Synthesis of Neoglycolipid Analogues of the Oligosaccharide Portion of Ganglioside GM3

Article abstract of DOI:10.1081/CAR-120025326

In order to elucidate the molecular specificity of the antimelanoma response induce by GM3 included in proteoliposome preparation, we designed syntheses of a series of neoglycolipids containing the trisaccharide portion of GM3 or its fragment. In the pres

Full text of DOI:10.1081/CAR-120025326

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Synthesis of Neoglycolipid Analogues of the
Oligosaccharide Portion of Ganglioside GM3
Freya González Núñez a , Maria T. Campos Valdes a , Elsa Aruca a , Richard R. Schmidt b
Vicente Verez Bencomo a
&
a Center for the Study of Synthetic Antigens , Facultad de Química , Universidad de La
Habana , Ciudad Habana, Cuba , 10400
b Fachbereich Chemie , Universität Konstanz , 78457, Konstanz, Germany
Published online: 23 Aug 2006.
To cite this article: Freya González Núñez , Maria T. Campos Valdes , Elsa Aruca , Richard R. Schmidt & Vicente Verez
Bencomo (2003) Synthesis of Neoglycolipid Analogues of the Oligosaccharide Portion of Ganglioside GM3 , Journal of
Carbohydrate Chemistry, 22:6, 395-406, DOI: 10.1081/CAR-120025326
To link to this article: http://dx.doi.org/10.1081/CAR-120025326
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JOURNAL OF CARBOHYDRATE CHEMISTRY
Vol. 22, No. 6, pp. 395–406, 2003
Synthesis of Neoglycolipid Analogues of the
Oligosaccharide Portion of Ganglioside GM3#
Freya Gonza´lez Nu´n˜ez,1 Maria T. Campos Valdes,1 Elsa Aruca,1
Richard R. Schmidt,2 and Vicente Verez Bencomo1,
*
1Center for the Study of Synthetic Antigens, Facultad de Qu´ımica,
Universidad de La Habana, Ciudad Habana, Cuba
2Fachbereich Chemie, Universita¨t Konstanz, Konstanz, Germany
ABSTRACT
In order to elucidate the molecular specificity of the antimelanoma response induce by
GM3 included in proteoliposome preparation, we designed syntheses of a series of
neoglycolipids containing the trisaccharide portion of GM3 or its fragment. In the
present paper, we synthesized two neoglicolipids containing as a lipid, a racemic
glycerol unit substituted by two aliphatic octadecyl ether chains. The di- and trisac-
charide derivatives were prepared as glycosides of the spacer by a sequence of iso-
propilidenation-benzylation-hydrolysis followed by sialylation. The condensation be-
tween the oligosaccharides and the lipid was performed by an amidation reaction.
Key Words: Neoglycolipids; Proteoliposome; GM3 analogues.
[1]
#See Ref.
*
Correspondence: Vicente Verez Bencomo, Center for the Study of Synthetic Antigens, Facultad
de Qu´ımica, Universidad de La Habana, Ciudad Habana, Cuba 10400; E-mail: vicente@fq.uh.cu.
395
DOI: 10.1081/CAR-120025326
0732-8303 (Print); 1532-2327 (Online)
www.dekker.com
Copyright D 2003 by Marcel Dekker, Inc.

396
Gonza´lez Nu´n˜ez et al.
INTRODUCTION
Gangliosides are important targets for passive or active specific immunotherapy
(ASI) of tumors.[2,3] Gangliosides exhibit low immunogenicity, therefore several gly-
coprotein conjugates or proteoliposomal constructs are usually employed in order to
promote an immune response useful for ASI.[4,5] The molecular specificity of the
response induced by this kind of conjugate and the role of the oligosaccharidic and
lipidic parts in defining their cross recognition of the parent gangliosides in tumors are
not fully understood.
Despite the limited knowledge currently available, the use of carbohydrate tumor-
associated antigens as glycolipids[6,7] for the specific activation of natural killer T cells
seems more promising now. The conventional use of neoglycoprotein conjugates for the
induction of a specific antibody response was very useful in vaccines against bacterial
pathogens but did not give the result expected[8] in the fight against tumor cells.
We selected GM3[9] as the ganglioside with the shortest oligosaccharide chain in
order to determine the individual contribution of each segment of the molecule to
recognition. Additionally, GM3 included in Very Small Size Proteoliposome (VSSP)
vesicles containing the Neisseria meningitidis outer membrane protein complex have
been shown[5] to induce a strong immune response against the parent ganglioside in
animals and also in melanoma patients. These antibodies failed to recognize the
trisaccharide Neu5Aca-(2-3)-Galb-(1-4)-Glc-b that represents the oligosaccharide
portion of GM3 in neoglycoproteins.[10]
In the present paper, we describe the synthesis of neoglycolipids containing the
trisaccharide or its fragments for further understanding of the specificity of anti-GM3
antibodies and also to elucidate the involvement of the CD1-receptor in the anti-
melanoma response.
RESULTS AND DISCUSSION
The disaccharide NeuAca-(2-3)-Gal-b was selected as the first candidate for the
synthesis of neoglycolipids. The azido spacer 1 at the reducing end of the oligo-
saccharide has been shown to allow not only an efficient conjugation to a protein, but
also to provide an easy route to the neoglycolipid having the intact oligosaccharide
structure, as previously reported.[11] The reaction of acetobromogalactose with 5-azido-
3-oxapentanol (1) in the presence of mercuric cyanide afforded the corresponding
glycoside 2 in 83% yield (Scheme 1).[18] The presence of the spacer in glycoside 2 and
also in the other derivatives was confirmed by the observation of a triplet at 3.4 ppm in
1
the H NMR spectrum. The 13C NMR spectrum also contained a characteristic signal
for the CH2N3 residue at 50.0 ppm.
The glycoside 2 was subsequently deacetylated by the Zemplen procedure with
sodium methoxide in methanol followed by isopropylidenation at the 3’, 4’-O-positions
by the method of Catelani et al.[12] Pure compound 3 was isolated by column chro-
matography in 70% yield. The position of the isopropylidene group was ascertained
by the absence of substitution at position C-6 as indicated by the observation of a
signal at 61.9 ppm in the 13C NMR spectrum, typical of an unsubstituted primary
carbon atom. The corresponding glycosyl acceptor 4 was obtained from 3 in 65% yield

Synthesis of Neoglycolipid Analogues
397
Scheme 1. Synthesis of disaccharide NeuAc a(2-3)-Gal neoglycolipid analogue 9.
after benzylation with benzyl chloride and sodium hydride in N,N-dimethylformamide
followed by the hydrolysis of the isopropylidene group with 80% acetic acid.
The sialylation of acceptor 4 with the thioglycoside 5[13] was performed with N-
iodosuccinimide and trimethylsilyl triflate[14] in acetonitrile at ꢀ 15°C to afford the a-
D-linked disaccharide 6 in an acceptable 35% yield. The structure was assigned by
1
observation of a signal in the H NMR spectrum at 2.52 ppm for the equatorial H-3,
characteristic[13] of a-linked sialic acid.
The protecting groups of compound 6 were removed in two steps prior to
the introduction of the lipid moiety. First, deacetylation with sodium methoxide in

398
Gonza´lez Nu´n˜ez et al.
methanol followed by saponification with aqueous 0.2 M KOH afforded disaccharide
derivative 7 that was further hydrogenolized with Pd/C in methanol-acetic acid.
Disaccharide derivative 8 was condensed with 2,3-di-O-octadecyloxypropyl
succinimidyl butanedioate[11] in the presence of triethylamine in dimethyl sulfoxide
at 60°C. The desired neoglycolipid 9 was obtained in 50% yield as a racemic mixture
1
after purification by reverse phase column chromatography. The H NMR spectrum
contained a signal for the CH2NHCO group at 3.30 ppm.
Once the strategy had been proven to be effective for the disaccharide Neu5Ac
(2 ! 3)-Gal, it was also applied to a lactose acceptor. The attempt to prepare the
lactoside 11 using acetobromolactose in the presence of mercuric cyanide gave the
orthoester as the main product. Therefore, the known trichloroacetimidate 10[15] was
synthesized from peracetylated lactose by selective removal of the anomeric acetyl
group with ethanolamine,[16] followed by reaction with trichloroacetonitrile in the
presence of potassium carbonate. The resulting product was an anomeric mixture that
contained mainly the a-anomer.
Coupling of this imidate[15] with spacer 1 proceeded smoothly under trimethylsilyl
triflate catalysis (Scheme 2). The desired lactoside 11 was obtained in 60% yield.
The product was de-O-acetylated under Zemplen conditions and then reacted with
2,2-dimethoxypropane and p-toluenesulphonic acid. The initial product contained some
mixed acetal attached to O-6. Hydrolysis afforded the 3’,4’-O-isopropylidene derivative
12 in 70% yield. Observation of signals for C-6 and C-6’ at chemical shifts cha-
racteristic of unsubstituted primary alcohols in the 13C NMR spectrum, together with
the deshielding of the C-3’ signal to 78.7 ppm indicated that the substitution took place
as expected, on the 3’,4’-diol.
In a similar fashion as for the galactoside acceptor 4, benzylation of 12 with benzyl
chloride and sodium hydride in N,N-dimethylformamide followed by the hydrolysis of
the isopropylidene group with 80% acetic acid gave acceptor 13 in 60% yield. Sialy-
lation of acceptor 13 with thioglycoside 5 was performed with N-iodosuccinimide and
trimethylsilyl triflate[14] in acetonitrile at ꢀ 15°C to afford the a-linked disaccharide 14
1
in 25% yield. The H NMR spectrum contained the typical signal for sialic acid H-
3eq[17] at 2.55 ppm.
The protecting groups of compound 14 were removed in a similar fashion to those
of 6, by deacetylation with sodium methoxide in methanol followed by saponification
with aqueous 0.2 M KOH to give the intermediate 15, that was further hydrogenolyzed
with Pd/C in methanol-acetic acid to give the spacered trisaccharide 16 in a combined
74% yield.
The trisaccharide derivative 16 was condensed with 2,3-di-O-octadecyloxypropyl
succinimidyl butanedioate[11] in the presence of triethylamine in dimethyl sulfoxide at
60°C. The desired racemic neoglycolipid 17 was obtained in 60% yield after puri-
1
fication by reverse phase column chromatography. The H NMR spectrum contained
signals corresponding to the oligosaccharide portion, two anomeric doublets at 4.52 and
4.38 ppm, and the signal for the equatorial H-3 of sialic acid at 2.61 ppm. The
characteristic pattern of 2,3-di-O-octadecyloxypropyl butanedioate at 1.30 and 0.90
ppm was also present.
In a preliminary experiment compounds 9 and 17 bind very well to ELISA plates.
The results of their recognition by anti-GM3 antibodies will be published in due course.

Synthesis of Neoglycolipid Analogues
399
Scheme 2. Synthesis of trisaccharide NeuAc a(2-3)-Gal b (1-3) Glc neoglycolipid analogue 17.

400
Gonza´lez Nu´n˜ez et al.
EXPERIMENTAL
General procedures. Optical rotations were measured at 25°C with a Polamat A
automatic polarimeter, using a 5-cm 5-mL cell. NMR spectra were recorded at 25°C
with a BRUKER AC-250F spectrometer. Chemical shifts (d) are given in ppm relative
1
to the signal for internal tetramethylsilane for H NMR spectra and indirectly to CDCl3,
d 77.03 for 13C NMR spectra. Assignments were performed on the basis of homo-and
heteronuclear correlation experiments. The following notation was used to define the
NMR signals, when more than one monosaccharide is present in the molecule: ’ for
galactose and @ for the sialyl moiety.
All compounds were purified by column chromatography on Kieselgel 60 or
reverse phase C18 silica gel and fractions were monitored by TLC on Kieselgel 60 F254
(Merck). Detection was effected by charring with a 5% solution of concentrated
sulfuric acid in ethanol after examination under UV light. Evaporations were conducted
under reduced pressure at 40°C (bath).
5-Azido-3-oxa-1-pentanol (1). Compound 1 was synthesized following a pub-
lished procedure.[18]
5-Azido-3-oxapentyl 2,3,4,6-tetra-O-acetyl- -D-galactopyranoside (2). A solu-
tion of 1 (1.72 g, 13.1 mmol) and 2,3,4,6-tetra-O-acetyl a-D-galactopyranosyl bromide
(5.85 g, 13.7 mmol) in anhydrous acetonitrile (10 mL) was stirred in the presence of
˚
molecular sieves (4A, 5.85 g) at rt for 5 min. Then mercury(II) cyanide (3.56 g, 14.1
mmol) was added. After 12 h, the mixture was filtered and the filtrate concentrated
under reduced pressure. The syrup was dissolved in dichloromethane (50 mL) and the
solution was successively washed with 10% KI (15 mL ꢁ 2), M NaHCO3 (15 mL) and
water (15 mL), dried (Na2SO4) and concentrated. The residue was purified by column
chromatography (hexane/ethyl acetate 5:1 v/v) to give 2 (5.15 g, 83%); [a]D + 33° (c
1
1.0, chloroform); H NMR (CDCl3) d 5.40 (m, 1H, H-4’), 5.21 (dd, 1H, J2,3 = 9.6 HZ,
H-2’), 5.05 (dd, 1H, J3’,4’ = 3.3 Hz, H-3’), 4.59 (d, 1H, J1’,2’ = 7.7 Hz, H-1’), 4.19 (m,
2H, H-5’, H-6’a), 3.89 (m, 1H, H-6’b), 3.8–3.6 (m, 6H, CH2O), 3.37 (t, 2H, CH2N3),
2.18, 2.15, 2.05, 1.98 (4s,CH3); 13C NMR (CDCl3) d 169.8–168.9 (C = 0), 100.7 (C-1),
70.6 (C-3), 70.0 (C-2), 69.9 (C-4), 69.0 (CH2O), 68.9 (CH2O), 68.7 (CH2O), 66.6 (C-
5), 61.2 (C-6), 50.7 (CH2-N3), 20.6–20.4 (CH3).
Anal. Calcd for C18H27O11N3 (461.2): C, 46.85; H, 5.90; N, 9.11. Found: C, 46.99;
H, 5.95; N 9.00.
5-Azido-3-oxapentyl 3,4-O-isopropylidene- -D-galactopyranoside (3). To a
solution of 2 (5.15 g, 11.2 mmol) in methanol (50 mL) was added sodium methoxide
(200 mg) and the reaction was stirred for 1 h at rt. The reaction mixture was
neutralized with Dowex-50 (H+) resin, filtered and the filtrate concentrated. The syrup
was dissolved in 2,2-dimethoxypropane (184 mL) containing p-toluenesulfonic acid
(430 mg). The mixture was stirred for 48 h. Triethylamine (0.5 mL) was then added
and the reaction mixture was stirred for 15 min. The mixture was concentrated to
dryness and coevaporated with toluene to remove traces of triethylamine. A solution of
the crude product in 10:1 MeOH-H2O (278 mL) was boiled under reflux for 3 h. The
solvents were evaporated and the residue was purified by flash chromatography (ethyl

Synthesis of Neoglycolipid Analogues
401
1
acetate/methanol 20:1 v/v) to give 3 (2.6 g, 70%); [a]D + 23° (c 1.0, chloroform); H
NMR (D2O) d 4.32 (d, 1H, J1’,2 7.6 Hz, H-1), 4.15–3.80 (m, 6H), 3.80–3.52 (m, 6H,
CH2O), 3.40 (t, 2H, CH2-N3), 1.55, 1.30 (2s, 6H, CH3); 13C NMR (CDCl3) d 110.0
[C(CH3)2], 102.3 (C-1), 78.7 (C-3), 73.5 (C-4), 73.3 (C-5), 70.1, 69.6 (CH2O), 68.3 (C-
2), 61.9 (C-6), 50.3 (CH2-N3), 27.8, 26.1 (CH3).
Anal. Calcd for C13H23O7N3 (333.3): C, 46.83; H, 6.96; N, 12.61. Found: C, 46.98;
H, 7.01; N, 12.51.
5-Azido-3-oxapentyl 2,6-di-O-benzyl- -D-galactopyranoside (4). Compound 3
(2.6 g, 7.8 mmol) was dissolved in N,N-dimethylformamide (17 mL) and sodium
hydride (1.25 g, 31.2 mmol) was added at 0°C. After 30 min, benzyl chloride (3.6
mL, 31.2 mmol) was added at 0°C and the mixture was stirred for an additional 24
h at 25°C. Methanol was then added to destroy the excess sodium hydride and the
mixture was concentrated. The resulting syrup was dissolved in dichloromethane (50
mL) and washed with water (15 mL), dried (Na2SO4) and the organic solvent
evaporated. The residue was dissolved in 80% acetic acid solution (130 mL) and was
boiled under reflux for 3 h. The mixture was concentrated to dryness and coeva-
porated with toluene. The residue was purified by flash chromatography (toluene/
1
acetone 8:1 v/v) to give 4 (1.56 g, 65%): [a]D + 13° (c 1.0, chloroform); H NMR
(CDCl3) d 7.45–7.20 (m, 10H, Ph), 4.98 (d, 1H, J1’,2’7.8 Hz, H-1), 4.55 (AB, 2H,
JAB = 12.1 Hz, CH2Ph), 4.52 (s, 2H, CH2Ph), 4.05–3.75 (m, 6H), 3.70–3.51 (CH2O),
3.35 (t, 2H, CH2-N3); 13C NMR (CDCl3) d 103.8 (C-1), 78.9 (C-2), 75.0 (C-5), 74.8
(CH2Ph), 74.1 (CH2Ph), 71.2 (CH2O), 70.9, 70.0 (C-3, C-4), 69.2, 69.0 (CH2O), 68.5
(C-6), 50.1 (CH2-N3).
Anal. Calcd for C24H31O7N3 (473.5): C, 60.88; H, 6.60; N, 8.87. Found: C, 61.04;
H, 6.66; N, 8.90.
Methyl (phenyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-thio-D-glycero-
-D-galacto-2-nonulopyranosid) onate (5). Compound 5 was synthesized following a
published procedure.[13,19]
5-Azido-3-oxapentyl 3-O-[methyl (5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-
dideoxy-D-glycero- -D-galacto-2-nonulopyranosyl) onate]-2,6-di-O-benzyl- -D-galac-
topyranoside (6). A solution of compounds 4 (0.5 g, 1.06 mmol) and 5 (0.74 g, 1.27
mmol) and N-iodosuccinimide (0.43 g, 1.9 mmol) in dry acetonitrile (10 mL) was
˚
stirred in the presence of molecular sieves (3A, 0.75 g) for 15 min. The solution was
cooled to ꢀ15°C under nitrogen. Trimethylsilyl triflate[14] (0.1 mL, 0.57 mmol) was
added and the mixture was stirred for 5 h. The mixture was diluted with di-
chloromethane (10 mL), filtered and the filtrate was successively washed with M
NaHCO3 (15 mL), aqueous sodium thiosulphate (15 mL) and water (15 mL), dried
(Na2SO4) and concentrated. The residue was purified by column chromatography
1
(toluene/acetone 10:1 v/v) to give 6 (0.35 g, 35%): [a]D + 12° (c 1.0, chloroform); H
NMR (CDCl3) d 7.45–7.11 (m, 10H, Ph), 5.40 (m, 1H, H-8@), 5.32 (m, 2H, H-7@, NH),
4.88 (m, 1H, H-4’), 4.75 (AB, 2H, JAB = 12.6 Hz, CH2Ph), 4.61 (s, 2H, CH2Ph), 4.51
(d, 1H, J1’,2’ = 7.5 Hz, H-1’), 4.15 (m, 2H, H-3’, H-5@), 3.85–3.52 (m, 10H, OCH3, H-
2’, CH2O), 3.39 (t, 2H, CH2-N3), 2.52 (dd, 1H, H-3eq@), 2.18–1.82 (5s, 15H, CH3).
Anal. Calcd for C44H58O19N4 (947.0): C, 55.79; H, 6.18. Found: C, 56.02; H, 6.22.

402
Gonza´lez Nu´n˜ez et al.
5-Azido-3-oxapentyl 3-O-(5-acetamido-3,5-dideoxy-D-glycero- -D-galacto-2-non-
ulopyranosylonic acid)-2,6-di-O-benzyl- -D-galactopyranoside (7). A solution of 6
(0.35 g, 0.46 mmol) in 0.74 M sodium methoxide (16.5 mL) was stirred at room
temperature. After 24 h, a 0.2 M KOH solution (4.6 mL) was added. The reaction
mixture was stirred for another 24 h, then neutralized with Dowex-50 (H+) resin,
filtered and the filtrate was concentrated. The residue was purified by reverse phase
column chromatography (H2O/MeOH 5:1 v/v) to give 7 (0.27 g, 95%): [a]D + 22° (c
1
1.0, methanol); H NMR (CD3OD) d 7.55–7.32 (m, 10H, Ph), 4.50 (d, 1H, J1’,2’ = 7.0
Hz, H-1’), 3.75 (m, 2H, CH2O), 3.65 (m, 1H, H-4@), 3.52–3.40 (m, 4H, CH2O), 3.28
(CH2-N3), 2.65 (dd, 1H, J3eq@,3ax@ = 12.9 Hz, J3eq@,4@ = 4.6 Hz, H-3@eq), 1.98 (s, 3H,
CH3), 1.85 (dd, 1H, J3ax@,4’ = 12.9 Hz, H-3ax@).
Anal. Calcd for C35H48O15N4 (764.8): C, 54.97; H, 6.33; N, 7.33. Found: C, 54.78;
H, 6.36; N, 7.37.
5-Amino-3-oxapentyl 3-O-(5-acetamido-3,5-dideoxy-D-glycero- -D-galacto-2-
nonulopyranosylonic acid)- -D-galactopyranoside (8). A solution of compound 7
(0.25 g, 0.34 mmol) in methanol (10 mL) and acetic acid (0.1 mL) was hydrogenated in
presence of 10% Pd-C (40 mg) for 12 h, then filtered and concentrated to give 8 (0.14g,
1
75% yield): [a]D + 8° (c 1.0, H2O); H NMR (D2O) d 4.49 (d, 1 H, H-1’), 3.15 (m, 2H,
CH2-NH2), 2.61 (dd, 1H, J3eq@,3ax@ = 12.9 Hz, J3eq@,4@ = 4.6 Hz, H-3@eq), 1.98 (s, 3H,
CH3), 1.75 (dd, 1H, J3ax@, 4’ = 12.9 Hz, H-3@ax).
5-(3-[(2,3-Dioctadecyloxypropyl)oxycarbonyl]propanoyl)-5-aza-3-oxapentyl 3-
O-[(5-acetamido-3,5-dideoxy-D-glycero- -D-galacto-2-nonulopyranosylonic acid)]- -
D-galactopyranoside (9). To a mixture of 8 (40 mg, 0.07 mmol) and 2,3-di-O-
octadecyloxypropyl succinimidyl butanedioate (56.7 mg, 0.07 mmol) was added
dimethyl sulfoxide (1 mL) and triethylamine (10 mL, 0.07 mmol) and the solution was
vigorously stirred at 60°C for 4 h. Then water (5 mL) was added and the resulting
suspension was centrifuged. The supernatant liquid was discarded and the pellet was
dissolved in chloroform/methanol 4:1 (v/v) and chromatographed using a small C 18
column eluting first with methanol-H2O 1:1 (v/v), then with methanol and methanol-
chloroform 3:1 (v/v). The fractions containing the desired compounds were concen-
1
trated: yield 50%; [a]D + 4° (c1, MeOH); H NMR (CD3OD-CDCl3) d 4.45 (d, 1 H,
J1,2 = 7.0 Hz, H-1’) 3.30 (m, 2 H, CH2NH2), 2.78 (t, 2H, CH2CO), 2.61 (m, 3 H,
CH2CO and H-3@eq), 1.89 (s, 3 H, Ac), 1.70 (m, 5 H, CH2CH2CO and H-3@ax), 1.30 (s,
60H, CH2), 0.90 (t, 6H, CH3).
Anal. Calcd for C64H117O20N2 (1234.6): C, 62.26; H, 9.55; N, 2.27. Found: C,
62.43; H, 9.60; N, 2.00.
4-O-(2,3,4,6-Tetra-O-acetyl- -D-galactopyranosyl)-2,3,6-tri-O-acetyl- -D-gluco-
pyranosyl trichloroacetimidate (10). 2-Aminoethanol (8 mL) was added to a
solution of lactose octaacetate (35.7 g, 52.6 mmol) in ethyl acetate (280 mL) and the
solution was stirred for 24 h, then was washed with aqueous NaCl and water until
neutral. The organic phase was dried (Na2SO4 anhydrous) and filtered, and the filtrate
was concentrated and dried in vacuo. The resulting syrup was stirred in the presence of
K2CO3 (17.6 g, 127.7 mmol) and trichloroacetonitrile (49.5 mL, 491.2 mmol) in dry
dichloromethane (100 mL) for 24 h. Then the suspension was filtered through Celite
and the filtrate concentrated in vacuo to give 10 (32.5 g, 85%): [a]D + 7.0° (c 1.0,

Synthesis of Neoglycolipid Analogues
403
1
chloroform); H NMR (CDCl3) d 8.65 (s, 1H, NH), 6.73 (d, 1H, J1,2 = 2.2 Hz, H-1),
6.13 (dd, 1H, J2’,3’ = 9.1 Hz, J3’,4’ = 9.0 Hz, H-3’), 5.78 (m, 2H, H-2’, H-4’), 5.59 (dd,
1H, J2.3 = 9.1 Hz, H-2), 5.43 (dd, 1H, J3.4 = 2.3 Hz, H-3), 4.96 (d, 1H, J1’,.2’ = 9.3 Hz,
H-1’), 4.62–3.71 (m, 6H, H-6a, H-6b, H-6’a, H-6’b, H-5, H-5’), 2.12–1.90 (7s, 21H,
CH3); 13C NMR (CDCl3) d 170.2–168.8 (C = 0), 160.5 (C-Cl3), 100.8 (C-1’), 92.5 (C-
1), 20.0–20.1 (CH3).
5-Azido-3-oxapentyl 4-O-(2,3,4,6-tetra-O-acetyl- -D-galactopyranosyl)-2,3,6-tri-
O-acetyl- -D-glucopyranoside (11). A solution of 10 (32.5 g, 41.7 mmol) and
5-azido-3-oxa-1-pentanol 1 (9.8 g, 75 mmol) in anhydrous dichloromethane (100 mL)
˚
was stirred in the presence of molecular sieves (4A, 37 g). The solution was cooled to
0°C under nitrogen and trimethylsilyl triflate (7.5 mL, 41.7 mmol) was added. The
mixture was stirred for 20 h, then diluted with dichloromethane (50 mL) and filtered.
The filtrate was washed successively with 1 M NaHCO3 (30 mL), and water (30 mL),
dried (Na2SO4) and concentrated. The residue was purified by column chromatography
1
(toluene/acetone 10:1 v/v) to give 11 (18.6g, 60%): [a]D + 26° (c 1.0, chloroform); H
NMR (CDCl3) d 5.81 (dd, 1H, J1’,2’ = 9.3 Hz, J2’,3’ = 9.1 Hz, H-2’), 5.72 (m, 2H, H-2,
H-4’), 5.48 (dd, 1H, J3,4 = 9.2 Hz, H-3), 5.37 (dd, 1H, J2’,3’ = 9.1 Hz, J3’,4’ = 2,3 Hz, H-
3’), 4.88 (d, 1H, J1,2 = 9.1 Hz, H-1), 4.81 (d, 1H, H-1’), 4.52 (m, 2H, H-6a, H-6’a), 4.26
(dd, 1H, J4,.5 = 9.2 Hz, H-4), 3.82–3.65 (m, 6H, CH2O), 3.35 (m, 2H, CH2N3), 2.15–
1.95 (7s, 21H, CH3); 13C NMR (CDCl3) d 170.2–168.8 (C = 0), 100.8 (C-1’), 100.6
(C-1), 77.2 (C-4), 71.4 (C-3’), 70.4 (C-2, C-3), 70.0 (CH2O), 69.9 (C-2’, C-4’), 69.0 (C-
5’), 68.9, 68.7 (CH2O), 68.4 (C-5), 62.1 (C-6), 61.2 (C-6’), 20.6–20.4 (CH3).
Anal. Calcd for C30H43O19N3 (749.7): C, 48.05; H, 5.78; N, 5.61. Found: C, 48.20;
H, 5.82; N, 5.64.
5-Azido-3-oxapentyl 4-O-(3,4-O-isopropylidene- -D-galactopyranosyl)-b-D-glu-
copyranoside (12). Compound 12 was synthesized following the procedure for
compound 3 using 11 (7.29 g, 9.73 mmol), as starting material in methanol (150 mL)
and 0.2 M sodium methoxide (80 mL). The syrup was dissolved in 2,2-dimethoxy-
propane (468 mL) containing p-toluenesulfonic acid (600 mg). The crude product was
dissolved in 10:1 MeOH-H2O (400 mL) and the residue was purified by flash
chromatography (ethyl acetate/methanol 20:1 v/v) to give 12 (5.5 g, 70%): [a]D + 21°
1
(c 1.0, chloroform); H NMR (CD3Cl) d 4.35 (d, 1H, J1’,2’ = 7.6 Hz, H-1’), 4.08 (d, 1H,
J1,2 = 7.6 Hz, H-1), 4.05–3.65 (m, 10H), 3.65–3.50 (m, 6H, CH2O), 3.41 (t, 2H,
CH2N3), 1.55, 1.32 (2s, 6H, CH3); 13C NMR (CDCl3) d 110.2 [C(CH3)2], 102.3 (C-1’),
101.0 (C-1), 78.7 (C-3’), 74,8 (C-3), 74.5 (C-4), 73.5 (C-4’), 73.3 (C-5’), 72.3, 69.9
(CH2O), 68.6, 68.3 (C-2’, C-2), 62.0, 61.9 (C-6’, C-6), 50.3 (CH2N3), 27.9–26.1 (CH3).
Anal. Calcd for C19H33O12N3 (495.5): C, 46.04; H, 6.72; N, 8.48. Found: C, 46.15;
H, 6.77; N, 8.51.
5-Azido-3-oxapentyl 4-O-(2,6-di-O-benzyl- -D-galactopyranosyl)-2,3,6-tri-O-
benzyl- -D-glucopyranoside (13). Compound 13 was synthesized following the
procedure for compound 4 using 12 (0.6 g, 1.21 mmol) in N,N-dimethylformamide (6
mL), sodium hydride (0.97 g, 24.2 mmol) and benzyl chloride (2.8 mL, 24.2 mmol).
The resulting syrup was dissolved in dichloromethane (60 mL) and washed with water
(20 mL), dried (Na2SO4) and evaporated. The residue was dissolved in 80% acetic acid
aqueous solution (60 mL). The residue was purified by flash chromatography (toluene/

404
Gonza´lez Nu´n˜ez et al.
1
acetone 10:1 v/v) to give 13 (0.59 g, 60%): [a]D + 11° (c 1.0, chloroform); H NMR
(CDCl3) d 7.38–7.20 (m, 25H, Ph), 4.65 (d, 1H, J1’,2’ = 8.1 Hz, H-1’), 4.60 (d, 1H,
J1,2 = 7.8 Hz, H-1), 4.13 (dd, 1H, J4’,5’ = 1.4 Hz, H-4’), 4.11 (dd, 1H, J3’,4’ = 7.7 Hz, H-
3’), 3.86 (dd, 1H, J4,5 = 9.7 Hz, H-4) 3.84 (m, 1H, J5’,6’a = 7.1 Hz, J5’,6’b = 5 Hz, H-5’),
3.76 (AB, 2H, 6a, 6b), 3.72–3.63 (m, 6H, CH2O), 3.62 (dd, 1H, H-6’b), 3.60 (dd, 1H,
J3,4 = 9.3 Hz, H-3), 3.59 (m, 1H, H-5), 3.47 (dd, 1H, J6’a,6’b = 10.3 Hz, H-6’a), 3.40
(dd, 1H, J2,3 = 9.1 Hz, H-2), 3.32 (dd, 1H, J2’,3’ = 6.3 Hz, H-2’), 3.25 (t, 2H, CH2N3);
13C NMR (CDCl3) d 138.8–138.2 (Ph), 128.2–126.7 (Ph), 103.5 (C-1’), 102.4 (C-1),
82.4, 81.3, 79.8 (C-2, C-3, C-2’), 78.4 (C-4), 75.5, 74.2, 74.0, 73.9, 70.2 (CH2Ph), 73.4,
72.4, 72.3, 71.6 (C-5, C-3’, C-4’, C-5’), 68.7 (C-6’), 67.9 (C-6), 50.4 (CH2N3).
Anal. Calcd for C51H57O12N3 (903.4): C, 67.74; H, 6.36; N, 4.65. Found: C, 67.86;
H, 6.42; N, 4.50.
5-Azido-3-oxapentyl 4-O-{3-O-[methyl (5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-
dideoxy-D-glycero- -D-galacto-2-nonulopyranosyl) onate]-(2,6-di-O-benzyl- -D-
galactopyranosyl)}-2,3,6-tri-O-benzyl- -D-glucopyranoside (14). Compound 14
was synthesized following the procedure for compound 6 using 13 (0.06 g, 0.07
mmol), sialyl donor 5 (0.05 g, 0.08 mmol), N-iodosuccinimide (0.03 g, 0.12 mmol) in
˚
dry acetonitrile (1 mL), molecular sieves (3A, 0.05 g) and trimethylsilyl triflate (2 mL,
0.012 mmol). The residue was purified by flash chromatography (toluene/acetone 10:1
1
v/v) to give 14 (0.04 g, 25%): [a]D + 9° (c 1.0, chloroform); H NMR (CDCl3) d 7.40–
7.15 (m, 25H, Ph), 5.75 (m, 1H, H-7@), 5.50 (m, 1H, H-8@), 5.32 (m, 1H, H-4@), 4.97
(dd, 2H, JAB = 12.5 Hz, CH2Ph), 4.87 (t, 2H, J = 10.4Hz, CH2Ph), 4.74 (dd, 2H,
JAB = 10Hz, CH2Ph), 4.69 (bb, 1H, NH), 4.62 (dd, 2H, JAB = 12.5 Hz, CH2Ph), 4.56
(d, 1H, J1’,2’ = 7.3 Hz, H-1’), 4.42 (m, 3H, CH2Ph, H-1), 4.25 (m, 1H, H-5@), 3.81 (s,
3H, OCH3), 3.76–3.60 (m, 6H, CH2O), 3.28 (t, 2H, CH2-N3), 2.55 (dd, 1H,
J3@eq,3@ax = 12.5 Hz, J3@eq,4@ = 4.6 Hz, H-3eq@), 2.20–1.88 (m, 13H, 4CH3, H-3ax@).
Anal. Calcd for C71H86O29N4 (1459.5): C, 58.43; H, 5.94; N, 3.84. Found: C,
58.57; H, 6.00; N, 3.86.
5-Azido-3-oxapentyl 4-O-[3-O-(5-acetamido-3,5-dideoxy-D-glycero-D-galacto-2-
nonulopyranosylonic acid)-2,6-di-O-benzyl- -D-galactopyranosyl]-2,3,6-tri-O-ben-
zyl- -D-glucopyranoside (15). Compound 15 was synthesized following the proce-
dure for compound 7 using 14 (0.04 g, 0.027 mmol) in 0.74 M sodium methoxide (2
mL) and 0.2 M KOH solution (0.5 mL). The residue was purified by column reverse
phase chromatography (H2O/MeOH 5:1 v/v) to give 7 (0.03 g, 92%): [a]D + 12° (c 1.0,
1
chloroform); H NMR (CD3OD) d 7.45–7.11 (m, 25H, Ph), 4.51 (d, 1H, J1’,2’ = 7.8 Hz,
H-1’), 4.45 (m, 3H, CH2Ph, H-1), 4.18 (d, 2H, JAB = 12.8 Hz, CH2Ph), 3.28 (t, 2H,
CH2-N3), 2.65 (dd, 1H, J3@eq,3@ax = 13.2 Hz, J3@eq,4@ = 4.15 Hz, H-3@eq), 1.95 (m, 4H,
H-3@ax, CH3).
Anal. Calcd for C62H76O20N4 (1197.3): C, 62.18; H, 6.40; N, 4.68. Found: C,
62.33; H, 6.36; N, 4.72.
5-Amino-3-oxapentyl 4-O-[3-O-(5-acetamido-3,5-dideoxy-D-glycero- -D-galacto-
2-nonulopyranosylonic acid)- -D-galactopyranosyl)]- -D-glucopyranoside (16).
solution of compound 15 (30 mg, 0.025 mmol) in MeOH (2 mL) and CH3COOH (0.01
A
mL) was hydrogenated in the presence of 10% Pd-C (25 mg) for 12 h, then filtered and

Synthesis of Neoglycolipid Analogues
405
1
concentrated to give 16 in a 80% yield. [a]D+ 4° (c 1.0, MeOH); H NMR (D2O) d
4.49 (d, 1H, J1,2 = 7.8 Hz, H-1), 4.40 (d, 1H, J1’,2’ = 8.0 Hz, H-1’), 3.18 (m, 2 H,
CH2NH2), 2.61 (dd, 1 H, J3@eq,4@ = 3.2, J3@eq,3@ax = 13.0 Hz, H-3@eq), 1.89 (s, 3 H, Ac),
1.75 (dd, 1 H, J3@ax,4@ = 13.0 Hz, H-3@ax).
Anal. Calcd for C27H48O20N2 (720.7): C, 45.00; H, 6.71; N, 3.89. Found: C, 45.38;
H, 6.75; N, 4.02.
5-(3-[(2,3-Dioctadecyloxypropyl)oxycarbonyl]propanoyl)-5-aza-3-oxapentyl 4-O-
[3-O-(5-acetamido-3,5-dideoxy-D-glycero- -D-galacto-2-nonulopyranosylonic acid)- -
D-galactopyranosyl]- -D-glucopyranoside (17). To a mixture of 16 (35 mg, 0.025 mmol)
and 2,3-di-O-octadecyloxypropyl succinimidyl butanedioate (19.8 mg, 0.025 mmol) was
addeddimethylsulfoxide(1mL) and triethylamine(3.6mL, 0.025mmol) andthesolution was
vigorously stirred at 60°C for 4 h. Then water (5 mL) was added and the resulting suspension
was centrifuged. The supernatant liquid was discarded and the pellet was dissolved in
chloroform/methanol 4:1 (v/v) and chromatographed in a small C18 column using methanol-
H2O, methanol and methanol-chloroform as eluent. The fractions containing the desired
compounds were concentrated: yield 60%; [a]D + 4° (c 1.0, MeOH); 1H NMR (CD3OD) d
4.52 (d, 1H, J1,2 = 7.5 Hz, H-1), 4.38 (d, 1H, J1’,2’ = 7.0 Hz, H-1’), 3.30 (m, 2 H, CH2NH2),
2.78 (t, 2H, CH2CO) 2.61 (m, 3 H, CH2CO, H-3@eq), 1.89 (s, 3 H, Ac), 1.60 (m, 5 H,
CH2CH2CO, H-3@ax), 1.30 (s, 60H, CH2), 0.90 (t, 6H, CH3).
Anal. Calcd for C70H127O25N2 (1396.8): C, 60.19; H, 9.16; N, 2.01. Found: C,
59.87; H, 9.29; N, 2.04.
ACKNOWLEDGMENT
Support of this work by the Volkswagen Foundation is gratefully acknowledged.
REFERENCES
1. Gonza´lez Nu´n˜ez, F.; Campos Valde´s, M.; Schmidt, R.R.; Verez Bencomo, V.
Proceeding of the Xth European Carbohydrate Symposium, Lisbon, Portugal, Sept.
2–7, 2001; Rauter, A.P., Ed.; Abstract 265.
2. Livingston, P.O.; Natoli, E.J.; Calves, M.J.; Stockert, E.; Oettgen, H.F.; Old, L.J.
Vaccines containing purified GM2 ganglioside elicit GM2 antibodies in melanoma
patients. Proc. Natl. Acad. Sci. U. S. A. 1987, 84 (9), 2911–2915.
3. Livingston, P.O.; Wong, G.Y.; Adluri, S.; Tao, Y.; Padavan, M.; Parente, R.;
Hanlon, C.; Calves, M.J.; Helling, F.; Ritter, G. Improved survival in stage III
melanoma patients with GM2 antibodies: a randomized trial of adjuvant vaccination
with GM2 ganglioside. J. Clin. Oncol. 1994, 12 (5), 1036–1044.
4. Livingston, P.O.; Ritter, G.; Srivastava, P.; Padavan, M.; Calves, M.J.; Oettgen,
H.F.; Old, L.J. Characterization of IgG and IgM antibodies induced in melanoma
patients by immunization with purified GM2 ganglioside. Cancer Res. 1989, 49,
7045–7050.
5. Estevez, F.; Carr, A.; Solo´rzano, L.; Valiente, O.; Mesa, C.; Barroso, O.; Sierra,
G.V.; Fernandez, L.E. Enhancement of the immune response to poorly

406
Gonza´lez Nu´n˜ez et al.
immunogenic gangliosides alters incorporation into very small size proteoliposomes
(VSSP). Vaccine 2000, 18, 190–197.
6. Kawano, T.; Cui, J.; Koezuka, Y.; Toura, I.; Kaneko, Y.; Motoki, K.; Ueno, H.;
Nakagawa, R.; Sato, H.; Kondo, E.; Koseki, H.; Taniguchi, M. CD1d-restricted and
TCR-mediated activation of Va14 NKT cells by glycosylceramides. Science 1997,
278, 1626–1629.
7. Fiedler, T.; Walter, W.; Reichert, T.E.; Meurer, M.T. Regulation of CD1d
expression by murine tumor cells: escape from immuno surveillance or arternative
target molecule. Int. J. Cancer 2002, 98 (3), 389–397.
8. Kirkwood, J.M.; Ibrahim, J.G.; Sosman, J.A.; Sondak, V.K.; Agarwala, S.S.;
Ernstoff, M.S.; Rao, U.J. High-dose interferon a-2b significantly prolongs relapse-
free and overall survival compared with the GM2-KLH/QS-21 vaccine in patients
with resected stage IIB/III melanoma: results on inter group trial E1694/S9512C
509801. J. Clin. Oncol. 2001, 19 (9), 2370–2380.
9. Nores, G.; Dohi, T.; Taniguchi, M.; Hakomori, S.I. Density-dependent recognition
of cell surface GM3 by a certain anti-melanoma antibody, and GM3 lactone as a
possible immunogen: requirement for tumor-associated antigen and immunogen. J.
Immunol. 1987, 139(9), 3171–3176.
10. Valiente, O.; Mesa, C.; Ferna´ndez, V.; Ve´rez, V.; Schmidt, R.R.; Ferna´ndez, L.E. 3’-
Sialyllactose neoglycoconjugates are not able to provide an immunological response
against N-acetyl GM3 ganglioside in mice and chickens. Biotecnol. Appl. in press.
11. Figueroa-Perez, S.; Verez-Bencomo, V. Synthesis of neoglycolipids containing
oligosaccharides based on 3,6-branched-a-D-mannopyranosides as the carbohydrate
moieties. J. Carbohydr. Chem. 1998, 17, 851–868.
12. Catelani, G.; Colonna, F.; Marra, A. An improved preparation of 3,4-O-
isopropylidene derivatives of a- and b-D-galactopyranosides. Carbohydr. Res.
1988, 182, 297–300.
13. Marra, A.; Sinay¨, P. Stereoselective synthesis of 2-thioglycosides of neuraminic
acid. Carbohydr. Res. 1989, 187, 35–42.
14. Kiso, M.; Ishida, H.; Ito, H. Special problems in glycosylations reactions:
sialydations. In Carbohydrates, Chemistry and Biology; Ernst, B., Hart, G.W.,
Sinay, P., Eds.; Wiley-VCH: New York, 2000; 346–361.
15. Schmidt, R.R.; Michel, J.; Roos, M. Glycosyl imidates, 12. Direct synthesis of O-a-
and O-b-glycosyl imidates. Liebigs Ann. Chem. 1984, 1343–1357.
16. Grynnkiewicz, G.; Fokt, I.; Szeja, W.; Fitak, H. Chemoselective deprotection of
1-O-acyl sugar derivatives. J. Chem. Res. (S) 1989, 152–153.
17. Hasegawa, A.; Nagahama, T.; Ohki, H.; Kiso, M. Synthetic studies on sialo-
glycoconjugates 41: a facile total synthesis of ganglioside GM2. J. Carbohydr.
Chem. 1992, 11 (6), 699–714.
18. Ferna´ndez-Santana, V.; Gonza´lez-Lio, R.; Sarracent-Pe´rez, J.; Verez-Bencomo, V.
Conjugation of 5-azido-3-oxapentyl glycosides with thiolated proteins through the
use of thiophilic amide derivatives. Glycoconj. J. 1998, 15, 549–553.
19. Kirchner, E.; Thiem, F.; Dernick, R.; Heukeshoven, J.; Thiem, J. Studies on the
glycosylation of N-acetylneuraminic acid. J. Carbohydr. Chem. 1988, 17, 453–486.
Received September 10, 2002
Accepted June 10, 2003