Synthesis of aminoethyl glycosides of the ganglioside GM1 and asialo-GM1 oligosaccharide chains

Article abstract of DOI:10.1023/B:RUBI.0000015775.24385.a0

4′-O-Glycosylation of 2-azidoethyl 2,3,6-tri-O-benzyl-4-O-(2,3-di-O- benzyl-6-O-benzoyl-β-D-galactopyranosyl)-β-D-glucopyranoside with a disaccharide donor, 4-trichloroacetamidophenyl 4,6-di-O-acetyl-2-deoxy-3-O-(2,3, 4,6-tetra-O-acetyl-β-D-galactopyranos

Full text of DOI:10.1023/B:RUBI.0000015775.24385.a0

Russian Journal of Bioorganic Chemistry, Vol. 30, No. 1, 2004, pp. 60–70. Translated from Bioorganicheskaya Khimiya, Vol. 30, No. 1, 2004, pp. 68–79.
Original Russian Text Copyright © 2004 by Cheshev, Khatuntseva, Tsvetkov, Shashkov, Nifantiev.
Synthesis of Aminoethyl Glycosides of the Ganglioside GM1
and Asialo-GM1 Oligosaccharide Chains
P. E. Cheshev, E. A. Khatuntseva, Yu. E. Tsvetkov, A. S. Shashkov, and N. E. Nifantiev1
Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Leninskii pr. 47, Moscow, 119991 Russia
Received December 10, 2002; in final form, January 20, 2003
Abstract—4'-O-Glycosylation of 2-azidoethyl 2,3,6-tri-é-benzyl-4-é-(2,3-di-é-benzyl-6-é-benzoyl-β-D-
galactopyranosyl)-β-D-glucopyranoside with a disaccharide donor, 4-trichloroacetamidophenyl 4,6-di-é-
acetyl-2-deoxy-3-é-(2,3,4,6-tetra-é-acetyl-β-D-galactopyranosyl)-1-thio-2-trichloroacetamido-β-D-galactopy-
ranoside, in dichloromethane in the presence of N-iodosuccinimide and trifluoromethanesulfonic acid resulted
in a tetrasaccharide, 2-azidoethyl (2,3,4,6-tetra-é-acetyl-β-D-galactopyranosyl)-(1
deoxy-2-trichloroacetamido-β-D-galactopyranosyl)-(1 4)-(2,3-di-é-benzyl-6-é-benzoyl-β-D-galactopy-
ranosyl)-(1 4)-2,3,6-tri-é-benzyl-β-D-glucopyranoside, in 69% yield. The complete removal of é-pro-
tecting groups in the tetrasaccharide, the replacement of N-trichloroacetyl by N-acetyl group, and the reduction
of the aglycone azide group to amine led to the target aminoethyl glycoside of β-D-Gal-(1 3)-β-D-Gal-
NAc-(1 4)-β-D-Gal-(1 4)-β-D-Glc-OëH2CH2NH2 containing the oligosaccharide chain of asialo-
3)-(4,6-di-é-acetyl-2-
GM1 ganglioside in 72% overall yield. Selective 3'-O-glycosylation of 2-azidoethyl 2,3,6-tri-é-benzyl-4-é-
(2,6-di-é-benzyl-β-D-galactopyranosyl)-β-D-glucopyranoside with thioglycoside methyl (ethyl 5-acetamido-
4,7,8,9-tetra-é-acetyl-3,5-dideoxy-2-thio-D-glycero-α-D-galacto-2-nonulopyranosyl)oate in acetonitrile in
the presence of N-iodosuccinimide and trifluoromethanesulfonic acid afforded 2-azidoethyl [methyl (5-aceta-
mido-4,7,8,9-tetra-é-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosyl)oate]-(2
3)-(2,6-di-
é-benzyl-β-D-galactopyranosyl)-(1 4)-2,3,6-tri-é-benzyl-β-D-glucopyranoside, the selectively pro-
tected derivative of the oligosaccharide chain of GM3 ganglioside, in 79% yield. Its 4'-é-glycosylation with a
disaccharide glycosyl donor, (4-trichloroacetophenyl-4,6-di-é-acetyl-2-deoxy-3-é-(2,3,4,6-tetra-é-acetyl-β-
D-galactopyranosyl) 1-thio-2-trichloroacetamido-β-D-galactopyranoside in dichloromethane in the presence
of N-iodosuccinimide and trifluoromethanesulfonic acid gave 2-azidoethyl (2,3,4,6-tetra-O-acetyl-β-D-galac-
topyranosyl)-(1
3)-(4,6-di-é-acetyl-2-deoxy-2-trichloroacetamido-β-D-galactopyranosyl)-(1
4)-
{[methyl (5-acetamido-4,7,8,9-tetra-é-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosyl)onate]-
(2
3)}-(2,6-di-é-benzyl-β-D-galactopyranosyl)-(1
4)-2,3,6-tri-é-benzyl-β-D-glucopyranoside in
85% yield. The resulting pentasaccharide was é-deprotected, its N-trichloroacetyl group was replaced by N-
acetyl group, and the aglycone azide group was reduced to afford in 85% overall yield aminoethyl glycoside of
β-D-Gal-(1
3)-β-D-GalNAc-(1
4)-[α-D-Neu5Ac-(2
3)]-β-D-Gal-(1
4)-β-D-Glc-
OëH2CH2NH2 containing the oligosaccharide chain of GM1 ganglioside.
Key words: aminoethyl glycosides, asialo-GM1, gangliosides, glycosylation, GM1, sialylation
1
INTRODUCTION
(asialo-GM1), which are necessary for studying the car-
bohydrate specificity of galectins. The aminoethyl
spacer group in the target compounds (I) and (XXII)
(see Schemes 1, 4) will be used for further conjugation
with carriers and probes.
Galectins, animal proteins specifically binding
galactose-containing structures, are involved in the pro-
cesses of cell adhesion, proliferation, and apoptosis [1–
4]. Galectins are known to selectively recognize car-
2
Syntheses of ganglioside GM1 and its derivatives
had previously been accomplished in a number of lab-
oratories [6–10]. Three different approaches to the syn-
thesis of the GM1 (I) pentasaccharide chain were used.
The first approach is based on sialylation of the corre-
sponding derivative of asialo-GM1 (Scheme 1, path B)
bohydrate fragments of gangliosides; e.g., a high galec-
tin-1 specificity to ganglioside GM1 was demonstrated
[5]. Herein, we report the synthesis of aminoethyl gly-
cosides of the pentasaccharide carbohydrate chain of
ganglioside GM1 and also of its asialo derivative
1
Corresponding author; phone/fax: +7 (095) 138-8784; e-mail:
[8]. The attachment of β-D-Gal-(1
3)-β-D-Gal-
nen@ioc.ac.ru
NAc disaccharide fragment to the selectively protected
sialyllactose block (GM3 chain) [6, 10] is used in the
second approach (Scheme 1, A). The third approach
includes galactosamination of the sialyllactose block,
2
Abbreviations: Bn, benzyl; Bz, benzoyl; CAN, ceric ammonium
nitrate; MPLC, medium pressure liquid chromatography; MPM,
p-methoxybenzyl; MS, molecular sieve; NIS, N-iodosuccinimide;
Tf, trifluoromethanesulfonyl; TsOH, p-toluenesulfonic acid.
1068-1620/04/3001-0060 © 2004 MAIK “Nauka /Interperiodica”

OAc
OAc
AcO
AcO
AcO
O
COOMe
O
OAc
O
OAc
O
OR
O
OR
O
AcO
AcNH
O
+
SPhNHC(O)CCl3
O
O
RO
N3
AcO
NHC(O)CCl3
AcO
OR
OR
OH
(II)
(III)
OAc
OAc
OR
O
A
CO2Me
SEt
OR
O
AcO
AcNH
HO
O
O
+
O
RO
N3
OR
OR
AcO
(IV)
OH
(V)
OH
COOH
O
OH
OH
O
OH
O
HO
AcNH
O
E
B
O
HO
O
A
NH2
HO
OH
OH
HO
HO
O
OH
O
OH
O
O
(I)
C
D
HO
AcNH
HO
B
OR
O
OR
OR
O
HO
O
O
OR
O
RO
O
OR
N3
AcO
AcO
AcO
O
OAc
O
OAc
O
(IV)
R'O
(II)
+
OR
O
+
O
RO
N3
O
OR
OR
HO
AcO
Cl3CC(O)NH
(VI)
(VII)
Scheme 1. A retrosynthetic analysis of the oligosaccharide structure of ganglioside GM with decomposition via (A) the GM chain and (B) the asialo-GM chain.
1
3
1

62
CHESHEV et al.
OAc
O
OAc
O
OAc
OAc
‡
AcO
AcO
O
AcO
O
O
AcO
O
AcO
O
R
OAc
AcO
OAc
OAc
Br
AcO
(VIII)
(IX) R = Cl
(X) R = N3
b
c, d
e, f
OR
O
OR
O
OR
O
OR
R1O
R2O
RO
R1O
O
O
O
O
RO
RO
N3
O
N3
OR
OR
OR
OR2
(XI), R = H; R1 + R2 = CMe2
(XII), R = Bn; R1 = R2 = H
(XIII), R = Bn; R1 = MPM; R2 = H
(XIV), R = H; R1 + R2 = CHPh
(XV), R = Bn; R1 = R2 = H
(XVI), R = Bn; R1 = H; R2 = Bz
i, d
h
i, d
h
Scheme 2. Reagents: a, ClCH CH OH, HgBr /Hg(CN) ; b, NaN , DMF, 18-crown-6; c, MeONa/MeOH; d, Me C(OMe) , TsOH;
2
2
2
2
3
2
2
e, NaH, BnBr, DMF; f, AcOH, t; g, Bu SnO/toluene, MPM-Cl/Py; h, PhCH(OMe) , TsOH; i, BzCl/Py.
2
2
OAc
OAc
COOMe
O
OBn
O
OBn
O
AcO
AcNH
‡
O
(IV) + (XII)
O
O
BnO
N3
AcO
OBn
OBn
OH
(XVII)
(II)
b
OAc
COOMe
O
OAc
OBn
O
OBn
AcO
AcNH
O
O
O
BnO
O
N3
AcO
AcO
OBn
OBn
AcO
AcO
OAc
O
OAc
O
O
O
AcO
Cl3CC(O)NH
c, d, e, f
(XVIII)
(I)
Scheme 3. Reagents: a, NIS/TfOH, CH CN; b, NIS/TfOH, CH Cl ; c, NaOH, MeOH–H O; d, Ac O, NaOH; e, H , Pd/C, Boc O;
3
2
2
2
2
2
2
f, CF COOH, H O–MeOH.
3
2
followed by galactosylation of the terminal GalNAc based on β-D-Gal-(1
3)-D-GalNAc disaccharide
[11] in the syntheses of natural carbohydrate chains. We
had previously reported the use of this compound in the
synthesis of a spacered derivative of Gb5 pentasaccha-
residue (assembling via GM2 chain) [7].
This work continues our studies devoted to the use
of the new trichloroacetamidophenyl glycoside block ride. In this work, the use of disaccharide (II) [11] in
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 30 No. 1 2004

SYNTHESIS OF AMINOETHYL GLYCOSIDES
63
OBn
OBn
R1O
O
O
O
AcO
AcO
AcO
O
OAc
O
OAc
O
BnO
O
N3
OR2
OBn
O
AcO
Cl3CC(O)NH
‡
(XIII) + (II)
(XIX), R1 = MPM, R2 = Bn
(XX), R1 = OH, R2 = Bn
(XXI), R1 = Bn; R2 = Bz
b
‡
(IV)
(XVI) + (II)
Ê
c, d, e, f
(XVIII)
OH
O
OH
HO
O
O
HO
HO
HO
OH
O
OH
O
HO
O
OH
NH2
OH
O
O
HO
AcNH
(XXII)
Scheme 4. Reagents: a, NIS/TfOH, CH Cl ; b, CAN, CH CN/H O; c, NaOH, MeOH–H O; d, Ac O, NaOH; e, H , Pd/C, Boc O;
2
2
3
2
2
2
2
2
f, CF COOH, H O–MeOH; g, NIS/TfOH, CH CN.
3
2
3
the synthesis of aminoethyl glycosides of pentasaccha- 82% yield. The total perbenzylation of (XI) and the
ride GM1 and tetrasaccharide asialo-GM1 is reported.
removal of isopropylidene group led to diol (ïII) in
98% yield.
Diol (ïII) was glycosylated with thioglycoside
(IV) [13] in anhydrous acetonitrile using NIS and
TfOH as promoters. Under these conditions, the target
trisaccharide (XVII), almost free of the undesirable β-
isomer, was obtained in 79% yield (Scheme 3). This
result was significantly higher than the previously
reported signal sialylation reactions of various lactose
derivatives with sialyl donors with non-participating
group at C3 when yields did not exceed 70% and the
process often proceeded with a low stereoselectivity
[15, 9]. The structure of (XVII) was confirmed by
NMR spectroscopy (Tables 1, 2). The direction of gly-
cosylation was confirmed by a lower field position of
the C3' signal at 76.1 ppm in the 13C NMR spectrum of
(XVII) (Table 2) in comparison with its position
(73.5 ppm) in the spectrum of diol (XII). The α-config-
uration of neuraminic acid residue was confirmed by
the position of the H4'' signal at 4.83 ppm, the value of
RESULTS AND DISCUSSION
Retrosynthetic analysis of the target compound (I)
(Scheme 1) found useful its preparation through the fol-
lowing synthetic blocks: disaccharide β-D-Gal-(1
3)-β-D-GalNAc (II), neuraminic acid (IV) [13], and, in
dependence on the assembling scheme used, di- or
monohydroxyl lactose derivatives (V) or (VII).
First, we investigated the scheme for pentasaccha-
ride (I) synthesis involving the preliminary obtaining of
a selectively protected derivative with the GM3 back-
bone (III) and the subsequent attachment of it to disac-
charide (II) (Scheme 1, Ä). Diol (XII) containing the
azidoethyl prespacer was used as the acceptor diol lac-
tose block (V) necessary for obtaining (III) (Scheme 2).
A series of reactions, shown in Scheme 2, was used for
the synthesis of (XII).
Glycosylation of chloroethanol with acetylated lac-
tosyl bromide (VIII) in the presence of mercury(II)
cyanide and bromide resulted in chloroethyl glycoside
(IX) in 74% yield (Scheme 2). Azido group was substi-
tuted for the chlorine atom in aglycone part of (IX) to
give azide (X). 2-Azidoethyl glycoside (X) was
deacetylated, and the resulting heptaol was selectively
''
''
J
7'', 8'' 7.6 Hz, and ∆δ (H9a –H9b ) of 0.36 ppm (Table 1).
These values are in complete agreement with the well-
known empiric rules [16], according to which the pen-
taacetylated α-bound Neu5Ac residue has δ of H4 =
4.80–4.93 ppm, J7, 8 = 6.2–8.5 Hz, and ∆δ (H9a–H9b) <
3',4'-isopropylidenated by the method [14] to (XI) in 0.5 ppm in 1H NMR spectra.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 30 No. 1 2004

64
CHESHEV et al.
1
Table 1. H NMR spectral data for (I) and (XXI) in D2O; (XIV) in CD3OD; and (IX)–(XIII), (XV)–(XX), and (XXII) in CDCl3
Chemical shift (δ), ppm
Coupling constant, Hz
J3, 4 J4, 5 J5, 6a J5, 6b J6a, 6b
7.8 8.6 8.1
Com-
pound
Residue
Glc
H1 H2
H3
H4
H5
H6a H6b J1, 2 J2, 3
3.57–4.0
(I)
4.50 3.32 3.68 3.62
4.46 3.34 4.14 4.12
4.77 4.04 3.80 4.15
4.48 3.53 3.63 3.92
1.9; 2.7 3.70
Gal
7.6 9.4
7.3 10.8
9.5
GalN
Gal'
Neu5Ac*
Glc
Gal
Glc
Gal
Glc
Gal
Glc
2.7
5.1 eq
9.2
3.5
9.2
3.0
(IX)
(X)
4.47 4.89 5.18 3.78
4.52 5.08 4.93 5.32
4.46 4.87 5.15 3.78
4.52 5.05 4.93 5.25
3.58
3.86
3.62
3.84
3.40
3.92
3.30
3.30
3.43
4.08
3.63
3.82
3.41
3.17
3.41
3.47
3.33
3.50
4.07
3.39
3.45
4.49 4.09 7.8 9.5
9.5
<1
9.3
<1
4.09
4.50 4.06 8.1 8.1
4.08 7.8 7.9
8.0 10.5
6.7
6.8
(XI)
(XII)
4.32 3.24
3.52
3.87 3.80 8.0 8.0
3.77 3.74 9.0 6.9
3.67 3.73 9.6
<1
4.1 12.0
10.4
4.34 3.42 4.03 4.18
4.33 3.34 3.52 3.91
4.37 3.32 3.37 3.85
4.45 3.45 3.70 4.05
4.48 3.62 3.35 – 3.42
4.55 3.48 3.76 3.82
6.9
2.3
<1
5.2
Gal
3.44 3.55 8.0
3.46 3.83 8.4
3.65–3.85 7.9
(XIII) Glc
Gal
(XIV)
(XV)
Glc
Gal
Glc
Gal
Glc
Gal
4.11
7.8 7.9
4.68
3.86
4.40
4.40 4.35 6.8
<1
4.45 3.49 3.61 3.96
4.40 3.62 3.35 3.92
4.44 3.50 3.63 4.03
4.45 3.62 3.39 3.91
4.36 3.41 3.55 3.95
4.56 3.53 4.08 3.82
2.0; 2.5 4.83
4.40 3.40 3.58 3.94
4.51 3.56 4.01 3.72
5.13 4.33 4.20 5.24
4.73 5.10 4.92 5.37
5.17
4.43 3.45 3.60 4.03
4.47 3.62 3.39 4.08
5.10 3.84 4.26 5.45
4.53 5.09 4.91 5.36
4.40 3.40 3.59 4.03
4.45 3.57 3.49 4.07
3.83 3.73 7.8
9.3
3.1
9.1
3.1
9.3 4.1 <1
<1
9.1 4.0 <1
10.8
10.8
3.63 3.60 7.3 9.3
3.85 3.73 7.8 9.1
4.54 4.29 7.6 8.9
(XVI)
<1
6.5 6.5 11.2
(XVII) Glc
3.49
3.67
4.0
8.0
Gal
Neu5Ac**
7.5 10.3
2.3
4.4 eq 11.1
(XVIII) Glc
3.70 4.00 7.7 8.7
3.42 3.69 7.1 9.6
4.06 4.20 8.6
4.10 4.18 7.9 10.4
3.82
Gal
GalN
Gal'
Neu5Ac
Glc
2.3
<1
3.0
3.85
3.92
3.40
3.42
3.82
3.87
3.38
(XIX)
(XX)
3.43 3.69 8.3 9.6 10.0
Gal
3.72–3.88 7.9 8.8
4.16–4.27 8.4
4.07 4.29 8.2 10.2
7.9 8.9
2.6
3.1
3.2
GalN
Gal'
Glc
Gal
GalN
Gal'
Glc
Gal
GalN
Gal'
5.12
5.45
3.80–4.10 8.4
2.8
2.4
9.0
2.5
3.0
3.2
4.61 5.07 4.86 5.33
4.40 3.44 3.59 4.02
4.42 3.57 3.38 3.96
5.09 3.72 4.37 5.40
4.54 5.06 4.86 5.35
4.15–4.27 8.1 10.1
3.70 3.78 8.2 8.7
4.12 4.57 8.4
(XXI)
3.40
3.42
3.70
3.83
4.2 10.7
6.5 6.6
3.86 4.07 8.3 10.4
4.18
9.4 7.9
8.2 10.1
7.4 10.2
9.5 10.5
7.5 9.8
(XXII) Glc
4.54 3.38
3.63
Gal
GalN
Gal'
4.44 3.42 3.78 4.12 3.60–3.80 3.72–4.0
4.70 4.03 3.90 4.17
4.46 3.54 3.62 3.92
2.1
3.0
3.1
Notes: * Signals: H7, H8, and H9, 3.57–4.0
** Signals: H7, 5.29 (J 7.6); H8, 5.38; H9 , 3.96; H9 , 4.32; and NHAc, 5.52.
7,8
a
b
Other signals: CH COO, 1.9–2.1; C H COO, 7.7–8.0; C H CH , 6.8–7.5; C H CH , 4.5–4.9; OCH CH Cl, 3.58; OCH CH Cl, 3.68–4.07;
3
6
2
5
3
6
5
2
3
6
5
2
2
2
2
2
2
2
OCH CH N , 3.38–3.58; OCH CH N , 3.67–4.10; NHC(O)CCl , 6.8; OCHCH NH , 3.26; OCH CH NH , 3.94 and 4.12; C(O)OCH , 3.7;
2
2
3
2
2
2
2
3
CH OPhCH , 3.84; and CH OPhCH , 6.85–6.95.
3
2
3
2
In CD OD: OCH CH N , 3.45; OCH CH N , 3.72 and 4.01; PhCH(OR)OR', 5.81; and (CH ) C(OR)OR', 1.57 and 1.30.
3
2
2
3
2
2
3
3 2
In D O: CH CONH, 2.0–2.1; OCH CH NH , 3.9–4.2; CH CH NH , 3.22–3.28.
2
3
2
2
2
2
2
2
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 30 No. 1 2004

SYNTHESIS OF AMINOETHYL GLYCOSIDES
65
Table 2. 13C NMR spectral data for (I) and (XXI) in D2O; (XIV) in CD3OD; and (IX), (XI)–(XIII), (XV)–(XX), and (XXII)
in CDCl3
Chemical shift (δ), ppm
Compound
(I)
Residue
Glc
C1
C2
C3
C4
C5
C6
102.9
103.5
71.3
74.0
75.8
75.9
79.4
77.4
Gal
73.0–77.0
61.0–65.0
GalN
Gal'
Neu5Ac
Glc
Gal
Glc
Gal
Glc
Gal
Glc
Gal
Glc
Gal
Glc
Gal
Glc
Gal
Glc
103.3
105.6
174.0
100.8
101.0
104.1
103.9
103.6
102.6
103.6
102.5
104.8
105.3
103.7
102.6
103.7
102.4
103.3
102.1
168.2
103.4
102.2
100.3
100.6
168.4
103.7
102.2
99.3
100.5
103.6
102.1
99.8
100.5
103.6
102.2
98.9
100.5
103.2
104.2
103.6
106.0
52.2
72.1
101.7
71.3
69.0
74.8
74.2
81.7
79.9
81.7
79.3
75.2
72.2
81.7
79.3
81.8
79.3
81.6
78.2
98.2
81.5
78.6
54.5
68.3
99.2
81.7
80.7
55.5
68.6
81.6
81.0
55.7
68.7
81.6
80.2
55.6
68.7
73.9
72.3
52.8
71.9
81.6
74.0
38.3
72.5
70.9
76.1
80.6
82.6
73.5
82.7
80.8
77.0
73.9
82.7
81.0
82.5
81.0
82.7
76.1
36.2
82.6
75.7
70.2
70.5
35.3
82.9
80.9
73.5
70.5
82.5
74.8
74.1
69.1
82.5
81.1
73.0
70.6
69.2
70.0
74.5
72.7
70.6
76.2
75.1
75.1
72.8
74.9
66.1
77.0
68.7
75.1
74.0
75.0
72.0
74.7
72.3
48.8
75.0
73.1
(IX)
76.1
66.6
80.7
74.6
76.5
68.8
76.5
72.8
80.5
77.8
76.7
67.1
76.6
66.5
76.1
67.7
68.9
76.3
68.3
68.0
66.5
69.0
76.3
72.8
69.1
66.8
75.7
73.6
68.7
66.8
76.0
72.4
68.9
66.9
79.6
77.3
69.3
69.8
61.8
60.7
61.7
62.1
68.2
68.6
68.4
68.2
62.2
70.6
68.2
62.3
68.3
63.0
68.2
68.2
72.5
69.0
68.0
61.9
61.6
71.7
69.0
68.3
61.0
62.1
68.3
69.2
62.1
61.1
68.2
63.5
62.0
61.1
(XI)
(XII)
(XIII)
(XIV)
(XV)
(XVI)
(XVII)
Gal
Neu5Ac*
Glc
(XVIII)
Gal
GalN
Gal'
Neu5Ac**
Glc
70.5
49.4
75.2
73.2
71.3
71.0
75.1
71.1
70.8
70.6
74.9
71.9
71.2
70.8
(XIX)
(XX)
Gal
GalN
Gal'
Glc
Gal
GalN
Gal'
Glc
(XXI)
(XXII)
Gal
GalN
Gal'
Glc
Gal
GalN
Gal'
73.7
81.0
73.7
75.4–76.3
61.2–62.2
Notes: * Signals: C7, 67.1; C8, 69.0; and C9, 62.1
** Signals: C7, 66.5; C8, 69.0; C9, 60.4
Other signals: OCH CH NH 64.8; OCH CH NH 38.4; OCH CH Cl 42.3; OCH CH Cl 69.8; CH CONH 22.6; C(O)OCH 52.7;
2
2
2
2
6
2
2
2
2
2
2
3
3
CH CONH 175.5; C(O)OCH 168.2; C H CH 127.5–128.5; C H CH 72.1–75.6; OCH CH N 51.0; OCH CH N 68.1–68.2;
3
3
5
2
6
5
2
2
2
3
2
2 3
C H COO 166.3; C H COO 129.8–139.0; CH COO 169.6–170.2; CH COO 20.5–20.7; CH OPhCH 55.2; CH OPhCH 113.8.
6
5
6
5
3
3
3
2
3
2
In CD OD: OCH CH N 51.8; OCH CH N 69.1 and 4.01; PhCH(OR)OR' 102.7; (CH ) C(OR)OR 28.1 and 26.2; (CH ) C(OR)OR' 110.8.
3
2
2
3
2
2
3
3 2
3 2
In D O: CH CONH 23.3; OCH CH NH 67.0; OCH CH NH 40.4; CH CONH 176.0; COOH 174.0.
2
3
2
2
2
2
2
2
3
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 30 No. 1 2004

66
CHESHEV et al.
1
The interaction of the resulting trisaccharide (XVII)
of J1'', 2'' = 8.4 Hz in the H NMR spectrum (Table 1),
whereas the position of glycosylation, by the low-field
position of the C4' signal at 72.8 ppm in the 13C NMR
spectrum (Table 2). The methoxybenzyl protecting
group in (XIï) was removed by the treatment with
CAN in aqueous acetonitrile, and monohydroxy deriv-
ative (Xï) was obtained in 90% yield.
with disaccharide glycosyl donor (II) led to the target
derivative of GM1 (XVIII) (Scheme 3) in 85% yield
(from the reacted acceptor). The configuration of the
resulting β-glycoside bond was confirmed by the char-
acteristic value of coupling constant J1'', 2'' of 8.6 Hz in
the 1H NMR spectrum of (XVIII) (Table 1).
The attempts of (Xï) sialylation with thioglycoside
(IV) under the conditions suggested by Hasegawa [15]
(anhydrous acetonitrile, NIS, catalytic amount of
TfOH, MS 3 Å, –30 to –40°C) did not result in the for-
mation of any noticeable amount of the glycosylation
product, but led to the elimination of ethanethiol from
the glycosyl donor, whereas the glycosyl acceptor
remained unreacted. Similar problems have already
been observed previously when synthesizing GM1
according to this scheme [8, 9]. It is likely that low
reactivity of glycosyl acceptors of the used type seems
to be due to the presence of a hydrogen bond between
the amide moiety in the GalN residue and the C3'-
hydroxyl, since the substitution of azide group for the
amide moiety in GalN enables the sialylation though in
a low yield [8].
All acyl groups of pentasaccharide (XVIII) were
removed by the treatment with 1 M NaOH in aqueous
methanol, followed by the N-acetylation of the result-
ing free amino group in the galactosamine residue with
acetic anhydride (Scheme 3). The removal of benzyl
groups and reduction of azide moiety were carried out
by hydrogenation in aqueous methanol over Pd/C in the
presence of Boc2O to fix the amine generated upon the
reaction [17]. The Boc protecting group was removed
by treatment with trifluoroacetic acid in aqueous meth-
anol, and the target derivative GM1 (I) was isolated by
column chromatography in overall 85% yield from pre-
cursor (XVIII). The structure of pentasaccharide (I),
namely, the configurations of its anomeric centers, the
directions of glycosidic bonds, and the presence of ami-
noethyl aglycone (δ CH2Nç2 of 38.4 ppm), was con-
firmed by its 1ç NMR (Table 1) and 13C NMR (Table 2)
spectra. In particular, the α-configuration of the
neuraminic acid residue follows from the chemical
shifts of H3eq (2.70 ppm) and H4 (3.70 ppm) protons,
which are in complete agreement with the empiric rules
[16]: according to them, H3eq of the α-bound
neuraminic acid residue resonates at 2.67 to 2.72 ppm,
whereas H4 at 3.6–3.8 ppm.
To obtain 2-aminoethyl glycoside of asialo-GM1, it
was necessary to synthesize the acceptor lactose block
of type (VII) containing the azidoethyl prespacer and
free hydroxyl group at C4'. For this purpose, it was sug-
gested to use lactoside (XVI), which was prepared
according to the sequence shown in Scheme 2. The
complete deacetylation of azide (X) and directed 4',6'-
é-benzylidenation of the resulting heptaol with benzal-
dehyde dimethyl acetal in the presence of TsOH
afforded pentaol (XIV) in 89% yield. This was perben-
zylated, the benzylidene group was removed, and the
resulting diol (XV) was regioselectively 6'-é-benzoyl-
ated [12] to give alcohol (XVI) in total yield of 79%
from pentaol (XIV). The presence of benzoyl group at
O6' in (XVI) was confirmed by a lower-field chemical
As mentioned above, we considered two approaches
to the synthesis of target GM1 derivative (I). Its synthe-
sis according to the path Ç (Scheme 1) includes the pre-
liminary obtaining of monohydroxy derivative of
asialo-GM1 (VI) and the subsequent sialylation. The
acceptor lactose block of type (VII) is necessary for
obtaining azidoethyl glycoside (VI). To this end, we
suggested the use of alcohol (XIII) containing a tempo-
rary methoxybenzyl protecting group at C3'. Diol (XII)
was treated with Bu2SnO; the resulting stannylidene
derivative was 3'-é-methoxybenzylated with MPM-Cl
to result in regioselective formation of 4'-hydroxy
derivative (XIII) in 93% yield. The presence of the
methoxybenzyl group at O3' in (XIII) was confirmed
by the chemical shift of the resonance of C3' atom
(81.7 ppm) in the 13C NMR spectrum down field to the
signal of this atom in diol (XII) (73.2 ppm). The pres-
ence of free hydroxy group at C4' was confirmed by the
presence of an additional coupling of the H4' signal
'
'
shift of the H6a (4.29 ppm) and H6b (4.54 ppm) sig-
nals in the 1H NMR spectrum (Table 1) in comparison
with the signals of these protons in diol (XV) (3.60–
3.65 ppm). The presence of free OH group at C4' fol-
lowed from the strong-field chemical shift of H4'
(3.92 ppm); which also had an additional splitting on
the proton of OH group.
The glycosylation of monohydroxy derivative
(XVI) with thioglycoside (II) [11] in dichloromethane
in the presence of NIS and TfOH resulted in asialo-
GM1 derivative (XXI) in 69% yield (Scheme 4). The
configuration of β-GalN residue in (XXI) was unam-
biguously confirmed by the characteristic value of J1'', 2''
1
with the hydroxyl proton in the H NMR spectrum of
of 8.7 Hz in the 1H NMR spectrum (Table 1), whereas
(XIII).
the (1
4)-binding followed from a low-field chem-
The glycosylation of acceptor (XIII) with glycosyl
donor (II) in dichloromethane in the presence of NIS
and TfOH (Scheme 4) gave a protected tetrasaccharide
(XIï) in 63% yield. The β-configuration of GalN resi-
ical shift of C4 of the Gal residue (72.4 ppm) in the 13C
NMR spectrum (Table 2).
Hydrolysis of the ester groups and the trichloroace-
due in (XIï) was confirmed by the characteristic value timidate moiety, hydrogenolysis, and reduction of azide
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 30 No. 1 2004

SYNTHESIS OF AMINOETHYL GLYCOSIDES
67
group in (XXI) were carried out under the conditions reaction mixture was diluted with dichloromethane
similar to those for transformation of (XVIII) to (I). (150 ml), washed with saturated NaHCO3 solution (2 ×
The target asialo-GM1 (XïII) was isolated by gel fil-
50 ml) and water (70 ml), and evaporated in a vacuum.
tration in overall yield of 72% from the starting (XXI). The residue was chromatographed in 2 : 1 toluene–
The structure of (XïII), in particular, the configuration ethyl acetate system to give 1.5 g (74%) of glycoside
of its anomeric centers, the direction of the interunit (IX) as a white foam, Rf 0.54 (3 : 2 toluene–ethyl ace-
bonds, and the presence of the aminoethyl aglycone
[δ(CH2Nç2) = 38.4 ppm] were confirmed by ç
(Table 1) and 13C (Table 2) NMR spectra.
tate), [α]D +9.0° (Ò 1, CHCl3).
1
2-Azidoethyl
2,3,6-tri-O-acetyl-4-O-(2,3,4,6-
tetra-O-acetyl-b-D-galactopyranosyl)-b-D-glucopy-
ranoside (X). A suspension of NaN3 (2.15 g, 33 mmol)
in a solution of chloroethyl glycoside (IX) ((2.81 g,
3.3 mmol) and 18-crown-6 (871 mg, 3.3 mmol) in
DMF (12 ml) was stirred for 18 h at 65°ë. The mixture
was diluted with ethyl acetate (350 ml), washed with
water (4 × 150 ml), filtered through a cotton layer, and
evaporated to give 2.27 g (97%) of azide (X) as a white
foam, Rf 0.62 (3 : 2 toluene–ethyl acetate), [α]D –16.5°
(Ò 2, ethyl acetate). Found, %: C 48.18, H 5.71, N 6.24;
calculated for C28H54O18N3, %: C 47.66, H 5.53, N
5.96.
Thus, we synthesized the aminoethyl glycosides of
the oligosaccharide chains of asialo-GM1 and GM1
gangliosides and demonstrated the effectiveness of the
use of the type (II) thioglycoside donors for obtaining
the oligosaccharide structures containing the gan-
gliotetraose chain. A highly effective glycosylation of
lactose diol (VIII) with thioglycoside sialyl donor (III)
resulting in the formation of the trisaccharide chain of
ganglioside GM3 was also carried out.
EXPERIMENTAL
2-Azidoethyl
4-O-(3,4-O-isopropylidene-b-D-
The procedures of solvent purification and the con-
ditions of registration of NMR spectra and determina-
tion of physicochemical constants are similar to those
reported in [18]. CAN, MPM-Cl, Bu2SnO, and NIS
galactopyranosyl)-b-D-glucopyranoside (XI).
A
solution of azidoethyl glycoside (X) (5.17 g,
7.33 mmol) in 0.1 N sodium methylate in methanol
(35 ml) was kept for 24 h, neutralized with cation
exchange resin KU-2 (ç+), and filtered. The resin was
washed with methanol (2 × 10 ml), and the combined
filtrate was concentrated in a vacuum. The residue was
dried for 2 h in a vacuum of an oil pump, dissolved in a
mixture of 2,2-dimethoxypropane (6 ml), acetone
(1.8 ml), and TsOH monohydrate (23 mg) and stirred
for 5 min at 60°ë. The mixture was cooled to room
temperature, the precipitate were filtered off and
washed with cold acetone (5 ml), and the combined fil-
trate was concentrated in a vacuum. The precipitated
solid was recrystallized from acetone (1 ml) and dried
in a vacuum of an oil pump to give 2.73 g (82%) of ace-
tonide (XI); colorless crystals; mp 167–169°ë; Rf 0.50
(3 : 1 ethyl acetate–methanol), [α]D + 19.8° (Ò 1, meth-
anol).
were purchased from Aldrich. 1ç and 13C NMR spectra
were registered on Bruker DRX-500 and Bruker AM-
300 spectrometers at 25°ë. Optical rotations were mea-
sured on a Jasco DIP-360 digital polarimeter at 18–
25°ë. Thin layer chromatography was performed on
Silica gel 60 precoated plates (Merck). Spots were
visualized by treatment with 10 vol % orthophosphoric
acid in ethanol or, in the case of amines, with ninhydrin
solution (3 g/l in 30 : 1 butanol–acetic acid) and subse-
quent heating at ~150°ë. Column chromatography was
carried out on Silica gel 60 (0.063–0.2 mm, Fluka). For
MPLC, Silasorb 600 (20 µm, Chemapol)-packed col-
umns were used. The reversed-phase chromatography
was carried out on a Chromsphere 5 C18 (Chrompack)
column using water as eluent. Gel chromatography was
performed on columns with Sephadex LH-20 (2 ×
40 cm) upon elution with methanol at a flow rate of 1
ml/min, Bio-Beads S-X3 (2.5 × 68 cm, Bio-Rad) eluted
with toluene, and TSK-HW40s (1.5 × 90 cm) upon elu-
tion with 0.1 M aqueous acetic acid at a flow rate of
1 ml/min. Hydrogenolysis was performed over 10%
Pd/C (Merck) under atmospheric pressure. TLC of free
aminoethyl glycosides was carried using BPH (1 : 2 : 1
butanol–propanol–0.1 M hydrochloric acid) and AMW
(1 : 1 : 1 acetonitrile–methanol–water) developing sys-
tems.
2-Azidoethyl 2,3,6-tri-O-benzyl-4-O-(2,6-di-O-
benzyl-b-D-galactopyranosyl)-b-D-glucopyranoside
(XII). Sodium hydride (430 mg of 60% oil suspension,
10.8 mmol) was added to a stirred solution of acetonide
(XI) (0.5 g, 1.07 mmol) in anhydrous DMF (10 ml)
cooled to 0°ë. After a gas liberation was over, benzyl
bromide (760 µl, 6.4 mmol) was added dropwise at stir-
ring. The mixture was kept for 4 h, the NaH excess was
quenched with methanol (2 ml), 90% acetic acid
(10 ml) was added, and the mixture was heated in oil
bath at 80°ë for 5 h. Then the reaction mixture was
diluted with ethyl acetate (100 ml) and washed with
water (2 × 100 ml) and saturated NaCl solution (50 ml).
The organic layer was separated and evaporated, and
the residue was chromatographed on a column eluted
with 3 : 1 toluene–ethyl acetate to isolate 900 mg (98%)
of disaccharide (XII); a white foam; Rf 0.23 (10 : 1
2-Chloroethyl
2,3,6-tri-O-acetyl-4-O-(2,3,4,6-
tetra-O-acetyl-b-D-galactopyranosyl)-b-D-glucopy-
ranoside (IX). A solution of acetylated lactosyl bro-
mide (VIII) (2.02 g, 2.9 mmol) in anhydrous 2-chloro-
ethanol (4 ml) was stirred at 60°ë with mercury(II)
cyanide (729 mg, 2.88 mmol) and mercury(II) bromide
(110 mg, 0.31 mmol) for 4 h at room temperature. The
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 30 No. 1 2004

68
CHESHEV et al.
dichloromethane–ethyl acetate); [α]D +15.2° (c 2, 69.16, H 6.36, N 5.12; calculated for C28H54O18N3, C
CHCl3).
68.29, H 6.38, N 4.88.
2-Azidoethyl 2,3,6-tri-O-benzyl-4-O-(2,3-di-O-
benzyl-6-O-benzoyl-b-D-galactopyranosyl)-b-D-glu-
copyranoside (XVI). Benzoyl chloride (50 µl,
0.5 mmol) was added to an intensively stirred solution
of diol (XV) (385 mg, 0.447 mmol) in a mixture of
anhydrous dichloromethane (10 ml) and anhydrous
pyridine (110 µl) cooled to 0°ë. The reaction mixture
was stirred for 2 h at 0°ë and diluted with dichlo-
romethane (50 ml) and water (10 ml). The organic layer
was separated; washed with 1 M H2SO4 (40 ml), water
(2 × 40 ml), and saturated NaHCO3 solution (20 ml);
filtered through a cotton layer; and evaporated. The res-
idue was chromatographed on a column eluted with
3 : 1 toluene–ethyl acetate to isolate 351 mg (81%) of
(XVI); Rf 0.36 (10 : 1 toluene–ethyl acetate); [α]D
+18.4° (Ò 2.8, ethyl acetate).
2-Azidoethyl 2,3,6-tri-O-benzyl-4-O-[2,6-di-O-
benzyl-3-O-(4-methoxybenzyl)-b-D-galactopyrano-
syl]-b-D-glucopyranoside (XIII). Dibutyltin oxide
(60 mg, 0.24 mmol) was added to a solution of diol
(XII) (170 mg, 0.2 mmol) in toluene (10 ml), and the
mixture was refluxed upon azeotropic distillation of
water for 1 h. p-Methoxybenzyl chloride (30 µl,
0.22 mmol) and Bu4NBr (64 mg, 0.2 mmol) were
added, and the mixture was refluxed for 1.5 h and evap-
orated. The residue was chromatographed on a column
eluted with 15 : 1 dichloromethane–ethyl acetate to iso-
late 180 mg (93%) of disaccharide (XIII); a white
foam; Rf 0.67 (10 : 1 dichloromethane–ethyl acetate),
[α]D +24.2° (c 2, CHCl3).
2-Azidoethyl 4-O-(4,6-O-benzylidene-b-D-galac-
topyranosyl)-b-D-glucopyranoside (XIV).A solution
of azidoethyl lactoside (X) (4.3 g, 6.1 mmol) in 0.1 N
sodium methylate in methanol (30 ml) was kept for
24 h, neutralized with KU-2 (H+), and filtered off. The
cation exchange resin was washed with methanol (2 ×
10 ml), and the combined filtrates were evaporated. The
residue was dried for 2 h in a vacuum of an oil pump,
dissolved in a mixture of anhydrous acetonitrile
(36 ml), benzaldehyde dimethyl acetal (1.5 ml), and
TsOH monohydrate (105 mg) and stirred for 25 min at
60°ë. The mixture was then cooled to room tempera-
ture, the crystalline precipitate was filtered off and
washed with ethyl acetate, and the combined filtrate
was concentrated in a vacuum and recrystallized from
ethyl acetate (2 ml). The crystalline precipitate was
dried in a vacuum of an oil pump to yield 2.57 g (84%)
of benzylidene derivative (XIV); colorless crystals; mp
205–207°ë, Rf 0.21 (ethyl acetate); [α]D –21.4° (Ò 1,
methanol). Column chromatography in ethyl acetate
allowed us to isolate another 163 mg (5%) of pentaol
(XIV) as a white foam.
2-Azidoethyl [methyl (5-acetamido-4,7,8,9-tetra-
O-acetyl-3,5-dideoxy-D-glycero-a-D-galacto-2-non-
ulopyranosyl)oate]-(2
galactopyranosyl)-(1
3)-(2,6-di-O-benzyl-b-D-
4)-2,3,6-tri-O-benzyl-b-D-
glucopyranoside (XVII). A solution of disaccharide
(XII) (300 mg, 0.35 mmol) and glycosyl donor (IV)
(373 mg, 0.7 mmol) in anhydrous acetonitrile (6 ml)
was stirred for 2 h at room temperature with preliminar-
ily heated MS 3 Å (600 mg) for 2 h at room tempera-
ture. NIS (190 mg, 0.84 mmol) was added, the mixture
was stirred for another 30 min at room temperature,
cooled to –40°ë, and treated with TfOH (10 µl). The
mixture was stirred for 2 h at –40 to –30°C, treated with
saturated NaHCO3 solution (2 ml) and 1M Na2S2O3
solution (2 ml), and stirred for another 15 min. The
reaction mixture was then filtered through a layer of
Celite, the filtrate was diluted with dichloromethane
(100 ml) and washed with NaHCO3 solution (100 ml).
The organic layer was separated and evaporated. The
residue was first chromatographed on Bio-Beads S-X3
gel to remove glycal generated as a by-product, and
then, the resulting mixture of di- and trisaccharides was
separated by column chromatography at elution with
2 : 1 toluene–ethyl acetate. The starting glycosyl accep-
tor (XII) (44 mg) was isolated; yield of (XVII) 313 mg
[79% from the reacted (XII)] of trisaccharide (XVII);
a white foam; Rf 0.4 (2 : 1 toluene–ethyl acetate);
[α]D +4.1° (Ò 1, CHCl3).
2-Azidoethyl 2,3,6-tri-O-benzyl-4-O-(2,3-di-O-
benzyl-b-D-galactopyranosyl)-b-D-glucopyranoside
(XV). Sodium hydride (122 mg of 60% oil suspension,
3.05 mmol) was added to a stirred solution of pentaol
(XIV) (230 mg, 0.51 mmol) in anhydrous DMF
(3.8 ml). After a gas liberation ceased, benzyl bromide
(360 µl, 3.05 mmol) was added dropwise. The mixture
was kept for 4 h; the NaH excess was decomposed with
methanol (1 ml), and the reaction mixture was diluted
with ethyl acetate (50 ml) and washed with water (2 ×
30 ml) and saturated aqueous NaCl (20 ml). The
organic layer was separated and evaporated, and the
residue was dissolved in a mixture of chloroform (5 ml)
and 90% trifluoroacetic acid (2 ml), kept for 30 min and
coevaporated in a vacuum with toluene. The residue
was chromatographed on a column eluted with 2 : 1 tol-
uene–ethyl acetate to give 426 mg (97%) of disaccha-
ride (XV); a white foam; Rf 0.19 (2 : 1 toluene–ethyl
acetate); [α]D + 24.5° (Ò 4, ethyl acetate). Found, %: C
General glycosylation procedure for obtaining
disaccharides (XVIII), (XIX), and (XXI). An
equimolar mixture of glycosyl donor (II) and a glyco-
syl acceptor was three times coevaporated with toluene
to remove the traces of water, dissolved (in argon atmo-
sphere) in freshly distilled anhydrous dichloromethane
and placed in argon atmosphere into the reaction flask
containing MS 4 Å preliminarily heated in a vacuum
(2 h at 200°ë). The reaction mixture was stirred for 2 h
at room temperature, then NIS (1.2–1.3 molar excess)
was added, and the mixture was stirred for another
30 min. The reaction mixture was cooled to –30°ë,
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 30 No. 1 2004

SYNTHESIS OF AMINOETHYL GLYCOSIDES
69
10 vol % TfOH in CH2Cl2 was added dropwise, and the evaporated. The residue was separated by column chro-
matography in 2 : 1 dichloromethane–ethyl acetate sys-
tem to give 163 mg (90%) of alcohol (XX); a white
foam; Rf 0.64 (5 : 1 dichloromethane–acetone).
resulting mixture was stirred for 1–1.5 h at –30°ë under
TLC monitoring. After glycosyl donor was exhausted,
the reaction was stopped by addition of saturated
NaHCO3 solution (2 ml) and 1 M Na2S2O3 (2 ml), cool-
ing was removed, and the mixture was stirred for
15 min at room temperature. The reaction mixture was
filtered through a layer of Celite, the filtrate was diluted
with dichloromethane (70 ml) and washed with
NaHCO3 solution (50 ml). The organic layer was sepa-
rated and evaporated. The reaction products were iso-
lated by chromatography.
2-Azidoethyl (2,3,4,6-tetra-O-acetyl-b-D-galac-
topyranosyl)-(1
3)-(4,6-di-O-acetyl-2-deoxy-2-
trichloroacetamido-b-D-galactopyranosyl)-(1
4)-(2,6-di-O-benzyl-6-O-benzoyl-b-D-galactopyrano-
syl)-(1
4)-2,3,6-tri-O-benzyl-b-D-glucopyrano-
side (XXI). The reaction of disaccharide (XVI)
(205 mg, 0.21 mmol) and glycosyl donor (II) (210 mg,
0.21 mmol) in anhydrous dichloromethane (5 ml) in the
presence of MS 4 Å (500 mg) was promoted with NIS
(57 mg, 0.25 mmol) and TfOH (150 µl of a solution in
CH2Cl2). After the standard treatment, the mixture was
chromatographed on a column eluted with 4 : 1 dichlo-
romethane–ethyl acetate to give 53 mg of the starting
glycosyl acceptor and 184 mg [69% from the reacted
(XVI)] of tetrasaccharide (XXI); a white foam; Rf 0.59
(10 : 3 dichloromethane–ethyl acetate); [α]D +5.9° (c 1,
CHCl3).
2-Azidoethyl (2,3,4,6-tetra-O-acetyl-b-D-galac-
topyranosyl)-(1
3)-(4,6-di-O-acetyl-2-deoxy-2-
trichloroacetamido-b-D-galactopyranosyl)-(1
4)-[methyl (5-acetamido-4,7,8,9-tetra-O-acetyl-3,5,-
dideoxy-D-glycero-a-D-galacto-2-nonulopyranosyl)-
oate]-(2
nosyl)-(1
3)]-(2,6-di-O-benzyl-b-D-galactopyra-
4)-2,3,6-tri-O-benzyl-b-D-glucopyra-
noside (XVIII). The reaction of trisaccharide (XVII)
(220 mg, 0.16 mmol) and thioglycoside (II) (163 mg,
0.16 mmol) in anhydrous dichloromethane (4 ml) in the
presence of molecular sieve MS 4 Å (400 mg) was pro-
moted with NIS (45 mg, 0.2 mmol) and TfOH (100 µl)
in CH2Cl2. After the standard treating, the mixture was
passed though the Silica gel layer (5 : 1 chloroform–
acetone) and then separated on a Bio-Beads S-X3 col-
umn to give 30 mg of unreacted glycosyl acceptor
(XVII) and 250 mg [85% from reacted (XVII)] of pen-
tasaccharide (XVIII); a white foam, Rf 0.2 (5 : 1 chlo-
roform–acetone); [α]D –2.8° (c 1, CHCl3). Found, %: C
55.39, H 5.59, N 3.41. Calculated for C95H114Cl3N5O39,
%: C 55.49, H 5.59, N 3.41.
2-Aminoethyl b-D-galactopyranosyl-(1
3)-2-
deoxy-2-acetamido-b-D-galactopyranosyl-(1
4)-b-D-galactopyranosyl-(1
4)-b-D-glucopyra-
noside (XXII). Tetrasaccharide (XXI) (72 mg,
0.043 mmol) was dissolved in 10% aqueous methanol
(5 ml), NaOH (200 mg) was added, and the mixture was
stirred up to complete dissolution and kept for 6 h at
40°ë and 14 h at room temperature. Then the mixture
was cooled to 0°ë, ÄÒ2é was added dropwise to pH 6
(control using universal pH-indicator, Merck), and the
mixture was deionized with cation exchange resin KU-
2 (H+). The cation exchanger was removed by filtration
and washed with methanol (3 × 10 ml), the filtrates were
combined and evaporated, and the residue was chro-
matographed on a Sephadex LH-20 column. The carbo-
hydrate-containing fractions were evaporated and dried
in a vacuum of an oil pump. The dry residue was dis-
solved in 10% aqueous ethanol, Boc2O (100 mg) and a
catalytic amount of Pd/C were added, and the mixture
was stirred for 14 h at room temperature in hydrogen
atmosphere up to complete removal of benzyl groups
(TLC monitoring). The reaction mixture was filtered
through a Celite column, which was washed with a
2-Azidoethyl (2,3,4,6-tetra-O-acetyl-b-D-galac-
topyranosyl)-(1
3)-(4,6-di-O-acetyl-2-deoxy-2-
trichloroacetamido-b-D-galactopyranosyl)-(1
4-[2,6-di-O-benzyl-3-O-(4-methoxybenzyl)-b-D-galac-
topyranosyl)-(1
4)-2,3,6-tri-O-benzyl-b-D-glu-
copyranoside (XIX). The reaction of disaccharide
(XIII) (180 mg, 0.18 mmol) and glycosyl donor (II)
(190 mg, 0.19 mmol) in anhydrous dichloromethane
(4 ml) in the presence of MS 4 Å (400 mg) was pro-
moted with NIS (52 mg, 0.23 mmol) and TfOH (100 µl
of a solution in CH2Cl2). After a standard treatment, the
mixture was chromatographed on a column eluted with
2 : 1 toluene–ethyl acetate to give 196 mg (63%) of tet-
rasaccharide (XIX); a white foam; Rf 0.45 (2 : 1 tolu-
ene–ethyl acetate).
100
0% methanol–water gradient (30 ml), and
90% CF3COOH (2 ml) was added. The mixture was
kept for 30 min, concentrated, and coevaporated with
water up to disappearance of TFA odor. The residue
was chromatographed on a TSK-HW-40s gel column to
give 23 mg (72%) of aminoethyl glycoside (XXII), Rf
0.23 (1 : 1 BPH–AMW), [α]D –13.8° (Ò 1, water).
2-Azidoethyl (2,3,4,6-tetra-O-acetyl-b-D-galac-
topyranosyl)-(11
3)-(4,6-di-O-acetyl-2-deoxy-2-
trichloroacetamido-b-D-galactopyranosyl)-(1
4)-(2,6-di-O-benzyl-b-D-galactopyranosyl)-(1
4)-2,3,6-tri-O-benzyl-b-D-glucopyranoside (XX). A
solution of tetrasaccharide (XIX) (196 mg, 0.12 mmol)
and CAN (315 mg, 0.57 mmol) in 9 : 1 acetonitrile–
water (5 ml) was stirred for 14 h at room temperature.
The reaction mixture was diluted with dichloromethane
(75 ml), washed with NaHCO3 solution (50 ml), and
2-Aminoethyl b-D-galactopyranosyl-(1
3)-
(2-acetamido-2-deoxy-b-D-galactopyranosyl)-(1
4)-[5-acetamido-3,5-dideoxy-D-glycero-a-D-galacto-
2-nonulopyranosyl-(2
syl-(1 4)-b-D-glucopyranoside (I). Pentasaccha-
ride (XVIII) (88 mg, 0.043 mmol) was deacetylated,
3)]-b-D-galactopyrano-
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 30 No. 1 2004

70
CHESHEV et al.
7. Hasegawa, A., Nagahama, T., and Kiso, M., Carbohydr.
Res., 1992, vol. 235, C13.
N-acetylated, and then subjected to hydrogenolysis and
reduction of azide group under the conditions similar to
those used in the synthesis of (XXII). Chromatography
on a Chromsphere 5 C18 column resulted in 38 mg
(85%) of aminoethyl glycoside (I), Rf 0.38 (1 : 1 BPH–
AMW); [α]D +8.5° (Ò 1, water).
8. Stauch, T., Greilich, U., and Schmidt, R.R., Liebigs Ann.
Chem., 1995, pp. 2101–2111.
9. Bhattacharya, S.K. and Danishefsky, S.J., J. Org. Chem.,
2000, vol. 65, pp. 144–151.
10. Ito, Y., Nunomura, S., Shibayama, S., and Ogawa, T.,
J. Org. Chem., 1992, vol. 57, pp. 1821–1831.
ACKNOWLEDGMENTS
11. Cheshev, P.E., Kononov, L.O., Tsvetkov, Yu.E., Shash-
kov, A.S., and Nifantiev, N.E., Bioorg. Khim., 2002,
vol. 28, pp. 462–473.
The work was supported by the Russian Foundation
for Basic Research, project no. 00-03-32815a, and by
the Foundation for Assistance for Home Science.
12. Cheshev, P.E., Khatuntseva, E.A., Gerbst, A.G., Tsvet-
kov, Yu.E., Shashkov, A.S., and Nifantiev, N.E., Bioorg.
Khim., 2003, vol. 29, pp. 408–418.
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