Synthesis and conjugation of a sulfated disaccharide involved in the aggregation process of the marine sponge Microciona prolifera

Article abstract of DOI:10.1016/S0957-4166(99)00585-6

The synthesis is reported of allyl (sodium 2-acetamido-2-deoxy-β-D-glucopyranosyl 3-sulfate)-(1→3)-α-L-fucopyranoside which represents an oligosaccharide fragment of the aggregation factor of the marine sponge Microciona prolifera. The title compound was

Full text of DOI:10.1016/S0957-4166(99)00585-6

TETRAHEDRON:
ASYMMETRY
Pergamon
Tetrahedron: Asymmetry 11 (2000) 539–547
Synthesis and conjugation of a sulfated disaccharide involved in
the aggregation process of the marine sponge Microciona
prolifera
Henricus J. Vermeer, Johannis P. Kamerling ∗ and Johannes F. G. Vliegenthart
Bijvoet Center, Department of Bio-Organic Chemistry, Utrecht University, PO Box 80075, NL-3508 TB Utrecht, The
Netherlands
Received 1 November 1999; accepted 13 December 1999
Abstract
The synthesis is reported of allyl (sodium 2-acetamido-2-deoxy-β-D-glucopyranosyl 3-sulfate)-(1→3)-α-L-
fucopyranoside which represents an oligosaccharide fragment of the aggregation factor of the marine sponge
Microciona prolifera. The title compound was obtained by coupling of 3-O-allyloxycarbonyl-2-deoxy-4,6-
O-isopropylidene-2-phthalimido-β-D-glucopyranosyl trichloroacetimidate with allyl 2,4-di-O-benzoyl-α-L-
fucopyranoside, followed by de-isopropylidenation, acetylation, de-allyloxycarbonylation, sulfation, de-acylation,
and finally N-acetylation. The allyl glycoside was eventually converted into a 3-(2-aminoethylthio)propyl
glycoside and then coupled to bovine serum albumin (BSA) using diethyl squarate as the bivalent linker, yielding
8 hapten molecules per molecule of BSA. © 2000 Elsevier Science Ltd. All rights reserved.
1. Introduction
The Ca2+-dependent reaggregation of dissociated cells of the marine sponge Microciona prolifera
is species-specific1 and occurs through proteoglycans.2 Cell surfaces are covered with large adhesion
proteoglycans, and multiple low affinity carbohydrate–carbohydrate interactions are responsible for the
interaction between the sponge cells.3 It is suggested that similar interactions occur during embryonic
development in higher animals and human beings, as well as between tumor cells. Characterization
of the aggregation factors revealed the existence of pyruvylated and sulfated glycans as part of these
proteoglycans.4,5 In order to develop a model system for mimicking cellular interactions, a programme
was started in our group to synthesize some of these aggregation factors.
Here, we present the synthesis of β-D-GlcpNAc3S-(1→3)-α-L-Fucp as its allyl glycoside 10 (a
preliminary report of 10 has appeared)6 and the subsequent conversion into a BSA-conjugate. During
∗
Corresponding author.
0957-4166/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(99)00585-6
tetasy 3219

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the preparation of this manuscript a study was published dealing with the synthesis of the corresponding
methyl glycoside.7 It should be noted that the anomeric configuration of fucose in the proteoglycan has
not yet been determined,5 so that the choice of the α-glycoside is arbitrary. Multivalent exposition of
the synthetic material via a spacer on the surface of a suitable carrier can resemble the natural sponge
surfaces.
2. Results and discussion
For the synthesis of 10 the acceptor allyl 2,4-di-O-benzoyl-α-L-fucopyranoside 4 and the donor
3-O-allyloxycarbonyl-2-deoxy-4,6-O-isopropylidene-2-phthalimido-β-D-glucopyranosyl trichloroacet-
imidate 58 were selected as precursors. The allyloxycarbonyl group at O-3 of 5 was chosen in view
of the sulfation of this position in a later stage of the synthesis. In the synthesis of 4, allyl α-L-
fucopyranoside9 was isopropylidenated at O-3 and O-4 with α,α-dimethoxypropane in the presence of
a catalytic amount of p-toluenesulfonic acid to give 1 (80%). Subsequent benzoylation with benzoyl
chloride in pyridine in the presence of a catalytic amount of 4-dimethylaminopyridine (→2, 96%),
followed by de-isopropylidenation using trifluoroacetic acid and water in dichloromethane, yielded 3
(Scheme 1). Compound 3 was selectively protected at O-4 by reaction with trimethylorthobenzoate and
p-toluenesulfonic acid, after which the resulting 3,4-dioxolane-type acetal was opened by treatment of
the residue with acetic acid–water10 (→4, 71%).
Scheme 1.
Coupling of 4 with 5 in dichloromethane, using trimethylsilyl trifluoromethanesulfonate as a catalyst,
gave disaccharide derivative 6 (84%). De-isopropylidenation of 6 with aqueous trifluoroacetic acid
in dichloromethane, followed by conventional acetylation (→7, 98%), and de-allyloxycarbonylation
using tetrakis(triphenylphosphine)palladium in tetrahydrofuran and morpholine afforded 8 (97%).11,12
Sulfation of O-3 was performed using sulfur trioxide trimethylamine complex, and the product was
purified on Sephadex LH-20, followed by silica gel chromatography (63%). De-phthaloylation/de-
acylation was performed by stirring 9 with methylamine13 in ethanol (8 days), followed by selective
N-acetylation (0°C) using acetic anhydride in methanol, affording disaccharide 10 (61%). The presence

H. J. Vermeer et al. / Tetrahedron: Asymmetry 11 (2000) 539–547
541
of the sulfate group on C-30 was established by 1H NMR analysis (GlcNAc H-3, δ 4.414). Other chemical
shifts were in good agreement with data of Spillmann et al.5
Michael addition of 10 with 2-aminoethanethiol (cysteamine) under UV-irradiation afforded the
corresponding 3-(2-aminoethylthio)propyl glycoside14,15 (→11, 86%). The structure of 11 was unam-
1
biguously ascertained by FAB mass spectrometry and H (1D and 2D TOCSY) NMR analysis (Table
1).
Table 1
500 MHz 1H NMR data (1D and 2D TOCSY) of compound 11
The oligosaccharide amine 11 was treated with 0.95 equivalents of diethyl squarate in ethanol and
sodium phosphate buffer, pH 7.0 (1:1 v/v).16,17 The resulting oligosaccharide–squarate adduct 12 was
left to react with bovine serum albumin (BSA) in 0.1 M sodium bicarbonate buffer (pH 9.0) at room
temperature for 5 days (Scheme 2).
Scheme 2.
The degree of incorporation of oligosaccharide onto the protein was determined using matrix assisted
laser desorption ionization/time of flight (MALDI-TOF) mass spectrometry (MS), revealing a distribu-
tion of 7–10 hapten molecules per molecule of BSA. Monosaccharide analysis18 showed an achieved
incorporation of 52% of 12 on BSA (n=8). In conclusion, the conjugation method was successfully
applied with only 1 mg of 12, revealing its efficiency even on small scales. The neoglycoconjugate

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H. J. Vermeer et al. / Tetrahedron: Asymmetry 11 (2000) 539–547
will be used to test the Ca2+-dependent self-binding capacity of the sulfated disaccharide in biological
experiments.
3. Experimental
3.1. General procedures
Reactions were monitored by TLC on Kieselgel 60 F254 (Merck); compounds were visualized under
UV light and by charring with aq. 50% H2SO4. Column chromatography was performed on Kieselgel 60
(Merck, <230 mesh). Solvents were evaporated under reduced pressure at 40°C (bath). Crystalline bovine
serum albumin (BSA) was obtained from Bayer Corporation. A Hanovia UV lamp (50 W) was used
for irradiations in photochemical reactions. Optical rotations were determined for solutions in CHCl3,
unless otherwise stated, at 20°C with a Perkin–Elmer 241 polarimeter using a 10 cm 1 mL cell. 1H NMR
spectra were recorded at 25°C with Bruker AC 300 or Bruker AMX 500 spectrometers. Two-dimensional
1
1
double-quantum filtered H–1H correlated spectra (2D DQF H–1H COSY spectra with a mixing time
1
of 100 ms) were recorded at 300 K using a Bruker AMX 500 spectrometer. H chemical shifts (δ) are
given in ppm relative to the signal for internal Me4Si for solutions in CDCl3 or CD3OD, or by reference
to acetone (δ 2.225) for solutions in D2O. J-Values are given in Hertz. Fast-atom-bombardment mass
spectrometry (FABMS) was performed on a JEOL JMS SX/SX 102A four-sector mass spectrometer
using m-nitrobenzyl alcohol or glycerol as the matrix, operated at 10 kV accelerating voltage, equipped
with a JEOL MS-FAB 10 D FAB gun operated at 10 mA emission current, producing a beam of 6
keV Xenon atoms. MALDI-TOF mass spectra were recorded on a Voyager-DE (PerSeptive Biosystems)
instrument using α-cyano-4-hydroxycinnamic acid as the matrix. Proteins were analyzed in the linear
mode with the acceleration voltage kept at 22.5 kV, and samples were prepared as follows: first, 1 µL
matrix solution was placed on the sample plate. After evaporation of the solvent, 1 µL sample solution
(7.5 pmol mL−1) in acetonitrile:water (1:1) acidified with 0.1% trifluoroacetic acid was applied to the
thin matrix film. Spectra were obtained by summing positive-ion signals of 183 to 188 laser shots. GLC
was performed using a CP-Sil5 CB WCOT fused-silica capillary column (25 m×0.34 mm, Chrompack).
3.2. Allyl 3,4-O-isopropylidene-α-L-fucopyranoside 1
To a solution of allyl α-L-fucopyranoside9 (1.0 g, 4.9 mmol) in dry DMF (17 mL) was added α,α-
dimethoxypropane (7.5 mL) and p-toluenesulfonic acid monohydrate (16.7 mg). The mixture was stirred
for 3 h at 20°C, when TLC (CH2Cl2:MeOH 97:3) showed a complete conversion into 1 (Rf=0.66). After
quenching with NEt3 (0.8 mL) and concentration, the residue was dissolved in CH2Cl2 (25 mL) and the
solution was extracted with aq. 5% NaCl (3×10 mL), dried (MgSO4), filtered, and concentrated. Column
chromatography (CH2Cl2:MeOH 98:2) of the residue gave 1, isolated as a colorless syrup (0.9 g, 80%);
[α]D= −120 (c=1); 1H NMR (300 MHz, CDCl3): δ 1.272 (d, 3H, J5,6=6.7 Hz, H-6,6,6), 1.313 and 1.470
(2 s, each 3H, C(CH3)2), 4.014 and 4.199 (2 m, each 1H, OCH2CH_CH2), 4.819 (d, 1H, J1,2=3.9 Hz, H-
1), 5.166 and 5.258 (2 m, each 1H, OCH2CH_CH2), 5.872 (m, 1H, OCH2CH_CH2); FABMS (positive):
calcd for C12H20O5 244.2, found m/z 245.1 ([M+H]+). Elemental analysis: found C, 58.74%; H, 8.35%;
calcd C, 59.00%; H, 8.25%.

H. J. Vermeer et al. / Tetrahedron: Asymmetry 11 (2000) 539–547
543
3.3. Allyl 2-O-benzoyl-3,4-O-isopropylidene-α-L-fucopyranoside 2
To a solution of 1 (0.92 g, 3.8 mmol) in pyridine (14 mL) were added benzoyl chloride (0.55 mL,
4.9 mmol) and a catalytic amount of 4-dimethylaminopyridine. The solution was stirred overnight, when
TLC (CH2Cl2:acetone 95:5) showed the complete disappearance of 1 and the formation of a new spot
(Rf=0.80). The mixture was diluted with CH2Cl2 (150 mL) and the solution was extracted with aq. 5%
NaHCO3 (50 mL). The organic phase was washed with aq. 5% NaHCO3 (50 mL) and water (50 mL),
dried (MgSO4), filtered, concentrated, and co-concentrated with toluene, EtOH and CH2Cl2 (each 3×75
mL). Column chromatography (CH2Cl2:acetone 98:2) of the residue gave 2, isolated as a colorless syrup
(1.28 g, 96%); [α]D= −110 (c=1); 1H NMR (300 MHz, CDCl3): δ 1.402 (d, 3H, J5,6=6.7 Hz, H-6,6,6),
1.364 and 1.568 (2 s, each 3H, C(CH3)2), 3.985 and 4.175 (2 m, each 1H, OCH2CH_CH2), 4.149 (dd,
1H, J3,4=5.4, J4,5=2.6 Hz, H-4), 4.520 (dd, 1H, J2,3=8.2 Hz, H-3), 5.074 (d, 1H, J1,2=3.6 Hz, H-1),
5.115 and 5.260 (2 m, each 1H, OCH2CH_CH2), 5.168 (dd, 1H, H-2), 5.825 (m, 1H, OCH2CH_CH2),
7.40–8.17 (m, 5H, Bz); FABMS (positive): calcd for C19H24O6 348.3, found m/z 349.1 ([M+H]+), 371.1
([M+Na]+).
3.4. Allyl 2,4-di-O-benzoyl-α-L-fucopyranoside 4
To a solution of 2 (1.25 g, 3.6 mmol) in CH2Cl2 (58 mL) were added CF3CO2H (3.4 mL, 44
mmol) and water (0.43 mL, 22.5 mmol). After 30 min TLC (CH2Cl2:acetone 9:1) indicated the de-
isopropylidenation to be completed, showing a new spot with Rf=0.12 (3). The mixture was concentrated
and toluene, EtOH, and CH2Cl2 (each 3×100 mL) were evaporated from the residue. To a solution
of 3 (1.02 g, 3.3 mmol) in dry CH2Cl2 (23 mL) were added trimethylorthobenzoate (3.2 mL, 18.9
mmol) and p-toluenesulfonic acid monohydrate (pH 3). The solution was stirred overnight, after which
TLC (CH2Cl2:acetone 9:1) showed the formation of a new product (Rf=0.88). Then, the solution was
concentrated to about 15 mL, and HOAc (8 mL) and water (15 mL) were added. The resulting mixture
was stirred for 15 min at 40°C, when TLC (CH2Cl2:acetone 9:1) showed the formation of 4 (Rf=0.72).
After cooling to room temperature, the mixture was diluted with EtOAc (200 mL), then washed with
aq. 10% NaHCO3 (100 mL) and water (50 mL), and the organic layer was dried (MgSO4), filtered,
concentrated, and co-concentrated with toluene, EtOH and CH2Cl2 (3×75 mL). Column chromatography
(CH2Cl2:acetone 95:5) of the residue gave 4, isolated as a white foam (0.96 g, 71%); [α]D= −160 (c=1);
1H NMR data (300 MHz, CDCl3): δ 1.224 (d, 3H, J5,6=6.6 Hz, H-6,6,6), 4.050 and 4.221 (2 m, each 1H,
OCH2CH_CH2), 4.267 (m, 1H, H-5), 4.518 (dd, 1H, J2,3=10.4, J3,4=3.6 Hz, H-3), 5.163 and 5.311 (2 m,
each 1H, OCH2CH_CH2), 5.249 (d, 1H, J1,2=3.7 Hz, H-1), 5.374 (dd, 1H, H-2), 5.553 (dd, 1H, J4,5=1.2
Hz, H-4), 5.873 (m, 1H, OCH2CH_CH2), 7.40–8.15 (m, 10H, 2 Bz). Elemental analysis for C23H24O7:
found C, 66.29%; H, 6.06%; calcd C, 66.98%; H, 5.87%.
3.5. Allyl (3-O-allyloxycarbonyl-2-deoxy-4,6-O-isopropylidene-2-phthalimido-β-D-glucopyranosyl)-
(1→3)-2,4-di-O-benzoyl-α-L-fucopyranoside 6
A
solution of 3-O-allyloxycarbonyl-2-deoxy-4,6-O-isopropylidene-2-phthalimido-β-D-gluco-
pyranosyl trichloroacetimidate8 (5; 0.45 g, 0.77 mmol) and 4 (0.24 g, 0.59 mmol) in dry CH2Cl2 (6.5
mL), containing molecular sieves (4 Å, 0.79 g), was stirred for 30 min under Ar. Then, trimethylsilyl
trifluoromethanesulfonate (4.9 µL, 26 µmol) was added, and the mixture was stirred for 10 min,
when TLC (n-hexane:EtOAc 3:2) showed the formation of a new spot (6; Rf=0.26). The reaction was
quenched by the addition of NEt3 until neutral pH, and the mixture was diluted with CH2Cl2 (50 mL),

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H. J. Vermeer et al. / Tetrahedron: Asymmetry 11 (2000) 539–547
filtered, washed with aq. 5% NaCl, dried (MgSO4), filtered, and concentrated. Column chromatography
(n-hexane:EtOAc 3:2) of the residue yielded 6, isolated as a colorless syrup (0.4 g, 84%); [α]D= −124
1
(c=0.5); H NMR (300 MHz, CDCl3): δ 1.052 (d, 3H, J5,6=6.5 0Hz, H-6,6,6), 1.353 and 1.364 (2 s,
0
0
0
0
each 3H, C(CH3)2), 4.095 (dd, 1H, J1 ,2 =8.3, J2 ,3 =10.2 Hz, H-2 ), 4.515 (dd, 1H, J2,3=10.3, J3,4=3.5
Hz, H-3), 4.896 and 4.988 (2 m, each 1H, C(O)OCH2CH_CH2), 5.142 and 5.323 (2 m, each 1H,
0
0
OCH2CH_CH2), 5.232 (d, 1H, J1,2=3.8 Hz, H-1), 5.342 (d, 1H, H-4), 5.373 (dd, 1H, J3 ,4 =9.0 Hz,
H-30), 5.392 (dd, 1H, H-2), 5.509 (m, 1H, C(O)OCH2CH_CH2), 5.587 (d, 1H, H-10), 5.844 (m, 1H,
OCH2CH_CH2), 7.23–8.16 (m, 14H, Phth and 2 Bz); FABMS (positive): calcd for C44H45O15N 827.4,
found m/z 828.2 ([M+H]+), 850.1 ([M+Na]+).
3.6. Allyl (4,6-di-O-acetyl-3-O-allyloxycarbonyl-2-deoxy-2-phthalimido-β-D-glucopyranosyl)-(1→3)-
2,4-di-O-benzoyl-α-L-fucopyranoside 7
To a solution of 6 (0.83 g, 1.02 mmol) in CH2Cl2 (15.8 mL) was added CF3CO2H (0.9 mL) and
water (0.1 mL). The mixture was stirred for 30 min when TLC (CH2Cl2:acetone 75:25) revealed the
de-isopropylidenation to be completed, showing a new spot at Rf=0.43. After concentration and co-
concentration with toluene, EtOH, and CH2Cl2 (each 3×50 mL), Ac2O (10 mL) and pyridine (10 mL)
were added, and the mixture was stirred overnight. Then, TLC (CH2Cl2:acetone 95:5) showed the
formation of 7 (Rf=0.54). After concentration and co-concentration with toluene, EtOH, and CH2Cl2
(each 3×50 mL), column chromatography (CH2Cl2:acetone 95:5) of the residue yielded 7, isolated as a
colorless syrup (0.64 g, 98%); [α]D= −120 (c=1); 1H NMR (300 MHz, CDCl3): δ 1.076 (d, 3H, J5,6=6.6
0
0
0
0
0
Hz, H-6,6,6), 1.997 and 2.016 (2 s, each 3H, 2 Ac), 4.212 (dd, 1H, J1 ,2 =8.4, J2 ,3 =10.5 Hz, H-2 ),
4.588 (dd, 1H, J2,3=10.4, J3,4=3.4 Hz, H-3), 4.908 and 4.993 (2 m, each 1H, C(O)OCH2CH_CH2),
0
0
0
0
0
5.035 (dd, 1H, J3 ,4 =9.3, J4 ,5 =10.0 Hz, H-4 ), 5.108 and 5.257 (2 m, each 1H, OCH2CH_CH2),
5.249 (d, 1H, J1,2=3.7 Hz, H-1), 5.382 (dd, 1H, H-2), 5.407 (dd, 1H, J4,5<1 Hz, H-4), 5.500 (m,
1H, C(O)OCH2CH_CH2), 5.552 (dd, 1H, H-30), 5.585 (d, 1H, H-10), 5.812 (m, 1H, OCH2CH_CH2),
7.19–8.16 (m, 14H, Phth and 2 Bz); FABMS (positive): calcd for C45H45O17N 871.4, found m/z 872.2
([M+H]+), 894.1 ([M+Na]+). Elemental analysis: found C, 61.36%; H, 4.90%; calcd C, 61.98%; H,
5.20%.
3.7. Allyl (4,6-di-O-acetyl-2-deoxy-2-phthalimido-β-D-glucopyranosyl)-(1→3)-2,4-di-O-benzoyl-α-L-
fucopyranoside 8
To a solution of 7 (0.59 g, 0.68 mmol) in THF (9.8 mL) and morpholine (0.35 mL) was added
tetrakis(triphenylphosphine)palladium (114 mg). The mixture was stirred and boiled under reflux until
the de-allyloxycarbonylation was complete (TLC, CH2Cl2:acetone 9:1; new spot at Rf=0.50). Then, the
mixture was diluted with CH2Cl2 (50 mL), washed with aq. 5% NaCl (25 mL), dried (MgSO4), filtered,
and concentrated. Column chromatography (CH2Cl2:acetone 9:1) of the residue yielded 8, isolated as
1
a syrup (0.52 g, 97%); [α]D= −123 (c=1); H NMR (300 MHz, CDCl3): δ 1.079 (d, 3H, J5,6=6.5
0
0
0
0
0
Hz, H-6,6,6), 2.005 and 2.041 (2 s, each 3H, 2 Ac), 4.032 (dd, 1H, J1 ,2 =8.4, J2 ,3 =10.5 Hz, H-2 ),
0
0
0
0
0
4.593 (dd, 1H, J2,3=10.5, J3,4=3.4 Hz, H-3), 4.765 (t, 1H, J3 ,4 =J4 ,5 =9.6 Hz, H-4 ), 5.104 and 5.257
(2 m, each 1H, OCH2CH_CH2), 5.248 (d, 1H, J1,2=3.8 Hz, H-1), 5.379 (dd, 1H, H-2), 5.420 (dd, 1H,
J4,5=0.8 Hz, H-4), 5.458 (d, 1H, H-10), 5.812 (m, 1H, OCH2CH_CH2), 7.17–8.16 (m, 14H, Phth and
2 Bz); FABMS (positive): calcd for C41H41O15N 787.4, found m/z 788.2 ([M+H]+), 810.1 ([M+Na]+).
Elemental analysis: found C, 61.78%; H, 5.33%; calcd C, 62.51%; H, 5.25%.

H. J. Vermeer et al. / Tetrahedron: Asymmetry 11 (2000) 539–547
545
3.8. Allyl (sodium 4,6-di-O-acetyl-2-deoxy-2-phthalimido-β-D-glucopyranosyl 3-sulfate)-(1→3)-2,4-di-
O-benzoyl-α-L-fucopyranoside 9
A solution of 8 (0.46 g, 0.59 mmol) and SO3–NMe3 complex (3.26 g, 23.4 mmol) in abs. DMF
(27 mL) was stirred at 55°C overnight. Then, TLC (CH2Cl2:acetone 9:1) showed the sulfation to be
complete (new spot at Rf=0.29). After cooling to room temperature and quenching with MeOH, the
suspension was concentrated and co-concentrated with toluene, EtOH, and CH2Cl2 (each 3×60 mL).
A solution of the residue in MeOH was stirred for 30 min with Dowex-Na+ resin. After filtration and
concentration, the residue was purified on Sephadex LH-20 (CH2Cl2:MeOH 1:1), followed by silica gel
column chromatography (CH2Cl2:acetone 9:1) to give 9, isolated as a white solid (0.32 g, 63%); [α]D=
−114 (c=1); 1H NMR (300 MHz, CD3OD): δ 1.038 (d, 3H, J5,6=6.5 Hz, H-6,6,6), 1.985 and 2.010 (2 s,
0
0
0
0
0
each 3H, 2 Ac), 3.963 (dd, 1H, J1 ,2 =8.5, J2 ,3 =10.4 Hz, H-2 ), 4.718 (dd, 1H, J2,3=10.6, J3,4=3.3 Hz,
0
0
0
0
0
0
H-3), 4.855 (dd, 1H, J3 ,4 =8.9, J4 ,5 =10.1 Hz, H-4 ), 5.098 (dd, 1H, H-3 ), 5.098 and 5.279 (2 m, each
1H, OCH2CH_CH2), 5.184 (d, 1H, J1,2=3.8 Hz, H-1), 5.352 (dd, 1H, H-2), 5.451 (d, 1H, H-4), 5.594
(d, 1H, H-10), 5.854 (m, 1H, OCH2CH_CH2), 7.22–8.17 (m, 14H, Phth and 2 Bz); FABMS (positive):
calcd for C41H40O18NSNa 889.4, found m/z 890.1 ([M+H]+), 912.0 ([M+Na]+).
3.9. Allyl (sodium 2-acetamido-2-deoxy-β-D-glucopyranosyl 3-sulfate)-(1→3)-α-L-fucopyranoside 10
A solution of 9 (38.4 mg, 44 µmol) in ethanolic 30% NH2Me (5 mL) was stirred for 5 days, when TLC
(n-butanol:EtOH:H2O:HOAc 4:2:2:1) showed an incomplete conversion of 9 into an intermediate amino
compound (Rf=0.56). Ethanolic 30% NH2Me (5 mL) was again added, and the mixture was stirred for 3
days, when TLC showed the de-acylation to be completed. After concentration, a solution of the residue
in MeOH (2 mL) and Ac2O (77 µL) was stirred for 2 h at 0°C, then concentrated and co-concentrated
with toluene and MeOH (1:1, 3×2 mL). The remaining oil was purified on Bio-Gel P-2 (H2O) followed
by silica gel column chromatography (CH2Cl2:methanol 8:2) to afford 10, isolated, after lyophilization,
1
as an amorphous white powder (12 mg, 61%); [α]D= −106 (c=0.4, H2O); H NMR (500 MHz, D2O):
0
0
0
0
δ 1.215 (d, 3H, J5,6=6.7 Hz, H-6,6,6), 2.012 (s, 3H, NAc), 3.824 (dd, J1 ,2 =8.6, J2 ,3 =10.4 Hz, H-
20), 3.892 (dd, 1H, J2,3=10.4 Hz, H-2), 4.046 (dd, 1H, J2,3=10.4, J3,4=3.4 Hz, H-3), 4.414 (dd, 1H,
0
0
0
0
J3 ,4 =8.8 Hz, H-3 ), 4.805 (d, 1H, H-1 ), 4.964 (d, 1H, J1,2=3.9 Hz, H-1), 5.266 and 5.362 (2 m, each
1H, OCH2CH_CH2), 5.973 (m, 1H, OCH2CH_CH2); FABMS (positive): calcd for C17H28O13NSNa
509.2, found m/z 532.0 ([M+Na]+).
3.10. 3-(2-Aminoethylthio)propyl (sodium 2-acetamido-2-deoxy-β-D-glucopyranosyl 3-sulfate)-(1→3)-
α-L-fucopyranoside 11
To a solution of 10 (5.0 mg, 10.3 µmol) in water (0.5 mL) was added cysteamine hydrochloride (6.4
mg, 56.6 µmol), and the mixture was irradiated for 2 h in a quartz vial using a Hanovia UV lamp.
Then, TLC (n-butanol:H2O:HOAc 2:1:1) showed a complete conversion of 10 into a lower moving
spot (Rf=0.18). The mixture was loaded directly on a Toyopearl HW-40SO column yielding, after
lyophilization, 11 as an amorphous white powder (5.7 mg, 86%); [α]D= −112 (c=0.6, H2O); 1H NMR
data (500 MHz) are given in Table 1; FABMS (positive): calcd for C19H35O13N2S2Na 586.2, found m/z
587.3 ([M+Na]+).

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H. J. Vermeer et al. / Tetrahedron: Asymmetry 11 (2000) 539–547
3.11. 3-[2-N-(3,4-Dione-2-ethoxycyclobutene)aminoethylthio]propyl (sodium 2-acetamido-2-deoxy-β-
D-glucopyranosyl 3-sulfate)-(1→3)-α-L-fucopyranoside 12
To a solution of compound 11 (1.0 mg, 2.1 µmol) in 0.75 mM sodium phosphate buffer (pH 7.0;
100 µL) was added a solution of 3,4-diethoxy-3-cyclobutene-1,2-dione (diethyl squarate; 0.28 µL, 2.0
µmol) in EtOH (100 µL). The mixture was stirred for 2.5 h when TLC (CH2Cl2:H2O:MeOH 120:12:7)
showed complete conversion to a higher moving spot (Rf=0.73). After concentration, a solution of the
crude residue in water (1 mL) was loaded on a C-18 Sep-Pak cartridge. The column was washed with
water (3×2 mL), then the product was eluted with MeOH (3×2 mL). The MeOH phase was evaporated
and a solution of the residue in water (2 mL) was lyophilized to yield 12 as a white powder. The material
was used directly for the preparation of neoglycoconjugate 13.
3.12. Pretreatment of bovine serum albumin
BSA was stirred in 0.1 M NaOAc buffer (pH 4.5; 40 mg/mL) containing 10 mM NaIO4 for 1.5 h at
rt to oxidize carbohydrate of glycoprotein contaminants.19 Excess of periodate was destroyed by adding
glycerol to a final concentration of 10 mM. The solution was dialyzed against water (three changes; Milli
Q), followed by lyophilization. After treatment of the material with an aq. solution of NaBH4 (catalytic
amount) for 1 h at rt, the solution was diluted with water (1 mL) and neutralized with 4 M HOAc. After
lyophilization, the quality of pretreated BSA was checked by SDS–polyacrylamide gel electrophoresis.
To verify the complete removal of sugar contaminants with GLC, a small amount of the protein material
(1 mg) was subjected to methanolysis (1.0 M methanolic HCl, 24 h, 85°C) followed by re-N-acetylation
and trimethylsilylation.18
3.13. Preparation of the BSA-glycoconjugate 13
Pretreated BSA (9.1 mg) was dissolved in 0.1 M NaHCO3 buffer (pH 9.0; 400 µL) and stirred for
30 min. Then, a solution of 12 in 0.1 M NaHCO3 buffer (pH 9.0, 400 µL) was added and the mixture
was stirred for 5 days at rt, when TLC (CH2Cl2:H2O:MeOH 120:12:7) showed complete transfer of 12
onto BSA (Rf=0.0). The mixture was purified by HiTrap gel filtration (aq. 5% NH4HCO3) to afford,
after lyophilization, neoglycoconjugate 13. The incorporation degree of 12 onto BSA was determined
by MALDI-TOF MS, revealing an average incorporation of 7–9 hapten molecules per molecule BSA.
The incorporation degree was also determined by GLC; a small amount of the neoglycoconjugate (1 mg)
was subjected to methanolysis (1.0 M methanolic HCl, 24 h, 85°C) followed by re-N-acetylation, and
trimethylsilylation,18 revealing the presence of 8 hapten molecules per molecule BSA.
Acknowledgements
The authors wish to thank Dr. P. H. Kruiskamp for recording NMR spectra, Mrs. A. C. H. T. M. van der
Kerk-van Hoof for recording FAB-mass spectra, and Dr. C. H. Grün for recording MALDI-TOF-mass
spectra.
References
1. Wilson, H. V. J. Exp. Zool. 1907, 5, 245–258.

H. J. Vermeer et al. / Tetrahedron: Asymmetry 11 (2000) 539–547
547
2. Jumblatt, J. E.; Schlup, V.; Burger, M. M. Biochemistry 1980, 19, 1038–1042.
3. Misevic, G. N.; Burger, M. M. J. Biol. Chem. 1990, 265, 20577–20584.
4. Spillmann, D.; Hård, K.; Thomas-Oates, J. E.; Vliegenthart, J. F. G.; Misevic, G.; Burger, M. M.; Finne, J. J. Biol. Chem.
1993, 268, 13378–13387.
5. Spillmann, D.; Thomas-Oates, J. E.; Van Kuik, J. A.; Vliegenthart, J. F. G.; Misevic, G.; Burger, M. M.; Finne, J. J. Biol.
Chem. 1995, 270, 5089–5097.
6. Vermeer, H. J.; Kamerling, J. P.; Vliegenthart, J. F. G. Abstracts of Papers, 18th Int. Carbohydr. Symp., Milano, Italy, 1996;
BP 251.
7. Guo, Z.-W.; Deng, S.-J.; Hui, Y.-Z. J. Carbohydr. Chem. 1996, 15, 965–974.
8. Slaghek, T. M.; Hyppönen, T. K.; Ogawa, T.; Kamerling, J. P.; Vliegenthart, J. F. G. Tetrahedron: Asymmetry 1994, 5,
2291–2301.
9. Garegg, P. J.; Norberg, T. Carbohydr. Res. 1976, 52, 235–240.
10. Takeo, K. I.; Aspinall, G. O.; Brennan, P. J.; Chatterjee, D. Carbohydr. Res. 1986, 150, 133–150.
11. Kunz, H.; Waldmann, H. Angew. Chem. 1984, 96, 49–50.
12. Hayakawa, Y.; Kato, H.; Uchiyama, M.; Kajino, H.; Noyori, R. J. Org. Chem. 1986, 51, 2400–2402.
13. Nakano, T.; Ito, Y.; Ogawa, T. Carbohydr. Res. 1993, 243, 43–69.
14. Auzanneau, F.-I.; Pinto, B. M. Bioorg. Med. Chem. 1996, 4, 2003–2010.
15. Hällgren, C.; Hindsgaul, O. J. Carbohydr. Chem. 1995, 14, 453–464.
16. Tietze, L. F.; Arlt, M.; Beller, M.; Glüsenkamp, K.-H.; Jähde, E.; Rajewsky, M. F. Chem. Ber. 1991, 124, 1215–1221.
17. Kamath, V. P.; Diedrich, P.; Hindsgaul, O. Glycoconjugate J. 1996, 13, 315–319.
18. Kamerling, J. P.; Vliegenthart, J. F. G. In Clinical Biochemistry — Principles, Methods, Applications, Vol. 1, Mass
Spectrometry; Lawson, A. M., Ed. Carbohydrates. Walter de Gruyter: Berlin, 1989; pp. 176–263.
19. Glass II, W. F.; Briggs, R. C.; Hnilica, L. S. Anal. Biochem. 1981, 115, 219–224.