Synthesis of variously sulfated biotinylated oligosaccharides from the linkage region of proteoglycans

Article abstract of DOI:10.1016/j.carres.2012.03.039

The synthesis of a collection, as biotinylated conjugates, of various sulfoforms of the trisaccharide β-d-GlcpA-(1→3)-β-d-Galp- (1→3)-β-d-Galp, structures encountered in the linkage region of proteoglycans, is reported herein for the first time. An effici

Full text of DOI:10.1016/j.carres.2012.03.039

Carbohydrate Research 353 (2012) 33–48
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Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Synthesis of variously sulfated biotinylated oligosaccharides from the
linkage region of proteoglycans
⇑
Katia Aït-Mohand, Chrystel Lopin-Bon, Jean-Claude Jacquinet
Institut de Chimie Organique et Analytique, UMR 7311 CNRS et Université d’Orléans, Pôle de Chimie, Université d’Orléans, BP 6759, 45067 Orléans Cedex 2, France
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis of a collection, as biotinylated conjugates, of various sulfoforms of the trisaccharide b-D-
Received 15 February 2012
Received in revised form 27 March 2012
Accepted 30 March 2012
Available online 6 April 2012
GlcpA-(1?3)-b-D-Galp-(1?3)-b-D-Galp, structures encountered in the linkage region of proteoglycans,
is reported herein for the first time. An efficient and stereocontrolled preparation was achieved using
common key intermediates in a divergent manner. These molecules should be useful probes to study
the substrate specificity of the glycosyltransferases involved at the bifurcation point in the biosynthesis
of proteoglycans.
Keywords:
Oligosaccharides
Sulfoforms
Ó 2012 Elsevier Ltd. All rights reserved.
Proteoglycans
Biotinylated sugars
Linkage region
1. Introduction
biological significance of these unique substitutions is not yet fully
understood, though they could act as biosynthetic signals.4
Proteoglycans (PGs) are biologically important macromolecules
composed of glycosaminoglycan (GAG) chains covalently linked to
a core protein. They are widespread on the cell surface and in the
extracellular matrix, and are involved in many biological processes
and pathologies,1 thus having a great potential as pharmacological
targets. PG assembly starts in the Golgi apparatus with the attach-
Phosphorylation of the D-Xyl unit was demonstrated to be a
transient phenomenon involved in the early steps of the biosynthe-
sis.5,6 Specific sulfation at C-6 of GalI was found to strongly accel-
erate the transfer of the D
-GlcA unit.6 But the exact role of these
sulfate substituents at the bifurcation of the biosynthesis of PGs
has not yet been clarified.
ment of
stepwise addition of two
one -Glucuronic acid ( -GlcA) unit provides the so-called common
D
-Xylose to a
L
-serine residue within the protein core. The
To address this issue, several syntheses of various glycoconju-
gates of the linkage region containing or not sulfate and/or phos-
phate groups have been reported,7 but no clear-cut biological
results were obtained with these synthetic molecules. Since phos-
D-Galactose units (
D
-GalI and -GalII) then
D
D
D
tetrasaccharide intermediate.
At this point, a possible divergence in the biosynthetic pathway
phorylation or not on the D-Xyl unit apparently has no effect at the
may occur (Fig. 1).2 Transfer of an
a
-D
-GlcNAc residue at O-4 of the
-GlcA unit initiates the formation of
glucosaminoglycans (heparin, heparan sulfate), whereas those of
a b- -GalNAc residue lead to galactosaminoglycans (chondroitin
divergence level in the biosynthesis, and to shed light on the exact
role of the sulfate substituents, we now report for the first time a
systematic preparation of all possible sulfoforms of the truncated
terminal non-reducing
D
D
structure b-D-GlcpA-(1?3)-b-D-Galp-(1?3)-b-D-Galp conjugated
sulfate and dermatan sulfate). GAG chains consist of hexosamines
and uronic acids arranged in alternating sequences, and these
repeating units are variously substituted by sulfate groups creating
a great degree of structural and functional diversity. The current
hypothesis that the linkage region should be common to all GAG
species strongly contrasts with the structural heterogeneity of
the GAG region. However, it has been reported that this linkage
to biotin to serve as potential substrates for the hexosaminyl trans-
ferases involved at the bifurcation point in the biosynthesis of PGs.
2. Results and discussion
For the synthesis of the non-sulfated target molecule 1 and the
four possible sulfoforms 2–5 (Fig. 2), two common key trisaccha-
ride intermediates 6 and 7 were designed. Diol 6 may be a precur-
sor of 1 and of the two species 2 and 3 sulfated at C-4 and C-6 on
GalI, whereas diol 7 may lead to the two corresponding species 4
and 5 sulfated on GalII. These later may be in turn prepared by ster-
eoselective assembly of two donors 8 and 9, which differ in their
region may be modified by sulfation at C-4 or C-6 of both
D
-Gal
units, and/or by phosphorylation at C-2 of the
D
-Xyl unit.3 The
⇑
Corresponding author. Tel.: +33 238 417072; fax: +33 238 417281.
E-mail address: Jean-Claude.Jacquinet@univ-orleans.fr (J.-C. Jacquinet).
0008-6215/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.carres.2012.03.039

34
K. Aït-Mohand et al. / Carbohydrate Research 353 (2012) 33–48
OH
SO3Na
O
HO
HO
Heparin
Heparan sulfate
O
AcNH
HN
HO
OH
COONa
HO
OH
OH
O
O
O
O
O
?
HO
O
O
O
HO
O
O
HO
HO
HN
HO
OH
HO
HO
O
Chondroitin sulfate
Dermatan sulfate
AcNH
Figure 1. The linkage region of proteoglycans and the bifurcation in their biosynthesis. The arrows indicate possible substitutions with sulfate groups.
O
HN
H
NH
H
1
OR
2
3
4
R O
OR
R O
H
N
O
COONa
O
O
O
O
HO
HO
O
S
N
H
O
HO
O
HO
HO
1: R1 = R2 = R3 = R4 = H
2: R1 = SO3Na, R2 = R3 = R4 = H
3: R1 = R3 = R4 = H, R2 = SO3Na
1
2
4
3
4: R = R = R = H, R = SO3Na
5: R1 = R2 = R3 = H, R4 = SO3Na
OH
OBz
OBz
OH
HO
BzO
BzO
HO
COOMe
COOMe
O
O
O
O
O
O
O
O
LevO
BzO
LevO
BzO
O
O
NHZ
NHZ
O
O
BzO
BzO
BzO
BzO
BzO
BzO
7
6
Si
O
O
6
4
OR
R O
COOMe
O
O
BzO
O
LevO
BzO
O
O
HO
NHZ
+
BzO
BzO
O
CCl3
10
8: R4 = R6 = Bz
9: R4 = R6 = ClAc
NH
Si
O
O
COOMe
O
O
LevO
BzO
OMP
HO
+
BzO
BzO
O
CCl3
12
11
NH
Figure 2. Retrosynthesis for target molecules 1–5.
substitution pattern at C-4 and C-6 of the
D
-Gal unit, with acceptor
all glycosidic linkages would rely on neighboring group assistance
by benzoyl groups, which serve also as permanent protective
groups. All glycosylation reactions will be performed using
Schmidt’s trichloroacetimidate procedure.8 As a prelude for further
10 still possessing a masked amine linker to allow further coupling
with biotin. These two donors may be obtained by stereoselective
coupling of donor 11 and acceptor 12. The b-selectivity in forming

K. Aït-Mohand et al. / Carbohydrate Research 353 (2012) 33–48
35
6
4
COOMe
O
OR
R O
COOMe
b
COOMe
LevO
BzO
O
O
O
a
RO
BzO
LevO
BzO
OMP
OMP
BzO
O
11
12
+
BzO
O
CCl3
BzO
BzO
11
13 : R = H
NH
a
4
6
18 : R , R = tBu2Si
14 : R = Lev
b
19 : R4 = R6 = H
20: R4 = R6 = Bz
21 : R4 = R6 = ClAc
Scheme 1. Reagents and conditions: (a) LevOH, DCC, DMAP, CH2Cl2, rt, 1 h; (b) CAN,
toluene/MeCN/H2O, rt, 30 min; then Cl3CCN, DBU, CH2Cl2, rt, 30 min.
c
d
possible chain elongation at C-4 of the D-GlcA moiety, an orthogo-
nal levulinoyl group was installed on this position from the begin-
ning of the synthesis.
e
Preparation of the
D-glucuronyl donor 11 was achieved as
follows (Scheme 1). Known alcohol 13,9 easily prepared in multi-
gram quantities by tin-mediated selective dibenzoylation of the
corresponding triol, was treated with levulinic acid, 1,3-dicyclo-
hexylcarbodiimide (DCC), and 4-dimethylaminopyridine (DMAP)
in dichloromethane to give 14 in 91% yield. Introduction of the
trichloroacetimidoyl group at C-1 was then achieved through
oxidative removal of the 4-methoxyphenyl group with Cerium(IV)
diammonium hexanitrate (CAN) followed by imidoylation of the
intermediate hemiacetal with trichloroacetonitrile and 1,8-diaza-
bicyclo[5,4,0]-undec-7-ene (DBU) in dichloromethane to afford
6
OR
4
R O
COOMe
O
O
LevO
BzO
O
BzO
BzO
O
CCl3
NH
8: R4 = R6 = Bz
the
a-imidate 11 in 74% overall yield, for which physical data
4
6
9: R = R = ClAc
matched those reported for the preparation of 11 following
another route.10
Scheme 3. Reagents and conditions: (a) Compound 11 (1.3 equiv), TMSOTf,
4 Å mol. sieves, CH2Cl2, rt, 40 min; (b) Et3Nꢀ3HF, THF, 0 °C, 3 h; (c) PhCOCl,
pyridine/CH2Cl2, 0 °C, 2 h; (d) (ClAc)2O, pyridine/CH2Cl2, 0 °C, 1 h; (e) CAN,
toluene/MeCN/H2O, rt, 30 min; then Cl3CCN, DBU, CH2Cl2, rt, 30 min.
The precursor of the central
D
-GalII unit 12 was prepared as
-GalI moiety was
reported previously,11 and the reducing
D
synthesized following a similar route (Scheme 2). Coupling reac-
tion of known12 2,3,4,6-tetra-O-acetyl-1-O-trichloroacetimidoyl-
a-D-galactopyranose 15 with 2-benzyloxycarbonylaminoethanol
convenience of the silylene acetal in oligosaccharide synthesis
compared to its 4,6-benzylidene congener was previously
demonstrated.14
Preparation of the activated disaccharide derivatives 8 and 9
was then achieved as follows (Scheme 3). Condensation of imidate
11 (1.3 equiv) with alcohol 12 in dichloromethane in the presence
of TMSOTf gave 18 (85%). The 1,2-trans configuration of the inter-
glycosidic linkage was deduced from its 1H NMR spectrum (J1,2
8.0 Hz, GlcA H-1). Treatment of 18 with triethylamine–trihydroflu-
oride complex in tetrahydrofuran afforded the crystalline diol 19 in
in the presence of trimethylsilyl triflate (TMSOTf) in dichlorometh-
ane gave 16 in 91% yield. Zemplèn transesterification of 16 gave
quantitatively the corresponding tetrol, which was directly treated
with 2,2-dimethoxypropane and 10-camphorsulfonic acid (CSA)
under thermodynamic control13 to give the intermediate 3,4,6-tri-
substituted bis-acetal that was benzoylated to afford the crystal-
line ester 17 in 76% overall yield. Acid hydrolysis of 17 gave the
corresponding triol derivative which was treated with di-tert-
butylsilyl ditriflate (DTBS ditriflate) and sym-collidine in
dichloromethane to provide alcohol 10 in 90% overall yield. The
OAc
AcO
OAc
AcO
a
O
O
O
AcO
AcO
NHZ
AcO
AcO
O
CCl3
15
16
NH
b, c, d
Si
O
O
OMe
O
O
e, f
O
O
O
O
O
NHZ
HO
NHZ
BzO
BzO
17
10
Scheme 2. Reagents and conditions: (a) 2-Benzyloxycarbonylaminoethanol, TMSOTf, CH2Cl2, 0 °C, 30 min; (b) MeONa, MeOH, rt, 1 h; (c) 2,2-dimethoxypropane (neat), CSA,
rt, 60 h; (d) PhCOCl, pyridine/CH2Cl2, 0 °C, 1 h; (e) 90% TFA, rt, 10 min; (f) DTBS ditriflate, sym-collidine, CH2Cl2, 0 °C, 90 min.

36
K. Aït-Mohand et al. / Carbohydrate Research 353 (2012) 33–48
D
d ꢁ0.4 ppm of the signals for GalI H-6a, 6b in 24 compared with
10
8
+
those of its non-sulfated precursor 6, and the presence of a telling
signal at d 5.57 ppm (GalI H-4). Sulfation of the low-reactive alco-
hol 23 required excess of reagent and higher temperature to go to
completion, and the sodium salt 25 was isolated in 83% yield. Com-
parison of the 1H NMR spectra of 25 and those of its precursor 23
a
6
OR
4
R O
OBz
BzO
showed the expected16 downfield shift
D
d ꢁ1 ppm of the signal for
COOMe
O
O
O
GalI H-4 in 25.
O
LevO
BzO
O
NHZ
O
BzO
The preparation of species selectively O-sulfonated at C-4 and
C-6 of GalII unexpectedly was more problematical (Scheme 5).
Coupling reaction between imidate 9 and alcohol 10 (1.5 equiv),
as reported for the preparation of 18, gave trisaccharide 26 in
49% yield, along with ꢁ15% of a corresponding orthoester deriva-
tive that could not be rearranged into 26 by addition of more
catalyst. Desilylation of 26 afforded readily the crystalline diol 27
in 81% yield, that was fully resistant to benzoylation or acetylation.
Changes of acylating reagent, catalyst, or temperature did not
allow the obtention of 35 in acceptable yields. These frustrating
results, probably due to a dramatic change of the conformation
of 27 in solution, prompt us to design a new strategy for the
preparation of the key intermediate 7. To achieve this goal, two
possible routes were explored. The first one involved a change in
the protective groups pattern in the acceptor (Scheme 6). To this
end, alcohol 10 was transformed into alcohol 30 through levuli-
noylation at O-3 to give 28, followed by desilylation and benzoyl-
ation to afford 29, and finally delevulinoylation with hydrazine
acetate to provide alcohol 30 in excellent overall yield. Coupling
BzO
6
BzO
4
22 : R , R = tBu2Si
b
4
6
6 : R = R = H
c
4
6
23 : R = H, R = Bz
d
4
6
24 : R = Ac, R = SO3Na
e
4
6
25 : R = SO3Na, R = Bz
Scheme 4. Reagents and conditions: (a) Compound 10 (1.5 equiv), TMSOTf,
4 Å mol. sieves, CH2Cl2, rt, 40 min; (b) Et3Nꢀ3HF, THF, 0 °C, 3 h; (c) PhCOCN,
pyridine, rt, 16 h; (d) Me3NꢀSO3, DMF, 40 °C, 10 h; then Ac2O, pyridine, rt, 16 h; (e)
Me3NꢀSO3, DMF, 60 °C, 16 h.
83% yield. At this point, diol 19 can either be benzoylated to give
crystalline 20 in 86% yield, the precursor of species non-sulfated
on GalII, or chlororacetylated to give crystalline 21 in 90% yield,
those of species sulfated on GalII. Disaccharide derivatives 20 and
21 were transformed, as reported for the preparation of 11, into
reactions between disaccharide imidate
9 and alcohol 30
(1.5 equiv), as reported above, afforded the crystalline trisaccha-
ride derivative 35 in 48% yield, the 1H NMR spectrum of which
was in full agreement with the expected structure. The second
route involved the preparation of the trisaccharide imidate 34
and coupling with the spacer (Scheme 7). Thus, coupling reaction
between imidate 9 and alcohol 12 (1.5 equiv) gave trisaccharide
31 in 61% yield, the structure of which was deduced from its 1H
NMR spectrum. Desilylation of 31 gave diol 32 in 88% yield, that
was benzoylated to afford the crystalline ester 33 in 83% yield.
Derivative 33 was transformed, as reported for the preparation of
the corresponding a-imidates 8 and 9.
Next was undertaken the preparation of intermediates selec-
tively O-sulfonated at C-4 and C-6 on GalI (Scheme 4). Coupling
reaction between donor 8 and acceptor 10 (1.5 equiv), as described
for the preparation of 18, proceeded readily and afforded 22 in 67%
yield, the structure of which was deduced from its 1H NMR spec-
trum (J1,2 8.0 Hz, GalII H-1). Desilylation of 22, as reported for the
preparation of 19, gave the crystalline diol 6 in 82% yield, the key
intermediate for the preparation of target molecules 1–3. Treat-
ment of 6 with benzoyl cyanide in pyridine afforded regioselec-
tively the crystalline 6-benzoate 23 in 77% yield, the precursor of
the 4-sulfated species. Controlled regioselective 6-O-sulfonation
of diol 6 with sulfur trioxide–trimethylamine complex in N,N-
dimethylformamide (DMF) at 40 °C,15 followed by direct acetyla-
tion and ion-exchange, gave the sodium salt 24 in 71% yield. The
1H NMR spectrum of 24 showed the expected16 downfield shift
11, into
a-imidate 34 in 38% overall yield. Coupling reaction
between trisaccharide imidate 34 and 2-benzyloxycarbonylamino-
ethanol, as reported above, provided 35 in 56% yield, whose
Si
O
O
a
O
O
10
LevO
NHZ
BzO
28
10
9
+
b
a
6
OR
4
OBz
R O
OAcCl
ClAcO
BzO
COOMe
O
O
O
O
O
LevO
BzO
O
O
NHZ
O
3
R O
NHZ
BzO
BzO
BzO
3
BzO
29 : R = Lev
26 : R4, R6 = tBu2Si
c
b
3
30 : R = H
27 : R4 = R6 = H
35
Scheme 6. Reagents and conditions: (a) LevOH, DCC, DMAP, CH2Cl2, rt, 1 h; (b)
Et3Nꢀ3HF, THF, 0 °C, 3 h; then PhCOCl, pyridine/CH2Cl2, 0 °C, 1 h; (c) hydrazine
acetate, pyridine, rt, 8 min.
Scheme 5. Reagents and conditions: (a) Compound 10 (1.5 equiv), TMSOTf,
4 Å mol. sieves, CH2Cl2, rt, 40 min; (b) Et3Nꢀ3HF, THF, 0 °C, 3 h.

K. Aït-Mohand et al. / Carbohydrate Research 353 (2012) 33–48
37
6
4
OR
R O
OAcCl
ClAcO
O
COOMe
O
a
O
O
R
LevO
BzO
9 + 12
O
BzO
BzO
BzO
1
R
1
4
6
31 : R = OMP, R = H, R ,R = tBu2Si
b
32 : R = OMP, R1 = H, R4 = R6 = H
c
1
4
6
33 : R = OMP, R = H, R = R = Bz
d
34 : R = H, R1 = OC(NH)CCl3, R4 = R6 = Bz
e
OBz
6
4
OR
R O
BzO
O
COOMe
f
O
O
O
O
LevO
9 + 30
NHZ
O
BzO
BzO
BzO
BzO
4
6
35 : R = R = ClAc
7 : R4 = R6 = H
g
h
4
6
i
36 : R = H, R = Bz
37 : R4 = Ac, R6 = SO3Na
j
4
6
38 : R = SO3Na, R = Bz
Scheme 7. Reagents and conditions: (a) Compound 12 (1.5 equiv), TMSOTf, 4 Å mol. sieves, CH2Cl2, rt, 40 min; (b) Et3Nꢀ3HF, THF, 0 °C, 3 h; (c) PhCOCl, pyridine/CH2Cl2, 0 °C,
1 h; (d) CAN, toluene/MeCN/H2O, rt, 30 min; then Cl3CCN, DBU, CH2Cl2, rt, 30 min; (e) 2-benzyloxycarbonylaminoethanol, TMSOTf, CH2Cl2, 0 °C, 30 min; (f) compound 30
(1.5 equiv), TMSOTf, 4 Å mol. sieves, CH2Cl2, rt, 40 min; (g) thiourea, pyridine/EtOH, 80 °C, 2 h; (h) PhCOCN, pyridine, rt, 16 h; (i) Me3NꢀSO3, DMF, 40 °C, 6 h; then Ac2O,
pyridine, rt, 16 h; (j) Me3NꢀSO3, DMF, 60 °C, 16 h.
physical data matched those of compound prepared by the first
route. Treatment of 35 with thiourea in pyridine/ethanol gave
the key diol 7 in 77% yield, which upon regioselective benzoylation
at O-6, as reported for the preparation of 23, afforded alcohol 36 in
80% yield. Controlled regioselective 6-O-sulfonation on 7, followed
by acetylation and ion-exchange, as reported for the preparation of
24, gave the sodium salt 37 in 76% yield, the 1H NMR spectrum of
which showed the same characteristic features as those reported
for 24. Sulfation of alcohol 36 at C-4 provided the sodium salt 38
in 77% yield, the 1H NMR spectrum of which was in full agreement
with the expected structure.
NMR spectra clearly showed the downfield shifts
the signals for Gal C-4 in 4-sulfated units, and those
for the signals of Gal C-6 in 6-sulfated units, compared with those
of the non-sulfated units.
D
d ꢁ9 ppm for
d ꢁ7 ppm
D
3. Conclusion
In conclusion, we have reported for the first time a stereo-
controlled approach toward the preparation of a set of sulfoforms
of biotinylated structures from the linkage region of proteoglycans.
Common precursors were used in a divergent manner which
allowed the obtention of target molecules with high purity. These
structures will be useful to check the substrate specificity of the
hexosaminyltransferases involved in the chain elongation and
sorting of proteoglycans. All these molecules are currently being
investigated in biological assays, the results of which will be
reported elsewhere in due course.
Access to the target molecules 1–5 was then achieved as follows
(Scheme 8). For the deprotections of diol 6 and sulfoforms 24, 25,
37, and 38, a two-step saponification process avoiding b-elimina-
tion reaction at the D
-GlcA moiety,17 with lithium hydroperoxide
followed by sodium hydroxide, provided the non-sulfated 39 in
69% yield, and the disodium salts 40–43 in 80–88% yields,
respectively. The 1H and 13C NMR spectra for 39–43 showed high
purity, and were fully consistent with the expected structures.
Finally, catalytic hydrogenation of 39–43 in water led to the corre-
sponding free amine derivatives, which were readily treated with
6-biotinylamidohexanoic acid N-hydroxysuccinimidoyl ester and
triethylamine in N,N-dimethylformamide/water to afford, after
purification on Sephadex LH-20 in water, the target molecules
1–5 in 60–79% yields. For all biotinylated molecules, the 13C
4. Experimental
4.1. General methods
Solvents were dried by standard methods, and molecular sieves
were activated prior to use by heating for 4 h at 500 °C. Melting
points were determined in capillary tubes with a Büchi apparatus

38
K. Aït-Mohand et al. / Carbohydrate Research 353 (2012) 33–48
6
OR
4
R O
OH
HO
O
COONa
O
O
O
O
O
HO
HO
NHZ
HO
HO
HO
39 : R4 = R6 = H
1
2
3
6
24
25
b
b
a
4
6
40 : R = H, R = SO3Na
41 : R4 = SO3Na, R6 = H
OH
6
4
OR
R O
HO
O
COONa
O
O
O
O
HO
HO
NHZ
O
HO
HO
HO
4
6
4
5
42 : R = H, R = SO3Na
a
37
38
43 : R4 = SO3Na, R6 = H
Scheme 8. Reagents and conditions: (a) LiOH/H2O2, THF/H2O, ꢄ10 °C ? rt, 8 h; then 4 M NaOH, rt, 16 h; (b) H2, Pd/C, H2O, rt, 16 h; then 6-biotinylamidohexanoic acid
N-hydroxysuccinimidoyl ester, Et3N, DMF/H2O, rt, 1 h.
and are uncorrected. Optical rotations were measured at 20 °C with
a Perkin–Elmer 341 polarimeter. 1H and 13C NMR spectra were re-
corded at 25 °C with a Bruker Advance II 400 instrument, with
Me4Si as internal standard, unless otherwise stated. Assignments
were based on homo- and heteronuclear correlations using the
supplier’s software. In the NMR data for oligomers, GalI (with ro-
4H, CH2CO), 2.08 (s, 3H, COCH3); ISMS: m/z 643 [M+Na]+. Anal.
Calcd for C33H32O12: C, 63.87; H, 5.35. Found: C, 63.71; H, 5.40.
4.3. Methyl 2,3-di-O-benzoyl-4-O-levulinoyl-1-O-
trichloroacetimidoyl-a-D-glucopyranuronate (11)
man numeral superscript) refer to ‘reducing’ D-galactose residue,
A mixture of 14 (2.92 g, 4.7 mmol) and CAN (13.7 g, 25 mmol) in
1:1.5:1 toluene/MeCN/H2O (140 mL) was stirred for 30 min at rt,
then was poured into ice-cold water (300 mL) and extracted with
EtOAc (3 ꢃ 50 mL). The organic layer was washed with brine, and
water, dried (MgSO4), and concentrated. Flash silica chromatogra-
phy (5:4 EtOAc/petroleum ether) gave the corresponding hemiace-
tal as a yellow foam. A mixture of the hemiacetal, Cl3CCN (4.5 mL,
and GalII to non-reducing residue, respectively. Low-resolution
mass spectra (ISMS) were obtained on a Perkin–Elmer SCIEX API
300 spectrometer operating in the ion-spray mode or on a Micro-
mass Quattro Ultima spectrometer equipped with a Z-spray ioniza-
tion source operating in the negative mode. High-resolution mass
spectra (HRESIMS) were performed on a Bruker maXis mass spec-
trometer by the ‘Fédération de Recherche ICOA/CBM (FR 2708)
platform. Flash-silica chromatography was performed on Silica
Gel 60 (0.040–0.063 mm, Merck, Darmstadt). The reactions were
monitored by TLC on coated aluminium sheets (Silica Gel 60
GF254, Merck), and spots were detected under UV light and by char-
ring with a 95/5 mixture of ethanol and sulfuric acid. Elemental
analyses were carried out at the Service Central d’Analyse du CNRS
(Institut des Sciences Analytique, Solaize, France).
45 mmol), and DBU (165 lL, 1.1 mmol) in anhyd CH2Cl2 (20 mL)
was stirred for 30 min at rt, then was concentrated. Flash silica
chromatography (3:2 petroleum ether/EtOAc, containing 0.1% of
Et3N) gave imidate 11 (2.28 g, 74%) as a white foam: ½a D20
ꢂ
+120 (c
1, CHCl3), lit.10 +113.3, 1H NMR spectral data matched that
reported.
4.4. 2-Benzyloxycarbonylaminoethyl 2,3,4,6-tetra-O-acetyl-b-D-
galactopyranoside (16)
4.2. Methyl (4-methoxyphenyl 2,3-di-O-benzoyl-4-O-levulinoyl-
b-
D
-glucopyranosid)uronate (14)
A mixture of imidate 1512 (4.93 g, 10 mmol), 2-benzyloxycar-
bonylaminoethanol (3.12 g, 16 mmol) and powdered 4 Å molecular
sieves (1 g) in anhyd CH2Cl2 (50 mL) was stirred at 0 °C under dry
Ar. A solution of Me3SiOTf in anhyd toluene (1 M, 1.6 mL) was
added, and the mixture was stirred for 30 min at 0 °C. Triethyl-
amine (1 mL) was added, and the mixture was filtered, and concen-
trated. Flash silica chromatography (1:1 petroleum ether/EtOAc,
containing 0.1% of Et3N) gave 16 (4.79 g, 91%) as a white foam:
A mixture of methyl (4-methoxyphenyl 2,3-di-O-benzoyl-b-D-
glucopyranosid)uronate 139 (3.13 g, 6 mmol), levulinic acid
(0.88 g, 7.5 mmol) and DMAP (122 mg, 1 mmol) in anhyd CH2Cl2
(40 mL) was treated in portions with DCC (1.55 g, 7.5 mmol), and
the mixture was stirred for 1 h at rt. The precipitated DCU was fil-
tered off, washed with CH2Cl2, and the filtrate was washed with
cold 0.1 M HCl, satd aq NaHCO3, and water, dried (MgSO4), and
concentrated. The solid residue was recrystallized from hot ace-
tone to give 14 (3.39 g, 91%) as a white cotton: mp 169–170 °C;
½
a 2D0
ꢂ
ꢄ3 (c 1, CHCl3); 1H NMR (CDCl3, 400 MHz): d 7.40–7.30 (m,
5H, Ar-H), 5.38 (dd, 1H, J3,4 3.5, J4,5 <1 Hz, H-4), 5.17 (dd, 1H, J1,2
8.0, J2,3 11.0 Hz, H-2), 5.15 (br s, 1H, NH), 5.10 (s, 2H, CH2-Ar),
4.99 (dd, 1H, H-3), 4.45 (d, 1H, H-1), 4.15–4.10 (m, 2H, H-6a,6b),
3.91–3.87 (m, 2H, H-5, OCH2), 3.70–3.66 (m, 1H, OCH2), 3.42–
3.38 (m, 2H, CH2N), 2.16, 2.10, 2.08, 2.07 (4s, 12H, OCOCH3); ISMS:
½
a 2D0 +58 (c 1, CHCl3); 1H NMR (CDCl3, 400 MHz): d 8.0–6.80 (m,
14H, Ar-H), 5.76 (dd, 1H, J2,3 = J3,4 = 9.0 Hz, H-3), 5.68 (dd, 1H, J1,2
7.0 Hz, H-2), 5.58 (dd, 1H, J4,5 9.5 Hz, H-4), 5.23 (d, 1H, H-1), 4.32
(d, 1H, H-5), 3.78, 3.76 (2s, 6H, OCH3, COOCH3), 2.70–2.40 (m,
ꢂ

K. Aït-Mohand et al. / Carbohydrate Research 353 (2012) 33–48
39
(0.14 mL) was added, and the mixture was filtered, and concen-
trated. Flash silica chromatography (2:1 petroleum ether/EtOAc,
containing 0.1% of Et3N) gave 18 (430 mg, 85%) as a white foam:
m/z 548 [M+Na]+. Anal. Calcd for C24H31NO12: C, 54.85; H, 5.95; N,
2.66. Found: C, 54.73; H, 5.99; N, 2.49.
½
a 2D0 +66.5 (c 1, CHCl3); 1H NMR (CDCl3, 400 MHz): d 7.85–6.67
ꢂ
4.5. 2-Benzyloxycarbonylaminoethyl 2-O-benzoyl-3,4-O-
(m, 19H, Ar-H), 5.76 (dd, 1H, J1,2 8.0, J2,3 10.0 Hz, Gal H-2), 5.55
(dd, 1H, J2,3 = J3,4 = 9.0 Hz, GlcA H-3), 5.49–5.42 (m, 2H, GlcA H-
2,4), 5.08 (d, 1H, J1,2 8.0 Hz, GlcA H-1), 4.87 (d, 1H, Gal H-1), 4.72
(dd, 1H, J3,4 3.0, J4,5 <1 Hz, Gal H-4), 4.29–4.27 (m, 2H, Gal H-
6a,6b), 4.15 (d, 1H, J4,5 10.0 Hz, GlcA H-5), 3.99 (dd, 1H, Gal H-3),
3.77 (s, 3H, COOCH3), 3.70 (s, 3H, OCH3), 3.53 (s, 1H, Gal H-5),
2.63–2.46 (m, 3H, CH2CO), 2.39–2.32 (m, 1H, CH2CO), 2.04 (s, 3H,
COCH3), 1.08, 1.07 (2s, 18H, (CH3)3C); 13C NMR (CDCl3, 100 MHz):
d 205.60 (1C, COCH3), 171.02, 167.00, 165.53, 164.67, 164.61 (5C,
GlcA C-6, C@O), 155.49-114.22 (24C, Ar-C), 101.1 (1C, Gal C-1),
101.36 (1C, GlcA C-1), 79.46 (1C, Gal C-3), 72.84 (1C, GlcA C-5),
72.62 (1C, Gal C-4), 72.35 (1C, GlcA C-3), 71.68 (1C, GlcA C-2),
71.52 (1C, Gal C-5), 70.66 (1C, Gal C-2), 69.61 (1C, GlcA C-4),
66.87 (1C, Gal C-6), 55.51 (1C, OCH3), 52.96 (1C, COOCH3), 37.58
(1C, CH2CO), 29.52 (1C, COCH3), 27.64 (1C, CH2CO), 27.43, 27.38,
27.28, 27.20, 23.34, 20.70 (6C, (CH3)3C); HRESIMS: Calcd for
isopropylidene-6-O-(2-methoxy-2-propyl)-b-D-
galactopyranoside (17)
A solution of 16 (4.20 g, 8 mmol) in MeOH (30 mL) was treated
for 1 h with methanolic sodium methoxide (1 M, 1 mL), then was
deionized with Amberlite IR-120 [H+] resin, filtered, and concen-
trated to give quantitatively the corresponding tetrol. A mixture
of the tetrol and CSA (120 mg) in 2,2-dimethoxypropane
(120 mL) was stirred for 60 h at rt. Triethylamine (2 mL) was
added, and the mixture was concentrated. Flash silica chromatog-
raphy (3:1 EtOAc/petroleum ether, containing 0.2% of Et3N) gave
the corresponding bis-acetal (3.08 g, 82%) as a colorless glass. Ben-
zoyl chloride (1.56 mL, 13 mmol) was added dropwise at 0 °C to a
solution of the above isolated alcohol in anhyd pyridine (7 mL) and
anhyd CH2Cl2 (20 mL), and the mixture was stirred for 1 h at 0 °C.
Methanol (1 mL) was added, and the mixture was diluted with
CH2Cl2 (100 mL), washed with water, satd aq NaHCO3, and water,
dried (MgSO4), and concentrated. Crystallization of the residue
from hot petroleum ether gave 17 (3.49 g, 76% from 16): mp
C
54H63O18Si [M+H]+: m/z 1027.37782. Found: 1027.37759. Anal.
Calcd for C54H62O18Si: C, 63.14; H, 6.08. Found: C, 63.24; H, 6.60.
4.8. 4-Methoxyphenyl O-(methyl 2,3-di-O-benzoyl-4-O-
117–118 °C; ½a 2D0
ꢂ
+22 (c 1, CHCl3); 1H NMR (CDCl3, 400 MHz): d
levulinoyl-b-D-glucopyranosyluronate)-(1?3)-2-O-benzoyl-b-D-
8.10–7.20 (m, 10H, Ar-H), 5.51 (br s, 1H, NH), 5.21 (dd, 1H, J1,2
8.0, J2,3 7.0 Hz, H-2), 5.02 (ABq, 2H, CH2-Ar), 4.47 (d, 1H, H-1),
4.32 (dd, 1H, J3,4 5.5 Hz, H-3), 4.23 (dd, 1H, J4,5 2.0 Hz, H-4), 3.96–
3.94 (m, 1H, H-5), 3.87–3.83 (m, 1H, OCH2), 3.80–3.70 (m, 3H, H-
6a,6b, OCH2), 3.40–3.30 (m, 2H, CH2N), 3.19 (s, 3H, OCH3), 1.62,
1.35, 1.31 (3s, 12H, (CH3)2C); ISMS: m/z 596 [M+Na]+. Anal. Calcd
for C30H39NO10: C, 62.81; H, 6.85; N, 2.44. Found: C, 62.71; H,
6.92; N, 2.31.
galactopyranoside (19)
A mixture of 18 (3.19 g, 3.1 mmol) and Et3Nꢀ3HF (1.12 mL,
6.8 mmol) in anhyd THF (35 mL) was stirred for 3 h at 0 °C, then
was concentrated. A solution of the residue in CH2Cl2 (200 mL)
was washed with water, satd aq NaHCO3, and water, dried
(MgSO4), and concentrated. Flash silica chromatography (19:1
CH2Cl2/MeOH) gave diol 19 (2.29 g, 83%) as a white solid: mp
215–216 °C (from hot EtOH); ½a D20
ꢂ
+66 (c 1, CHCl3); 1H NMR (CDCl3,
4.6. 2-Benzyloxycarbonylaminoethyl 2-O-benzoyl-4,6-O-di-tert-
400 MHz): d 7.82–6.68 (m, 19H, Ar-H), 5.68 (dd, 1H, J1,2 8.0, J2,3
10.0 Hz, Gal H-2), 5.61 (dd, 1H, J2,3 = J3,4 = 9.0 Hz, GlcA H-3), 5.45
(dd, 1H, J4,5 10.0 Hz, GlcA H-4), 5.41 (dd, 1H, J1,2 7.0 Hz, GlcA H-
2), 5.02 (d, 1H, GlcA H-1), 4.95 (d, 1H, Gal H-1), 4.230–4.28 (m,
1H, Gal H-4), 4.26 (d, 1H, GlcA H-5), 4.11–4.09 (m, 1H, Gal H-5),
4.05 (dd, 1H, J3,4 3.0, J4,5 <1 Hz, Gal H-3), 3.92–3.88 (m, 1H, Gal
H-6a), 3.79 (s, 3H, COOCH3), 3.78–3.74 (m, 1H, Gal H-6b), 3.69 (s,
3H, OCH3), 3.26 (s, 1H, Gal HO-4), 2.66–2.48 (m, 3H, CH2CO),
2.41–2.33 (m, 2H, Gal HO-6, CH2CO), 2.03 (s, 3H, COCH3); 13C
NMR (CDCl3, 100 MHz): d 206.41 (1C, COCH3), 171.11, 167.38,
165.23, 164.83, 164.75 (5C, GlcA C-6, C@O), 154.85–113.81 (24C,
Ar-C), 101.15 (1C, GlcA C-1), 100.87 (1C, Gal C-1), 80.92 (1C, Gal
C-3), 74.75 (1C, Gal C-5), 71.69, 71.46 (2C, Gal C-2,4), 71.07 (1C,
GlcA C-4), 70.36 (1C, GlcA C-3), 69.09 (1C, GlcA C-2), 67.95 (1C,
GlcA C-5), 60.60 (1C, Gal C-6), 47.75 (1C, OCH3), 47.54 (1C,
COOCH3), 36.86 (1C, CH2CO), 28.56 (1C, COCH3), 27.08 (1C, CH2CO);
HRESIMS: Calcd for C46H46NaO18 [M+Na]+: m/z 909.25764. Found:
909.25813. Anal. Calcd for C46H46O18: C, 62.30; H, 5.23. Found: C,
62.24; H, 5.60.
butylsilylene-b-D-galactopyranoside (10)
A solution of 17 (2.17 g, 3.78 mmol) in TFA/H2O (9:1, 10 mL)
was stirred for 10 min at rt, then was diluted with water (20 mL),
concentrated, evaporated with water (3 ꢃ 10 ml), and dried in va-
cuo to give quantitatively the corresponding triol. Di-tert-butylsilyl
ditriflate (1.53 mL, 4.5 mmol) was added at 0 °C to a suspension of
the triol and sym-collidine (1.25 mL, 9.5 mmol) in anhyd CH2Cl2
(15 mL), and the mixture was stirred for 90 min at 0 °C. The clear
solution was washed with water, satd aq NaHCO3, and water, dried
(MgSO4), and concentrated. Flash silica chromatography (3:2
petroleum ether/EtOAc, containing 0.1% of Et3N) gave alcohol 10
(2.05 g, 90% from 17) as a white foam: ½a D20
ꢂ
+10 (c 1, CHCl3); 1H
NMR (CDCl3, 400 MHz): d 8.10–7.20 (m, 10H, Ar-H), 5.34 (dd, 1H,
J1,2 8.0, J2,3 11.0 Hz, H-2), 5.21 (br s, 1H, NH), 5.02 (ABq, 2H, CH2-
Ar), 4.54 (d, 1H, H-1), 4.45 (dd, 1H, J3,4 3.5, J4,5 0.8 Hz, H-4), 4.30–
4.26 (m, 2H, H-6a,6b), 3.87–3.84 (m, 1H, OCH2), 3.74–3.68 (m,
2H, H-3, OCH2), 3.51–3.49 (m, 1H, H-5), 3.36–3.30 (m, 2H, CH2N),
2.68 (d, 1H, J3,OH 3.5 Hz, HO-3), 1.09, 1.06 (2s, 18H, (CH3)3C); ISMS:
m/z 624 [M+Na]+. Anal. Calcd for C31H43NO9Si: C, 61.87; H, 7.20; N,
2.33. Found: C, 61.65; H, 7.11; N, 2.21.
4.9. 4-Methoxyphenyl O-(methyl 2,3-di-O-benzoyl-4-O-
levulinoyl-b-D-glucopyranosyluronate)-(1?3)-2,4,6-tri-O-
benzoyl-b- -galactopyranoside (20)
D
4.7. 4-Methoxyphenyl O-(methyl 2,3-di-O-benzoyl-4-O-
levulinoyl-b-
O-di-tert-butylsilylene-b-
D
-glucopyranosyluronate)-(1?3)-2-O-benzoyl-4,6-
-galactopyranoside (18)
A mixture of diol 19 (0.56 g, 0.63 mmol) and benzoyl chloride
(0.29 mL, 2.5 mmol) in anhyd pyridine and CH2Cl2 (1:1, 10 mL)
was stirred for 2 h at 0 °C. Methanol (1 mL) was added, and the
mixture was diluted with CH2Cl2 (50 mL), washed with water, satd
aq NaHCO3, and water, dried (MgSO4), and concentrated. Flash sil-
ica chromatography (1:1 EtOAc/petroleum ether) gave 20 (590 mg,
86%) as a white solid: mp 183–184 °C (from EtOAc/petroleum
D
A mixture of imidate 11 (428 mg, 0.65 mmol), alcohol 12
(265 mg, 0.5 mmol) and powdered 4 Å molecular sieves (0.2 g) in
anhyd CH2Cl2 (6 mL) was stirred for 40 min at rt under dry Ar. A
solution of Me3SiOTf in anhyd toluene (1 M, 0.12 mL) was added,
and the mixture was stirred for 40 min at rt. Triethylamine
ether); ½a 2D0
ꢂ
+83.5 (c 1, CHCl3); 1H NMR (CDCl3, 400 MHz): d

40
K. Aït-Mohand et al. / Carbohydrate Research 353 (2012) 33–48
8.15–6.53 (m, 29H, Ar-H), 5.90 (dd, 1H, J3,4 3.0, J4,5 <1 Hz, Gal H-4),
5.82 (dd, 1H, J1,2 8.0, J2,3 10.0 Hz, Gal H-2), 5.50–5.39 (m, 2H, GlcA
H-3,4), 5.26 (dd, 1H, J1,2 7.0, J2,3 9.0 Hz, GlcA H-2), 5.00 (d, 1H, Gal
H-1), 4.98 (d, 1H, GlcA H-1), 4.61 (dd, 1H, J5,6a 4.5, J6a,6b 12.0 Hz, Gal
H-6a), 4.46 (dd, 1H, J5,6b 8.0 Hz, Gal H-6b), 4.39 (dd, 1H, Gal H-3),
4.27–4.24 (m, 1H, Gal H-5), 4.14 (d, 1H, J4,5 10.0 Hz, GlcA H-5),
3.73 (s, 3H, COOCH3), 3.64 (s, 3H, OCH3), 2.61–2.29 (m, 4H, CH2CO),
2.01 (s, 3H, COCH3); 13C NMR (CDCl3, 100 MHz): d 205.59 (1C,
COCH3), 170.88, 166.80, 166.05, 165.74, 165.47, 164.52, 164.26
(7C, GlcA C-6, C@O), 155.43–114.26 (36C, Ar-C), 100.96, 100.80
(2C, Gal C-1, GlcA C-1), 76.80 (1C, Gal C-3), 72.79 (1C, GlcA C-5),
72.10 (2C, GlcA C-3, Gal C-5), 71.52 (1C, GlcA C-2), 71.39 (1C, Gal
C-2), 69.59 (1C, Gal C-4), 69.29 (1C, GlcA C-4), 62.90 (1C, Gal C-
6), 55.46 (1C, OCH3), 52.96 (1C, COOCH3), 37.61 (1C, CH2CO),
29.50 (1C, COCH3), 27.61 (1C, CH2CO). HRESIMS: Calcd for
100 MHz): d 205.60 (1C, COCH3), 170.92, 166.95, 166.01, 165.54,
165.46, 164.99, 164.26, 160.30 (8C, GlcA C-6, C@O, C@N),
133.37–127.99 (30C, Ar-C), 100.89 (1C, GlcA C-1), 93.56 (1C, Gal
C-1), 90.76 (1C, CCl3), 73.27 (1C, Gal C-3), 72.86 (1C, GlcA C-5),
72.03 (1C, GlcA C-3), 71.76 (1C, GlcA C-2), 70.30 (1C, Gal C-4),
69.92 (1C, Gal C-5), 69.85 (1C, Gal C-2), 69.21 (1C, GlcA C-4),
62.87 (1C, Gal C-6), 53.01 (1C, COOCH3), 37.63 (1C, CH2CO), 29.51
(1C, COCH3), 27.64 (1C, CH2CO). HRESIMS: Calcd for
C
55H48Cl3NNaO19 [M+Na]+: m/z 1154.17783. Found: 1154.17922.
Anal. Calcd for C55H48Cl3NO19: C, 58.29; H, 4.27; N, 1.24. Found:
C, 58.15; H, 4.36; N, 0.88.
4.12. O-(Methyl 2,3-di-O-benzoyl-4-O-levulinoyl-b-
anosyluronate)-(1?3)-2-O-benzoyl-4,6-di-O-chloroacetyl-1-O-
trichloroacetimidoyl-b- -galactopyranoside (9)
D-glucopyr-
D
C
60H55O20 [M+H]+: m/z 1095.32812. Found: 1095.32863. Anal.
Calcd for C60H54O20: C, 65.81; H, 4.97. Found: C, 65.64; H, 4.75.
Compound 21 (2.05 g, 1.97 mmol) was treated as described for
the preparation of 11. Flash silica chromatography (3:2 petroleum
ether/EtOAc, containing 0.1% of Et3N) gave imidate 9 (1.40 g, 62%
from 21) as a white solid: mp 180–181 °C (from petroleum
4.10. 4-Methoxyphenyl O-(methyl 2,3-di-O-benzoyl-4-O-
levulinoyl-b-
di-O-chloroacetyl-b-
D
-glucopyranosyluronate)-(1?3)-2-O-benzoyl-4,6-
-galactopyranoside (21)
D
ether/EtOAc); ½a D20
ꢂ
+107 (c 1, CHCl3); 1H NMR (CDCl3, 400 MHz):
d 8.54 (s, 1H, C@NH), 7.83–7.09 (m, 15H, Ar-H), 6.59 (d, 1H, J1,2
3.5 Hz, Gal H-1), 5.75 (dd, 1H, J3,4 3.0, J4,5 <1 Hz, Gal H-4), 5.57
(dd, 1H, J2,3 9.0, J3,4 10.0 Hz, GlcA H-3), 5.49 (dd, 1H, J2,3 10.0 Hz,
Gal H-2), 5.45 (dd, 1H, J4,5 10.0 Hz, GlcA H-4), 5.31 (dd, 1H, J1,2
7.0 Hz, GlcA H-2), 5.01 (d, 1H, GlcA H-1), 4.55–4.52 (m, 2H, Gal
H-3,5), 4.35 (dd, 1H, J5,6a 5.0 Hz, J6a,6b 11.0 Hz, Gal H-6a), 4.28–
4.19 (m, 4H, GlcA H-5, Gal H-6b, CH2Cl), 4.07 (s, 2H, CH2Cl), 3.84
(s, 3H, COOCH3), 2.65–2.47 (m, 3H, CH2CO), 2.41–2.33 (m, 1H,
CH2CO), 2.04 (s, 3H, COCH3); 13C NMR (CDCl3, 100 MHz): d
205.57 (1C, COCH3), 171.00, 166.81, 165.47, 164.74, 164.56,
160.23 (8C, GlcA C-6, C@O, C@N), 133.46–128.13 (18C, Ar-C),
100.95 (1C, GlcA C-1), 93.32 (1C, Gal C-1), 90.65 (1C, CCl3), 72.82
(1C, Gal C-3), 72.73 (1C, GlcA C-5), 71.77, 71.56 (2C, GlcA C-2,3),
70.90 (1C, Gal C-4), 69.32, 69.13, 69.07 (3C, Gal C-2,5, GlcA C-4),
63.32 (1C, Gal C-6), 53.19 (1C, COOCH3), 40.61, 40.49 (2C, CH2Cl),
37.59 (1C, CH2CO), 29.52 (1C, COCH3), 27.60 (1C, CH2CO). HRESIMS:
Calcd for C43H41Cl2NaO19 [MꢄCl3CC(NH)+Na]+): m/z 955.15896.
Found: 955.15969. Anal. Calcd for C45H42Cl5NO19: C, 50.13; H,
3.93; N, 1.30. Found: C, 50.09; H, 4.10; N, 0.86.
A mixture of diol 19 (709 mg, 0.8 mmol) and freshly recrystal-
lized chloroacetic anhydride (547 mg, 3.2 mmol) in anhyd pyridine
and CH2Cl2 (1:1, 10 mL) was stirred for 1 h at 0 °C, then was poured
into ice-cold water (100 mL) and extracted with CH2Cl2. The organ-
ic layer was washed with water, satd aq NaHCO3, and water, dried
(MgSO4), and concentrated. Flash silica chromatography (22:1
CH2Cl2/acetone) gave 21 (749 mg, 90%) as a white solid: mp 234–
236 °C (from EtOAc/petroleum ether); ½a D20
ꢂ
+76 (c 1, CHCl3); 1H
NMR (CDCl3, 400 MHz): d 7.83–6.69 (m, 19H, Ar-H), 5.63–5.58
(m, 2H, Gal H-2,4), 5.23 (dd, 1H, J2,3 9.0 Hz, J3,4 9.5 Hz, GlcA H-3),
5.43 (dd, 1H, J4,5 10.0 Hz, GlcA H-4), 5.30 (dd, 1H, J1,2 7.0 Hz, GlcA
H-2), 4.94 (d, 1H, J1,2 8.0 Hz, Gal H-1), 4.88 (d, 1H, GlcA H-1),
4.38–4.34 (m, 3H, Gal H-6a,6b, CH2Cl), 4.27–4.23 (m, 2H, Gal H-3,
CH2Cl), 4.12 (d, 1H, GlcA H-5), 4.09 (s, 2H, CH2Cl), 4.10–4.07 (m,
1H, Gal H-5), 3.82 (s, 3H, COOCH3), 3.71 (s, 3H, OCH3), 2.64–2.45
(m, 3H, CH2CO), 2.38–2.31 (m, 1H, CH2CO), 2.03 (s, 3H, COCH3);
13C NMR (CDCl3, 100 MHz): d 205.57 (1C, COCH3), 170.96, 167.04,
166.84, 166.72, 165.48, 164.56, 164.35 (7C, GlcA C-6, C@O),
155.67–114.39 (24C, Ar-C), 100.83, 100.80 (2C, Gal C-1, GlcA C-
1), 76.04 (1C, Gal C-3), 72.73, 71.82 (2C, GlcA C-3,5), 71.42, 70.97,
70.69, 70.53 (4C, Gal C-2,4,5, GlcA C-2), 69.31 (1C, GlcA C-4),
63.46 (1C, Gal C-6), 55.55 (1C, OCH3), 53.18 (1C, COOCH3), 40.74,
40.53 (2C, CH2Cl), 37.57 (1C, CH2CO), 29.51 (1C, COCH3), 27.57
(1C, CH2CO). HRESIMS: Calcd for C50H49Cl2O20 [M+H]+: m/z
1039.21888. Found: 1039.21743. Anal. Calcd for C50H48Cl2O20: C,
57.75; H, 4.65. Found: C, 57.50; H, 4.62.
4.13. 2-Benzyloxycarbonylaminoethyl O-(methyl 2,3-di-O-
benzoyl-4-O-levulinoyl-b-
(2,4,6-tri-O-benzoyl-b- -galactopyranosyl)-(1?3)-2-O-benzoyl-
4,6-O-di-tert-butylsilylene-b- -galactopyranoside (22)
D-glucopyranosyluronate)-(1?3)-
D
D
A mixture of imidate 8 (681 mg, 0.6 mmol) and alcohol 10
(542 mg, 0.9 mmol) was treated as described for the preparation
of 18. Flash silica chromatography (18:1 CH2Cl2/acetone containing
4.11. O-(Methyl 2,3-di-O-benzoyl-4-O-levulinoyl-b-
glucopyranosyluronate)-(1?3)-2,4,6-tri-O-benzoyl-1-O-
trichloroacetimidoyl-b- -galactopyranose (8)
D
-
0.1% of Et3N) gave 22 (632 mg, 67%) as a white foam: ½a D20
ꢂ
+62 (c 1,
CHCl3); 1H NMR (CDCl3, 400 MHz): d 8.09–6.91 (m, 35H, Ar-H),
5.83 (dd, 1H, J3,4 3.0, J4,5 <1 Hz, GalII H-4), 5.51 (dd, 1H, J1,2 8.0,
J2,3 10.0 Hz, GalII H-2), 5.39–5.37 (m, 3H, GalI H-2, GlcA H-3,4),
5.16 (dd, 1H, J1,2 7.0, J2,3 8.5 Hz, GlcA H-2), 5.05 (br s, 1H, NH),
4.95 (ABq, 2H, CH2-Ar), 4.92 (d, 1H, GalII H-1), 4.79 (d, 1H, GlcA
H-1), 4.69 (dd, 1H, J3,4 3.0, J4,5 <1 Hz, GalI H-4), 4.74–4.39 (m, 2H,
GalII H-6a,b), 4.34–4.31 (m, 1H, GalII H-3), 4.31 (d, 1H, J1,2 7.5 Hz,
GalI H-1), 4.16-4.06 (m, 2H, GalII H-5, GalI H-6a), 4.08 (d, 1H, J4,5
9.0 Hz, GlcA H-5), 4.02–3.98 (m, 1H, GalI H-6b), 3.74–3.68 (m,
5H, GalI H-3, OCH2, COOCH3), 3.52–3.47 (m, 1H, OCH2), 3.21–3.17
(m, 3H, GalI H-5, NCH2), 2.60–2.41 (m, 3H, CH2CO), 2.34–2.26 (m,
1H, CH2CO), 2.01 (s, 3H, COCH3), 1.01, 0.95 (2s, 18H, (CH3)3C); 13C
NMR (CDCl3, 100 MHz): d 205.63 (1C, COCH3), 170.86, 166.89,
165.87, 165.49, 165.41, 164.64, 164.14, 164.11, 156.29 (9C, GlcA
C-6, C@O), 133.67–127.75 (42C, Ar-C), 102.26 (1C, GalII C-1),
101.52 (1C, GalI C-1), 100.51 (1C, GlcA C-1), 80.16 (1C, GalI C-3),
D
Compound 20 (2.16 g, 1.97 mmol) was treated as described for
the preparation of 11. Flash silica chromatography (2:1 petroleum
ether/EtOAc, containing 0.1% of Et3N) gave imidate 8 (1.40 g, 62%
from 20) as a white foam: mp 110–115 °C (from petroleum
ether/EtOAc); ½a D20
ꢂ
+114 (c 1, CHCl3); 1H NMR (CDCl3, 400 MHz):
d 8.48 (s, 1H, C@NH), 8.10–7.04 (m, 25H, Ar-H), 6.74 (d, 1H, J1,2
3.5 Hz, Gal H-1), 6.05 (dd, 1H, J3,4 3.0, J4,5 <1 Hz, Gal H-4), 5.72
(dd, 1H, J2,3 10.0 Hz, Gal H-2), 5.55 (dd, 1H, J2,3 9.0, J3,4 10.0 Hz, GlcA
H-3), 5.45 (dd, 1H, J4,5 10.0 Hz, GlcA H-4), 5.27 (dd, 1H, J1,2 7.0 Hz,
GlcA H-2), 5.12 (d, 1H, GlcA H-1), 4.72–4.68 (m, 2H, Gal H-3,5),
4.52 (dd, 1H, J5,6a 5.0, J6a,6b 12.0 Hz, Gal H-6a), 4.41 (dd, 1H, J5,6b
7.5 Hz, Gal H-6b), 4.26 (d, 1H, GlcA H-5), 3.77 (s, 3H, COOCH3),
2.62–2.33 (m, 4H, CH2CO), 2.03 (s, 3H, COCH3); 13C NMR (CDCl3,

K. Aït-Mohand et al. / Carbohydrate Research 353 (2012) 33–48
41
75.83 (1C, GalII C-3), 72.68 (1C, GalI C-4), 72.63 (1C, GlcA C-5),
72.08, 71.96, 71.64, 71.52, 71.37, 70.51, 69.70, 69.25 (8C, GalI C-
2,5, GalII C-2,4,5, GlcA C-2,3,4), 68.65 (1C, OCH2), 66.69 (1C, GalI
C-6), 66.28 (1C, CH2-Ar), 62.85 (1C, GalII C-6), 52.93 (1C, COOCH3),
40.84 (1C, NCH2), 37.57 (1C, CH2CO), 29.51 (1C, COCH3), 27.58 (1C,
CH2CO), 27.38, 27.25 (4C, (CH3)3C), 23.18, 20.59 (2C, (CH3)3C). HRE-
SIMS: Calcd for C84H89NNaO27Si [M+Na]+: m/z 1594.52834. Found:
1594.52825. Anal. Calcd for C84H89NO27Si: C, 64.15; H, 5.70; N,
0.89. Found: C, 64.15; H, 5.83; N, 0.81.
1H, OCH2), 3.26–3.13 (m, 2H, NCH2), 2.30 (s, 1H, GalI HO-4),
2.60–2.41 (m, 3H, CH2CO), 2.35–2.27 (m, 1H, CH2CO), 2.01 (s,
3H, COCH3); 13C NMR (CDCl3, 100 MHz): d 205.56 (1C, COCH3),
170.82, 166.76, 166.10, 166.02, 165.81, 165.35, 164.60, 164.11,
164.06, 156.21 (10C, GlcA C-6, C@O), 133.36–127.80 (48C, Ar-C),
101.51 (1C, GalII C-1), 101.15, 100.53 (2C, GalI C-1, GlcA C-1),
80.15 (1C, GalI C-3), 75.69 (1C,GalII C-3), 72.69 (1C, GlcA C-5),
72.22, 72.14, 71.92, 71.31, 71.28, 70.54, 69.65, 69.19 (8C, GalI C-
2,5, GalII C-2,4,5, GlcA C-2,3,4), 68.54 (1C, OCH2), 68.29 (1C, GalI
C-4), 66.26 (1C, CH2-Ar), 63.59 (1C, GalI C-6), 62.68 (1C, GalII C-
6), 52.94 (1C, COOCH3), 40.72 (1C, NCH2), 37.54 (1C, CH2CO),
29.47 (1C, COCH3), 27.54 (1C, CH2CO). HRESIMS: Calcd for
4.14. 2-Benzyloxycarbonylaminoethyl O-(methyl 2,3-di-O-
benzoyl-4-O-levulinoyl-b-
(2,4,6-tri-O-benzoyl-b- -galactopyranosyl)-(1?3)-2-O-benzoyl-
b- -galactopyranoside (6)
D-glucopyranosyluronate)-(1?3)-
D
C
24H26NaO10 [M+Na]+: m/z 1558.45243. Found: 1558.45225. Anal.
D
Calcd for C24H26O10: C, 64.88; H, 5.05; N, 0.91. Found: C, 64.67; H,
5.07; N, 0.84.
Compound 22 (1.03 g, 0.65 mmol) was treated as described for
the preparation of 19. The solid residue was recrystallized from
EtOAc/petroleum ether to give diol 6 (771 mg, 82%); mp 194–
4.16. 2-Benzyloxycarbonylaminoethyl O-(methyl 2,3-di-O-
benzoyl-4-O-levulinoyl-b-
(2,4,6-tri-O-benzoyl-b- -galactopyranosyl)-(1?3)-4-O-acetyl-2-
O-benzoyl-6-O-sodium sulfonato-b- -galactopyranoside (24)
D-glucopyranosyluronate)-(1?3)-
195 °C; ½a 2D0
ꢂ
+56 (c 1, CHCl3); 1H NMR (CDCl3, 400 MHz): d 8.10–
D
6.99 (m, 35H, Ar-H), 5.81 (dd, 1H, J3,4 3.0, J4,5 <1 Hz, GalII H-4),
5.51 (dd, 1H, J1,2 8.0, J2,3 10.0 Hz, GalII H-2), 5.37–5.29 (m, 3H, GalI
H-2, GlcA H-3,4), 5.16–5.12 (m, 2H, GlcA H-2, NH), 4.92 (ABq, 2H,
CH2-Ar), 4.82 (d, 1H, J1,2 7.0 Hz, GlcA H-1), 4.76 (d, 1H, GalII H-1),
4.62 (dd, 1H, J5,6a 4.0, J6a,6b 12.0 Hz, GalII H-6a), 4.44 (dd, 1H, J5,6b
8.0 Hz, GalII H-6b), 4.33 (d, 1H, J1,2 8.5 Hz, GalI H-1), 4.27 (dd, 1H,
GalII H-3), 4.18 (m, 1H, GalII H-5), 4.05–4.03 (m, 2H, GalI H-4, GlcA
H-5), 3.78 (dd, 1H, J2,3 10.0, J3,4 3.0 Hz, GalI H-3), 3.82–3.70 (m, 5H,
GalI H-6a, OCH2, COOCH3), 3.57–3.47 (m, 2H, GalI H-6b, OCH2),
3.96–3.92 (m, 1H, GalI H-5), 3.23–3.20 (m, 2H, NCH2), 2.19 (s, 1H,
GalI HO-4), 2.60–2.41 (m, 3H, CH2CO), 2.34–2.23 (m, 2H, GalI HO-
6, CH2CO), 2.00 (s, 3H, COCH3); 13C NMR (CDCl3, 100 MHz): d
205.61 (1C, COCH3), 170.84, 166.80, 166.07, 165.84, 165.35,
164.11, 164.08 (9C, GlcA C-6, C@O), 133.48–127.86 (42C, Ar-C),
101.65 (1C, GalII C-1), 101.44 (1C, GalI C-1), 100.52 (1C, GlcA C-
1), 80.66 (1C, GalI C-3), 75.66 (1C, GalII C-3), 74.39 (1C, GalI C-5),
72.66 (1C, GlcA C-5), 72.13, 71.89, 71.29, 71.25, 70.53, 69.74,
69.17 (7C, GalI C-2, GalII C-2,4,5, GlcA C-2,3,4), 68.79 (1C, GalI C-
4), 68.68 (1C, OCH2), 66.36 (1C, CH2-Ar), 62.74 (1C, GalII C-6),
62.03 (1C, GalI C-6), 52.96 (1C, COOCH3), 40.82 (1C, NCH2), 37.52
(1C, CH2CO), 29.46 (1C, COCH3), 27.52 (1C, CH2CO). HRESIMS: Calcd
for C76H74NO27 [M+H]+: m/z 1432.44427. Found: 1432.44471. Anal.
Calcd for C76H73NO27: C, 63.73; H, 5.14; N, 0.98. Found: C, 62.10; H,
5.13; N, 0.88.
D
A mixture of diol 6 (199 mg, 0.14 mmol) and Me3NꢀSO3 (39 mg,
0.28 mmol) in anhyd DMF (5 mL) was stirred for 6 h at 40 °C. More
reagent (19.5 mg, 0.14 mmol) was then added, and the mixture
was stirred for 4 h at 40 °C, then was cooled. Methanol (1 mL)
was added, and the mixture was concentrated. A mixture of the
residue, pyridine (4 mL), and Ac2O (2 mL) was stirred overnight
at rt, then was concentrated. Flash silica chromatography (9:1
CH2Cl2/MeOH) followed by elution from a column (1.5 ꢃ 30 cm)
of Sephadex SP-C25 [Na+] with 9:5:1 CH2Cl2/MeOH/water gave
the sodium salt 24 (156 mg, 71%) as a white powder: ½a D20
ꢂ
+58 (c
1, MeOH); 1H NMR (CD3OD/CDCl3, 400 MHz): d 8.09–6.94 (m,
35H, Ar-H), 5.86 (dd, 1H, J3,4 3.0, J4,5 <1 Hz, GalII H-4), 5.57 (dd,
1H, J3,4 3.0, J4,5 <1 Hz, GalI H-4), 5.43–5.38 (m, 2H, GalII H-2, GlcA
H-3), 5.29 (dd, 1H, J3,4 = J4,5 = 10.0 Hz, GlcA H-4), 5.18 (dd, 1H, J1,2
8.0, J2,3 10.0 Hz, GalI H-2), 5.10 (dd, 1H, J1,2 7.5, J2,3 9.0 Hz, GlcA
H-2), 4.95 (d, 1H, GlcA H-1), 4.84 (d, 1H, J1,2 8.0 Hz, GalII H-1),
4.84 (ABq, 2H, CH2-Ar), 4.50 (dd, 1H, J5,6a 6.5, J6a,6b 12.0 Hz, GalII
H-6a), 4.44 (d, 1H, GalI H-1), 4.40 (dd, 1H, J5,6b 8.0 Hz, GalII H-6b),
4.28 (dd, 1H, J2,3 10.0 Hz, GalII H-3), 4.22–4.18 (m, 2H, GalII H-5,
GlcA H-5), 4.12–4.06 (m, 2H, GalI H-3,6a), 3.97–3.90 (m, 2H, GalI
H-5,6b), 3.79–3.73 (m, 1H, OCH2), 3.65 (s, 3H, COOCH3), 3.52–
3.47 (m, 1H, OCH2), 3.15–3.12 (m, 2H, NCH2), 2.58–2.54 (m, 2H,
CH2CO), 2.44–2.26 (m, 2H, CH2CO), 2.06 (s, 3H, COCH3), 1.97 (s,
3H, OCOCH3); 13C NMR (CD3OD/CDCl3, 100 MHz): d 207.99 (1C,
COCH3), 172.31, 171.36, 168.36, 167.44, 167.00, 166.51, 165.66,
165.60, 158.04 (10C, GlcA C-6, C@O), 134.23–128.41 (42C, Ar-C),
102.24 (1C, GalII C-1), 101.99 (1C, GalI C-1), 101.57 (1C, GlcA C-
1), 78.25 (1C, GalII C-3), 77.68 (1C, GalI C-3), 73.37, 73.18, 73.01,
72.42, 72.33, 72.27, 72.13 (7C, GalI C-2,5, GalII C-2,5, GlcA C-
2,3,5), 71.11, 71.01 (2C, GalI C-4, GalII C- 4), 70.33 (1C, GlcA C-4),
69.14 (1C, OCH2), 67.16 (1C, CH2-Ar), 67.09 (1C, GalI C-6), 63.32
(1C, GalII C-6), 53.40 (1C, COOCH3), 41.31 (1C, NCH2), 38.14 (1C,
CH2CO), 29.55 (1C, COCH3), 28.42 (1C, CH2CO), 20.85 (1C, OCOCH3).
4.15. 2-Benzyloxycarbonylaminoethyl O-(methyl 2,3-di-O-
benzoyl-4-O-levulinoyl-b-
(2,4,6-tri-O-benzoyl-b- -galactopyranosyl)-(1?3)-2,6-di-O-
benzoyl-b- -galactopyranoside (23)
D-glucopyranosyluronate)-(1?3)-
D
D
A mixture of 6 (250 mg, 0.17 mmol) and benzoyl cyanide
(46 mg, 0.35 mmol) in anhyd pyridine (5 mL) was stirred for
16 h at rt. Methanol (1 mL) was added, and the mixture was con-
centrated. Flash silica chromatography (12:1 CH2Cl2/acetone)
gave alcohol 23 (207 mg, 77%) as a white solid: mp 192–193 °C
(from EtOAc/petroleum ether); ½a D20
ꢂ
+66.5 (c 1, CHCl3); 1H NMR
HRESIMS: Calcd for
Found: 1552.40024.
C78H74NO31
S
[MꢄNa]ꢄ: m/z 1552.39710.
(CDCl3, 400 MHz): d 8.10–7.03 (m, 40H, Ar-H), 5.81 (dd, 1H, J3,4
3.5, J4,5 <1 Hz, GalII H-4), 5.52 (dd, 1H, J1,2 8.0, J2,3 10.0 Hz, GalII
H-2), 5.36–5.31 (m, 3H, GalI H-2, GlcA H-3,4), 5.15 (dd, 1H, J1,2
7.0, J2,3 9.0 Hz, GlcA H-2), 5.0 (br s, 1H, NH), 4.87 (ABq, 2H,
CH2-Ar), 4.83 (d, 1H, GlcA H-1), 4.78 (d, 1H, GalII H-1), 5.58 (dd,
1H, J5,6a 4.5 Hz, J6a,6b 12.0 Hz, GalII H-6a), 4.50–4.41 (m, 2H, GalI
H-6a, GalII H-6b), 4.37–4.33 (m, 1H, GalI H-6b), 4.33 (d, 1H, J1,2
8.0 Hz, GalI H-1), 4.26 (dd, 1H, GalII H-3), 4.19–4.16 (m, 1H, GalII
H-5), 4.13 (dd, 1H, J3,4 3.0, J4,5 <1 Hz, GalI H-4), 4.04 (d, 1H, J4,5
10.0 Hz, GlcA H-5), 3.82 (dd, 1H, J2,3 10.0 Hz, GalI H-3), 3.73 (s,
3H, COOCH3), 3.72–3.67 (m, 2H, GalI H-5, OCH2), 3.51–3.46 (m,
4.17. 2-Benzyloxycarbonylaminoethyl O-(methyl 2,3-di-O-
benzoyl-4-O-levulinoyl-b- -glucopyranosyluronate)-(1?3)-
(2,4,6-tri-O-benzoyl-b- -galactopyranosyl)-(1?3)-2,6-di-O-
benzoyl-4-O-sodium sulfonato-b- -galactopyranoside (25)
D
D
D
A mixture of alcohol 23 (189 mg, 0.12 mmol) and Me3NꢀSO3
(171 mg, 1.2 mmol) in anhyd DMF (3 mL) was stirred overnight
at 60 °C, then was cooled. Methanol (2 mL) was added, and the
mixture was directly eluted from
a
column (3 ꢃ 80 cm) of

42
K. Aït-Mohand et al. / Carbohydrate Research 353 (2012) 33–48
Sephadex LH-20 with 1:1 CH2Cl2/MeOH. Fractions containing the
product were submitted to flash silica chromatography (12:1
CH2Cl2/MeOH) followed by elution from a column (1.5 ꢃ 30 cm)
of Sephadex SP-C25 [Na+] with 9:5:1 CH2Cl2/MeOH/water to give
4.19. 2-Benzyloxycarbonylaminoethyl O-(methyl 2,3-di-O-
benzoyl-4-O-levulinoyl-b- -glucopyranosyluronate)-(1?3)-(2-
O-benzoyl-4,6-di-O-chloroacetyl-b- -galactopyranosyl)-(1?3)-
2-O-benzoyl-b- -galactopyranoside (27)
D
D
D
the sodium salt 25 (177 mg, 83%) as a white powder: ½a D20
ꢂ
+49
(c 1, MeOH); 1H NMR (CD3OD/CDCl3, 400 MHz): d 8.27–6.96
(m, 40H, Ar-H), 5.91 (dd, 1H, J3,4 3.2, J4,5 <1 Hz, GalII H-4), 5.91
(dd, 1H, J1,2 8.0, J2,3 9.0 Hz, GalII H-2), 5.45 (dd, 1H,
J2,3 = J3,4 = 9.0 Hz, GlcA H-3), 5.36 (dd, 1H, J4,5 10.0 Hz, GlcA H-
4), 5.19 (dd, 1H, J1,2 7.5, J2,3 10.0 Hz, GalI H-2), 5.14–5.10 (m,
2H, GalI H-4, GlcA H-2), 4.97 (d, 1H, J1,2 7.5 Hz, GlcA H-1),
4.88–4.80 (m, 3H, GalII H-1, CH2-Ar), 4.69–4.52 (m, 3H, GalI H-
6a,6b, GalII H-6a), 4.47–4.42 (m, 2H, GalI H-1, GalII H-6b), 4.32
(dd, 1H, GalII H-3), 4.26–4.21 (m, 2H, GalII H-5, GlcA H-5), 4.02
(dd, 1H, J3,4 2.0 Hz, GalI H-3), 3.81–3.78 (m, 1H, GalI H-5), 3.69
(s, 3H, COOCH3), 3.69–3.64 (m, 1H, OCH2), 3.54–3.48 (m, 1H,
OCH2), 3.14–3.10 (m, 2H, NCH2), 2.59–2.28 (m, 3H, CH2CO),
2.47–2.39 (m, 1H, CH2CO), 1.98 (s, 3H, COCH3); 13C NMR
(CD3OD/CDCl3, 100 MHz): d 206.48 (1C, COCH3), 170.83, 166.86,
166.08, 165.92, 165.65, 165.55, 165.23, 165.12, 164.31 (10C, GlcA
C-6, C@O), 133.02–127.22 (48C, Ar-C), 101.57 (1C, GalII C-1),
100.63 (2C, GalI C-1, GlcA C-1), 76.78, 76.74 (2C, GalI C-3, GalII
C-3), 75.49 (1C, GalI C-4), 72.22 (1C, GalI C-5), 71.93, 71.86,
71.54 (3C, GalII C-5, GlcA C-2,5), 71.34 (1C, GalI C-2), 70.41,
69.22 (2C, GalII C-2, GlcA C-3), 68.70 (1C, GalII C-4), 68.11 (1C,
GlcA C-4), 68.07 (1C, OCH2), 65.95 (1C, CH2-Ar), 63.38 (1C, GalI
C-6), 62.00 (1C, GalII C-6), 52.39 (1C, COOCH3), 40.11 (1C,
NCH2), 37.02 (1C, CH2CO), 28.68 (1C, COCH3), 27.14 (1C, CH2CO).
HRESIMS: Calcd for C83H76NNaO31S [MꢄNa]ꢄ: m/z 1614.41275.
Found: 1614.41347.
Compound 26 (473 mg, 0.31 mmol) was treated as described for
the preparation of 19. The residue was crystallized from hot EtOH
to give 27 (348 mg, 81%): mp 230–232 °C; ½a D20
ꢂ
+48 (c 1, CHCl3); 1H
NMR (CDCl3, 400 MHz): d 7.78–7.01 (m, 25H, Ar-H), 5.54 (dd, 1H,
J3,4 3.0, J4,5 <1 Hz, GalII H-4), 5.42–5.34 (m, 2H, GlcA H-3,4), 5.32–
5.27 (m, 2H, GalI H-2, GalII H-2), 5.20–5.13 (m, 2H, GlcA H-2,
NH), 4.93 (ABq, 2H, CH2-Ar), 4.73, 4.71 (2d, 2H, J1,2 7.0 Hz, GalII
H-1, GlcA H-1), 4.41 (d, 1H, J1,2 8.0 Hz, GalI H-1), 4.35–4.26 (m,
2H, GalII H-6a,6b), 4.26 (ABq, 2H, CH2Cl), 4.13 (s, 2H, CH2Cl),
4.13–4.10 (m, 2H, GalI H-4, GalII H-3), 4.05–3.92 (m, 3H, GalI H-
6a, GalII H-5, GlcA H-5), 3.84 (dd, 1H, J2,3 9.5, J3,4 3.0 Hz, GalI H-
3), 3.79 (s, 3H, COOCH3), 3.79–3.70 (m, 2H, GalI H-6b, OCH2),
3.62–3.52 (m, 2H, GalI H-5, OCH2), 3.26–3.17 (m, 2H, NCH2), 2.80
(s, 1H, GalI HO-4), 2.63–2.43 (m, 4H, GalI HO-6, CH2CO), 2.34–
2.27 (m, 1H, CH2CO), 2.01 (s, 3H, COCH3); 13C NMR (CDCl3,
100 MHz): d 206.30 (1C, COCH3), 170.95, 167.07, 166.97, 166.84,
165.36, 164.75, 164.49, 164.47 (9C, GlcA C-6, C@O), 156.55–
127.47 (30C, Ar-C), 101.45, 101.18, 100.42 (3C, GalI C-1, GalII C-1,
GlcA C-1), 80.26 (1C, GalI C-3), 75.35 (1C, GalII C-3), 74.47 (1C, GalI
C-5), 72.18, 71.86, 71.58, 71.09, 70.69, 70.65, 70.50, 69.05 68.36,
(9C, GalI C-2,4, GalII C-2,4,5, GlcA C-2,3,4,5), 68.25 (1C, OCH2),
66.14 (1C, CH2-Ar), 63.08 (1C, GalII C-6), 60.87 (1C, GalI C-6),
52.92 (1C, COOCH3), 40.45, 40.35, 40.29 (3C, CH2Cl, NCH2), 37.21
(1C, CH2CO), 29.10 (1C, COCH3), 27.26 (1C, CH2CO). HRESIMS: Calcd
for C66H68Cl2NO27 [M+H]+: m/z 1376.33503. Found: 1376.33502.
Anal. Calcd for C66H67Cl2NO27: C, 57.56; H, 4.90; N, 1.02. Found:
C, 57.31; H, 4.93; N, 0.94.
4.18. 2-Benzyloxycarbonylaminoethyl O-(methyl 2,3-di-O-ben
zoyl-4-O-levulinoyl-b-
benzoyl-4,6-di-O-chloroacetyl-b-
benzoyl-4,6-O-di-tert-butylsilylene-b-
D
-glucopyranosyluronate)-(1?3)-(2-O-
-galactopyranosyl)-(1?3)-2-O-
-galactopyranoside (26)
4.20. 2-Benzyloxycarbonylaminoethyl 2-O-benzoyl-4,6-O-di-
tert-butylsilylene-3-O-levulinoyl-b-D-galactopyranoside (28)
D
D
Compound 10 (1.0 g, 1.66 mmol) was treated as described for
the preparation of 14. Flash silica chromatography (2:1 petroleum
A mixture of imidate 9 (158 mg, 0.15 mmol) and alcohol 10
(133 mg, 0.22 mmol) was treated as described for the preparation
of 18. Flash silica chromatography (15:1 CH2Cl2/acetone containing
ether/EtOAc) gave 28 (1.09 g, 94%) as a white foam: ½a D20
ꢂ
+32 (c 1,
CHCl3); 1H NMR (CDCl3, 400 MHz): d 8.01–7.31 (m, 10H, Ar-H),
5.62 (dd, 1H, J1,2 8.0, J2,3 10.0 Hz, H-2), 5.21 (br s, 1H, NH), 5.01
(ABq, 2H, CH2-Ar), 4.94 (dd, 1H, J2,3 11.0, J3,4 3.0 Hz, H-3), 4.66
(dd, 1H, H-4), 4.57 (d, 1H, H-1), 4.29–4.23 (m, 2H, H-6a, H-6b),
3.86–3.82 (m, 1H, OCH2), 3.71–3.32 (m, 1H, OCH2), 3.54–3.52 (m,
1H, H-5), 3.35–3.31 (m, 2H, NCH2), 2.59–2.55 (m, 4H, CH2CO),
2.04 (s, 3H, COCH3), 1.10, 1.04 (2s, 18H, (CH3)3C). HRESIMS: Calcd
for C36H50NO11Si [M+H]+: m/z 700.31476. Found: 700.31471. Anal.
Calcd for C36H49NO11Si: C, 61.78; H, 7.06; N, 2.00. Found: C, 61.48;
H, 7.15; N, 2.09.
0.1% of Et3N) gave 26 (68 mg, 49%) as a white solid: ½a D20
ꢂ
+53 (c 1,
CHCl3); 1H NMR (CDCl3, 400 MHz): d 7.78–6.97 (m, 25H, Ar-H),
5.51 (dd, 1H, J3,4 3.5, J4,5 <1 Hz, GalII H-4), 5.43–5.33 (m, 3H, GalI
H-2, GlcA H-3,4), 5.30 (dd, 1H, J1,2 8.0, J2,3 10.5 Hz, GalII H-2),
5.20 (dd, 1H, J1,2 7.0, J2,3 8.5 Hz, GlcA H-2), 5.07 (br s, 1H, NH),
4.94 (ABq, 2H, CH2-Ar), 4.92 (d, 1H, GalII H-1), 4.69 (d, 1H, GlcA
H-1), 4.61 (dd, 1H, J3,4 3.0, J4,5 <1 Hz, GalI H-4), 4.38 (d, 1H, J1,2
8.0 Hz, GalI H-1), 4.26–4.05 (m, 9H, GalI H-6a,6b, GalII H-3,6a,6b,
CH2Cl), 4.02 (d, 1H, J4,5 10.0 Hz, GlcA H-5), 3.91 (dd, 1H, J5,6a 6.0,
J5,6b 7.0 Hz, GalII H-5), 3.79 (s, 3H, COOCH3), 3.78–3.70 (m, 2H, GalI
H-3, OCH2), 3.54–3.49 (m,1H, OCH2), 3.39–3.36 (m, 1H, GalI H-5),
3.23–3.20 (m, 2H, NCH2), 2.62–2.27 (m, 4H, CH2CO), 2.02 (s, 3H,
COCH3), 1.02, 0.99 (2s, 18H, (CH3)3C); 13C NMR (CDCl3, 100 MHz):
d 205.59 (1C, COCH3), 170.88, 166.85, 166.68, 165.38, 164.69,
164.43, 163.97, 156.25 (9C, GlcA C-6, C@O), 136.57–127.69 (30C,
Ar-C), 101.52 (1C, GalI C-1), 101.23 (1C, GalII C-1), 100.63 (1C, GlcA
C-1), 79.13 (1C, GalI C-3), 75.86 (1C, GalII C-3), 72.74 (1C, GalI C-4),
72.52 (1C, GlcA C-5), 71.74, 71.42, 71.23, 70.97, 70.76, 70.72, 69.43,
69.23 (8C, GalI C-2,5, GalII C-2,4,5, GlcA C-2,3,4), 68.70 (1C, OCH2),
66.72 (1C, GalII C-6), 66.25 (1C, CH2-Ar), 66.46 (1C, GalI C-6), 52.07
(1C, COOCH3), 40.77, 40.60, 40.57 (3C, CH2Cl, NCH2), 37.47 (1C,
CH2CO), 29.45 (1C, COCH3), 27.49 (1C, CH2CO), 27.40, 27.17 (6C,
(CH3)3C), 23.20, 20.51 (2C, (CH3)3C). ISMS: m/z 1518.5 [M+H]+.
Anal. Calcd for C74H83Cl2NO27Si: C, 58.57; H, 5.51; N, 0.92. Found:
C, 58.43; H, 5.39; N, 0.87.
4.21. 2-Benzyloxycarbonylaminoethyl 2,4,6-tri-O-benzoyl-3-O-
levulinoyl-b-D-galactopyranoside (29)
Compound 28 (1.07 g, 1.53 mmol) was treated as described for
the preparation of 19 to give the crude intermediate diol (0.83 g,
96%) as a white foam: HRESIMS: Calcd for C28H34O11N [M+H]+:
m/z 560.21264. Found: 560.21235. A mixture of the above isolated
diol (284 mg, 0.51 mmol) and benzoyl chloride (235lL, 2.04 mmol)
in anhyd pyridine and CH2Cl2 (1:1, 6 mL) was stirred at 0 °C for 1 h.
Methanol (1 mL) was added, and the mixture was diluted with
CH2Cl2 (30 mL), washed with water, satd aq NaHCO3, and water,
dried (MgSO4), and concentrated. Flash silica chromatography
(3:2 petroleum ether/EtOAc) gave 29 (366 mg, 94% from 28) as a
white foam: ½a 2D0
ꢂ
+24 (c 1, CHCl3); 1H NMR (CDCl3, 400 MHz): d

K. Aït-Mohand et al. / Carbohydrate Research 353 (2012) 33–48
43
8.15–7.28 (m, 20H, Ar-H), 5.82 (dd, 1H, J3,4 3.0, J4,5 <1 Hz, H-4), 5.56
4.24. 4-Methoxyphenyl O-(methyl 2,3-di-O-benzoyl-4-O-
levulinoyl-b- -glucopyranosyluronate)-(1?3)-(2-O-benzoyl-
4,6-di-O-chloroacetyl-b- -galactopyranosyl)-(1?3)-2-O-
benzoyl-b- -galactopyranoside (32)
(dd, 1H, J1,2 7.5, J2,3 10.0 Hz, H-2), 4.35 (dd, 1H, H-3), 5.14 (br s, 1H,
NH), 4.97 (ABq, 2H, CH2-Ar), 4.71 (d, 1H, H-1), 4.39 (dd, 1H, J5,6a 7.0,
J6a,6b 12.0 Hz, H-6a), 4.39 (dd, 1H, J5,6b 6.5 Hz, H-6b), 4.22–4.20 (m,
1H, H-5), 3.97–3.93 (m, 1H, OCH2), 3.73–3.68 (m, 1H, OCH2), 3.46–
3.30 (m, 2H, NCH2), 2.62–2.31 (m, 4H, CH2CO), 1.92 (s, 3H, COCH3).
HRESIMS: Calcd for C42H43 NO13 [M+H]+: m/z 768.26507. Found:
768.26602. Anal. Calcd for C42H42NO13: C, 65.70; H, 5.38; N, 1.82.
Found: C, 65.21; H, 5.51; N, 1.74.
D
D
D
Compound 31 (1.10 g, 0.76 mmol) was treated as described for
the preparation of 19. Flash silica chromatography (22:1 CH2Cl2/
MeOH) gave 32 (0.87 g, 88%) as a white foam: ½a D20
ꢂ
+66 (c 1, CHCl3);
1H NMR (CD3OD/CDCl3, 400 MHz): d 7.79–6.65 (m, 24H, Ar-H), 5.54
(dd, 1H, J1,2 8.0, J2,3 9.5 Hz, GalI H-2), 5.52 (dd, 1H, J3,4 3.0, J4,5 <1 Hz,
GalII H-4), 5.42–5.36 (m, 2H, GlcA H-3,4), 5.33 (dd, 1H, J1,2 8.0, J2,3
10.0 Hz, GalII H-2), 5.18 (dd, 1H, J1,2 7.5, J2,3 9.0 Hz, GlcA H-2), 4.87
(d, 1H, GalI H-1), 4.75 (d, 1H, GalII H-1), 4.71 (d, 1H, GlcA H-1),
4.33–4.28 (m, 2H, GalII H-6a,6b), 4.27 (ABq, 2H, CH2Cl), 4.17 (dd,
1H, J3,4 3.0, J4,5 <1 Hz, GalI H-4), 4.14 (s, 2H, CH2Cl), 4.12 (dd, 1H,
GalII H-3), 4.04–4.01 (m, 2H, GalII H-5, GlcA H-5), 3.98 (dd, 1H,
J5,6a 4.5, J6a,6b 12.5 Hz, GalI H-6a), 3.90 (dd, 1H, GalI H-3), 3.89–
3.81 (m, 2H, GalI H-5,6b), 3.79 (s, 3H, COOCH3), 3.68 (s, 3H,
OCH3), 2.96 (s, 1H, GalI HO-4), 2.63–2.42 (m, 3H, CH2CO), 2.36–
2.27 (m, 2H, CH2CO, GalI HO-6), 2.01 (s, 3H, COCH3); 13C NMR
(CD3OD/CDCl3, 100 MHz): d 206.46 (1C, COCH3), 171.09, 167.99,
167.92, 167.04, 165.19, 164.84, 164.65, 164.44 (8C, GlcA C-6,
C@O), 151.0–113.85 (30C, Ar-C), 101.32 (1C, GlcA C-1), 100.92
(1C, GalI C-1), 100.19 (1C, GalII C-1), 80.31 (1C, GalI C-3), 74.66,
71.89, 70.97, 70.51, 70.34, 70.18, 67.99 (11C, GalI C-2,4,5, GalII C-
2,3,4,5, GlcA C-2,3,4,5), 63.01 (1C, GalII C-6), 60.41 (1C, GalI C-6),
54.83 (1C, OCH3), 52.55 (1C, COOCH3), 40.12, 40.04 (2C, CH2Cl),
36.93 (1C, CH2CO), 28.60 (1C, COCH3), 27.06 (1C, CH2CO). HRESIMS:
4.22. 2-Benzyloxycarbonylaminoethyl 2,4,6-tri-O-benzoyl-b-D-
galactopyranoside (30)
A solution of 29 (320 mg, 0.42 mmol) in pyridine (3 mL) was
treated for 8 min at rt with 12:8:1 pyridine/AcOH/hydrazine hy-
drate (9 mL), then was diluted with CH2Cl2 (30 mL), washed with
water, satd aq NaHCO3, and water, dried (MgSO4), and concen-
trated. Flash silica chromatography (3:2 petroleum ether/EtOAc)
gave alcohol 30 (263 mg, 94%) as a white foam: ½a D20
ꢂ
+13.5 (c 1,
CHCl3); 1H NMR (CDCl3, 400 MHz): d 8.16–7.29 (m, 20H, Ar-H),
5.76 (dd, 1H, J3,4 3.0, J4,5 <1 Hz, H-4), 5.34 (dd, 1H, J1,2 8.0, J2,3
10.0 Hz, H-2), 5.15 (br s, 1H, NH), 4.98 (ABq, 2H, CH2-Ar), 4.69 (d,
1H, H-1), 4.56 (dd, 1H, J5,6a 7.0, J6a,6b 12.0 Hz, H-6a), 4.44 (dd, 1H,
J5,6b 6.0 Hz, H-6b), 4.16–4.09 (m, 2H, H-3,5), 3.98–3.93 (m, 1H,
OCH2), 3.76–3.71 (m, 1H, OCH2), 3.46–3.30 (m, 2H, NCH2), 2.70
(d, 1H, J3,OH 6.0 Hz, HO-3). HRESIMS: Calcd for C37H36NO11
[M+H]+: m/z 670.22829. Found: 670.22879. Anal. Calcd for
C37H35NO11: C, 66.36; H, 5.27; N, 2.09. Found: C, 66.15; H, 5.37;
Calcd for
C
63H62Cl2NaO26 [M+Na]+: m/z 1327.27986. Found:
N, 2.07.
1327.28076. Anal. Calcd for C63H62Cl2O26: C, 57.94; H, 4.78. Found:
C, 57.64; H, 4.60.
4.23. 4-Methoxyphenyl O-(methyl 2,3-di-O-benzoyl-4-O-
4.25. 4-Methoxyphenyl O-(methyl 2,3-di-O-benzoyl-4-O-
levulinoyl-b-
4,6-di-O-chloroacetyl-b-
benzoyl-4,6-O-di-tert-butylsilylene-b-
D
-glucopyranosyluronate)-(1?3)-(2-O-benzoyl-
-galactopyranosyl)-(1?3)-2-O-
-galactopyranoside (31)
levulinoyl-b-
4,6-di-O-chloroacetyl-b-
benzoyl-b- -galactopyranoside (33)
D
-glucopyranosyluronate)-(1?3)-(2-O-benzoyl-
D
D
-galactopyranosyl)-(1?3)-2,4,6-tri-O-
D
D
A mixture of imidate 9 (1.34 g, 1.24 mmol) and alcohol 12
(1.0 g, 1.88 mmol) was treated as described for the preparation
of 18. Flash silica chromatography (22:1 CH2Cl2/acetone contain-
Compound 32 (153 mg, 0.12 mmol) was benzoylated as de-
scribed for the preparation of 29. Flash silica chromatography
(19:1 CH2Cl2/acetone) gave 33 (143 mg, 83%) as a white solid:
ing 0.1% of Et3N) gave 31 (1.10 g, 61%) as a white foam: ½a D20
ꢂ
mp 179–180 °C (from EtOAc/petroleum ether); ½a D20
ꢂ
+58 (c 1,
+64.5 (c 1, CHCl3); 1H NMR (CDCl3, 400 MHz): d 7.87–6.65 (m,
24H, Ar-H), 5.63 (dd, 1H, J1,2 8.0, J2,3 9.5 Hz, GalI H-2), 5.52 (dd,
1H, J3,4 3.0, J4,5 <1 Hz, GalII H-4), 5.43–5.31 (m, 3H, GalII H-2, GlcA
H-3,4), 5.20 (dd, 1H, J1,2 7.5, J2,3 9.0 Hz, GlcA H-2), 4.94 (d, 1H, J1,2
7.5 Hz, GalII H-1), 4.81 (d, 1H, GalI H-1), 4.70 (d, 1H, GlcA H-1),
4.66 (dd, 1H, J3,4 2.5, J4,5 <1 Hz, GalI H-4), 4.25–4.20 (m, 4H, GalI
H-6a,6b, GalII H-6a,6b), 4.15 (ABq, 2H, CH2Cl), 4.10 (s, 2H, CH2Cl),
4.07 (dd, 1H, J2,3 10.0 Hz, GalII H-3), 4.01 (d, 1H, J4,5 9.0 Hz, GlcA
H-5), 3.94–3.91 (m, 1H, GalII H-5), 3.87 (dd, 1H, GalI H-3), 3.79
(s, 3H, COOCH3), 3.69 (s, 3H, OCH3), 3.47–3.44 (m, 1H, GalI H-5),
2.62–2.43 (m, 3H, CH2CO), 2.35–2.27 (m, 1H, CH2CO), 2.01 (s,
3H, COCH3), 1.04, 1.03 (2s, 18H, (CH3)3C); 13C NMR (CDCl3,
100 MHz): d 205.56 (1C, COCH3), 170.91, 166.89, 166.72, 166.70,
165.41, 164.56, 164.48, 164.02 (8C, GlcA C-6, C@O), 155.45–
114.19 (30C, Ar-C), 101.70 (1C, GalI C-1), 101.27 (1C, GalII C-1),
100.65 (1C, GlcA C-1), 79.22 (1C, GalI C-3), 75.84 (1C, GalII C-3),
72.73, 72.57 (2C, GalI C-4, GlcA C-5), 71.79, 71.49, 71.27, 71.03,
70.82, 70.77, 69.27 (8C, GalI C-2,5, GalII C-2,4,5, GlcA C-2,3,4),
66.84, 63.48 (2C, GalI C-6, GalII C-6), 55.49 (1C, OCH3), 53.09
(1C, COOCH3), 40.60 (2C, CH2Cl), 37.51 (1C, CH2CO), 29.49 (1C,
COCH3), 27.53 (1C, CH2CO), 27.41, 27.30 (6C, (CH3)3C), 23.25,
20.63 (2C, (CH3)3C). HRESIMS: Calcd for C71H79Cl2O26Si [M+H]+:
m/z 1445.40004. Found: 1445.40082. Anal. Calcd for C71H78Cl2O26-
Si: C, 58.96; H, 5.44. Found: 58.57; H, 5.68.
CHCl3); 1H NMR (CDCl3, 400 MHz): d 8.09–6.48 (m, 34H, Ar-H),
5.79 (dd, 1H, J3,4 3.0, J4,5 <1 Hz, GalI H-4), 5.69 (dd, 1H, J1,2 8.0, J2,3
9.0 Hz, GalI H-2), 5.45 (dd, 1H, J3,4 3.0, J4,5 <1 Hz, GalII H-4), 5.39–
5.30 (m, 2H, GlcA H-3,4), 5.20–5.12 (m, 2H, GalII H-2, GlcA H-2),
4.94 (d, 1H, GalI H-1), 4.77 (d, 1H, J1,2 7.5 Hz, GalII H-1), 4.69–
4.65 (m, 1H, GalI H-6a), 4.66 (d, 1H, J1,2 7.0 Hz, GlcA H-1),
4.42–4.35 (m, 1H, GalI H-6b), 4.22–4.16 (m, 4H, GalI H-3, GalII H-
5,6a,6b), 4.16 (s, 2H, CH2Cl), 4.04 (dd, 1H, J2,3 10.0 Hz, GalII H-3),
3.99 (d, 1H, J4,5 9.0 Hz, GlcA H-5), 3.97 (ABq, 2H, CH2Cl), 3.91 (dd,
1H, J5,6a 6.0, J5,6b 6.5 Hz, GalI H-5), 3.77 (s, 3H, COOCH3), 3.63 (s,
3H, OCH3), 2.61–2.41 (m, 3H, CH2CO), 2.34–2.25 (m, 1H, CH2CO),
2.01 (s, 3H, COCH3); 13C NMR (CDCl3, 100 MHz): d 205.56 (1C,
COCH3), 170.89, 166.94, 166.82, 166.69, 166.06, 166.02, 165.40,
164.44, 164.39 (10C, GlcA C-6, C@O), 155.36–114.19 (42C, Ar-C),
101.11 (1C, GalII C-1), 100.85 (1C, GalI C-1), 100.53 (1C, GlcA C-
1), 77.35 (1C, GalII C-3), 75.55 (1C, GalI C-3), 72.58 (1C, GlcA C-5),
72.20 (1C, GalI C-5), 71.76, 71.27, 71.15, 70.77, 70.71, 70.60,
69.97, 69.23 (8C, GalI C-2,4, GalII C-2,4,5, GlcA C-2,3,4), 63.29 (2C,
GalI C-6, GalII C-6), 55.44 (1C, OCH3), 53.10 (1C, COOCH3), 40.75,
40.49 (2C, CH2Cl), 37.53 (1C, CH2CO), 29.49 (1C, COCH3), 27.53
(1C, CH2CO). HRESIMS: Calcd for C77H71Cl2O28 [M+H]+: m/z
1513.35034. Found: 1513.34906. Anal. Calcd for C77H70Cl2O28: C,
61.07; H, 4.66. Found: C, 61.39; H, 4.62.

44
K. Aït-Mohand et al. / Carbohydrate Research 353 (2012) 33–48
4.26. O-(Methyl 2,3-di-O-benzoyl-4-O-levulinoyl-b-
glucopyranosyluronate)-(1?3)-(2-O-benzoyl-4,6-di-O-
chloroacetyl-b- -galactopyranosyl)-(1?3)-2,4,6-tri-O-benzoyl-
1-O-trichloroacetimidoyl- -galactopyranose (34)
D
-
69.20 (2C, GlcA C-3,4), 68.92 (1C, OCH2), 66.34 (1C, CH2-Ar),
63.25, 63.13 (2C, GalI C-6, GalII C-6), 53.10 (1C, COOCH3), 40.71
(2C, CH2Cl), 40.48 (1C, NCH2), 37.50 (1C, CH2CO), 29.47 (1C,
COCH3), 27.50 (1C, CH2CO). HRESIMS: Calcd for C80H75Cl2NNaO29
[M+Na]+: m/z 1606.36940. Found: 1606.37004. Anal. Calcd for
D
a
-D
Compound 33 (143 mg, 0.1 mmol) was treated as described for
the preparation of 11. Flash silica chromatography (1:1 EtOAc/
petroleum ether, containing 0.1% of Et3N) gave imidate 34
(94 mg, 38%) as a white powder: mp 187–189 °C (from EtOAc/
C80H75Cl2NO29: C, 60.61; H, 4.77; N, 0.88. Found: C, 60.60; H,
4.73; N, 0.87.
4.27.2. Procedure B
A mixture of trisaccharide imidate 34 (79 mg, 52 lmol) and 2-
benzyloxycarbonylaminoethanol (20.5 mg, 105 lmol) was treated
petroleum ether);
½
a 2D0
ꢂ
+69 (c 1, CHCl3); 1H NMR (CDCl3,
400 MHz): d 8.43 (s, 1H, C@NH), 8.05–6.91 (m, 30H, Ar-H), 6.65
(d, 1H, J1,2 3.5 Hz, GalI H-1), 5.94 (dd, 1H, J3,4 3.0, J4,5 <1 Hz, GalI
H-4), 5.63 (dd, 1H, J2,3 10.0 Hz, GalI H-2), 5.50 (dd, 1H, J3,4 3.0,
J4,5 <1 Hz, GalII H-4), 5.39–5.32 (m, 2H, GlcA H-3,4), 5.20–5.12 (m,
2H, GalII H-2, GlcA H-2), 4.88 (d, 1H, J1,2 8.0 Hz, GalII H-1), 4.69
(d, 1H, J1,2 7.0 Hz, GlcA H-1), 4.64–4.61 (m, 1H, J5,6a 7.0, J5,6b
4.5 Hz, GalI H-5), 4.56 (dd, 1H, J6a,6b 12.0 Hz, GalI H-6a), 4.48 (dd,
1H, GalI H-3), 4.34 (dd, 1H, GalI H-6b), 4.26–4.22 (m, 2H, GalII H-
6a,6b), 4.16 (s, 2H, CH2Cl), 4.07–3.99 (m, 3H, GalII H-3,5, GlcA H-
5), 4.00 (ABq, 2H, CH2Cl), 3.79 (s, 3H, COOCH3), 2.64–2.42 (m, 3H,
CH2CO), 2.34–2.27 (m, 1H, CH2CO), 2.01 (s, 3H, COCH3); 13C NMR
(CDCl3, 100 MHz): d 205.55 (1C, COCH3), 170.88, 166.99, 166.83,
166.69, 166.04, 165.82, 165.39, 164.82, 164.36, 163.60, 160.127
(11C, GlcA C-6, C@O, C@N), 133.35–127.96 (36C, Ar-C), 101.26
(1C, GalII C-1), 100.58 (1C, GlcA C-1), 93.51 (1C, GalI C-1), 75.67
(1C, GalII C-3), 74.07 (1C, GalI C-3), 72.61, 71.74, 71.30, 70.84,
70.75, 70.57, 70.49, 70.30, 69.19, 69.13 (10C, GalI C-2,4,5, GalII
C-2,4,5, GlcA C-2,3,4,5), 63.34, 63.23 (2C, GalI C-6, GalII C-6),
53.13 (1C, COOCH3), 40.70, 40.50 (2C, CH2Cl), 37.53 (1C, CH2CO),
29.49 (1C, COCH3), 27.52 (1C, CH2CO). HRESIMS: Calcd for
as described for the preparation of 18. Flash silica chromatography
(12:1 CH2Cl2/acetone, containing 0.1% of Et3N) gave 35 (46.5 mg,
56%) as a white solid. Physical data matched those reported above.
4.28. 2-Benzyloxycarbonylaminoethyl O-(methyl 2,3-di-O-
benzoyl-4-O-levulinoyl-b-
O-benzoyl-b- -galactopyranosyl)-(1?3)-2,4,6-tri-O-benzoyl-b-
-galactopyranoside (7)
D-glucopyranosyluronate)-(1?3)-(2-
D
D
A mixture of 35 (639 mg, 0.4 mmol) and thiourea (123 mg,
1.6 mmol) in pyridine and EtOH (1:1, 9 mL) was stirred for 2 h at
80 °C, then was cooled. The mixture was concentrated and diluted
with CH2Cl2, washed with water, brine, and water, dried (MgSO4),
and concentrated. Flash silica chromatography (5:1 CH2Cl2/ace-
tone) gave diol 7 (443 mg, 77%) as a white foam: mp 188–190 °C
(from EtOAc/petroleum ether); ½a D20
ꢂ
+44 (c 1, CHCl3); 1H NMR
(CDCl3, 400 MHz): d 8.11–6.88 (m, 35H, Ar-H), 5.9 (dd, 1H, J3,4
3.0, J4,5 <1 Hz, GalI H-4), 5.49 (dd, 1H, J1,2 8.0, J2,3 9.5 Hz, GalI H-
2), 5.42 (dd, 1H, J2,3 = J3,4 = 9.0 Hz, GlcA H-3), 5.34 (dd, 1H, J4,5
9.0 Hz, GlcA H-4), 5.24–5.20 (m, 2H, GalII H-2, GlcA H-2), 5.01–
4.91 (m, 2H, NH, CH2-Ar), 4.82–4.79 (m, 2H, GalII H-1, CH2-Ar),
4.72 (d, 1H, J1,2 8.0 Hz, GlcA H-1), 4.52–4.48 (m, 2H, GalI H-1,6a),
4.340–4.36 (m, 1H, GalI H-6b), 4.10 (d, 1H, GlcA H-5), 4.09–4.03
(m, 3H, GalI H-3,5, GalII H-4), 3.88–3.83 (m, 2H, GalII H-3,6a),
3.80–3.77 (m, 1H, OCH2), 3.72 (s, 3H, COOCH3), 3.67–3.64 (m, 1H,
GalII H-6b), 3.60–3.58 (m, 1H, GalII H-5), 3.53–3.48 (m, 1H,
OCH2), 3.27–3.15 (m, 2H, NCH2), 2.98 (br s, 1H, GalII HO-4), 2.63–
2.43 (m, 4H, GalII HO-6, CH2CO), 2.35–2.28 (m, 1H, CH2CO), 2.01
(s, 3H, COCH3); 13C NMR (CDCl3, 100 MHz): d 205.60 (1C, COCH3),
171.03, 167.22, 167.13, 166.11, 165.33, 164.74, 164.54, 163.85,
156.18 (9C, GlcA C-6, C@O), 136.51–127.72 (42C, Ar-C), 101.96
(1C, GlcA C-1), 101.55 (1C, GalI C-1), 100.81 (1C, GalII C-1), 80.21
(1C, GalII C-3), 78.38 (1C, GalI C-3), 72.27 (1C, GalII C-5), 72.27
(1C, GlcA C-5), 71.73, 71.58, 71.24, 70.95 (4C, GalI C-2, GalII C-2,
GlcA C-2,3), 70.23 (1C, GalI C-4), 69.11, 69.02, 68.95 (3C, GalI C-5,
GalII C-4, GlcA C-4), 66.30 (1C, OCH2), 62.88 (1C, CH2-Ar), 62.82
(1C, GalI C-6), 62.63 (1C, GalII C-6), 53.76 (1C, COOCH3), 40.65
(1C, NCH2), 37.46 (1C, CH2CO), 29.48 (1C, COCH3), 27.51 (1C,
CH2CO). HRESIMS: Calcd for C76H73NNaO27 [M+Na]+: m/z
1454.42622. Found: 1454.42746. Anal. Calcd for C76H73NO27: C,
63.73; H, 5.14; N, 0.98. Found: C, 63.79; H, 5.07; N, 0.89.
C
72H64Cl5NNaO27 [M+Na]+: m/z 1572.20006. Found: 1572.19878.
Anal. Calcd for C72H64Cl5NO27: C, 55.70; H, 4.15; N, 0.90. Found:
C, 55.31; H, 4.11; N, 0.81.
4.27. 2-Benzyloxycarbonylaminoethyl O-(methyl 2,3-di-O-
benzoyl-4-O-levulinoyl-b-
D
-glucopyranosyluronate)-(1?3)-(2-
-galactopyranosyl)-(1?3)-
D-galactopyranoside (35)
O-benzoyl-4,6-di-O-chloroacetyl-b-
2,4,6-tri-O-benzoyl-b-
D
4.27.1. Procedure A
A mixture of disaccharide imidate 9 (129 mg, 0.12 mmol) and
alcohol 30 (130 mg, 0.19 mmol) was treated as described for the
preparation of 18. Flash silica chromatography (3:2 EtOAc/petro-
leum ether, containing 0.1% of Et3N) gave 35 (92 mg, 48%) as a
white solid: mp 166–168 °C (from EtOAc/petroleum ether); ½a D20
ꢂ
+48.5 (c 1, CHCl3); 1H NMR (CDCl3, 400 MHz): d 8.05–6.96 (m,
35H, Ar-H), 5.76 (dd, 1H, J3,4 3.0, J4,5 <1 Hz, GalI H-4), 5.45 (dd,
1H, J3,4 3.0, J4,5 <1 Hz, GalII H-4), 5.42 (dd, 1H, J1,2 8.0, J2,3 9.5 Hz, GalI
H-2), 5.36–5.33 (m, 2H, GlcA H-3,4), 5.16–5.11 (m, 2H, GalII H-2,
GlcA H-2), 4.98 (br s, 1H, NH), 4.88 (ABq, 2H, CH2-Ar), 4.74 (d,
1H, J1,2 8.0 Hz, GalII H-1), 4.65 (d, 1H, J1,2 7.5 Hz, GlcA H-1), 4.56
(dd, 1H, J5,6a 5.0, J6a,6b 12.0 Hz, GalI H-6a), 4.51 (d, 1H, GalI H-1),
4.37 (dd, 1H, J5,6b 6.0 Hz, GalI H-6b), 4.20–4.17 (m, 2H, GalII H-
6a,6b), 4.14 (s, 2H, CH2Cl), 4.11–4.05 (m, 2H, GalI H-5, GalII H-3),
4.03–3.98 (m, 2H, GalI H-3, GlcA H-5), 3.97 (ABq, 2H, CH2Cl),
3.93–3.90 (m, 1H, GalII H-5), 3.80–3.76 (m, 1H, OCH2), 3.77 (s,
3H, COOCH3), 3.55–3.50 (m, 1H, OCH2), 3.29–3.14 (m, 2H, NCH2),
2.63–2.61 (m, 3H, CH2CO), 2.33–2.26 (m, 1H, CH2CO), 2.00 (s, 3H,
4.29. 2-Benzyloxycarbonylaminoethyl O-(methyl 2,3-di-O-
benzoyl-4-O-levulinoyl-b-
(2,6-di-O-benzoyl-b- -galactopyranosyl)-(1?3)-2,4,6-tri-O-
benzoyl-b- -galactopyranoside (36)
D-glucopyranosyluronate)-(1?3)-
D
D
Compound 7 (202 mg, 0.14 mmol) was treated as described for
the preparation of 23. Flash silica chromatography (12:1 CH2Cl2/
acetone) gave alcohol 36 (175 mg, 80%) as a white solid: mp
COCH3); 13C NMR (CDCl3, 100 MHz):
d 205.57 (1C, COCH3),
170.87, 166.935, 166.82, 166.69, 166.17, 165.88, 165.38, 164.59,
164.36, 163.57, 156.19 (11C, GlcA C-6, C@O), 136.51–127.85
(42C, Ar-C), 101.36 (1C, GlcA C-1), 101.03 (1C, GalI C-1), 100.52
(1C, GalII C-1), 77.02 (1C, GalI C-3), 75.52 (1C, GalII C-3), 72.55
(1C, GlcA C-5), 72.02, 71.73 (2C, GalI C-2,5), 71.29, 71.24, 70.72,
70.69 (4C, GalII C-2,4,5, GlcA C-2), 70.57 (1C, GalI C-4), 70.07,
148–150 °C (from EtOAc/petroleum ether);); ½a D20
ꢂ
+47.5 (c 1,
CHCl3); 1H NMR (CDCl3, 400 MHz): d 8.09–6.91 (m, 40H, Ar-H),
5.80 (dd, 1H, J3,4 3.0, J4,5 <1 Hz, GalI H-4), 5.46–5.33 (m, 3H, GalI
H-2, GlcA H-3,4), 5.30–5.21 (m, 2H, GalII H-2, GlcA H-2), 4.99
(br s, 1H, NH), 4.88 (ABq, 2H, CH2-Ar), 4.81 (d, 1H, J1,2 7.0 Hz, GalII

K. Aït-Mohand et al. / Carbohydrate Research 353 (2012) 33–48
45
H-1), 4.70 (d, 1H, J1,2 8.0 Hz, GlcA H-1), 4.62–4.59 (m, 2H, GalI H-6a,
GalII H-6a), 4.46–4.38 (m, 3H, GalI H-1, GalI H-6b, GalII H-6b), 4.20–
4.16 (m, 2H, GalI H-3, GalII H-4), 4.09 (d, 1H, J4,5 10.0 Hz, GlcA H-5),
3.96–3.93 (m, 1H, GalI H-5), 3.86–3.75 (m, 3H, GalII H-3,5, OCH2),
3.66 (s, 3H, COOCH3), 3.52–3.47 (m, 1H, OCH2), 3.25–3.13 (m, 2H,
NCH2), 2.89 (br s, 1H, GalII HO-4), 2.63–2.44 (m, 3H, CH2CO),
2.36–2.29 (m, 1H, CH2CO), 2.02 (s, 3H, COCH3); 13C NMR (CDCl3,
100 MHz): d 205.60 (1C, COCH3), 167.01, 166.23, 166.04, 165.55,
165.36, 164.58, 164.51, 163.88, 156.21 (10C, GlcA C-6, C@O),
136.54–127.77 (48C, Ar-C), 101.46 (1C, GalI C-1), 100.99, 100.90
(2C, GalII C-1, GlcA C-1), 80.27 (1C, GalII C-3), 75.30 (1C, GalI C-3),
72.27 (1C, GlcA C-5), 72.15, 71.96, 71.63 (3C, GalI C-5, GalII C-5,
GlcA C-3), 71.19, 70.43 (2C, GalII C-2, GlcA C-2), 69.97 (1C, GalI
C-4), 69.15 (2C, GalI C-2, GlcA C-4), 68.88 (1C, OCH2), 68.17 (1C,
GalII C-4), 66.33 (1C, CH2-Ar), 62.99, 62.78 (2C, GalI C-6, GalII
C-6)), 53.03 (1C, COOCH3), 40.68 (1C, NCH2), 37.48 (1C, CH2CO),
29.51 (1C, COCH3), 27.53 (1C, CH2CO). HRESIMS: Calcd for
GlcA H-1), 4.87–4.77 (m, 4H, GalI H-6a, GalII H-1, CH2-Ar), 4.64
(dd, 1H, J5,6b 4.5, J6a,6b 12.0 Hz, GalI H-6b), 4.58 (d, 1H, J1,2 8.0 Hz,
GalI H-1), 4.36 (d, 1H, GlcA H-5), 4.33 (dd, 1H, J2,3 11.0 Hz, GalI
H-3), 4.26–4.13 (m, 4H, GalI H-5, GalII H-3,6a,6b), 3.99–3.96 (m,
1H, GalII H-5), 3.74–3.70 (m, 4H, OCH2, COOCH3), 3.54–3.49 (m,
1H, OCH2), 3.12–3.09 (m, 2H, NCH2), 2.59–2.55 (m, 2H, CH2CO),
2.46–2.38 (m, 1H, CH2CO), 2.35–2.28 (m, 1H, CH2CO), 1.95 (s, 3H,
COCH3); 13C NMR (CD3OD/CDCl3, 100 MHz): d 208.38 (1C, COCH3),
172.89, 169.40, 167.75, 167.48, 167.11, 166.84, 166.80, 166.61,
166.35, 158.46 (10C, GlcA C-6, C@O), 138.12–128.65 (48C, Ar-C),
102.97 (1C, GalII C-1), 102.63, 102.57 (2C, GalI C-1, GlcA C-1),
78.98 (1C, GalII C-3), 78.12 (1C, GalI C-3), 78.09 (1C, GalII C-4),
77.06 (1C, GalI C-5), 73.85 (1C, GlcA C-3), 73.32, 73.09 (2C, GalII
C-5, GlcA C-5), 73.06, 73.01 (2C, GalI C-2, GlcA C-2), 72.41 (1C, GalI
C-4), 72.09 (1C, GalII C-2), 70.60 (1C, GlcA C-4), 69.54 (1C, OCH2),
67.28 (1C, CH2-Ar), 65.11 (1C, GalI C-6), 64.30 (1C, GalII C-6),
53.59 (1C, COOCH3), 41.59 (1C, NCH2), 38.29 (1C, CH2CO), 29.47
C
83H77NNaO28 [M+Na]+: m/z 1558.45243. Found: 1558.45250.
(1C, COCH3), 28.73 (1C, CH2CO). HRESIMS: Calcd for C83H76NO31
S
Anal. Calcd for C83H77NO28: C, 64.88; H, 5.05; N, 0.91. Found: C,
64.61; H, 4.99; N, 0.80.
[MꢄNa]ꢄ: m/z 1614.41275. Found: 1614.41567.
4.32. 2-Benzyloxycarbonylaminoethyl O-(sodium b-
glucopyranosyluronate)-(1?3)-(b- -galactopyranosyl)-(1?3)-
b- -galactopyranoside (39)
D-
4.30. 2-Benzyloxycarbonylaminoethyl O-(methyl 2,3-di-O-
D
benzoyl-4-O-levulinoyl-b-
D-glucopyranosyluronate)-(1?3)-(4-
D
O-acetyl-2-O-benzoyl-6-O-sodium sulfonato-b-
D
-
galactopyranosyl)-(1?3)-2,4,6-tri-O-benzoyl-b-
D
-
A solution of 6 (191 mg, 0.13 mmol) in THF/water (3:1, 3 mL)
galactopyranoside (37)
was treated at ꢄ10 °C with a freshly prepared mixture of H2O2
(30 wt % in water, 0.35 mL) and LiOH (1 M, 0.7 mL), and the mix-
ture was stirred for 8 h at rt, then was cooled to 0 °C. Sodium
hydroxide (4 M, 2.3 mL) was added, and the mixture was stirred
overnight at rt, then was adjusted to pH 3.5 (pH meter monitoring)
with Amberlite IR-120 [H+] resin, filtered, concentrated, and dried.
The residue was extracted several times with EtOH, then was taken
up in water (2 mL), and the pH of the solution was adjusted to pH
8.5 with 0.1 M NaOH. The solution was eluted from a column
(3 ꢃ 80 cm) of Sephadex LH-20 with water to give the sodium salt
39 (66 mg, 69%) as a white powder: R = 0.42 (4:2:1 EtOAc/MeOH/
Compound 7 (174 mg, 0.12 mmol) was treated as described for
the preparation of 24 to give the sodium salt 37 (145 mg, 76%) as a
white powder: ½a D20
ꢂ
+53.5 (c 1, CHCl3); 1H NMR (CD3OD/CDCl3,
400 MHz): d 8.01–6.94 (m, 35H, Ar-H), 5.82 (dd, 1H, J3,4 3.0,
J4,5 <1 Hz, GalI H-4), 5.49 (dd, 1H, J3,4 3.0, J4,5 <1 Hz, GalII H-4),
5.45–5.37 (m, 2H, GalI H-2, GlcA H-3), 5.25 (dd, 1H, J3,4 9.5, J4,5
10.0 Hz, GlcA H-4), 5.13–5.09 (m, 2H, GalII H-2, GlcA H-2), 4.84
(ABq, 2H, CH2-Ar), 4.78, 4.76 (2d, 2H, J1,2 7.5 Hz, GalII H-1, GlcA
H-1), 4.63 (d, 1H, J1,2 8.0 Hz, GalI H-1), 4.50–4.46 (m, 1H, GalI H-
6a), 4.40–4.35 (m, 2H, GalI H-3,6b), 4.24–4.21 (m, 1H, GalI H-5),
4.14 (d, 1H, GlcA H-5), 4.10–4.03 (m, 3H, GalII H-5,6a,6b), 3.97
(dd, 1H, J2,3 10.0 Hz, GalII H-3), 3.80–3.75 (m, 4H, OCH2, COOCH3),
3.56–3.53 (m, 1H, OCH2), 3.18–3.16 (m, 2H, NCH2), 2.59–2.28 (m,
4H, CH2CO), 1.99 (s, 6H, COCH3, OCOCH3); 13C NMR (CD3OD/CDCl3,
100 MHz): d 207.68 (1C, COCH3), 172.25, 171.95, 168.75, 167.30,
167.14, 166.28, 166.02, 165.56, 165.03 (10C, GlcA C-6, C@O),
137.20–128.27 (42C, Ar-C), 102.08 (1C, GalI C-1), 101.91, 101.39
(2C, GalII C-1, GlcA C-1), 78.03 (1C, GalII C-3), 76.88 (1C, GalI C-3),
72.87, 72.74, 72.57 (3C, GalI C-5, GalII C-5, GlcA C-5), 72.51, 72.42
(2C, GalI C-2, GlcA C-3), 72.04 (1C, GalI C-4), 71.99, 71.53 (2C, GalII
C-2, GlcA C-2), 70.30 (1C, GlcA C-4), 70.14 (1C, GalII C-4), 69.37 (1C,
OCH2), 66.98 (1C, CH2-Ar), 66.25 (1C, GalII C-6), 64.07 (1C, GalI C-6),
53.62 (1C, COOCH3), 41.20 (1C, NCH2), 37.98 (1C, CH2CO), 29.59
(1C, COCH3), 28.22 (1C, CH2CO), 20.65 (1C; OCOCH3). HRESIMS:
water); ½a 2D0
ꢂ
ꢄ4 (c 1, water); 1H NMR (D2O, internal HOD, dH
4.79, 400 MHz): d 7.48–7.40 (m, 5H, Ar-H), 5.14 (s, 2H, CH2-Ar),
4.69 (d, 1H, J1,2 8.0 Hz, GlcA H-1), 4.67 (d, 1H, J1,2 8.0 Hz, GalII
H-1), 4.43 (d, 1H, J1,2 7.5 Hz, GalI H-1), 4.21, 4.19 (2dd, 2H, J3,4
2.0, J4,5 <1 Hz, GalI H-4, GalII H-4), 3.98–3.93 (m, 1H, OCH2), 3.84–
3.80 (m, 2H, GalI H-3, GalII H-3), 3.80–3.65 (m, 10H, GalI H-
2,5,6a,6b, GalII H-2,5,6a,6b, GlcA H-5, OCH2), 3.54–3.52 (m, 2H,
GlcA H-3,4), 3.44 (dd, 1H, J2,3 9.0 Hz, GlcA H-2), 3.42–3.37 (m,
2H, NCH2); 13C NMR (D2O, internal acetone, dC 30.83, 100 MHz):
d 176.55 (1C, GlcA C-6), 159.02 (1C, NHC(O)O), 137.11–128.38
(6C, Ar-C), 104.60 (1C, GalII C-1), 104.19 (1C, GlcA C-1), 103.31
(1C, GalI C-1), 83.01, 82.89 (2C, GalI C-3, GalII C-3), 76.80, 75.94,
75.41, 75.36, 73.78, 72.39, 70.78, 70.51 (8C, GalI C-2,5, GalII C-2,5,
GlcA C-2,3,4,5), 69.50 (1C, OCH2), 69.00, 68.65 (2C, GalI C-4, GalII
C-4), 67.55 (1C, CH2-Ar), 61.57, 61.48 (2C, GalI C-6, GalII C-6),
41.16 (1C, NCH2). HRESIMS: Calcd for C28H41NNaO19 [M+H]+: m/z
718.21650. Found: 718.21580.
Calcd for
C
78H74NO31
S
[MꢄNa]ꢄ: m/z 1552.39710. Found:
1552.39847.
4.31. 2-Benzyloxycarbonylaminoethyl O-(methyl 2,3-di-O-benzoyl
-4-O-levulinoyl-b- -glucopyranosyluronate)-(1?3)-(2,6-di-O-ben
zoyl-4-O-sodium sulfonato-b- -galactopyranosyl)-(1?3)-2,4,6-tri-
O-benzoyl-b- -galactopyranoside (38)
4.33. 2-Benzyloxycarbonylaminoethyl O-(sodium b-
glucopyranosyluronate)-(1?3)-(b- -galactopyranosyl)-(1?3)-
6-O-sodium sulfonato-b- -galactopyranoside (40)
D-
D
D
D
D
D
Compound 24 (203 mg, 0.13 mmol) was treated as described for
the preparation of 39 to give the disodium salt 40 (91 mg, 86%) as a
Compound 36 (148 mg, 98 lmol) was treated as described for
the preparation of 25 to give the sodium salt 38 (122 mg, 77%) as
white powder: R = 0.27 (4:2:1 EtOAc/MeOH/water); ½a D20
ꢄ34 (c 1,
ꢂ
a white powder: ½a D20
ꢂ
+48 (c 1, MeOH); 1H NMR (CD3OD/CDCl3,
water); 1H NMR (D2O, internal HOD, dH 4.79, 400 MHz): d 7.46–
7.42 (m, 5H, Ar-H), 5.15 (s, 2H, CH2-Ar), 4.70 (d, 1H, J1,2 7.5 Hz, GlcA
H-1), 4.67 (d, 1H, J1,2 7.5 Hz, GalII H-1), 4.45 (d, 1H, J1,2 8.0 Hz, GalI
H-1), 4.25, 4.21 (2dd, 2H, J3,4 3.0, J4,5 <1 Hz, GalI H-4, GalII H-4),
4.24–4.19 (m, 2H, GalI H-6a,6b), 3.98–3.90 (m, 2H, GalI H-3,
400 MHz): d 8.18–6.97 (m, 40H, Ar-H), 5.81 (dd, 1H, J3,4 3.0,
J4,5 <1 Hz, GalI H-4), 5.34 (dd, 1H, J2,3 9.0, J3,4 9.5 Hz, GlcA H-3),
5.44 (dd, 1H, J4,5 10.0 Hz, GlcA H-4), 5.41–5.37 (m, 2H, GalI H-2,
GlcA H-2), 5.17–5.12 (m, 2H, GalII H-2,4), 4.99 (d, 1H, J1,2 7.5 Hz,

46
K. Aït-Mohand et al. / Carbohydrate Research 353 (2012) 33–48
OCH2), 3.85–3.65 (m, 9H, GalI H-2,5, GalII H-2,3,5,6a,6b, GlcA H-5,
OCH2), 3.57–3.51 (m, 2H, GlcA H-3,4), 3.46 (dd, 1H, J2,3 9.0 Hz, GlcA
H-2), 3.42–3.36 (m, 2H, NCH2); 13C NMR (D2O, internal acetone, dC
30.83, 100 MHz): d 176.52 (1C, GlcA C-6), 150.02 (1C, NHC(O)O),
137.16–128.40 (6C, Ar-C), 104.66 (1C, GalII C-1), 104.21 (1C, GlcA
C-1), 103.24 (1C, GalI C-1), 83.04, 82.71 (2C, GalI C-3, GalII C-3),
75.94, 75.40, 73.78, 72.99, 72.39, 70.83, 70.78, 70.39 (8C, GalI C-
2,5, GalII C-2,5, GlcA C-2,3,4,5), 69.56 (1C, OCH2), 68.76, 68.65
(2C, GalI C-4, GalII C-4), 67.75 (1C, GalI C-6), 67.54 (1C, CH2-Ar),
61.54 (1C, GalII C-6), 41.20 (1C, NCH2). HRESIMS: Calcd for
white powder: R = 0.28 (4:2:1 EtOAc/MeOH/water); ½a D20
ꢄ63 (c 1,
ꢂ
water); 1H NMR (D2O, internal HOD, dH 4.79, 400 MHz): d 7.50–
7.44 (m, 5H, Ar-H), 5.13 (s, 2H, CH2-Ar), 4.79–4.77 (m, 2H, GalI
H-1, GalII H-4), 4.68 (d, 1H, J1,2 7.5 Hz, GalII H-1), 4.42 (d, 1H, J1,2
7.5 Hz, GlcA H-1), 4.17 (dd, 1H, J3,4 2.0, J4,5 <1 Hz, GalI H-4), 4.06
(dd, 1H, J2,3 10.0, J3,4 3.0 Hz, GalII H-3), 3.96–3.92 (m, 1H, OCH2),
3.85–3.66 (m, 11H, GalI H-2,3,5,6a,6b, GalII H-2,5,6a,6b, GlcA H-5,
OCH2), 3.57–3.49 (m, 2H, GlcA H-3,4), 3.42 (dd, 1H, J2,3 9.0 Hz, GlcA
H-2), 3.40–3.33 (m, 2H, NCH2); 13C NMR (D2O, internal acetone, dC
30.83, 100 MHz): d 176.55 (1C, GlcA C-6), 159.02 (1C, NHC(O)O),
137.09–128.32 (6C, Ar-C), 104.71 (1C, GalII C-1), 103.71, 103.29
(2C, GalI C-1, GlcA C-1), 83.00 (1C, GalII C-3), 77.81 (1C, GalII C-4),
77.16 (1C, GalI C-3), 76.77, 75.96, 75.35, 75.07, 73.69, 72.46,
71.62, 70.50 (8C, GalI C-2,5, GalII C-2,5, GlcA C-2,3,4,5), 69.48 (1C,
OCH2), 68.90 (1C, GalI C-4), 67.52 (1C, CH2-Ar), 61.52, 61.48 (2C,
GalI C-6, GalII C-6), 41.12 (1C, NCH2). HRESIMS: Calcd for
C28H39NNaO22S [MꢄNa]ꢄ: m/z 796.15876. Found: 796.15886.
C
28H39NNaO22S [MꢄNa]ꢄ: m/z 796.15876. Found: 796.15841.
4.34. 2-Benzyloxycarbonylaminoethyl O-(sodium b-
glucopyranosyluronate)-(1?3)-(b- -galactopyranosyl)-(1?3)-
4-O-sodium sulfonato-b- -galactopyranoside (41)
D-
D
D
Compound 25 (167 mg, 0.1 mmol) was treated as described for
the preparation of 39 to give the disodium salt 41 (68 mg, 80%) as a
white powder: R = 0.28 (4:2:1 EtOAc/MeOH/water); ½a D20
ꢂ
ꢄ14 (c 1,
4.37. 2-(6-Biotinylamidohexanoyl)aminoethyl O-(sodium b-
glucopyranosyluronate)-(1?3)-(b- -galactopyranosyl)-(1?3)-
b- -galactopyranoside (1)
D-
water); 1H NMR (D2O, internal HOD, dH 4.79, 400 MHz): d 7.47–
7.42 (m, 5H, Ar-H), 5.14 (s, 2H, CH2-Ar), 4.86 (dd, 1H, J3,4 3.0,
J4,5 <1 Hz, GalI H-4), 4.70 (d, 1H, J1,2 7.5 Hz, GlcA H-1), 4.69 (d,
1H, J1,2 7.5 Hz, GalII H-1), 4.46 (d, 1H, J1,2 7.5 Hz, GalI H-1), 4.18
(dd, 1H, J3,4 2.0, J4,5 <1 Hz, GalII H-4), 4.03–3.94 (m, 2H, GalI H-3,
OCH2), 3.85–3.68 (m, 11H, GalI H-2,5,6a,6b, GalII H-2,3,5,6a,6b,
GlcA H-5, OCH2), 3.56–3.51 (m, 2H, GlcA H-3,4), 3.45 (dd, 1H, J2,3
9.0 Hz, GlcA H-2), 3.42–3.39 (m, 2H, NCH2); 13C NMR (D2O, internal
acetone, dC 30.83, 100 MHz): d 176.63 (1C, GlcA C-6), 159.12 (1C,
NHC(O)O), 137.21–128.40 (6C, Ar-C), 104.65, 104.37 (2C, GalII C-
1, GlcA C-1), 103.45 (1C, GalI C-1), 83.03 (1C, GalI C-3), 79.13 (1C,
GalII C-3), 78.11 (1C, GalI C-4), 76.88, 75.99, 75.61, 75.18, 73.83,
72.47, 71.07, 70.84 (8C, GalI C-2,5, GalII C-2,5, GlcA C-2,3,4,5),
69.75 (1C, OCH2), 69.08 (1C, GalII C-4), 67.62 (CH2-Ar), 62.76,
62.61 (2C, GalI C-6, GalII C-6), 42.18 (1C, NCH2). HRESIMS: Calcd
for C28H39NNaO22S [MꢄNa]ꢄ: 796.15876. Found: 796.15884.
D
D
A solution of 39 (23.5 mg, 33 mol) in water (8 mL) was hydroge-
nated in the presence of 10% Pd/C catalyst (20 mg) overnight at rt.
The catalyst was removed by filtration through a Celite pad, and
the filtrate was concentrated to give the intermediate amine. A
solution of the amine and 6-biotinylamidohexanoic acid N-
hydroxysuccinimidoyl ester (45 mg, 98 mol) in DMF/water (9:1,
0.85 mL) was stirred for 1 h at rt, then was concentrated. The
resulting solid was washed several times with absolute EtOH,
and a solution of the residue in water (2 mL) was eluted from a col-
umn (3 ꢃ 80 cm) of Sephadex LH-20 with water and lyophilized to
afford biotinylated 1 (19.3 mg, 64%) as a white powder: R = 0.32
(2:1:1 EtOAc/MeOH/water); ½a D20
ꢂ
+17 (c 1, water); 1H NMR (D2O,
internal HOD, dH 4.79, 400 MHz): d 4.70 (d, 1H, J1,2 8.0 Hz, GalII
H-1), 4.68 (d, 1H, J1,2 8.0 Hz, GlcA H-1), 4.62 (ddd, 1H, Jb,c 8.0
Jc,d <1.0, Jc,d 5.0 Hz, H-c), 4.48 (d, 1H, J1,2 8.0 Hz, GalI H-1), 4.44
0
4.35. 2-Benzyloxycarbonylaminoethyl O-(sodium b-
glucopyranosyluronate)-(1?3)-(6-O-sodium sulfonato-b-
galactopyranosyl)-(1?3)-b- -galactopyranoside (42)
D
-
D
-
(dd, 1H, Jb,e 4.5 Hz, H-b), 4.22–4.20 (m, 2H, GalI H-4, GalII H-4),
4.02–3.97 (m, 1H, OCH2), 3.86–3.69 (m, 12 H, GalI H-2,3,5,6a,6b,
GalII H-2,3,5,6a,6b, GlcA H-5, OCH2), 3.54–3.52 (m, 2H, GlcA H-
3,4), 3.48–3.43 (m, 3H, GlcA H-2, NCH2), 3.38–3.34 (m, 1H, H-e),
D
Compound 37 (135 mg, 85 lmol) was treated as described for
3.21–3.18 (m, 2H, NCH2), 3.01 (dd, 1H, Jd,d 13.0 Hz, H-d0), 2.80
0
the preparation of 39 to give the disodium salt 42 (56.5 mg, 80%)
as a white powder: R = 0.32 (4:2:1 EtOAc/MeOH/water); ½a D20
ꢂ
+5
(dd, 1H, H-d), 2.31–2.28 (m, 4H, CH2CO), 1.79–1.29 (m, 12H,
CCH2); 13C NMR (D2O, internal acetone, dC 30.83, 100 MHz): d
177.75, 177.24, 176.54, (3C, GlcA C-6, C@O), 165.96 (1C, C-a),
104.62, 104.23 (2C, GalI C-1, GalII C-1), 103.25 (1C, GlcA C-1),
83.04, 82.96 (2C, GalI C-3, GalII C-3), 76.88, 76.00, 75.48, 73.83,
72.45, 70.85 (8C, GalI C-2,5, GalII C-2,5, GlcA C-2,3,4,5), 69.11 (1C,
OCH2), 69.08, 68.70, (2C, GalI C-4, GalII C-4), 62.77 (1C, C-b),
61.63 (2C, GalI C-6, GalII C-6), 60.93 (1C, C-c), 56.08 (1C, C-e),
40.39 (1C, C-d), 39.98, 39.78 (2C, NCH2), 36.30, 36.22 (2C, CH2CO),
28.72, 28.54, 28.37, 26.24, 25.91, 25.65 (6C, CCH2). HRESIMS: Calcd
for C36H59N4O20S [MꢄNa]ꢄ: m/z 899.34488. Found: 899.34481.
(c 1, water); 1H NMR (D2O, internal HOD, dH 4.79, 400 MHz): d
7.46–7.42 (m, 5H, Ar-H), 5.14 (s, 2H, CH2-Ar), 4.69 (d, 1H, J1,2
8.0 Hz, GlcA H-1), 4.65 (d, 1H, J1,2 7.0 Hz, GalII H-1), 4.42 (d, 1H,
J1,2 7.5 Hz, GalI H-1), 4.26–4.25 (m, 2H, GalI H-4, GalII H-4), 4.22–
4.20 (m, 2H, GalII H-6a,6b), 3.98–3.92 (m, 2H, GalII H-5, OCH2),
3.86–3.65 (m, 9H, GalI H-2,3,5,6a,6b, GalII H-2,3, GlcA H-5, OCH2),
3.54–3.52 (m, 2H, GlcA H-3,4), 3.45 (dd, 1H, J2,3 9.0 Hz, GlcA H-
2), 3.42–3.36 (m, 2H, NCH2); 13C NMR (D2O, internal acetone, dC
30.83, 100 MHz): d 172.55 (1C, GlcA C-6), 159.02 (1C, NHC(O)O),
138.39–129.35 (6C, Ar-C), 104.41, 104.23 (2C, GalII C-1, GlcA C-1),
103.25 (1C, GalI C-1), 83.33, 82.82 (2C, GalI C-3, GalII C-3), 75.95,
75.63, 73.80, 73.16, 72.42, 70.66, 70.37, 70.11 (8C, GalI C-2,5, GalII
C-2,5, GlcA C-2,3,4,5), 69.49 (1C, OCH2), 69.08, 68.58 (2C, GalI C-4,
GalII C-4), 68.49 (1C, GalII C-6), 67.57 (1C, CH2-Ar), 61.86 (1C, GalI
4.38. 2-(6-Biotinylamidohexanoyl)aminoethyl O-(sodium b-
glucopyranosyluronate)-(1?3)-(b- -galactopyranosyl)-(1?3)-
6-O-sodium sulfonato-b- -galactopyranoside (2)
D-
D
D
C-6), 41.05 (1C, NCH2). HRESIMS: Calcd for C28H41NNaO22
S
[MꢄNa+2H]+: m/z 798.17331. Found: 798.17149.
Compound 40 (32.8 mg, 40 lmol) was treated as described for
the preparation of 1 to give biotinylated 2 (29.2 mg, 71%) as a
4.36. 2-Benzyloxycarbonylaminoethyl O-(sodium b-
glucopyranosyluronate)-(1?3)-(4-O-sodium sulfonato-b-
galactopyranosyl)-(1?3)-b- -galactopyranoside (43)
D
-
white powder: R = 0.39 (3:2:1 EtOAc/MeOH/water); ½a D20
ꢂ
+19 (c 1,
D
-
water); 1H NMR (D2O, internal HOD, dH 4.79, 400 MHz): d 4.70
(d, 1H, J1,2 8.0 Hz, GalII H-1), 4.68 (d, 1H, J1,2 8.0 Hz, GlcA H-1),
D
0
4.63 (ddd, 1H, Jc,d <1.0, Jc,d 5.0, Jb,c 8.00 Hz, H-c), 4.50 (d, 1H, J1,2
Compound 38 (113.5 mg, 70
the preparation of 39 to give the disodium salt 43 (50 mg, 88%) as a
l
mol) was treated as described for
8.0 Hz, GalI H-1), 4.44 (dd, 1H, Jb,e 4.5 Hz, H-b), 4.27 (dd, 1H, J3,4
3.0, J4,5 <1 Hz, GalI H-4), 4.24 (dd, 1H, J3,4 3.0, J4,5 <1 Hz, GalII

K. Aït-Mohand et al. / Carbohydrate Research 353 (2012) 33–48
47
H-4), 4.23–4.21 (m, 2H, GalI H-6a,6b), 3.99-3.96 (m, 2H, GalI H-5,
OCH2), 3.88–3.69 (m, 9 H, GalI H-2,3, GalII H-2,3,5,6a,6b, GlcA H-
5, OCH2), 3.55–3.53 (m, 2H, GlcA H-3,4), 3.48–3.43 (m, 3H, GlcA
H-2, NCH2), 3.36–3.33 (m, 1H, H-e), 3.22–3.18 (m, 2H, NCH2),
(1C, C-a), 104.38, 104.22 (2C, GalII C-1, GlcA C-1), 103.16 (1C,
GalI C-1), 83.34, 82.85 (2C, GalI C-3, GalII C-3), 75.88, 75.60,
73.71, 73.08, 72.32, 70.59, 70.32, 70.25 (8C, GalI C-2,5, GalII C-
2,5, GlcA C-2,3,4,5), 69.01 (1C, OCH2), 68.50 (2C, GalI C-4, GalII
C-4), 68.38 (1C, GalII C-6), 62.68 (1C, C-b), 61.86 (1C, GalI C-6),
60.84 (1C, C-c), 55.98 (1C, C-e), 40.30 (1C, C-d), 39.90, 39.68
(2C, NCH2), 36.20, 36.12 (2C, CH2CO), 28.61, 28.45, 28.27,
26.14, 25.81, 25.55 (6C, CCH2). HRESIMS: Calcd for
3.02 (dd, 1H, Jd,d 13.0 Hz, H-d0), 2.80 (dd, 1H, H-d), 2.31–2.25 (m,
0
4H, CH2CO), 1.79–1.32 (m, 12H, CCH2); 13C NMR (D2O, internal ace-
tone, dC 30.83, 100 MHz): d 177.67, 177.18 (3C, GlcA C-6, C@O),
165.88 (1C, C-a), 104.57, 104.14 (2C, GalII C-1, GlcA C-1), 103.12
(1C, GalI C-1), 82.96, 82.67 (2C, GalI C-3, GalII C-3), 75.86, 75.33,
73.70, 73.00, 72.32, 70.71, 70.32 (8C, GalI C-2,5, GalII C-2,5, GlcA
C-2,3,4,5), 69.24 (1C, OCH2), 68.73, 68.57 (2C, GalI C-4, GalII C-4),
67.87 (1C, GalI C-6), 62.64 (1C, C-b), 61.47 (1C, GalII C-6), 60.81
(1C, C-c), 55.93 (1C, C-e), 40.27 (1C, C-d), 39.92, 39.65 (2C,
NCH2), 36.18, 36.10 (2C, CH2CO), 28.55, 28.40, 28.23, 26.09,
25.79, 25.43 (6C, CCH2). HRESIMS: Calcd for C36H58N4O23S2
[Mꢄ2Na]2ꢄ: m/z 489.14721. Found: 489.14818.
C
36H58N4NaO23S2 [MꢄNa]ꢄ: m/z 1001.28364. Found: 1001.28399.
4.41. 2-(6-Biotinylamidohexanoyl)aminoethyl O-(sodium b-
glucopyranosyluronate)-(1?3)-(4-O-sodium sulfonato-b-
galactopyranosyl)-(1?3)-b- -galactopyranoside (5)
D-
D-
D
Compound 43 (33.4 mg, 40.7 lmol) was treated as described for
the preparation of 1 to give biotinylated 5 (25 mg, 60%) as a white
powder: R = 0.30 (3:2:1 EtOAc/MeOH/water); ½a D20
ꢂ
+12 (c 1, water);
4.39. 2-(6-Biotinylamidohexanoyl)aminoethyl O-(sodium b-
glucopyranosyluronate)-(1?3)-(b- -galactopyranosyl)-(1?3)-
4-O-sodium sulfonato-b- -galactopyranoside (3)
D-
1H NMR (D2O, internal HOD, dH 4.79, 400 MHz): d 4.81 (dd, 1H, J3,4
2.0, J4,5 <1 Hz, GalII H-4), 4.79 (d, 1H, J1,2 7.5 Hz, GlcA H-1), 4.70 (d,
D
1H, J1,2 8.0 Hz, GalII H-1), 4.62 (ddd, 1H, Jc,d <1.0, Jc,d 5.0, Jb,c 7.5 Hz,
0
D
H-c), 4.47 (d, 1H, J1,2 8.0 Hz, GalI H-1), 4.43 (dd, 1H, Jb,e 5.0 Hz, H-b),
4.19 (dd, 1H, J3,4 2.0, J4,5 <1 Hz, GalI H-4), 4.06 (dd, 1H, J2,3 10.0, GalII
H-3), 4.00–3.95 (m, 1H, OCH2), 3.85-3.69 (m, 11H, GalI H-
2,3,5,6a,6b, GalII H-2,5,6a,6b, GlcA H-5, OCH2), 3.58–3.49 (m, 2H,
GlcA H-3,4), 3.48–3.36 (m, 3H, GlcA H-2, NCH2), 3.35–3.33 (m,
Compound 41 (32.8 mg, 40 lmol) was treated as described for
the preparation of 1 to give biotinylated 3 (32.5 mg, 79%) as a
white powder: R = 0.24 (3:2:1 EtOAc/MeOH/water); ½a D20
ꢂ
+16 (c 1,
water); 1H NMR (D2O, internal HOD, dH 4.79, 400 MHz): d 4.88
(dd, 1H, J3,4 2.5, J4,5 <1 Hz, GalI H-4), 4.71 (d, 1H, J1,2 8.0 Hz, GlcA
0
1H, H-e), 3.20–3.16 (m, 2H, NCH2), 3.00 (dd, 1H, Jd,d 13.0 Hz, H-
H-1), 4.69 (d, 1H, J1,2 8.0 Hz, GalII H-1), 4.63 (ddd, 1H, Jc,d <1.0, Jc,d
d0), 2.79 (dd, 1H, H-d), 2.29–2.24 (m, 4H, CH2CO), 1.77–1.29 (m,
12H, CCH2). 13C NMR (D2O, internal acetone, dC 30.83, 100 MHz):
d 177.72, 177.69, 176.51 (3C, GlcA C-6, C@O), 165.89 (1C, C-a),
104.69 (1C, GalII C-1), 103.42 (1C, GlcA C-1), 103.19 (1C, GalI C-
1), 83.06 (1C, GalI C-3), 77.79 (1C, GalII C-4), 77.16 (1C, GalII C-3),
76.76, 75.95, 75.38, 75.05, 73.68, 72.45, 71.62, 70.49 (8C, GalI C-
2,5, GalII C-2,5, GlcA C-2,3,4,5), 69.00 (1C, GalI C-4), 68.90 (1C,
OCH2), 62.68 (1C, C-b), 61.59, 61.53 (2C, GalI C-6, GalII C-6), 60.84
(1C, C-c), 55.98 (1C, C-e), 40.30 (1C, C-d), 39.88, 39.68 (2C, NCH2),
36.20, 36.11 (2C, CH2CO), 28.61, 28.43, 28.27, 26.14, 25.81, 25.55
(6C, CCH2). HRESIMS: Calcd for C36H58N4NaO23S2 [MꢄNa]ꢄ: m/z
1001.28364. Found: 1001.28430.
0
5.5, Jb,c 7.5 Hz, H-c), 4.50 (d, 1H, J1,2 8.0 Hz, GalI H-1), 4.45 (dd, 1H,
Jb,e 4.5 Hz, H-b), 4.18 (dd, 1H, J3,4 2.0, J4,5 <1 Hz, GalII H-4), 4.04 (dd,
1H, J2,3 10.0 Hz, GalI H-3), 4.03–3.97 (m, 1H, OCH2), 3.85–3.68 (m,
11H, GalI H-2,5,6a,6b, GalII H-2,3,5,6a,6b, GlcA H-5, OCH2), 3.55–
3.52 (m, 2H, GlcA H-3,4), 3.47–3.41 (m, 3H, GlcA H-2, NCH2),
0
3.36–3.33 (m, 1H, H-e) 3.24–3.16 (m, 2H, NCH2), 3.02 (dd, 1H, Jd ,c
5.0, Jd,d 13.0 Hz, H-d0), 2.81 (dd, 1H, H-d), 2.31–2.25 (m, 4H,
0
CH2CO), 1.77–1.31 (m, 12H, CCH2); 13C NMR (D2O, internal acetone,
dC 30.83, 100 MHz): d 177.65, 177.12, 176.50 (3C, GlcA C-6, C@O),
165.85 (1C, C-a), 104.50, 104.22 (2C, GalII C-1, GlcA C-1), 103.16
(1C, GalI C-1), 82.89 (1C, GalII C-3), 79.03 (1C, GalI C-3), 77.99
(1C, GalI C-4), 75.84, 75.45, 75.06, 73.66, 72.31, 70.94, 70.69 (8C,
GalI C-2,5, GalII C-2,5, GlcA C-2,3,4,5), 69.07 (1C, OCH2), 68.92
(1C, GalII C-4), 62.64 (1C, C-b), 61.61, 61.53 (2C, GalI C-6, GalIIC-
6), 60.80 (1C, C-c), 55.95 (1C, C-e), 40.27 (1C, C-d), 39.82, 39.64
(2C, NCH2), 36.17, 36.08 (2C, CH2CO), 28.57, 28.41, 28.23, 26.08,
25.77, 25.51 (6C, CCH2). HRESIMS: Calcd for C36H58N4NaO23S2
[MꢄNa]ꢄ: m/z 1001.28364. Found: 1001.28269.
Acknowledgements
This research was supported by the Agence Nationale de la
Recherche (programs ANR-08-BLAN ‘Glycocart’ and ‘GAGNet-
work’). The authors are grateful for support by the Ministère de
l’Enseignement Supérieur et de la Recherche through a studentship
to K.A.-M.
4.40. 2-(6-Biotinylamidohexanoyl)aminoethyl O-(sodium b-
glucopyranosyluronate)-(1?3)-(6-O-sodium sulfonato-b-
galactopyranosyl)-(1?3)-b- -galactopyranoside (4)
D-
D
-
Supplementary data
D
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.carres.2012.03.
039.
Compound 42 (33.9 mg, 41 lmol) was treated as described
for the preparation of 1 to give biotinylated 4 (27.4 mg, 65%)
as a white powder: R = 0.37 (3:2:1 EtOAc/MeOH/water); ½a D20
ꢂ
+15 (c 1, water); 1H NMR (D2O, internal HOD, dH 4.79,
400 MHz): d 4.70 (d, 1H, J1,2 8.0 Hz, GalII H-1), 4.67 (d, 1H, J1,2
References
1. Sugahara, K.; Miyami, T.; Uyuma, T.; Mizoguchi, S.; Nomura, K.; Kitagawa, H.
Curr. Opin. Struct. Biol. 2003, 13, 612–620.
0
7.5 Hz, GlcA H-1), 4.62 (ddd, 1H, Jc,d <1.0, Jc,d 5.0, Jb,c 8.0 Hz, H-
c), 4.48 (d, 1H, J1,2 8.0 Hz, GalI H-1), 4.44 (dd, 1H, Jb,e 4.5 Hz,
H-b), 4.28, 4.24 (2dd, 2H, J3,4 2.5, J4,5 <1 Hz, GalI H-4, GalII H-
4), 4.22–4.20 (m, 2H, GalII H-6a,6b), 4.02–3.97 (m, 1H, OCH2),
3.95–3.92 (m, 1H, J5,6a 5.5, J5,6b 6.0 Hz, GalII H-5), 3.87–3.68 (m,
9H, GalI H-2,3,5,6a,6b, GalII H-2,3, GlcA H-5, OCH2), 3.55–3.52
(m, 2H, GlcA H-3,4), 3.48–3.41 (m, 3H, GlcA H-2, NCH2), 3.36–
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3.33 (m, 1H, H-e), 3.21–3.17 (m, 2H, NCH2), 3.01 (dd, 1H, Jd,d
13.0 Hz, H-d0), 2.80 (dd, 1H, H-d), 2.30–2.24 (m, 4H, CH2CO),
1.77–1.30 (m, 12H, CCH2); 13C NMR (D2O, internal acetone, dC
30.83, 100 MHz): d 177.70, 177.1 (3C, GlcA C-6, C@O), 165.90

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