Synthesis of oligosaccharides containing β-d-Gal-(1→3)-O-(6-O-sulfo-β-d-GlcNAc) as a terminal unit

Article abstract of DOI:10.1016/S0008-6215(98)00166-9

The chemical synthesis of β-D-Gal-(1→3)-6-O-SO₃Na-β-D-GlcNAc-(1→6)-α-D-Man-O-C₆H₄NO₂ (1) and β-D-Gal-(1→3)-6-O-SO₃Na-β-D-GlcNAc-(1→2)-α-D-Man-OMe (2) is reported using a key glycosyl donor, phenyl O-(

Full text of DOI:10.1016/S0008-6215(98)00166-9

Carbohydrate Research 311 (1998) 165±169
Note
Synthesis of oligosaccharides containing
ꢀ-d-Gal-(1!3)-O-(6-O-sulfo-ꢀ-d-GlcNAc) as a
terminal unit1
Bao-Guo Huanga,2, Rakesh K. Jaina,3, Walter A. Tabaczynskib,
James L. Alderferb, Khushi L. Mattaa,*
a Department of Gynecologic Oncology, Roswell Park Cancer Institute, Elm & Carlton Streets, Bu€alo,
NY 14263, USA
b Department of Biophysics, Roswell Park Cancer Institute, Elm & Carlton Streets, Bu€alo, NY 14263,
USA
Received 10 February 1998; accepted 29 May 1998
Abstract
The chemical synthesis of ꢀ-d-Gal-(1!3)-6-O-SO3Na-ꢀ-d-GlcNAc-(1!6)-ꢁ-d-Man-O-
C6H4NO2 (1) and ꢀ-d-Gal-(1!3)-6-O-SO3Na-ꢀ-d-GlcNAc-(1!2)-ꢁ-d-Man-OMe (2) is reported
using a key glycosyl donor, phenyl O-(2,3,4,6-tetra-O-acetyl-ꢀ-d-galactopyranosyl)-(1!3)-4,6-di-
O-chloroacetyl-2-deoxy-2-phthalimido-1-thio-ꢀ-d-glucopyranoside (3). # 1998 Published by
Elsevier Science Ltd. All rights reserved
Carcinoembryonic antigen (CEA) has been shown
to contain numerous N-linked oligosaccharides of
both high mannose and complex type [2]. Yama-
shita [3] reported the ꢀ-d-Gal-(1!3)-6-O-SO3-ꢀ-d-
GlcNAc moiety to be present in the complex type
N-linked oligosaccharide structure of CEA. This
moiety is expressed in adult colonic endothelial
cells. This ®nding prompted us to develop the che-
mical synthesis of ꢀ-d-Gal-(1!3)-6-O-SO3Na-ꢀ-d-
GlcNAc-(1!6)-ꢁ-d-Man-O-C6H4NO2(p) (1) and
ꢀ-d-Gal-(1!3)-6-O-SO3Na-ꢀ-d-GlcNAc-(1!2)-ꢁ-
d-Man-OMe (2), which will be used for examining
the biosynthetic pathway of the ꢀ-d-Gal-(1!3)-6-
O-SO3-GlcNAc moiety in CEA. The assembly of
complex carbohydrate structures bearing the 6-O-
SO3-GlcNAcꢀ1!2/6Manꢁ determinant will be
established by con®rming the reactions shown in
Reaction 1.
The p-nitrophenyl derivative 1 can also be
employed in immunological studies after reducing
the nitro group and subsequently linking to protein.
This communication reports the chemical synth-
esis of target molecules 1 and 2 through the
employment of a key glycosyl donor; phenyl O-
(2,3,4,6-tetra-O-acetyl-ꢀ-d-galactopyranosyl)-(1!3)-
4,6-di-O-chloroacetyl-2-deoxy-2-phthalimido-1-thio-
ꢀ-d-glucopyranoside (3). The building block 3 was
prepared from known compound 6 [4] in two steps
(Scheme 1). Cleavage of the benzylidene group in 6
* Corresponding author. Fax: +1-716-845-7608.
1
Synthetic studies in carbohydrates, Part 106. For Part
105 see ref. [1].
2
Present address: Occidental Chemical Corporation,
Technology Center, 2801 Long Road, Grand Island, NY
14072, USA.
3
Present address: TransCell Technologies, Inc., 2000
Cornwall Road, Monmouth Junction, NJ 08852, USA.
0008-6215/98/$Ðsee front matter # 1998 Published by Elsevier Science Ltd. All rights reserved
PII S0008-6215(98)00166-9

166
B.-G. Huang et al./Carbohydrate Research 311 (1998) 165±169
with 60% aq acetic acid followed by treatment
with chloroacetic anhydride±NaHCO3±DMF [5]
a€orded 3 in 63% yield, after silica gel chromato-
graphy. Glycosylation of 4-nitrophenyl 2,3,4-tri-O-
acetyl-ꢁ-d-mannopyranoside (4) with 3 in CH2Cl2
promoted by N-iodosuccinimide-tri¯ic acid [6] gave
the ꢀ-linked trisaccharide 8 in 48% yield. Selective
removal of chloroacetyl groups in 8 with thiourea
and 2,6-lutidine in EtOH±CH2Cl2 (1:1, v/v) pro-
vided the diol 9 in 75% yield after chromato-
graphic puri®cation. Selective sulfation of 9 with
SO3±pyridine complex in DMF followed by
removal of the phthalimido group and N-acetyl-
ation with MeOH±Ac2O±triethyl amine produced
the target molecule 1 in 33% yield. Similarly, reaction
of methyl 3,4,6-tri-O-benzyl-ꢁ-d-mannopyranoside
(5) [7,8] with 3 in the presence of NIS-tri¯ic acid
provided the trisaccharide 11 (Scheme 2). Treat-
ment of 11 in a manner analogous to that described
in the synthesis of 1 from 8 a€orded compound 2
in 39% yield after hydrogenolysis to remove O-
benzyl groups.
The products 1 and 2 were characterized unam-
biguously by NMR spectroscopy (see Tables 1 and
2), FAB mass spectroscopy and elemental analysis
1
(see Experimental section). The H and 13C NMR
assignments for 1 and 2 were con®rmed through
the application of the following two-dimensional
NMR techniques: H,H-COSY (H-assignment),
H,C-COSY (C-assignment) and long-range H,C-
COSY (glycosidic linkage). The C-6 resonances of
both Man (ꢂ 68.1) and GlcNAc (ꢂ 66.3) in com-
pound 1 exhibited down®eld shifts, con®rming
that they are substitution sites. Similarly, the C-2
(ꢂ 75.7) of Man and C-6 of GlcNAc (ꢂ 66.0) in
compound 2 displayed down®eld shifts, con®rm-
ing these positions as sites of glycosylation and
sulfation.
Scheme 1. Reagents and conditions: (a) 60% HOAc, 60 ꢀC,
1 h; (b) ClAc2O, NaHCO3, DMF, 0 ꢀC±room temperature,
3 h, 63%; (c) 4 (1.0 equiv), NIS (3.0 equiv)-tri¯ic acid, MS-4
A, CH2Cl2, 20 ꢀC, 0.5 h, 37%; (d) thiourea, 2,6-lutidine,
EtOH±CH2Cl2 (1:1), 80 ꢀC, 12 h, 75%; (e) SO3±pyridine,
DMF, 0 ꢀC, 4 h; (f) 85% hydrazine hydrate, EtOH, 80 ꢀC, 3 h;
MeOH±TEA±Ac2O (4:2:1), 0 ꢀC±room temperature, 2 h; Na+
resin, 33% from 9.
Scheme 2. Reagents and conditions: (a) NIS (3.0 equiv)-tri¯ic
acid, MS-4 A, CH2Cl2, 0 ꢀC, 2 h; (b) thiourea, 2,6-lutidine,
ꢀ
EtOH±CH2Cl2 (1:1), 80 C, 12 h, 14% for a and b; (c) SO3±
Pyridine complex, DMF, 0 ꢀC, 3.5 h; (d) 85% hydrazine
ꢀ
ꢀ
hydrate, EtOH, 80 C, 2 h; MeOH±TEA±Ac2O (4:2:1), 0 C±
room temperature, 2 h; (e) 10% Pd/C, MeOH±H2O (9:1), H2,
room temperature, 12 h; Na+ resin, 39% from 12.

B.-G. Huang et al./Carbohydrate Research 311 (1998) 165±169
167
1. Experimental
General methods.ÐOptical rotations were
measured at 25 ꢀC with a Perkin±Elmer 241
Polarimeter. TLC was conducted on glass plates,
precoated with a 0.25 mm layer of Silica Gel 60F-
254 (Analtech GHLF uniplates). The compounds
were visualized by exposure to UV light and/or by
spraying with 5% H2SO4 in EtOH and charring.
Baker Analyzed (60±200 mesh) silica gel was used
for column chromatography. The solvents used for
chromatography are given below: Solvent A,
hexanes±EtOAc=1:1; Solvent B, CH2Cl2±ace-
tone=9:1; Solvent C, CH2Cl2±acetone=4:1;
Solvent D, CH2Cl2±MeOH=4:1; Solvent E,
CH2Cl2±MeOH±H2O=13:6:1; Solvent F, CH2Cl2±
MeOH±H2O=5:4:1. NMR spectra were recorded
ꢀ
1
at 30 C. H NMR and 13C NMR spectra were
obtained with a Bruker AM-400 (400 MHz) and
Bruker AMX-600 (600 MHz) NMR spectrometers.
Solutions in organic solvents were generally dried
over anhydrous Na2SO4. Dichloromethane and
DMF were kept dry over molecular sieves (4 A).
Elemental analyses were performed by Robertson
Laboratories, Madison, NJ.
Phenyl O-(2,3,4,6-tetra-O-acetyl-b-d-galacto-
pyranosyl)-(1!3)-4,6-di-O-chloroacetyl-2-deoxy-2-
phthalimido-1-thio-b-d-glucopyranoside (3).ÐCom-
pound 6 (410 mg, 0.5 mmol) in 60% aq acetic acid
Table 1
1H and 13C chemical shift assignments (ppm)a of 1 and 2
Gal
GlcNAc
Man
Assignment
H
C
H
C
H
C
Compound 1
1
2
3
4
4.42
3.56
3.66
3.94
3.72
3.77,3.81
102.5
69.7
71.6
67.6
74.3
60.0
4.58
100.3
53.4
81.3
67.7
72.2
66.3
21.3
5.76
96.8
68.7
69.4
65.9
71.8
68.1
3.75
3.76
3.43
3.66
4.07,4.32
2.03
4.22
4.09
3.72
3.89
5
6
NHCOCH3
3.84,4.08
Compound 2
1
2
3
4
4.49
3.56
3.67
3.95
3.73
3.77,3.82
102.4
69.7
71.5
67.6
74.3
60.0
4.67
3.90
3.86
3.66
3.73
4.28,4.40
2.08
98.4
53.4
80.5
67.3
72.4
66.0
21.4
4.80
4.11
3.81
3.53
3.61
3.66,3.94
97.0
75.7
68.6
66.3
71.7
60.6
5
6
NHCOCH3
OMe
3.45
53.8
a
Obtained from spectra of aqueous (D2O) solutions of 1 or 2. Expressed relative to TSP for 1H, and relative to acetone (at
29.2 ppm) for 13C.

168
B.-G. Huang et al./Carbohydrate Research 311 (1998) 165±169
Table 2
1H±1H coupling constants (Hz)a in 1 and 2
temperature, satd NaHCO3 was added and the
mixture was ®ltered through a bed of Celite. The
organic layer was successively washed with H2O,
satd NaHCO3 and 10% Na2S2O3, dried, and con-
centrated. The residue was subjected to column
chromatography to provideꢀ8 (440 mg, 37%). Rf
Gal
GlcNAc
Man
Compound 1
3J1,2
7.7
9.9
3.5
1.0
4.0
8.2
11.8
8.3
1.9
3.5
9.7
9.8
6.2
1.8
11.7
b
3J2,3
3J3,4
8.6
10.0
6.0
2.2
11.2
1
0.35 (Solvent B), [ꢁ]d=+32 (c 0.40, CHCl3), H
3J4,5
NMR (CD2Cl2): ꢂ 8.21±7.10 (m, 8H, arom.), 5.44
(d, 1H, J=1.82 Hz, H-1), 4.16 (2 s, 4H, 2ÂCH2Cl),
2.20±1.84 (cluster of s, 21H, 7ÂOAc). Anal. Calcd
for C50H54Cl2N2O28: C, 49.97; H, 4.53; N, 2.33;
Found: C, 49.74; H, 4.34; N, 2.16.
3J5,6
3
0
J5,6
J6,6
2
0
Compound 2
3J1,2
7.8
10.0
0.4
3.4
1.1
3.9
8.1
11.8
8.3
10.4
1.8
3.5
3J2,3
4J2,4
c
c
4-Nitrophenyl O-(2,3,4,6-tetra-O-acetyl-b-d-gal-
actopyranosyl)-(1!3)-O-(2-deoxy-2-phthalimido-
b-d-glucopyranosyl)-(1!6)-2,3,4-tri-O-acetyl-a-d-
manno-pyranoside (9).ÐA mixture of compound 8
(330 mg, 0.27 mmol), thiourea (206 mg, 2.7 mmol)
and 2,6-lutidine (0.16 mL, 1.38 mmol) in EtOH±
CH2Cl2 (40 mL, 1:1) was stirred at 80 ꢀC overnight.
The solvents were evaporated and the residue was
taken up in CH2Cl2 (20 mL) and washed with H2O,
dried, and concentrated. The residue was subjected
to column chromatography to provide 9 (216 mg,
75%). Rf 0.18 (Solvent D), [ꢁ]d=+46ꢀ (c 1.40,
3J3,4
8.3
10.0
5.3
2.2
11.3
9.6
9.7
7.3
2.1
3J4,5
3J5,6
3
0
J5,6
J6,6
3
0
11.8
a
Obtained by ®rst-order analysis of spectra acquired on aqu-
eous (D2O) solutions of 1 or 2. Probable error is ‹ 0.1 Hz.
Couplings through three bonds are assumed to be positive,
and those through two or four bonds are assumed to be
negative.
b
Complex patterns exhibited by H-2 and H-3 of GlcNAc
were not amenable to ®rst-order analysis.
c
Not observed.
1
CHCl3), H NMR (CD2Cl2): ꢂ 8.20±7.11 (m, 8H,
arom.), 5.48 (d, 1H, J=1.74 Hz, H-1), 2.21±1.40
(cluster of s, 21H, 7ÂOAc). Anal. Calcd for
C46H52N2O26: C, 52.67; H, 5.00; N, 2.67; Found:
C, 52.90; H, 4.92; N, 2.59.
ꢀ
(20 mL) was stirred at 60 C for 1 h. The solvents
were evaporated and coevaporated with toluene.
The resulting residue (7) was treated with chlor-
oacetyl anhydride (427 mg, 2.50 mmol) in DMF
(10 mL) containing sodium bicarbonate (504 mg,
6 mmol) at 0 ꢀC, then kept at room temperature for
3 h. The mixture was poured into ice water and the
white solid product was collected by ®ltration.
Chromatographic puri®cation using Solvent A as
eluent gave 3 (280 mg, 63%). Rf 0.26 (Solvent A),
4-Nitrophenyl O-(b-d-galactopyranosyl)-(1!3)-
O-(2-acetamido-2-deoxy-6-O-sulfo-b-d-glucopyrano-
syl sodium salt)-(1!6)-a-d-mannopyranoside (1).Ð
To a stirred solution of compound 9 (220 mg,
0.20 mmol) in dry DMF (10 mL) at 0 ꢀC was added
SO3±pyridine complex (38 mg, 0.24 mmol). After
2 h, more reagent (38 mg) was added to the mixture
and stirring was continued for an additional 2 h at
[ꢁ]d=+11ꢀ (c 0.54, CHCl3), H NMR (CD2Cl2): ꢂ
1
ꢀ
7.91±7.23 (m, 9H, arom.), 5.50 (d, 1H, J=10.6 Hz,
H-1), 4.16 (m, 4H, 2ÂCH2Cl), 2.14±1.84 (4 s, 12H,
4ÂOAc). Anal. Calcd for C38H39Cl2NO17S: C, 51.59;
H, 4.44; N, 1.58; Found: C, 51.30; H, 4.23; N, 1.55.
4-Nitrophenyl O-(2,3,4,6-tetra-O-acetyl-b-d-gal-
actopyranosyl)-(1!3)-4,6-di-O-chloroacetyl-2-deoxy-
2-phthalimido-b-d-glucopyranosyl)-(1!6)-2,3,4-tri-
O-acetyl-a-d-mannopyranoside (8).ÐA solution of
compound 3 (700 mg, 1.1 mmol) and 4 (320 mg,
1.0 mmol) in CH2Cl2 (20 mL) was stirred with 4 A
0 C. Methanol and pyridine were added to con-
sume excess reagent. The solvents were evaporated
and the residue was subjected to column chroma-
tography to provide 10. Compound 10 was then
treated with 85% hyꢀdrazine hydrate (4 mL) in
ethanol (20 mL) at 80 C for 3 h. The solvent was
evaporated and coevaporated with toluene. To a
solution of this compound in methanol (20 mL)
containing triethylamine (10 mL) was added acetic
ꢀ
anhydride (5 mL) at 0 C. After the mixture was
ꢀ
molecular sieves (5.0 g) at 20 C for 30 min, after
stirred at room temperature for 2 h, the solvent was
evaporated and the product was puri®ed by col-
umn chromatography with Solvent F as the eluent.
The fractions corresponding to the product were
combined and concentrated, and the residue so
which NIS (680 mg, 3.0 mmol) was added to the
mixture. A solution of tri¯uoromethanesulfonic
acid (0.1 mL) in 20 mL of CH2Cl2 was then added
dropwise. After stirring for 30 min at the same

B.-G. Huang et al./Carbohydrate Research 311 (1998) 165±169
169
ꢀ
obtained was dissolved in a small amount of water
and passed through Amberlite IR-120P (Na+)
cation-exchange resin. Lyophilization of the frac-
tions corresponding to 1 gave a hygroscopic
amorphous solid (50 mg, 33%). Rf 0.26 (CH2Cl2±
MeOH±H2O=5:4:1), [ꢁ]d=+45ꢀ (c 0.73, H2O),
anhydride (7.5 mL) at 0 C. After the mixture was
stirred at room temperature for 2 h, the solvents
were evaporated and the product was puri®ed by
column chromatography with Solvent E as the
eluent. The fractions corresponding to the product
(14) were combined and concentrated, and the
residue (ꢁ100 mg) so obtained was hydrogenolyzed
in MeOH±H2O (20 mL, 9:1) in the presence of 10%
Pd/C (300 mg) overnight. The suspension was ®l-
tered through a bed of Celite. The solids were
thoroughly washed with MeOH±H2O. The ®ltrate
and washings were combined and evaporated
under reduced pressure. The residue was puri®ed
by column chromatography using Solvent F as the
eluent. The fractions corresponding to the product
were combined and concentrated, and passed
through Amberlite IR-120P (Na+) cation-
exchange resin. Lyophilization of the fractions
corresponding to 2 gave a hygroscopic amorphous
solid (70 mg, 39% from 12). Rf 0.20 (Solvent F),
1
for H and 13C NMR data, see Table 1; MS m/z,
745.1 (M Na) ; Anal. Calcd for C26H37N2
NaO21S: C, 40.63; H, 4.85; N, 3.64; Found: C,
40.52; H, 5.09; N, 3.42.
Methyl O-(2,3,4,6-tetra-O-acetyl-b-d-galacto-
pyranosyl)-(1!3)-O-(2-deoxy-2-phthalimido-b-d-
glucopyranosyl)-(1!2)-3,4,6-tri-O-benzyl-a-d-
mannopyranoside (12).ÐA solution of compound 3
(2.30 g, 2.60 mmol) and 5 (1.09 g, 2.35 mmol) in
CH2Cl2 (100 mL) ꢀwas stirred with 4 A molecular
sieves (12 g) at 0 C for 30 min, after which time
NIS (1.60 g) was added to the mixture. A solution
of tri¯uoromethanesulfonic acid (0.2 mL) in 20 mL
of CH2Cl2 was then ꢀadded dropwise and the mix-
ture was stirred at 0 C for an additional 1 h. Satd
NaHCO3 was added and the mixture was ®ltered
through a bed of Celite. The organic layer was
successively washed with H2O, satd NaHCO3 and
10% Na2S2O3, dried, and concentrated. The resi-
due (11) was treated with thiourea (2.8 g) and 2,6-
lutidine (2.0 mL) in EtOH±CH2Cl2 (50 mL, 1:1) at
80 ꢀC overnight. The solvents were evaporated and
the residue was subjected to column chromato-
graphy to provide 12 (350 mg, 14% from 5). Rf
[ꢁ]d=+8ꢀ (c 0.55, H2O), for H and 13C NMR
data see Table 1; MS m/z 638.2 (M Na) .
1
Acknowledgements
The authors thank Mr. Conrad F. Piskorz for
his help in preparing this manuscript. These inves-
tigations were supported by Grant Nos CA-63218
and CA-16056 awarded by the National Cancer
Institute.
ꢀ
1
0.43 (Solvent C), [ꢁ]d=+15 (c 0.57, CHCl3), H
NMR (CD2Cl2): ꢂ 7.67±7.16 (m, 19H, arom.), 3.13
(s, 3H, OCH3), 2.12±1.55 (cluster of s, 12H,
4ÂOAc). Anal. Calcd for C56H63NO21: C, 61.93;
H, 5.85; N, 1.29; Found: C, 62.30; H, 6.02; N, 1.22.
Methyl O-(b-d-galactopyranosyl)-(1!3)-O-(2-
acetamido-2-deoxy-6-O-sulfo-b-d-glucopyranosyl
sodium salt)-(1!2)-a-d-mannopyranoside (2).Ð
To a stirred solution of compound 12 (300 mg,
0.27 mmol) in dry DMF (10 mL) at 0 ꢀC was added
SO3±pyridine complex (50 mg, 0.32 mmol). After
2 h, more reagent (50 mg) was added to the mixture
and stirring continued for an additional 16 h at
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ꢀ
0 C. Methanol and pyridine were added to con-
sume excess reagent. The solvents were evaporated
and the residue was subjected to column chroma-
tography to provide 13. The compound 13 was
then treated with 85% hydrazine hydrate (8 mL) in
ethanol (40 mL) at 80 ꢀC for 2 h. The solvents were
evaporated and coevaporated with toluene. To a
solution of this compound in methanol (30 mL)
containing triethylamine (15 mL) was added acetic