Chemoenzymatic synthesis of the 3-sulfated Lewisa pentasaccharide

Article abstract of DOI:10.1016/j.carres.2005.10.004

The sulfated pentasaccharide benzyl O-(3-O-sulfo-β-d-galactopyranosyl) -(1→3)-O-[(α-l-fucopyranosyl)-(1→4)]-O-(2-acetamido-2-deoxy- β-d-glucopyranosyl)-(1→3)-O-(β-d-galactopyranosyl)-(1→4) -O-β-d-glucopyranoside sodium salt was synthesized using a chemo-e

Full text of DOI:10.1016/j.carres.2005.10.004

Carbohydrate
RESEARCH
Carbohydrate Research 341 (2006) 29–34
Chemoenzymatic synthesis of the 3-sulfated Lewisa pentasaccharide
Annie Malleron, Yael Hersant and Christine Le Narvor*
¨
ˆ
´
Laboratoire de Chimie Organique Multifonctionnelle, UMR 8614, GDR 2590, Bat. 420, Universite de Paris Sud,
F-91405 Orsay, France
Received 12 July 2005; accepted 13 October 2005
Available online 4 November 2005
Abstract—The sulfated pentasaccharide benzyl O-(3-O-sulfo-b-D-galactopyranosyl)-(1!3)-O-[(a-L-fucopyranosyl)-(1!4)]-
O-(2-acetamido-2-deoxy-b-D-glucopyranosyl)-(1!3)-O-(b-D-galactopyranosyl)-(1!4)-O-b-D-glucopyranoside sodium salt was
synthesized using a chemo-enzymatic approach. Lacto-N-tetraose, obtained from two disaccharides [4-methoxybenzyl O-(2,3,4,6-
tetra-O-acetyl-b-D-galactopyranosyl)-(1!3)-4,6-O-benzylidene-2-deoxy-2-phtalimido-b-D-glucopyranoside and benzyl 2,6-di-O-
acetyl-b-D-galactopyranosyl-(1!4)-2,3,6-tri-O-acetyl-b-D-glucopyranoside], was regioselectively sulfated at the 3 OH position of
the terminal galactose using the stannylene procedure. The fucosylation of the sulfated tetrasaccharide was performed using soluble
or immobilized fucosyltransferase FucT-III to give the title compound.
Ó 2005 Elsevier Ltd. All rights reserved.
Keywords: Chemo-enzymatic synthesis; Fucosyltransferase; Sulfated Lewisa pentasaccharide
1. Introduction
enzymes for the synthesis of these compounds, has been
cloned but is not easily available to be of use in a prepara-
tive scale. Thus, its use in synthesis has only been reported
once5 and the chemical synthesis remains the best way
to obtain Gal-b-(1!3)-GlcNAc linkage. This has been
demonstrated in our group where Lubineau et al. have
reported the chemical synthesis of the 3IV-sulfated Lewisa
pentasaccharide, the most powerful monovalent ligand
for human E-selectin known until now.6,7
Sulfated and sialylated Lewisa and Lewisx compounds
are known to be good ligands for selectins, a family of
adhesion molecules that mediates the interaction of cir-
culating leukocytes with endothelial cells, a key step in
their recruitment to sites of inflammation.1 The synthe-
sis of these compounds has been a great challenge for
many groups.2
Due to their regio- and stereoselectivity, enzymes have
proved to be powerful tools as catalysts in carbohydrate
chemistry avoiding the protection and deprotection
steps.3 In this respect, the enzymatic syntheses of these
Lewis derivatives, or of some mimics, have been an
intensive field of research.
Our interest in the chemo-enzymatic synthesis of oli-
gosaccharides4b,8 led us to use the fucosyltransferase
FucT-III in this synthesis since it was already reported
that natural and cloned fucosyltransferases were able
to accept sulfated oligosaccharides as substrates.9,10
We thus describe here a chemo-enzymatic approach
to the title compound based on the enzymatic fucosyl-
ation of the sulfated tetrasaccharide lacto-N-tetraose
11 using the FucT-III.
The enzymatic synthesis of the sialylated Lewisx com-
pounds was made easier by the availability of the three
enzymes necessary to their synthesis: (a) b-(1!4) galac-
tosyl transferase, (b) a-(2!3) sialyltransferase and (c)
a-(1!3/4) fucosyltransferase.4
2. Results and discussion
Regarding the Lewisa derivatives, the situation is quite
different. b-(1!3) galactosyltransferase, one of the key
The synthetic strategy adopted in this paper was
based on the enzymatic fucosylation of tetrasaccharide
11 obtained from the two disaccharides (4 and 8).
*
Corresponding author. E-mail: chlenarv@icmo.u-psud.fr
0008-6215/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2005.10.004

30
A. Malleron et al. / Carbohydrate Research 341 (2006) 29–34
Disaccharide 4 was synthesized with 71% yield by
yield after purification on DEAE-Sephadex and elution
with triethylammonium hydrogen carbonate buffer
(Scheme 2).
glycosylation of the acceptor 211 with the 2,3,4,6-tetra-
O-acetyl-galactopyranosyl a-trichloroacetimidate 312 in
the presence of trimethylsilyl trifluoromethane sulfonate
(Scheme 1).13
The fucosylation of 11 was then achieved using a
recombinant FucT-III, expressed in CHO cells.18 Two
sites of fucosylation are available on the sulfated tetra-
saccharide 11 leading to an hexasaccharide. Indeed,
disaccharide Gal-b-(1!3)-GlcNAc and the lactose resi-
due are substrates for the enzyme, but we can expect to
stop the reaction at the pentasaccharide stage, as the
Gal-b(1!3)-GlcNAc residue is six times better substrate
at 1 mM than disaccharide lactose according to de
Vries.19
In order to minimize the formation of the hexasaccha-
ride, fucosylation of acceptor 11 was therefore moni-
tored by RP-HPLC at 1 mM using 1 equiv of GDP-
fucose with 1 mU of enzyme for 1 lmol of substrate.
The enzyme was used as a solution or immobilized on
Ni2+–NTA–Agarose through its 6His tag, as described
previously in our group.20
After 18 h of incubation, we found 34% of starting
material 11, 64% of pentasaccharide 1 and 2% of hexa-
saccharide for both enzymatic preparations (soluble and
immobilized). After 42 h, 80% of pentasaccharide 1,
10% of tetrasaccharide 11 but also 10% of hexasaccha-
ride could be detected.
For a preparative scale (20 lmol of tetrasaccharide),
we chose to perform the incubations at 1 mM with sol-
uble and immobilized enzyme and to stop the reaction
after 18 h. After purification on reverse phase chroma-
tography (C-18 column), 1 was isolated in 51% yield in
the case of soluble enzyme and 62% yield with immobi-
lized one. In each case, the unreacted tetrasaccharide 11
A p-methoxybenzyl group at the anomeric position
was chosen for its facile introduction and the possibility
to remove it before the activation step for tetrasaccha-
ride synthesis. However during its cleavage (!4), we
obtained a small quantity of the 4,6-diol by-product
derived from the hydrolysis of the benzylidene group.
We thus chose to remove this protecting group and to
acetylate the diol. In this way, the cleavage of the
p-methoxybenzyl group using ammonium and cerium
nitrate in MeCN–water gave disaccharide 6 with 87%
yield. Then, 6 was transformed in the usual way into
trichloroacetimidate 7 (93%).
Glycosylation of 7 with the known protected deriva-
tive lactose 8,14 catalyzed by trimethylsilyl trifluoro-
methane sulfonate according to the Schmidt procedure,15
provided tetrasaccharide 9 in 50% yield.
Conversion of the phtalimido group into the acetam-
ido group under standard conditions afforded tetrasac-
charide 10 in 86% yield after purification by reverse
phase chromatography (C-18 column).
The sulfate group was then regioselectively introduced
at position 3 of the galactose moiety by stannylene-
directed sulfation.6,16,17 Compound 10, having 12 free
hydroxyl groups, was thus heated at reflux with 1 equiv
of dibutyltin oxide in 1:5 DMF–benzene for 16 h, then
after removal of the benzene, 1.1 equiv of sulfur triox-
ide–trimethylamine complex was added. The 3IV-sul-
fated tetrasaccharide 11 was thus obtained in 54%
OR1
AcO
AcO
OAc
O
AcO
AcO
OAc
O
O
Ph
R2O
O
+
a
O
O
O
HO
OMPM
NPht
OMPM
AcO
AcO
O
NH
CCl3
NPht
4 R1,R2= PhCH
5 R1=R2=Ac
2
3
OAc
O
AcO
AcO
OAc
O
OAc
O
AcO
AcO
OAc
O
b
c
AcO
O
AcO
O
NH
CCl3
OH
O
AcO
AcO
NPht
NPht
6
7
OH
HO
OAc
O
OAc
O
O
AcO
AcO
AcO
OAc
O
OBn
OAc
OH
OAc
OAc
OAc
AcO
O
O
OAc
8
O
O
O
d
O
OAc
AcO
OBn
NPht
OAc
OAc
9
Scheme 1. Synthesis of tetrasaccharide. Reagents and conditions: (a) 2 (1 equiv), 3 (1.5 equiv), Me3SiOTf (0.1 equiv), CH2Cl2, rt, 3 h, 71%; (b) CAN,
CH3CN/H2O, 0 °C, 15 min, 87%; (c) CCl3CN, NaH, CH2Cl2, 0 °C, 30 min, 93%; (d) 7 (1.15 equiv), 8 (1 equiv), Me3SiOTf, CH2Cl2, ꢀ10 °C, 5 h, 50%.

A. Malleron et al. / Carbohydrate Research 341 (2006) 29–34
31
HO
HO
OH
OH
O
OH
a
OH
O
HO
OH
O
O
O
9
O
O
OH
HO
OBn
NHAc
OH
OH
10
b
HO
OH
O
OH
O
OH
OH
HO
O
OH
NaO3SO
O
O
O
O
OH
HO
OBn
NHAc
OH
OH
11
c
HO
OH
OH
O
HO
OH
OH
OH
OH
O
O
O
O
OH
NaO3SO
O
O
O
O
OH
HO
OBn
NHAc
OH
OH
1
Scheme 2. Reagents and conditions: (a) (i) CH2CH2NH2, EtOH, reflux, 20 h, (ii) Ac2O, MeOH, 86%; (b) (i) Bu2SnO (1.1 equiv), DMF–benzene
reflux, 16 h, (ii) SO3ÆMe3N (1.1 equiv) 54%; (c) GDP-fucose (0.95 equiv), soluble FucT-III or immobilized FucT-III MnCl2 (20 mM), 100 mM MES
buffer pH 6.4, 37 °C, 7 h, 50–60%.
(45%) was recovered. Only traces of hexasaccharide
were isolated.
The chemical shifts spectra are given relative to Me4Si
in CDCl3 and to acetone (d 2.22 and 30.5 ppm) for spec-
tra performed in D2O. Mass spectra were obtained with
a Finnigan MATT 95 apparatus using ESI. Elemental
analyses were performed at the CNRS (Gif sur Yvette,
France).
FucT-III was obtained within a French network (G3)
devoted to the production and studies of recombinant
glycosyltransferases. GDP-fucose was synthesized
according to published procedures.21
In conclusion, the pentasaccharide 3-sulfated Lewisa
has been synthesized using
a
chemo-enzymatic
approach. We have shown that enzymatic fucosylation
of the intermediate tetrasaccharide could be achieved
with a minimum formation of the hexasaccharide by-
product. We are currently extending the use of these
conditions to the synthesis of Lewisa oligosaccharide
on a soluble support.
3.2. 4-Methoxybenzyl O-(2,3,4,6-tetra-O-acetyl-b-D-
galactopyranosyl)-(1!3)-4,6-O-b-benzylidene-2-
deoxy-2-phtalimido-b-D-glucopyranoside (4)
3. Experimental
3.1. General methods
A soln of Me4SiOTf (66 lL in 3.3 mL of CH2Cl2) was
added dropwise to a cooled suspension (0 °C) of the
alcohol 2 (3.34 g, 9.74 mmol), 2,3,4,6-tetra-O-acetyl-a-
D-galactopyranosyl a-D-galactopyranosyl trichloroace-
All moisture-sensitive reactions were performed under
an argon atmosphere using oven-dried glassware. All
solvents were dried over standard drying agents and
freshly distilled prior to use. Flash column chromatogra-
phy 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. Optical rotations were measured
on a Jasco DIP 370 digital polarimeter. IR spectra were
recorded on a Bruker IF S66. NMR spectra were
recorded with Bruker AM-250 or AMX-360 spectro-
meters; 13C spectra were performed at 60 or 100 MHz.
˚
timidate 3 (4.8 g, 9.74 mmol) and 4 A powdered mole-
cular sieves in CH2Cl2 (20 mL). After 3 h at rt, the
reaction mixture was neutralized with Et3N. The solvent
was evaporated and the residue was purified by flash
chromatography (2:3–29:21 EtOAc–petroleum ether)
to give 4 (3.9 g, 71.2%) as a powder. Rf 0.30 (2:1 toluene–
29
EtOAc); ½aꢁD ꢀ32 (c 2, CHCl3); 1H NMR (CDCl3,
250 MHz): d 7.75 (m, 4H, arom NPht), 7.60–7.40 (m,
5H, arom), 6.90 (d, 2H, J 9 Hz, H-9, H-90), 6.45 (d,
2H, J 9 Hz, H-10, H-100), 5.60 (s, 1H, H-7), 5.19 (d,

32
A. Malleron et al. / Carbohydrate Research 341 (2006) 29–34
1H, J3,4 3.5 Hz, H-4II), 5.12 (d, J1,2 9 Hz, H-1I), 4.99 (dd,
1H, J1,2 8.5 Hz; J2,3 10.5 Hz, H-2II), 4.74 (d, 1H, J
12.5 Hz, Ph–CH), 4.73 (dd, 1H, J2,3 11 Hz, J3,4 3.5 Hz,
H-3II), 4.73 (dd, 1H, J2,3 11 Hz, J3,4 10 Hz, H-3I), 4.5
(d, 1H, J1,2 8.5 Hz, H-1II), 4.42 (d, 1H, J1,2 12.5 Hz,
A mixture of 6 (2.43 g, 3.36 mmol), CCl3CN (3.4 mL,
3.36 mmol) and NaH (cat) in CH2Cl2 (8 mL) was stirred
for 30 min at 0 °C. Then, the mixture was filtered
through a silica gel column (1:1 petroleum ether–EtOAc
containing 0.1% Et3N) to give 7 (2.7 g, 93%) as a pow-
29
Ph–CH), 4.41 (dd, 1H, J6,6 11 Hz, J5,6 4.5 Hz, H-6I),
der. Rf 0.36 (1:1 EtOAc–petroleum ether); ½aꢁD +23 (c
0
4.31 (dd, 1H, J1,2 9 Hz, J2,3 11 Hz, H-2I), 4.06 (dd,
1.2, CH2Cl2); H NMR (CDCl3, 200 MHz): d 8.6 (s,
1
1H, J6,6 11 Hz, J5,6 9 Hz, H-6II), 3.67 (s, OCH3), 3.63
1H, C@NH), 7.95–7.73 (m, 4H, Ph), 6.33 (d, 1H, J1,2
9 Hz, H-1I), 5.25 (d, 1H, J3,4 3.5 Hz, H-4II), 5.19 (dd,
1H, J3,4 = J4,5 10 Hz, H-4I), 4.98 (dd, 1H, J1,2 8 Hz,
J2,3 10 Hz, H-2II), 4.85 (dd, 1H, J2,3 10.5 Hz, H-3I),
4.67 (dd, 1H, H-3II), 4.61 (dd, 1H, H-2I), 4.25 (d, 1H,
H-1II), 2.13, 2.12, 2.08, 2.07, 1.87 (4s, 12H, CH3CO).
0
(ddd, 1H, J5,6 4.5, J5,6 10 Hz, J4,5 9 Hz, H-5I), 3.48
0
(dd, 1 H, J5,6 6 Hz, J5,6 8.5 Hz, H-5II), 2.07, 1.91,
0
1.84, 1.52 (4s, 12H, CH3CO). 13C (CDCl3, 60 MHz): d
170.17, 169.95, 168.75 (C@O), 159.02 (C–Ph), 113.43
(2C–Ph), 101.47 (C-7), 100.40 (C-1II), 97.26 (C-1I),
80.74 (C-4I), 60.67 (C-6II), 55.24 (C-2I), 54.90 (CH3O),
20.57, 20.51, 20.40, 20.01 (CH3CO); HRMS Calcd for
C44H45NO17 [M+Na+]: 870.2580. Found 870.2589.
13C (CDCl3, 60 MHz):
d 170.84, 170.25, 170.08,
169.25, 169.05 (C@O), 160.67 (OC@N), 100.49 (C-1II),
93.60 (C-1I), 90.07 (CCl3), 61,79, 60.48 (C-6I, C-6II),
54.46 (C-2I), 20.70–20.26 (CH3CO).
3.3. 4-Methoxybenzyl O-(2,3,4,6-tetra-O-acetyl-b-D-
galactopyranosyl)-(1!3)-4,6-di-O-acetyl-2-deoxy-2-
phtalimido-b-D-glucopyranoside (5)
3.5. Benzyl O-(2,3,4,6-tetra-O-acetyl-b-D-galactopyran-
osyl)-(1!3)-4,6-di-O-acetyl-2-deoxy-2-phtalimido-b-D-
glucopyranosyl-(1!3)-2,6-di-O-acetyl-b-D-galactopyr-
anosyl-(1!4)-2,3,6-tri-O-acetyl-b-D-glucopyranoside (9)
A soln of 4 (3.5 g, 4.13 mmol) in 70% AcOH (60 mL)
was stirred at 50 °C for 4.5 h. The solvent was evapo-
rated and coevaporated with toluene. The residue was
dissolved in pyridine (35 mL) and Ac2O (15 mL) over-
night at rt. Coevaporation with toluene followed by
flash chromatography (1:1 petroleum ether–EtOAc)
gave 5 (3.26 g, 94%) as a powder. Rf 0.26 (2:1 EtOAc–
A
mixture of 7 (1.085 g, 1.25 mmol), 8 (0.69 g,
˚
1.08 mmol) and powdered molecular sieves 4 A in
CH2Cl2 (5 mL) was stirred during 15 min at rt. Then,
the temperature was decreased at ꢀ30 °C and a soln of
Me3SiOTf (11 lL, 0.05 mL) in CH2Cl2 (430 lL) was
added dropwise and the mixture was stirred at ꢀ10 °C
during 5 h. Et3N was added and the mixture was con-
centrated. Flash chromatography (1:1 petroleum
ether–EtOAc) gave 9 (715 mg, 50%) as a syrup. Rf
29
1
petroleum ether); ½aꢁD ꢀ27 (c 1.4, CH2Cl2); H NMR
(CDCl3, 200 MHz): d 7.80 (m, 4H, arom NPht), 6.92
(d, 2H, J 9 Hz, H-9, H-90), 6.52 (d, 2H, J 9 Hz, H-10,
H-100), 5.20 (d, 1H, J3,4 3.5 Hz, H-4II), 5.08 (t, 1H,
J3,4 = J4,5 10 Hz, H-4I), 5.00 (d, 1H, J1,2 8 Hz, H-1I),
4.93 (dd, 1H, J1,2 8 Hz, J2,3 10 Hz, H-2II), 4.72 (d, 1H,
CH2Ph), 4.67 (dd, 1H, J2,3 9 Hz, H-3I), 4.61 (dd, 1H,
H-3II), 4.40 (d, 1H, Ph–CH), 4.13 (d, 1H, H-1II), 3.80–
3.72 (m, 2H, H-5, H-5II), 3.70 (s, 3H, OCH3), 2.14,
2.11, 2.05, 1.86, 1.84 (4s, 12H, CH3CO). 13C (CDCl3,
60 MHz): d 170.70, 170.13, 169.98, 169.10, 168.95
(C@O), 159.01 (C–Ph), 113.43 (2C–Ph), 100.23 (C-1II),
96.40 (C-1I), 60.48 (C-6II), 55.33 (C-2I), 54.85 (CH3O),
20.59, 20.37, 20.23, 20.12 (CH3CO). Anal. Calcd for
C43H45NO17: C, 56.92; H, 5.38; N, 1.66; O, 36.04.
Found: C, 56.42; H, 5.41; N, 1.48; O, 36.33.
29
0.27 (1:2 petroleum ether–EtOAc); ½aꢁD ꢀ3 (c 1.1,
CH2Cl2); 1H NMR (CDCl3, 250 MHz): d 8–7.80 (m,
4H, arom Ph), 7.40–7.20 (m, 5H, Ph), 5.30 (d, 1H, J3,4
3.5 Hz, H-4IV), 5.15 (d, 1H, J1,2, H-1III), 4.43 (d, 1H,
J1,2 7.5 Hz, H-1IV), 3.94 (d, 1H, J3,4 3 Hz, H-4IV),
2.13, 2.10, 2.09, 2.07, 2.06, 1.99, 1.97, 1.87, 1.60 (9s,
18H, CH3CO). 13C (CDCl3, 60 MHz): d 171.35–169.14
(C@O), 100.21, 100.12, 98.96, 98.33 (C-1I, C-1II,
C-1III, C-1IV), 62.80, 62.15, 61.78, 60.54 (C-6I, C-6II,
C-6III, C-6IV), 55.16 (C-2I), 20.62–20.00 (CH3CO). Anal.
Calcd for C61H73NO33: C, 54.33; H, 5.46; N, 1.04; O,
39.17. Found: C, 54.21; H, 5.46; N, 0.96; O, 39.19.
3.4. O-(2,3,4,6-Tetra-O-acetyl-b-D-galactopyranosyl)-
(1!3)-4,6-di-O-acetyl-2-deoxy-2-phtalimido-b-D-
glucopyranosyl trichloroacetimidate (7)
3.6. Benzyl O-b-D-galactopyranosyl-(1!3)-O-
(2-acetamido-2-deoxy-b-D-glucopyranosyl)-(1!3)-O-b-D-
galactopyranosyl-(1!4)-O-b-D-glucopyranoside (10)
Cerium ammonium nitrate (21.8 g, 3.86 mmol, 10 equiv)
was added to a vigorously stirred soln of 5 (3.26 g,
3.86 mmol) in 9:1 MeCN–water (25 mL) at 0 °C. After
15 min, the reaction mixture was diluted with CH2Cl2
then washed with KHCO3 and water, dried and concen-
trated to give after flash chromatography 6 (2.43 g,
3.36 mmol, 87%) as a powder.
A mixture of ethylene diamine (2 mL) and 9 (0.67 g,
0.49 mmol) in EtOH (8 mL) was refluxed for 20 h. After
cooling, the soln was concentrated and the residue was
extracted with water, dried and treated with Ac2O
(1.5 mL) in MeOH (8 mL). After addition of Et3N
(50 lL), the mixture was stirred for 3 h at rt, then con-
centrated. Purification on a C-18 column (1:0–9:1

A. Malleron et al. / Carbohydrate Research 341 (2006) 29–34
33
water–MeOH) gave 10 (338 mg, 0.42 mmol, 86.5%) as a
were incubated at 37 °C for 7 h in 100 mM MES buffer
(20 mL, pH 6.4). After addition of EtOH, the precipitate
was removed and the supernatant was concentrated.
Purification on C-18 column (1:0–49:1 water–MeOH)
gave 1, which was converted to the sodium salt by pass-
ing through a column of Bio-Rad AG 50W-X8 resin
29
powder. ½aꢁD ꢀ3 (c 1.3, water); 1H NMR (D2O,
200 MHz): d 7.50–7.3 (m, 5H, arom Ph), 4.88 (d, 2H,
CH2Ph), 4.69 (d, 2H, CH2Ph), 4.66 (d, 1H, J1,2 8 Hz,
H-1III), 4.50 (d, 1H, J1,2 8.5 Hz, H-1I), 4.39 (d, 2H,
J1,2 8 Hz, H-1II, H-1IV), 4.10 (d, 1H, J3,4 3 Hz, H-4II),
3.29 (t, 1H, H-2I), 1.96 (s, 3H, CH3CO). 13C (D2O,
(Na+ form). The freeze-dried eluate afforded
1
60 MHz):
d
172.80 (C@O), 126.55 (Ph), 101.30
(10.8 mg, 51.5%). Tetrasaccharide 11 (9 mg, 45%) was
also recovered.
(C-1IV), 100.73, 100.36 (C-1II, C-1III), 98.82 (C-1I), 79.80
(C-3II), 76.17 (C-4I), 58.80 (C-6II, C-6IV), 58.30
(C-6III), 57.90 (C-6I), 52.50 (C-2III), 20.03 (CH3CO);
LRMS Calcd for C33H51N21 [M+Na+]: 820.27. Found
820.2.
3.8.2. Method B (using immobilized FucT-III). Tetra-
saccharide 11 (20 mg, 20 lmol), GDP-fucose (15 mg,
19 lmol), immobilized FucT-III (15 mU), and MnCl2
(80 mg) were incubated at 37 °C for 7 h in 100 mM
MES buffer (20 mL, pH 6.4). After centrifugation, the
supernatant was concentrated. Purification on column
C-18 (!1:0–98:2 water–MeOH) gave 1, which was con-
verted to the sodium salt by passing through a column
of Bio-Rad AG 50W-X8 resin (Na+ form). The freeze-
dried eluate afforded 1 (13 mg, 62%) 6.5 mg of tetrasac-
charide 11 (45%) was recovered.
3.7. Benzyl O-(3-sulfo-b-D-galactopyranosyl)-(1!3)-O-
(2-acetamido-2-deoxy-b-D-glucopyranosyl)-(1!3)-O-b-D-
galactopyranosyl-(1!4)-O-b-D-glucopyranoside sodium
salt (11)
A mixture of 10 (152 mg, 0.19 mmol) and dibutyltinox-
ide (53 mg, 0.21 mmol) in DMF–benzene (1:5, 30 mL)
was boiled for 16 h under reflux with continual removal
of water using a Dean–Stark apparatus. Then, benzene
was removed and the dibutylstannylene derivative was
treated with the Me3N–sulfur trioxide complex (29 mg,
0.21 mmol) at rt for 28 h. The reaction mixture was then
diluted with MeOH and neutralized with NaHCO3.
After evaporation, the compound was extracted with
water and purified on DEAE Sephadex A-25 column,
eluted with a 0.1 M triethylammonium hydrogen car-
bonate buffer (pH 8). Fractions containing the starting
compound were pooled to give 10 (21 mg, 14%). Frac-
tions containing the sulfated tetrasaccharide 11
(99.7 mg, 54%) were pooled and twice lyophilized.
29
Compound 1: ½aꢁD ꢀ33 (c 0.6, water); 1H NMR (D2O,
400 MHz): d 7.50–7.35 (m, 5H, arom Ph), 5.02 (d, 1H,
J1,2 3.5 Hz, H-1V), 4.93 (d, 2H, CH2Ph), 4.86 (q, 1H,
H-5V), 4.75 (d, 2H, CH2Ph), 4.70 (d, 1H, J1,2 8 Hz, H-
1III), 4.60 (d, 1H, J1,2 8 Hz, H-1IV), 4.55 (d, 1H, J1,2
8 Hz, H-1I), 4.42 (d, 1H, J1,2 8 Hz, H-1II), 4.32–4.25
(m, 2H, H-4IV, H-3IV), 4.15 (d, 1H, J3,4 3.5 Hz, H-4II),
3.30 (t, 1H, H-2I), 2.04 (s, 3H, CH3CO), 1.17 (d, 3H,
CH3). 13C (D2O, 100 MHz): d 128.76 (Ph), 102.51,
102.12 (C-1II, C-1III, C-1IV), 100.56 (C-1V), 97.53
(C-1I), 81.62 (C-3II), 79.76 (C-3IV), 77.90 (C-4I), 75.51
(C-3III), 61.08, 60.52, 59.62, 59.00 (C-6I, C-6II, C-6III
,
C-6IV), 55.42 (C-2III), 21.87 (CH3CO), 14.89 (C-6V).
LRMS (negative mode) calcd for C39H60NO28SNa
[MꢀNa] 1022.3. Found 1022.5.
29
1
½aꢁD ꢀ8 (c 0.266, water); H NMR (D2O, 250 MHz): d
7.50–7.30 (m, 5H, arom Ph), 4.95 (d, 2H, CH2Ph),
4.77 (d, 2H, CH2Ph), 4.74 (d, 1H, J1,2 8 Hz, H-1III),
4.57 (d, 2H, J1,2 8 Hz, H-1I, H-1IV), 4.44 (d, 1H, J1,2
8 Hz, H-1II), 4.33 (dd, 1H, J2,3 10 Hz, J3,4 3.5 Hz, H-
3IV), 4.30 (d, 1H, H-4IV), 4.15 (d, 1H, J3,4 3.5 Hz, H-
3II), 3.35 (m, 1H, H-2I), 2.04 (s, 3H, CH3CO). 13C
(D2O, 60 MHz): d 126.22 (Ph), 100.94 (C-1IV), 100.66,
100.21 (C-1II, C-1III), 98.76 (C-1I), 80.17 (C-3II), 77.86
(C-3IV), 76.2 (C-4I), 58.80 (C-6II, C-6IV), 58.30 (C-6III),
57.80 (C-6I), 52.31 (C-2III), 19.95 (CH3CO); LRMS
(negative mode) Calcd for C33H50NO24SNa [MꢀNa+]
876.24. Found 876.4.
Acknowledgements
The authors thank Prof. Lubineau for helpful discus-
sions, CNRS and University of Paris-Sud, for financial
supports.
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