Synthesis of mono-, di- and trisulfated Lewis x trisaccharides

Article abstract of DOI:10.1016/S0957-4166(98)00246-8

3'-Sulfated and 3',6-disulfated Lewis x trisaccharides have been prepared through selective sulfation of methyl 2-acetamido-6-O-benzyl-2- deoxy-4-O-β-D-galactopyranosyl-3-O-(2,3,4-tri-O-benzyl-α-L-fucopyranosyl)- β-Dglucopyranoside, followed by catalytic

Full text of DOI:10.1016/S0957-4166(98)00246-8

TETRAHEDRON:
ASYMMETRY
Pergamon
Tetrahedron: Asymmetry 9 (1998) 2451–2464
Synthesis of mono-, di- and trisulfated Lewis x trisaccharides
Yong-Min Zhang, Annie Brodzky and Pierre Sinaÿ ∗
Ecole Normale Supérieure, Département de Chimie, Associé au CNRS, 24, rue Lhomond, 75231 Paris Cedex 05, France
Received 12 May 1998; accepted 17 June 1998
Abstract
30-Sulfated and 30,60-disulfated Lewis x trisaccharides have been prepared through selective sulfation of methyl
2-acetamido-6-O-benzyl-2-deoxy-4-O-β-D-galactopyranosyl-3-O-(2,3,4-tri-O-benzyl-α-L-fucopyranosyl)-β-D-
glucopyranoside, followed by catalytic hydrogenolysis. In a similar manner, 30,6-disulfated and 30,6,60-
trisulfated Lewis x trisaccharides have been selectively obtained from methyl 2-acetamido-2-deoxy-4-O-β-D-
galactopyranosyl-3-O-(2,3,4-tri-O-benzyl-α-L-fucopyranosyl)-β-D-glucopyranoside. © 1998 Elsevier Science
Ltd. All rights reserved.
1. Introduction
Sulfated Lewis x are structural trisaccharidic entities which have been found to be present at the
extremity of sugar chains (capping oligosaccharides). They are frequently sialylated. A typical example
is 60-sulfated sialyl Lewis x, hereafter designated structure A, which is a main capping group1 of the high
endothelial venules (HEV) associated glycoprotein GLYCAM-1. The structure B, wherein the primary
hydroxyl group of the GlcNAc residue is sulfated (6-sulfated sialyl Lewis x), and the 6,60-disulfated
sialyl Lewis x, may also1 be determinants on GLYCAM-1.
These variously sulfated oligosaccharides are of current interest as potential physiological L-selectin
ligands,1 thus playing a role in the recruitment of leukocytes to sites of acute or chronic inflammation2
and also in the process of lymphocyte recirculation.3 On the other hand, non sialylated 30-sulfated
∗
Corresponding author. E-mail: pierre.sinay@ens.fr
0957-4166/98/$ - see front matter © 1998 Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(98)00246-8

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Y.-M. Zhang et al. / Tetrahedron: Asymmetry 9 (1998) 2451–2464
Lewis x (structure C) has also been identified in human foetal meconium4 glycoproteins and in an
ovarian cystodenoma glycoprotein.5 Structure C has been shown to be a strong ligand for the inducible
endothelial adhesion molecule E-selectin, which is involved in the leucocyte recruitment to inflammation
sites.6 Interestingly the structure of bovine articular cartilage keratan sulfate is partially made out of
repeating units of sulfated Lewis x.7
It thus appears that the homogeneous linear poly (N-acetyl lactosamine) backbone, which is present
in many glycoconjugates, is amenable to considerable structural heterogeneity, not only after α-L-
fucosylation on position 3 of N-acetyl glucosamine or sialylation of the non-reducing end, but also
through sulfation on various hydroxyl groups, especially the primary ones. Whether this heterogeneity,
as in the well known case of heparin/heparan sulfate, is a key feature for the setting-up of a ‘poly N-
acetyl lactosamine’ derived biologically relevant secret code, is so far an open question. For this reason,
the synthesis of variously sulfated Lewis x containing oligosaccharides has recently been worked out.8
On the other hand, close mimics have also been prepared,9 together with more remote non fucosylated
analogs.10 We would like to report our own achievements in this area.
2. Results and discussion
2.1. Synthesis of the mono- and disulfated Lewis x derivatives 9 and 10
The selected starting material for the synthesis of the Lewis x skeleton was the disaccharide 1.
It has previously been efficiently prepared using a novel glycosylation reaction developed in our
group.11 Reaction of 1 with methanol in acetonitrile, promoted by tris (4-bromophenyl) ammoniumyl
hexachloroantimonate,12 provided 2 in high yield (Scheme 1). The β configuration of the newly intro-
1
duced anomeric carbon was confirmed by the H NMR spectrum that showed the H-1 as a doublet at
δ 5.31 (J1,2=8.5 Hz). Regioselective reductive opening of the benzylidene moiety with NaCNBH3–HCl
(gas in ether) in THF13 gave the alcohol 3 in 86% yield.
Scheme 1. Reaction conditions: (i) 7 equiv. of MeOH, 1.5 equiv. of tris (4-bromophenyl) ammoniumyl hexachloroantimonate,
4 Å MS, CH3CN, rt, 20 min, 90%; (ii) 10 equiv. of NaCNBH3, HCl·Et2O, 4 Å MS, THF, 0°C, 1 h, 86%
Condensation of 3 with 2,3,4,6-tetra-O-benzoyl-α-D-galactopyranosyl bromide 4,14 in the presence
of silver triflate, furnished the desired trisaccharide 5 in crystalline form in 87% yield (Scheme 2). The

Y.-M. Zhang et al. / Tetrahedron: Asymmetry 9 (1998) 2451–2464
2453
β configuration of the newly introduced anomeric carbon was confirmed by the 1H NMR spectrum that
showed the H-10 (galactose unit) as a doublet at δ 5.03 (J1 ,2 =8.2 Hz). Treatment of 5 first with hydrazine
hydrate in boiling ethanol, then with acetic anhydride in pyridine at room temperature, and finally with
sodium methoxide in methanol at room temperature, afforded the trisaccharide 6 in crystalline form in
80% overall yield from 5.
0
0
Scheme 2. Reaction conditions: (i) 1 equiv. of 3, 2 equiv. of 4, 3 equiv. of AgOTf, 1.9 equiv. of s-collidine, 4 Å MS, CH2Cl2,
toluene, −20°C, 0.5 h, 87%; (ii) hydrazine hydrate, 90% aqueous ethanol, reflux 18 h; Ac2O, pyridine, rt, 16 h; cat. NaOMe,
MeOH, 80%
Sulfation was now accomplished using the stannylene procedure.15 Thus, 6 was heated at reflux with
one equivalent of dibutyltin oxide in methanol for 2 h then the residue was reacted, after removal of
methanol, with two equivalents of trimethylamine–sulfur trioxide complex in dry THF, to give selectively
the monosulfated derivative 7 (79%), which was easily separated from the disulfated derivative 8 (13%).
Catalytic hydrogenolysis of 7 and 8, followed by purification of the products on Sephadex G-10-120,
and chromatography with a cation exchange resin (Na+) provided quantitatively, after freeze-drying,
the monosodium salt 9 and disodium salt 10, respectively, as amorphous white powders (Scheme 3).
Their structures were fully confirmed by NMR spectroscopy using COSY experiments. Sulfation of 30-
OH (galactose unit) caused a downfield shift of the signal of H-30 of 0.64 and 0.68 ppm for 9 and 10
respectively, and sulfation of 60-OH (galactose unit) caused a downfield shift of the signal of H-60 of
0.42 ppm for 10, compared to the non-sulfated parent compound Lewis x trisaccharide-β-OMe.16
Scheme 3. Reaction conditions: (i) 1 equiv. of Bu2SnO, MeOH, reflux, 2 h; then 2 equiv. of Me3N·SO3 complex, THF, rt, 40 h,
Dowex resin (50x8-200, Na+): 7 (79%), 8 (13%); (ii) H2, 10% Pd–C, MeOH, 15°C, 15 h, quantitative
2.2. Synthesis of the di- and trisulfated Lewis x derivatives 23 and 24
Glycosylation of 12, prepared as shown in Scheme 4, with 2,3,4,6-tetra-O-acetyl-β-D-
galactopyranosyl fluoride 1317 in the presence of AgOTf–SnCl2 selectively provided the β 1-→4
11
linked disaccharide 14 in 70% yield. (Scheme 5). The β configuration of the newly introduced anomeric

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Y.-M. Zhang et al. / Tetrahedron: Asymmetry 9 (1998) 2451–2464
1
centre was confirmed by the H NMR spectrum that showed the H-10 as a doublet at δ 4.59 ppm
0
0
(J1 ,2 =8.0 Hz). The preparation of 15 from 14 was achieved as described for 2 in 95% yield.
Scheme 4. Reaction conditions: (i) cat. NaOMe, MeOH; (ii) 0.75 equiv. of (Bu3Sn)2O, toluene, reflux with continuous removal
of water, 14 h; (iii) 1.5 equiv. of BzCl, rt, 5 h, 92%
Scheme 5. Reaction conditions: (i) 1 equiv. of 12, 2 equiv. of 13, 1 equiv. of AgOTf, 1 equiv. of SnCl2, 4 Å MS, CH2Cl2:toluene
(5:1), −15°C to rt, 2 h, 70%; (ii) 10 equiv. of MeOH, 2 equiv. of tris(4-bromophenyl) ammoniumyl hexachloroantimonate, 4 Å
MS, CH3CN, rt, 1 h, 95%
Glycosylation of 15 with the fucosyl donor 16,11 in the presence of TMSOTf, gave the crystalline
trisaccharide 17 in 81% yield (Scheme 6). Treatment of 17 with hydrazine hydrate in aqueous ethanol at
85°C followed by N-acetylation with acetic anhydride in dichloromethane–methanol afforded 18 in 70%
yield.
Scheme 6. Reaction conditions: (i) 1 equiv. of 15, 1.1 equiv. of 16, 1.1 equiv. of TMSOTf, Et2O, 0°C, 40 min, 81%; (ii) hydrazine
hydrate, 90% aqueous ethanol, reflux 18 h; Ac2O, CH2Cl2:MeOH (1:1), rt, 3 h, 70%
The sulfation of 18 was achieved as described earlier for 7, after stirring for 40 h with 2 equivalents of
trimethylamine–sulfur trioxide complex in dry THF at room temperature. The THF was evaporated, and
the residue was further reacted with 2 equivalents of trimethylamine–sulfur trioxide complex in dry DMF
at room temperature for 60 h. Four compounds were separated and identified as 19 (26%), 20 (11%), 21
(52%), and 22 (7%). Reaction of the stannylene acetal intermediate with 6 equivalents of pyridine–sulfur
trioxide complex in DMF gave the trisulfated compound 22 in 66% yield. Catalytic hydrogenolysis of
19 and 21 followed by purification of the products on Sephadex G-10-120, followed by chromatography
with cation exchange resin (Na+) provided quantitatively, after freeze-drying, the monosodium salt 9 and
disodium salts 10, identical with the samples prepared from compounds 7 and 8. Debenzylation of 20
gave a trisaccharide disodium salt, characterized as 23; in a similar manner, 22 was converted into the
trisodium salt 24 (Scheme 7). The structures of 23 and 24 were confirmed by NMR spectroscopy using
COSY experiments. In particular, sulfation of 30-OH in the galactose unit caused a downfield shift of

Y.-M. Zhang et al. / Tetrahedron: Asymmetry 9 (1998) 2451–2464
2455
0.64 and 0.69 ppm for 23 and 24 respectively, sulfation of 60-OH in the galactose unit caused a downfield
shift of the signal of H-60 of 0.41 ppm for 24, and sulfation of 6-OH in the glucosamine unit caused a
downfield shift of the signal of H-6 of 0.49 and 0.52 ppm for 23 and 24 respectively, compared to the
non-sulfated parent compound Lewis x trisaccharide-β-OMe.16
Scheme 7. Reaction conditions: (i) 1 equiv. of Bu2SnO, MeOH, reflux 2 h; after evaporation of MeOH in vacuo: (a) 2 equiv.
of Me3N·SO3 complex, THF, rt, 40 h, then evaporation of THF, 2 equiv. of Me3N·SO3 complex, DMF, rt, 60 h, Dowex resin
(50x8-200, Na+): 19 (26%), 20 (11%), 21 (52%), 22 (7%); (b) 6 equiv. of Py·SO3 complex, DMF, rt, 5 days, Dowex resin
(50x8-200, Na+): 22 (66%); (ii) H2 (170 kPa), 10% Pd–C, MeOH, rt, 16 h, 97%; (iii) H2 (180 kPa), 10% Pd–C, MeOH, rt, 20
h, 90%
3. Experimental
3.1. General
Melting points (m.p.) were determined with a Büchi model 510 m.p. apparatus and are uncorrected.
Optical rotations were measured at 20ꢀ2°C with a Perkin–Elmer Model 241 digital polarimeter, using
a 10 cm, 1 ml cell. CI (ammonia) mass spectra were obtained with a Nermag R10-10 spectrometer.
Electrospray Ionisation (EI) mass spectra were recorded on a VG-Platform mass spectrometer (Fisons
Instruments) in negative mode. Elemental analyses were performed by Service Central d’Analyse du
C.N.R.S., BP 22, 69390 Vernaison, France, or by Service de Microanalyse de l’Université Pierre et
1
Marie Curie, 4, Place Jussieu, 75005 Paris, France. H NMR spectra were recorded with a Bruker AC
250 and a Bruker AM 400 spectrometer for solutions in CDCl3, CD3OD or D2O at ambient temperature.
Assignments were aided by COSY experiments. 13C NMR spectra were recorded at 62.89 MHz with
a Bruker AC 250 and at 100.57 MHz with a Bruker AM 400 for solutions in CDCl3 adopting 77.00
ppm for the central line of CDCl3. Assignments were aided by the J-mod technique and proton–carbon
correlation. For trisaccharide, a single prime refers to the hydrogens or carbons of the galactose unit;
and a double prime refers to the hydrogens or carbons of the fucose unit. Reactions were monitored by
thin-layer chromatography (TLC) on a precoated plate of silica gel 60 F254 (layer thickness, 0.2 mm; E.
Merck, Darmstadt, Germany) and detection by charring with sulfuric acid. Flash column chromatography
was performed on silica gel 60 (230–400 mesh, Merck).

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Y.-M. Zhang et al. / Tetrahedron: Asymmetry 9 (1998) 2451–2464
3.2. Methyl 4,6-O-benzylidene-2-deoxy-2-phthalimido-3-O-(2,3,4-tri-O-benzyl-α-L-fucopyranosyl)-β-
D-glucopyranoside 2
A suspension of 1 (1.2 g, 1.4 mmol), dry methanol (0.4 ml, 10 mmol) and molecular sieves (4
Å, 2 g) in dry acetonitrile (20 ml) was stirred for 0.5 h under argon at room temperature. Tris (4-
bromophenyl) ammoniumyl hexachloroantimonate (1.63 g, 2 mmol) was added. The mixture was kept
at room temperature for 20 min, when TLC showed the disappearance of 1. The mixture was neutralized
with triethylamine, diluted with dichloromethane, filtered through Celite, and concentrated. The residue
was eluted from a column of silica gel with cyclohexane:ethyl acetate (4:1) to give 2 as an amorphous
1
solid (1.05 g, 90%). Rf=0.36 (cyclohexane:ethyl acetate=3:1). [α]D −19 (c 1, CHCl3). H NMR (400
MHz, CDCl3): δ 7.83–7.76, 7.65–7.62, 7.57–7.54, 7.39–7.21, 7.08–7.06 (5m, 24H, arom. H), 5.60 (s,
0
0
0
1H, PhCH), 5.31 (d, 1H, J1,2=8.5 Hz, H-1), 4.87 (d, 1H, J1 ,2 =3.1 Hz, H-1 ), 4.84, 4.54 (2d, 2H, J=11.6
Hz, PhCH2), 4.67 (dd, 1H, J3,4=8.3, J2,3=10.3 Hz, H-3), 4.49 (dd, 1H, J6a,5=4.5, J6a,6b=10.5 Hz, H-6a),
4.47, 4.42 (2d, 2H, J=11.5 Hz, PhCH2),04.37 (dd, 1H, H-2), 4.30, 3.88 (2d, 2H, J=12.7 Hz, PhCH2),
0
0
0
0
0
0
0
4.11 (dq, 1H, J5 ,6 =6.5, J5 ,4 <1 Hz, H-5 ), 3.73 (dd, 1H, J2 ,3 =10.1 Hz, H-2 ), 3.50 (s, 3H, OMe), 0.92
(d, 3H, J6 ,5 =6.5 Hz, H-6 ). 13C NMR (62.89 MHz, CDCl3): 168.21 (2O_C, Phth), 138.91, 138.59,
138.37, 137.21 (4C, Ph), 133.78, 132.11, 129.10, 128.79, 128.55–127.38, 126.10, 123.18 (arom. C),
101.15 (PhCH), 99.86 (C-1), 99.57 (C-10), 82.23, 79.85, 78.09, 75.85, 75.63, 66.28 (ring C), 74.78,
73.15, 72.70 (3PhCH2), 68.71 (C-6), 67.33 (C-50), 57.08 (OMe), 55.70 (C-2), 16.49 (C-60). MS (CI)
0
0
0
+
m/z: 845 (40%) M+NH4 . Anal. calcd for C49H49NO11: C, 71.08; H, 5.97; N, 1.69. Found: C, 70.97; H,
6.16; N, 1.61.
3.3. Methyl 6-O-benzyl-2-deoxy-2-phthalimido-3-O-(2,3,4-tri-O-benzyl-α-L-fucopyranosyl)-β-
D-glucopyranoside 3
To a cold (0°C), stirred mixture of 2 (530 mg, 0.64 mmol), sodium cyanoborohydride (402 mg),
molecular sieves (4 Å, 1 g), and methyl orange (0.1 g) in dry THF (10 ml) was added dropwise, a
saturated solution of HCl in ether until the red color ceased to be discharged (about 2 ml), and stirring was
continued for 30 min. The mixture was diluted with dichloromethane, and the solid was filtered through
a Celite bed. The filtrate was washed with cold water, cold saturated NaHCO3 solution, water, dried,
and concentrated. The residue was applied to a column of silica gel and eluted with cyclohexane:ethyl
acetate (3:1) to give 3 as an amorphous solid (457 mg, 86%). Rf=0.32 (cyclohexane:ethyl acetate=3:1).
[α]D +34 (c 1, CHCl3). 1H NMR (400 MHz, CDCl3): δ 7.89–7.87, 7.73–7.67, 7.61–7.57 (3m, 4H, Phth),
7.45–7.23, 7.02–6.99 (2m, 20H, 4Ph), 5.30 (d, 1H, J1,2=8.4 Hz, H-1), 4.90, 4.56 (2d, 2H, J=11.5 Hz,
0
0
0
PhCH2), 4.72, 4.67 (2d, 2H, J=12.0 Hz, PhCH2), 4.66 (d, 1H, J1 ,2 =3.3 Hz, H-1 ), 4.64, 4.57 (2d, 2H,
J=11.8 Hz, PhCH2), 4.30 (dd, 1H, J2,3=10.8 Hz, H-2), 4.29 (d, 1H, JOH,4=1 Hz, OH), 4.20 (dd, 1H,
0
0 0 0 0
3,4=8.1 Hz, H-3), 4.15, 3.46 (2d, 2H, J=13 Hz, PhCH2), 4.12 (dq, 1H, J5 ,4 <1, J5 ,6 =6.4 Hz, H-5 ),
J
0
0
0
0
0
3.95 (dd, 1H, J6a,5=1.8, J6a,6b=10.7 Hz, H-6a), 3.82 (dd, 1H, J3 ,4 =2.5, J3 ,2 =10.2 Hz, H-3 ), 3.80 (dd,
1H, J6b,5=5.5 Hz, H-6b), 3.75 (dd, 1H, H-20), 3.71 (ddd, 1H, J5,4=9.6 Hz, H-5), 3.60 (ddd, 1H, H-4), 3.54
(dd, 1H, H-40), 3.53 (s, 3H, OMe), 1.11 (d, 3H, H-60). 13C NMR (62.89 MHz, CDCl3): δ 168.91, 168.11
(2C_O, Phth), 138.75, 138.49, 138.32, 138.14 (4C, Ph), 133.61 (2CH, Phth), 132.59, 132.02 (2C, Phth),
128.44–127.51 (CH, Ph), 122.95 (2CH, Phth), 100.80 (C-10), 98.23 (C-1), 83.19 (C-3), 78.89 (C-30),
77.92 (C-40), 75.27 (C-5), 74.76 (PhCH2), 73.99 (C-20), 73.48 (PhCH2), 73.43 (PhCH2), 72.39 (PhCH2),
71.25 (C-4), 69.29 (C-6), 68.47 (C-50), 56.78 (OMe), 54.55 (C-2), 16.47 (C-60). MS (CI): m/z 847 (65%)
+
M+NH4 . Anal. calcd for C49H51NO11: C, 70.91; H, 6.19; N, 1.69. Found: C, 70.66; H, 6.25; N, 1.60.

Y.-M. Zhang et al. / Tetrahedron: Asymmetry 9 (1998) 2451–2464
2457
3.4. Methyl 6-O-benzyl-2-deoxy-2-phthalimido-4-O-(2,3,4,6-tetra-O-benzoyl-β-D-galactosyl)-3-O-(2,
3,4-tri-O-benzyl-α-L-fucopyranosyl)-β-D-glucopyranoside 5
A solution of silver triflate (642 mg, 2.5 mmol) and s-collidine (0.25 ml) in dichloromethane (4.5
ml) and toluene (3 ml) was added dropwise at −20°C to a stirred solution of 3 (700 mg, 0.84 mmol)
and 2,3,4,6-tetra-O-benzoyl-α-D-galactopyranosyl bromide 4 (1.11 g, 1.68 mmol) in toluene (7.5 ml)
containing ground molecular sieves (4 Å, 2 g) under argon. When TLC indicated a complete reaction
(0.5 h), 10% aqueous sodium thiosulfate (10 ml) and toluene (20 ml) were added. The mixture was
filtered through Celite. The organic layer was separated and washed with water, dried, and concentrated.
The residue was flash chromatographed on a column of silica gel (cyclohexane:ethyl acetate=2.5:1) to
yield 5 (1.03 g, 87%) as a white solid. Rf=0.34 (cyclohexane:ethyl acetate=2:1). M.p. 178–179°C (from
ether). [α]D +3.7 (c 1, CHCl3). 1H NMR (400 MHz, CDCl3): δ 8.18–7.07 (m, 44H, arom. H), 5.84 (dd,
0
0
0
0
0
0
0
0
0
0
0
1H, J4 ,5 <1, J4 ,3 =3.7 Hz, H-4 0)0, 5.75 (dd, 1H, J2 ,1 =8.2, J2 ,3 =10.3 Hz, H-2 ), 5.38 (dd, 1H, H-3 ),
0
00 00
5.10 (d, 1H, J1 ,2 =3.5 Hz, H-1 ), 5.03 (d, 1H, H-1 ), 4.98 (d, 1H, J1,2=8.6 Hz, H-1), 4.93, 4.50 (2d,
00 00
00 00
2H, J=12.1 Hz, PhCH2), 4.82 (dd, 1H, J3,4=8.9, J3,2=10.5 Hz, H-3), 4.76 (dq, 1H, J5 ,4 <1, J5 ,6 =6.5
Hz, H-500), 4.72, 4.24 (2d, 2H, J=11.2 Hz, PhCH2), 4.40 (dd, 1H, H-2), 4.30 (dd, 1H, J4,5=9.6 Hz, H-4),
00,200
00 00
3.95 (dd, 1H, J3 ,4 =2.6, J3
=10.2 Hz, H-300), 3.83 (dd, 1H, H-200), 3.60 (dd, 1H, H-400), 3.37 (s, 3H,
OMe), 1.46 (d, 3H, H-600). 13C NMR (100.57 MHz, CDCl3): δ 167.79 (2O_C, Phth), 165.84, 165.74,
165.27, 164.70 (4O_C, Bz), 138.94, 138.82, 138.21, 137.78, 133.98, 133.50, 133.32, 133.24, 131.75,
129.84, 129.63, 129.60, 129.42, 129.19, 128.98, 128.76, 128.66, 128.63, 128.60, 128.48, 128.31, 128.24,
128.14, 127.96, 127.95, 127.86, 127.77, 127.16, 127.06, 126.89, 126.63, 123.45 (arom. C), 99.79 (C-10),
99.07 (C-1), 96.71 (C-100), 79.25 (C-400), 79.15 (C-300), 75.66, 75.37, 74.68, 72.36, 71.70, 71.28, 69.78,
68.24 (ring C), 75.05, 73.69, 72.66, 71.96, (4PhCH2), 67.54 (C-6), 66.62 (C-500), 61.30 (C-60), 56.62
(OMe), 56.06 (C-2), 16.81 (C-600). MS (CI): m/z 1425 (100%) M+NH4 . Anal. calcd for C83H77NO20:
+
C, 70.78; H, 5.51; N, 0.99. Found: C, 70.70; H, 5.46; N, 0.99.
3.5. Methyl 2-acetamido-6-O-benzyl-2-deoxy-4-O-β-D-galactopyranosyl-3-O-(2,3,4-tri-O-benzyl-α-L-
fucopyranosyl)-β-D-glucopyranoside 6
Hydrazine hydrate (9 ml) was added to a stirred solution of compound 5 (450 mg, 0.319 mmol) in
90% aqueous ethanol (90 ml) and refluxed for 20 h. The solution was concentrated and the residue
was acetylated using acetic anhydride (15 ml) and pyridine (30 ml) at room temperature overnight. The
solution was concentrated and coevaporated with toluene. Then the residue was stirred with sodium (10
mg) in methanol:dichloromethane (25 ml, 3:2) overnight at room temperature. The base was neutralized
by an IR-120 cation exchange resin. The resin was filtered off and thoroughly washed with methanol.
The combined filtrate and washings were concentrated. The residue was flash chromatographed on a
column of silica gel (dichloromethane:ethyl acetate:methanol=5:10:2) to yield 6 as a white solid (231 mg,
80%). Rf=0.38 (dichloromethane:ethyl acetate:methanol=5:10:2). M.p. 95°C (dichloromethane–ether).
1
[α]D −76 (c 1, MeOH). H NMR (400 MHz, CD3OD): δ 7.62–7.45 (m, 20H, arom. H), 5.53 (d, 1H,
00,200
J1
=3.8 Hz, H-100), 5.08–4.98 (m, 5H, H-500, PhCH2, 2H from 2PhCH2), 4.87, 4.75 (2d, 2H, J=11.8
0
0
0
Hz, PhCH2), 4.77–4.73 (m, 2H from 2PhCH2), 4.61 (d, 1H, J1 ,2 =7.7 Hz, H-1 ), 4.50 (d, 1H, J1,2=7.9
00
00 00
00 00
Hz, H-1), 4.36 (m, 1H, H-4), 4.30 (dd, 1H, J3 ,4 =2.8, J3 ,2 =10.2 Hz, H-3 ), 4.23 (dd, 1H, J6a,5=3.4,
J
6a,6b=11.2 Hz, H-6a), 4.22 (dd, 1H, J2,3=8.2 Hz, H-2), 4.22–4.20 (m, 1H, H-3), 4.16 (d, 1H, H-200), 4.13
(dd, 1H, J6 a,5 =7.6, J6 a,6 b=11.8 Hz, H-600a), 4.10 (m, 1H, H-400), 4.02 (dd, 1H, J6b,5=1.9 Hz, H-6b),
0
0
0
0
0
0
0
0
0
0
0
3.90 (dd, 1H, J4 ,5 <1, J4 ,3 =3.40 Hz, H-4 ), 3.77 (dd, 1H, J6 b,5 =3.7 Hz, H-6 b), 3.75 (m,01H, H-5),
0
0
0
3.67 (dd, 1H, J2 ,3 =9.1 Hz, H-2 ), 3.64 (s, 3H, OMe), 3.52 (dd, 1H, H-3 ), 3.50 (m, 1H, H-5 ), 2.17 (s,

2458
Y.-M. Zhang et al. / Tetrahedron: Asymmetry 9 (1998) 2451–2464
00
3H, NHAc), 1.37 (d, 3H, J6 ,5 =6.5 Hz, H-6 ). 13C NMR (100.57 MHz, CD3OD=49.30 ppm): δ 173.53
(O_C, NHAc), 140.75, 140.52, 139.95, 139.80 (4C, Ph), 129.84, 129.73, 129.47, 129.44, 128.96, 128.93,
128.85, 128.71, 128.63 (CH, Ph), 104.13 (C-10), 103.80 (C-1), 98.05 (C-100), 80.24, 80.24, 77.57, 77.41,
76.86, 75.86, 75.21, 74.85, 73.07, 70.41, 68.05 (11CH, ring C), 76.68, 74.39, 73.93, 73.91 (4PhCH2),
69.61 (C-6), 63.70 (C-60), 57.52 (C-2), 57.30 (OMe), 23.73 (CH3, NHAc), 17.20 (C-600). MS (CI): m/z
00 00
+
921 (40%) M+NH4 , 904 (100%) M+H+. Anal. calcd for C49H61NO15·2H2O: C, 62.60; H, 6.97; N, 1.49.
Found: C, 62.63; H, 6.80; N, 1.45.
3.6. Methyl 2-acetamido-6-O-benzyl-2-deoxy-4-O-(3-O-sodium sulfonato-β-D-galactopyranosyl)-
3-O-(2,3,4-tri-O-benzyl-α-L-fucopyranosyl)-β-D-glucopyranoside 7 and methyl 2-acetamido-6-O-
benzyl-2-deoxy-4-O-(3,6-di-O-sodium sulfonato-β-D-galactopyranosyl)-3-O-(2,3,4-tri-O-benzyl-α-L-
fucopyranosyl)-β-D-glucopyranoside 8
A mixture of compound 6 (135 mg, 0.15 mmol) and dibutyltin oxide (37 mg, 0.15 mmol) in dry
methanol (1.5 ml) was heated to reflux for 2 h (the solution becames clear) and then the solvent was
distilled at 80°C to give a yellowish syrup, which was evaporated to dryness in vacuo (2 h). Sulfur
trioxide–trimethylamine complex (41 mg, 0.3 mmol) and dry tetrahydrofuran (1.5 ml) were introduced.
The mixture was stirred under argon for 40 h at room temperature. Methanol (1.5 ml) was added,
after filtration, the filtrate was concentrated. Flash chromatography of the residue on a column of
silica gel (dichloromethane:methanol=5:1) followed by cation exchange chromatography (Dowex 50X8-
200, Na+ form) using methanol afforded first 7 as a white amorphous mass (120 mg, 79%). Rf=0.36
1
(dichloromethane:methanol=5:1). [α]D −58 (c 1, MeOH). H NMR (250 MHz, CD3OD): δ 7.41–7.30
(m, 20H, arom. H), 5.52 (d, 1H, J1
=3.8 Hz, H-100), 4.87, 4.74 (2d, 2H, J=12 Hz, PhCH2), 4.67 (d,
00,200
0
0
0
00 00
1H, J1,2=7.7 Hz, H-1), 4.48 (d, 1H, J1 ,2 =7.9 Hz, H-1 ), 3.65 (s, 3H, OMe), 1.33 (d, 3H, J6 ,5 =6.5 Hz,
H-600). 13C NMR (62.89 MHz, CD3OD=49.40 ppm): δ 173.70 (O_C, NHAc), 140.85, 140.60, 139.93,
139.87 (4C, Ph), 129.96, 129.91, 129.83, 129.61, 129.60, 129.53, 129.23, 129.12, 129.03, 128.89, 128.81,
128.71 (CH, Ph), 104.38 (C-10), 103.58 (C-1), 98.17 (C-100), 82.54 (C-30), 80.34, 80.18, 77.36, 77.25,
76.80, 75.87, 74.81, 74.54, 74.06, 73.87, 71.52, 69.59, 68.78, 68.15 (4PhCH2, C-6, ring C), 63.64 (C-60),
57.54 (C-2), 57.54 (OMe), 23.83 (CH3, NHAc), 17.24 (C-600). Anal. calcd for C49H60NO18SNa·2.5H2O:
C, 55.99; H, 6.23; N, 1.33. Found: C, 55.95; H, 6.17; N, 1.52.
Compound 8 was eluted second (after ion exchange), as a syrup (21 mg, 13%). Rf=0.18
(dichloromethane:methanol=4:1). This compound was characterized after debenzylation.
3.7. Methyl 2-acetamido-2-deoxy-3-O-α-L-fucopyranosyl-4-O-(3-O-sodium sulfonato-β-D-galacto-
pyranosyl)-β-D-glucopyranoside 9
A solution of 7 (85 mg, 84.5 µmol) in methanol (8 ml) was reacted over Pd/C (10%, 160 mg) at
15°C under H2 (160 kPa) for 15 h, filtered, and evaporated. The residue was purified on a Sephadex
column (G10-120), using water as eluant. After ion exchange with Dowex 50X8-200 (Na+ form) and
freeze-drying, compound 9 was obtained as a white amorphous solid (54 mg, 99%). Rf=0.53 (ethyl
acetate:isopropanol:water=3:3:2). [α]D −63 (c 0.75, MeOH). 1H NMR (400 MHz, D2O): δ 5.10 (d, 1H,
00 00 0
00 00 00 00 0 0
=4.0 Hz, H-1 ), 4.81 (dq, 1H, J5 ,4 <1, J5 ,6 =6.5 Hz, H-5 ), 4.56 (d, 1H, J1 ,2 =7.8 Hz, H-1 ),
00,200
J1
0
0
0
0
0
0
0
4.46 (d, 1H, J1,2=8.1 Hz, H-1), 4.32 (dd, 1H, J3 ,4 =3.3, J3 ,2 =9.9 Hz, H-3 ), 4.26 (dd, 1H, J4 ,5 <1 Hz,
H-40), 4.01 (dd, 1H, J6a,5=2.0, J6a,6b=12.0 Hz, H-6a), 3.91 (dd, 1H, J2,3=10.0 Hz, H-2), 3.87 (dd, 1H,
00
6b,5=4.0 Hz, H-6b), 3.73 (m, 2H, H-60a, H-60b), 3.68 (dd, 1H, J2 ,3 =10.5 Hz, H-2 ), 3.62 (dd, 1H,
00 00
J
H-20), 3.50 (s, 3H, OMe), 2.02 (s, 3H, NHAc), 1.17 (d, 3H, H-600). 13C NMR (100.57 MHz, D2O): δ

Y.-M. Zhang et al. / Tetrahedron: Asymmetry 9 (1998) 2451–2464
2459
175.70 (C_O, NHAc), 103.05 (C-1), 102.74 (C-10), 99.89 (C-100), 81.46 (C-30), 76.51, 76.11, 75.89,
74.74, 73.16, 70.48, 70.37, 68.96, 67.97, 67.97 (10CH, ring C), 62.63 (C-60), 60.95 (C-6), 58.44 (OMe),
56.92 (C-2), 23.50 (CH3, NHAc), 16.54 (C-600). MS (EI) calcd for C21H36NO18SNa: 645.38. Found: m/z
622 (M–Na)−. Anal. calcd for C21H36NO18SNa·3.5H2O: C, 35.59; H, 6.12; N, 1.98. Found: C, 35.58;
H, 6.38; N, 2.07.
3.8. Methyl 2-acetamido-2-deoxy-3-O-α-L-fucopyranosyl-4-O-(3,6-di-O-sodium sulfonato-β-
D-galactopyranosyl)-β-D-glucopyranoside 10
A solution of 8 (33 mg, 30 µmol) in methanol (5 ml) was reacted over Pd/C (10%, 140 mg) at
15°C under H2 (160 kPa) for 15 h, filtered, and evaporated. The residue was purified on a Sephadex
column (G10-120), using water as eluant. After ion exchange with Dowex 50X8-200 (Na+ form) and
freeze-drying, compound 10 was obtained as a white amorphous solid (22 mg, 99%). Rf=0.34 (ethyl
acetate:isopropanol:water=3:3:2). [α]D −41 (c 0.54, MeOH). 1H NMR (400 MHz, D2O): δ 5.10 (d, 1H,
00 00 0
00 00 00 00 0 0
=3.9 Hz, H-1 ), 4.82 (dq, 1H, J5 ,6 =6.5, J5 ,4 <1 Hz, H-5 ), 4.60 (d, 1H, J1 ,2 =7.8 Hz, H-1 ),
00,200
J1
0
0
0
0
0
0
0
4.48 (d, 1H, J1,2=7.8 Hz, H-1), 4.36 (dd, 1H, J3 ,4 =3.4, J3 ,2 =9.7 Hz, H-3 ), 4.33 (dd, 1H, J4 ,5 <1 Hz,
0
H-40), 4.19 (dd, 1H, J6 a,5 =6.0, J6 a,6 b=11.0 Hz, H-60a), 4.15 (dd, 1H, J6 b,5 =7.0 Hz, H-6 b), 4.03 (dd,
0
0
0
0
0
0
1H, J6a,5=2.1, J6a,6b=12.0 Hz, H-6a), 3.91 (dd, 1H, J2,3=10.030 Hz, H-2), 3.90 (ddd, 1H, H-50), 3.89 (dd,
0
00 00
1H, J6b,5=5.0 Hz, H-6b), 3.66 (dd, 1H, J2 ,3 =10.2 Hz, H-2 ), 3.65 (dd, 1H, H-2 ), 3.51 (s, 3H, OMe),
2.03 (s, 3H, NHAc), 1.18 (d, 3H, H-600). 13C NMR (100.57 MHz, D2O): δ 175.69 (C_O, NHAc), 103.04
(C-1), 102.71 (C-10), 99.87 (C-100), 81.15 (C-30), 76.57, 76.32, 75.14, 73.26, 73.20, 70.41, 70.28, 69.12
(8CH, ring C), 68.25 (C-60), 67.96 (C-500), 67.57 (C-40), 61.05 (C-6), 58.41 (OMe), 56.93 (C-2), 23.50
(CH3, NHAc), 16.59 (C-600). MS (EI) calcd for C21H35NO21S2Na2: 747.64. Found: m/z 724 (M–Na)−,
350.3 [(M–2Na)/2]2−, 622 (M–NaSO3–Na+H)−.
3.9. Phenyl 6-O-benzoyl-2-deoxy-2-phthalimido-1-thio-β-D-glucopyranoside 12
Sodium (23 mg) was added to a solution of phenyl 2-deoxy-2-phthalimido-1-thio-3,4,6-tri-O-acetyl-
β-D-glucopyranoside 11 (1.05 g, 2 mmol) in methanol (20 ml). After stirring for 2 h at room temperature,
the mixture was neutralized by Amberlite resin (IR120, H+ form), filtered and concentrated to dryness.
The obtained amorphous solid was heated to reflux in toluene (40 ml) with bis (tributyltin) oxide (1
g, 1.68 mmol) for 15 h, then allowed to cool. Benzoyl chloride (472 mg, 3.36 mmol) was added and
the mixture was stirred at room temperature for 5 h. Methanol (0.5 ml) was introduced. After 1 h of
stirring at room temperature, the solvent was evaporated. The residue was flash chromatographed on a
column of silica gel (dichloromethane:ethyl acetate=2:1) to yield 12 (930 mg, 92%) as a white solid.
Rf=0.25 (dichloromethane:ethyl acetate=2:1). M.p. 113°C (from cyclohexane:ethyl acetate). [α]D +11.5
(c 1, CHCl3). 1H NMR (400 MHz, CDCl3): δ 8.08–7.15 (m, 14H, arom. H), 5.66 (d, 1H, J1,2=10.5 Hz,
H-1), 4.73 (dd, 1H, J6a,5=5.0, J6a,6b=12.2 Hz, H-6a), 4.66 (dd, 1H, J6b,5=2.4 Hz, H-6b), 4.44 (ddd, 1H,
J
3,OH=4.7, J3,4=8.8, J3,2=10.2 Hz, H-3), 4.23 (dd, 1H, H-2), 3.82 (ddd, 1H, J5,4=9.8 Hz, H-5), 3.73 (d,
1H, JOH,4=4.4 Hz, OH-4), 3.54 (ddd, 1H, H-4), 3.28 (d, 1H, OH-3). 13C NMR (100.57 MHz, CDCl3):
δ 168.26, 167.83 (2O_C, Phth), 166.77 (O_C, Bz), 134.09, 133.13, 132.22, 131.94, 131.28, 129.68,
129.39, 128.63, 128.25, 127.53, 123.65, 123.23 (arom. C), 83.19 (C-1), 77.66, 72.43, 71.35 (C-3, 4, 5),
+
64.06 (C-6), 55.33 (C-2). MS (CI): m/z 523 (100%) M+NH4 . Anal. calcd for C27H23NO7S: C, 64.15;
H, 4.59. Found: C, 64.10; H, 4.61.

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Y.-M. Zhang et al. / Tetrahedron: Asymmetry 9 (1998) 2451–2464
3.10. Phenyl 6-O-benzoyl-2-deoxy-2-phthalimido-4-O-(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)-
1-thio-β-D-glucopyranoside 14
A solution of 12 (252 mg, 0.5 mmol) and 2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl fluoride 13 (350
mg, 1 mmol) in 5 ml of CH2Cl2 and 1 ml of toluene was stirred with 4 Å ground molecular sieves (0.5 g)
for 30 min at room temperature under an argon atmosphere. A mixture of stannous chloride (95 mg, 0.5
mmol) and silver triflate (128 mg, 0.5 mmol) was added at −15°C, then the reaction mixture was allowed
to gradually warm to room temperature, and the stirring was continued for an additional 2 h. The mixture
was filtered through Celite and the solids were washed with CH2Cl2. The combined filtrate and washings
were washed with a saturated NaHCO3 solution, then with water, dried over MgSO4, and concentrated.
The residue was applied to a column of silica gel and eluted with cyclohexane:ethyl acetate (1:1) to afford
an amorphous solid (292 mg, 70%). Rf=0.34 (cyclohexane:ethyl acetate=1:1). [α]D +17 (c 1, CHCl3). 1H
NMR (250 MHz, CDCl3): δ 8.08–7.13 (m, 14H, arom. H), 5.66 (d, 1H, J1,2=10.5 Hz, H-1), 5.38 (0dd, 1H,
0
0
0
0
0
0
0
0
0
0
J4 ,5 <1, J4 ,3 =3.4 Hz, H-4 ), 5.28 (dd, 1H, J2 ,1 =8.0, J2 ,3 =10.4 Hz, H-2 ), 4.97 (dd, 1H, H-3 ), 4.67
(dd, 1H, J6a,5=2, J6a,6b=11.8 Hz, H-6a), 4.59 (d, 1H, H-10), 4.48 (ddd, 1H, J3,OH<1, J3,4=8.1, J3,2=10.2
Hz, H-3), 4.43 (d, 1H, OH-3), 4.33 (dd, 1H, J6b,5=5 Hz, H-6b), 4.28 (dd, 1H, H-2), 4.11–3.93 (m, 4H, H-
5, 50, 60a, 60b), 3.68 (dd, 1H, J4,5=9.7 Hz, H-4), 2.15, 2.12, 1.98, 1.85 (4s, 12H, 4Ac). 13C NMR (100.57
MHz, CDCl3): δ 169.98, 169.72, 169.58, 169.26 (4O_C, Ac), 167.80, 167.17 (2O_C, Phth), 165.77
(O_C, Bz), 133.98, 133.07, 132.49, 131.39, 131.29, 131.26, 129.41, 129.31, 128.52, 128.26, 127.69,
123.29, 123.07 (arom. C), 101.61 (C-10), 83.01, 82.82 (C-1, C-4), 75.59, 71.04, 70.79, 70.53, 68.39,
66.55 (C-3, 5, 20, 30, 40, 50), 63.02, 61.50 (C-6, 60), 54.47 (C-2), 20.33, 20.23, 20.15, 19.80 (4CH3, Ac).
+
MS (CI): m/z 853 (100%) M+NH4 . Anal. calcd for C41H41NO16S: C, 58.92; H, 4.94. Found: C, 58.86;
H, 5.15.
3.11. Methyl 6-O-benzoyl-2-deoxy-2-phthalimido-4-O-(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)-
β-D-glucopyranoside 15
A suspension of 14 (400 mg, 0.48 mmol), dry methanol (0.2 ml, 4.9 mmol) and molecular sieves
(4 Å, 0.6 g) in dry acetonitrile (15 ml) was stirred for 0.5 h under argon at room temperature. Tris
(4-bromophenyl) ammoniumyl hexachloroantimonate (800 mg, 0.98 mmol) was added. The reaction
mixture was stirred at room temperature for 1 h, TLC showed the disappearance of 14. The mixture was
neutralized with triethylamine, diluted with dichloromethane, filtered through Celite, and concentrated.
The residue was eluted from a column of silica gel with dichloromethane:ethyl acetate (4:1) to give 15
as an amorphous solid (345 mg, 95%). Rf=0.26 (dichloromethane:ethyl acetate=5:1). [α]D +5.5 (c 1.5,
CHCl3). 1H NMR (400 MHz, CDCl3): δ 8.11–7.52 (m, 9H, arom. H), 5.38 (dd, 1H, J4 ,5 =1, J4 ,3 =3.4
0
0
0
0
0
Hz, H-40), 5.30 (dd, 1H, J2 ,1 =8.1, J2 ,3 =10.5 Hz, H-2 ), 5.28 (d, 1H, J1,2=8.6 Hz, H-1), 4.97 (dd, 1H,
0
0
0
0
H-30), 4.66 (dd, 1H, J6a,5=1.7, J6a,6b=11.8 Hz, H-6a), 4.63 (d, 1H, H-10), 4.49 (ddd, 1H, J3,OH=1.8,
J
3,4=8.2, J3,2=10.7 Hz, H-3), 4.39 (d, 1H, OH-3), 4.38 (dd, 1H, J6b,5=4.6 Hz, H-6b), 4.24 (dd, 1H, H-
2), 4.12 (dd, 1H, J6 a,5 =4.5, J6 a,6 b=11.1 Hz, H-60a), 4.07 (dd, 1H, J6 b,5 =8.0 Hz, H-6 b), 4.01 (ddd,
1H, H-50), 3.93 (ddd, 1H, J5,4=9.7 Hz, H-5), 3.76 (dd, 1H, H-4), 2.18, 2.13, 2.00, 1.89 (4s, 12H, 4Ac).
13C NMR (100.57 MHz, CDCl3): δ 170.15, 169.85, 169.73, 169.45 (4O_C, Ac), 167.95 (2O_C, Phth),
166.02 (O_C, Bz), 133.95, 133.19, 131.57, 129.47, 129.45, 128.41, 123.19 (arom. C), 101.79 (C-10),
99.03 (C-1), 83.34 (C-4), 71.86 (C-5), 71.13 (C-50), 70.67 (C-30), 69.81 (C-3), 68.49 (C-20), 66.61 (C-
40), 62.98 (C-6), 61.61 (C-60), 56.69 (OMe), 55.54 (C-2), 20.46, 20.38, 20.28, 19.95 (4CH3, Ac). MS
0
0
0
0
0
0
0
+
(CI): m/z 775 (80%) M+NH4 . Anal. calcd for C36H39NO17·H2O: C, 55.74; H, 5.31; N, 1.80. Found: C,
55.53; H, 5.01; N, 1.75.

Y.-M. Zhang et al. / Tetrahedron: Asymmetry 9 (1998) 2451–2464
2461
3.12. Methyl 6-O-benzoyl-2-deoxy-2-phthalimido-4-O-(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)-
3-O-(2,3,4-tri-O-benzyl-α-L-fucopyranosyl)-β-D-glucopyranoside 17
To a cooled (0°C), stirred mixture of 15 (225 mg, 0.3 mmol), isopropenyl 2,3,4-tri-O-benzyl-α,β-
L-fucopyronosyl carbonate 16 (171 mg, 0.33 mmol), activated 4 Å ground molecular sieves (0.6 g),
and dry ether (9 ml) was added dropwise TMSOTf (64 µl, 0.33 mmol). Stirring was continued for 40
min at 0°C, then the mixture was neutralized with Et3N, diluted with CH2Cl2, filtered through Celite,
and concentrated. The residue was eluted from a column of silica gel with cyclohexane:ethyl acetate
(1.5:1) to give 17. Crystallization in ether gave a white solid (285 mg, 81%). Rf=0.5 (cyclohexane:ethyl
1
acetate=1:1). M.p. 190–191°C (from ether). [α]D +15 (c 1, CHCl3). H NMR (250 MHz, CDCl3): δ
0
0
0
0
0
0
0
8.12–7.07 (m, 24H, arom. H), 5.32 (dd, 1H, J4 ,5 <1, J4 ,3 =3.4 Hz, H-4 ), 5.17 (dd, 1H, J2 ,1 =8.3,
0
0
0
J2 ,3 =10.3 Hz, H-2 ), 5.12 (d, 1H, J1,2=8.5 Hz, H-1), 3.40 (s, 3H, OMe), 2.14, 2.01, 1.98, 1.89 (4S, 12H,
4Ac), 1.27 (d, 3H, J6 ,5 =6.5 Hz, H-6 ). 13C NMR (100.57 MHz, CDCl3): δ 169.91, 169.82, 169.70,
168.84 (4O_C, Ac), 165.78 (O_C, Bz), 138.70, 138.51, 137.97, 134.07, 133.45, 131.69, 129.59, 129.43,
128.62, 128.26, 128.14, 128.10, 128.03, 127.96, 127.94, 127.76, 127.37, 127.23, 127.11, 126.88, 123.50
(arom. C), 99.91 (C-10), 99.08 (C-1), 97.61 (C-100), 79.65, 77.06, 76.23, 74.49, 73.21, 72.62, 70.86,
70.66, 68.85, 66.55, 66.51 (11CH, ring C), 74.11, 72.94, 72.29 (3PhCH2), 62.59, 60.15 (C-6, C-60),
56.68 (OMe), 56.00 (C-2), 20.64, 20.47, 20.41, 20.39 (4CH3, Ac), 16.67 (C-600). MS (CI): m/z 1191
00
00 00
+
(100%) M+NH4 . Anal. calcd for C63H67NO21: C, 64.44; H, 5.75; N, 1.19. Found: C, 64.32; H, 5.85; N,
1.10.
3.13. Methyl 2-acetamido-2-deoxy-4-O-β-D-galactopyranosyl-3-O-(2,3,4-tri-O-benzyl-α-L-fucopyran-
osyl)-β-D-glucopyranoside 18
Hydrazine hydrate (4 ml) was added to a stirred solution of compound 17 (220 mg, 0.19 mmol) in
90% aqueous ethanol (42 ml) and refluxed for 18 h. The solution was concentrated and the residue
was taken by dichloromethane (2×30 ml), filtered and evaporated to dryness. The residue was stirred
with acetic anhydride (1 ml) in 10 ml of dichloromethane:methanol (1:1) at room temperature for 3 h.
The solution was concentrated and coevaporated with ethanol. The residue was flash chromatographed
on a column of silica gel (dichloromethane:acetone:methanol=3:6:1) to yield 18 as a white amorphous
solid (108 mg, 70%). Rf=0.26 (dichloromethane:acetone:methanol=3:6:1). [α]D −75 (c 2.2, MeOH). 1H
00
00 00
NMR (400 MHz, CD3OD): δ 7.59–7.42 (m, 15H, arom. H), 5.52 (d, 1H, J1 ,2 =3.8 Hz, H-1 ), 5.06 (m,
1H, H-500), 5.04, 4.75 (2d, 2H, J=11.3 Hz, 0PhCH2), 5.01 (m, 2H, PhCH2), 5.00, 4.75 (2d, 2H, J=12.3
0
0
00 00
Hz, PhCH2), 4.68 (d, 1H, J1 ,2 =7.1 Hz, H-1 ), 4.50 (d, 1H, J1,2=7.5 Hz, H-1), 4.31 (dd, 1H, J3 ,4 =2.8,
00,200
0
J3
0
=10.3 Hz, H-300), 4.22–4.11 (m, 8H, H-2, H-4, H-5, H-6a, H-6b, H-200, H-400, H-60a), 3.96 (dd, 1H,
J4 ,5 <1, J4 ,3 =2.8 Hz, H-4 ), 3.81 (dd, 1H, J6 b,5 =3.7, J6 b,6 a=11.8 Hz, H-60b), 3.71 (dd, 1H, J2 ,3 =9.7
0
0
0
0
0
0
0
0
0
Hz, H-20), 3.67 (dd, 1H, H-30), 3.66 (m, 10H0 , H-50), 3.65 (s, 3H, OMe), 3.61 (m, 1H, H-3), 2.17 (s,
3H, NHAc), 1.38 (d, 3H, J6 ,5 =6.5 Hz, H-6 ). 13C NMR (100.57 MHz, CD3OD=49.30 ppm): δ 173.59
00 00
(O_C, NHAc), 140.75, 140.53, 139.80 (3C, Ph), 129.87, 129.54, 129.49, 129.45, 128.95, 128.81, 128.72,
128.63-1 (CH, Ph), 104.26 (C-1), 103.99 (C-10), 98.18 (C-100), 80.25, 80.23, 77.66, 77.58, 77.39, 75.93,
75.20, 74.88, 73.21, 70.45, 68.06 (11CH, ring C), 76.72, 73.96, 73.88 (3PhCH2), 63.72 (C-60), 61.64 (C-
6), 57.57 (C-2), 57.38 (OMe), 23.75 (CH3, NHAc), 17.19 (C-600). Anal. calcd for C42H55NO15·2H2O:
C, 59.35; H, 7.00; N, 1.65. Found: C, 59.58; H, 6.94; N, 1.59.

2462
Y.-M. Zhang et al. / Tetrahedron: Asymmetry 9 (1998) 2451–2464
3.14. Methyl
2-acetamido-2-deoxy-4-O-(3-O-sodium
sulfonato-β-D-galactopyranosyl)-3-O-(2,3,
4-tri-O-benzyl-α-L-fucopyranosyl)-β-D-glucopyranoside 19; methyl 2-acetamido-2-deoxy-6-O-
sodium sulfonato-4-O-(3-O-sodium sulfonato-β-D-galactopyranosyl)-3-O-(2,3,4-tri-O-benzyl-α-
L-fucopyranosyl)-β-D-glucopyranoside 20; methyl 2-acetamido-2-deoxy-4-O-(3,6-di-O-sodium
sulfonato-β-D-galactopyranosyl)-3-O-(2,3,4-tri-O-benzyl-α-L-fucopyranosyl)-β-D-glucopyranoside
21 and methyl 2-acetamido-2-deoxy-6-O-sodium sulfonato-4-O-(3,6-di-O-sodium sulfonato-β-D-
galactopyranosyl)-3-O-(2,3,4-tri-O-benzyl-α-L-fucopyranosyl)-β-D-glucopyranoside 22
A mixture of compound 18 (105 mg, 0.13 mmol) and dibutyltin oxide (32 mg, 0.13 mmol) in dry
methanol (1 ml) was heated to reflux for 2.5 h (the solution became clear), then the methanol was
distilled at 80°C to give a yellowish syrup, which was evaporated to dryness in vacuo (2 h). Sulfur
trioxide–trimethylamine complex (36 mg, 0.26 mmol) and dry tetrahydrofuran (1 ml) were introduced,
and the mixture was stirred under argon for 40 h at room temperature. THF was evaporated and
more sulfur trioxide–trimethylamine complex (36 mg, 0.26 mmol) was added, the mixture was stirred
in 2 ml DMF for 60 h. Methanol (0.2 ml) was added, after filtration, the filtrate was concentrated.
Flash chromatography of the residue on a column of silica gel (dichloromethane:methanol=2:1, then
dichloromethane:methanol:H2O=4:2:0.1) followed by cation exchange chromatography (Dowex 50X8-
200, Na+ form) using methanol afforded 4 compounds.
19: 31 mg (26%), Rf=0.57 (dichloromethane:methanol=3:1). Compound 19 (14 mg) was
transformed into a triethylammonium salt by passage down a column of silica gel, eluted with
dichloromethane:methanol:triethylamine (3.5:1:0.05) to give a dichloromethane soluble derivative
1
(16 mg). H NMR 0(0400 MHz, CDCl3): δ 7.41–7.26 (m, 15H, 3Ph), 6.32 (br, 1H, NH), 5.26 (d, 1H,
0 0
0 0 0 0 0 0
=2.5 Hz, H-1 ), 4.37 (dd, 1H, J3 ,4 =3.1, J3 ,2 =9.7 Hz, H-3 ), 4.31 (dd, 1H, J4 ,5 <1 Hz, H-4 ),
00,200
J1
3.44 (s, 3H, OMe), 3.15 (q, J=7.4 Hz, CH2, Et3N), 1.79 (s, 3H, Ac), 1.37 (t, CH3, Et3N), 1.18 (d, 3H,
=6.5 Hz, H-600). 13C NMR (62.89 MHz, CDCl3): δ 170.61 (O_C, NHAc), 138.77, 138.66, 138.19
00,500
J6
(3C, Ph), 128.21, 128.09, 127.91, 127.89, 127.85, 127.51, 127.20, 127.13, 127.08 (CH, Ph), 101.89,
101.75 (C-1, C-10), 97.14 (C-100), 80.17 (C-30), 79.19, 77.61, 75.83, 75.59, 75.19, 75.07, 74.54, 70.27,
67.56, 66.61 (10CH, ring C), 74.81, 73.04, 71.75 (3PhCH2), 61.96, 60.52 (C-6, C-60), 57.11 (OMe),
56.70 (C-2), 46.08 (CH2, Et3N), 23.27 (CH3, NHAc), 16.47 (C-600), 8.52 (CH3, Et3N). Debenzylation
of 19 gave a monosulfated trisaccharide identical with compound 9 prepared from 7.
20: 15 mg (11%), Rf=0.22 (dichloromethane:methanol=3:1). Compound 20 (11 mg) was
transformed into a triethylammonium salt by passage down a column of silica gel, eluted with
dichloromethane:methanol:triethylamine (3:1:0.05) to give a dichloromethane soluble derivative (15
1
mg). H NMR (400 MHz, CDCl3): δ 7.41–7.25 (m, 15H, 3Ph), 6.64 (br, 1H, NH), 5.36 (d, 1H,
00,200
J1
=2.5 Hz, H-100), 3.41 (s, 3H, OMe), 3.14 (q, J=7.4 Hz, CH2, from Et3N), 1.79 (s, 3H, Ac), 1.38
00
00 00
(t, CH3, from Et3N), 1.16 (d, 3H, J6 ,5 =6.5 Hz, H-6 ). Further characterization of this compound was
performed after debenzylation into the disulfated trisaccharide 23.
21: 68 mg (52%), Rf=0.19 (dichloromethane:methanol=3:1). 1H NMR (250 MHz, CD3OD): δ
00
00 00
7.36–7.08 (m, 15H, 3Ph), 5.08 0(0d, 1H, J1 ,2 =3.0 Hz, H-1 ), 3.38 (s, 3H, OMe), 1.87 (s, 3H, Ac),
+
00 00
1.08 (d, 3H, J6 ,5 =6.4 Hz, H-6 ). Debenzylation of 21, followed by ion exchange (Na form), gave a
disulfated trisaccharide identical to compound 10 prepared from 8.
1
22: 10 mg (7%), Rf=0.31 (dichloromethane:methanol:H2O=4:2:0.1). H NMR (400 MHz, CD3OD):
00
00 00
0
0
0
0
δ 7.35–7.10 (m, 15H, 3Ph), 5.07 (d, 1H, J1 ,2 =3.6 Hz, H-1 ), 4.25 (dd, 1H, J3 ,4 =3.0, J3 ,2 =9.7 Hz,
H-30), 3.37 (s, 3H, OMe), 1.87 (s, 3H, Ac), 1.07 (d, 3H, J6 ,5 =6.5 Hz, H-6 ). 13C NMR (100.57 MHz,
CD3OD=49.30 ppm): δ 174.00 (O_C, NHAc), 140.77, 140.52, 139.74 (3C, Ph), 130.07, 130.03, 129.82,
129.58, 129.45, 129.41, 129.33, 129.06, 128.91, 128.79, 128.74, 128.37 (CH, Ph), 104.02, 103.45 (C-1,
00
00 00

Y.-M. Zhang et al. / Tetrahedron: Asymmetry 9 (1998) 2451–2464
2463
C-10), 98.83 (C-100), 81.25 (C-30), 80.17, 79.63, 76.16, 76.08, 75.61, 75.31, 74.08, 71.27, 68.08, 68.07
(10CH, ring C), 76.66, 73.75, 73.29 (3PhCH2), 67.58, 67.45 (C-6, C-60), 57.78 (OMe), 57.48 (C-2), 23.79
(CH3, NHAc), 17.14 (C-600). The trisulfated trisaccharide 22 was also obtained as the main compound
by the following procedure.
A mixture of compound 18 (90 mg, 0.11 mmol) and dibutyltin oxide (27 mg, 0.11 mmol) in dry
methanol (1 ml) was heated to reflux for 2.5 h (the solution became clear), then the methanol was
distilled at 80°C to give a yellowish syrup, which was evaporated to dryness in vacuo (3 h). Sulfur
trioxide–pyridine complex (57 mg, 0.36 mmol) and dry DMF (1.5 ml) were introduced and the mixture
was stirred under argon at room temperature for 3 days. TLC showed complete disappearance of
starting material, a mixture of 19, 20, 21, and 22 was formed. More sulfur trioxide–pyridine complex
(57 mg, 0.36 mmol) was added, and stirring was continued for 40 h at room temperature. DMF was
evaporated under reduced pressure. Flash chromatography of the residue on a column of silica gel
(dichloromethane:methanol:H2O=4:2:0.2) followed by cation exchange chromatography (Dowex 50X8-
200, Na+ form) using methanol afforded 22 as an amorphous solid (82 mg, 66%).
3.15. Methyl 2-acetamido-2-deoxy-3-O-α-L-fucopyranosyl-6-O-sodium sulfonato-4-O-(3-O-sodium
sulfonato-β-D-galactopyranosyl)-β-D-glucopyranoside 23
A solution of 20 (10 mg, 9.8 µmol) in methanol (3 ml) was reacted over Pd/C (10%, 30 mg) at
15°C under H2 (170 kPa) for 16 h, filtered, and evaporated. The residue was purified on a Sephadex
column (G10-120), using water as eluant. After ion exchange with Dowex 50X8-200 (Na+ form) and
freeze-drying, compound 23 was obtained as a white amorphous solid (7.3 mg, 97%). Rf=0.37 (ethyl
1
acetate:isopropanol:water=3:3:2). [α]D −19 (c 0.38, H2O). H NMR (400 MHz, D2O): δ 5.10 (d, 1H,
00 00 0
00 00 00 00 0 0
=3.9 Hz, H-1 ), 4.80 (dq, 1H, J5 ,4 <1, J5 ,6 =6.5 Hz, H-5 ), 4.64 (d, 1H, J1 ,2 =7.8 Hz, H-1 ),
00,200
J1
0
0
0
0
0
4.50 (d, 1H, J1,2=7.8 Hz, H-1), 4.39 (m, 2H, H-6a, H-6b), 4.32 (dd, 1H, J3 ,4 =3.3, J3 ,2 =9.8 Hz, H-3 ),
0
0
0
4.27 (dd, 1H, J4 ,5 <1 Hz, H-4 ), 3.90 (dd, 1H, J2,3=8.8 Hz, H-2), 3.81 (m, 1H, H-5), 3.67 (dd, 1H,
=10.4 Hz, H-200), 3.62 (dd, 1H, H-20), 3.50 (s, 3H, OMe), 2.02 (s, 3H, Ac), 1.17 (d, 3H, H-600).
00,300
J2
13C NMR (100.57 MHz, D2O): δ 175.72 (C_O, NHAc), 103.08 (C-1), 102.50 (C-10), 99.87 (C-100),
81.49 (C-30), 76.02, 75.96, 74.22, 74.20, 73.20, 70.52, 70.45, 69.05, 68.04, 68.03 (10CH, ring C), 67.28
(C-6), 62.82 (C-60), 58.58 (OMe), 56.79 (C-2), 23.55 (CH3, NHAc), 16.59 (C-600). MS (EI) calcd for
C21H35NO21S2Na2: 747.64. Found: m/z 724 (M–Na)−, 350.2 [(M–2Na)/2]2−, 622 (M–NaSO3–Na+H)−.
3.16. Methyl 2-acetamido-2-deoxy-3-O-α-L-fucopyranosyl-6-O-sodium sulfonato-4-O-(3,6-di-O-
sodium sulfonato-β-D-galactopyranosyl)-β-D-glucopyranoside 24
A solution of 22 (14 mg, 12.5 µmol) in methanol (5 ml) was reacted over Pd/C (10%, 35 mg) at
room temperature under H2 (170 kPa) for 20 h, filtered, and evaporated. The residue was purified on a
Sephadex column (G10-120), using water as eluant. Further purification was made by anion exchange
chromatography on DEAE-Sephacel eluting with a linear gradient of 4 mM to 2 M pyridine:AcOH (1:1).
After lyophilization from water, the pyridinium salt was converted to the sodium salt by ion exchange
with Dowex 50X8-200 (Na+ form) and freeze-drying, compound 24 was obtained as a white amorphous
solid (9.6 mg, 90%). Rf=0.24 (ethyl acetate:isopropanol:water=3:3:2). [α]D −34 (c 0.35, H2O). 1H NMR
00 00
00 00 00 00
=4.0 Hz, H-1 ), 4.85 (dq, 1H, J5 ,4 <1, J5 ,6 =6.5 Hz, H-5 ),
00,200
(500 MHz, D2O): δ 5.16 (d, 1H, J1
0
0
0
4.70 (d, 1H, J1 ,2 =7.8 Hz, H-1 ), 4.58 (d, 1H, J1,2=7.7 Hz, H-1), 4.49 (dd, 1H, J6a,5=20.3, J6a,6b=11 Hz,
0
0
0
0
H-6a), 4.45 (dd,01H, J6b,5=4.4 Hz, H-6b), 4.43 (dd, 1H, J3 ,4 =3.4, J3 ,2 =9.8 Hz, H-3 ), 4.38 (dd, 1H,
0
J4 ,5 <1 Hz, H-4 ), 4.24 (dd, 1H, J6 a,5 =5.7, J6 a,6 b=10 Hz, H-60a), 4.21 (dd, 1H, J6 b,5 =6.5 Hz, H-6 b),
0
0
0
0
0
0
0
0

2464
Y.-M. Zhang et al. / Tetrahedron: Asymmetry 9 (1998) 2451–2464
4.08 (m, 1H, H-4), 3.97, 3.87 (2m, 5H, H-2, 3, 300, 400, 50), 3.91 (m, 1H, H-5), 3.72 (dd, 1H, J2
=10.4
00,300
Hz, H-200), 3.70 (dd, 1H, H-20), 3.57 (s, 3H, OMe), 2.10 (s, 3H, Ac), 1.25 (d, 3H, H-600). 13C NMR (75
MHz, D2O): δ 175.66 (C_O, NHAc), 103.04 (C-1), 102.67 (C-10), 99.92 (C-100), 81.14 (C-30), 76.24,
74.94, 74.39, 73.31, 73.22, 70.44, 70.36, 69.20, 68.03, 67.63 (10CH, ring C), 68.12 (C-60), 67.46 (C-6),
58.49 (OMe), 56.76 (C-2), 23.52 (CH3, NHAc), 16.63 (C-600). MS (EI) calcd for C21H34NO24S3Na3:
849.69. Found: m/z 401 [(M–2Na)/2]2−, 260 [(M–3Na)/3]3−
.
Acknowledgements
The authors would like to thank C. Picard and F. Uzabiaga for recording the electrospray ionisation
mass spectra of 9, 10, 23 and 24.
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