Synthesis and structural study of two new heparin-like hexasaccharides.

Article abstract of DOI:10.1039/b303115b

Two new heparin-like hexasaccharides, 5 and 6, have been synthesised using a convergent block strategy and their solution conformations have been determined by NMR spectroscopy and molecular modelling. Both hexasaccharides contain the basic structural motif of the regular region of heparin but with negative charge distributions which have been designed to get insight into the mechanism of fibroblast growth factors (FGFs) activation.

Full text of DOI:10.1039/b303115b

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Synthesis and structural study of two new heparin-like
hexasaccharides
Ricardo Lucas, Jesús Angulo, Pedro M. Nieto and Manuel Martín-Lomas*
Grupo de Carbohidratos, Instituto de Investigaciones Químicas, CSIC, Américo Vespucio s/n,
Isla de la Cartuja, 41092 Sevilla, Spain
Received 20th March 2003, Accepted 20th May 2003
First published as an Advance Article on the web 2nd June 2003
Two new heparin-like hexasaccharides, 5 and 6, have been synthesised using a convergent block strategy and
their solution conformations have been determined by NMR spectroscopy and molecular modelling. Both
hexasaccharides contain the basic structural motif of the regular region of heparin but with negative charge
distributions which have been designed to get insight into the mechanism of fibroblast growth factors (FGFs)
activation.
sequence, have a different charge distribution and their syn-
Introduction
theses were designed for the final products to display the
sulfate groups only on one side of the expected helical
structures (Fig. 1). We have also investigated the solution con-
formation of these molecules using NMR spectroscopy and
molecular dynamics simulations which confirmed the predicted
helix-like three dimensional structures.11,12 These compounds
have shown different behaviour in inducing the mitogenic
activity of FGF-1,11,13 which clearly indicates the importance
of size and charge distribution in the regulation of the FGF
system and strongly suggest that a previously proposed GAG
induced FGF dimerisation14 is not an absolute requirement for
biological activity.
We report in this paper the synthesis of two new hexa-
saccharides, 5 and 6, and a NMR study of their solution con-
formation. Compounds 5 and 6 have the same sequence as
1–4 but their charge distribution has been designed to gain
structural and biological information on the importance of
sulfation at positions 2 of the -iduronate units and 6 of the
-glucosamine residues. With the same number of negative
charges, compounds 5 and 6 would present different three
dimensional charge distributions which would also be different
from those of 1 and 3. The N and 2-O-sulfo groups have been
reported to be essential for binding to FGF-215 whereas bind-
ing to FGF-1 also required the presence of 6-O-sulfo groups.16
Binding to FGFRs seems to require N, 2 and 6 sulfation as
well.17 Fig. 1 schematically shows the different charge distri-
bution and orientation of hexasaccharides 1 and 3 and those
expected for 5 and 6.
The family of fibroblast growth factors (FGFs) presently com-
prises more than twenty signalling polypeptides which play a
significant role in important biological functions such as cell
proliferation, differentiation and angiogenesis.1 These functions
are initiated after binding of FGFs to specific receptors
(FGFRs) at the cell surface.2 FGF-1 and FGF-2 are the proto-
typical members of the family. FGFs and FGFRs are heparin
binding proteins3,4 and, therefore, the FGF system is tightly
regulated by heparan sulfate glycosaminoglycans (HS-GAGs).5
In structural terms HS is recognised as a family of closely
related linear polysaccharides (heparin is just a member) con-
sisting of alternating units of -glucuronic or -iduronic acid
and -glucosamine. These units may be unsulfated and vari-
ously sulfated at specific positions (typically position 2 of
the uronic acid units and N and 6 of the glucosamine units)
and appear distributed in different domains along the poly-
saccharide chain.5 The overall conformation of these bio-
molecules has been reported to be a well defined helical
structure in which the heterogeneity of sequences and patterns
of sulfation result in a diversity of charge distributions and
orientations.6 The charge orientation is further modulated by
the flexibility of the internal -iduronate units, which may
adopt the C4 or the SO conformation.6,7 Since the binding of
these molecules to FGFs and FGFRs is thought to be primarily
electrostatic in nature, the diversity of charge distributions and
orientations is believed to be related to the specificity of their
interactions with the diverse and complex FGF system.2,8 This
complexity and diversity have been held responsible for appar-
ently conflicting evidence.2 It is generally agreed that the avail-
ability of homogeneous oligosaccharides with precisely defined
structure may contribute a key step in deciphering the struc-
tural and biological consequences of this diversity.2 These
homogeneous oligosaccharides can be obtained by synthesis.9,10
To gain insight into the biological process using these synthetic
molecules, their solution conformation has to be carefully
investigated in order to determine the orientation of the neg-
ative charges.6 Having this information, binding and biological
studies with these synthetic oligosaccharides may provide a
rigorous insight into the molecular basis of the HS-GAGs
mediated regulation of the FGF system.
1
2
Results and discussion
For the synthesis of 1–4 we previously developed a con-
vergent modular approach from key disaccharide structures
operating as glycosyl donors and as glycosyl acceptors. These
disaccharide modules were endowed with protective group
patterns permitting stereochemical control during the building
block assembly process and allowing subsequent sulfation at
the required positions.11,12 The synthesis of 5 and 6 has now
been carried out similarly thus validating the general design of
the synthetic approach and contributing further knowledge
towards a much needed automation of these processes. The
retrosynthetic analysis is shown in Scheme 1. A common
hexasaccharide precursor (14) is prepared by stereoselective
assembly of three modular structures that will constitute the
reducing end (11) the inner region (12) and the nonreducing
end (13) of the hexasaccharide skeleton. The sequence of
In the framework of a project on the activation of FGFs we
are presently using the above approach. We have previously
synthesised hexasaccharides 1 and 3 and octasaccharides 2 and
4.11,12 Compounds 1 and 2 contain the sequence (GlcNα1
4IdoA) and the charge distribution of the regular region of
heparin.5 Compounds 3 and 4, with the same disaccharide
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 3
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 2 2 5 3 – 2 2 6 6
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Fig. 1 Oligosaccharides 1–6. Formulae and schematic representation, small and large circles indicate carboxylate and sulfate groups respectively,
plain or shadowed filling indicate opposite spatial orientations.
deprotection steps preceding sulfation is a crucial aspect of the
synthesis allowing the preparation of either 5 or 6 from
the same common intermediate (14). The reducing end build-
ing block (11) appears as an isopropyl glycoside as, according
to previous experience,11,12,18,19 this grouping provides a con-
venient structural environment for the subsequent structural
and binding studies of the final products. Also in agreement
with extensive previous experience,11,12 the stereoselective
assembly of 11, 12, and 13, which is also a crucial step for the
success of the synthesis, was designed to be performed by the
trichloroacetimidate glycosylation procedure.20 These three
building blocks are also prepared from a common precursor
(10) which derives from the key disaccharide 9 which is in turn
synthesised11 from the monosaccharide derivatives 7 and 8
obtained in multigram quantities from diacetoneglucose and
-glucosamine hydrochloride respectively. For some time we
have been preparing the -iduronic acid diol (7) in reasonable
amounts from -glucuronolactone.21 However, the synthesis of
7 reported by Bonnaffé et al.22 has obvious advantages over
ours21 and this procedure has been used in the present work.
Trichloroacetimidate 823 is prepared in a straightforward
manner by a sequence involving a diazo transfer reaction24
and benzylidenation.25 This sequence is being routinely used
by us11,12 to prepare a variety of starting materials and a
number of different intermediates are currently in stock in our
laboratory.
the inner region building block (12) in almost quantitative yield.
This has been used as starting material for the preparation of
the reducing end building block (11) as well. Reaction of 12
with isopropyl alcohol gave 16 in 70% yield. The sequence
16 17 18 leading to the reducing end building block (11)
formally consists of a transacetalation reaction. For practical
reasons, primarily based on the availability of starting benzyl-
idene acetal derivatives and the synthetic potentiality of 17 in
the modular synthesis of other GAG oligosaccharides, this
route to 18, rather than direct p-methoxybenzylidenation of the
starting monosaccharide building block, was preferred. Treat-
ment of 16 with EtSH and BF3OEt2 27 afforded diol 17 in almost
quantitative yield. Compound 17 was reacted with p-methoxy-
benzaldehyde dimethyl acetal in the presence of TSOH to give
18 in 85% yield. Regioselective reductive opening of the the
p-methoxybenzylidene ring in 18 using NaBH3CN and THF in
DMF28 yielded the reducing end building block 11 (69%).
Scheme 2 also shows the synthesis of the non reducing end
building block 13. Reductive opening of the benzylidene ring in
29
10 using BH3NHMe2in the presence of BF3OEt2 gave 19 in
80% yield. Treatment of 19 with p-methoxybenzyl trichloro-
acetimidate at Ϫ20Њ gave 20 in almost quantitative yield. Di-
saccharide 13 was obtained after removal of the silyl group26 in
20 to give 21 (83%) and anomeric activation in the usual
conditions.20
The assembly of these building blocks involved first the
glycosylation of the reducing end disaccharide acceptor 11 with
the inner region disaccharide donor 12 in dichloromethane at
Ϫ20 ЊC. (Scheme 3). In these experimental conditions the donor
was reactive enough and the acceptor sufficiently stable as to
afford tetrasaccharide 22 in 85% yield. The reaction was highly
stereoselective with the configuration of the newly formed
We have previously reported11 that reaction of diol 721,22 with
trichloroacetimidate 823 under carefully controlled experi-
mental conditions results in the regio- and stereoselective glyco-
sylation to give disaccharide 9.11 This key disaccharide has been
the basic structure from which all the successful HS-GAG
oligosaccharide syntheses so far performed by us11,12 have been
developed. The synthesis of the inner region and the reducing
end building blocks starting from disaccharide 9 is shown in
Scheme 2. Conventional benzoylation of 9 gave 10 in 95% yield.
Removal of the silyl group in 10 with (HF)nؒPy complex26
afforded 15 in 80% yield. Activation of the anomeric position
of 15 as a trichloroacetimidate in the usual conditions20 gave
1
glycosidic linkage being exclusively α as indicated by the H
NMR spectrum. This tetrasaccharide derivative was then trans-
formed into glycosyl acceptor 25 following the sequence
22 23 24 25. Also in this case the route involving removal
of the benzylidene acetal group27 in 22 to give diol 23 (85%),
acetalation of 23 with p-methoxybenzaldehyde dimethyl acetal
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 2 2 5 3 – 2 2 6 6
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Scheme 1 Retrosynthesis of 5 and 6.
to give 24 in almost quantitative yield and stereoselective reduc-
construction of larger conveniently protected oligosaccharide
tive opening28 to 25 (52%) was preferred in spite of the moder-
ate yield of the last step. The removal of the benzylidene group
in 22 with EtSH27 should now be carried out in the presence of
CSA. The crude reaction mixture after reductive opening
(24 25) permits the recovery of 40% unreacted 24. Glyco-
sylation of 25 with the non reducing end building block 13 in
the conditions of the above discussed glycosylation step gave
the hexasaccharide derivative 14 in 79% yield with excellent
stereoselectivity. The protective group pattern in 14 permits
access to the two target hexasaccharides depending on the
deprotection sequence as presented in the next paragraph. As
in our previous syntheses of oligosaccharides 1–4 using a
similar strategy, this modular approach may permit also the
skeletons allowing access to a variety of oligosaccharides of
different sizes, charge distributions and orientations. The
plethora of existing protecting groups,30 the accessibility of
building blocks from 7, 8 and derivatives thereof and the
effectiveness11,12 of the trichloroacetimidate procedure20 with
regard to yield and selectivity in the building block assembly,
provide a promising platform for the development of solid
phase synthesis and automation. This would contribute a key
step in the elucidation of the molecular basis of these GAG
regulated biological processes.
For the synthesis of 5, removal of the p-methoxybenzyl
groups in 14 was the first step. This was achieved using 10%
TFA31 to give 26 in almost quantitative yield. Compound 26
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 2 2 5 3 – 2 2 6 6
2255

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Scheme 2 Synthesis of building blocks. Reagents and conditions: a) BzCl, Py, 95%; b) (HF)nPy, THF, Ϫ15 ЊC, 80%; c) K2CO3, Cl3CCN, CH2Cl2,
70%; d) iPrOH, TMSOTf, CH2Cl2, 70%; e) EtSH, PTSA, CH2Cl2, 95%; f ) p-MeOPhCH(OMe)2, PTSA, DMF, 85%; g) NaBH3CN, TFA, DMF, 69%;
h) BH3NHMe2, BF3OEt2, CH2Cl2, Ϫ15 ЊC, 81%; i) PMBOC(NH)CCl3, BF3OEt2, CH2Cl2, 0 ЊC, 96%; j) (HF)nPy, THF, Ϫ4 ЊC, 83%; k) K2CO3,
Cl3CCN, CH2Cl2, 88%.
was treated with the SO3ؒPy complex in pyridine32 to give 27 in
98% yield. The removal of the benzoyl and methoxycarbonyl
groups in 27 was then performed with LiOOH and then KOH
in order to minimise elimination.33 Compound 28, thus
obtained in quantitative yield, was then hydrogenated in the
presence of 10% Pd/C to yield 29 that was immediately submit-
ted to N-sulfation with the SO3ؒPy complex in water at a con-
stant pH value of 9.5. Hexasaccharide 5 was purified by gel
permeation and ion exchange chromatography as reported for
other synthetic heparin derived oligosaccharides.34,35 For the
preparation of 6 the ester groups in 14 were first removed as
above to give 30 in almost 90% yield. O-Sulfation afforded 31
which was hydrogenated to 32 and this N-sulfated to give 6
whcih was purified as described for 5.
charge distribution to binding and biological activity data.
The key structural features, conformational equilibria of the
-iduronate units and geometry and flexibility of the glycosidic
linkages, can be conveniently described by NMR spectroscopy
1
parameters.6,36 The H and 13C NMR spectra of 5 and 6 were
assigned using standard two-dimensional techniques by identi-
fying the spin systems and the interresidue NOEs as previously
reported.11,12 The spectra of both molecules showed consider-
able overlapping as a result of the identical substitution
patterns of the repeating units. The values of chemical shifts
(Table 1) were as expected for heparin derived oligosaccharides
with those patterns of sulfation.37
The coupling constants (Table 2), which permitted estab-
lishment of the 1C4–2SO conformational equilibria of the
-iduronate units7,36,38,39 (Fig. 2), were measured from the 2D
dqf-COSY cross peaks by recursive deconvolution in the fre-
quency domain. The presence of the 2SO conformation was also
The solution three-dimensional structures of 5 and 6 have
been studied to determine the precise orientation of the neg-
ative charges as compared with 1–4 in order to correlate this
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 2 2 5 3 – 2 2 6 6
2256

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Scheme 3 Assembly of building blocks. Reagents and conditions: a) TMSOTf, CH2Cl2, Ϫ20 ЊC, 85%; b) EtSH, CSA, CH2Cl2, 85%;
c) p-MeOPhCH(OMe)2, CSA, CH2Cl2, 95%; d) NaBH3CN, TFA, DMF, 52%; e) TMSOTf, CH2Cl2, Ϫ20 ЊC, 79%; f ) 10% TFA, CH2Cl2, 96%;
g) SO3Py, Py, Dowex 50WX4 (Na+), 98%; h) H2O2, LiOH aq., THF, KOH aq., MeOH, 98%; i) 10% Pd/C, H2, 9 : 1 MeOH–H2O; j) SO3Py, H2O,
pH 9.5, 73%; k) H2O2, LiOH aq., THF, 0 ЊC, KOH aq., MeOH, 5 ЊC, Dowex 50WX4 (Na+), 87%; l) SO3Py, Py, Dowex 50WX4 (Na+), 86%; m) 10%
Pd/C, H2, 9 : 1 MeOH–H2O; n) SO3Py, H2O, pH 9.5, 73%.
compounds 1–411,12 and heparin fragments,40–42 were entirely
compatible with an overall helical secondary structure. This is
dictated by syn-ψ type glycosidic conformations characterised
by H1Ј–H4, H1Ј–H3 and H5Ј–H4 exclusive NOEs around the
GlcN–IdoA glycosidic linkage and by H1Ј–H4 and H1Ј–H6
proR exclusive NOEs around the IdoA–GlcN bonds40 (Table
4). The absence of H5Ј–H3 and H1Ј–H5 NOEs for the GlcN–
IdoA and the IdoA–GlcN linkages respectively permitted us to
discard any contributions from arrangements of the anti-ψ
Fig. 2 Iduronate conformational equilibrium.
indicated by the H2–H5 exclusive NOE. As previously observed
for compounds 1–4, no significant contribution of the 4C1 form
was observed for the -iduronate units at the reducing end thus
confirming the suitability of this isopropyl glycoside structure
for structural and binding studies.11,12 The conformer popu-
lations in the conformational equilibria were quantified by least
type.
The already reported strong anisotropic hydrodynamic
behaviour shown by heparin fragments longer than penta-
saccharide,43 which prevents the straightforward quantification
of NOE data, was also observed for 5 and 6. We have also
described a similar behaviour for the synthetic oligosaccharides
1–411,12 where, as for 5 and 6, different relative intensities of the
H1–H2 and H2–H4 NOEs were observed for similar H1–H2
(2.4 Å) and H2–H4 (2.5 Å) distances of the GlcN units.12,43 The
orthogonal H1–H2 and H2–H4 vectors have in this series dif-
ferent sensitivity to parallel and perpendicular correlation time
which is a characteristic feature of prolate ellipsoid rigid mole-
cules. This is the result of combining a linear molecular shape
with a significant rigidity of the glycosidic linkages which are
not usual in carbohydrate molecules other than GAGs.36,44
This structural study was complemented with molecular
modelling data. Calculations were performed using, as in the
3
squares fitting of the experimental JH,H values with those cal-
culated for the canonical forms (Table 3). The calculated 75 : 25
1C4–2SO conformational distribution indicated that, within the
experimental error, the sulfation pattern and the -iduronate
position along the saccharide chain did not significantly influ-
ence in this case the conformational populations. This is in
agreement with previous results described for heparin and
heparan sulfate fragment analogues to 5 and 6.7,38,39
The overall three dimensional structure of both hexa-
saccharides was determined from the observed interresidue
NOE patterns. These, which were similar to those observed for
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 2 2 5 3 – 2 2 6 6
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1
2
Table 1 Proton and carbon chemical shift in ppm for hexasaccharide 5
and 6 at 25 ЊC
Table 3 Population of C4, and SO iduronate conformers estimated
from the experimental 3JHH values and fitting error
5
6
IdoA-a
1C4
IdoA-c
1C4
IdoA-e
1C4
2SO
χ2
2SO
χ2
2SO
χ2
a
1
2
3
4
5
4.94
3.56
4.07
4.00
4.50
101.4
71.9
70.9
77.6
71.1
5.23
4.16
4.19
4.00
4.51
99.5
78.3
70.6
78.2
70.5
5
6
72
78
28
22
0.8
1.2
75
71
25
29
1.4
2.7
75
76
25
24
1.4
2.3
Table 4 Observed interglycosidic NOE for hexasaccharides 5 and 6
b
1
2
3
4
5
6
6Ј
5.34
3.23
3.65
3.71
3.98
4.31a
4.18b
98.1
60.2
72.2
79.8
71.4
68.7
5.31
3.21
3.69
3.68
3.85
3.86
3.84
99.4
60.6
71.9
79.8
73.5
62.0
Glycosidic linkage
5
6
f–e
H1Ј–H3
H1Ј–H3
H1Ј–H4
H1Ј–H4
H5Ј–H4a
H1Ј–H4
H5Ј–H4a
H1Ј–H4
e–d
c
1
2
3
4
5
5.00
3.77
4.10
4.03
4.80
104.5
70.9
70.0
76.8
70.9
5.25
4.32
4.22
4.01
4.85
101.4
76.7
69.9
78.0
70.5
H1Ј–H6proR
H2Ј–H6proR
H1Ј–H6proSa
H5Ј–H3a
H1Ј–H3
H1Ј–H6proR
H2Ј–H6proR
d–c
c–b
H1Ј–H3
H1Ј–H4
H5Ј–H4
H1Ј–H4
H1Ј–H4
d
1
2
3
4
5
6
6Ј
5.31
3.22
3.64
3.72
3.99
4.32a
4.18b
98.1
60.2
72.2
79.8
71.1
68.7
5.27
3.22
3.69
3.68
3.85
3.86
3.84
99.7
60.5
71.9
79.8
73.5
62.0
H1Ј–H4
H1Ј–H6proR
H2Ј–H6proR
H1Ј–H6proSa
H2Ј–H6proSa
H5Ј–H3a
H1Ј–H6proR
H2Ј–H6proR
b–a
H1Ј–H3
H1Ј–H4
H1Ј–H3
H1Ј–H4
H5Ј–H4
e
f
1
2
3
4
5
4.99
3.76
4.08
4.04
4.79
104.5
70.8
70.0
76.8
70.9
5.24
4.31
4.22
4.02
4.84
101.5
76.7
69.9
77.9
70.5
a Weak NOE peaks.
1
2
1
2
3
4
5
6
6Ј
5.33
3.19
3.61
3.54
3.91
4.34a
4.14b
98.1
60.2
73.6
71.5
72.2
68.7
5.28
3.20
3.64
3.44
3.80
3.80
3.76
99.7
60.5
73.4
72.3
74.1
62.7
the -iduronate units both in the C4 and in the SO conform-
ation, based on the previous heparin model.40 The resulting
eight structures were subjected to several cycles of molecular
dynamics runs and energy minimisation in the presence of the
adequate number of counterions and including explicit water
molecules in the calculations. The final structures (Fig. 3) main-
tained the helical structure and their backbone could be super-
imposed with that of heparin fragments.40 The interprotonic
distances were in all cases compatible with the interglycosidic
NOEs.
a pro-R. b pro-S.
Table 2 Iduronate residues endocyclic 3JH4H (Hz) observed for 5 and 6
1
2
and calculated for canonical C4, SO, and C1 structures for iduronate
and 2-O-sulfoiduronate
Conclusions
Residue
3J1,2
3J2,3
3J3,4
3J4,5
In conclusion, compounds 5 and 6 present three dimensional
structures and dynamics as compounds 1 and 311,12 and all of
them are structural models of GAGs which reproduce heparin
basic structural features. The different negative charge distri-
bution and orientation in these molecules result in a different
biological behaviour that will be reported in due course.
IdoA-a
5
3.2
2.5
2.1
3.6
2.1
3.6
2.1
7.9
6.9
2.2
7.9
7.1
5.1
4.8
3.9
3.6
4.2
4.5
4.2
3.6
2.9
9.9
6.8
3.0
9.9
6.6
2.9
2.8
2.5
3.7
2.5
3.5
1.0
4.5
3.0
1.0
4.4
3.4
6
IdoA-c
5
5.4a
6
4.3
5.4a
4.2
3.0
IdoA-e
5
6
IdoAؒ2OSO3
1C4
4C1
2SO
1C4
4C1
2SO
Experimental
10.0
10.4
3.1
10.0
10.4
General procedures
IdoA
Thin layer chromatography (TLC) analyses were performed on
silica gel F254 precoated on aluminum plates (Merk). The com-
pounds were detected by staining with sulfuric acid–ethanol
(1 : 9) or anisaldehyde solution (25 : 25 : 450 : 1 anisaldehyde–
sulfuric acid–ethanol–acetic acid) followed by heating at over
200 ЊC. Column chromatography was carried out on silica gel
60 (0.2–0.5 mm, 0.2–0.063 mm, or 0.040–0.015 mm; Merck)
and distilled solvents were used. All solvent and reagents used
in the synthesis were purified and dried according to standard
procedures. Optical rotations were determined at room temper-
a Averaged due to overlapping.
case of 111 and 3,12 the AMBER force field45 with the GLY-
CAM_93 modification for carbohydrates46 and specific param-
eters for the sulfate and sulfamate groups.47 The energetic
landscape of IdoA–GlcN and GlcN–IdoA glycosidic linkages
was extensively explored resulting in a densely populated syn-ψ
central region possibly with several accessible local sub-
minima.40,41 The alternative anti-ψ arrangement was clearly
unfavourable. Models for 1, 3, 5 and 6 were constructed, with
1
ature in a 1 dm cell on a Perkin-Elmer 341 polarimeter. H
and 13C NMR spectra were acquired on a Bruker DRX-500
spectrometer and chemical shifts are given in ppm relative to
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 2 2 5 3 – 2 2 6 6
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Fig. 3 Relaxed structures for hexasaccharides 1, 3, 5 and 6 corresponding to all iduronate residues in 1C4 conformation (left) and 2SO one (right).
TMS. Elemental analyses were performed with a Leco CHNS-
932 apparatus, after drying analytical samples over phos-
phorous pentoxide for 24 h. MALDI-TOF mass spectra were
recorded with a MALDI-TOF GSG System spectrometer.
Samples of the intermediate products were dissolved in EtOAc
or MeOH at mM concentrations and 2,5-dihydroxybenzoic
acid was used as the matrix. Gel filtration chromatography
(Sephadex LH-20 and G-25; Pharmacia) and ion-exchange
chromatography (Dowex 50WX4 Naϩ; Fluka) were used in
order to achieve purification of the final products.
6.0 program,53 modified for carbohydrate molecules by the
GLYCAM_93 parameter set.46 Specific parameters for sulfate
and sulfamate groups were also included, as described by
Altona and Huige47 Calculations were carried out using
periodic boundary conditions with TIP3P water molecules.
The initial structure of the hexasaccharides 5 and 6 were built
manually using the ꢀ/ψ values found for a minimised structure
of the regular heparin-like synthetic hexasaccharide 1.11 The
relaxed structures were neutralised by adding sodium atoms
using “addions” as implemented in xleap from AMBER 5.0
according to the solute electrostatic potential and further radi-
ally displaced allowing appropriate solvation in the next step.
Finally, water TIP3P type molecules were added. The resulting
systems have the following characteristics: 3724 water mole-
cules in a box whose dimensions were 50 Å × 50 Å × 51 Å for
NMR measurements
1D and 2D experiments with 5 and 6 were recorded in D2O at
298 K on Bruker AVANCE 800 and 500 MHz instruments. The
sample concentration was nearly 5mM and the pH* was
adjusted to 7.0. Chemical shifts are in ppm with respect to the
proton signal of the tetramethylsilane and the calibration has
been made using the manufacturers software from the nucleus
base frequency and the corresponding frequency ratio. DQF-
COSY,48,49 TOCSY,50 NOESY,51 and HSQC52 experiments were
recorded using standard z pulsed field gradient enhanced or
selected pulse sequences when possible. Phase-sensitive experi-
ments were performed in all cases using the TPPI (Time
Proportional Phase Increment) method. Data were trans-
formed into phase-sensitive modes after zero filling and weight-
ing with shifted square sine-bell functions, incrementing
the number of experiments in the indirect dimension of the
heteronuclear experiments by linear prediction according to
the manufacturers software.
1
the 5 C4 model; 3689 water molecules in a box whose dimen-
2
sions were 50 Å × 50 Å × 51 Å for the SO model for 5; 3759
water molecules in a box whose dimensions were 50 Å × 52 Å ×
1
49 Å for the 6 C4 model and 3833 water molecules in a box
whose dimensions were 51 Å × 51 Å × 51 Å for the 2SO model
for 6. All these boxes are 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
calculation. The initial water configuration was subjected to
1000 cycles of energy minimization, with the conformation
of the sugar and counterions frozen (5000 Kcal molϪ1
Å
Ϫ1).
Following this step, a 25 ps volume constant MD simu-
lation was performed, in which only the water molecules were
allowed to move. After this pre-equilibration of the solvent
all the further steps were carried out using Particle Mesh
Ewald (PME) electrostatic treatment with 1 Å grid and cubic
B-spline interpolation. The initial velocities were assigned using
a Maxwellian distribution at the corresponding temperature.
Molecular modelling
Molecular modelling was performed using AMBER force
field25 (parameter set “parm91”) as integrated in the AMBER
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 2 2 5 3 – 2 2 6 6
2259

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Using these conditions the protocol continues with a 25 ps
molecular dynamics run with the solute atoms restrained by
O-(Methyl
4-O-(2-azido-3-O-benzyl-4,6-O-benzylidene-2-
deoxy-ꢀ-D-glucopyranosyl)-2-O-benzoyl-3-O-benzyl-ꢀ,ꢁ-L-ido-
pyranosyluronate) trichloroacetimidate (12). To a solution of 15
(600 mg, 0.78 mmol) in dry CH2Cl2 (6 mL), CCl3CN (1.17
mL, 11.7 mmol) and activated K2CO3 (108.5 mg, 0.82 mmol)
were added. After stirring for 3 h the residue was filtered and
concentrated to dryness. The residue was purified by flash
chromatography (3 : 1 hexane–EtOAc), to yield 12 (707 mg,
95%), as a mixture of anomers α–β. TLC α–β, 0.2–0.36 (3 : 1
hexane–EtOAc); 1H-NMR (500 MHz, CDCl3), β anomer;
δ 8.68 (s, 1 H, NH ); 8.20–7.08 (m, 20 H, Ph); 6.58 (s, 1 H, H1);
5.52 (s, 1 H, Ph–CH–); 5.34 (s, 1 H, H2); 5.03 (s, 1 H, H5); 4.93
(d, 1 H, Jgem = 11.5 Hz, CH2Ph); 4.76 (d, 1 H, Jgem = 11.5 Hz,
CH2Ph); 4.73 (d, 1 H, J1Ј,2Ј = 3.5 Hz, H1Ј); 4.37–4.34 (m, 2 H,
CH2Ph, H6aЈ); 4.25 (s, 1 H, H3); 4.18 (s, 1 H, H4); 4.00–3.94 (m,
1 H, H5Ј); 3.84 (d, 1 H, Jgem = 11.5 Hz, CH2Ph); 3.78 (s, 3 H,
COOCH3); 3.62 (t, 1 H, J3Ј,2Ј = J3Ј,4Ј = 9.5 Hz, H3Ј); 3.58–3.45 (m,
2 H, H6Јb, H4Ј); 3.22 (dd, 1 H, J2Ј,1Ј = 3.5 Hz, J2Ј,3Ј = 9.5 Hz, H2Ј);
α anomer; δ 8.66 (s, 1 H, NH ); 8.20–7.10 (m, 20 H, Ph); 6.32
(s, 1 H, H1); 5.50 (s, 1 H, Ph–CH–); 5.42 (s, 1 H, J2,1 = 2.0 Hz,
H2); 4.90 (d, 1 H, Jgem = 11.5 Hz, CH2Ph); 4.78 (d, 1 H,
Jgem = 11.5 Hz, CH2Ph); 4.71 (d, 1 H, J5,4 = 1.5 Hz, H5); 4.69 (d,
1 H, J1Ј,2Ј = 3.5 Hz, H1Ј); 4.38 (t, 1 H, J3,4 ≈ J3,2 = 3 Hz, H3); 4.33
(dd, 1 H, J6Јa,5Ј = 5.0 Hz, J6Јa,6Јb = 10.0 Hz, H6Јa); 4.18 (s, 1 H, H4);
4.03 (m, 1 H, H5Ј); 3.78 (d, 4 H, CH2Ph, COOCH3); 3.61 (t,
1 H, J4Ј,3Ј = J4Ј,5Ј = 9.5 Hz, H4Ј); 3.54–3.49 (m, 2 H, H6Јb, H3Ј); 3.20
(dd, 1 H, J2Ј,1Ј = 3.5 Hz, J2Ј,3Ј = 9.5 Hz, H2Ј). Anal. calcd. for
C43H41N4O12Cl3: C, 56.61; H, 4.53; N, 6.14; found: C, 56.24; H,
4.51; N, 6.12%.
500 Kcal molϪ1
Å
Ϫ1. This restrain was weakened to 25 Kcal
molϪ1 ÅϪ1 during a 1000 cycles of relaxation and 3 ps of
molecular dynamics. The obtained structures were subjected to
five consecutive runs of 600 cycles of minimisation with the
positional restrains 20, 15, 10, 5 and 0 Kcal molϪ1 ÅϪ1
respectively.
Methyl (dimethyltexylsilyl-4-O-(2-azido-3-O-benzyl-4,6-O-
benzylidene-ꢀ-D-glucopyranosyl)-2-O-benzoyl-ꢁ-idopyranosyl)
uronate (10). To a solution of 911 (3.66 g, 4.54 mmol) in Py (38
mL) benzoyl chloride (2.63 mL, 22 mmol) and catalytic DMAP
were added at 0 ЊC. The mixture was stirred at room temper-
ature for 18 h and then diluted with CH2Cl2 (200 mL), and
washed with water and with 1 M HCl (100 mL). The aqueous
phases were washed with CH2Cl2 (2 × 100 mL) and the organic
phases dried on Mg2SO4 and concentrated. The residue was
purified by column chromatography (25 : 1 toluene–EtOAc) to
give pure 10 (3.92 g., 95%). Rf 0.20 (25 : 1 toluene–EtOAc). [α]D20
Ϫ18.1 (c 1.0, CHCl3). 1H-NMR (500 MHz, CDCl3): δ 8.15–7.08
(m, 20 H, Ph); 5.52 (s, 1 H, Ph–CH ); 5.20 (br s, 1 H, H1); 5.10
(br s, 1 H, H2); 4.85 (d, 1 H, Jgem = 12.0 Hz, CH2Ph); 4.76 (d,
1 H, Jgem = 11.0 Hz, CH2Ph); 4.72 (d, 1 H, J1Ј,2Ј = 3.5 Hz, H1Ј);
4.49 (br s, 1 H, H5); 4.40 (dd, 1 H, J6Јa,6Јb = 10.0 Hz, J6Јa,5Ј
=
5.0 Hz, H6Јa); 4.24 (m, 1 H, H3); 4.20 (m, 2 H, CH2Ph, H5Ј); 4.07
(br s, 1 H, H4); 3.77–3.68 (m, 4 H, CH2Ph, COOCH3); 3.60 (t,
1 H, J3Ј,2Ј = J3Ј,4Ј = 9.5 Hz, H3Ј); 3.58–3.49 (m, 2 H, H6Јa, H4Ј); 3.11
(dd, 1 H, J2Ј,1Ј = 3.5 Hz, J2Ј,3Ј = 9.5 Hz, H2Ј); 1.57 (m, 1 H,
CH(CH3)2); 0.82–0.77 (m, 12 H, CH(CH3)2, C(CH3)2); 0.31–
0.15 (2 s, 6 H, Si(CH3)2). 13C-NMR (125 MHz, CDCl3): δ 172.5,
Methyl (isopropyl 4-O-(2-azido-3-O-benzyl-4,6-O-benzyl-
idene-2-deoxy-ꢀ-D-glucopyranosyl)-3-O-benzyl-2-O-benzoyl-ꢀ-
L-idopyranosyl) uronate (16). To a cooled (0 ЊC) solution of 12
(2.7 g, 2.95 mmol) in dry CH2Cl2 (30 mL) was added isopropyl
alcohol (0.56 mL, 7.39 mmol) and TMSOTf (25.7 µL, 88.7
µmol). After 15 min, the mixture was neutralised with Et3N
(1 mL) and concentrated in vacuo. The residue was purified by
flash chromatography (6 : 1 hexane–EtOAc) to yield the
α anomer 16 (1.57 g, 70%) and the β anomer (235 mg, 11%):
[α]2D0 ϩ22.5 (c 1.1, CHCl3); TLC 0.21 (3 : 1 hexane–EtOAc);
1H-NMR (500 MHz, CDCl3): δ 8.15–7.1 (m, 20 H, Ph); 5.50 (s,
1 H, Ph–CH–); 5.30 (s, 1 H, H1); 5.10 (s, 1 H, H2); 4.93 (d, 1 H,
Jgem = 11.5 Hz, CH2Ph); 4.90 (d, 1 H, J1Ј,2Ј = 4.0 Hz, H1Ј); 4.76 (d,
1 H, J5,4 = 4.0 Hz, H5); 4.72 (d, 1 H, Jgem = 11.5 Hz, CH2Ph);
168.9 (C᎐O); 138.1–126.3 (Ph); 101.3 (Ph–CH–); 99.8 (C );
᎐
1Ј
94.2 (C1); 82.4 (C4Ј); 76.3 (C3Ј); 75.3 (C4); 75.0 (C3); 74.7
(CH2Ph); 73.4 (C5); 73.1 (CH2Ph); 68.7 (C2); 68.4 (C6Ј); 63.4
(CH2Ph); 63.0 (C2Ј); 52.2 (COOCH3); 20.2, 20.0, 18.6, 18.5,
18.6, 14.3, Ϫ1.8, Ϫ3.4 (OTDS). MALDI-TOF m/z 932.1 (M ϩ
Naϩ), 948.1 (M ϩ Kϩ). Anal: calcd for: C49H59N3O12Siؒ1H2O:
C, 64.66; H, 6.53; N, 4.61; found: C, 64.70; H, 6.05; N, 3.88%.
Methyl 4-O-(2-azido-3-O-benzyl-4,6-O-benzylidene-2-deoxy-
ꢀ-D-glucopyranosyl)-3-O-benzyl-2-O-benzoyl-ꢀ,ꢁ-L-idopyrano-
syluronate (15). To a solution of 10 (1.75 g, 1.92 mmol) in dry
THF (40 mL) an excess of (HF)nؒPy (5.0 mL) was added at
Ϫ10 ЊC. The reaction was warmed to 0 ЊC and stirred for 48 h
under an argon atmosphere. The mixture was diluted with
CH2Cl2 (2 × 100 mL) and washed with H2O (2 × 100 mL) and
saturated NaHCO3 solution until neutral pH. The aqueous
layer was extracted with CH2Cl2 (2 × 150 mL) and the organic
layers were dried (MgSO4) and concentrated in vacuo. The resi-
due was purified by flash chromatography (2 : 1 hexane–
EtOAc), to yield 15 (1.18 g, 80%) as a mixture of anomers α–β.
TLC 0.24 (2 : 1 hexane–EtOAc). 1H-NMR (500 MHz, CDCl3):
δ 8.20–7.05 (m, 40 H, Ph α and β); 5.51 (m, 3 H, Ph–CH– α and
β, H1α); 5.24 (d, 1 H, J1,OH = 10.0 Hz, H1 β); 5.08 (s, 2 H, H2
α and β); 4.98 (d, 2 H, J5,4 = 2.0 Hz, H5 α); 4.92–4.75 (m, 4 H,
CH2Ph α and β); 4.66–4.58 (m, 3 H, H1Ј α CH2Ph, H1Ј β); 4.40–
4.25 (m, 6 H, H3, H6Јa, CH2Ph, OH) α; 4.05 (s, 2 H, H4 α and β);
4.00–3.89 (m, 3 H, H5Ј α and β, CH2Ph); 3.84–3.77 (m, 8 H,
CH2Ph, OH β, COOCH3 α and β); 3.66–3.59 (m, 3 H, H6Јb
α and β, CH2Ph); 3.52–3.45 (m, 4 H, H3Ј, H4Ј α and β); 3.22 (m,
2 H, H2Ј α and β). 13C-NMR (125 MHz, CDCl3): δ 169.4, 168.7,
4.41 (d, 1 H, Jgem = 11.0 Hz, CH2Ph); 4.33 (dd, 1 H, J6Јa,5Ј
=
5.0 Hz, J6Јa,6Јb = 10.0 Hz, H6aЈ); 4.14 (t, 1 H, J3,4 = J3,2 = 9.5 Hz,
H3); 4.09 (s, 1 H, H4); 4.03–3.95 (m, 2 H, H5Ј, CH(CH3)2); 3.97
(d, 1 H, Jgem = 11.0 Hz, CH2Ph); 3.75 (s, 3 H, COOCH3); 3.65–
3.60 (m, 2 H, H3Ј, H6Јb); 3.54 (t, 1 H, J4Ј,3Ј = J4Ј,5Ј = 9.5 Hz, H4Ј);
3.21 (dd, 1 H, J2Ј,1Ј = 4.0 Hz, J2Ј,3Ј = 10.0 Hz, H2Ј); 1.24–1.18 (d,
6 H, CH(CH3)2).13C-NMR (125 MHz, CDCl3): δ 170.1, 165.9
(C᎐O); 138.1–126.4 (Ph); 101.7 (Ph–CH–); 100.1 (C ); 97.7
᎐
5
(C1); 82.7 (C4Ј); 76.8 (C3Ј); 76.3 (C4); 75.0 (CH2Ph); 73.8 (C3);
72.5 (CH2Ph); 70.1 (C5Ј, CH(CH3)2); 68.9 (C2); 68.7 (C6Ј); 67.8
(C1Ј); 63.7 (C2Ј); 52.5 (COOCH3); 23.6–21.8 (CH(CH3)2).
MALDI-TOF m/z 832.2 (M ϩ Naϩ), 848.2 (M ϩ Kϩ). Anal.
calcd. for C44H47N3O12: C, 65.24; H, 5.84; N, 5.18; found: C,
64.70; H, 5.81; N, 5.59%.
Methyl (isopropyl 4-O-(2-azido-3-O-benzyl-2-deoxy-ꢀ-D-
glucopyranosyl)-3-O-benzyl-2-O-benzoyl-ꢀ-L-idopyranosyl)
uronate (17). To a solution of 16 (1.0 g, 1.23 mmol) in dry
CH2Cl2 (12 mL), EtSH (0.45 mL, 6.17 mmol) and BF3ؒOEt2
(5 µL, 37 µmol) were added at 0 ЊC. After stirring for 1 h under
an argon atmosphere, the reaction was neutralised with satur-
ated NaHCO3 solution (2 mL), diluted with CH2Cl2 (200 mL)
and washed with H2O (2 × 50 mL). The organic layer was dried
with MgSO4and concentrated to dryness. The residue was puri-
fied by flash chromatography (1 : 1 hexane–EtOAc) to yield 17
(840 mg, 95%). TLC 0.26 (1 : 1 hexane–EtOAc). [α]2D0 Ϫ28.5
165.9, 165.6 (C᎐O); 137.8–127.9 (Ph); 101.3 (Ph–CH–); 100.4
᎐
(C1Ј α); 100.3 (C1Ј β); 93.9 (C1 α); 92.6 (C1 β); 82.3 (C4Ј α); 82.2
(C4Ј β); 76.9, 76.7 (C3Јα and β); 76.1, 75.8 (CH2Ph); 74.7 and
74.6 (C4 β and α); 75.1 (CH2Ph); 73.5 (C3 α and β); 73.0, 72.9
(CH2Ph); 68.7, 68.5, 67.4 (C2 α and β, C6Ј α and β); 66.9
(C5 α and β); 63.5, 63.5, 63.2 (C5Ј, C2Ј α and β); 52.5–52.4
(COOCH3 α and β). MALDI-TOF m/z 790 (M ϩ Naϩ); 806
(M ϩ Kϩ). Anal. calcd. for C41H41N3O12: C 64.13; H, 5.38; N,
5.47; found C, 63.96; H, 5.21; N, 5.76%.
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 2 2 5 3 – 2 2 6 6
2260

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(c 1.0, CHCl3). 1H-NMR (500 MHz, CDCl3): δ 8.15–7.16 (m, 15
H, Ph); 5.30 (s, 1 H, H1); 5.09 (s, 1 H, H2); 4.93 (d, 1 H, Jgem
2 H, Jortho = 11.0 Hz, MeOPh); 7.38–7.19 (m, 15 H, Ph); 6.90 (d,
=
2 H, Jortho = 11.0 Hz, MeOPh); 5.30 (br s, 1 H, H1); 5.10 (br s,
1 H, H2); 4.90 (d, 1 H, Jgem = 11.5 Hz, CH2Ph); 4.87 (d, 1 H,
J5,4 = 2.5 Hz, H5); 4.79 (d, 1 H, J1Ј2Ј = 3.5 Hz, H1Ј); 4.71 (d, 1 H,
Jgem = 11.5 Hz, CH2Ph); 4.50 (d, 1 H, Jgem = 12.0 Hz, CH2-
PhOMe); 4.41 (d, 1 H, Jgem = 12.0 Hz, CH2PhOMe); 4.37 (d,
1 H, Jgem = 11.0 Hz, CH2Ph); 4.22 (d, 1 H, Jgem = 11.0 Hz,
CH2Ph); 4.13 (br s, 1 H, H3); 4.08 (br s, 1 H, H4); 4.04–3.98 (m,
1 H, CH(CH3)2); 3.82 (m, 1 H, H5Ј); 3.78 (s, 3 H, PhOCH3); 3.71
(s, 4 H, H6Јa, COOCH3); 3.64 (t, 1 H, J4Ј,3Ј ≈ J4Ј,5Ј = 9.0 Hz, H4Ј);
3.55 (dd, 1 H, J6Јb,5Ј = 5.0 Hz, J6Јb,6Јa = 10 Hz, H6Јb); 3.48 (t, 1 H,
11.5 Hz, CH2Ph); 4.90 (d, 1 H, J5,4 = 2.0 Hz, H5); 4.77 (d, 1 H,
J1Ј2Ј = 3.5 Hz, H1Ј); 4.72 (d, 1 H, Jgem = 11.5 Hz, CH2Ph); 4.26 (d,
1 H, Jgem = 11.0 Hz, CH2Ph); 4.16–4.08 (m, 3 H, H3, H4,
CH2Ph); 4.04–3.98 (m, H, CH(CH3)2); 3.87–3.78 (m, 2 H, H5Ј,
H6aЈ); 3.77 (s, 3 H, COOCH3); 3.76–3.68 (m, 1 H, H6Јb); 3.49–
3.40 (m, 2 H, H4Ј, H3Ј); 3.09 (dd, 1 H, J2Ј,1Ј = 3.5 Hz, J2Ј,3Ј = 10.0
Hz, H2Ј); 2.35 (br s, 1 H, OH4); 2.25 (br s, 1 H, OH6); 1.23–1.17
(d, 6 H, CH(CH3)2). 13C-NMR (125 MHz, CDCl3): δ 170.2,
165.6 (C᎐O); 137.8–127.7 (Ph); 99.0 (C ); 97.4 (C ); 79.9 (C );
᎐
5
1
4Ј
75.4 (C4); 74.7 (CH2Ph); 73.4 (C3); 72.6 (CH2Ph); 72.3 (C5Ј);
71.2 (C3Ј); 70.7 (CH(CH3)2); 68.7 (C2); 67.6 (C1Ј); 63.2 (C2Ј); 62.4
(C6Ј); 52.3 (COOCH3); 23.3–21.0 (CH(CH3)2). MALDI-TOF
m/z 745 (M ϩ Naϩ), 761 (M ϩ Kϩ). Anal. calcd for C37H43-
N3O12.1H2O: C, 60.00; H, 6.13; N, 5.67; found: C, 59.85; H,
6.30; N, 5.46%.
J3Ј,2Ј ≈ J3Ј,4Ј = 9.5 Hz, H3Ј); 3.15 (dd, 1 H, J2Ј,1Ј = 3.5 Hz, J2Ј,3Ј =
9.5Hz, H2Ј); 2.56 (br s, 1 H, OH4); 1.25–1.18 (2d, 6 H,
CH(CH3)2). 13C-NMR (125 MHz, CDCl3): δ 171.0, 164.5
(C᎐O); 159.0 (MeOPh); 137.4–127.4 (Ph); 113.6 (MeOPh); 99.1
᎐
(C1Ј); 97.3 (C1); 79.5 (C4Ј); 75.2 (C4); 74.7 (CH2Ph); 73.6 (C3);
73.4 (CH2PhOMe); 72.7 (C3Ј); 72.4 (CH2Ph); 70.7 (CH(CH3)2);
70.4 (C5Ј); 69.3 (C6Ј); 69.0 (C2); 68.1 (C5); 63.0 (C2Ј); 55.3
(CH3OPh); 52.3 (COOCH3); 23.3, 21.5 (CH(CH3)2). MALDI-
TOF m/z 866 (M ϩ Naϩ); 882 (M ϩ Kϩ). Anal. calcd. for
C37H43N3O12.ؒ½ H2O: C, 62.17; H,6.26; N, 4.83; found C, 62.20;
H, 6.45; N, 4.62%.
Methyl (isopropyl 4-O-(2-azido-3-O-benzyl-4,6-O-(p-methoxy-
benzylidene)-2-deoxy-ꢀ-D-glucopyranosyl)-3-O-benzyl-2-O-
benzoyl-ꢀ-L-idopyranosyl) uronate (18). To a solution of 17
(825 mg, 1.14 mmol) in dry DMF (10 mL), 4-methoxybenzalde-
hyde dimethyl acetal (292 µL, 1.71 mmol) and a catalytic
amount of p-toluenesulfonic acid monohydrate were added.
After stirring for 3 h under argon atmosphere, the reaction was
neutralised with saturated NaHCO3 solution (10 mL), diluted
with EtOAc (100 mL) and washed with H2O. The organic layer
was dried with MgSO4 and concentrated to dryness. The
residue was purified by flash chromatography (6 : 1 hexane–
EtOAc), affording compound 18 (790 mg, 85%). TLC 0.27 (3 : 1
hexane–EtOAc). [α]2D0 Ϫ65.4 (c 1.0, CHCl3). 1H-NMR (500
MHz, CDCl3): δ 8.14 (d, 2 H, Jortho = 11.0 Hz, MeOPh); 7.40–
7.11 (m, 15 H, Ph); 6.90 (d, 2 H, Jortho = 11.0 Hz, MeOPh); 5.43
(s, 1 H, MeOPh–CH–); 5.30 (s, 1 H, H1); 5.11 (s, 1 H, H2); 4.93
(d, 1 H, Jgem = 11.5 Hz, CH2Ph); 4.90 (d, 1 H, J5,4 = 2.0 Hz, H5);
4.75 (d, 1 H, J1Ј2Ј = 3.5 Hz, H1Ј); 4.72 (d, 1 H, Jgem = 11.5 Hz,
CH2Ph); 4.41 (d, 1 H, Jgem = 11.0 Hz, CH2Ph); 4.30 (dd, 1 H,
Methyl (dimethylthexylsilyl 4-O-(2-azido-3,4-di-O-benzyl-2-
deoxy-ꢀ-D-glucopyranosyl)-3-O-benzyl-2-O-benzoyl-ꢀ,ꢁ-L-ido-
pyranosyl) uronate (19). To a solution of 10 (100 mg, 0.10
mmol) and the complex BH3ؒNHMe2 (33 mg, 0.54 mmol) in dry
CH2Cl2 (2 mL), at Ϫ15 ЊC, BF3ؒEt2O (68.5 µL, 0.54 mmol) was
added. After stirring for 30 min at this temperature under an
argon atmosphere, the mixture was neutralised with saturated
NaHCO3 solution (15 mL), diluted with EtOAc (75 mL) and
washed with H2O (2 × 50 mL). The aqueous layer was extracted
with EtOAc (2 × 100 mL), and the organic layers were dried
over MgSO4 and concentrated in vacuo. The residue was puri-
fied by flash cromatography (3 : 1 hexane–EtOAc), to yield 19
(81 mg, 81%). TLC 0.26 (2 : 1 hexane–EtOAc), [α]2D0 Ϫ2.9 (c 1,
CHCl3). 1H-NMR (500 MHz, CDCl3): δ 8.12–7.07 (m, 20 H, 4
Ph); 5.16 (d, 1 H, J1,2 = 1.5 Hz, H1); 5.11 (br s, 1 H, H2); 4.84 (d,
1 H, Jgem = 11.5 Hz, CH2Ph); 4.76 (d, 1 H, Jgem = 11.5 Hz,
J6Јa,5Ј = 5.0 Hz, J6Јa,6Јb = 10.0 Hz, H6Јa); 4.13 (t, 1 H, J3,4 ≈ J3,2
=
2.5 Hz, H3); 4.09 (m, 1 H, H4); 4.04–3.96 (m, 3 H, CH(CH3)2,
H5Ј, CH2Ph); 3.82 (s, 3 H, CH3OPh); 3.75 (s, 3 H, COOCH3);
3.67–3.58 (m, 2 H, H3Ј, H6Јb); 3.52 (t, 1 H, J4Ј,3Ј = J4Ј,5Ј = 9.5 Hz,
H4Ј); 3.21 (dd, 1 H, J2Ј,1Ј = 3.5 Hz, J2Ј,3Ј = 10.0 Hz, H2Ј); 1.25–1.18
(2 d, 6 H, CH(CH3)2). 13C-NMR (125 MHz, CDCl3): δ 170.1,
CH2Ph); 4.72–4.65 (m, 2 H, CH2Ph, H1Ј); 4.58 (d, 1 H, Jgem
=
11.0 Hz, CH2Ph); 4.48 (d, 1 H, J5,4 = 1.5 Hz, H5); 4.22 (t, 1 H,
J3,2 ≈ J3,4 = 2.5 Hz, H3); 4.02 (m, 1 H, Jgem = 11.0 Hz, CH2Ph);
3.98 (br s, 1 H, H4); 3.96–3.92 (m, 1 H, H5Ј); 3.90–3.82 (m, 2 H,
H6Јa, CH2Ph); 3.77 (s, 3 H, COOMe); 3.71–3.66 (m, 1 H, H6Јb);
165.9 (C᎐O); 160.4 (MeOPh); 137.4–127.4 (Ph); 113.4
᎐
(MeOPh); 101.4 (MeOPh–CH–); 99.7 (C1Ј); 97.4 (C1); 82.4
(C4Ј); 76.5 (C3Ј); 76.0 (C4); 74.7 (CH2Ph); 73.5 (C3); 72.2
(CH2Ph); 70.6 (CH(CH3)2); 68.5 (C6Ј); 68.4 (C2); 67.6 (C5); 63.4
(C5Ј); 63.2 (C2Ј); 55.3 (MeOPh); 52.5 (COOCH3); 23.3, 21.5
(CH(CH3)2). MALDI-TOF m/z 863 (M ϩ Naϩ); 880 (M ϩ
Kϩ). Anal. calcd. for C45H49N3O13: C, 64.35; H, 5.88; N, 5.00;
found C, 64.63; H, 6.02; N, 4.95%.
3.48 (t, 1 H, J3Ј,2Ј ≈ J3Ј,4Ј = 9.5 Hz, H3Ј); 3.37 (t, 1 H, J4Ј,3Ј ≈ J4Ј, 5Ј =
9.5 Hz, H4Ј); 3.09 (dd, 1 H, J2Ј,1Ј = 3.5 Hz, J2Ј,3Ј = 10.0 Hz); 1.83
(t, 1 H, OH ); 1.55 (m, 1 H, CH(CH3)2); 0.76–0.74 (m, 12 H,
CH(CH3)2, C(CH3)2); 0.24, 0.12 (2 s, 6 H, Si(CH3)2). 13C-NMR
(125 MHz, CDCl ): δ 169.0, 166.5 (C᎐O); 138.0–127.6 (Ph);
᎐
3
99.7 (C1Ј); 94.0 (C1); 79.7 (C3Ј); 77.7 (C4Ј); 75.7 (C4); 75.1 (C3);
74.8 (CH2Ph); 74.6 (CH2Ph); 73.4 (C5); 73.0 (CH2Ph); 72.7
(C5Ј); 68.6 (C2); 63.7 (C2Ј); 61.5 (C6Ј); 52.2 (COOCH3); 34.0,
24.7, 20.1, 19.8, 18.5, 18.3, Ϫ2.0, Ϫ3.4 (OTDS). MALDI-TOF
m/z935 (M ϩ Naϩ); 951.0 (M ϩ Kϩ). Anal. calcd. for C49H61-
N3O12Si: C, 64.54; H, 6.74; N, 4.60; found C, 64.33; H, 7.03; N,
4.64%.
Methyl (isopropyl 4-O-(2-azido-3-O-benzyl-6-O-(4-methoxy-
benzyl)-2-deoxy-ꢀ-D-glucopyranosyl)-3-O-benzyl-2-O-benzoyl-
ꢀ-L-idopyranosyl) uronate (11). A solution at 0 ЊC of TFA
(1.17 mL, 15.22 mmol) in dry DMF (8 mL) cooled at 0 ЊC was
added dropwise to a stirred mixture containing compound 18
(640 mg, 0.76 mmol) and NaBH3CN (1 M solution in THF
11.4 mL, 11.41 mmol). After 3 h an identical amount of NaB-
H3CN was added in the same conditions. Three more additions
in the same conditions were made at three hour intervals. After
stirring at 35 ЊC for 24 h, the mixture was neutralised, at 0 ЊC
with saturated NaHCO3 solution (15 mL), diluted with EtOAc
(100 mL) and washed with H2O (2 × 100 mL). The aqueous
layer was extracted with EtOAc (2 × 100 mL) and the organic
layers were dried (MgSO4) and concentrated in vacuo. The resi-
due was purified by flash cromatography (3 : 1 hexane–EtOAc),
to yield 11 (442.3 mg, 69%) as well as non-reacted starting
material (160.5 mg, 25%). TLC 0.33 (2 : 1 hexane–EtOAc). [α]2D0
Ϫ25.2Њ(c 1.0, CHCl3).1H-NMR (500 MHz, CDCl3): δ 8.14 (d,
Methyl dimethylthexylsilyl 4-O-(2-azido-3,4-di-O-benzyl-2-
deoxy-[6-O-(4-methoxybenzyl)]-ꢀ-D-glucopyranosyl)-3-O-
benzyl-2-O-benzoyl-ꢀ,ꢁ-L-idopyranosyluronate (20). To a solu-
tion of 19 (180 mg, 0.19 mmol) and 4-methoxybenzyl trichloro-
acetimidate (55 mg, 0.59 mmol), in dry CH2Cl2 (3 mL) at
Ϫ20 ЊC, was added BF3ؒOEt2 (6 µL, 5.91 µmol, 1 M solution in
THF). After stirring for 10 min at this temperature, the mixture
was neutralised with Et3N (0.5 mL) and concentrated in vacuo.
The residue was purified by flash chromatography (4 : 1
hexane–EtOAc), to yield 20 (186 mg, 96%). TLC 0.20 (14 : 1
toluene–EtOAc), [α]2D0 ϩ6.6 (c 0.8, CHCl3). 1H-NMR (500
MHz, CDCl3): δ 8.12–7.08 (m, 20 H, Ph); 6.80 (d, 2 H, Jortho
=
8.5 Hz, MeOPh); 5.19 (br s, 1 H, H1); 5.11 (br s, 1 H, H2); 4.85
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 2 2 5 3 – 2 2 6 6
2261

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(d, 1 H, Jgem = 11.0 Hz, CH2Ph); 4.80–4.74 (m, 2 H, CH2Ph,
H1Ј); 4.62–4.57 (2 d, 2 H, Jgem = 11.0 Hz, CH2Ph, CH2PhOMe);
4.50 (br s, 1 H, H5); 4.44–4.36 (2 d, 2 H, Jgem = 11.0 Hz,CH2Ph,
CH2PhOMe); 4.24 (t, 1 H, J3,2 ≈ J3,4 = 2.5 Hz, H3); 4.04–3.98 (m,
3 H, CH2Ph, H4, H5Ј); 3.85 (d, 1 H, CH2Ph); 3.81 (dd, 1 H,
J6Јa,6Јb = 11.0 Hz, J6Јa,5Ј = 2 Hz, H6Јa,); 3.78–3.72 (2 s, 6 H,
CH3OPh, COOCH3); 3.68 (dd, 1 H, J6Јb,6Јa = 11.0 Hz, J6Јb,5Ј = 1.5
Hz, H6Јb); 3.64 (t, 1 H, J4Ј,3Ј ≈ J4Ј,5Ј = 9.5 Hz, H4Ј); 3.50 (t, 1 H,
J3Ј,2Ј ≈ J3Ј,4Ј = 9.5 Hz, H3Ј); 3.18 (dd, 1 H, J2Ј,1Ј = 3.5 Hz, J2Ј,3Ј = 9.5
Hz, H2Ј); 1.57 (m, 1 H, CH(CH3)2); 0.78–0.75 (m, 12 H,
CH(CH3)2, C(CH3)2); 0.26–0.15 (2 s, 6 H, Si(CH3)2). 13C-NMR
CDCl ): δ 168.6, 165.4 (C᎐O); 160.3 (C᎐NH); 159.3 (MeOPh);
᎐ ᎐
3
138.4–127.6 (Ph, MeOPh); 113.7 (MeOPh); 100.1 (C1Ј); 96.1
(C1); 80.8 (C3Ј); 77.6 (C4Ј); 75.7 (C4); 74.7 (CH2Ph); 74.6
(CH2PhOMe); 73.1, 72.5 (CH2Ph); 72.2 (C3); 71.8 (C5Ј); 69.0
(C5); 67.2 (C6Ј); 65.8 (C2); 63.7 (C2Ј); 55.2 (CH3OPh); 52.45
(COOCH3). Anal. calcd. for C51H51N4O13Cl3: C, 59.22; H, 4.96;
N, 5.41; found: C, 59.05; H, 5.12; N, 5.68%.
Methyl (isopropylO-(2-azido-3-O-benzyl-4,6-O-benzylidene-
2-deoxy-ꢀ-D-glucopyranosyl)-(1 4)-O-(methyl 2-O-benzoyl-3-
O-benzyl-ꢀ-L-idopyranosyluronate)-(1 4)-O-(2-azido-3-O-
benzyl-2-deoxy-6-O-(p-methoxybenzyl)-ꢀ-D-glucopyranosyl)-
(1 4)-(2-O-benzoyl-3-O-benzyl-ꢀ-L-idopyranosyl) uronate (22).
A mixture of 12 (172.5 mg, 0.18 mmol) and acceptor 11 (114.3
mg, 0.13 mmol) was dissolved in dry CH2Cl2 (3 mL) and cooled
at Ϫ20 ЊC under an argon atmosphere. A solution of TMSOTf
(21.5 µL of 0.55 M in dry CH2Cl2) was added dropwise and the
solution was stirred for 15 min at the same temperature, and
then neutralised with triethylamine. The solvent was concen-
trated in vacuo and the obtained residue was purified by flash
chromatography (4 : 1 hexane–EtOAc), affording compound 22
(185 mg, 85%) and a mixture of 11–12, which was purified with
(10 : 1 toluene–acetone) affording compound 11 (10 mg, 8.5%)
and 12 (20 mg, 14%). TLC 0.29 (3 : 1 hexane–EtOAc). [α]D20
(125 MHz, CDCl ): δ 168.5, 166.5 (C᎐O); 159.3 (MeOPh);
᎐
3
138.4–127.8 (Ph, MeOPh); 113.8 (MeOPh); 100.0 (C1Ј); 93.9
(C1); 79.8 (C3Ј); 77.8 (C4Ј); 75.6 (C4); 75.0 (C3); 74.8 (CH2Ph);
74.5 (CH2PhOMe); 73.5 (C5); 73.2–73.0 (CH2Ph); 71.7 (C5Ј);
68.6 (C2); 67.2 (C6Ј); 63.7 (C2Ј); 55.2 (CH3OPh); 52.2 (CO-
OCH3); 34.0, 24.8, 20.2, 19.9, 18.6, 18.4, Ϫ1.8, Ϫ3.4 (OTDS).
MALDI-TOF m/z 1054.0 (M ϩ Naϩ); 1070.2 (M ϩ Kϩ). Anal.
calcd. for C57H69N3O13Si: C, 66.32; H, 6.73; N, 4.07; found C,
66.21; H, 6.69; N, 3.87%.
Methyl 4-O-(2-azido-3,4-di-O-benzyl-2-deoxy-6-O-(p-methoxy-
benzyl)]-ꢀ-D-glucopyranosyl)-2-O-benzoyl-3-O-benzyl-ꢀ,ꢁ-L-
idopyranosyluronate (21). To a solution of 20 (200 mg, 0.19
mmol) at Ϫ15 ЊC in dry THF (6 mL) an excess of (HF)nؒPy
(0.7 mL) was added. The reaction was warmed to 0 ЊC and
stirred for 48 h under argon atmosphere. The mixture was
diluted with CH2Cl2 (50 mL) and washed with H2O (2 × 25 mL)
and saturated NaHCO3 solution until neutral pH. The aqueous
layer was extracted with CH2Cl2 (2 × 50 mL) and the organic
layers were dried over MgSO4and concentrated in vacuo. The
residue was purified by flash chromatography (2 : 1 hexane–
EtOAc), to yield 21 (140 mg, 83%) as a mixture of anomers
1
Ϫ24.8 (c 1.0, CHCl3). H-NMR (500 MHz, CDCl3): δ 8.2–6.7
(m, 39 H, Ph, PhOMe), 5.51 (s, 2 H, Ph–CH–, H1c); 5.22 (d, 1 H,
J1,2 = 2.0 Hz, H1a); 5.16 (t, 1 H, J2,3 ≈ J2,1≈ 4.5 Hz, H2c); 5.06 (t,
1 H, J2,3 ≈ J2,1 ≈ 2.5 Hz, H2a); 4.88 (d, 1 H, Jgem = 11.5 Hz, CH2-
Ph); 4.84 (d, 1H, J1,2 = 3.5 Hz, H1b); 4.81–4.76 (m, 3 H,CH2-
PhOMe, H5c); 4.73 (d, 1H, J1,2 = 3.5 Hz, H1d); 4.70 (d, 1 H,
Jgem = 11.5 Hz, CH2Ph); 4.63 (d, 1 H, J5,4 = 3.5 Hz, H5a); 4.57 (d,
1 H, Jgem = 11.5 Hz, CH2Ph); 4.47 (d, 1 H, Jgem = 11.5 Hz,
CH2Ph); 4.37 (s, 1 H, CH2Ph); 4.27–4.20 (m, 2 H, H6d, CH2Ph);
1
α–β. TLC 0.23 (2 : 1 hexane–EtOAc). H-NMR (500 MHz,
4.18 (t, 1 H, J3,4 = J3,2 = 5.0 Hz, H3c); 4.08 (t, 1 H, J3,4 ≈ J3,2 =
CDCl3): δ 8.11–7.03 (m, 44 H, Ph, MeOPh α and β); 6.78–6.72
(d, 4 H, MeOPh); 5.43 (d, 1 H, J1α,OH = 9.0 Hz, H1α); 5.24 (d,
1 H, J1β, OH = 11.5 Hz, H1β); 5.05 (s, 2 H, H2 α and β); 4.88–4.84
(m, 3 H,CH2Ph, H5α); 4.80–4.72 (m, 2 H, CH2Ph α and β); 4.69
(d, 1 H, J1Ј,2Јα = 3.5 Hz, H1Јα); 4.64 (d, 1 H, J1Ј,2Јβ = 3.0 Hz, H1Ј
β); 4.60–4.50 (m, 5 H, H5β, CH2Ph α and β); 4.40–4.28 (m, 6 H,
H3, CH2Ph α and β); 4.19 (d, 1 H, J1α,OH = 9.0 Hz, OH α); 4.03–
3.94 (m, 5 H, H6Јa,, H4,CH2Ph, α and β); 3.85–3.75 (m, 5 H, H5Ј,
H6Јb, CH2Ph, α and β); 3.73 (s, 6 H, CH3OPh α and β); 3.71 (s,
6 H, COOCH3 α and β); 3.65–3.55 (m, 4 H, CH2Ph, OH β, H4Ј
α and β); 3.42–3.34 (m, 2 H, H3Ј α and β); 3.24–3.18 (m, 2 H, H2Ј
α and β). MALDI-TOF m/z 913 (M ϩ Naϩ); 928.5 (M ϩ Kϩ).
Anal. calcd. for C49H51N3O13: C, 66.13; H, 5.57; N, 4.72; found:
C, 66.08; H, 6.34; N, 5.04%.
3.5 Hz, H3a); 4.00–3.92 (m, 6 H, H4c, H4a, CH(CH3)2, CH2Ph,
H5b, H5d); 3.71–3.67 (m, 4 H, PhOCH3, H3b); 3.67–3.59 (m, 3 H,
H4d, H6Јd, H6b); 3.57–3.45 (m, 9 H, H3d, H4b, H6Јb, COOCH3);
3.27–3.21 (m, 2 H, H2d, H2b); 1.25–1.15 (2 d, 6 H, CH(CH3)2).
13C-NMR (125 MHz, CDCl3) δ 169.9, 169.2, 165.7, 165.2
(C᎐O), 159.0 (MeOPh); 137.9–126.1 (Ph); 113.6 (MeOPh);
᎐
101.5 (Ph–CH–); 99.8 (C1b), 99.1 (C1d), 98.4 (C1c), 97.2 (C1a),
82.4 (C3d); 78.5 (C4b); 76.0 (C4d); 75.6 (C4c); 75.4 (C3c); 75.2
(C4a); 74.7 (C5b); 74.2 (CH2Ph); 73.9 (C5d); 73.2 (C3a); 73.0
(CH2Ph); 72.2, 71.2 (C3b); 70.5 (C2c); 70.2 (CH(CH3)2); 69.6
(C5a); 68.6 (C2a); 68.5 (C6d); 67.8 (C5c); 67.1 (C6b); 63.5 (C2b);
63.3, 62.9 (C2d); 55.2 (PhOCH3); 52.1, 52.0 (COOCH3); 23.3,
21.5 (CH(CH3)2). MALDI-TOF m/z 1615.5 (M ϩ Naϩ); 1631.5
(M ϩ Kϩ). Anal. calcd. for C86H90N6O24: C, 64.85; H, 5.69; N,
5.27; found C, 65.09; H, 6.16; N, 5.05%.
O-(Methyl 4-O-(2-azido-3,4-di-O-benzyl-2-deoxy-6-O-
(p-methoxybenzyl)-ꢀ-D-glucopyranosyl)-2-O-benzoyl-2-O-benzyl-
ꢀ,ꢁ-L-idopyranosyluronate) trichloroacetimidate (13). To a solu-
tion of 21 (140 mg, 0.15 mmol) in dry CH2Cl2(2 mL), CCl3CN
(236 µL, 2.35 mmol) and activated K2CO3 (24 mg, 0.17 mmol)
were added. After stirring for 6 h the residue was filtered and
concentrated to dryness. The residue was purified by flash
chromatography (3 : 1 hexane–EtOAc), to yield 13 (142 mg,
88%) as a mixture of α–β anomers. TLC 0.31, 0.15 β–α (3 : 1
hexane–EtOAc). 1H-NMR (500 MHz, CDCl3): β anomer;
δ 8.65 (s, 1 H, NHβ); 8.10–7.05 (m, 22 H, Ph, MeOPh); 6.77 (d,
2 H, Jortho = 8.5 Hz, MeOPh); 6.53 (s, 1 H, H1); 5.32 (br s, 1 H,
H2); 4.99 (d, 1 H, J5,4 = 1.5 Hz, H5); 4.90 (d, 1 H, Jgem = 11.5 Hz,
Methyl (isopropylO-(2-azido-3-O-benzyl-2-deoxy-ꢀ-D-gluco-
pyranosyl)-(1 4)-O-(methyl 2-O-benzoyl-3-O-benzyl-ꢀ-L-
idopyranosyluronate)-(1 4)-O-(2-azido-3-O-benzyl-2-deoxy-6-
O-(4-methoxybenzyl)-ꢀ-D-glucopyranosyl)-(1 4)-(2-O-benzoyl-
3-O-benzyl-ꢀ-L-idopyranosyl) uronate (23). To a solution of
22 (500 mg, 0.31 mmol) in dry CH2Cl2(8 mL), EtSH (0.34 mL,
4.70 mmol) and catalytic amount camphor sulfonic acid were
added. After stirring for 24 h, the reaction was neutralised with
triethylamine (2 mL). The mixture was concentrated in vacuo
and the obtained residue was purified by flash chromatography
(1 : 1 hexane–EtOAc), to yield 23 (400 mg, 85%) as well as
non-reacted starting material 22 (61 mg, 12%). TLC 0.28 (1 : 1
hexane–EtOAc). [α]2D0 Ϫ6.8 (c 1.0, CHCl3). 1H-NMR (500 MHz,
CDCl3): δ 8.2–6.7 (m, 34 H, Ph), 5.51 (d, 1 H, J1,2 = 4.0 Hz, H1c);
5.26 (s, 1 H, H1a); 5.17 (t, 1 H, J2,3 = J2,1 = 4.5 Hz, H2c); 5.06 (m,
1 H, H2a); 4.88 (d, 1 H, Jgem = 11.5 Hz, CH2Ph); 4.85 (d, 1H,
J1,2 = 3.5 Hz, H1b); 4.79 (m, 3 H, CH2PhOMe, H5c); 4.73 (d, 1H,
J1,2 = 3.5 Hz, H1d); 4.69 (d, 1 H, Jgem = 11.5 Hz, CH2Ph); 4.64
(d, 1 H, J5,4 = 4.0 Hz, H5a); 4.45 (m, 5 H, CH2Ph); 4.18 (t, 1 H,
J3,4 ≈ J3,2 = 5 Hz, H3c); 4.09 (m, 1 H, H3a); 4.00–3.93 (m, 5 H,
CH2Ph); 4.78 (d, 1 H, J1Ј2Ј = 3.0 Hz, H1Ј); 4.74 (d, 1 H, Jgem
=
11.5 Hz,CH2Ph); 4.60–4.51 (2 d, 2 H, Jgem = 11.0 Hz, CH2Ph,
CH2PhOMe); 4.39–4.34 (2 d, 2 H, Jgem = 11.0 Hz, CH2Ph,
CH2PhOMe); 4.23 (br s, 1 H, H3); 4.16 (br s, 1 H, H4); 4.06 (d,
1 H, Jgem = 11.5 Hz, CH2Ph); 3.90–3.85 (m, 2 H, H5Ј, CH2Ph);
3.78–3.70 (m, 7 H, H6Јa,, CH3OPh, COOCH3); 3.65–3.58 (m,
2 H, H4Ј, H6Јb); 3.47 (t, 1 H, J3Ј,4Ј = J3Ј,2Ј = 10.0 Hz, H3Ј); 3.21 (dd,
1 H, J2Ј,1Ј = 3.0 Hz, J2Ј,3Ј = 10.0 Hz, H2Ј). 13C-NMR (125 MHz,
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 2 2 5 3 – 2 2 6 6
2262

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H4c, H4a, H5d, CH2Ph, CH(CH3)2); 3.71–3.67 (m, 7 H, PhOMe,
H5b, H6d, H4b, H4d); 3.62 (m, 1 H, H6b); 3.53–3.44 (m, 10 H, H6Јd
H6Јb, H3b, H3d, 2 COOCH3); 3.24 (dd, 1 H, J2,1 = 3.5 Hz, J2,3
(15 mL), diluted with EtOAc (75 mL) and washed with H2O
,
=
(2 × 50 mL). The aqueous layer was extracted with EtOAc
(2 × 100 mL) and the organic layers were dried (MgSO4) and
concentrated in vacuo. The residue was purified by flash chrom-
atography (3 : 1 hexane–EtOAc), to yield 25 (156 mg, 54%) as
well as non-reacted starting material 24 (122 mg, 42%). TLC
0.22 (2 : 1 hexane–EtOAc). [α]2D0 Ϫ2.8 (c 1.0, CHCl3). 1H-NMR
(500 MHz, CDCl3): δ 8.09–7.14 (m, 34 H, Ph, MeOPh); 6.85–
10.5 Hz, H2d); 3.10 (dd, 1 H, J2,1 = 3.5 Hz, J2,3 = 10.0 Hz, H2b);
1.20–1.14 (2 d, 6 H, CH(CH3)2). 13C-NMR (125 MHz, CDCl3) δ
169.8, 169.4, 165.7, 165.3 (C᎐O), 159.2 (MeOPh); 138.0–127.3
᎐
(Ph); 113.7 (MeOPh); 99.2 (C1b), 98.9 (C1d), 98.3 (C1c), 97.2
(C1a), 79.6 (C3d); 78.4 (C3b); 75.4 (C4c, C4a); 75.3 (C3c); 75.0
(CH2Ph); 74.7, 74.3, 73.8 (CH2Ph); 72.3 (C3a); 73.3, 72.2
(CH2Ph); 71.7; 71.3, 71.1 (C4d, C4b); 71.1 (C6d); 70.6
(CH(CH3)2); 70.4 (C2c); 69.6 (C5a); 69.0 (C2a); 68.1 (C5c); 67.3
(C6b); 63.2 (C2d); 62.9 (C2b); 62.1, 55.2 (PhOMe); 52.0 (CO-
OCH3); 23.3, 21.5 (CH(CH3)2). MALDI-TOF m/z 1527 (M ϩ
Naϩ), 1543 (M ϩ Kϩ). Anal. calcd. for C79H86N6O24: C, 63.10;
H, 5.76; N, 5.58; found: C, 63.14; H, 5.85; N, 5.46%.
6.79 (2 d, 4 H, Jortho = 8.5 Hz, MeOPh); 5.54 (d, 1 H, J1,2
4.5 Hz, H1c); 5.25 (d, 1 H, J1,2 = 2.0 Hz, H1a); 5.18 (t, 1 H, J2,3
=
≈
J2,1 = 5.0 Hz, H2c); 5.05 (m, 1 H, H2a); 4.92 (d, 1H, J1,2 = 3.5 Hz,
H1b); 4.87 (d, 1 H, Jgem = 11.5 Hz, CH2Ph); 4.79 (d, 1 H, Jgem
=
11.5 Hz, CH2Ph); 4.77 (d, 1 H, J5,4 = 4.0 Hz, H5c); 4.75–4.70 (m,
2 H,CH2Ph, H1d); 4.69 (d, 1 H, Jgem = 11.5 Hz, CH2Ph); 4.57 (d,
1 H, J5,4 = 4.5 Hz, H5a); 4.54 (d, 1 H, Jgem = 11.5 Hz,CH2Ph);
4.50–4.40 (m, 6 H, CH2Ph); 4.19 (t, 1 H, J3,4 ≈ J3,2≈ 5.5 Hz, H3c);
4.07 (t, 1 H, J3,4 ≈ J3,2≈ 5.5 Hz, H3a); 4.01–3.91 (m, 5 H, H4a,
H4c,CH2Ph, CH(CH3)2, H5d); 3.77 (s, 3 H, MeOPh); 3.75 (m,
1 H, H4b); 3.71 (s, 3 H, CH3OPh); 3.70–3.64 (m, 3 H, H4d,
Methyl (isopropylO-(2-azido-3-O-benzyl-2-deoxy-4,6-O-
(p-methoxybenzylidene)-ꢀ-D-glucopyranosyl)-(1 4)-O-(methyl
2-O-benzoyl-3-O-benzyl-ꢀ-L-idopyranosyluronate)-(1 4)-O-(2-
azido-3-O-benzyl-2-deoxy-6-O-(p-methoxybenzyl)-ꢀ-D-gluco-
pyranosyl)-(1 4)-(2-O-benzoyl-3-O-benzyl-ꢀ-L-idopyranosyl)
uronate (24). To a solution of 23 (799 mg, 0.53 mmol) in dry
DMF (8 mL), 4-methoxybenzaldehyde dimethyl acetal (182.0
µL, 1.06 mmol) and a catalytic amount of camphor sulfonic
acid were added. After stirring for 3 h under an argon atmos-
phere, the reaction was neutralised with saturated NaHCO3
solution (10 mL), diluted with EtOAc (100 mL) and washed
with H2O. The organic layer was dried with MgSO4 and concen-
trated to dryness. The residue was purified by flash chromato-
graphy (3 : 1 hexane–EtOAc), affording compound 24 (820 mg,
95%). TLC 0.26 (3 : 1 hexane–EtOAc). [α]2D0 Ϫ26.0 (c 1.0,
CHCl3). 1H-NMR (500 MHz, CDCl3): δ 8.2–6.7 (m, 38 H, Ph,
PhOMe), 5.52 (d, 1 H, J1,2 = 4.0 Hz, H1c); 5.47 (s, 1 H, MePh–
H6d, H5b); 3.61 (m, 1 H, H6b); 3.54–3.49 (m, 5 H, H3b, H6Јd
COOCH3); 3.48–3.44 (m, 5 H, H3d, H6Јb, COOCH3); 3.23
(dd, 1 H, J2,1 = 3.5 Hz, J2,3 = 10.0 Hz, H2d); 3.17 (dd, 1 H, J2,1
,
=
3.5 Hz, J2,3 = 10.0 Hz, H2b); 2.11 (br s, 1 H, OH4); 1.20–1.13 (2 d,
6 H, CH(CH3)2). 13C-NMR (125 MHz, CDCl3): δ 169.8, 169.3,
165.6, 165.2 (C᎐O); 159.4, 159.2 (MeOPh); 138.0, 127.2 (Ph);
᎐
113.9, 113.7 (MeOPh); 99.2 (C1b); 98.9 (C1d); 98.2 (C1c); 97.2
(C1a); 79.2 (C3b); 78.4 (C3d); 75.8 (C3c); 75.6 (C5d); 75.4 (C4c);
74.9 (C4a); 74.7, 74.3, 74.0, 73.4 (CH2Ph); 73.2 (C3a); 72.6, 72.3
(CH2Ph); 71.3, 71.0 (C2c); 70.6 (C4b); 70.4 (CH(CH3)2); 70.3
(C5a); 69.3 (C6d); 69.0 (C2a); 68.1 (C5c); 67.1 (C6b); 63.4, 62.7
(C2b); 64.1 (C2d); 55.3, 55.2 (MeOPh); 51.9, 51.8 (COOCH3);
23.3, 21.5 (CH(CH3)2). MALDI-TOF m/z 1646.5 (M ϩ Naϩ);
1662.7 (M ϩ Kϩ). Anal. calcd. for C87H94N6O25: C, 64.31; H,
5.80; N, 5.17; found C, 64.26; H, 6.26; N, 5.70%.
CH–); 5.27 (d, 1 H, J1,2 = 4.5 Hz, H1a); 5.18 (t, 1 H, J2,3 ≈ J2,1
=
4.5 Hz, H2c); 5.07 (m, 1 H, H2a); 4.89 (d, 1 H, Jgem = 11.5 Hz,
CH2Ph); 4.85 (d, 1H, J1,2 = 3.5 Hz, H1b); 4.79 (m, 3 H,
CH2PhOMe, H5c); 4.75 (d, 1H, J1,2 = 3.5 Hz, H1d); 4.70 (d, 1 H,
Jgem = 11.5 Hz, CH2Ph); 4.64 (d, 1 H, J5,4 = 4.0 Hz, H5a); 4.58 (d,
1 H, Jgem = 11.5 Hz,CH2Ph); 4.48 (d, 1 H, Jgem = 11.5 Hz,
CH2Ph); 4.45 (s, 2 H, CH2Ph); 4.27–4.19 (m, 3 H, CH2Ph, H6d,
H3c); 4.09 (m, 1 H, H3a); 4.01–3.87 (m, 6 H, H4a, H4c,CH2Ph,
H5b, CH(CH3)2, H5d); 3.82 (s, 3 H, CH3OPh); 3.70–3.61 (m, 6 H,
CH3OPh, H,3b, H4d H6b); 3.59–3.48 (m, 5 H, COOCH3, H6d,
H4b); 3.49–3.46 (m, 5 H, H3d, H6Јb, COOCH3); 3.24 (m, 2 H, H2d,
H2b); 1.18–1.14 (2 d, 6 H, CH(CH3)2). 13C-NMR (125 MHz,
Methyl (isopropylO-(2-azido-3,4-di-O-benzyl-2-deoxy-6-O-
(p-methoxybenzyl)-ꢀ-D-glucopyranosyl)-(1 4)-O–(2-O-benzoyl-
3-O-benzyl-ꢀ-L-idopyranosyluronate)-(1 4)-O-(2-azido-3-O-
benzyl-2-deoxy-6-O-(p-methoxybenzyl)-ꢀ-D-glucopyranosyl)-
(1 4)-O–(3-O-benzyl-2-O-benzoyl–ꢀ-L-idopyranosyluronate)-
(1 4)-O-(2-azido-3-O-benzyl-2-deoxy-6-O-(p-methoxybenzyl)-
ꢀ-D-glucopyranosyl)-(1 4)-2-O-benzoyl-3-O-benzyl-ꢀ-L-ido-
pyranosyl) uronate (14). A mixture of acceptor 25 (156 mg,
96 µmol) and donor 13 (170 mg, 0.16 mmol) was dissolved in
dry CH2Cl2 (1.5 mL) and cooled at Ϫ20 ЊC under an argon
atmosphere. A solution of TMSOTf ((25.8 µL, 4.8 µmol, 1.8 M
in dry CH2Cl2) was added dropwise and the solution was stirred
for 15 min at the same temperature, and then neutralised with
Et3N (0.5 mL). The solvent was evaporated and the obtained
residue was purified by flash chromatography (4 : 1 hexane–
EtOAc), affording compound 14 (190 mg, 79%) TLC 0.29 (2 : 1
CDCl ): δ 169.9, 169.1, 165.7, 165.2 (C᎐O), 160.1, 159.2
᎐
3
(MeOPh); 138.0–127.3 (Ph); 113.7, 113.6 (MeOPh); 101.4
(MeOPh–CH–); 99.8 (C1b); 98.9 (C1b); 98.4 (C1c); 97.2 (C1a);
82.4 (C4b); 78.5 (C3d); 76.1 (C4d); 75.6 (C5b); 75.5 (C3c); 75.4
(C4c); 75.3, 74.7, 74.2 (C4a); 73.9 (CH2Ph); 73.4 (C3a); 73.2 (C5d);
72.3, 71.7 (CH2Ph); 71.3 (C3b); 70.7 (CH(CH3)2); 70.3 (C2c);
69.7 (C5a); 69.0 (C2a); 68.5 (C6d); 68.1 (C5c); 67.3 (C6b); 63.6
(C2b); 63.4 (C2d); 60.0, 55.3–55.2 (CH3OPh); 52.0–51.9 (CO-
OCH3); 23.3–21.5 (CH(CH3)2). MALDI-TOF m/z 1646.4
(M ϩ Naϩ), 1662.8 (M ϩ Kϩ). Anal. calcd. for C87H93N6O25: C,
64.39; H, 5.77; N, 5.17; found C, 64.26; H, 5.77; N, 5.15%.
hexane–EtOAc). [α]2D0 Ϫ1.7 (c 1, CHCl3). H-NMR (500 MHz,
1
CDCl3): δ 8.08–7.03 (m, 56 H, Ph, MeOPh); 6.78 (m, 6 H,
MeOPh); 5.51 (d, 1 H, J1,2 = 5.0 Hz, H1e); 5.48 (d, 1 H, J1,2
=
4.5 Hz, H1c); 5.24 (d, 1 H, J1,2 = 2.0 Hz, H1a); 5.15 (t, 2 H, H2e,
H2c); 5.05 (t, 1 H, J2,1 = 2.5 Hz, H2a); 4.94 (d, 1 H, J1,2 = 3.0 Hz,
H1d); 4.90–4.86 (m, 2 H, H1f, CH2Ph); 4.80–4.64 (m, 10 H, H1b,
CH2Ph, CH2PhOMe, H5a, H5c, H5e); 4.53–4.26 (m, 12 H,
CH2Ph, CH2PhOMe); 4.19 (t, 1 H, J3,2≈ J3,4 = 5 Hz, H3c); 4.15 (t,
1 H, J3,2 ≈ J3,4 = 6.0 Hz, H3e); 4.06 (t, 1 H, J3,2 ≈ J3,4 = 3.5 Hz,
H3a); 4.02 (t, 1 H, J4,5 ≈ J4,3 = 5.0 Hz, H4c); 4.00–3.92 (m, 4 H,
H4a, H4f, CH2Ph, CH(CH3)2); 3.92–3.85 (m, 2 H, H4d, H4e); 3.81
(m, 1 H, H5d); 3.71–3.69 (m, 10 H, 3 CH3OPh, H6d); 3.66–3.52
(m, 8 H, H5b, H6b, H3d, H4d, H6Јd, H3f, H5f, H6f); 3.48–3.46 (2 s, 6
H, 2 COOCH3); 3.44–3.40 (m, 2 H, H3b, H6Јb); 3.33 (m, 1 H,
H6Јf); 3.27 (s, 3 H, COOCH3); 3.26–3.20 (m, 3 H, H2f, H2d, H2b);
1.19–1.13 (2 d, 6 H, CH(CH3)2). 13C-NMR (125 MHz, CDCl3):
Methyl (isopropylO-(2-azido-3-O-benzyl-2-deoxy-6-O-
(p-methoxybenzyl)-ꢀ-D-glucopyranosyl)-(1 4)-O-(methyl 2-O-
benzoyl-3-O-benzyl-ꢀ-L-idopyranosyluronate)-(1 4)-O-(2-
azido-3-O-benzyl-2-deoxy-6-O-(4-methoxybenzyl)-ꢀ-D-gluco-
pyranosyl)-(1 4)-(2-O-benzoyl-3-O-benzyl-ꢀ-L-idopyranosyl)
uronate (25). A solution of TFA (1.17 mL, 15.22 mmol) in dry
DMF (4 mL) was added dropwise at 0 ЊC to a stirred mixture
containing compound 24 (290 mg, 0.17 mmol) and NaBH3CN
(1 M solution in THF 3.55 mL, 3.55 mmol). A new addition of
NaBH3CN and the solution of TFA in DMF at 0 ЊC was made
after three hours. Three more identical additions were made at
three hours intervals. After stirring at 35 ЊC for 24 h, the mix-
ture was neutralised at 0 ЊC, with saturated NaHCO3 solution
δ 169.83, 169.26, 169.22, 165.62, 165.20, 165.17 (C᎐O); 159.3,
᎐
159.2 (MeOPh); 138.2–127.5 (Ph, MeOPh); 113.8, 113.8, 113.7
(MeOPh); 99.0 (C1d); 98.9 (C1f); 98.8 (C1b); 98.3 (C1e); 98.1 (C1c);
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 2 2 5 3 – 2 2 6 6
2263

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97.2 (C1a); 55.2, 55.2 (CH3OPh); 51.9, 51.6 (COOCH3); 23.2,
21.5 (CH(CH3)2). MALDI-TOF m/z 2516.6 (M ϩ Naϩ); 2532.5
(M ϩ Kϩ). Anal. calcd. for C136H143N9O37: C, 65.46; H, 5.73; N,
5.05; found: C, 65.32; H, 5.62; N, 5.24%.
Hϩ), filtered and concentrated. The residue was purified on
Sephadex LH-20 (9 : 1 MeOH–H2O), to yield 28 (40.1 mg,
98%). TLC 0.33 (12 : 5 : 3 : 1 EtOAc–Py–H2O–AcOH).
1H-NMR (500 MHz, MeOD): δ 7.46–7.19 (m, 35 H, Ph); 5.34–
5.30 (m, 2 H, H1e, H1c); 5.10–5.00 (m, 4 H, H1a, H1b, H1d, H1f);
3.59–3.51 (m, 4 H, H2a, H2f, H2b, H2d); 1.19 (m, 6 H, CH(CH3)2).
Methyl (isopropyl-O-(2-azido-3,4-di-O-benzyl-2-deoxy-ꢀ-D-
glucopyranosyl)-(1 4)-O–(2-O-benzoyl-3-O-benzyl-ꢀ-L-ido-
pyranosyluronate)-(1 4)-O-(2-azido-3-O-benzyl-2-deoxy-ꢀ-
D-glucopyranosyl)-(1 4)-O-(2-O-benzoyl-2-O-benzyl-ꢀ-L-ido-
pyranosyluronate)-(1 4)-O-(2-azido-3-O-benzyl-2-deoxy-ꢀ-
D-glucopyranosyl)-(1 4)-2-O-benzoyl-3-O-benzyl-ꢀ-L-ido-
pyranosyl) uronate (26). To a stirred solution of 14 (63 mg,
25 µmol) in dry CH2Cl2 (6 mL), CF3COOH (138 µL) and H2O
(3.0 µL) were added at room temperature. After stirring for
15 min at this temperature, the reaction mixture became purple.
Then, the reaction was neutralised with Et3N (150 µL) and con-
centrated in vacuo. The residue was purified by flash chromato-
graphy (1 : 1 hexane–EtOAc), to yield 26 (51.3 mg, 96%). TLC
0.23 (1 : 1 hexane–EtOAc). 1H-NMR (500 MHz, CDCl3):
δ 8.20–7.1 (m, 50 H, Ph); 5.44 (m, 2 H, H1e, H1c); 5.26 (br s, 1 H,
H1a); 5.13 (m, 2 H, H2e, H2c); 5.05 (m, 1 H, H2a); 4.91–4.69 (m,
13 H, H1b, H1d, H1f, H5e, H5c, CH2Ph); 4.64–4.58 (m, 3 H, H5a,
CH2Ph); 4.46–4.31 (2 d, 2 H, CH2Ph); 4.20 (m, 2 H, CH2Ph);
4.10 (m, 3 H, H3e, H3a, H3c); 4.01–3.92 (m, 5 H, H4a, H4c, H4e,
CH2Ph, CH(CH3)2); 3.62, 3.54 and 3.42 (3 s, 9 H, COOCH3);
3.25–3.16 (3 dd, 3 H, J2,1 = 3.5 Hz, J2,3 = 10 Hz, H2f, H2d, H2b);
2.04 (br s, 3 H, 3 OH6); 1.20–1.14 (2 d, 6 H, CH(CH3)2).
13C-NMR (125 MHz, CDCl3): δ 170.0, 169.6, 169.4, 165.6,
IsopropylO-(2-deoxy-2-sulfamide-6-O-sulfo-ꢀ-D-glucopyrano-
syl)-(1 4)-O-(ꢀ-L-idopyranosyluronic acid)-(1 4)-O-(2-
deoxy-2-sulfamide-6-O-sulfo-ꢀ-D-glucopyranosyl)-(1 4)-O-(-ꢀ-
L-idopyranosyluronic acid)-(1 4)-O-(2-deoxy-2-sulfamide-6-O-
sulfo-ꢀ-D-glucopyranosyl)-(1 4)-ꢀ-L-idopyranosyluronic acid
nonasodium salt (5). A solution of 28 (25 mg, 11.1 µmol) in
MeOH–H2O (1.5 mL, 9 : 1) was hydrogenated in the presence
of 10% Pd/C. After 24 h, the suspension was filtered and con-
centrated to give 29 as homogenous product by TLC 0.35 (4 : 5 :
3 : 1 EtOAc–Py–H2O–AcOH). This compound was used
directly for N-sulfation. The crude product was dissolved in
H2O (1 mL) and the pH of the solution was adjusted to 9.5 with
1 M solution of NaOH. The pyridine–sulfur trioxide complex
(24 mg, 0.145 mmol, 10 eq. for each amine group) was added in
portions during 1 h and the pH was maintained at 9.5. Sub-
sequent additions of the complex were made after stirring for 2,
4, and 6 h, respectively. After 24 h, the mixture was neutralised
with 0.1 M solution of HCl and then subjected to chromato-
graphy over a Sephadex G-25 column with 0.9% solution of
NaCl. The appropriate fractions were pooled and passed
through a column of Dowex 50WX4-Naϩ (9 × 1.2 cm) with
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 hexasaccharide, were lyophilized to give 5 (11.1 mg, 73%
from 28). TLC 0.35 (4 : 5 : 3 : 1 EtOAc–Py–H2O–AcOH).
1H-NMR (500 MHz, D2O): δ 5.33 (d, 2 H, H1f, H1b); 5.31 (d,
1 H, J1,2 = 3.5 Hz, H1d); 4.99 (br s, 2 H, H1c, H1e); 4.94 (d, 1 H,
J1,2 = 3.0 Hz, H1a); 4.79 (br s, 2 H, H5c, H5e); 4.49 (d, 1 H,
J5,4 = 2.5 Hz, H5a); 3.24–3.18 (m, 3 H, H2b, H2d, H2f); 1.19–1.15
(m, 6 H, CH(CH3)2).
165.2, 165.1 (C᎐O); 137.7–127.3 (Ph); 99.1 (C ); 98.8 (C ); 98.5
᎐
1d
1f
(C1b); 98.1 (C1e); 98.0 (C1c); 97.3 (C1a); 52.2, 52.0 (COOCH3);
23.2, 21.4 (CH (CH3)2).
Methyl (isopropylO-(2-azido-3,4-di-O-benzyl-2-deoxy-6-O-
sulfo-ꢀ-D-glucopyranosyl)-(1 4)-O–(2-O-benzoyl-3-O-benzyl-
ꢀ-L-idopyranosyluronate)-(1 4)-O-(2-azido-3-O-benzyl-2-
deoxy-6-O-sulfo-ꢀ-D-glucopyranosyl)-(1 4)-O-(2-O-benzoyl-3-
O-benzyl-ꢀ-L-idopyranosyluronate)-(1 4)-O-(2-azido-3-O-
benzyl-2-deoxy-6-O-sulfo-ꢀ-D-glucopyranosyl)-(1 4)-2-O-
benzoyl-3-O-benzyl-ꢀ-L-idopyranosyl) uronate trisodium salt
(27). A mixture of 26 (50 mg, 23.0 µmol) and the SO3ؒPy com-
plex (56 mg, 0.35 mmol) in dry Py (2.0 mL) was stirred under an
argon atmosphere. After 1 h, the mixture was cooled and
MeOH (1 mL) and CH2Cl2 (1 mL) were added. The solution
was eluted through a Sephadex LH-20 (1 : 1 MeOH–CH2Cl2).
Fractions containing the hexasaccharide 27 were concentrated
and passed through a Dowex 50WX4-Naϩ (2 : 1 MeOH–H2O),
to yield pure 27 (55 mg, 98%). TLC 0.25 (18 : 5 : 3 : 1 EtOAC–
IsopropylO-(2-azido-3,4-di-O-benzyl-2-deoxy-6-O-
(p-methoxybenzyl]-ꢀ-D-glucopyranosyl)-(1 4)-O-(3-O-benzyl-
ꢀ-L-idopyranosyluronic acid)-(1 4)-O-(2-azido-3-O-benzyl-2-
deoxy-6-O-(p-methoxybenzyl)-ꢀ-D-glucopyranosyl)-(1 4)-O-
(3-O-benzyl-ꢀ-L-idopyranosyluronic acid)-(1 4)-O-(2-azido-3-
O-benzyl-2-deoxy-6-O-(p-methoxybenzyl)-ꢀ-D-glucopyranosyl)-
(1 4)-3-O-benzyl-ꢀ-L-idopyranosyl) uronic acid (30). To a solu-
tion of 14 (30 mg, 12.0 µmol) in THF (4 mL) at Ϫ5 ЊC, H2O2
30% (0.4 mL) and 1.25 M aqueous solution of LiOH (0.70 mL)
were added. After stirring for 24 h at 5 ЊC, MeOH (1.1 mL) and
a 3 M aqueous solution of KOH (2 mL) was added. After
stirring for 24 h more at the same temperature, the reaction was
neutralized with acidic resin (IRA-120 Hϩ), filtered and concen-
trated. The residue was purified by Sephadex LH-20 (1 : 1
CH2Cl2–MeOH), to yield 30 (23.1 mg, 87%). TLC 0.48 (9 : 1
CH2Cl2–MeOH). 1H-NMR (500 MHz, MeOD): δ 7.41–7.00
(m, 41 H, Ph, MeOPh); 6.87–6.81 (m, 6 H, MeOPh); 5.20 (d,
1 H, J1,2 = 2.5 Hz, H1e); 5.18 (d, 1 H, J1,2 = 3.0 Hz, H1c); 5.12
(d, 1 H, J1,2 = 3.5 Hz, H1f); 5.10–5.07 (m, 2 H, H1b, H1d); 5.04 (d,
1 H, J1,2 = 2.5 Hz, H1a); 3.70, 3.69 and 3.67 (3 s, 9 H, CH3OPh);
1.21–1.16 (m, 6 H, CH(CH3)2). MALDI-TOF m/z 2161.9
(M ϩ Naϩ); 2178.7 (M ϩ Kϩ).
1
Py–H2O–AcOH). H-NMR (500 MHz, CD3OD): δ 8.20–7.08
(m, 50 H, Ph); 5.55 (d, 1 H, J1,2 = 2.5 Hz, H1e); 5.48 (br s, 1 H,
H1c); 5.25 (br s, 1 H, H1a); 5.17 (t, 1 H, J2,1 ≈ J2,3 = 3.5 Hz, H2e);
5.14 (br s, 1 H, H2c); 4.98 (s, 1 H, H2a); 4.90–4.73 (m, 13 H, H1b,
H1d, H1f, H5e, H5c, H5a, CH2Ph); 4.44–4.35 (2 d, 2 H, CH2Ph);
4.32–4.27 (m, 2 H, CH2Ph); 4.22–4.15 (m, 5 H, H3c, H3e,
CH2Ph); 4.12–4.08 (m, 3 H, H3a, CH2Ph); 4.02 (m, 1 H, H4a);
3.99–3.90 (m, 3 H, CH(CH3)2, H4c, H4e); 3.72, 3.46 and 3.39 (3 s,
9 H, COOCH3); 3.32–3.22 (m, 3 H, H2f, H2b, H2d); 1.19–1.15 (2
d, 6 H, CH(CH3)2).
Isopropyl O-(2-azido-3,4-di-O-benzyl-2-deoxy-6-O-sulfo-ꢀ-D-
glucopyranosyl)-(1 4)-O–(3-O-benzyl-ꢀ-L-idopyranosyluronic
acid)-(1 4)-O-(2-azido-3-O-benzyl-2-deoxy-6-O-sulfo-ꢀ-D-
glucopyranosyl)-(1 4)-O–(3-O-benzyl-ꢀ-L-idopyranosyluronic
acid)-(1 4)-O-(2-azido-3-O-benzyl-2-deoxy-6-O-sulfo-ꢀ-D-
glucopyranosyl)-(1 4)-3-O-benzyl-ꢀ-L-idopyranosyluronic acid
hexasodium salt (28). To a solution of 27 (50 mg, 20.4 µmol) in
THF (8 mL) at Ϫ5 ЊC, 30% H2O2 (0.6 mL) and a 1.25 M
aqueous solution of LiOH (0.70 mL) were added. After stirring
for 24 h at room temperature, MeOH (1.1 mL) and a 3 M
aqueous solution of KOH (2 mL) were added. After stirring for
24 h more the reaction was neutralised with resin (IRA-120
IsopropylO-(2-azido-3,4-di-O-benzyl-2-deoxy-6-O-(p-methoxy-
benzyl)-ꢀ-D-glucopyranosyl)-(1 4)-O-(3-O-benzyl-2-O-
sulfo-ꢀ-L-idopyranosyluronic acid)-(1 4)-O-(2-azido-3-O-
benzyl-2-deoxy-6-O-(p-methoxybenzyl)-ꢀ-D-glucopyranosyl)-
(1 4)-O–(3-O-benzyl-2-O-sulfo-ꢀ-L-idopyranosyluronic acid)-
(1 4)-O-(2-azido-3-O-benzyl-2-deoxy-6-O-(p-methoxybenzyl]-
ꢀ-D-glucopyranosyl)-(1 4)-3-O-benzyl-2-O-sulfo-ꢀ-L-idopyrano-
syl) uronic acid hexasodium salt (31). A mixture of 30 (18.5 mg,
8.38 µmol) and SO3ؒPy complex (20 mg, 0.12 mmol) in dry Py
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 2 2 5 3 – 2 2 6 6
2264

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10 C. A. A. van Boeckel and M. Petitou, Angew. Chem., Int. Ed. Engl.,
(1.5 mL) was stirred under an argon atmosphere. After
stirring for 8 h, the mixture was cooled and MeOH (1 mL) and
CH2Cl2 (1 mL) were added. The solution was eluted through a
Sephadex LH-20 (1 : 1 MeOH–CH2Cl2). Fractions containing
the hexasaccharide were concentrated and passed through a
Dowex 50WX4-Naϩ (2 : 1 MeOH–H2O), to yield 31 (18 mg,
86%). TLC 0.44 (14 : 5 : 3:1 AcOEt–Py–H2O–AcOH).
1H-NMR (500 MHz, CD3OD): δ 7.40–7.09 (m, 41 H, Ph,
MeOPh); 6.81–6.77 (m, 6 H, MeOPh); 5.42 (d, 1 H, H1e); 5.38
(d, 1 H, H1c); 5.12 (d, 1 H, H1f); 5.17–5.14 (m, 3 H, H1b, H1d,
H1a); 3.72 (s, 6 H, CH3OPh); 3.65 (s, 3 H, CH3OPh); 1.21–1.17
(m, 6 H, CH(CH3)2).
1993, 32, 1671–1690.
11 J. L. de Paz, J. Angulo, J.-M. Lassaletta, P. M. Nieto, M. Redondo-
Horcajo, R. M. Lozano, G. Giménez-Gallego and M. Martín-
Lomas, ChemBioChem., 2001, 2, 673–685.
12 R. Ojeda, J. Angulo, P. M. Nieto and M. Martín-Lomas, Can. J.
Chem., 2002, 80, 917–936.
13 R. Ojeda, Ph D. Thesis, University of Seville, 2001.
14 G. Waksman and A. B. Herr, Nat. Struct. Biol., 1998, 5, 527–
530.
15 L. Lundin, H. Larsson, J. Kreuger, S. Kanda, U. Lindahl,
M. Salmivirta and L. Claesson-Welsh, J. Biol. Chem., 2000, 275,
24653–24660.
16 J. Kreuger, M. Salmivirta, L. Sturiale, G. Giménez-Gallego and
U. Lindahl, J. Biol. Chem., 2001, 276, 30744–30752.
17 A. K. Powell, D. G. Fernig and J. E. Turnbull, J. Biol. Chem., 2002,
277, 28554–28562.
IsopropylO-(2-deoxy-2-sulfamido-ꢀ-D-glucopyranosyl)-
(1 4)-O-(2-O-sulfo-ꢀ-L-idopyranosyluronic acid)-(1 4)-O-(2-
deoxy-2-sulfamido-ꢀ-D-glucopyranosyl)-(1 4)-O-(2-O-sulfo-ꢀ-
L-idopyranosyluronic acid)-(1 4)-O-(2-deoxy-2-sulfamido-ꢀ-D-
glucopyranosyl)-(1 4)-2-O-sulfo-ꢀ-L-idopyranosyl) uronic acid
nonasodium salt (6). A solution of 31 (18 mg, 7.16 µmol) in
MeOH–H2O (1.5 mL, 9 : 1) was hydrogenated in the presence
of 10% Pd/C. After 24 h, the suspension was filtered and con-
centrated to give 32 which was homogenous on TLC (6 : 5 : 3 : 1
EtOAc–Py–H2O–AcOH. This compound was directly submit-
ted to N-sulfation. The hydrogenated hexasaccharide was dis-
solved in H2O (1 mL) and the pH of the solution was adjusted
to 9.5 with a 1 M solution of NaOH. A pyridine–sulfur trioxide
complex (24 mg, 0.145 mmol, 10 eq. for each amine group) was
added in portions during 1 h and the pH was maintained at 9.5.
Subsequent additions of the pyridine–sulfur trioxide complex
were made after stirring for 2, 4, and 6 h, respectively. After
24 h, the mixture was neutralised with 0.1 M solution of HCl
and then subjected to chromatography over a Sephadex G-25
column with 0.9% solution of NaCl. The appropriate fractions
were pooled and passed through a column of Dowex 50WX4-
Naϩ (9 × 1.2 cm) with a 0.5 M solution of NaCl and then a
column of Sephadex G-25 with H2O–EtOH (9 : 1). The frac-
tions, which contained the final hexasaccharide, were lyophil-
18 J. Angulo, J.-L. de Paz, P. M. Nieto and M. Martín-Lomas, Isr. J.
Chem., 2000, 40, 289–299.
19 F. Chevalier, J. Angulo, R. Lucas, P. M. Nieto and M. Martín-
Lomas, Eur. J. Org. Chem., 2002, 2367–2376.
20 R. R. Schmidt and W. Kinzy, Adv. Carbohydr. Chem. Biochem.,
1994, 50, 21–123.
21 R. Ojeda, J.-L. de Paz, M. Martín-Lomas and J.-M. Lassaletta,
Synlett, 1999, 8, 1316–1318.
22 A. Lubineau, O. Gavard, J. Alais and D. Bonnaffé, Tetrahedron
Lett., 2000, 41, 307–311.
23 M. Martín-Lomas, M. Flores-Mosquera and J. L. Chiara, Eur. J.
Org. Chem., 2000, 1547–1562.
24 A. Vasella, C. Witzig, J. L. Chiara and M. Martín-Lomas,
Helv. Chim. Acta, 1991, 74, 2073–2077.
25 S. Y. Lou, S. R. Thopate, C. Y. Hsu and S. C. Hung, Tetrahedron
Lett., 2002, 43, 4889–4992.
26 K. C. Nicolaou, R. A. Daines and T. K. Chakraborty, J. Am. Chem.
Soc., 1987, 109, 2208–2210.
27 K. C. Nicolaou, C. A. Veale, C. K. Hwang, J. Hutchison, C. V. C.
Prasad and W. W. Olgiwie, Angew. Chem., Int., Ed. Engl., 1991, 30,
299.
28 R. Johansson and B. Samuelsson, J. Chem. Soc., Perkin Trans. 1,
1984, 2371–2374.
29 M. Oikawa, W. C. Liu, Y. Nakay, S. Koshida, K. Fukase and
S. Kusumoto, Synlett, 1996, 1179–1180.
30 P. J. Kociénski, in Protecting Groups, Thieme, Stuttgart-New York,
1994.
31 Y. Oikawa, T. Yoshioka and O. Yonemitsu, Tetrahedron Lett., 1982,
885–892.
32 M. Petitou, G. Jaurand, M. Derrien and J. Choay, Biorg. Med.
Chem. Lett., 1991, 1, 95–98.
33 J. Kiss, Adv. Carbohydr. Chem., 1974, 29, 229–303.
34 P. D. J. Grootenhuis and C. A. A. van Boeckel, J. Am. Chem. Soc.,
1991, 113, 2743–2747.
1
ized to give 6 (9.1 mg, 73% from 30). H-NMR (500 MHz,
D2O): δ 5.18–5.05 (d, 1 H, J1,2 = 3.5 Hz, H1b); 5.28 (m, 2 H, H1d,
H1f); 5.26–5.22 (m, 3 H, H1e, H1c, H1a); 4.85 (br s, 2 H, H5c, H5e);
4.51 (d, 1 H, J5,4 = 2.5 Hz, H5a); 3.44 (t, 1 H, J4,3 = J4,5 = 9.5 Hz,
H4f); 2.84–2.80 (m, 3 H, H2b, H2d, H2f); 1.20–1.16 (m, 6 H,
CH(CH3)2).
35 N. Sakairi, J. E. M. Basten, G. A. van der Marel, C. A. A. van
Boeckel and J. H. van Boom, Chem. Eur. J., 1997, 2, 1007–
1013.
36 M. Hricovini, P. M. Nieto and G. Torri in Spectroscopy of
Glycoconjugates, eds. J. Jiménez-Barbero and T. Peters, Wiley-VCH,
2003, pp. 189–229.
37 E. A. Yates, F. Santini, B. De Cristofano, N. Payre, C. Cosentino,
M. Guerrini, A. Naggi, G. Torri and M. Hricovini, Carbohydr. Res.,
2000, 329, 239–247.
Acknowledgements
We thank DGICYT (grant PB96–0820) for financial support
and the NMR facilities of the Universidad de Barcelona for the
800 MHz NMR experiments. R.L. and J.A. thank CSIC and
Fundación Ramón Areces respectively for fellowships.
38 D. R. Ferro, A. Provasoli, M. Ragazzi, B. Casu, G. Torri,
V. Bossennec, B. Perly, P. Sïnay, M. Petitou and J. Choay, Carbohydr.
Res., 1990, 195, 157–1167.
39 C. A. A. A. van Boeckel, S. F. Van Aelst, G. N. Wagenhaars,
G. R. Mellema, H. Paulsen, T. Peters, A. Pollex and V. Sinnwell,
Rec. Trav. Chim. Pays-Bas, 1987, 106, 19–29.
40 B. Mulloy, M. J. Forster, C. Jones and D. B. Davis, Biochem. J., 1993,
293, 849–858.
41 D. Mikhailov, K. H. Mayo, I. R. Vlahov, T. Toida, A. Pervin and
R. J. Lindhardt, Biochem. J., 1996, 318, 93–102.
42 D. Mikhailov, R. J. Lindhardt and K. H. Mayo, Biochem. J., 1997,
328, 51–61.
43 M. Hricovini and G. Torri, Carbohydr. Res., 1995, 268, 159–175.
44 G. Wildman in NMR Spectroscopy of Glycoconjugates,
eds. J. Jiménez-Barbero and T. Peters, Wiley-VCH, Weinheim, 2003,
pp 3–22.
45 S. J. Weiner, P. A. Kollman, D. T. Nguyen and D. A. Case, J. Comput.
Chem., 1986, 7, 230–252.
46 R. J. Woods, R. A. Dwek, C. J. Edge and B. Frasier-Reid, J. Phys
Chem., 1995, 99, 3832–3846.
47 C. Altona and C. J. M. J. Huige, J. Comput. Chem., 1995, 16,
56–79.
References
1 T. Nishimura, Y. Nakataye, M. Konishi and N. Itoh, Biochem.
Biophys. Acta, 2000, 1492, 203–206.
2 S. Faham, R. J. Lindhardt and D. C. Rees, Curr. Opin. Struct. Biol.,
1998, 8, 578–586.
3 H. E. Conrad in Heparin Binding Proteins, Academic Press, San
Diego, 1998.
4 I. Capila and R. J. Lindhardt, Angew. Chem., Int. Ed., 2002, 41, 390–
412.
5 B. Casu and U. Lindahl, Adv. Carbohydr. Chem. Biochem., 2001, 57,
159–206.
6 B. Mulloy and M. J. Forster, Glycobiology, 2000, 10, 1147–1156.
7 D. R. Ferro, A. Provasoli, M. Ragazzi, G. Torri, B. Casu, G. Gatti,
J.-C. Jaquinet, P. Sinaÿ and M. Petitou, J. Am. Chem. Soc., 1986,
108, 6773–6778.
8 B. Casu, M. Petitou, A. Provasoli and P. Sinaÿ, Trends Biochem.
Sci., 1988, 13, 221–225.
9 J. Tamura, Trends Glycosci. Glycotechnol., 2001, 13, 65–88.
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 2 2 5 3 – 2 2 6 6
2265

View Article Online
48 A. L. Davis, E. D. Laue, J. Keeler, D. Moskau and J. Lohman,
J. Magn. Reson., 1991, 94, 637.
49 A. A. Show, C. Salaun, J. F. Dauphin and B. Ancian, J. Magn.
Reson. Ser., A., 1996, 120, 110–115.
50 A. Bax and D. G. Davis, J. Magn. Reson., 1985, 65, 355–366.
51 D. Neuhaus and M. Williamson, The Nuclear Overhauser
Effect in Structural and Conformational Analysis, VCH, New York,
1989.
52 J. Schleucher, M. Schwendinger, M. Sattler, P. Scmidt,
O. Schedletzky, S. L. Glaser, O. W. Sorensen and C. Griesenger,
J. Biomol. NMR, 1994, 4, 301–306.
53 D. A. Case, D. A. Pearlman, J. T. Cadwell, T. E. Cheatham III,
W. S. Ross, C. L. Simmerling, T. A. Darden, K. M. Merz, R. V.
Staton, A. L. Cheng, J. J. Vincent, M. Crowley, D. M. Ferguson,
R. J. Radmer, G. L. Seibel, U. C. Singh, P. K. Weiner and P. A.
Kollman, AMBER 5, University of California, San Francisco, 1997.
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 2 2 5 3 – 2 2 6 6
2266