Synthesis of a spacer-armed disulfated tetrasaccharide of SB1a, a carbohydrate hapten associated with human hepatocellular carcinoma

Article abstract of DOI:10.1016/S0008-6215(02)00291-4

A disulfated tetrasaccharide fragment with a spacer arm of human hepatocellular carcinoma carbohydrate antigen SB_1a, namely, 2-aminoethyl 3-O-sulfo-β-D-galactopyranosyl-(1→3)-2-acetamido-2-deoxy-β-D- galactopyranosyl-(1→4)-3-O-sulfo-β-D-galactopyranosyl-(1→4)- β-D-glucopyranoside was synthesized via a [2+1+1] block building mode. In the last coupling step toward the trisaccharide acceptor 8, benzoyl protected galactosyl bromide donor 14 was found to be much more reactive than the acetyl-protected donors.

Full text of DOI:10.1016/S0008-6215(02)00291-4

Carbohydrate Research 337 (2002) 1929–1934
www.elsevier.com/locate/carres
Synthesis of a spacer-armed disulfated tetrasaccharide of SB1a, a
carbohydrate hapten associated with human hepatocellular
carcinoma
Qin Li, Hui Li, Qing Li, Qing-Hua Lou, Bin Su, Meng-Shen Cai, Zhong-Jun Li*
Department of Chemical Biology, School of Pharmaceutical Sciences, Peking Uni6ersity, Beijing 100083, China
Received 22 March 2002; accepted 17 July 2002
Dedicated to Professor Derek Horton on the occasion of his 70th birthday
Abstract
A disulfated tetrasaccharide fragment with a spacer arm of human hepatocellular carcinoma carbohydrate antigen SB1a,
namely, 2-aminoethyl 3-O-sulfo-b- -galactopyranosyl-(1“3)-2-acetamido-2-deoxy-b- -galactopyranosyl-(1“4)-3-O-sulfo-b-
galactopyranosyl-(1“4)-b- -glucopyranoside was synthesized via a [2+1+1] block building mode. In the last coupling step
D
D
D-
D
toward the trisaccharide acceptor 8, benzoyl protected galactosyl bromide donor 14 was found to be much more reactive than the
acetyl-protected donors. © 2002 Elsevier Science Ltd. All rights reserved.
Keywords: SB1a, HCC carbohydrate antigen; Disulfated tetrasaccharide; Synthesis
1. Introduction
important cancer-associated carbohydrate antigens of
HCC.5,6
Aberrant cell-surface glycosylation is often closely
associated with tumor progression and malignancy.1 In
most cases, carbohydrate antigens may be rather spe-
cific to a certain type of tumor and are not overex-
pressed or recognized by the immune system in normal
tissues.2 Therefore, carbohydrate antigens have been
greatly mesmerizing scientists in relevant fields because
of their potential applications in tumor immunother-
apy.3 SB1a, a glycosphinolipid with a disulfated tetra-
saccharide moiety, was first isolated from rat kidney by
Tadano and Ishizuka.4 The normal human liver con-
tains essentially no detectable amount of SB1a. How-
In order to elucidate the functions of SB1a in detail,
especially its mechanism involved in the onset, progres-
sion, and metastasis of HCC, and hence pursue optimal
carbohydrate-based anticancer vaccines for HCC, we
have synthesized the disulfated tetrasaccharide moiety
of the SB1a determinant, namely compound 1, in which
a 2-aminoethyl group is attached to the reducing termi-
nal as a spacer arm, which could facilitate further
formation of immunogenic glycoconjugates by the
coupling of the spacer amino group and a carrier
protein.
ever, studies have shown that
a
remarkable
accumulation of SB1a exists, not only in the cultured
human hepatocellular carcinoma (HCC) cell lines, but
also in glycolipid fractions extracted from HCC tissues.
Therefore, it is suggested that SB1a is one of the most
* Corresponding author. Tel.: +86-10-6209-1504; fax: +
86-10-6236-7134
E-mail address: zjli@bjmu.edu.cn (Z.-J. Li).
0008-6215/02/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved.
PII: S0008-6215(02)00291-4

1930
Q. Li et al. / Carbohydrate Research 337 (2002) 1929–1934
D
-lactose.7 In the synthesis of 3, the p-methoxybenzyl
2. Results and discussion
group (PMB) was introduced to the 3-OH position of
the galactosyl moiety via a dibutyltin oxide-mediated
procedure,8 followed by addition of p-methoxybenzyl
chloride and tetrabutylammonium bromide in boiling
toluene (Scheme 1).9
Of the various approaches available for the prepara-
tion of oligosaccharides, we adopted the stepwise syn-
thetic strategy to build the target molecule. The
reducing terminal
D-lactosyl building block 3 of the
Standard glycosylation of 3 and the glycosyl donor
target molecule was first synthesized in a good yield
3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-b-D-galacto-
(89.6%) via the regioselective etherification of the 3%-OH
pyranosyl trichloroacetimidate (4)10 in toluene at
−40 °C gave the desired b-linked trisaccharide 5
(83.2%). Dephthaloylation of compound 5 with 1,2-
diaminoethane11 in n-butanol at 75 °C, followed by
acetylation, resulted in the formation of 6. Subsequent
O-deacetylation and benzylidenation at the C-4¦ and
C-6¦ hydroxy groups with benzaldehyde dimethyl acetal
in acetonitrile under acidic conditions provided the
trisaccharide acceptor 8 in excellent yield.
of 2-azidoethyl 2,3,6-tri-O-benzyl-2,6-di-O-benzyl-b-
D-
galactopyranosyl-(1“4)-b-
D
-glucopyranoside (2),
which was prepared steadily through several steps from
However, for the assembly of the tetrasaccharide
backbone, some interesting results occurred. In our
initial design, ethyl 2,4,6-tri-O-acetyl-3-O-p-methoxy-
benzyl-1-thio-b- -galactopyranoside (9) or the corre-
D
sponding glycosyl bromide 10 was chosen as the
glycosyl donor to couple with the trisaccharide acceptor
8. No reaction occurred when 9 and 8 were mixed and
stirred at room temperature in nitromethane or DMF
employing Bu4NBr–CuBr2 as the promoter. Neither
did the coupling reaction of 9 and 8 using methyl
triflate as the promoter in dichloromethane or diethyl
ether. We next investigated the glycosylation of the
donor 10 with the trisaccharide acceptor 8, no desired
tetrasaccharide was obtained when silver triflate was
chosen to promote the coupling reaction. The main
product is the asymmetric (1“1)-linked disaccharide
11, in which two galactosyl groups were condensed to
each other by a and b configurations at the anomeric
center, respectively.
Scheme 1. Synthesis of the trisaccharide acceptor 8. (i) (a)
Bu2SnO, MeOH, reflux; (b) toluene, p-CH3OC6H4CH2Cl,
Bu4NBr, 4 A MS (89.6%); (ii) TMSOTf, toluene, −40 °C
(83.2%); (iii) (a) 1,2-diaminoethane, n-butanol, 75 °C; (b)
Ac2O–Py (91.2%); (iv) NaOMe, MeOH; (v) PhCH(OMe)2,
camphorsulfonic acid, CH3CN (90%).
,
After a series of failures in the building of the
tetrasaccharide backbone, we selected another type of
glycosyl donor containing a benzoyl group at C-2 for
coupling with acceptor 8. Therefore, we chose the gly-
cosyl bromide 14 as the glycosyl donor. Compound 14
was synthesized by the in situ transformation of ethyl
4-O-acetyl-2,6-di-O-benzoyl-3-O-chloroacetyl-1-thio-b-
D
-galactopyranoside (13), which was prepared from
chloroacetylation of the known ethyl 4-O-acetyl-2,6-di-
O-benzoyl-1-thio-b-
-galactopyranoside (12).12 To our
surprise, the silver triflate-promoted glycosylation with
8 using donor 14 in dichloromethane at −20 °C gave
the desired tetrasaccharide 15 in very high yield (89%)
(Scheme 2).
Scheme 2. Synthesis of the target tetrasaccharide. (i) ClAcCl,
CH2Cl2–pyridine (83.7%); (ii) Br2, CH2Cl2, 0 °C“rt; (iii)
D
,
AgOTf, 4 A MS, CH2Cl2, −20 °C (89%); (vi) thiourea,
2,6-lutidine, CH2Cl2–EtOH, 58 °C (76.4%); (v) CAN,
CH3CN–H2O, rt, (92.4%); (vi) SO3·Pyr, Pyr (95%); (vii) H2,
10% Pd/C, MeOH–H2O, 1 M HCl; (viii) 0.012 M NaOMe–
MeOH, rt; (ix) 0.5 M NaOMe–MeOH, 0 °C.

Q. Li et al. / Carbohydrate Research 337 (2002) 1929–1934
1931
Deblocking of 15 to the target tetrasaccharide 1
includes several steps as in the following. At first,
selective removal of the chloroacetyl group at the 3§-
OH position and the p-methoxybenzyl group at 3%-OH
position with thiourea and cerium(IV) ammonium ni-
trate (CAN), respectively, gave 17. Then, treatment of
the diol 17 with sulfur trioxide·pyridine complex in
pyridine furnished the disulfated compound 18 in 95%
yield. But deprotection of 18 was rather complicated.
Catalytic hydrogenolysis, using palladium-on-charcoal
in different solvents (AcOH, 2:1 MeOH–AcOH) was
sluggish and the yield was low. This problem may be
ascribed to the catalyst passiveness due to the interac-
tion with the aminoethyl fragment formed. A similar
phenomenon has been observed by Spijker et al.13 and
Stahl et al.14 To avoid this inhibitory effect, hydrochlo-
ric acid was added to the reaction mixture to convert
the formed amine to its hydrochloride salt. This greatly
increased the hydrogenolysis rate and yield. Then, deac-
ylation of 19 with 0.012 M sodium methoxide in MeOH
at room temperature provided product 20 with the
2§-O-benzoyl group retained. Increasing base concen-
tration and prolonging reaction time only led to decom-
position of the product. When the O-deacylation was
carried out with ammonia in MeOH, no O-deacylation
but O-desulfonation was observed. Finally, the sapon-
ification of 19 was completed with 0.5 M sodium
methoxide in MeOH at 0 °C for 6 h to give the title
compound 1 in 90% yield.
dry MeOH (200 mL) was added Bu2SnO (1.77 g, 7.11
mmol), and the mixture was stirred overnight at 60 °C
under Ar. After cooling to room temperature, the mix-
ture was concentrated, and the residue was dissolved in
dry toluene (200 mL). Bu4NBr (0.588 g, 1.8 mmol) and
,
powdered 4 A molecular sieves (5 g) were added, and
the mixture was stirred for 1 h at room temperature
under Ar. Then, p-methoxybenzyl chloride (1.77 mL)
was added, and the mixture was stirred for 4 h at
120 °C, at the end of which time TLC (5:1 toluene–
EtOAc) showed the disappearance of 2 and the forma-
tion of 3. After cooling to room temperature, MeOH
(10 mL) and Et3N (2.5 mL) were added, and the
stirring was continued for 15 min. After filtration
through Celite, the filtrate was concentrated. Column
chromatography (4:1 petroleum ether–acetone) of the
residue afforded 3 as a white needles (3.60 g, 89.6%).
mp 86.0–87.0 °C. [h]D +15.8° (c 1.58, CHCl3). 1H
NMR (CDCl3): l 7.51–6.91 (m, 29 H, Ar-H), 5.12–
3.43 (m, 32 H, sugar H, 5×PhCH2, CH3OC6H4CH2,
OCH2CH2N3), 2.64 (bs, 1 H, 4%-OH). 13C NMR: l
159.1, 113.6 (CH3OC6H4CH2), 138.9, 138.5, 138.4,
138.0, 129.8, 129.2, 128.2, 127.9, 127.8, 127.5, 127.4,
127.3, 127.1 (Ar-C), 103.4, 102.4 (C-1, C-1%), 82.6, 81.6,
79.2, 76.3, 75.1, 75.0, 74.9, 73.3, 72.9, 72.7, 71.7, 68.3,
68.0, 67.9, 65.9 (sugar C, 5×PhCH2, CH3OC6H4CH2,
OCH2CH2N3), 55.0 (OCH3), 50.7 (CH2N3). MALDI-
TOFMS: m/z 1002.8 [M+Na]+. Anal. Calcd for
C17H29N3O17: C, 69.72; H, 6.42; N, 3.79. Found: C,
69.40; H, 6.56; N, 3.79.
2-Azidoethyl
imido-i- -galactopyranosyl-(1“4)-2,6-di-O-benzyl-3-
O-p-methoxybenzyl-i- -galactopyranosyl-(1“4)-2,3,6-
tri-O-benzyl-i- -glucopyranoside (5).—To a solution
of 3 (1.00 g) and 3,4,6-tri-O-acetyl-2-deoxy-2-phthal-
imido-b-
-galactopyranosyl trichloroacetimidate (4,10
3,4,6-tri-O-acetyl-2-deoxy-2-phthal-
3. Experimental
D
D
General methods.—All moisture-sensitive reactions
were performed under argon atmosphere, and organic
solvents were dried over standard drying agents and
freshly distilled prior to use. Optical rotations were
measured at 25 °C with an Optical Activity LTD AA-
10R polarimeter in a 5-cm, 1-mL cell. Melting points
were uncorrected. NMR spectra were recorded at room
temperature with a JEOL 300, Bruker AM 400, and
INOVA-600 spectrometers. Chemical shifts were ex-
pressed in ppm downfield from the signal for internal
Me4Si for solutions in CDCl3, CD3OD and DMSO-d6,
or DSS in case of D2O. MALDI-TOFMS analyses were
performed with an LDI-1700 mass spectrometer.
Column chromatography was performed on silica gel H
60, and fractions were monitored by TLC on silica gel
60 GF254 with detection by UV light and/or by charring
with 10% H2SO4 in EtOH. Solutions were concentrated
at or below 40 °C and dried with anhydrous Na2SO4.
2-Azidoethyl 2,6-di-O-benzyl-3-O-p-methoxybenzyl-i
D
D
,
1.00 g) in dry toluene (50 mL) were added 4 A molecu-
lar sieves (0.93 g), and the mixture was stirred for 1 h
under argon. The mixture was cooled to −40 °C, and
a solution of TMSOTf (60 mL) in dry CH2Cl2 (1 mL)
was added. The mixture was stirred at −40 °C for 3 h
and then overnight at room temperature. Et3N (0.5
mL) was added, and the mixture was diluted with
EtOAc (100 mL) and filtered (Celite). The filtrate was
washed with water (100 mL), aq NaHCO3 (100 mL)
and water (100 mL), dried, concentrated. Column chro-
matography (4:1:0.1 C6H12–CHCl3–acetone) afforded
5, isolated as a colorless foam (1.12 g, 83.2%). [h]D
1
+8.5° (c 1.42, CHCl3). H NMR (CDCl3): l 7.87–6.79
(m, 33 H, 5×PhCH2, Phth, CH3OC6H4CH2O), 6.61
(dd, 1 H, J3§,4¦ 3.90 Hz, J2%,3¦ 11.70 Hz, H-3¦), 5.55 (d,
1 H, H-4¦), 5.35 (d, 1 H, J1¦,2¦ 8.40 Hz, H-1¦), 3.79 (s,
3 H, CH3O), 2.20, 2.03, 1.85 (3 s, 3 H each, 3×OAc).
13C NMR (CDCl3): l 170.4, 170.3, 169.8, 168.2, 167.4
(Phth, 3×OAc), 159.4, 113.7 (CH3OC6H4CH2O),
139.1, 138.9, 138.7, 138.4, 138.3, 134.0, 133.7, 132.6,
-
D
-galactopyranosyl-(1“4)-2,3,6-tri-O-benzyl-i-
glucopyranoside (3).—To a solution of 2-azidoethyl 2,6-
di-O-benzyl-b- -galactopyranosyl-(1“4)-2,3,6-tri-O-
benzyl-b-
-glucopyranoside (2,7 3.53 g, 4.10 mmol) in
D-
D
D

1932
Q. Li et al. / Carbohydrate Research 337 (2002) 1929–1934
131.7, 130.1, 130.0, 129.7, 28.3, 128.2, 128.0, 127.9,
127.5, 127.4, 127.3, 123.4, 123.3 (Ar-C), 103.7, 102.1
(C-1, C-1%), 99.8 (C-1¦), 82.7, 81.7, 80.6, 80.1, 77.2, 76.4,
76.1, 75.7, 75.3, 75.1, 74.6, 73.2, 73.1, 72.4, 70.4, 68.9,
68.2, 68.1, 67.4, 66.6, 61.2 (sugar C, CH3OC6H4CH2,
5×PhCH2, OCH2CH2N3), 55.3 (CH3O), 51.5 (C-2¦),
50.9 (CH2N3), 20.7, 20.5, 20.4 (3×OAc). Anal. Calcd
for C77H82N4O21: C, 66.04; H, 5.87; N, 4.01. Found: C,
65.90; H, 6.13; N, 3.74.
82.4, 81.9, 81.5, 80.6, 76.5, 75.9, 75.5, 75.1, 74.7, 74.4,
73.1, 73.0, 72.7, 68.8, 68.0, 67.1, 61.1 (sugar C,
CH3OC6H4CH2, 5×PhCH2, OCH2CH2N3), 55.2, 55.1
(CH3O, C-2¦), 50.8 (CH2N3), 22.4 (NHAc). MALDI-
TOFMS: 1294.0 [M+Na]+, 1310 [M+K]+. Anal.
Calcd for C72H80N4O17: C, 67.92; H, 6.29; N, 4.40.
Found: C, 67.76; H, 6.50; N, 4.39.
Ethyl 4-O-acetyl-2,6-di-O-benzoyl-3-O-chloroacetyl-
1-thio-i- -galactopyranoside (13).—Monochloroacetyl
D
2-Azidoethyl 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-
chloride (0.53 mL, 6.67 mmol) in dry CH2Cl2 (10 mL)
i-
methoxybenzyl-i-
O-benzyl-i- -glucopyranoside (6).—A solution of 5
D
-galactopyranosyl-(1“4)-2,6-di-O-benzyl-3-O-p-
was added dropwise to a cooled (0 °C) solution of ethyl
D
-galactopyranosyl-(1“4)-2,3,6-tri-
4-O-acetyl-2,6-di-O-benzoyl-1-thio-b-D-galactopyranos-
D
ide (12,12 2.22 g, 4.26 mmol) in 5:1 CH2Cl2–pyridine
(60 mL). After 2 h the solution was washed with H2O,
dried, filtered and concentrated. After column chro-
matography (7:1 petroleum ether–EtOAc) 13 was ob-
(2.30 g, 1.65 mmol) and 1,2-diaminoethane (32 mL) in
n-butanol (117 mL) was stirred overnight at 75 °C
under argon. After cooling to room temperature, the
mixture was co-evaporated with toluene (3×50 mL). A
solution of the residue in 1:1 Ac2O–pyridine (130 mL)
was stirred overnight at room temperature, then the
mixture was co-evaporated with toluene (3×30 mL).
Column chromatography of the residue (2.3:1
petroleum ether–acetone) afforded 6, isolated as a col-
orless solid (1.96 g, 91.2%). [h]D +12.6° (c 1.27,
CHCl3). 1H NMR (CDCl3): l 7.47–6.90 (m, 29 H,
5×PhCH2, CH3OC6H4CH2), 3.84 (s, 3 H, CH3O),
2.18, 2.00, 1.93 (3 s, 3 H each, 3×OAc), 1.69 (s, 3 H,
NHAc). 13C NMR (CDCl3): l 170.3, 170.2, 169.7 (4×
Ac), 159.8, 114.3 (CH3OC6H4CH2), 103.5, 103.1, 102.5
(C-1, 1%, 1¦), 55.3 (CH3O), 50.8, 50.7 (C-2¦, CH2N3),
23.2 (NHAc), 20.8, 20.6, 20.5 (3×OAc). Anal. Calcd
for C71H82N4O20: C, 65.04; H, 6.26; N, 4.27. Found: C,
65.39; H, 6.38; N, 4.21.
1
tained: [h]D +7.1° (c 1.13, CHCl3). H NMR (CDCl3):
l 7.99–7.40 (m, 10 H, Ar-H), 5.60–5.52 (m, 2 H, H-2,
4), 5.36 (dd, 1 H, J2,3 9.90 Hz, J3,4 3.30 Hz, H-3), 4.72
(d, 1 H, J1,2 9.90 Hz, H-1), 4.54 (dd, 1 H, J5,6a 6.60 Hz,
J6a,6b11.40 Hz, H-6a), 4.32 (dd, 1 H, J5,6b 6.90 Hz,
H-6b), 3.88 (m, 2 H, ClCH2CO), 2.77–2.68 (m, 2 H,
CH3CH2S), 2.19 (s, 3 H, Ac), 1.23 (t, 3 H, CH3CH2S).
13C NMR (CDCl3): l 170.4, 166.6, 165.9, 165.2 (CO),
133.5 (2 C), 129.8, 129.6, 129.2, 129.0, 128.5 (Ar-C),
84.2 (C-1), 74.4, 73.6, 67.7, 67.3, 61.7 (C-2, 3, 4, 5, 6),
40.3 (ClCH2CO), 24.6 (CH3CH2S), 20.7 (Ac), 14.8
(CH3CH2S).
2-Azidoethyl
4-O-acetyl-2,6-di-O-benzoyl-3-O-
chloroacetyl-i-
D
-galactopyranosyl-(1“3)-2-acetam-
ido-4,6-O-benzylidene-2-deoxy-i-
“4)-2,6-di-O-benzyl-3-O-p-methoxybenzyl-i-
topyranosyl-(1“4)-2,3,6-tri-O-benzyl-i- -glucopyran-
D-galactopyranosyl-(1
D
-galac-
2-Azidoethyl
oxy-i- -galactopyranosyl-(1“4)-2,6-di-O-benzyl-3-
O-p-methoxybenzyl-i- -galactopyranosyl-(1“4)-2,3,6-
tri-O-benzyl-i- -glucopyranoside (8).—To a solution
2-acetamido-4,6-O-benzylidene-2-de-
D
D
oside (15).—To a solution of 13 (402 mg, 0.731 mmol)
in dry CH2Cl2 (16 mL) was added Br2 (35 mL, 0.731
mmol) at 0 °C. The solution was stirred at 0 °C for 40
min, and the solvent was subsequently evaporated.
After co-evaporation twice with benzene, the residue
was dissolved in CH2Cl2 (16 mL) and added to a
mixture of 8 (560 mg, 0.440 mmol), AgOTf (268 mg),
2,6-di-tert-butyl-4-methylpyridine (94.6 mg) and
D
D
of 6 (1.30 g, 0.992 mmol) in dry MeOH (40 mL) was
added NaOMe (108 mg). The mixture was stirred at
room temperature for 14 h, then neutralized with
cation-exchange resin (H+ form), and filtered. The
filtrate was concentrated affording 7 (1.25 g, quant). To
a solution of 7 (1.25 g) and a,a-dimethoxytoluene (354
mL) in dry CH3CN (15 mL) was added camphorsul-
fonic acid until pH 4 was attained. The mixture was
stirred at room temperature for 24 h, then Et3N (0.5
mL) was added, and the solvent was removed under
reduced pressure. The residue was chromatographed
(2:1 petroleum ether–acetone) to give 8 (1.21 g, 90%):
1H NMR (CDCl3): l 7.59–6.79 (m, 34 H, Ar-H), 5.59
(s, 1 H, PhCH), 4.93–3.35 (m, 40 H, sugar H, 5×
PhCH2, CH3OC6H4CH2, OCH2CH2N3), 1.65 (s, 3 H,
NHAc). 13C NMR (CDCl3): l 173.6 (NHAc), 159.9,
114.1 (CH3OC6H4CH2), 138.5, 138.4, 138.1, 137.9,
137.8, 130.2, 129.6, 128.8, 128.7, 128.6, 128.5, 128.4,
128.2, 128.0, 127.9, 127.7, 127.5, 127.2, 126.4 (Ar-C),
101.1 (PhCH), 103.5, 102.9, 102.5 (C-1, C-1%, C-1¦),
,
crushed 4 A molecular sieves (950 mg) in CH2Cl2 (16
mL), which had been stirred under argon for 40 min,
and then cooled to −20 °C. The mixture was allowed
to warm to room temperature and to stir overnight.
The reaction mixture was diluted with CH2Cl2 (100
mL) and filtered through Celite. The filtrate was
washed with aq NaHCO3, 10% Na2S2O3 and H2O. The
organic layer was dried and concentrated. Column
chromatography (2.3:1 petroleum ether–acetone) of the
residue afforded 15 (690 mg, 89.0%) as a white solid.
[h]D +41.2° (c 0.97, CHCl3). 1H NMR (CDCl3): l
8.08–6.73 (m, 44 H, Ar-H), 5.56–3.23 (m, 50 H, PhCH,
5×PhCH2, CH3OC6H4CH2, ClCH2CO, OCH2CH2N3
and sugar H), 2.24 (s, 3 H, OAc), 1.30 (s, 3 H, NHAc).
13C NMR (CDCl3): l 171.4, 170.9, 167.0, 166.3, 165.1

Q. Li et al. / Carbohydrate Research 337 (2002) 1929–1934
1933
(2×PhCO, ClCH2CO, NHAc, OAc), 159.6, 114.1
(CH3OC6H4CH2), 138.9, 138.7, 138.6, 138.5, 133.9,
130.7, 130.2, 130.0, 129.8, 129.7, 129.0, 128.9, 128.8,
128.7, 128.6, 128.4, 128.0, 127.9, 127.6126.7 (Ar-C),
104.0, 102.8, 102.6, 101.1, 99.9 (C-1, C-1%, C-1¦, C-1§,
PhCH), 83.2, 82.0, 80.4, 76.6, 75.6, 75.4, 73.0, 72.5,
71.1, 69.6 (sugar C, 5×PhCH2, CH3OC6H4CH2,
OCH2CH2N3), 55.6 (CH3O), 54.8 (C-2¦), 51.3
(OCH2CH2N3), 40.7 (ClCH2CO), 23.7 (NHAc), 21.2
(OAc). MALDI-TOFMS: m/z 1783.5 [M+Na]+. Anal.
Calcd for C96H102ClN4O26: C, 65.45; H, 5.80; N, 3.18.
Found: C, 65.41; H, 6.01; N, 3.15.
17, isolated as a colorless solid (600 mg, 92.4%). [h]D
+21.3 (c 2.07, CHCl3). H NMR (CDCl3): l 8.04–6.81
1
(m, 40 H, Ar-H), 5.48–3.11 (m, 43 H, sugar H, 5×
PhCH2, PhCH, OCH2CH2N3), 2.09 (s, 3 H, OAc), 1.30
(s, 3 H, NHAc). 13C NMR (CDCl3): l 171.4, 171.0,
165.9 (2×PhCO, OAc, NHAc), 103.5, 102.2, 101.9,
100.4, 99.0 (C-1, C-1%, C-1¦, C-1§, PhCH), 54.7 (C-2¦),
50.9 (OCH2CH2N3), 23.1 (NHAc), 20.8 (OAc). Anal.
Calcd for C86H92N4O24: C, 65.98; H, 5.88; N, 3.58.
Found: C, 65.69; H, 6.17; N, 3.33.
2-Azidoethyl 4-O-acetyl-2,6-di-O-benzoyl-3-O-sulfo-
i-
zylidene-2-deoxy-i-
O-benzyl-3-O-sulfo-i-
tri-O-benzyl-i- -glucopyranoside (18).—To a solution
D
-galactopyranosyl-(1“3)-2-acetamido-4,6-O-ben-
-galactopyranosyl-(“4)-2,6-di-
-galactopyranosyl-(1“4)-2,3,6-
D
2-Azidoethyl
4-O-acetyl-2,6-di-O-benzoyl-3-O-
D
chloroacetyl -i -
D
-galactopyranosyl-(1“3)-2- acetam-
D
ido-4,6-O-benzylidene-2-deoxy-i-
“4)-2,6-di-O-benzyl-i- -galactopyranosyl-(1“4)-
2,3,6-tri-O-benzyl-i- -glucopyranoside (16).—To a so-
D-galactopyranosyl-(1
of 17 (220 mg, 0.639 mmol) in dry pyridine was added
sulfur trioxide·pyridine complex (668 mg, 4.20 mmol),
and the mixture was stirred at room temperature for 36
h. MeOH (1 mL) was added, and stirring was contin-
ued for 10 min. The mixture was concentrated, and the
residue was purified by flash chromatography (10:1
D
D
lution of 15 (575 mg, 0.327 mmol) in 1:1 CH2Cl2–abs
EtOH (16 mL) was added thiourea (115 mg) and 2,6-lu-
tidine (69 mL). After stirring for 10 h at 58 °C, the
mixture was cooled to room temperature and diluted
with CH2Cl2 and washed with water. The organic layer
was dried and concentrated. Purification of the residue
by chromatography (2:1:0.1 petroleum ether–EtOAc–
EtOH) gave 16 (420 mg, 76.4%). [h]D +21.1° (c 1.14,
CHCl3). 1H NMR (CDCl3): l 8.06–6.80 (m, 44 H,
Ar-H), 5.45–3.12 (m, 48 H, PhCH2, PhCH,
CH3OC6H4CH2, OCH2CH2N3, sugar H), 2.14 (s, 3 H,
OAc), 1.41 (s, 3 H, NHAc). 13C NMR (CDCl3): l 171.0
(2 C), 166.4, 166.0 (2×PhCO, OAc, NHAc), 159.3,
113.8 (CH3OC6H4CH2), 138.7, 138.6, 138.4, 138.3,
133.4, 130.4, 129.9, 129.7, 128.6, 128.4, 128.3, 128.2,
128.1, 128.0, 127.9, 127.5, 127.3, 126.4, 126.3 (Ar-C),
103.6, 102.5, 101.9, 100.6, 99.0 (C-1, C-1%, C-1¦, C-1§,
PhCH), 82.8, 81.7, 81.2, 80.1, 76.2, 76.1, 75.6, 75.3,
75.2, 75.0, 73.4, 73.3, 73.2, 73.1, 72.2, 71.7, 71.2, 70.0,
68.9, 68.5, 68.0, 66.3, 62.7 (sugar C, 5×PhCH2, PhCH,
CH3O C6H4CH2, OCH2CH2N3), 55.3 (CH3O), 54.6 (C-
2¦), 51.0 (OCH2CH2N3), 23.6 (NHAc), 20.9 (OAc).
MALDI-TOFMS: m/z 1708.9 [M+Na]+, 1724.2 [M+
K]+. Anal. Calcd for C94H100N4O25: C, 66.98; H, 5.94;
N, 3.32. Found: C, 66.69; H, 6.21; N, 3.11.
CHCl3–MeOH) to give 18 (250 mg, 94.3%). [h]D
+
1
43.4° (c 1.29, MeOH). H NMR (CD3OD): l 8.12–7.13
(m, 40 H, Ar-H), 5.85 (bs, H, H-4§), 5.46
1
(t, 1 H, J2§,3§ 8.62 Hz, H-2§), 5.17 (s, 1 H, PhCH), 5.16
(d, 1 H, J1§,2§ 7.64 Hz, H-1§), 4.98 (d, 1 H, H-3§),
4.88–4.18 (m, 18 H), 3.96–3.80 (m, 1 H, OCH2CH2N3),
3.80–3.34 (m, 19 H), 3.22 (t, 1 H, J1,2 8.10 Hz, H-2).
13C NMR (CDCl3): l 174.9, 172.3, 167.8, 167.7 (2×
PhCO, OAc, NHAc), 140.1, 140.0, 139.5, 139.4, 134.7,
1‘34.4, 131.7, 131.3, 131.2, 131.1, 130.3, 129.9, 129.7,
129.6, 129.5, 129.3, 129.2, 129.1, 129.0, 128.7, 128.3,
127.9, 127.7 (Ar-C), 104.7 (C-1%, C-1¦), 104.2 (C-1§),
103.9 (C-1), 101.9 (PhCH), 83.9, 82.9, 81.9, 79.6,
78.0, 77.1, 76.7, 76.3, 76.1, 76.0, 75.3, 74.7, 74.2,
74.1, 72.5, 71.6, 71.0, 69.3, 69.2, 67.7, 67.5, 64.7 (sugar
C, 5×PhCH2, OCH2CH2N3), 52.2 (C-2¦), 49.9
(OCH2CH2N3), 23.1 (NHAc), 21.0 (OAc). MALDI-
TOFMS: m/z 1742.8 [M−2H+Na]−, 1758.9
[M−2H+K]− (negative-ion mode).
2-Aminoethyl 2-O-benzoyl-3-O-sulfo-i-
ranosyl-(1“3)-2-acetamido-2-deoxy-i-
ranosyl-(1“4)-3-O-sulfo-i- -galactopyranosyl-(1“4)-
i- -glucopyranoside (20).—A solution of 18 (100 mg)
D-galactopy-
D
-galactopy-
D
2-Azidoethyl
galactopyranosyl - (1“3) - 2 - acetamido - 4,6 - O - benzyli-
dene-2-deoxy-i- -galactopyranosyl-(1“4)-2,6-di-O-
benzyl-i- -galactopyranosyl-(1“4)-2,3,6-tri-O-ben-
zyl-i- -glucopyranoside (17).—To a solution of 16
4-O-acetyl-2,6-di-O-benzoyl-i-D-
D
in 10:1 MeOH–H2O (15 mL) and HCl (1 M, 160 mL)
was hydrogenolysed at 0.42 MPa in the presence of
palladium-on-charcoal (10%, 100 mg) for 60 h. The
mixture was then filtered through Celite, and the solid
was washed thoroughly with MeOH and water. The
filtrate was then concentrated. Flash chromatography
(5:4:0.6:1 CHCl3–MeOH–H2O–HOAc) of the residue
afforded 2-aminoethyl 4-O-acetyl-2,6-di-O-benzoyl-3-O-
D
D
D
(700 mg) in CH3CN (27 mL) and water (3 mL) was
added ammonium cerium(IV) nitrate (675 mg), and the
mixture was stirred for 1.5 h at room temperature. TLC
(1.3:1 petroleum ether–acetone) then showed the disap-
pearance of 16 and the formation of 17. The mixture
was diluted with CH2Cl2 (100 mL) and washed with aq
NaHCO3 (3×50 mL). The organic layer was dried,
filtered, and concentrated. Column chromatography
(1.3:1 petroleum ether–acetone) of the residue afforded
sulfo - b -
deoxy-b-
galactopyranosyl-(1“4)-b-
D
- galactopyranosyl - (1“3) - 2 - acetamido - 2-
-galactopyranosyl-(1“4)-3-O-sulfo-b-
-glucopyranoside (19, 55
D
D-
D
mg, 88%) as a white solid. MALDI-TOFMS: m/z
1184.2 [M−2H+Na]− (negative-ion mode).

1934
Q. Li et al. / Carbohydrate Research 337 (2002) 1929–1934
To a solution of 19 (50 mg) in dry MeOH was added
8.17 Hz, H-2), 3.28 (t, 2 H, OCH2CH2NH2), 2.10 (s, 3
H, NHAc). 13C NMR (D2O): l 177.8 (NHAc), 107.3
(C-1§), 105.4 (2 C, C-1, C-1¦), 104.8 (C-1%), 83.0 (C-3§),
82.7 (C-3¦), 82.3 (C-3%), 81.2 (C-3), 77.6 (C-2§), 77.4,
77.3, 77.0, 76.9, 76.6 (C-4%, C-5, C-5%, C-5¦, C-5§), 75.5
(C-2), 75.3 (C-2%), 71.6 (C-4), 70.6 (C-4¦), 69.6 (C-4§),
68.7 (OCH2CH2NH2), 63.8, 63.7, 63.5, 62.7 (C-6, C-6%,
C-6¦, C-6§), 54.0 (C-2¦), 42.3 (OCH2CH2NH2), 25.3
(NHAc). MALDI-TOFMS: m/z 932.6 [M−2H+Na]−
(negative-ion mode).
NaOMe (10 mg). The mixture was stirred overnight at
room temperature, then neutralized with HOAc until
pH 7 was reached. The solution was then concentrated.
Purification of the residue by passage through a Sep-
hadex LH-20 column using water as eluent afforded,
after lypophilization, 20 (44 mg, quant) as a white solid.
[h]D +7.4° (c 0.5, water). 1H NMR (D2O): l 8.10–7.60
(m, 5 H, PhCO), 5.32 (t, 1 H, J2§,3§ 8.42 Hz, H-2§), 4.96
(d, 1 H, J1§,2§ 7.61 Hz, H-1§), 4.71 (dd, 1 H, J3§,4§ 3.30
Hz, J2§,3§ 6.47 Hz, H-3§), 4.57 (d, 1 H, J1¦,2¦ 8.06 Hz,
H-1¦), 4.53 (d, 1 H, J1%,2% 8.06 Hz, H-1%), 4.49 (d, 1 H,
J1,2 7.69 Hz, H-1), 4.34–4.31 (m, 3 H, H-3, H-4, H-4§),
4.24 (d, 1 H, J3¦,4¦ 2.20 Hz, H-4¦), 4.13–4.10 (m, 1 H,
OCH2CH2NH2), 3.96–3.71 (m, 13 H), 3.67–3.57 (m, 9
Acknowledgements
This project was supported by the National Natural
Science Foundation of P. R. China (NSFC) and a grant
from the Ministry of Science and Technology of P. R.
China.
H), 3.36 (t,
2 H, H-2, H-2%), 3.27 (t, 2 H,
OCH2CH2NH2), 1.20 (s, 3 H, NHAc). 13C NMR
(D2O): l 177.2 (NHAc), 170.3 (PhCO), 137.1, 132.9,
131.7, 131.5 (Ar-C), 105.6 (C-1%), 105.4 (C-1), 105.2
(C-1§), 104.8 (C-1¦), 83.2 (C-3¦), 82.1 (C-3), 81.2 (C-3%),
80.7 (C-3§), 77.0 (C-4), 75.5 (C-2), 73.3 (C-2§), 72.1
(C-2%), 70.6 (C-4¦), 68.7 (OCH2CH2NH2), 63.7 (C-6,
6§), 63.6 (C-6%), 63.4 (C-6¦), 53.6 (C-2¦), 42.3
(OCH2CH2NH2), 24.3 (NHAc). MALDI-TOFMS: m/z
1035.9 [M−2H+Na]− (negative-ion mode).
References
1. (a) Hakomori, S.; Zhang, Y. Chem. Biol. 1997, 4, 97–104;
(b) Kim, Y. J.; Varki, A. Glycoconjugate J. 1997, 14,
569–576.
2. Hakomori, S. Cancer Res. 1996, 56, 5309–5318.
3. Livingston, P. O. Immunol. Re6. 1995, 145, 147–166.
4. Tadano, K.; Ishizuka, I. J. Biol. Chem. 1982, 57, 13413–
13420.
5. Hiraiwa, N.; Iida, N.; Ishizuka, I. Cancer Res. 1988, 48,
6769–6774.
6. Hiraiwa, N.; Fukudas, Y.; Imura, H. Cancer Res. 1990,
50, 2917–2928.
7. Chernyak, A.; Oscarson, S.; Turek, D. Carbohydr. Res.
2000, 329, 309–316.
8. Nashed, M. A.; Anderson, L. Carbohydr. Res. 1977, 56,
419–422.
9. Kameyama, A.; Ishida, H.; Kiso, M.; Hasegawa, A.
Carbohydr. Res. 1990, 200, 269–285.
10. (a) Grundler, G.; Schmidt, R. R. Carbohydr. Res. 1985,
135, 203;
(b) Pougny, J. R.; Nasser, M. A. M.; Naulet, N.; Sinay¨,
P. Nou6. J. Chim. 1978, 2, 389.
11. Kanie, O.; Crawley, S. C.; Palcic, M. M.; Hindsgaul, O.
Carbohydr. Res. 1993, 243, 139–164.
12. Sarbajna, S.; Roy, N. Carbohydr. Res. 1998, 306, 401–
407.
13. Spijker, N. M.; Keuning, C. A.; Hooglugt, M. Tetra-
hedron 1996, 52, 5945–5960.
2-Aminoethyl 3-O-sulfo-i-
3)-2-acetamido-2-deoxy-i- -galactopyranosyl-(1“4)-
3-O-sulfo-i- -galactopyranosyl-(1“4)-i- -glucopy-
D-galactopyranosyl-(1“
D
D
D
ranoside (1).—A solution of 19 (50 mg, 0.0422 mmol)
in 0.5 M NaOMe–MeOH (10 mL) was stirred at 0 °C
for 6 h. Work-up of the reaction mixture as described
for compound 20 afforded 1 (35.2 mg, 90%) as a white
1
solid. [h]D +10.2° (c 0.8, water). H NMR (D2O): l
4.69 (d, 1 H, J1¦,2¦ 8.52 Hz, H-1¦), 4.58 (d, 1 H, J1§,2§
7.83 Hz, H-1§), 4.563 (d, 1 H, J1%,2% 8.00 Hz, H-1%), 4.560
(d, 1 H, J1,2 7.83 Hz, H-1), 4.43 (dd, 1 H, J3%,4% 2.92 Hz,
H-4%), 4.40 (dd, 1 H, J2%,3% 9.89 Hz, H-3%), 4.33 (dd, 1 H,
J
3§,4§ 3.35 Hz, H-3§), 4.30 (bs, 1 H, H-4§), 4.18 (d, 1 H,
J3¦,4¦ 3.10 Hz, H-4¦), 4.15–4.12 (m, H,
OCH2CH2NH2), 4.10–4.08 (m, 1 H), 4.07 (dd, 1 H,
J2¦,3¦ 10.88 Hz, H-2¦), 4.01–3.98 (m, H,
1
1
OCH2CH2NH2), 3.93 (dd, 1 H, H-3¦), 3.87–3.62 (m, 15
H), 4.16–4.12 (m, 1 H, OCH2CH2NH2), 4.08 (dd, 1 H,
H-2), 4.01–3.99 (m, 1 H, OCH2CH2NH2), 3.87–3.62
(m, 15 H, H-2§, H-3, H-4, H-5, H-5%, H-5¦, H-5§, H-6,
H-6%, H-6¦, H-6§), 3.53 (dd, 1 H, H-2%), 3.38 (t, 1 H, J2,3
14. Stahl, W.; Sprengard, U.; Kretschmar, G.; Kunz, H.
Angew. Chem., Int. Ed. Engl. 1994, 33, 2096–2098.