Chemo-enzymatic supported synthesis of the 3-sulfated Lewisa pentasaccharide on a multimeric polyethylene glycol

Article abstract of DOI:10.1016/j.carres.2008.01.005

The 3-sulfated Lewis^a pentasaccharide was synthesized on multimeric-based polyethylene glycol support. Coupling of O-(2,3,4,6-tetra-O-acetyl-β-d-galactopyranosyl)-(1→3)-4,6-di-O -acetyl-2-deoxy-2-phthalimido-β-d-glucopyranosyl trichloroacetimidate with (2,6-di-O-acetyl-β-d-galactopyranosyl)-(1→4)-(2,3,6-tri-O-acet yl-β-d-glucopyranoside) bound onto the polymer afforded lacto-N-tetraose, which was then regioselectively sulfated at the 3-OH position of the terminal galactose using the stannylene procedure. Fucosylation of the sulfated tetrasaccharide was performed using an immobilized fucosyltransferase FucTIII to give the title compound after cleavage.

Full text of DOI:10.1016/j.carres.2008.01.005

Available online at www.sciencedirect.com
Carbohydrate Research 343 (2008) 970–976
Note
Chemo-enzymatic supported synthesis of the 3-sulfated
Lewisa pentasaccharide on a multimeric polyethylene glycol
Annie Malleron and Christine Le Narvor*
Laboratoire de Chimie Organique Multifonctionnelle, Equipe Glycochimie Mole´culaire et Macromole´culaire,
ˆ
CNRS, UMR 8182, Bat. 420, Univ Paris -Sud, Orsay F-91405, France
Received 3 April 2007; received in revised form 21 December 2007; accepted 7 January 2008
Available online 15 January 2008
Abstract—The 3-sulfated Lewisa pentasaccharide was synthesized on multimeric-based polyethylene glycol support. Coupling of
O-(2,3,4,6-tetra-O-acetyl-b-D-galactopyranosyl)-(1?3)-4,6-di-O-acetyl-2-deoxy-2-phthalimido-b-D-glucopyranosyl trichloroacetimi-
date with (2,6-di-O-acetyl-b-D-galactopyranosyl)-(1?4)-(2,3,6-tri-O-acetyl-b-D-glucopyranoside) bound onto the polymer afforded
lacto-N-tetraose, which was then regioselectively sulfated at the 3-OH position of the terminal galactose using the stannylene pro-
cedure. Fucosylation of the sulfated tetrasaccharide was performed using an immobilized fucosyltransferase FucTIII to give the title
compound after cleavage.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords: Chemo-enzymatic synthesis; Multimeric support; Glycosyltransferase; Selectins; Cell adhesion; Inflammation
Sulfated and sialylated Lewisa and Lewisx compounds
are known to be good ligands for selectins, a family of
adhesion molecules that mediates the interaction
between circulating leucocytes and endothelial cells, a
key step in their recruitment to the sites of inflamma-
tion.1 A lot of attention has therefore been focused on
the chemical synthesis of these oligosaccharides to study
their mechanism of action and to develop inhibitors.2
Enzymes are becoming powerful tools as catalysts in
carbohydrate chemistry avoiding protection and depro-
tection steps due to the regio- and stereoselectivity of the
reaction, they catalyze,3 and they appear to be attractive
reagents for solid phase synthesis.4 However, enzymatic
synthesis on solid support raises extra problems, such as
the accessibility of the enzyme to the interior of a solid
matrix5 or the compatibility of the support with both
aqueous and organic solvents. A solution would be to
use soluble supports, which combine the advantages of
both liquid phase (for the accessibility) and solid phase
(for the purification) strategies. Polyethylene glycol
(PEG) has been among the most studied soluble poly-
mers for oligosaccharide synthesis.6 In previous papers,
we described the use of a modified PEG support. Our
approach was to increase the loading capacity of the
polymer by coupling a pentaerythritol derivative at both
extremities of a PEG 6000 molecule, giving a soluble
polymeric support with a loading capacity similar to
that of a resin (1 mmol/g). Using this polymer, we have
synthesized the Lewisx trisaccharide and lacto-N-
tetraose by an enzymatic approach.7,8
Our interest in the 3-sulfated Lewisa pentasaccharide
led us to envisage its synthesis on our support using
our expertise in the conventional liquid phase chemo-
enzymatic synthesis of this pentasaccharide.9
The supported tetrasaccharide 8, obtained by glyco-
sylation of the two disaccharides 6 and 7, was enzymat-
ically fucosylated after deprotection and 1 was obtained
by cleavage from the multimeric support (Scheme 1).
In a previous work, we have shown that the coupling
of a thiosugar derivative onto our multimeric support 3
was very efficient.7 The lactose unit 2 was thus installed
on the PEG support as 1-thiolactose (Scheme 2). Radi-
cal coupling onto compound 3 was realized under
254 nm UV irradiation using a 0.03 M solution of 3 in
water and 1.3 equiv of sugar per allyl group. Purification
*
Corresponding author. E-mail: chlenarv@icmo.u-psud.fr
0008-6215/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2008.01.005

A. Malleron, C. Le Narvor / Carbohydrate Research 343 (2008) 970–976
971
HO
OH
OH
O
OH
HO
NaO3SO
OH
HO
O
OH
O
OH
O
O
O
O
O
O
OH
HO
OH
NHAc
OH
OH
1
OAc
OAc
OAc
O
HO
OAc
O
O
OAc
AcO
O
AcO
O
AcO
O
O
OAc
S
OAc
NPht
OAc
8
HO
HO
OAc
AcO
AcO
OAc
O
OAc
AcO
O
OAc
O
O
NH
CCl3
O
O
O
+
AcO
AcO
S
NPht
OAc
OAc
6
7
= modified PEG
Scheme 1.
pylidene lactose 5 (Scheme 3). Acetylation of the free
alcohols followed by acid hydrolysis of the isopropyli-
dene group and precipitation of the polymer using tert-
butyl methyl ether gave 6 in 96% yield based on precipi-
tated support. The disappearance of the 3,4-O-isopropyl-
OH
OH
O
OH
O
O
HO
O
+
HO
SH
OH
OH
6 mol/ PEG
2
3
1
idene group was complete as confirmed by H NMR.
a,b
Glycosylation of 6 with 7 was performed using
0.6 equiv boron trifluoride diethyl etherate as promotor
at 0 °C in dry dichloromethane and led to the b(1?3)
HO
HO
OH
OH
O
O
O
O
S
HO
OH
HO
OH
OH
O
OH
4
O
HO
3.7 lactose /PEG
O
HO
S
O
OH
OH
O
4
O
O
O
O
3.7 lactose/PEG
O
O
O
O
O
O
a
n=134 +/- 20
6
O
OH
O
OH
O
O
Scheme 2. Reagents and conditions: (a) hm (254 nm), H2O, 10 h, rt; (b)
O
HO
S
O
dialysis.
OH
OH
5
3.7 lactose/PEG
b
was easily carried out by dialysis and the amount of the
disaccharide covalently bound to support 4 was quanti-
fied by the phenol sulfuric acid method after cleavage of
an aliquot using mercury(II) trifluoroacetate.10 We
found 3.7 mol of disaccharide/ mol of PEG
(0.46 mmol/g of support).
The reaction of 4 with 2,2-dimethoxypropane in the
presence of p-toluene sulfonic acid monohydrate as de-
scribed by Catelani11 gave the supported 3,4-O-isopro-
HO
HO
OAc
O
OAc
O
O
S
O
AcO
OAc
OAc
6
3.7 lactose/PEG
Scheme 3. Reagents and conditions: (a) (i) 2,2-dimethoxypropane, cat.
p-toluene-sulfonic acid monohydrate, 55 °C, 3 h; (ii) dialysis; (iii)
MeOH–H2O (9:1), reflux, 24 h; (b) (i) Ac2O–pyridine, rt, 16 h; acetic
acid 70%, reflux, 3 h; (ii) precipitation with tert-butylmethyl ether.

972
A. Malleron, C. Le Narvor / Carbohydrate Research 343 (2008) 970–976
linked polymer-bound tetrasaccharide 8 that was puri-
fied by precipitation with tert-butyl methyl ether.
Then, the removal of the phthalimido group along
with O-deacetylation of 8, using ethylene diamine, and
subsequent N-acetylation, using acetic anhydride in
methanol, afforded the tetrasaccharide bound on sup-
port 9 (Scheme 4).
Cleavage of an aliquot of 9 with mercury(II) trifluoro-
acetate gave, after purification on a gel filtration column,
tetrasaccharide 10 with 50% yield based on the initial
loading of lactose in compound 4. The spectral data of
10 were identical to those already published.12 Com-
pound 10 was also reduced and peracetylated. The
NMR data were identical to those described for
the 2,3,4,6-tetra-O-acetyl-b-D-galactopyranosyl-(1?3)-
4,6-di-O-acetyl-2-deoxy-2-acetyl-b-D-glucopyranosyl-
(1?3)-2,6-di-O-acetyl-b-D-galactopyranosyl-(1?4)-1,2,
3,6-tetra-O-acetyl-glucitol tetrasaccharide.13
complex was added. After 28 h at room temperature, the
mixture was neutralized, dialyzed and lyophilized to give
the sulfated tetrasaccharide bound onto polymer 11.
Cleavage of an aliquot of 11 with mercury(II) trifluoro-
acetate gave, after purification on a DEAE-Sephadex
column and elution with triethylammonium hydrogen
carbonate buffer, the known 3-sulfated tetrasaccharide
12 with 54% yield based on tetrasaccharide loading in
9 (Scheme 5).15
Fucosylation of 11 was achieved by incubation with
recombinant FucT-III adsorbed on Ni2+–agarose in
25 mM, pH 6.4, MES buffer and GDP-fucose.16 Using
the results found in our previous work,9 GDP-fucose
was introduced in less than stoichiometric amount to
fucosylate only the O-4 of the GlcNAc residue without
secondary fucosylation on the O-3 of the Glc residue.
After 3 days, the enzyme was removed by filtration
and the filtrate was dialyzed, lyophilized and treated
with mercury(II) trifluoroacetate. After filtration and
evaporation, the supernatant was passed through a
DEAE-Sephadex column. First, nonsulfated com-
pounds (lactose, tetrasaccharide, pentasaccharide) were
eluted with water and then, elution with a triethylammo-
nium hydrogen carbonate buffer gradient (0–1 M)
allowed the separation of mono-sulfated compounds in
The sulfate group was then regioselectively introduced
on the O-3 of the galactose moiety by stannylene-direc-
ted sulfation.14 Compound 9, having 12 free hydroxyl
groups per tetrasaccharide residue, was heated at reflux
in the presence of 1 equiv of dibutyltin(II) oxide in a 3:5
DMF/benzene mixture for 16 h. The benzene was then
removed and 1.1 equiv of sulfur trioxide-trimethylamine
HO
OAc
O
OAc
O
HO
O
AcO
S
O
OAc
OAc
6
3.7 lactose / PEG
1.8 mol / PEG
a
HO
HO
OAc
O
OAc
OAc
O
OAc
O
AcO
S
O
AcO
AcO
OAc
O
HO
OAc
OAc
O
AcO
O
OAc
O
O
O
O
OAc
AcO
S
O
NPht
OAc
OAc
1.8 mol / PEG
1.8 mol / PEG
8
b
HO
HO
OH
OH
O
O
O
HO
S
OH
OH
O
HO
HO
OH
O
OH
O
HO
O
OH
OH
HO
O
O
O
O
OH
S
HO
O
NHAc
OH
OH
1.8 mol / PEG
9
Scheme 4. Reagents and conditions: (a) (i) 6 (1 equiv), 7 (2.3 equiv/lactose), BF3ÁEt2O, CH2Cl2, 0 °C, 4 h; (ii) precipitation with tert-butylmethyl
ether; (b) (i) NH2CH2CH2NH2, EtOH, reflux, 16 h; (ii) Ac2O, MeOH, rt, 3 h; (iii) dialysis.

A. Malleron, C. Le Narvor / Carbohydrate Research 343 (2008) 970–976
973
HO
OH
OH
O
HO
O
O
HO
S
OH
OH
O
O
1.8 mol / PEG
HO
HO
OH
HO
OH
OH
O
OH
HO
O
O
O
O
O
HO
O
OH
S
NHAc
OH
OH
9
1.8 mol / PEG
HO
HO
a
OH
HO
OH
OH
O
HO
O
OH
O
O
O
O
O
OH
HO
S
NHAc
OH
S
OH
O
OH
OH
O
O
O
HO
O
HO
OH
HO
NaO3SO
0.9 mol / PEG
0.9 mol / PEG
0.9 mol / PEG
HO
OH
OH
HO
OH
OH
O
HO
O
OH
O
O
O
O
O
OH
HO
OH
S
HO
NHAc
O
O
S
OH
OH
O
O
OH
O
HO
OH
NaSO3O
OH
0.9 mol / PEG
11
Cleavage of an aliquot
with Hg(OTFA)2
HO
OH
HO
O
OH
HO
OH
OH
O
OH
O
HO
O
HO
HO
O
OH
O
O
OH
O
OH
O
HO
OH
OH
HO
O
O
NaO3SO
O
O
NHAc
O
HO
OH
OH
OH
OH
NHAc
OH
OH
10
Ratio 3-Sulfated tetrasaccharide 12 / unsulfated tetrasaccharide 10 1/1
12
3-Sulfated lactose / Lactose 1/1
Scheme 5. Reagents and conditions: (a) (i) Bu2SnO (1.1 equiv), DMF–benzene, reflux, 16 h; (ii) SO3ÁMe3N (1.1 equiv); (iii) dialysis.
function of their molecular weight (pentasaccharide,
tetrasaccharide, lactose). Compound 1 was obtained
with a 10% overall yield based on the supported lactose.
The individual yield of the enzymatic fucosylation step
was estimated to 36%. In these conditions, only penta-
saccharide 1 was obtained, and no trace of hexasaccha-
ride resulting from the fucosylation onto the glucose
residue was detected (Scheme 6).
1. Experimental
1.1. General methods
All moisture-sensitive reactions were performed under
an argon atmosphere using oven-dried glassware. All sol-
vents were dried over standard drying agents and freshly
distilled prior to use. Flash column chromatography was
performed on Silica Gel 60 A C.C (6–35 lm). Reactions
were monitored by TLC on Silica Gel 60F254 plates with
detection by UV at 254 nm and by charring with 10%
H2SO4 in EtOH or 2% orcinol in 10% H2SO4 in EtOH.
NMR spectra were recorded with Bruker AM-250,
AMX-360, DRX-400 spectrometers. The chemical shifts
spectra are given relative to Me4Si in CDCl3 and to ace-
tone (d 2.22 and 30.5 ppm) for spectra performed in
D2O. Mass Spectra were carried out with a Finnigan
MATT 95 apparatus using ESI.
The 3-sulfated Lewisa pentasaccharide has been
totally synthesized on a soluble polymer using a
chemo-enzymatic approach. The yield for each step
was similar to the yield obtained in soluble phase. Our
results demonstrated the possibility to extend solution-
phase chemo-enzymatic synthesis methods to supported
synthesis. The use of a soluble support greatly facilitated
the purification of the different intermediates while a sin-
gle, final chromatographical purification allowed easy
isolation of pentasaccharide 1.

974
A. Malleron, C. Le Narvor / Carbohydrate Research 343 (2008) 970–976
HO
HO
OH
O
HO
O
OH
O
OH
O
OH
O
HO
0.9 mol / PEG
O
O
O
OH
HO
NHAc
OH
S
OH
S
OH
OH
O
O
HO
O
HO
O
OH
O
0.9 mol / PEG
HO
HO
NaO3SO
OH
OH
O
HO
OH
O
OH
O
OH
O
HO
O
O
O
S
O
OH
OH
S
HO
O
OH
OH
NHAc
OH
O
OH
HO
O
HO
OH
NaSO3O
0.9 mol / PEG
0.9 mol / PEG
11
a,b
HO
OH
OH
O
OH
OH
OH HO
OH
O
O
OH
O
O
O
NHAc
NaO3SO
O
O
O
OH
HO
OH
OH
OH
1
Scheme 6. Reagents and conditions: (a) (i) GDP-fucose, immobilized FucT-III, MnCl2 (20 mM), 25 mM MES buffer pH 6.4, 37 °C, 3 days; (ii)
filtration and dialysis; (b) (i) Hg(OTFA)2, H2O, 16 h, 55 °C; (ii) DEAE-Sephadex, elution Et3NHCO3 buffer pH 8 (0–1 M).
FucT-III was obtained within a French network (G3)
devoted to the production and studies of recombinant
glycosyltransferases.17 GDP-fucose was synthesized
according to published procedures.18
Carbohydrate content was determinated according to
a modification of the Dubois method by the phenol
sulfuric acid assay.10
nation using a germicide lamp for 16 h at rt. At the
end of the reaction, the mixture was diluted with
H2O (20 mL), dialyzed and lyophilized to give 4
(993 mg).
1H NMR (D2O, 250 MHz,): 4.53 (d, J1,2 7.5 Hz, H-1I),
4.42 (d, J1,2 7.0 Hz, H-1II), 3.70–3.50 (m, CH2CH2O),
2.90–2.70 (m, CH2–S), 2.00–1.80 (m, CH2–CH2).
An aliquot (10.7 mg) was hydrolyzed with mercuric(II)
trifluoroacetate (5.3 mg) in H2O (1 mL) during 4 h at rt.
A phenol sulfuric detection gave 3.7 mmol of fixed
disaccharide/ mmol of PEG.
1.2. Yield and recuperation of PEG-supported compounds
The yields of the PEG-supported compounds were
determined by weighing, with the assumption that the
MW of the PEG fragment was 7463 for 5 as determined
by MALDI-TOF MS (commercial PEG 6000, found for
the central peak 6952).
1.4. [(2,6-Di-O-acetyl-b-D-galactopyranosyl)-(1?4)-
(2,3,6-tri-O-acetyl-1-thio-b-D-glucopyranoside
supported)] (6)
After reaction, the PEG-supported compounds were
purified by dialysis using a regenerated cellulose mem-
brane (Spectra/Por 6 MWCO 2000) or by precipita-
tion. In the latter, PEG-supported compounds were
precipitated after dissolution with dichloromethane fol-
lowed by the addition of a 10-fold excess of tert-
butylmethyl ether at 0 °C with vigorous stirring or by
crystallization from absolute ethanol. This precipitate
was filtered, dried under vacuo and used in the follow-
ing step.
A soln of 4 (980 mg, 0.110 mmol) in 2,2-dimethoxypro-
pane (20 mL) containing p-toluene-sulfonic acid mono-
hydrate (10 mg) was heated at 55 °C for 3 h, then the
mixture was neutralized with Et3N, concentrated to dry-
ness under reduced pressure. The residue was dissolved
with H2O (20 mL), dialyzed and concentrated. The
resultant residue was refluxed with 9:1 MeOH–H2O
for 24 h and the solvents were evaporated. The mixture
was acetylated with 1:2 Ac2O–pyridine (30 mL) during
16 h. A soln of the previous compound in 70% aq
AcOH (50 mL) was kept for 3 h under reflux. After
evaporation and coevaporation with toluene, the resi-
due was dissolved in CH2Cl2 and tert-butyl methyl ether
was added at 0 °C with stirring. The precipitated solid
was collected by filtration to give 6 (1.03 g, 0.107 mmol,
96%).
1.3. (b-D-Galactopyranosyl-(1?4)-O-1-thio-b-D-gluco-
pyranoside supported) (4)
A solution of 3 (790 mg, 0.106 mmol) in water (4 mL)
was allowed to react with b-D-1-thiolactose
2
(340 mg, 0.951 mmol, 1.5 equiv/allyl) under UV illumi-

A. Malleron, C. Le Narvor / Carbohydrate Research 343 (2008) 970–976
975
1.5. [(2,3,4,6-Tetra-O-acetyl-b-D-galactopyranosyl)-
(1?3)-4,6-di-O-acetyl-2-deoxy-2-phthalimido-b-D-gluco-
pyranosyl-(1?3)-2,6-di-O-acetyl-b-D- galactopyranosyl-
(1?4)-2,3,6-tri-O-acetyl-1-thio-b-D-glucopyranoside
supported] (8)
of water using a Dean–Stark apparatus. Then, the
benzene was removed and the dibutylstannylene deriva-
tive was treated with Me3NÁsulfur trioxide complex
(42 mg., 0.30 mmol, 3.9 equiv) at rt for 28 h. The reac-
tion mixture was then diluted with MeOH and neutral-
ized with NaHCO3. After evaporation, the residue was
dissolved in H2O and dialyzed to give 11 (673 mg) after
lyophilization.
A mixture of 6 (1 g, 0.104 mmol) and 7 (0.75 g,
0.865 mmol, 2.3 equiv/lactose) in CH2Cl2 (20 mL) was
stirred during 15 min at rt, then cooled at À10 °C and
BF3ÁEt2O (35 lL) was added. The reaction mixture
was stirred at 0 °C for 4 h. Et3N was added and the mix-
ture was concentrated. The residue was dissolved in
CH2Cl2 and tert-butyl methyl ether was added at 0 °C
with stirring. The precipitated solid was collected by
filtration to give 8 (1.30 g).
1.9. 3-Sulfo-b-D-galactopyranosyl-(1?3)-(2-acetamido-2-
deoxy-b-D-glucopyranosyl)-(1?3)-b-D-galactopyranosyl-
(1?4)-D-glucopyranose sodium salt (12)
An aliquot (100 mg) was hydrolyzed with mercuric(II)
trifluoroacetate (40 mg) in H2O (5 mL) during 4 h at
rt, then the mixture was purified on DEAE-Sephadex
A-25 column, eluted with a 0.1 M Et3NÁHCO3 buffer
(pH 8). Fractions containing the starting compound
were pooled to give 10 (6.8 mg, 10 lmol). Fractions con-
taining the sulfated tetrasaccharide 12 (9 mg, 11 lmol,
54%) were pooled and twice lyophilized.
1.6. [b-D-Galactopyranosyl-(1?3)-(2-acetamido-2-deoxy-
b-D-glucopyranosyl)-(1?3)-b-D-galactopyranosyl-(1?4)-
1-thio-b-D-glucopyranoside supported] (9)
A mixture of ethylene diamine (2.5 mL) and 8 (1.17 g,
1.07 mmol) in EtOH (25 mL) was refluxed for 20 h.
After cooling and concentration, the residue was dia-
lyzed, dried and treated with Ac2O (8 mL) in MeOH
(25 mL). The mixture was stirred for 3 h at rt, then
concentrated to give 9 (887 mg, 94 lmol).
1H NMR (D2O, 400 MHz): 5.10 (d, 0.33H, J1,2
3.5 Hz, H-1Ia), 4.61 (d, 1H, J1,2 8.0 Hz, H-1III ), 4.55
(d, 0.66H, J1,2 7.5 Hz, H-1Ib), 4.43 (d, 1H, J1,2 7.5 Hz,
H-1IV), 4.30 (d, 1H, J1,2 8.0 Hz, H-1II), 4.20–4.17 (m,
2H, H-4IV, H-3IV), 4.03 (d, 1H, J3,4 3.5 Hz, H-4II),
3.20–3.15 (m, 1H, H-2I), 1.90 (s, 3H, CH3CO).
1.7. b-D-Galactopyranosyl-(1?3)-(2-acetamido-2-deoxy-
b-D-glucopyranosyl)-(1?3)-b-D-galactopyranosyl-(1?4)-
D-glucopyranose (10)
1.10. 3-Sulfo-b-D-galactopyranosyl-(1?3)-[(a-L-fuco-
pyranosyl)-(1?4)]-(2-acetamido-2-deoxy-b-D-gluco-
pyranosyl)-(1?3)-b-D-galactopyranosyl-(1?4)-D-gluco-
pyranose sodium salt (1)
An aliquot of 8 (98 mg) was refluxed in EtOH contain-
ing ethylene diamine (0.5 mL) for 16 h. After cooling,
the soln was concentrated and the residue was acetylated
with Ac2O (1.7 mL) in MeOH (5 mL). The product
(65 mg) was cleaved using mercuric(II) trifluoroacetate
(20 mg) in H2O (4.5 mL), then the mixture was purified
on a Biogel P2 gel filtration column (48 Â 2.2 cm) to
give 12.5 lmol of tetrasaccharide 10 (8.9 mg, condensa-
tion yield, 55%). This result agreed with the colorimetric
determination, and spectral data were identical to those
already published.19
Compound 11 (371 mg), GDP-fucose (43 mg, 55 lmol),
immobilized FucT-III (40 mU), MnCl2 (217 mg) were
incubated at 37 °C for 67 h in 25 mM MES buffer
(55 mL, pH 6.4). After centrifugation, the supernatant
was dialyzed and lyophilized to give 14 (377 mg).
Compound 14 (377 mg) was hydrolyzed with mercu-
ric(II) trifluoroacetate (140 mg, 0.33 mmol) in H2O
(15 mL) in the presence of BaCO3 during 16 h at
55 °C. After filtration, the supernatant was concentrated
and the mixture was purified on DEAE-Sephadex A-25
column, eluted with a gradient of Et3N HCO3 buffer, pH
8 (0–1 M). The fractions containing compound 1 were
lyophilized and the freeze-dried eluate was passed
through a column of Bio-Rad AG 50W-X8 resin (Na+
form) to give 1 (14.3 mg, 13.8 lmol, 36%). LRMS (neg-
ative mode): calcd for C32H54NO28NaS [MÀNa]À,
932.3; found, 932.5.
1H NMR (D2O, 360 MHz): d 5.18 (d, 0.33H, J1,2
4.0 Hz, H-1I a), 4.68 (d, 1H, J1,2 8.0 Hz, H-1III), 4.62
(d, 0.66H, J1,2 7.5 Hz, H-1Ib), 4.40 (d, 2H, J1,2 8.0 Hz,
H-1II, H-1IV), 4.11 (d, 1H, J3,4 3.5 Hz, H-4II), 3.30–
3.25 (m, 1H, H-2I), 1.98 (s, 3H, CH3CO).
1.8. [(3-Sulfo-b-D-galactopyranosyl)-(1?3)-(2-acet-
amido-2-deoxy-b-D-glucopyranosyl)-(1?3)-b-D-galacto-
pyranosyl-(1?4)-1-thio-b-D-glucopyranoside sodium
salt supported] (11)
The spectral data were identical to those already
published.13
Compound 1: 1H NMR (D2O, 400 MHz): 5.22 (d,
0.33H, J1,2 3.5 Hz, H-1Ia), 5.03 (d, 1H, J1,2 4.0 Hz,
H-1V), 4.87 (m, 1H, H-5V), 4.71 (d, 1H, J1,2 8.0 Hz,
H-1III), 4.67 (d, 0.66H, J1,2 7.5 Hz, H-1Ib), 4.61 (d,
A mixture of 9 (722 mg, 77 lmol) and dibutyltin(II)-
oxide (75 mg, 0.30 mmol) in DMF–benzene (3:5,
80 mL) was refluxed for 16 h with continuous removal

976
A. Malleron, C. Le Narvor / Carbohydrate Research 343 (2008) 970–976
1H, J1,2 7.5 Hz, H-1IV), 4.44 (d, 1H, J1,2 8.0 Hz, H-1II),
4.32–4.27 (m, 2H, H-4IV, H-3IV), 4.16 (d, 1H, J3,4
3.5 Hz, H-4II), 3.30–3.25 (m, 1H, H-2I), 2.05 (s, 3H,
CH3CO), 1.19 (d, 3H, J5,6 6.5 Hz, CH3).
Ojeda, R.; Terenti, O.; de Paz, J. L.; Martin-Lomas, M.
Glycoconjugate J. 2004, 21, 179–195; (d) Blattner, R.;
Furneaux, R. H.; Ludewig, M. Carbohydr. Res. 2006, 341,
299–321.
7. Lubineau, A.; Malleron, A.; Le Narvor, C. Tetrahedron
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´
8. Renaudie, L.; Daniellou, R.; Auge, C.; Le Narvor, C.
Acknowledgements
Carbohydr. Res. 2004, 339, 693–698.
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167.
The authors thank Professor Lubineau for helpful dis-
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sity of Paris-Sud for financial support.
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