 3.82 (m, 1H, H-5′),
3.76 (s, 3H, COOCH3), 3.70 (m, 1H, H-3′), 3.40 (m, 1H, H-4′),
3.20 (dd, 1H, H-2′, J2,1 = 3.4 Hz, J2,3 = 10.1 Hz), 2.83
© 2002 NRC Canada

926
Can. J. Chem. Vol. 80, 2002
(d, 1H, OH-4, JOH,4 = 3.4 Hz), 2.07 (s 3H, OCOCH3), 1.21
(s, 9H, C(CH3)3), 1.19 and 1.14 (2 d, 6H, J = 6.0 Hz,
CH(CH3)2). 13C NMR (125 MHz, CDCl3) δ: 177.3, 172.0,
170.0 (C=O), 98.4 (C-1), 97.2 (C-1′), 78.8, 76.0, 75.1, 74.1,
73.1; 71.3, 70.9, 70.7, 70.6, 70.5, 70.1, 62.7, 52.2
(COOCH3), 38.8, 27.2 (OPiv), 20.8 (OCOCH3), 23.3, 21.8
(CH(CH3)2). Anal. calcd. for C37H49N3O13 (%): C 59.74,
H 6.64, N 5.65; found (%): C 59.74, H 6.52, N 5.57.
CH(CH3)2, C(CH3)2), 0.26 and 0.19 (2 s, 6H, Si(CH3)2).
13C NMR (125 MHz, CDCl3) δ: 170.4, 168.8 (C=O), 101.4
(Ph-CH-), 98.2 (C-1), 93.8 (C-1′), 82.8, 74.2, 73.7, 73.4,
72.9, 72.1, 68.7, 68.5, 63.6, 52.6 (C-2′), 52.1 (COOCH3),
23.0 (NHCOCH3), 34.0, 25.1, 20.3, 20.1, 18.6, 18.4, –1.9,
–3.6 (OTDS). Anal. calcd. for C44H59NO12Si·0.5H2O (%):
C 63.59, H 7.28, N 1.68; found (%): C 63.37, H 7.53,
N 1.53.
Methyl dimethylthexylsilyl 2-O-acetyl-4-O-(2-azido-3-O-
benzyl-4,6-O-benzylidene-2-deoxy-α-D-glucopyranosyl)-3-
O-benzyl-β-L-idopyranuronate (16)
Methyl (isopropyl 4-O-(2-azido-3,6-di-O-benzyl-2-deoxy-
α-D-glucopyranosyl)- 3-O-benzyl-2-O-pivaloyl-β-L-
idopyranosyl) uronate (8)
Compound 9 (1 g, 1.24 mmol) was dissolved in Py
(10 mL) at 0°C and acetic anhydride (5 mL) was added and
was stirred for 48 h. The reaction was diluted with CH2Cl2
(250 mL) and washed with 1 N HCl (125 mL). The aqueous
layer was extracted with CH2Cl2 (2 × 125 mL) and the or-
ganic layers were dried (MgSO4) and concentrated in vacuo.
The residue was coevaporated with toluene twice and then
purified by flash chromatography (hexane–EtOAc, 4:1), to
yield 16 (1 g, 93%). TLC: 0.38 (hexane–EtOAc, 4;1). [α]D23
+29.5° (c 1.0, CHCl3). 1H NMR (500 MHz, CDCl3) δ: 7.45–
7.24 (m, 15H, 3 Ph), 5.51 (s, 1H, Ph-CH-), 5.07 (b.s., 1H,
H-1), 4.97 (b. s., 1H, H-2), 4.76 (d, 1H, H-1′, J1,2 = 3.5 Hz),
4.88−4.70 (2 d, 2H, 2 CH2Ph, Jgem = 11.1 Hz), 4.74−4.66
(2 d, 2H, 2 CH2Ph, Jgem = 11.8 Hz), 4.47 (b. s., 1H, H-5),
4.30 (dd, 1H, H-6′a, J6a,5 = 4.9 Hz, J6a,6b = 10.1 Hz), 3.97–
3.92 (m, 4H, H-3′, H-4, H-5′, H-3), 3.75 (s, 3H, COOCH3),
To a solution of 12 (550 mg, 0.696 mmol) in dry THF
(20 mL), NaBH3CN (7 mL of a solution of 1 M NaBH3CN
in THF) was added, and was stirred for 10 h. Afterwards, a
solution of 2.5 M HCl in Et2O was added dropwise until the
mixture became acidic. The reaction was neutralized with
saturated NaHCO3 solution, diluted with CH2Cl2 (25 mL),
and washed with H2O (15 mL). The aqueous layer was ex-
tracted with CH2Cl2 (2 × 20 mL) and the organic layers
were dried (MgSO4) and concentrated in vacuo. The residue
was purified by flash chromatography (hexane–EtOAc, 4:1)
to yield 8 (505 mg, 92%). TLC: 0.32 (hexane–EtOAc, 4:1).
1
[α]2D3 − 4.4° (c 1.0, CH2Cl2). H NMR (500 MHz, CDCl3) δ:
7.39–7.24 (m, 15 H, 3 Ph), 5.21 (d, 1H, H-1, J1,2 = 4.8 Hz),
5.05 (d, 1H, H-1′, J1,2 =3.4 Hz), 4.93 (t, 1H, H-2, J2,1 ≈ J2,3
5.1 Hz), 4.84 (d, 1H, 1 CH2Ph, Jgem = 11.1 Hz), 4.81 (d, 1H,
1 CH2Ph, Jgem = 11.2 Hz), 4.76 (d, 1H, 1 CH2Ph, Jgem
11.2 Hz), 4.74 (d, 1H, H-5, J5,4 = 5.0 Hz), 4.70 (d, 1H,
1 CH2Ph, Jgem = 11.1 Hz), 4.57 (d, 1H, 1 CH2Ph, Jgem
12 Hz), 4.51 (d, 1H, 1 CH2Ph, Jgem = 12 Hz), 4.14 (t, 1H,
H-4, J4,3 ≈ J4,5 = 5.3 Hz), 3.97 (t, 1H, H-3, J3,2 ≈ J3,4
=
=
3.64–3.59 (m, 2H, H-4′, H-6′b), 3.28 (t, 1H, H-2′, J2,1 ≈ J2,3
=
9.9 Hz), 2.03 (d, 3H, OCOCH3), 1.61 (m, 1H, CH(CH3)2),
0.86–0.83 (m, 12H, CH(CH3)2, C(CH3)2), 0.24 and 0.14 (2s,
6H, Si(CH3)2). 13C NMR (125 MHz, CDCl3) δ: 170.7, 168.8
(C=O), 137.8–126.1 (Ph), 101.6 (Ph-CH-), 98.4 (C-1′), 93.7
(C-1), 82.6, 76.0, 74.8 (CH2Ph), 74.3, 73.6, 73.4 (C-5),
72.9 (CH2Ph), 68.5 (C-6′), 67.7, 63.2, 63.1, 52.1
(COOCH3), 20.9 (OCOCH3), 34.0, 24.8, 20.2, 18.4, –2.0,
–3.6 (OTDS).
=
=
5.6 Hz), 3.94 (m, 1 H, CH(CH3)2, J 6.0 Hz), 3.81 (m, 1H, H-5′),
3.72 (s, 3H, COOCH3), 3.71–3.67 (m, 3H, H-3′, H-4′, H-6′a),
3.57 (dd, 1H, H-6′b, J6b,6a = 10.2 Hz, J6b,5 = 4.3 Hz), 3.21
(dd, 1H, H-2′, J2,1 = 3.4 Hz, J2,3 = 9.4 Hz), 2.57 (s, 1H, OH-4),
1.21 (s, 9H, C(CH3)3), 1.19 and 1.14 (2 d, 6H, J = 6.0 Hz,
CH(CH3)2). 13C NMR (125 MHz, CDCl3) δ: 177.3, 170.0
(C=O), 98.2 (C-1′), 97.2 (C-1), 79.1, 76.2, 74.8, 73.7, 73.6,
72.4, 71.3, 71.0, 70.6, 69.6, 62.6, 52.1 (COOCH3), 38.8,
27.2 (OPiv), 23.3, 21.8 (CH(CH3)2). Anal. calcd. for
C42H53N3O12 (%): C 63.70, H 6.75, N 5.30; found (%):
C 63.62, H 6.70, N 5.26.
Methyl (dimethylthexylsilyl 4-O-(2-acetamido-3-O-
benzyl-4,6-O-benzylidene-2-deoxy-α-D-glucopyranosyl)-2-
O-acetyl-3-O-benzyl-β-L-idopyranosid) uronate (17)
(a) A solution of 16 (4.3 g, 5.07 mmol) in thiolacetic acid
(55 mL) was stirred for 30 h at room temperature. After con-
centrating to dryness, the residue was purified by flash chro-
matography (hexane–EtOAc, 1:1) to yield 17 (3.19 g, 73%).
(b) Compound 15 (790 mg, 0.98 mmol) was dissolved in
Py (10 mL) at 0°C and acetic anhydride (5 mL) and catalytic
DMAP were added and was stirred for 48 h. The reaction
was diluted with CH2Cl2 (200 mL) and washed with 1 N
HCl (100 mL). The aqueous layer was extracted with
CH2Cl2 (2 × 100 mL) and the organic layers were dried
(MgSO4) and concentrated in vacuo. The residue was coeva-
porated with toluene twice and then purified by flash
chromatography (hexane–EtOAc, 1:1) to yield 17 (617 mg,
77%). TLC: 0.39 (hexane–EtOAc, 1:1). [α]2D3 +46.3° (c 1.0,
CH2Cl2). MALDI-TOF (m/z): 887 (M + Na+), 903 (M + K+).
1H NMR (500 MHz, CDCl3) δ: 7.43–7.22 (m, 15H, 3 Ph),
6.25 (d, 1H, NH, JNH,2 = 9.6 Hz), 5.52 (s, 1H, Ph-CH-), 5.04
Methyl dimethylthexylsilyl 4-O-(2-acetamido-3-O-
benzyl-4,6-O-benzylidene-2-deoxy-α-D-glucopyranosyl)-3-
O-benzyl-β-L-idopyranuronate (15)
A solution of 9 (255 mg, 0.316 mmol) in thiolacetic acid
(2 mL) was stirred for 16 h at room temperature. After con-
centrating to dryness, the residue was purified by flash chro-
matography (hexane–EtOAc, 1:1) to yield 15 (639 mg,
90%). TLC: 0.35 (Hexane–EtOAc, 1:1). [α]2D3 +59.5° (c 1.0,
CH2Cl2). MALDI-TOF (m/z): 845 (M + Na+), 861 (M + K+).
1H NMR (500 MHz, CDCl3) δ: 7.45–7.15 (m, 15H, 3 Ph),
6.82 (d, 1H, NH, JNH,2 = 9.5 Hz), 5.50 (s, 1H, Ph-CH-), 5.00
(s, 1H, H-1), 4.83 (d, 1H, 1 CH2Ph, Jgem = 12.1 Hz),
4.64−4.52 (m, 5H, 3 CH2Ph, H-1′, H-5), 4.30 (m, 1H, H-2′),
4.21 (dd, 1H, H-6′a, J6a,5 = 4.6 Hz, J6a,6b = 10.1 Hz), 3.94
(s, 1H, H-4), 3.82 (s, 3 H, COOCH3), 3.74–3.59 (m, 6H, H-2,
H-3, H-3′, H-4′, H-5′, H-6′b), 2.63 (s, 1H, OH-2), 1.85 (s, 3 H,
NHCOCH3), 1.64 (m, 1H, CH(CH3)2), 0.90–0.88 (m, 12H,
(s, 1H, H-1), 4.98 (s, 1H, H-2), 4.83 (d, 1H, 1 CH2Ph, Jgem
=
12.2 Hz), 4.76 (d, 1H, H-5, J5,4 = 3.6 Hz), 4.67 (d, 1H, 1
CH2Ph, Jgem = 11.8 Hz), 4.60 −4.53 (m, 3H, 2 CH2Ph,
© 2002 NRC Canada

Ojeda et al.
927
H-1′), 4.29 (m, 1H, H-2′), 4.19 (m, 1H, H-6′a), 3.87 (s, 1H,
H-4), 3.85 (s, 1H, H-3), 3.74 (s, 3H, COOCH3), 3.72–3.66
(m, 3H, H-4′, H-5′, H-6′b), 3.52 (m, 1H, H-3′), 1.91 (s, 3H,
NHCOCH3), 1.81 (s, 3H, OCOCH3), 1.58 (m, 1H, CH(CH3)2),
0.83–0.82 (m, 12H, CH(CH3)2, C(CH3)2), 0.19 and 0.10 (2s,
6H, Si(CH3)2). 13C NMR (125 MHz, CDCl3) δ: 170.0, 169.9,
168.4 (C=O), 101.4 (Ph-CH-), 96.4 (C-1), 93.0 (C-1′),
82.4, 76.1, 74.0, 73.3, 73.1, 71.8, 70.3, 68.6, 67.8, 63.4,
52.2 (C-2′), 52.0 (COOCH3), 23.3, 20.9 (OCOCH3,
NHCOCH3), 33.9, 24.9, 20.1, 19.8, 18.6, 18.4, –1.9,
–3.6 (OTDS). Anal. calcd. for C46H61NO13Si (%):
C 63.94, H 7.11, N 1.62; found (%): C 63.64, H 6.82,
N 1.59.
7.22 (m, 30H, 6 Ph (α and β)), 6.34 (s, 1H, H-1 (α)), 6.22 (s,
1H, H-1 (β)), 5.87 (d, 1H, NH (β), JNH,2 = 9.3 Hz), 5.72 (d,
1H, NH (α), JNH,2 = 9.4 Hz), 5.53 (s, 1H, Ph-CH-α), 5.52 (s,
1H, Ph-CH-β), 5.25 and 5.12 (m, 2 H, H-2′ (α and β)), 4.98
(s, 1H, H-5 (α)), 4.87−4.51 (m, 13H, 4 CH2Ph, H-1′ (α
and β), H-2 (α and β), H-5), 4.30 (m, 2H, H-2′ (α and β)),
4.24− 4.17 (m, 2H, H-6′a (α and β)), 4.11− 4.04 (m, 2H, H-
4 (α), H-3 (β)), 3.96 (m, 1H, H-4 (β)), 3.86 (s, 1H, H-3 (α)),
3.77 (s, 3H, COOCH3 (α)), 3.76 (s, 3H, COOCH3 (β)), 3.81–
3.64 (m, 6H, H-4′, H-5′, H-6′b (α and β)), 3.52 (m, 2H,
H-3′ (α and β)), 1.94 (s, 3H, OCOCH3 (α)), 1.92 (s, 3H,
OCOCH3 (β)), 1.81 (s, 3H, NHCOCH3 (α)), 1.75 (s, 3H,
NHCOCH3 (β)). 13C NMR (125 MHz, CDCl3) δ: 171.1,
170.0, 169.9, 169.7, 169.4, 168.3, 167.6 (C=O), 160.5,
160.1 (C=NH), 138.4–126.0 (Ph), 101.5 (Ph-CH-), 97.6 (C-1′
(β)), 96.2 (C-1′ (α)), 95.0 (C-1 (α)), 94.5 (C-1 (β)), 82.4,
82.3, 76.1, 75.6, 74.1, 73.9, 72.5, 72.2, 72.0, 70.4, 69.9,
69.1, 68.7, 68.6, 66.9, 65.8, 63.7, 63.5, 60.4, 52.5 (C-2′ (α
and β)), 52.3 (COOCH3 (α and β)), 23.4, 23.2, 21.0, 20.7
(OCOCH3, NHCOCH3 (α and β)). Anal. calcd. for
C40H43N2O13Cl3 (%): C 55.47, H 5.00, N 3.23; found
(%): C 55.78, H 5.09, N 3.52.
Methyl 4-O-(2-acetamido-3-O-benzyl-4,6-O-benzylidene-
2-deoxy-α-D-glucopyranosyl)-2-O-acetyl-3-O-benzyl-α ,β-
L-idopyranuronate (18)
To a solution of 17 (2.75 g, 3.18 mmol) at –15°C in dry
THF (90 mL) an excess of (HF)n·Py (8.8 mL) was added.
The reaction was warmed to 0°C and stirred for 24 h under
an argon atmosphere. The mixture was diluted with CH2Cl2
(2 × 400 mL) and washed with H2O (2 × 200 mL) and satu-
rated NaHCO3 solution until neutral pH. The aqueous layer
was extracted with CH2Cl2 (2 × 200 mL) and the organic
layers were dried (MgSO4) and concentrated in vacuo. The
residue was purified by flash chromatography (hexane–
EtOAc, 1:3) to yield 18 (1.8 g, 78%) as a mixture of α and β
anomers. TLC: 0.16 (hexane–EtOAc, 2:3). MALDI-TOF
Methyl (isopropyl O-(2-acetamido-3-O-benzyl-4,6-O-
benzylidene-2-deoxy-α-D-glucopyranosyl)-(1→4)-O-
(methyl 2-O-acetyl-3-O-benzyl-α-L-idopyranosyluronate)-
(1→4)-O-(6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-α-D-
glucopyranosyl)-(1→4)-3-O-benzyl-2-O-pivaloyl-α-L-
idopyranosyl) uronate (24)
1
(m/z): 774 (M + Na+), 760 (M + K+). H NMR (500 MHz,
CDCl3) δ: 7.44–7.21 (m, 30H, 6 Ph (α and β)), 5.87 (d, 1H,
To a solution of 14 (235 mg, 0.32 mmol) and 7 (362 mg,
0.42 mmol) in dry CH2Cl2 (0.5 mL) at room temperature,
TMSOTf (20 µL of a 0.93 M solution of TMSOTf in dry
CH2Cl2) was added under an argon atmosphere. After stir-
ring for 7 h 30 min, saturated NaHCO3 solution (2 mL) was
added and diluted with CH2Cl2 (10 mL). The residue was
washed with H2O (7.5 mL) and then the aqueous layer was
extracted with CH2Cl2 (2 × 5 mL) and the organic layers
were dried (MgSO4) and concentrated in vacuo. The residue
was purified by flash chromatography (hexane–EtOAc, 1:2)
to yield 24 (150 mg, 33%) and nonreacting acceptor 14
(156 mg, 43%). TLC: 0.24 (hexane–EtOAc, 1:1). [α]D23
+46.3° (c 1.0, CHCl3). MALDI-TOF (m/z): 1470 (M + Na+),
NH (α), JNH,2 = 9.0 Hz), 5.71 (d, 1H, NH (β), JNH,2
=
9.2 Hz), 5.54 (s, 1H, Ph-CH- β), 5.53 (s, 1H, Ph-CH-α), 5.23
(d, 1H, H-1 (β), J2,1 = 5.7 Hz), 5.16 (s, 1H, H-1 (α)),
4.91− 4.54 (m, 14H, 4 CH2Ph, H-1′, H-2, H-5 (α and β)),
4.27 (m, 2H, H-2′ (α and β)), 4.23−4.19 (m, 2H, H-6′a (α
and β)), 3.99–3.91 (m, 5H, H-4, H-3 (α and β), OH-1 (α)),
3.78 and 3.77 (2s, 6H, COOCH3 (β and α)), 3.74–3.63 (m,
7H, H-4′, H-5′, H-6′b (α and β), OH-1 (β)), 3.53–3.48 (m,
2H, H-3′ (α and β)), 1.97 (s, 3H, OCOCH3 (α)), 1.91 (s,
3H, OCOCH3 (β)), 1.80 (s, 3H, NHCOCH3 (α)), 1.76 (s,
3H, NHCOCH3 (β)). 13C NMR (125 MHz, CDCl3) δ: 170.1,
170.0, 169.8, 169.0, 168.6 (C=O), 138.3–127.9 (Ph), 101.5
(Ph-CH-), 97.1 (C-1′ (α)), 96.7 (C-1′ (β)), 93.1 (C-1 (α and
β)), 82.4, 75.9, 75.8, 73.5, 73.2, 73.1, 71.4, 71.2, 68.9, 68.6,
67.6, 67.5, 63.6, 63.5, 52.4 (C-2′ (α and β)), 52.3 (COOCH3
(α and β)), 23.3, 20.8 (OCOCH3, NHCOCH3 (α and β )).
Anal. calcd. for C38H43NO13·0.5H2O (%): C 62.45, H 6.07,
N 1.92; found (%): C 62.49, H 6.02, N 2.20.
1
1486 (M + K+). H NMR (500 MHz, CDCl3) δ: 7.42–7.22
(m, 25H, 5 Ph), 6.57 (d, 1H, NH, JNH,2 = 9.2 Hz), 5.54 (s,
1H, Ph-CH-), 5.13 (m, 2H, H-1a, H-1c), 4.98− 4.49 (m,
15H, H-1b, H-1d, H-2a, H-2c, H-5a, H-5c, H-6b, 8 CH2Ph),
4.29 (m, 1H, H-2d), 4.17 (m, 1H, H-6d), 4.09 (m, 4H, H-3c,
H-4a, H-4b, H-6′b), 3.93 (m, 3H, H-3a, H-4c, CH(CH3)2),
3.74 (s, 3H, COOCH3), 3.60 (s, 3H, COOCH3), 3.79–3.70
O-(Methyl 4-O-(2-acetamido-3-O-benzyl-4,6-O-benzyl-
idene-2-deoxy-α-D-glucopyranosyl)-2-O-acetyl-3-O-
(m, 4H, H-3b, H-4d, H-5b, H-5d), 3.65 (dd, 1H, H-6′d, J6′,6
9.3 Hz, J6′,5 = 3.8 Hz), 3.54 (t, 1H, H-3d, J3,2 ≈ J3,4
=
=
benzyl-α ,β-L-idopyranosyluronate) trichloroacetimidate (7)
To a solution of 18 (620 mg, 0.86 mmol) in dry CH2Cl2
(14 mL), Cl3CCN (1.3 mL, 13.05 mmol) and activated
K2CO3 (130 mg, 0.95 mmol) were added. After stirring for
1 h the residue was filtered and concentrated to dryness. The
residue was purified by flash chromatography (hexane–
EtOAc, 1:2) to yield 7 (701 mg, 94%) as a mixture of α and
β anomers. TLC (α and β): 0.51 and 0.62 (toluene–EtOAc,
1:2). 1H NMR (500 MHz, CDCl3) δ: 8.73 (s, 1H,
O(C=NH)CCl3 α), 8.66 (s, 1H, O(C=NH)CCl3 (β)), 7.44–
9.6 Hz), 3.33 (dd, 1H, H-2b, J2,1 = 3.2 Hz, J2,3 = 10.2 Hz),
2.12 (s, 3H, NHCOCH3), 2.04 (s, 3H, OCOCH3), 1.87 (s,
3H, OCOCH3), 1.21 (s, 9H, C(CH3)3), 1.19 and 1.16 (2d,
6H, J = 6.5 Hz, CH(CH3)2). 13C NMR (125 MHz, CDCl3) δ:
177.6, 171.7, 170.3, 170.1, 169.8, 169.0 (C=O), 137.7–
127.7 (Ph), 101.4 (Ph-CH-), 98.8 (C-1c), 97.2 (C-1b), 96.9
(C-1a), 96.2 (C-1d), 82.4, 78.1, 76.3, 75.9, 75.2, 74.5, 74.0,
73.9, 73.1, 72.9, 72.1, 71.2, 70.1, 69.9, 69.8, 69.5, 68.7,
63.3, 62.9, 61.6, 52.4, 52.2, 51.9, 29.7, 27.2, 23.3, 23.1,
21.7, 21.3, 20.8 (OPiv, OAc, NHCOCH3, O-i-Pr). Anal.
© 2002 NRC Canada

928
Can. J. Chem. Vol. 80, 2002
calcd. for C75H90N4O25·2.5H2O (%): C 60.35, H 6.41,
N 3.75; found: C 60.36, H 6.25, N 3.25.
(d, 1H, H-1b, J1,2 = 3.5 Hz), 4.90 (m, 2H, H-2a, H-2c), 4.84
(d, 1H, H-1d, J1,2 = 3.1 Hz), 4.81− 4.49 (m, 12H, H-5a,
H-5c, 10 CH2Ph), 4.29 (m, 1H, H-2d), 4.15 (dd, 1H, H-6d J6,5
4.4 Hz, J6,6′ = 10 Hz), 4.09 (t, 1H, H-4a, J4,3 ≈ J4,5
=
=
Methyl (isopropyl O-(2-acetamido-3-O-benzyl-4,6-O-
benzylidene-2-deoxy-α-D-glucopyranosyl)-(1→4)-O-
(methyl 3-O-benzyl-α-L-idopyranosyluronate)-(1→4)-O-
(2-azido-3-O-benzyl-2-deoxy-α-D-glucopyranosyl)-(1→4)-
3-O-benzyl-2-O-pivaloyl-α-L-idopyranosyl) uronate (25)
To a solution of 24 (280 mg, 0.19 mmol) in dry MeOH
(3 mL), MeONa (90 µL of a 0.43 M solution of MeONa in
MeOH) was added and stirred for 24 h. The reaction was di-
luted with CH2Cl2 (10 mL) and washed with saturated
NH4Cl solution (200 mL) and H2O (5 mL). The organic
layer was dried (MgSO4) and concentrated to dryness. The
mixture was purified by flash chromatography (hexane–
EtOAc, 1:3) to yield 25 (224 mg, 85%). TLC: 0.38 (hexane–
EtOAc, 1:2). [α]2D3 +20.4° (c 1.0, CH2Cl2). MALDI-TOF
(m/z): 1386 (M + Na+), 1402 (M + K+). 1H NMR (500 MHz,
5.3 Hz), 4.01 (t, 1H, H-4b J4,3 ≈ J4,5 = 9.5 Hz), 3.94–3.91
(m, 3H, H-3a, H-3c, CH(CH3)2), 3.86 (m, 1H, H-4c), 3.80
(d, 1H, H-5b, J5,4 = 10 Hz), 3.72–3.59 (m, 6H, H-3b, H-6b,
H-6′b, H-4d, H-5d, H-6′d), 3.67 (s, 3H, COOCH3), 3.50 (t,
1H, H-3d, J3,2 ≈ J3,4 = 9.6 Hz), 3.41 (s, 3H, COOCH3), 3.30
(dd, 1H, H-2b, J2,1 = 3.6 Hz, J2,3 = 10.3 Hz), 1.83 (s, 3H,
OCOCH3), 1.63 (s, 3H, NHCOCH3), 1.19 (s, 9H, C(CH3)3),
1.18 and 1.14 (2d, 6H, J = 6.5 Hz, CH(CH3)2). 13C NMR
(125 MHz, CDCl3) δ: 177.3, 170.1, 169.8, 169.4, 168.8
(C=O), 138.4–126.3 (Ph), 101.4 (Ph-CH-), 98.3 (C-1b), 97.6
(C-1c), 97.1 (C-1a), 82.4, 78.0, 76.3, 74.9, 74.2, 74.1, 73.9,
73.7, 73.6, 73.4, 73.2, 71.6, 71.3, 70.7, 70.4, 70.1, 69.5,
68.7, 67.3, 65.6, 63.7, 62.9, 52.3 (C-2d), 52.1, 51.9
(COOCH3), 38.8, 27.2, 23.2, 21.8, 20.8 (OPiv, NHCOCH3,
O-i-Pr). Anal. calcd. for C80H94N4O24 (%): C 64.24, H 6.33,
N 3.75; found (%): C 63.89, H 6.36, N 3.51.
CDCl3) δ: 7.42–7.20 (m, 25H, 5 Ph), 6.21 (d, 1H, NH, JNH,2
=
7.7 Hz), 5.50 (s, 1H, Ph-CH-), 5.17–5.15 (m, 2 H, H-1c,
H-1a), 5.04 (d, 1H, H-1b, J2,1 = 3.6 Hz), 4.98 (d, 1H, H-1d,
J1,2 = 3.8 Hz), 4.92 (t, 1H, H-2a, J2,1 ≈ J2,3 = 5.1 Hz), 4.86
(d, 1H, H-5c, J5,4 = 2.5 Hz), 4.77− 4.52 (m, 8H, 8 CH2Ph),
4.72 (d, 1H, H-5a, J5,4 = 4.6 Hz), 4.18− 4.09 (m, 3H, H-2d,
H-4a, H-6d), 4.01–3.86 (m, 5H, H-3a, H-3c, H-4b, H-4c,
CH(CH3)2), 3.69 (s, 3H, COOCH3), 3.78–3.65 (m, 8H, H-2c,
H-3b, H-4d, H-5b, H-5d, H-6′d, H-6b, H-6′b), 3.62 (t, 1H,
H-3d, J3,2 ≈ J3,4 = 9.8 Hz), 3.45 (s, 3H, COOCH3), 3.26 (dd,
1H, H-2b, J2,1 = 3.7 Hz, J2,3 = 10.2 Hz), 1.76 (s, 3H,
NHCOCH3), 1.18 (s, 9H, C(CH3)3), 1.19 and 1.17 (2d, 6H,
J = 6.5 Hz, CH(CH3)2). 13C NMR (125 MHz, CDCl3) δ:
177.3, 170.8, 170.0, 169.4 (C=O), 138.5–126.1 (Ph), 101.5
(Ph-CH-), 100.7 (C-1c), 98.3 (C-1b), 97.3 (C-1a), 96.9
(C-1d), 82.3, 78.2, 76.3, 75.9, 74.8, 74.3, 74.1, 74.0, 73.6,
73.2, 72.8, 72.5, 72.3, 71.3, 70.9, 68.6, 68.4, 68.1, 63.5, 63.4
(C-2c), 61.2, 52.8 (C-2a), 52.2, 52.1 (COOCH3), 27.2, 23.3,
22.9, 22.8, 21.8, 21.3 (OPiv, NHCOCH3, O-i-Pr). Anal.
calcd. for C71H86N4O23 (%): C 62.54, H 6.36, N 4.11; found
(%): C 62.45, H 6.51, N 4.12.
Methyl (isopropyl O-(2-acetamido-3-O-benzyl-4,6-O-
benzylidene-2-deoxy-α-D-glucopyranosyl)-(1→4)-O-
(methyl 3-O-benzyl-α-L-idopyranosyluronate)-(1→4)-O-
(2-azido-3,6-di-O-benzyl-2-deoxy-α-D-glucopyranosyl)-(1
→4)-3-O-benzyl-2-O-pivaloyl-α-L-idopyranosyl) uronate
(20)
To a solution of 19 (460 mg, 0.307 mmol) in dry MeOH
(10 mL), MeONa (73 µL of 1.2 M MeONa in MeOH) was
added and stirred for 24 h. The reaction was neutralized with
resin (IRA-120 H+) and filtered. The residue was purified by
flash chromatography (hexane–EtOAc, 1:2) to yield 20
(429 mg, 96%). TLC: 0.28 (hexane–EtOAc, 1:1). [α]D23
+14.0° (c 1.0, CH2Cl2). MALDI-TOF (m/z): 1475 (M +
1
Na+), 1491 (M + K+). H NMR (500 MHz, CDCl3) δ: 7.42–
7.21 (m, 30H, 6 Ph), 6.11 (d, 1H, NH, JNH,2 = 8.2 Hz), 5.50
(s, 1H, Ph-CH-), 5.17 (d, 1H, H-1a, J1,2 = 4.7 Hz), 5.11 (b.s.,
1H, H-1c), 5.06 (d, 1H, H-1b, J1,2 = 3.6 Hz), 4.96 (d, 1H,
H-1d, J1,2 = 3.8 Hz), 4.92 (t, 1H, H-2a, J2,1 ≈ J2,3 = 5.0 Hz),
4.87 (d, 1H, H-5c, J5,4 = 2.3 Hz), 4.83 (d, 1H, 1 CH2Ph,
Jgem = 12.2 Hz), 4.75 (2d, 2H, 2 CH2Ph, Jgem = 11 Hz), 4.72
Methyl (isopropyl O-(2-acetamido-3-O-benzyl-4,6-O-
benzylidene-2-deoxy-α-D-glucopyranosyl)-(1→4)-O-
(methyl 2-O-acetyl-3-O-benzyl-α-L-idopyranosyluronate)-
(1→4)-O-(2-azido-3,6-di-O-benzyl-2-deoxy-α-D-glucopy-
ranosyl)-(1→4)-3-O-benzyl-2-O-pivaloyl-α-L-idopyranosyl)
uronate (19)
To a solution of 8 (425 mg, 0.54 mmol) and 7 (600 mg,
0.693 mmol) in dry CH2Cl2 (0.5 mL) at room temperature,
TMSOTf (8.7 µL, 48 µmol) was added under an argon atmo-
sphere. After stirring for 4 h 30 min, saturated NaHCO3 so-
lution (2 mL) was added and diluted with CH2Cl2 (25 mL).
The residue was washed with H2O (20 mL) and then the
aqueous layer was extracted with CH2Cl2 (2 × 15 mL) and
the organic layers were dried (MgSO4) and concentrated in
vacuo. The residue was purified by flash chromatography
(hexane–EtOAc, 1:1), to yield 19 (401 mg, 50%) and
nonreacting acceptor 8 (195 mg, 46%). TLC: 0.33 (hexane–
EtOAc, 1:1). [α]D23 +10.0° (c 1.0, CH2Cl2). 1H NMR
(500 MHz, CDCl3) δ: 7.43–7.20 (m, 30H, 6 Ph), 5.77 (d,
1H, NH, JNH,2 = 9.5 Hz), 5.52 (s, 1H, Ph-CH-), 5.18 (d, 1H,
H-1a, J1,2 = 4.7 Hz), 5.16 (d, 1H, H-1c, J1,2 = 3.7 Hz), 5.03
(d, 1H, H-5a, J5,4 = 4.7 Hz), 4.68 (d, 1H, 1 CH2Ph, Jgem
=
11 Hz), 4.63 (2d, 2H, 2 CH2Ph, Jgem = 11.5 Hz), 4.56− 4.52
(m, 4H, 4 CH2Ph), 4.18− 4.11 (m, 3H, H-2d, H-4a, H-6d),
3.99–3.91 (m, 4H, H-4b, H-3a, H-4c, CH(CH3)2), 3.84 (m,
1H, H-3c), 3.72–3.65 (m, 6H, H-3b, H-6b, H-6′b, H-4d,
H-5d, H-6′d), 3.63 (s, 3H, COOCH3), 3.61–3.55 (m, 3H,
H-2c, H-3d, H-5b), 3.46 (m, 1H, H-3d), 3.40 (s, 3H,
COOCH3), 3.30 (dd, 1H, H-2b, J2,1 = 3.6 Hz, J2,3
=
10.3 Hz), 2.60 (d, 1H, OH-2c, JOH,2 = 4.7 Hz), 1.73 (s, 3H,
NHCOCH3), 1.19 (s, 9H, C(CH3)3), 1.18 and 1.13 (2d, 6H,
J = 6.5 Hz, CH(CH3)2). 13C NMR (125 MHz, CDCl3) δ:
177.3, 170.7, 170.0, 169.3 (C=O), 138.4–126.1 (Ph), 101.5
(Ph-CH-), 100.8 (C-1c), 98.3 (C-1b), 97.2 (C-1a), 96.8
(C-1d), 82.3, 78.2, 76.2, 75.2, 75.0, 74.1, 74.0, 73.8, 73.6,
73.2, 72.8, 72.1, 71.4, 71.3, 70.9, 70.4, 69.3, 68.6, 68.3,
68.0, 67.6, 63.4, 63.2, 52.6 (C-2d), 52.2, 52.0 (COOCH3),
38.8, 29.1, 27.2, 23.3, 22.9, 21.8 (OPiv, NHCOCH3, O-i-Pr).
Anal. calcd. for C78H92N4O23·H2O (%): C 63.66, H 6.43,
N 3.81; found (%): C 63.51, H 6.19, N 4.01.
© 2002 NRC Canada

Ojeda et al.
929
(hexane–EtOAc, 1:3). [α]2D3 +23.2° (c 1.0, CH2Cl2).
MALDI-TOF (m/z): 1477 (M + Na+), 1493 (M + K+).
1H NMR (500 MHz, CDCl3) δ: 7.34–7.16 (m, 30H, 6 Ph),
Methyl (isopropyl O-(2-acetamido-3-O-benzyl-4,6-O-
benzylidene-2-deoxy-α-D-glucopyranosyl)-(1→4)-O-
(methyl 2,3-di-O-benzyl-α-L-idopyranosyluronate)-
(1→4)-O-(2-azido-3,6-di-O-benzyl-2-deoxy-α-D-glucopy-
ranosyl)-(1→4)-3-O-benzyl-2-O-pivaloyl-α-L-idopyran-
osyl) uronate (21)
(a) To a solution of 25 (170 mg, 0.124 mmol), freshly pre-
pared Ag2O (90 mg, 0.388 mmol) and 4 Å molecular sieves
in dry DMF (0.5 mL), freshly distilled benzyl bromide
(100 µL, 0.84 mmol) was added. After stirring for 40 h, the
solution was filtered through Celite. The residue was puri-
fied by flash chromatography (toluene–EtOAc, 5:2) to yield
21 (101 mg, 52%).
6.19 (d, 1H, NH, JNH,2 = 9.6 Hz), 5.29 (d, 1H, H-1c, J2,1
=
3.1 Hz), 5.16 (d, 1H, H-1a, J2,1 = 4.5 Hz), 5.04 (d, 1H,
H-1b, J2,1 = 3.5 Hz), 4.91 (t, 1H, H-2a, J2,1 ≈ J2,3 = 4.8 Hz),
4.86 (d, 1H, 1 CH2Ph, Jgem = 10.8 Hz), 4.78− 4.74 (m, 3H,
H-1d, H-5c, 1 CH2Ph), 4.73 (d, 1H, H-5c, J5,4 = 4.5 Hz),
4.69− 4.40 (m, 10H, 10 CH2Ph), 4.22 (m, 1H, H-2d), 4.12 (t,
1H, H-4a, J4,3 ≈ J4,5 = 5.2 Hz), 4.04 (t, 1H, H-4b, J4,3 ≈ J4,5
=
9.5 Hz), 3.94 (t, 1H, H-3a, J3,2 ≈ J3,4 = 5.5 Hz), 3.92–3.90
(m, 2H, H-4c, CH(CH3)2), 3.85 (m, 1H, H-5b), 3.81 (t, 1H,
H-3c, J3,2 ≈ J3,4 = 4.4 Hz), 3.75 (t, 1H, H-3b, J3,2 ≈ J3,4
=
9.8 Hz), 3.72–3.57 (m, 5H, H-4d, H-5d, H-6d, H-6b, H-6′b),
3.64 (s, 3H, COOCH3), 3.46–3.35 (m, 3H, H-3d, H-2c,
(b) To a solution of 20 (440 mg, 0.303 mmol) and freshly
prepared Ag2O (161 mg, 0.7 mmol) in dry DMF (0.5 mL),
freshly distilled benzyl bromide (100 µL, 0.84 mmol) was
added. After stirring for 30 h, the solution was filtered through
Celite. The residue was purified by flash chromatography
(hexane–EtOAc, 1:1) to yield 21 (411 mg, 88%). TLC: 0.32
(hexane–EtOAc, 1:1). [α]2D3 +47.3° (c 1.0, CHCl3). MALDI-
H-6′b), 3.42 (s, 3H, COOCH3), 3.31 (dd, 1H, H-2b, J2,1
=
3.7 Hz, J2,3 = 10.3 Hz), 1.51 (s, 3H, NHCOCH3), 1.20
(s, 9H, C(CH3)3), 1.18 and 1.14 (2d, 6H, J = 6.1 Hz,
CH(CH3)2). 13C NMR (125 MHz, CDCl3) δ: 177.3, 170.1,
170.0, 169.3 (C=O), 138.2–127.6 (Ph), 98.7 (C-1c), 98.4
(C-1b), 97.5 (C-1d), 97.3 (C-1a), 80.4, 78.2, 76.2, 74.9,
74.2, 74.1, 74.0, 73.9, 73.7, 73.3, 73.2, 72.5, 72.4, 72.2,
71.8, 71.3, 70.7, 70.4, 70.2, 69.2, 63.1, 62.4 (C-2b), 52.1,
52.0 (COOCH3), 51.8 (C-2d), 38.8, 27.2, 23.3, 22.8,
21.8 (OPiv, NHCOCH3, O-i-Pr). Anal. calcd. for
C78H94N4O23·0.5H2O (%): C 63.96, H 6.54, N 3.82; found
(%): C 63.60, H 6.48, N 3.80.
1
TOF (m/z): 1566 (M + Na+ + H), 1581 (M + K+). H NMR
(500 MHz, CDCl3) δ: 7.44–7.15 (m, 35H, 7 Ph), 5.92 (d,
1H, NH, JNH,2 = 9.7 Hz), 5.50 (s, 1H, Ph-CH-), 5.29 (d, 1H,
H-1c, J2,1 = 3.7 Hz), 5.16 (d, 1H, H-1a, J2,1 = 4.6 Hz),
5.04 (d, 1H, H-1b, J2,1 = 3.6 Hz), 4.91 (t, 1H, H-2a, J2,1
≈
J2,3 = 5.0 Hz), 4.87 (d, 1H, 1 CH2Ph, Jgem = 10.7 Hz), 4.81
(d, 1H, H-1d, J2,1 = 3.8 Hz), 4.78− 4.64 (m, 7H, H-5c, H-
5a, 5 CH2Ph), 4.57 (d, 1H, 1 CH2Ph, Jgem = 11.9 Hz),
4.52− 4.47 (m, 3H, 3 CH2Ph), 4.43 (d, 1H, 1 CH2Ph, Jgem
11.9 Hz), 4.42 (d, 1H, 1 CH2Ph, Jgem = 12.1 Hz), 4.31 (m,
1H, H-2d), 4.16 (dd, 1H, H-6d, J6,5 = 4.5 Hz, J6,6′
10.2 Hz), 4.12 (t, 1H, H-4a, J4,3 ≈ J4,5 = 5.2 Hz), 4.04 (t, 1H,
H-4b, J4,3 ≈ J4,5 = 9.5 Hz), 3.96–3.91 (m, 3H, H-3a, H-4c,
CH(CH3)2), 3.85–3.82 (m, 2H, H-5b, H-3c), 3.78–3.52 (m,
7H, H-3b, H-3d, H-4d, H-5d, H-6′d, H-6b, H-6′b), 3.62 (s,
3H, COOCH3), 3.47 (s, 3H, COOCH3), 3.44 (t, 1H, H-2c,
=
Methyl (isopropyl O-(2-acetamido-6-O-benzoyl-3-O-
benzyl-2-deoxy-α-D-glucopyranosyl)-(1→4)-O-(methyl
2,3-di-O-benzyl-α-L-idopyranosyluronate)-(1→4)-O-
(2-azido-3,6-di-O-benzyl-2-deoxy-α-D-glucopyranosyl)-
(1→4)-3-O-benzyl-2-O-pivaloyl-α-L-idopyranosyl)
uronate (23)
To a solution of 22 (330 mg, 0.227 mmol) in dry CH3CN
(4 mL) at − 40°C, BzCN (262 µL of a 0.9 M solution of
BzCN in dry CH3CN) and a few drops of Et3N were added.
After 7 h, MeOH was added and the mixture was allowed to
reach room temperature. The solvent was evaporated and the
residue was dissolved in MeOH and concentrated to dryness.
The residue was purified by flash chromatography (hexane–
EtOAc, 1:1) to yield 23 (312 mg, 88%). TLC: 0.29 (hexane–
EtOAc, 1:1). [α]2D3 +43.2° (c 1.0, CH2Cl2). MALDI-TOF
(m/z): 1581 (M + Na+), 1597 (M + K+). 1H NMR (500 MHz,
=
J
2,1 ≈ J2,1 = 4.1 Hz), 3.30 (dd, 1H, H-2b, J2,1 = 3.6 Hz, J2,3
=
10.3 Hz), 1.51 (s, 3H, NHCOCH3), 1.20 (s, 9H, C(CH3)3),
1.19 and 1.14 (2d, 6H, J = 6.5 Hz, CH(CH3)2). 13C NMR
(125 MHz, CDCl3) δ: 177.3, 170.0, 169.1 (C=O), 138.6,–
126.1 (Ph), 101.4 (Ph-CH-), 98.7 (C-1c), 98.4 (C-1b), 98.3
(C-1d), 97.2 (C-1a), 82.3, 78.1, 76.2, 76.0, 75.8, 74.2, 74.1,
73.7, 73.4, 73.2, 71.8, 71.3, 70.7, 70.3, 69.5, 68.7 (C-6d),
67.7, 65.4, 63.6, 63.0 (C-2b), 52.2 (C-2d), 52.1, 51.9
(COOCH3), 29.7, 27.2, 23.3, 22.8, 21.8 (OPiv, NHCOCH3,
Oi-Pr). Anal. calcd. for C85H98N4O23 (%): C 66.13, H 6.40,
N 3.63; found (%): C 65.90, H 6.40, N 3.42.
CDCl3) δ: 8.03–7.13 (m, 35H, 7 Ph), 6.16 (d, 1H, NH, JNH,2
9.7 Hz), 5.29 (d, 1H, H-1c, J1,2 = 3.0 Hz), 5.17 (d, 1H, H-1a,
J1,2 = 4.6 Hz), 5.05 (d, 1H, H-1b, J1,2 = 3.6 Hz), 4.92 (t, 1H,
=
H-2a, J2,1 ≈ J2,3 = 5.0 Hz), 4.84 (d, 1H, 1 CH2Ph, Jgem
10.5 Hz), 4.83 (s, 1H, H-1d), 4.81 (d, 1H, H-5c, J5,4
3.3 Hz), 4.76 (d, 1H, 1 CH2Ph, Jgem = 11.2 Hz), 4.73 (d, 1H,
H-5a, J5,4 = 4.5 Hz), 4.70− 4.47 (m, 10H, H-6d, 9 CH2Ph),
4.39 (d, 1H, 1 CH2Ph, Jgem = 12.0 Hz), 4.38 (dd, 1H, H-6′d,
J6′,5 = 1.8 Hz, J6′,6′ = 12.0 Hz), 4.26 (m, 1H, H-2d), 4.12 (t,
=
=
Methyl (isopropyl O-(2-acetamido-3-O-benzyl-2-deoxy-α-
D-glucopyranosyl)-(1→4)-O-(methyl 2,3-di-O-benzyl-α-L-
idopyranosyluronate)-(1→4)-O-(2-azido-3,6-di-O-benzyl-
2-deoxy-α-D-glucopyranosyl)-(1→4)-3-O-benzyl-2-O-
pivaloyl-α-L-idopyranosyl) uronate (22)
To a solution of 21 (450 mg, 0.291 mmol) in dry CH2Cl2
(9 mL), EtSH (108 µL, 1.46 mmol), and catalytic PTSA
were added. After stirring for 3 h under an argon atmo-
sphere, the reaction was neutralized with solid NaHCO3, di-
luted with CH2Cl2 (40 mL), and washed with H2O (40 mL).
The organic layer was dried (MgSO4) and concentrated to
dryness. The residue was purified by flash chromatography
(hexane–EtOAc, 1:3) to yield 22 (351 mg, 83%). TLC: 0.23
1H, H-4a, J4,3 ≈ J4,5 = 5.2 Hz), 4.04 (t, 1H, H-4b, J4,3 ≈ J4,5 =
9.6 Hz), 3.99 (t, 1H, H-4c, J4,3 ≈ J4,5 = 3.9 Hz), 3.95 (t, 1H,
H-3a, J3,2 ≈ J3,4 = 5.4 Hz), 3.93 (m, 1H, CH(CH3)2, J =
6.2 Hz), 3.84 (m, 1H, H-5b), 3.82 (t, 1H, H-3c, J3,2 ≈ J3,4
=
4.3 Hz), 3.74 (t, 1H, H-3b, J3,2 ≈ J3,4 = 10.0 Hz), 3.69–3.57
(m, 4H, H-4d, H-5d, H-6b, H-6′b), 3.63 (s, 3H, COOCH3),
3.43 (s, 3H, COOCH3), 3.44–3.40 (m, 2H, H-3d, H-2c), 3.31
(dd, 1H, H-2b, J2,1 = 3.6 Hz, J2,3 = 10.3 Hz), 1.52 (s, 3H,
© 2002 NRC Canada

930
Can. J. Chem. Vol. 80, 2002
NHCOCH3), 1.20 (s, 9H, C(CH3)3), 1.19 and 1.14 (2d, 6H,
J = 6.1 Hz, CH(CH3)2). 13C NMR (125 MHz, CDCl3) δ:
177.3, 170.0, 169.2, 166.9 (C=O), 138.3–127.6 (Ph), 98.7
(C-1c), 98.4 (C-1b), 97.2 (C-1a), 97.1 (C-1d), 80.2, 78.1,
76.4, 74.9, 74.5, 74.2, 74.1, 73.7, 73.3, 73.2, 73.1, 73.0,
71.9, 71.8, 71.3, 71.0, 70.7, 70.3, 69.7, 69.1, 67.9, 63.1,
63.0, 52.0, 51.9 (COOCH3, C-2d), 38.8, 27.2, 23.3, 22.8,
21.8 (OPiv, NHCOCH3, O-i-Pr). Anal. calcd. for
C85H98N4O24·0.5H2O (%): C 65.08, H 6.36, N 3.57, found
(%): C 64.83, H 6.36, N 3.57.
calcd. for C124H141N7O35 (%): C 65.05, H 6.21, N 4.28;
found (%): C 64.85, H 6.18, N 4.23.
Isopropyl O-(2-azido-3-O-benzyl-4,6-di-O-benzylidene-2-
deoxy-α-D-glucopyranosyl)-(1→4)-O-(3-O-benzyl-α-L-
idopyranosyluronic acid)-(1→4)-O-(2-acetamido-3-O-
benzyl-2-deoxy-α-D-glucopyranosyl)-(1→4)-O-(2,3-di-O-
benzyl-α-L-idopyranosyluronic acid)-(1→4)-O-(2-azido-
3,6-di-O-benzyl-2-deoxy-α-D-glucopyranosyl)-(1→4)-3-O-
benzyl-α-L-idopyranosyluronic acid (26)
To a solution of 5 (39 mg, 17.0 µmol) in THF (2 mL) at
–5°C, 30% H2O2 (0.7 mL) and a 1 M aqueous solution of
LiOH (1.1 mL) were added. After stirring for 24 h at room
temperature MeOH (1 mL) and a 3 N aqueous solution of
KOH (2 mL) were added. After stirring for 24 h more the re-
action was neutralized with acidic resin (IRA-120 H+), fil-
tered, and concentrated. The residue was purified by
Sephadex LH-20 (MeOH–CH2Cl2, 1:1) to yield 26 (33 mg,
98%). TLC: 0.45 (CH2Cl2–MeOH, 8:1). 1H NMR
(500 MHz, MeOD) δ: 7.44–7.10 (m, 50 H, 10 Ph), 5.56 (s,
1H, Ph-CH-), 5.37 (b.s., 1H, H-1c), 5.05–5.01 (m, 3H, H-1a,
H-1b, H-1e), 4.89 (m, 1H, H-1f ), 4.83 (m, 1H, H-1d), 1.48
(m, 3H, NHCOCH3), 1.17–1.16 (m, 6H, CH(CH3)2).
Methyl (isopropyl O-(2-azido-3-O-benzyl-4,6-O-
benzylidene-2-deoxy-α-D-glucopyranosyl)-(1→4)-O-
(methyl 3-O-benzyl-2-O-pivaloyl-α-L-idopyranosy-
luronate)-(1→4)-O-(2-acetamido-6-O-benzoyl-3-O-
benzyl-2-deoxy-α-D-glucopyranosyl)-(1→4)-O-(methyl
2,3-di-O-benzyl-α-L-idopyranosyluronate)-(1→4)-O-(2-
azido-3,6-di-O-benzyl-2-deoxy-α-D-glucopyranosyl)-
(1→4)-3-O-benzyl-2-O-pivaloyl-α-L-idopyranosyl)
uronate (5)
To a solution of 23 (90 mg, 57.7 µmol) and 6 (68 mg,
75.7 µmol) in dry CH2Cl2 (0.5 mL) at room temperature,
TMSOTf (25 µL of a 0.1 M solution of TMSOTf in CH2Cl2)
was added under an argon atmosphere. After stirring for 2 h
30 min, saturated NaHCO3 solution (1 mL) was added and
diluted with CH2Cl2 (20 mL). The residue was washed with
H2O (15 mL) and then the aqueous layer was extracted with
CH2Cl2 (2 × 10 mL) and the organic layers were dried
(MgSO4) and concentrated in vacuo. The residue was puri-
fied by flash chromatography (hexane–EtOAc, 2:3) to yield
5 (85 mg, 65%) and nonreacting acceptor 23 (27 mg, 30%).
TLC: 0.33 (hexane–EtOAc, 3:2). [α]2D3 +10.4° (c 1.1,
CH2Cl2). 1H NMR (500 MHz, CDCl3) δ: 8.09–7.13 (m,
50H, 10 Ph), 5.90 (d, 1H, NH, JNH,2 = 9.8 Hz), 5.48 (s, 1H,
Ph-CH-), 5.30 (d, 1H, H-1c, J1,2 = 3.4 Hz), 5.25 (d, 1H,
H-1e, J1,2 = 3.8 Hz), 5.17 (d, 1H, H-1a, J1,2 = 4.6 Hz), 5.05
(d, 1H, H-1b, J1, 2 = 3.7 Hz), 4.93− 4.90 (m, 3H, H-2a, H-2e,
H-1f ), 4.85 (d, 1H, 1 CH2Ph, Jgem = 10.9 Hz), 4.83 (d, 1H,
1 CH2Ph, Jgem = 11 Hz), 4.79 (d, 1H, H-1d, J1,2 = 3.7 Hz),
4.76− 4.47 (m, 16H, H-5a, H-5c, H-5e, H-6d, 12 CH2Ph),
4.42 (m, 1H, H-6′d), 4.39 (d, 1H, 1 CH2Ph, Jgem = 12 Hz),
4.28 (m, 1H, H-2d), 4.27 (d, 1H, 1 CH2Ph, Jgem = 10.9 Hz),
4.19 (dd, 1H, H-6f, J6,5 = 4.9 Hz, J6,6′ = 10.1 Hz), 4.12 (dd,
1H, H-4a, J4,3 = 4.8 Hz, J4,5 = 5.9 Hz), 4.05–3.99 (m, 3H,
H-4b, H-4d, H-4e), 3.99–3.90 (m, 4H, H-3a, H-3e, H-4c,
CH(CH3)2), 3.88–3.82 (m, 3H, H-3f, H-5b, H-5f ), 3.80–3.72
Isopropyl O-(2-azido-3-O-benzyl-4,6-di-O-benzylidene-2-
deoxy-α-D-glucopyranosyl)-(1→4)-O-(3-O-benzyl-2-O-
sulfo-α-L-idopyranosyluronic acid)-(1→4)-O-(2-
acetamido-3-O-benzyl-2-deoxy-6-O-sulfo-α-D-gluco-
pyranosyl)-(1→4)-O-(2,3-di-O-benzyl-α-L-idopyranosyl-
uronic acid)-(1→4)-O-(2-azido-3,6-di-O-benzyl-2-deoxy-
α-D-glucopyranosyl)-(1→4)-3-O-benzyl-2-O-sulfo-α-L-
idopyranosyl)uronic acid hexasodium salt (27)
A mixture of 26 (33 mg, 16.7 µmol) and complex SO3·Py
(40 mg, 0.25 mmol) in dry Py (1.5 mL) was stirred under an
argon atmosphere. After 24 h, the mixture was cooled and
MeOH (1 mL) and CH2Cl2 (1 mL) were added. The solution
was eluted through a Sephadex LH-20 (MeOH–CH2Cl2,1:1).
Fractions containing the hexasaccharide were concentrated
and passed through a Dowex 50WX4-Na+ (MeOH–H2O,
2:1) to yield 27 (28 mg, 72%). TLC: 0.65 (EtOAc–Py–H2O–
1
AcOH, 8:5:3:1). H NMR (500 MHz, MeOD) δ: 7.42–7.10
(m, 50H, 10 Ph), 5.56 (s, 1H, Ph-CH-), 5.46 (s, 1H, H-1a or e),
5.32 (s, 1H, H-1c), 5.28 (s, 1H, H-1a or e), 5.15 (d, 1H,
H-1b or f ), 5.06 (d, 1H, H-1b or f ), 4.82 (m, 1H, H-1d),
1.26 (m, 3H, NHCOCH3), 1.20 (d, 6H, CH(CH3)2, J =
6.0 Hz). 13C NMR (125 MHz, MeOD) δ: 101.4 (Ph-CH-),
98.8 (C-1c), 98.1 (C-1a or e), 97.5 (C-1a or e), 96.1 (C-1b
or f ), 95.7 (C-1b or f ), 95.5 (C-1d).
(m, 3H, H-3c, H-3b, H-5d), 3.67 (dd, 1H, H-6b, J6,5
=
3.0 Hz, J6,6′ = 11.7 Hz), 3.63 (s, 3H, COOCH3), 3.61–3.55
(m, 3H, H-4f, H-6′b, H-6′f ), 3.45–3.41 (m, 2H, H-2c, H-3d),
3.39 (s, 3H, COOCH3), 3.32 (s, 3H, COOCH3), 3.32–3.30
Isopropyl O-(2-deoxy-2-sulfamide-α-D-glucopyranosyl)-
(1→4)-O-(2-O-sulfo-α-L-idopyranosyluronic acid)-(1→4)-
O-(2-acetamido-2-deoxy-6-O-sulfo-α-D-glucopyranosyl)-(1
→4)-O-(α-L-idopyranosyluronic acid)-(1→4)-O-(2-deoxy-
2-sulfamide-α-D-glucopyranosyl)-(1→4)-2-O-sulfo-α-L-
idopyranosyluronic acid octasodium salt (3)
To a solution of 27 (17 mg, 7.24 µmol) in MeOH–H2O
(1.5 mL, 9:1) was hydrogenolyzed in the presence of 10%
Pd/C. After 24 h, the suspension was filtered and concentrated
to give the desired product which was directly used in the
next step without further purification.
(m, 1H, H-2b), 3.27 (dd, 1H, H-2f, J2,1 = 3.8 Hz, J2,3
=
10.0 Hz), 1.41 (s, 3H, NHCOCH3), 1.20 (s, 9H, C(CH3)3),
1.19 and 1.13 (2d, 6H, J = 6.1 Hz, CH(CH3)2), 1.11 (s, 9H,
C(CH3)3). 13C NMR (125 MHz, CDCl3) δ: 177.4, 177.3,
170.0, 169.8, 169.3, 169.0, 166.1 (C=O), 138.4–126.1 (Ph),
101.5 (Ph-CH-), 99.4 (C-1f ), 98.7 (C-1b, C-1c), 98.0 (C-1e),
97.2 (C-1a, C-1d), 82.5, 78.1, 76.3, 76.0, 75.0, 74.9, 74.8,
74.6, 74.5, 74.1, 74.0, 73.7, 73.5, 73.3, 73.2, 71.8, 71.3,
71.1, 70.3, 70.1, 69.9, 69.6, 68.5, 67.8, 63.2, 63.0, 62.9,
62.2, 52.0, 51.9 (COOCH3), 51.7 (C-2d), 38.8, 38.7, 27.2,
27.1, 23.3, 22.8, 21.8 (OPiv, NHCOCH3, O-i-Pr). Anal.
© 2002 NRC Canada

Ojeda et al.
931
The hydrogenolyzed hexasaccharide was dissolved in H2O
(1mL) and the pH of the solution was adjusted to 9.5 with
1 N solution of NaOH. Complex SO3·Py (24 mg, 10 equiv
for each amine group) was added in three portions during
1 h and the pH was maintained at 9.5 by subsequent addition
of a 1 N solution of NaOH. A second, third, and fourth por-
tion of complex SO3·Py were added after 2, 4, and 6 h stir-
ring, respectively. After 24 h, the mixture was neutralized
with 0.1 N HCl to pH 7 and then passed through a column
of Sephadex G-25 with 0.9% NaCl. The appropriate frac-
tions were pooled and passed through a column of Dowex
50WX4-Na+ with 0.5 M NaCl and then a column of
Sephadex G-25 with H2O–EtOH (9:1). The fractions which
contained the final hexasaccharide were lyophilized to give 3
(9.1 mg, 75% from 27). Before NMR studies, it was useful
to make a last elution on a column of Dowex 50WX4-Na+ to
avoid the formation of calcium salts instead of sodium salts
and get a better resolution in the spectra. 1H NMR
(500 MHz, D2O) δ: 5.38 (d, 1H, H-1f, J1,2 = 3.7 Hz), 5.32
H-2 (α and β), H-4 (α and β), H-5 (β )), 3.61 (t, 1H, H-3 (β),
J3,2 ≈ J3,4 = 9.0 Hz).
Methyl dimethylthexylsilyl 2-O-acetyl-4-O-(2-azido-6-O-
benzoyl-3,4-di-O-benzyl-2-deoxy-α-D-glucopyranosyl)-3-
O-benzyl-β-L-idopyranuronate (31)
To a solution of 11 (600 g, 0.95 mmol) in dry CH2Cl2
(20 mL) at 0°C TMSOTf (8.6 µL, 0.048 mmol) was added
under an argon atmosphere. A solution of 29 (720 mg,
1.63 mmol) in dry CH2Cl2 (30 mL) was added dropwise for
30 min. The reaction was neutralized with saturated
NaHCO3 solution and diluted with CH2Cl2 (100 mL). The
organic layer was washed with H2O (75 mL), dried
(MgSO4), and concentrated in vacuo. The residue was puri-
fied by flash chromatography (hexane–EtOAc, 4:1) to yield
a mixture of isomers containing 30 which was used directly
in the next step.
To a solution of this mixture in Py (10 mL), acetic anhy-
dride (5 mL) was added and was stirred for 24 h. The reac-
tion was diluted with CH2Cl2 (100 mL) and washed with
H2O (80 mL) and 1 N HCl solution (80 mL). The organic
layer was dried (MgSO4) and concentrated to dryness. The
residue was purified by flash chromatography (hexane–
EtOAc, 6:1) to yield 31 (391 mg, 43% from 11). TLC: 0.36
(hexane–EtOAc, 6:1). [α]2D3 +76.0° (c, 1.0, CH2Cl2). 1H
NMR (500 MHz, CDCl3) δ: 7.99–7.20 (m, 20H, 4 Ph), 5.06
(d, 1H, H-1, J1,2 = 1.5 Hz), 4.99 (b.s., 1H, H-2), 4.90 (d, 1H,
H-1′, J1,2 = 3.4 Hz), 4.86 (d, 1H, 1 CH2Ph, Jgem = 11.2 Hz),
4.82 (s, 2H, 2 CH2Ph), 4.74− 4.58 (m, 4H, 3 CH2Ph, H-6′a),
(d, 1H, H-1b, J1,2 = 3.7 Hz), 5.22 (d, 1H, H-1a, J1,2
=
3.0 Hz), 5.18 (d, 1H, H-1e, J1,2 = 3.0 Hz), 5.15 (d, 1H,
H-1d, J1,2 = 4.0 Hz), 4.89 (d, 1H, H-1c, J1,2 = 3.5 Hz), 1.99
(m, 3H, NHCOCH3), 1.17 (d, 3H, CH(CH3)2, J = 6.2 Hz),
1.16 (d, 3H, CH(CH3)2, J = 6.2 Hz). 13C NMR (125 MHz,
D2O) δ: 102.9 (C-1c), 99.8 (C-1e), 97.8 (C-1a), 97.6 (C-1b),
97.3 (C-1f ), 94.9 (C-1d).
2-Azido-6-O-benzoyl-3,4-di-O-benzyl-2-deoxy-α ,β-D-
glucopyranose trichloroacetimidate (29)
4.47 (d, 1H, H-5, J5,4 = 1.2 Hz), 4.41 (dd, 1H, H-6′b, J6b,5
=
To a solution of 28 (1 g, 2.12 mmol) in dry MeOH
(8 mL), solid KOH (119 mg, 2.12 mmol) was added, and
was stirred for 20 min. The solution was diluted with
CH2Cl2 (200 mL) and saturated NH4Cl solution (2 ×
100 mL) and H2O (2 × 100 mL). The organic layer was
dried (MgSO4) and concentrated in vacuo. The residue was
directly used in the next step without further purification.
To this residue dissolved in Py (10 mL), BzCl (2 mL) was
added and stirred at room temperature for 24 h. The reaction
was diluted with CH2Cl2 (100 mL), washed with H2O
(75 mL) and 1 N HCl solution (75 mL), dried (MgSO4), and
concentrated to dryness. The residue was purified by flash
chromatography and used in the next step.
2.6 Hz, J6b,6a = 12.3 Hz), 4.22 (m, 1H, H-5′), 4.11 (b.s., 1H,
H-4), 3.99–3.95 (m, 2H, H-3′, H-3), 3.73 (s, 3H, COOMe),
3.70 (m, 1H, H-4′), 3.28 (dd, 1H, H-2′, J2,1 = 3.4 Hz, J2,3
=
10.3 Hz), 2.04 (d, 3H, OCOCH3), 1.60 (m, 1H, CH(CH3)2),
0.86–0.83 (m, 12H, CH(CH3)2, C(CH3)2), 0.24 and 0.14 (2s,
6H, Si(CH3)2). 13C NMR (125 MHz, CDCl3) δ: 170.6,
168.9, 166.1 (C=O), 137.8–127.5 (Ph), 97.4 (C-1′), 93.6
(C-1), 80.0 (C-3′), 77.9 (C-4′), 75.5, 74.9, 74.3, 73.2, 72.9,
72.8, 69.9 (C-5), 67.2 (C-2), 63.5 (C-2′), 62.8 (C-6), 52.1
(COOMe), 34.1, 24.8, 20.9, 20.2, 19.8 18.6, –2.1, –3.5 (OTDS,
OCOCH3). Anal. calcd. for C51H63N3O13Si·0.5H2O (%):
C 63.60, H 6.70, N 4.36; found (%): C 63.64, H 6.40,
N 4.34.
To a solution of this residue (1.2 g, 2.04 mmol) in dry
Et2O (10 mL), benzylamine (1.2 mL, 10.9 mmol) was added
and was stirred for 4 h. The residue was concentrated to dry-
ness and purified by flash chromatography (hexane–EtOAc,
3:1) and then to a solution of this compound (625 mg,
1.27 mmol) in dry CH2Cl2 (7 mL), Cl3CCN (1.9 mL,
19 mmol) and catalytic DBU were added. After stirring for
1 h the residue was concentrated to dryness. The residue was
purified by flash chromatography (hexane–EtOAc (4:1) +
1% Et3N) to yield 29 (703 mg, 87%) as a mixture of
anomers α and β. TLC (α and β): 0.50 and 0.43 (hexane–
Methyl dimethylthexylsilyl 4-O-(2-acetamido-6-O-
benzoyl-3,4-di-O-benzyl-2-deoxy-α-D-glucopyranosyl)-2-
O-acetyl-3-O-benzyl-β-L-idopyranuronate (32)
A solution of 31 (375 mg, 0.39 mmol) in thiolacetic acid
(6 mL) was stirred for 30 h at room temperature. After con-
centrating to dryness, the residue was purified by flash chro-
matography (hexane–EtOAc, 1:1) to yield 32 (259 mg,
68%). TLC: 0.45 (hexane–EtOAc, 1:1). [α]2D3 +70.5° (c, 1.0,
δ
CH2Cl2). 1H NMR (500 MHz, CDCl3): : 8.00–7.18 (m,
20H, 4 Ph), 6.29 (d, 1H, NH, JNH,2 = 9.8 Hz), 5.06 (b.s., 1H,
H-1), 4.98 (b.s., 1H, H-2), 4.84− 4.77 (m, 2H, H-1′, 2
CH2Ph), 4.84− 4.77 (m, 5H, 4 CH2Ph, H-5), 4.48 (d, 1H, H-
6′a, J6a,6b = 12.1 Hz), 4.40 (dd, 1H, H-6′b, J6b,6a = 12.1 Hz,
1
EtOAc, 3:1). H NMR (500 MHz, CDCl3) δ: 8.71 (s, 1H,
O(C=NH)CCl3 (α)), 8.69 (s, 1H, O(C=NH)CCl3 (β)), 7.29–
7.24 (m, 30H, 3 Ph (α and β)), 6.42 (d, 1H, H-1 (α), J1,2
=
4.1 Hz), 5.65 (d, 1H, H-1 (β), J1,2 = 8.5 Hz), 4.99− 4.84 (m,
6H, 6 CH2Ph (α and β)), 4.65− 4.45 (m, 6H, 2 CH2Ph (α and
β), H-6a (α and β), H-6b (α and β)), 4.17 (m, 1H, H-5 α),
4.09 (t, 1H, H-3 (α), J3,2 ≈ J3,4 = 9.5 Hz), 3.80–3.70 (m, 5H,
J6b,5 = 3.4 Hz), 4.33 (m, 1H, H-2′), 3.97 (b.s., 1H, H-4), 3.93
(b.s., 1H, H-3), 3.85 (m, 1H, H-5′), 3.78 (s, 3H, COOMe),
3.72 (t, 1H, H-4′, J4,3 ≈ J4,5 = 9.1 Hz), 3.58 (t, 1H, H-3′, J3,2
≈ J3,4 = 9.6 Hz), 2.00 (d, 3H, OCOCH3), 1.79 (d, 3H,
© 2002 NRC Canada

932
Can. J. Chem. Vol. 80, 2002
NHCOCH3), 1.59 (m, 1H, CH(CH3)2), 0.86–0.82 (m, 12H,
CH(CH3)2, C(CH3)2), 0.21 and 0.12 (2 s, 6H, Si(CH3)2). 13C
NMR (125 MHz, CDCl3) δ: 170.0, 169.8, 168.5, 166.2
(C=O), 138.1–127.8 (Ph), 95.3 (C-1′), 93.1 (C-1), 81.0 (C-3
′), 77.6 (C-4′), 75.4, 74.9, 73.3, 73.0, 71.4, 70.3, 69.5, 67.9
(C-2), 63.0 (C-6′), 52.3 (COOMe), 52.2 (C-2′), 34.0, 24.9,
23.4, 20.8, 20.1, 19.8, 18.6, 13.4, –2.0, –3.5 (OTDS, OCOCH3).
(m, 13H, H-5 (β), 6 CH2Ph (α and β)), 4.54−4.36 (m, 6H,
H-2′ (α), H-2′ (β), H-6a′ (α), H-6a′ (β), H-6b′ (α), H-6b′
(β)), 4.17–4.15 (m, 2 H, H-3 (β), H-4 (α)), 4.03 (m, 1H, H-4
(β)), 3.92 (m, 1H, H-3 (α)), 3.80–3.78 (m, 8H, H-5′ (α), H-
5′ (β), COOMe (α and β)), 3.71 (t, 2H, H-4′ (α), H-4′ (β),
J4,3 ≈ J4,5 = 8.5 Hz), 3.61–3.54 (m, 2H, H-3′ (α), H-3′ (β)),
1.98, 1.96, 1.80, 1.76 (m, 12H, OCOCH3 (α), OCOCH3 (β),
NHCH3 (α), NHCH3 (β)). 13C NMR (125 MHz, CDCl3) δ:
170.1, 169.8, 169.6, 169.3, 168.5, 167.7, 166.4, 166.1
(C=O), 160.5, 160.1 (C=NH), 138.0–127.6 (Ph), 96.3
(C-1′ (α or β)), 95.0 (C-1 (α)), 94.8 (C-1′ (α or β)), 94.5
(C-1 (β)), 80.9 (C-3′ (α)), 80.8 (C-3′ (β)), 77.6 (C-4′ (β)),
77.5 (C-4′ (α)), 75.5, 75.4, 75.0, 74.9, 73.9, 73.6, 72.5, 71.3,
70.5, 70.4, 69.1, 69.0, 66.9 (C-2 (α and β)), 66.7, 62.9 (C-6′
(α and β)), 52.6, 52.5, 52.4, 52.3 (C-2′ (α and β), COOMe),
23.5, 23.3, 20.7, 20.6 (OCOCH3 (α), OCOCH3 (β), NHCH3
(α), NHCH3 (β)).
Methyl 4-O-(2-acetamido-6-O-benzoyl-3,4-di-O-benzyl-2-
deoxy-α-D-glucopyranosyl)-2-O-acetyl-3-O-benzyl-β-L-
idopyranuronate (33)
To a solution of 32 (220 mg, 3.18 mmol) at –15°C in dry
THF (7 mL) an excess of (HF)n·Py (0.7 mL) was added. The
reaction was warmed to 0°C and stirred for 24 h under an ar-
gon atmosphere. The mixture was diluted with CH2Cl2 (2 ×
100 mL) and washed with H2O (2 × 75 mL) and saturated
NaHCO3 solution until neutral pH. The aqueous layer was
extracted with CH2Cl2 (2 × 100 mL) and the organic layers
were dried (MgSO4) and concentrated in vacuo. The residue
was purified by flash chromatography (CH2Cl2–MeOH,
20:1) to yield 33 (153 mg, 81%) as a mixture of anomers α
and β. TLC (α and β): 0.41 and 0.29 (hexane–EtOAc, 1:2).
1H NMR (500 MHz, CDCl3) δ: 8.00–7.18 (m, 40H, 4 Ph (α
and β)), 6.51 (d, 1H, NH (β), JNH,2 = 9.7 Hz), 6.05 (d, 1H,
NH (α), JNH,2 = 9.7 Hz), 5.25 (b.s., 1H, H-1 (α)), 5.18 (d,
Methyl (isopropyl O-(2-azido-3,6-di-O-benzyl-2-deoxy-α-
D-glucopyranosyl)-(1→4)-O-(methyl 3-O-benzyl-2-O-
pivaloyl-α-L-idopyranosyluronate)-(1→4)-O-(2-
acetamido-6-O-benzoyl-3-O-benzyl-2-deoxy-α-D-
glucopyranosyl)-(1→4)-O-(methyl 2,3-di-O-benzyl-α-L-
idopyranosyluronate)-(1→4)-O-(2-azido-3,6-di-O-benzyl-
2-deoxy-α-D-glucopyranosyl)-(1→4)-3-O-benzyl-2-O-
pivaloyl-α-L-idopyranosyl) uronate (35)
1H, H-1 (β), J1,2 = 3.0 Hz), 4.96 (d, 1H, H-2 (β), J1,2
=
2.6 Hz), 4.93 (b.s., 1H, H-2 (β)), 4.84–4.75 (m, 11H, H-1′ (α
and β), H-5 (β), 4 CH2Ph (α and β)), 4.64–4.38 (m, 17H,
H-5 (α), H-6a′ (α and β), H-6b′ (α and β), 8 CH2Ph (α and
β)), 4.37–4.30 (m, 1H, H-2′ (α and β)), 4.07 (m, 1H, H-3
(β)), 3.99 (m, 2H, H-3 (α), H-4 (α)), 3.80 (s, 3H, COOMe
(α)), 3.79 (s, 3H, COOMe (β)), 3.79–3.66 (m, 4H, H-4′ (α
and β), H-5′ (α and β)), 3.54 (m, 2H, H-3′ (α and β)), 1.99
(d, 3H, OCOCH3 (β)), 1.95 (d, 3H, OCOCH3 (α)), 1.81 (d,
3H, NHCOCH3 (β)), 1.75 (d, 3H, NHCOCH3 (α)). 13C NMR
(125 MHz, CDCl3) δ: 170.4, 170.2, 170.1, 169.8, 169.4,
169.2 (C=O), 138.9–127.6 (Ph), 95.4 (C-1′ (α)), 94.2 (C-1′
(β)), 93.1 (C-1 (α)), 80.7, 77.6, 77.5, 75.3, 74.9, 73.3, 73.2,
72.9, 70.5, 70.4, 69.6, 69.3, 68.3, 67.6, 63.0, 62.9, 60.4,
52.6, 52.5, 52.3, 23.3, 23.2, 20.8, 20.7 (OCOCH3
(α and β), NHCH3 (α and β)). Anal. calcd. for
C45H49NO14·0.5H2O (%): C 64.58, H 6.02, N 1.67, found
(%): C 64.63, H 5.94, N 1.67.
To a solution of 5 (175 mg, 0.076 mmol) in dry THF
(2 mL), NaBH3CN (1 mL of a solution of 1 M NaBH3CN in
THF) was added, and was stirred for 24 h. Afterwards, a so-
lution of 2.5 M HCl in Et2O was added dropwise until the
mixture became acidic. The reaction was neutralized with
saturated NaHCO3 solution (2 mL), diluted with CH2Cl2
(10 mL), and washed with H2O (7 mL). The aqueous layer
was extracted with CH2Cl2 (2 × 10 mL) and the organic lay-
ers were dried (MgSO4) and concentrated in vacuo. The resi-
due was purified by flash chromatography (hexane–EtOAc,
3:2) to yield 35 (115 mg, 66%). TLC: 0.51 (hexane–EtOAc,
1:1). [α]2D3 –9.8° (c 1.1, CH2Cl2). MALDI-TOF (m/z): 2311
1
(M + Na+ – H), 1486 (M + K+ – H). H NMR (500 MHz,
CDCl3) δ: 8.09–7.14 (m, 50H, 10 Ph), 5.92 (d, 1H, NH,
JNH,2 = 9.7 Hz), 5.31 (d, 1H, H-1e, J1,2 = 4.6 Hz), 5.28 (d,
1H, H-1c, J1,2 = 3.4 Hz), 5.17 (d, 1H, H-1a, J1,2 = 4.5 Hz),
5.05 (d, 1H, H-1b, J1,2 = 3.5 Hz), 4.97 (d, 1H, H-1f, J1,2
=
3.4 Hz), 4.95 −4.91 (m, 2H, H-2a, H-2e), 4.85 (d, 1H,
1 CH2Ph, Jgem = 10.9 Hz), 4.82 (d, 1H, H-1b, J1,2 = 3.3 Hz),
4.76−4.40 (m, 21H, 16 CH2Ph, H-5a, H-5c, H-5e, H-6d,
H-6′d), 4.33 (d, 1H, 1 CH2Ph, Jgem = 11.3 Hz), 4.29 (m, 1H,
H-2d), 4.14−4.02 (m, 4H, H-4a, H-4b, H-4d, H-4e), 3.96–
3.91 (m, 4H, H-3a, H-3e, H-4c, CH(CH3)2), 3.85–3.81 (m,
11H, H-3b, H-3c, H-3f, H-4f, H-5b, H-5d, H-5f, H-6b,
H-6′b, H-6f, H-6′f ), 3.63 (s, 3H, COOCH3), 3.46–3.40 (m,
2H, H-2c, H-3d), 3.41 (s, 3H, COOCH3), 3.32 (s, 3H,
COOCH3), 3.32–3.30 (m, 1H, H-2b), 3.19 (dd, 1H, H-2f,
J2,1 = 3.4 Hz, J2,3 = 10.0 Hz), 1.43 (s, 3H, NHCOCH3), 1.20
(s, 9H, C(CH3)3), 1.16 (s, 9H, C(CH3)3), 1.22–1.14 (m, 6H,
CH(CH3)2). 13C NMR (125 MHz, CDCl3) δ: 177.3, 177.2,
170.0, 169.8, 169.3, 169.2, 166.1 (C=O), 138.2–127.2 (Ph),
98.9 (C-1e), 98.4 (C-1b), 98.3 (C-1f ), 97.8 (C-1c), 97.3
(C-1a), 97.0 (C-1d), 79.3, 78.6, 78.1, 76.3, 75.9, 75.1, 74.9,
74.8, 74.5, 74.2, 74.0, 73.7, 73.6, 73.4, 73.3, 73.2, 72.4,
72.3, 71.8, 71.3, 70.8, 70.6, 70.4, 70.1, 69.5, 69.4, 67.8,
O-(Methyl 4-O-(2-acetamido-6-O-benzoyl-3,4-di-O-
benzyl-2-deoxy-α-D-glucopyranosyl)-2-O-acetyl-3-O-
benzyl-α,β-L-idopyranuronate) trichloroacetimidate (34)
To a solution of 33 (120 mg, 0.15 mmol) in dry CH2Cl2
(1.5 mL), Cl3CCN (0.22 mL, 2.18 mmol) and activated
K2CO3 (24 mg, 0.17 mmol) were added. After stirring for
3 h the residue was filtered and concentrated to dryness. The
residue was purified by flash chromatography (hexane–
EtOAc, 1:2) to yield 34 (100 mg, 71%) as a mixture of
anomers α and β. TLC (α and β) 0.37 and 0.20 (hexane–
1
EtOAc, 1:1). H NMR (500 MHz, CDCl3) δ: 8.70 (s, 1H,
O(C=NH)CCl3 (α)), 8.67 (s, 1H, O(C=NH)CCl3 (β)), 7.99–
7.19 (m, 40H, 4 Ph (α and β)), 6.36 (d, 1H, H-1 (α)), 6.22
(d, 1H, H-1 (β), J1,2 = 2.3 Hz), 6.20 (d, 1H, NH (β), JNH,2
9.7 Hz), 5.87 (d, 1H, NH (α), JNH,2 = 9.7 Hz), 5.25 (m, 1H,
H-2 (β)), 4.99 (b.s., 1H, H-1 (α)), 5.02 (d, 1H, H-5 (α), J1,2
2.0 Hz), 4.87−4.85 (m, 2H, H-1′ (α), H-1′ (β)), 4.84−4.57
=
=
© 2002 NRC Canada

Ojeda et al.
933
63.0, 62.7, 62.3, 60.4, 51.9 (C-2d), 51.7 (COOCH3), 51.7
(C-2d), 38.8, 27.2, 27.1, 23.3, 22.8, 21.8, 21.0 (OPiv,
NHCOCH3, O-i-Pr). Anal. calcd. for C124H143N7O35·1.5H2O
(%): C 64.23, H 6.34, N 4.23; found (%): C 64.32, H 6.64, N
4.29.
Methyl (isopropyl O-(2-acetamido-6-O-benzoyl-3,4-di-O-
benzyl-2-deoxy-α-D-glucopyranosyl)-(1→4)-O-(methyl 3-
O-benzyl-α-L-idopyranosyluronate)-(1→4)-O-(2-azido-
3,6-di-O-benzyl-2-deoxy-α-D-glucopyranosyl)-(1→4)-O-
(methyl 3-O-benzyl-2-O-pivaloyl-α-L-
idopyranosyluronate)-(1→4)-O-(2-acetamido-6-O-
benzoyl-3-O-benzyl-2-deoxy-α-D-glucopyranosyl)-(1→4)-
O-(methyl 2,3-di-O-benzyl-α-L-idopyranosyluronate)-
(1→4)-O-(2-azido-3,6-di-O-benzyl-2-deoxy-α-D-
glucopyranosyl)-(1→4)-3-O-benzyl-2-O-pivaloyl-α-L-
idopyranosyl) uronate (37)
Methyl (isopropyl O-(2-acetamido-6-O-benzoyl-3,4-di-O-
benzyl-2-deoxy-α-D-glucopyranosyl)-(1→4)-O-(methyl 2-
O-acetyl-3-O-benzyl-α-L-idopyranosyluronate)-(1→4)-O-
(2-azido-3,6-di-O-benzyl-2-deoxy-α-D-glucopyranosyl)-
(1→4)-O-(methyl 3-O-benzyl-2-O-pivaloyl-α-L-idopyrano-
syluronate)-(1→4)-O-(2-acetamido-6-O-benzoyl-3-O-
benzyl -2-deoxy-α-D-glucopyranosyl)-(1→4)-O-(methyl
2,3-di-O-benzyl-α-L-idopyranosyluronate)-(1→4)-O-
(2-azido-3,6-di-O-benzyl-2-deoxy-α-D-glucopyranosyl)-
(1→4)-3-O-benzyl-2-O-pivaloyl-α-L-idopyranosyl)
uronate (36)
To a solution of 19 (460 mg, 0.307 mmol) in dry MeOH
and CH2Cl2 (1:1, 4 mL), MeONa (56 µL of a 0.178 M so-
lution of MeONa in MeOH) was added and stirred for
40 h. The reaction was neutralized with resin (IRA-120
H+) and filtered. The residue was purified by flash chro-
matography (hexane–EtOAc, 1:2) to yield 37 (61 mg,
80%). TLC: 0.45 (hexane–EtOAc, 1:2). [α]2D3 +34.2° (c 1.0,
1
To a solution of 35 (130 mg, 56.7 µmol) and 34 (103 mg,
106 µmol) in dry CH2Cl2 (0.7 mL) at room temperature,
TMSOTf (55 µL of a 0.22 M solution of TMSOTf in
CH2Cl2) was added under an argon atmosphere. After stir-
ring for 3 h, saturated NaHCO3 solution (0.2 mL) was added
and diluted with CH2Cl2 (20 mL). The residue was washed
with H2O (15 mL) and then the aqueous layer was extracted
with CH2Cl2 (2 × 10 mL) and the organic layers were dried
(MgSO4) and concentrated in vacuo. The residue was puri-
fied by flash chromatography (hexane–EtOAc, 1:2) to yield
36 (60 mg, 40%) and nonreacting acceptor 35 (70 mg,
54%). TLC: 0.40 (hexane–EtOAc, 1:2). [α]2D3 +33.3° (c
1.0, CH2Cl2). MALDI-TOF (m/z): 3119 (M + Na+), 3135
CH2Cl2). H NMR (500 MHz, CDCl3) δ: 8.07–7.12 (m,
70H, 14 Ph), 6.19 (d, 1H, NH h, JNH,2 = 8.6 Hz), 5.96 (d,
1H, NH d, JNH,2 = 9.8 Hz), 5.29 (d, 1H, H-1c, J1,2
=
3.1 Hz), 5.25 (d, 1H, H-1e, J1,2 = 4.3 Hz), 5.17 (d, 1H,
H-1a, J1,2 = 4.5 Hz), 5.04 (m, 2H, H-1g, H-1b), 4.98 (d,
1H, H-1f, J1,2 = 3.3 Hz), 4.94−4.90 (m, 2H, H-2a, H-2e),
4.86 (d, 1H, H-1h, J1,2 = 3.1 Hz), 4.79 (m, 1H, H-1d),
4.88–4.38 (m, 31H, 23 CH2Ph, H-5a, H-5c, H-5e, H-5g,
H-6d, H-6′d, H-6h, H-6′h), 4.31–4.26 (m, 2H, H-2d, 1 CH2Ph),
4.19 (m, 1H, H-2h), 4.11 (t, 1H, H-4a, J4,3 ≈ J4,5
=
5.6 Hz), 4.07–3.91 (m, 9H, H-3a, H-3e, H-4b, H-4c, H-4d,
H-4e, H-4g, H-4h, CH(CH3)2), 3.84–3.64 (m, 11H, H-3b,
H-3c, H-3g, H-4f, H-5b, H-5d, H-5h, H-6b, H-6′b, H-6f,
H-6′f ), 3.61 (s, 3H, COOCH3), 3.58–3.52 (m, 4H, H-2g,
H-3f, H-3h, H-5f ), 3.44–3.39 (m, 2H, H-2c, H-3d), 3.38
(s, 3H, COOCH3), 3.33 (s, 3H, COOCH3), 3.32–3.27 (m,
2H, H-2b, H-2f ), 3.18 (s, 3H, COOCH3), 2.45 (d, 1H,
OH-2g, JOH,2 = 8.3 Hz), 1.65 (s, 3H, NHCOCH3), 1.41
(s, 3H, NHCOCH3), 1.19–1.13 (s, 24H, 2 C(CH3)3,
CH(CH3)2). 13C NMR (125 MHz, CDCl3) δ: 177.3, 170.5,
170.1, 169.9, 169.2, 166.2 (C=O), 138.1–127.1 (Ph),
100.9 (C-1g), 99.1 (C-1c), 98.8 (C-1b), 98.7 (C-1f ), 98.1
(C-1e), 97.6 (C-1a), 97.4 (C-1d), 97.2 (C-1h), 80.0, 78.7,
78.2, 78.0, 77.3, 77.1, 76.8, 75.9, 75.3, 74.9, 74.8, 74.6,
74.0, 73.7, 73.5, 73.3, 73.2, 72.8, 71.8, 71.3, 70.6, 70.5,
70.2, 70.1, 69.9, 68.2, 67.7, 63.3, 62.9, 62.6, 52.5, 52.1,
52.0, 51.9, 51.6, 38.8, 29.7, 27.2, 27.1, 23.3, 22.9, 22.8,
21.8 (OPiv, NHCOCH3, O-i-Pr).
1
(M + K+). H NMR (500 MHz, CDCl3) δ: 8.08–7.12 (m,
70H, 14 Ph), 5.99 (d, 1H, NH h, JNH,2 = 9.6 Hz), 5.90 (d,
1H, NH d, JNH,2 = 9.4 Hz), 5.31 (d, 1H, H-1e, J1,2
=
4.8 Hz), 5.29 (d, 1H, H-1c, J1,2 = 3.1 Hz), 5.17 (d, 1H, H-
1a, J1,2 = 4.2 Hz), 5.12 (d, 1H, H-1g, J1,2 = 3.2 Hz), 5.04
(d, 1H, H-1b, J1,2 = 3.0 Hz), 4.96 (d, 1H, H-1f, J1,2
=
3.2 Hz), 4.83 (m, 1H, H-1h), 4.80 (m, 1H, H-1d), 4.96–
4.90 (m, 2H, H-2a, H-2e), 4.90–4.23 (m, 33H, 24 CH2Ph,
H-2g, H-5a, H-5c, H-5e, H-5g, H-6d, H-6′d, H-6h, H-6′h),
4.36–4.23 (m, 2H, H-2d, H-2h), 4.12 (t, 1H, H-4a, J4,3
4,5 = 5.2 Hz), 4.09–3.86 (m, 10H, H-3a, H-3e, H-3g, H-4b,
≈
J
H-4c, H-4d, H-4e, H-4g, H-5h, CH(CH3)2), 3.85–3.64 (m,
10H, H-3b, H-3c, H-4f, H-4h, H-5b, H-5d, H-6b, H-6′b,
H-6f, H-6′f ), 3.62 (s, 3H, COOCH3), 3.60–3.49 (m, 3H,
H-3f, H-3h, H-5f ), 3.46–3.36 (m, 2H, H-2c, H-3d), 3.41
(s, 6H, 2 COOCH3), 3.34–3.21 (m, 2H, H-2b, H-2f ), 3.24
(s, 3H, COOCH3), 1.83 (s, 3H, OCOCH3), 1.61 (s, 3H,
NHCOCH3), 1.42 (s, 3H, NHCOCH3), 1.20–1.12 (s, 24H,
2 C(CH3)3, CH(CH3)2). 13C NMR (125 MHz, CDCl3) δ:
177.3, 170.1, 170.0, 169.4, 169.3, 169.2, 168.8, 166.2,
166.1, 166.0 (C=O), 138.0–127.2 (Ph), 99.2 (C-1c), 98.6
(C-1b), 98.6 (C-1f), 98.1 (C-1e), 97.8 (C-1g), 97.6 (C-1a),
97.5 (C-1d), 97.1 (C-1h), 80.8, 78.6, 78.0, 77.3, 77.0,
76.8, 75.3, 75.0, 74.8, 74.6, 74.4, 74.2, 74.0, 73.8,
73.7, 73.4, 73.2, 72.4, 71.8, 71.5, 71.3, 70.8, 70.5, 70.4,
69.7, 63.2, 63.0, 62.6, 52.4, 52.0, 51.6, 51.7 (C-2d), 38.8,
29.7, 27.2, 27.1, 23.3, 23.1, 22.8, 21.8, 20.7 (OPiv,
NHCOCH3, O-i-Pr). Anal. calcd. for C169H192N8O49·H2O
(%): C 65.07, H 6.20, N 3.59; found (%): C 65.08, H 6.44,
N 3.57.
Methyl (isopropyl O-(2-acetamido-6-O-benzoyl-3,4-di-O-
benzyl-2-deoxy-α-D-glucopyranosyl)-(1→4)-O-(methyl 2,3-
di-O-benzyl-α-L-idopyranosyluronate)-(1→4)-O-(2-azido-
3,6-di-O-benzyl-2-deoxy-α-D-glucopyranosyl)-(1→4)-O-
(methyl 3-O-benzyl-2-O-pivaloyl-α-L-idopyranosyluro-
nate)-(1→4)-O-(2-acetamido-6-O-benzoyl-3-O-benzyl-2-
deoxy-α-D-glucopyranosyl)-(1→4)-O-(methyl 2,3-di-O-
benzyl-α-L-idopyranosyluronate)-(1→4)-O-(2-azido-3,6-di-
O-benzyl-2-deoxy-α-D-glucopyranosyl)-(1→4)-3-O-benzyl-
2-O-pivaloyl-α-L-idopyranosyl) uronate (38)
To a solution of 37 (55 mg, 0.018 mmol) and freshly pre-
pared Ag2O (10.2 mg, 0.044 mmol) in dry DMF (0.1 mL),
freshly distilled benzyl bromide (24 µL, 0.2 mmol) was
added. After stirring for 24 h, the mixture was filtered
© 2002 NRC Canada

934
Can. J. Chem. Vol. 80, 2002
through Celite and new Ag2O (10.2 mg, 0.044 mmol) and
benzyl bromide (24 µL, 0.2 mmol) were added. After stir-
ring for 24 h more, the mixture was filtered through Celite
and concentrated to dryness. The residue was purified by
flash chromatography (hexane–EtOAc, 1:1) to yield 38
(41 mg, 72%). TLC: 0.28 (hexane–EtOAc, 1:1). [α]D23
Isopropyl O-(2-acetamido-3,4-di-O-benzyl-2-deoxy-6-O-
sulfo-α-D-glucopyranosyl)-(1→4)-O-(2,3-di-O-benzyl-α-L-
idopyranosyluronic acid)-(1→4)-O-(2-azido-3-O-benzyl-
2-deoxy- -D-glucopyranosyl)-(1 4)-O-(3-O-benzyl-2-O-
α
→
sulfo-α-L-idopyranosyluronic acid)-(1→4)-O-(2-
acetamido-3-O-benzyl-2-deoxy-6-O-sulfo-α-D-glucopy-
ranosyl)-(1→4)-O-(2,3-di-O-benzyl-α-L-idopyranosyl-
uronic acid)-(1→4)-O-(2-azido-3,6-di-O-benzyl-2-deoxy-
α-D-glucopyranosyl)-(1→4)-3-O-benzyl-2-O-sulfo-α-L-
idopyranosyluronic acid octasodium salt (40)
1
+30.5° (c 1.0, CH2Cl2). H NMR (500 MHz, CDCl3) δ:
8.07–7.09 (m, 75H, 15 Ph), 6.05 (d, 1H, NH d, JNH,2
=
9.8 Hz), 5.88 (d, 1H, NH h, JNH,2 = 9.5 Hz), 5.30 (d, 1H,
H-1c, J1,2 = 2.8 Hz), 5.27 (d, 1H, H-1e, J1,2 = 3.7 Hz),
A mixture of 39 (32 mg, 11.4 µmol) and complex SO3·Py
(37 mg, 0.23 mmol) in dry Py (1.5 mL) was stirred under an
argon atmosphere. After 24 h, the mixture was cooled and
MeOH (1 mL) and CH2Cl2 (1 mL) were added. The solution
was eluted through a Sephadex LH-20 (MeOH–CH2Cl2,
1:1). Fractions containing the hexasaccharide were concen-
trated and passed through a Dowex 50WX4-Na+ (MeOH–
H2O, 2:1). Finally, the product was purified by reverse phase
HPLC to yield 40 (23 mg, 64%). TLC: 0.62 (EtOAc–Py–
H2O–AcOH, 8:5:3:1). 1H NMR (500 MHz, CD3OD) δ:
7.42–7.00 (m, 65H, 13 Ph), 5.45–5.15 (5H, H-1a, H-1b, H-
1e, H-1f, H-1c or g), 5.05–4.70 (3H, H-1d, H-1h, H-1c or g),
1.56 (m, 3H, NHCOCH3), 1.46 (m, 3H, NHCOCH3), 1.18
(d, 6H, CH(CH3)2, J = 6.0 Hz).
5.24 (d, 1H, H-1g, J1,2 = 1.1 Hz), 5.17 (m, 1H, H-1a, J1,2
=
4.5 Hz), 5.05 (m, 1H, H-1b, J1,2 = 3.2 Hz), 4.97 (d, 1H,
H-1f, J1,2 = 3.2 Hz), 4.95–4.91 (m, 2H, H-2a, H-2e), 4.86–
4.38 (m, 35H, 25 CH2Ph, H-1d, H-1h, H-5a, H-5c, H-5e,
H-5g, H-6d, H-6′d, H-6h, H-6′h), 4.36–4.26 (m, 3H,
H-2d, H-2h, 1 CH2Ph), 4.12 (t, 1H, H-4a, J4,3 ≈ J4,5
=
4.8 Hz), 4.10–3.91 (m, 9H, H-3a, H-3e, H-4b, H-4c, H-4e,
H-4f, H-4g, H-4h, CH(CH3)2), 3.85–3.66 (m, 12H, H-3b,
H-3c, H-3g, H-4d, H-4f, H-5b, H-5d, H-5f, H-5h, H-6b,
H-6f, H-6′f ), 3.62 (s, 3H, COOCH3), 3.61–3.49 (m,
3H, H-3d, H-3f, H-6′b), 3.45–3.41 (m, 2H, H-2c, H-3h),
3.44 (s, 3H, COOCH3), 3.41 (s, 3H, COOCH3), 3.32–3.27
(m, 2H, H-2b, H-2f ), 3.18 (s, 3H, COOCH3), 1.49 (s, 3H,
NHCOCH3), 1.41 (s, 3H, NHCOCH3), 1.19–1.13 (m,
24H, 2 C(CH3)3, CH(CH3)2). 13C NMR (125 MHz,
CDCl3) δ: 177.3, 170.1, 170.1, 169.8, 169.2, 166.2, 166.1
(C=O), 138.1–127.1 (Ph), 99.1 (C-1c), 98.7 (C-1b, C-1f,
C-1g), 98.2 (C-1e), 97.6 (C-1a, C-1d), 97.4 (C-1h), 78.3,
78.0, 77.3, 77.0, 76.8, 76.3, 75.4, 74.9, 74.5, 74.0, 73.9,
73.7, 73.3, 73.2, 73.1, 73.0, 72.2, 71.8, 71.3, 70.8,
70.3, 70.1, 67.8, 63.2, 62.8, 52.5, 51.9, 51.65, 38.8,
29.7, 27.2, 27.1, 23.3, 22.9, 22.8, 21.8 (OPiv, NHCOCH3,
O-i-Pr).
Isopropyl O-(2-acetamido-2-deoxy-6-O-sulfo-α-D-
glucopyranosyl)-(1→4)-O-(α-L-idopyranosyluronic acid)-
(1→4)-O-(2-deoxy-2-sulfamide-α-D-glucopyranosyl)-
(1→4)-O-(2-O-sulfo-α-L-idopyranosyluronic acid)-(1→4)-
O-(2-acetamido-2-deoxy-6-O-sulfo-α-D-glucopyranosyl)-(1
→4)-O-(α-L-idopyranosyluronic acid)-(1→4)-O-(2-deoxy-
2-sulfamide-α-D-glucopyranosyl)-(1→4)-2-O-sulfo-α-L-
idopyranosyluronic acid decasodium salt (4)
To a solution of 40 (23 mg, 7.15 µmol) in MeOH–H2O
(1.5 mL, 9:1) was hydrogenolyzed in the presence of 10%
Pd/C. After 24 h, the suspension was filtered and concen-
trated to give the desired product which was directly used in
the next step without further purification.
Isopropyl O-(2-acetamido-3,4-di-O-benzyl-2-deoxy-α-D-
glucopyranosyl)-(1→4)-O-(2,3-di-O-benzyl-α-L-idopy-
ranosyluronic acid)-(1→4)-O-(2-azido-3,6-di-O-benzyl-2-
deoxy-α-D-glucopyranosyl)-(1→4)-O-(3-O-benzyl-α-L-
idopyranosyluronic acid)-(1→4)-O-(2-acetamido-3-O-
benzyl-2-deoxy-α-D-glucopyranosyl)-(1→4)-O-(2,3-di-O-
benzyl-α-L-idopyranosyluronic acid)-(1→4)-O-(2-azido-
3,6-di-O-benzyl-2-deoxy-α-D-glucopyranosyl)-(1→4)-3-O-
benzyl-α-L-idopyranosyluronic acid (39)
To a solution of 38 (39 mg, 12.4 µmol) in THF (2 mL) at
–5°C, H2O2 30% (0.7 mL) and a 1 M aqueous solution of
LiOH (0.8 mL) were added. After stirring for 24 h at room
temperature MeOH (0.8 mL) and a 3 N aqueous solution of
KOH (2 mL) were added. After stirring for 24 h more, the
reaction was neutralized with acidic resin (IRA-120 H+), fil-
tered, and concentrated. The residue was purified by
Sephadex LH-20 (MeOH–CH2Cl2, 1:1) to yield 39 (32 mg,
94%). TLC: 0.47 (CH2Cl2–MeOH, 8:1). 1H NMR
(500 MHz, CD3OD) δ: 7.37–7.06 (m, 65H, 13 Ph), 5.30–
5.19 (m, 3H, H-1b, H-1f, H-1a or e), 5.06 (s, 1H, H-1a or e),
5.00 (d, 1H, H-1c or g, J1,2 = 3.4 Hz), 4.98 (d, 1H, H-1c or
g, J1,2 = 3.2 Hz), 4.79 (m, 2H, H-1d, H-1h), 1.61 (s, 6H, 2
NHCOCH3), 1.30–1.15 (m, 6H, CH(CH3)2). 13C NMR
(125 MHz, CD3OD) δ: 99.7 (C-1a or e), 98.2 (C-1b or f ),
97.6 (C-1a or e), 97.3 (C-1b or f ), 96.6, 96.4 (C-1d, C-1h),
95.1 (C-1c, C-1g).
The hydrogenolyzed hexasaccharide was dissolved in H2O
(1 mL) and the pH of the solution was adjusted to 9.5 with a
1 N solution of NaOH. Complex SO3·Py (24 mg, 10 equiv
for each amine group) was added in three portions over 1 h
and the pH was maintained at 9.5 by subsequent addition of
a 1 N solution of NaOH. A second, third, and fourth portion
of complex SO3·Py were added after 2, 4, and 6 h stirring,
respectively. After 24 h, the mixture was neutralized with a
0.1 N solution of HCl to pH 7 and then chromatographed on
a column of Sephadex G-25 with a 0.9% solution of NaCl.
The appropriate fractions were pooled and passed through a
column of Dowex 50WX4-Na+ with a 0.5 M solution of
NaCl and then a column of Sephadex G-25 with H2O–EtOH
(9:1). The fractions which contained the final hexa-
saccharide were lyophilized to give 4 (10.4 mg, 66% from
40). Before NMR studies, it was useful to make a last elu-
tion on a column of Dowex 50WX4-Na+ to avoid the forma-
tion of calcium salts instead of sodium salts and get a better
1
resolution in the spectra. H NMR (500 MHz, D2O) δ: 5.38
(d, 1H, H-1f, J1,2 = 3.3 Hz), 5.30 (d, 1H, H-1b, J1,2
3.3 Hz), 5.23 (d, 1H, H-1a, J1,2 = 1.8 Hz), 5.19 (d, 1H, H-1e,
1,2 = 2.7 Hz), 5.16 (d, 1H, H-1h, J1,2 = 3.8 Hz), 5.14 (d, 1H,
=
J
H-1d, J1,2 = 3.3 Hz), 4.88–4.86 (m, 2H, H-1c, H-1g), 1.99
© 2002 NRC Canada

Ojeda et al.
935
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1f), 94.4 (C-1d), 94.3 (C-1h).
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This work was supported by DGES (Grant PB96–0820).
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