Synthesis of neoglycoproteins containing O-methylated trisaccharides related to excretory/secretory antigens of Toxocara larvae

Article abstract of DOI:10.1016/S0008-6215(02)00355-5

The disaccharides allyl β-D-galactopyranosyl-(1→3)-2-acetamido-2-deoxy-β- and α-D-galactopyranoside 10a and 10b and the trisaccharides allyl 2-O-methyl-α-L-fucopyranosyl-(1→2)-β-D-galactopyranosyl- (1→3)-2-acetamido-2-deoxy-β- and α-D-galactopyranoside 18a and 18b have been prepared using stepwise assembly of the sugar units. The glycosidic linkages were formed employing the trichloroacetimidate procedure for the attachment of the galactopyranosyl residue and N-iodosuccinimide/triflic acid activation of an ethyl 1-thiofucopyranoside donor for fucosylation. Deprotection furnished the allyl glycosides which were converted into cysteamine-spacered ligands, activated with thiophosgene and subsequently linked to bovine serum albumin. The neoglycoproteins serve as immunoreagents to determine epitope specificities of monoclonal antibodies directed against highly immunogenic O-glycans located at the surface of Toxocara larvae.

Full text of DOI:10.1016/S0008-6215(02)00355-5

Carbohydrate Research 338 (2003) 35–45
www.elsevier.com/locate/carres
Synthesis of neoglycoproteins containing O-methylated
trisaccharides related to excretory/secretory antigens of Toxocara
larvae
Hassan Amer, Andreas Hofinger, Paul Kosma*
Institute of Chemistry, Uni6ersity of Agricultural Sciences, Muthhasse 18, A-1190 Vienna, Austria
Received 8 July 2002; accepted 23 September 2002
Abstract
The disaccharides allyl b-
D
-galactopyranosyl-(1“3)-2-acetamido-2-deoxy-b- and a-
D-galactopyranoside 10a and 10b and the
-fucopyranosyl-(1“2)-b- -galactopyranosyl-(1“3)-2-acetamido-2-deoxy-b- and a-D-
trisaccharides allyl 2-O-methyl-a-
L
D
galactopyranoside 18a and 18b have been prepared using stepwise assembly of the sugar units. The glycosidic linkages were
formed employing the trichloroacetimidate procedure for the attachment of the galactopyranosyl residue and N-iodosuccinimide/
triflic acid activation of an ethyl 1-thiofucopyranoside donor for fucosylation. Deprotection furnished the allyl glycosides which
were converted into cysteamine-spacered ligands, activated with thiophosgene and subsequently linked to bovine serum albumin.
The neoglycoproteins serve as immunoreagents to determine epitope specificities of monoclonal antibodies directed against highly
immunogenic O-glycans located at the surface of Toxocara larvae. © 2002 Elsevier Science Ltd. All rights reserved.
Keywords: Neoglycoprotein; Toxocara; Antigen; O-Glycan; Fucose
1. Introduction
methylated at the 2-position. Furthermore, ꢀ50% of
the glycans contained a 4-O-methyl group at the
galactopyranosyl unit, which is also the prominent
structure found in T. cati glycans. The anomeric
configuration of the GalpNAc residue has not yet been
determined. Monoclonal anti-carbohydrate antibodies
recognizing the TES antigens bind to different sites
within the larval parasite.4 Previously, three disaccha-
ride ligands which are O-methylated either at position 2
of the fucopyranosyl moiety or at position 4 of the
galactopyranosyl unit or at both positions have been
synthesized.5 For a more detailed immunochemical
characterization of those monoclonal antibodies which
should also include the anomeric region of the reducing
end as part of the potential epitope, we report on the
synthesis of both anomers of the corresponding Gal-
NAc-containing disaccharide allyl glycoside as well as
two anomeric trisaccharide ligands containing the ter-
Toxocara canis and Toxocara cati are parasitic round-
worms usually occurring in dogs and cats, which may
cause severe infections in a human host affecting eyes,
liver and the central nervous system.1 The surface coat
of the parasitic nematode larvae may induce a major
immunological response of the host immune system.2 In
particular, glycoproteins being present in multiple
copies at the outer layer elicit a strong immune reac-
tion, are subsequently released from the surface and
divert the immune attack from the mobile nematode.
The structure of the excreted and secreted glycoproteins
(TES antigens) from Toxocara canis investigated by MS
analysis of alditol acetates revealed the presence of the
trisaccharide backbone a-
L
-Fucp-(1“2)-b-D-Galp-
(1“3)-
D
-GalpNAc,3 with the fucose part being O-
minal 2-O-methyl-a-L-fucopyranosyl residue. The allyl
glycosides were further converted into spacer-elongated
derivatives and coupled to BSA6 for immunochemical
studies.
* Corresponding author. Tel.: +43-1-360066055; fax.: +
43-1-360066059
E-mail address: pkosma@edv2.boku.ac.at (P. Kosma).
0008-6215/03/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved.
PII: S0008-6215(02)00355-5

36
H. Amer et al. / Carbohydrate Research 338 (2003) 35–45
2. Results and discussion
complex.21 Thus, compounds 8a and 8b were isolated in
40% and 45% yield, respectively. Unreacted acceptor
derivatives could be recovered in 40–43% yields
(Scheme 2).
The anomeric allyl 2-acetamido-2-deoxy-galactopyran-
oside derivatives 3a and 3b were prepared separately via
epimerization at C-4 of the readily available N-acetyl-
glucosamine precursors 1a and 1b.6,7 Thus, reaction of
1a and 1b with pivaloyl chloride in pyridine afforded
the 3,6-di-O-pivaloyl derivatives 2a and 2b in 74% and
85% yield, respectively. Following conversion into the
corresponding intermediate 4-O-triflate derivatives,
triflate displacement in the presence of water proceeded
smoothly with subsequent acyl migration in the case of
the b-allyl glycoside 2a to furnish the known allyl
2-acetamido-2-deoxy-4,6-di-O-pivaloyl-b-D-galactopy-
ranoside (3a) in 82% yield.8 By contrast, the intermedi-
ate 4-O-triflate derivative of a-glycoside 2b had to be
reacted with NaOH to effect epimerization at C-4.
Furthermore, the 3,4-di-O-pivaloyl derivative 5 was
formed as a byproduct, which was separated by column
chromatography. For structural confirmation of the
substitution pattern, an aliquot of 3b was O-acetylated
Scheme 2. Reagents and conditions: (a) BF3-etherate, CH2Cl2,
−25 °C, 2 h; (b) NaOMe, rt, 3 h; (c) 40% aq Bu4NOH, 9:1
dioxane–H2O, rt, 15 h.
The b-anomeric configuration of the galactopyranose
residue was confirmed by the value of the coupling
constant J1%,2% (7.6 Hz for 8a and 7.9 Hz for 8b).
Selective removal of the acetyl groups was achieved by
treatment with sodium methoxide, which produced 4,6-
di-O-pivaloyl derivatives 9a and 9b in 87% and 97%
yield, respectively. Finally, the pivaloyl groups were
cleaved in the presence of tetrabutylammonium hydrox-
(acetic
anhydride/pyridine/4-N,N-dimethylaminopy-
ridine) to give the 3-O-acetyl derivative 4, which dis-
played a downfield shift of the signal of H-3 to 5.18
1
ppm in the H NMR spectrum (Scheme 1).
ide in aq dioxane to furnish allyl b-
(1“3)-2-acetamido-2-deoxy-b- -galactopyranoside
(10a) and allyl b- -galactopyranosyl-(1“3)-2-acetam-
-galactopyranoside (10b) in 75% and
D-galactopyranosyl-
D
D
ido-2-deoxy-a-
D
89% yield, respectively.
For selective fucosylation of the O-2% position, com-
pounds 9a and 9b were reacted with 4-methoxyben-
zaldehyde dimethylacetal/toluenesulfonic acid in DMF
to give the 4%,6%-O-acetals 11a and 11b in 72% and 75%
yield, respectively. Regioselective protection of the 3%-
OH group was accomplished by reaction of 11a and
11b with N-benzoylimidazole,22,23 which produced
mainly the corresponding 3%-O-benzoates 12a (72%)
and 12b (87% yield) and only minor amounts of disub-
stituted products. The position of the benzoyl group
was confirmed upon subsequent O-acetylation (acetic
anhydride/pyridine) which afforded the 2%-O-acetyl
Scheme 1. Reagents and conditions: (a) pivaloyl chloride, 1:2
CH2Cl2–pyridine, 0 °C, 3 h; (b) Tf2O, CH2Cl2, pyridine,
−35 °C“0 °C, 2 h; then H2O, reflux, 3 h (3a) or 1.5 M
NaOH, reflux, 15 h (3b); (c) (Ac)2O, pyridine, DMAP (cat.),
rt, 15 h.
Numerous syntheses for b-Galp-(1“3)-GalpNAc
disaccharide units have been reported (for examples see
Refs. 9–14) as holds true for fucosylated oligosaccha-
rides of the globo H hexasaccharide.15–19 For the syn-
thesis of the disaccharide derivatives 8a and 8b, the
readily accessible 2,3,4,6-tetra-O-acetyl-galactopyran-
osyl trichloroacetimidate 6 (Ref. 20) was first coupled
with the glycosyl acceptor derivative 3a in the presence
of boron trifluoride etherate, which produced mainly
the exo-orthoester derivative 7 (31%) in addition to a
small proportion of the disaccharide 8a (10%). The
presence of the orthoester was deduced from the upfield
shifts of the methyl group signal at 1.63 ppm as well as
the H-2% signal (4.31 ppm). Better yields for the b-linked
disaccharide derivatives were obtained by adding a
solution of donor 6 to a preformed catalyst: acceptor
1
derivatives 13a in 95% yield. H NMR data showed the
expected downfield shift for H-2% (from 3.55–3.75 ppm
in 12a to 5.40–5.50 ppm in 13a) (Scheme 3).
The disaccharide acceptor derivatives 12a and 12b
were then glycosylated using the previously reported
2-O-methyl ethyl-1-thio fucopyranoside donor 14.5,24
Whereas iodonium–dicollidinium perchlorate did not
effect trisaccharide formation, promotion by N-iodo-
succinimide/triflic acid furnished a mixture of a- and
b-(1“2) linked trisaccharide derivatives which were
separated by column chromatography. Thus, the a-fu-

H. Amer et al. / Carbohydrate Research 338 (2003) 35–45
37
Scheme 4. Reagents and conditions: (a) 90% TFA, CH2Cl2,
rt, 1 h, then (Ac)2O, pyridine, DMAP (cat.), rt, 15 h; (b)
NaOMe, rt, 15 h, then 40% aq Bu4NOH, 9:1 dioxane-H2O, rt,
13 h; (c) cysteamine hydrochloride, H2O, UV-light (254 nm),
rt, 3 h; (d) thiophosgene, 0.1 M aq NaHCO3, rt, 2 h; then
BSA, 0.1 M aq NaHCO3, 0.3 M NaCl, rt, 48 h.
pound 18a) and 76.34 ppm (compound 18b), respec-
tively (Table 1) (Scheme 4).
For the preparation of neoglycoproteins, the addition
of cysteamine to the allylic double bond provided
spacer-elongated ligands containing a terminal amino
group for further modification.25,26 Reaction of 18a and
18b with cysteamine hydrochloride for 3–6 h afforded
the 3-(2-aminoethylthio)propyl glycosides 19a and 19b
in 70% and 71% yields after purification on a cation-ex-
change resin followed by desalting on Sephadex LH-20.
The structure of the thioether group was based on the
NMR spectral characteristics which indicated the ab-
sence of the allyl protons and the presence of CH2Nꢀ
and CH2Sꢀ at 3.03, 2.73 and 2.54 ppm for 19a and
3.09, 2.76 and 2.64 ppm for compound 19b, respec-
tively. Prolonged reaction times of compound 18a for 2
days, however, led also to formation of N-addition
products with subsequent dimerization to a disulfide
derivative. The spectrum of the dimer showed two
CH2Nꢀ groups in the 1H NMR spectrum (3.39 and 3.24
ppm) as well as in the 13C NMR assignments (38.96
and 34.47 ppm).
Finally, the trisaccharide ligands were reacted with
thiophosgene to give intermediate isothiocyanate
derivatives, which were coupled to bovine serum albu-
min.27 The neoglycoproteins were purified on Sephadex
G-10 and dialyzed against water. According to MALDI
MS data, the glyococonjugates 20a and 20b contained
6.8 and 3.1 mol ligand/mol protein. Immunochemical
results obtained with the neoglycoproteins will be re-
ported elsewhere.
Scheme 3. Reagents and conditions: (a) anisaldehyde
dimethylacetal, TsOH, DMF, rt, 15 h; (b) benzoylimidazole,
CHCl3, reflux, 48 h; (c) (Ac)2O, pyridine, DMAP (cat.), rt, 15
h; (d) NIS, TfOH, CH2Cl2-Et2O, 5 °C, 15 h.
cosylated derivatives 15a and 15b were isolated in 42%
and 48% yield, respectively, in addition to the b-(1“2)-
linked isomers 16a (19%) and 16b (20% yield). The
assignment of the anomeric configuration was based on
the small value of the coupling constant J1¦,2¦ for the
a-fucosyl derivatives, whereas the anomeric protons of
the fucopyranosyl units of the b-isomers 16a and 16b
displayed a spacing of 7.6 and 7.7 Hz, respectively.
Deprotection of the trisaccharide compounds 15a and
15b was performed 6ia removal of the methoxybenzyli-
dene group in 90% aqueous trifluoroacetic acid fol-
lowed by O-acetylation (acetic anhydride/pyridine/4-
dimethylaminopyridine) to afford the tetra-O-acetyl
derivatives 17a and 17b in 94% and 90% yield, respec-
tively.
The acyl groups were finally removed by treatment
with tetrabutylammonium hydroxide in aqueous diox-
ane to give the target allyl trisaccharide derivatives 18a
and 18b in 82% and 73% yield after final purification of
Sephadex LH 20. The structural assignments of the
deprotected allyl glycosides were fully confirmed by the
NMR data based on COSY, HMQC and HMBC corre-
lations. Thus, glycosylation of O-3 of the galactosamine
residue resulted in pronounced downfield shifts of the
13C NMR chemical shift of C-3 (80.78 ppm for 10a and
77.63 ppm for 10b), whereas the carbon signals of C-2%
3. Experimental
3.1. General methods
Thin layer chromatography was performed on E.
Merck precoated plates (5×10 cm, layer thickness 0.25
of the b-galactopyranosyl residues displayed
a
downfield shift upon fucosylation to 76.04 ppm (com-

38
H. Amer et al. / Carbohydrate Research 338 (2003) 35–45
Table 1
13C NMR data a for disaccharides 10a, 10b and trisaccharides 18a and 18b
Residue
Carbon
l (ppm)
10a
10b
18a
97.14
18b
97.38
a-2MeFucp-(1“2)
1
2
3
4
5
6
78.08
69.39
72.49
67.35
16.04
78.29
69.68
72.74
67.52
16.35
b-Galp-(1“3)
1
2
3
4
5
6
105.58
71.43
73.32
68.87
75.62
61.75 b
105.10
71.01
72.92
68.99
75.37
61.38 b
103.01
76.04
74.46
70.08
75.68
61.76 b
103.16
76.34
74.65
70.26
75.93
61.99 b
a- or b-GalpNAc
1
2
3
4
5
6
101.12
52.07
80.78
96.81
49.01
77.63
102.52
52.09
77.67
69.39
75.78
61.83 b
71.33
134.48
118.45
58.25
23.08
174.61
96.96
50.43
75.09
70.18
71.66
62.22 b
69.44
134.79
118.64
58.50
22.94
174.68
69.41
69.14
75.81
71.01
61.83 b
71.19
61.58 b
68.83
OCH2
ꢀCHꢁ
CH2ꢁ
OMe
NHAc
CO
134.23
119.05
134.09
118.27
23.05
175.64
22.38
174.98
a Spectra (75.47 MHz) were recorded at 297 K and referenced to 1,4-dioxane (l 67.40).
b Assignments within a column may be reversed.
mode using CH3CN or H2O, both with 2% 2,5-dihy-
droxybenzoic acid as matrix, by Dr F. Altmann, Insti-
tut fu¨r Chemie, University of Agricultural Sciences,
mm, Silica Gel 60 F254). The spots were detected by UV
light and by spraying with anisaldehyde reagent fol-
lowed by heating to 200 °C. Amine derivatives were
additionally detected by ninhydrin spray reagent.
Column chromatography was performed on silica gel
(0.040–0.063 mm) under normal pressure. Solvents
Vienna. Optical rotations were measured with
a
Perkin–Elmer 243 B polarimeter. Melting points were
determined with a Kofler hot stage and are uncor-
rected. Elemental analyses were provided by Dr J.
Theiner, Mikroanalytisches Laboratorium, Institut fu¨r
Physikalische Chemie, Universita¨t Wien.
,
were dried, distilled and kept dry over 4 A molecular
sieves. Evaporation of solvents was performed under
reduced pressure at 25–40 °C. Reagents and dry sol-
vents were added 6ia oven-dried syringes through
septa.1H NMR spectra were recorded at 300 MHz at
297 K with a Bruker DPX 300F instrument using
CDCl3 as solvent and Me4Si or sodium 3-(trimethylsi-
lyl)-propionate-2,2,3,3-d4 (for solutions in D2O) as the
internal standard.13C NMR spectra were recorded at
75.47 MHz for solutions in D2O at 24 °C. Chemical
shifts (l) were measured relative to that of 1,4-dioxane,
set at l 67.40 relative to Me4Si. Homo- and hetero-nu-
clear 2D NMR spectroscopy was performed with
Bruker standard NMR software. MALDI-TOF-MS-
ionisation mass spectra were recorded on a Dynamo
(Thermo BioAnalysis) instrument in the positive ion
3.2. Allyl 2-acetamido-2-deoxy-3,6-di-O-pivaloyl-b-D-
glucopyranoside (2a)
A mixture of 1a (2.3 g, 8.8 mmol), pivaloyl chloride
(3.25 mL, 26.8 mmol) in CH2Cl2 (10 mL) and pyridine
(20 mL) was stirred for 3 h at 0 °C under Ar. Methanol
(1.0 mL) was added and the mixture was diluted with
CH2Cl2 (50 mL). The organic phase was washed with
5% aq HCl, water, 5% aq NaHCO3, dried (Na2SO4),
and concentrated. Purification of the residue by column
chromatography (1:1 C6H5CH3–EtOAc) furnished 2a
as colorless crystals. Yield: 2.8 g (74%); mp 139–140 °C

H. Amer et al. / Carbohydrate Research 338 (2003) 35–45
39
(n-hexane–EtOAc), lit. mp 135–137 °C,8 [h]D20 −39° (c
3.5. Allyl 2-acetamido-2-deoxy-4,6-di-O-pivaloyl-a-
galactopyranoside (3b) and allyl 2-acetamido-2-deoxy-
3,4-di-O-pivaloyl-a- -galactopyranoside (5)
D-
1.0, CHCl3), lit. [h]2D0 −42° (c 1.0, CHCl3).8 1H NMR
(CDCl3): l 5.87 (d, 1 H, JNH,2 8.8 Hz, NH), 5.86 (m, 1
H, ꢀCHꢁ), 5.25 (dq, 1 H, ꢁCH2trans), 5.17 (dq, 1 H,
ꢁCH2cis), 5.10 (dd, 1 H, J2,3 10.7, J3,4 8.6 Hz, H-3), 4.54
(d, 1 H, J1,2 8.4 Hz, H-1), 4.45–4.35 (m, 2 H, H-6a,
H-6b), 4.31 (ddt,1 H, OCH2), 4.07 (ddt, 1 H, OCH2),
3.98 (ddd, 1 H, H-2), 3.60–3.45 (m, 2 H, H-4, H-5),
3.19 (br s,1 H, OH), 1.93 (s, 3 H, Ac), 1.23 and 1.20 (2
s, 18 H, 2 t-Bu). Anal. Calcd for C21H35NO8: C, 58.73;
H, 8.21; N, 3.26. Found: C, 58.82; H, 8.21; N, 3.28.
D
Compound 2b (500 mg, 1.16 mmol) was dissolved in
CH2Cl2 (10 mL) and pyridine (0.5 mL) under Ar at
−35 °C. Triflic anhydride (0.23 mL, 1.4 mmol) was
added dropwise. The mixture was allowed to warm to
0 °C and was stirred for 2 h. Aq NaOH (1.5 M, 1.0 mL)
was added and the mixture was refluxed overnight, then
diluted with CH2Cl2 (50 mL). The organic phase was
washed with 5% aq HCl, water, 5% aq NaHCO3, and
dried (Na2SO4). Purification of the residue on silica gel
(40:40:1 EtOAc–n-hexane–EtOH) furnished 3b as crys-
tals. Yield: (225 mg, 45%); mp 120 °C (n-hexane–
3.3. Allyl 2-acetamido-2-deoxy-4,6-di-O-pivaloyl-b-D-
galactopyranoside (3a)
1
Compound 2a (500 mg, 1.16 mmol) was dissolved in
CH2Cl2 (10 mL) and pyridine (0.5 mL) under Ar at
−35 °C. Triflic acid anhydride (0.23 mL, 1.4 mmol)
was added dropwise. The mixture was allowed to warm
to 0 °C and was stirred for 2 h. Water (1 mL) was
added and the mixture was refluxed for 3 h. The
solution was diluted with CH2Cl2 (50 mL), washed with
5% aq HCl, water, and 5% aq NaHCO3. The organic
phase was dried (Na2SO4) and concentrated. Purifica-
tion of the residue on silica gel (40:40:1 EtOAc–n-hex-
ane–EtOH) furnished 3a as a syrup. Yield: (410 mg,
82%); [h]2D0 −29° (c 0.7, CHCl3), lit. [h]2D0 −32.5° (c
1.05, CHCl3).8 1H NMR (CDCl3): l 5.92 (m, 1 H,
ꢀCHꢁ), 5.72 (d, 1 H, JNH,2 4.9 Hz, NH), 5.32 (dq, 1 H,
ꢁCH2trans), 5.26 (dq, 1 H, ꢁCH2cis), 5.30 (dd, 1 H, J3,4
3.8 Hz, H-4), 4.59 (d, 1 H, J1,2 8.3 Hz, H-1), 4.44–4.30
(m, 2 H, OCH2, OH), 4.20–4.00 (m, 4 H, H-3, H-6a,
H-6b, OCH2), 3.88 (dd, 1 H, J5,6a=J5,6b 6.8 Hz, H-5),
3.68 (ddd, 1 H, J2,3 10.4 Hz, H-2), 2.05 (s, 3 H, Ac),
1.27 and 1.20 (2 s, 18 H, 2 t-Bu). Anal. Calcd for
C21H35NO8: C, 58.73; H, 8.21; N, 3.26. Found: C,
58.23; H, 8.17; N, 3.22.
EtOAc); [h]2D0 +16° (c 0.7, CHCl3). H NMR (CDCl3):
l 5.98–5.82 (m, 2 H, NH, ꢀCHꢁ), 5.32 (d, 1 H, J3,4 3.3
Hz, H-4), 5.30 (dq, 1 H, ꢁCH2trans), 5.26 (dq, 1 H,
ꢁCH2cis), 4.95 (d, 1 H, J1,2 3.7 Hz, H-1), 4.37 (ddd, 1 H,
J2,3 11.3, J2,NH 9.7 Hz, H-2), 4.22 (ddt, 1 H, OCH2),
4.18–3.92 (m, 5 H, H-3, H-5, H-6a, H-6b, OCH2), 3.21
(br s, 1 H, OH), 2.06 (s, 3 H, Ac), 1.28 and 1.20 (2 s, 18
H, 2 t-Bu). Anal. Calcd for C21H35NO8: C, 58.73; H,
8.21; N, 3.26. Found: C, 58.26; H, 8.12; N, 3.20.
Further elution afforded 5 as crystals. Yield: 150 mg
(30%); mp 127 °C (n-hexane–EtOAc). [h]2D0 +109° (c
0.9, CHCl3). 1H NMR (CDCl3): l 5.82 (m, 1 H, ꢀCHꢁ),
5.58 (d, 1 H, JNH,2 9.8 Hz, NH), 5.28–5.10 (m, 4 H,
H-3, H-4, ꢁCH2trans, ꢁCH2cis), 4.85 (d, 1 H, J1,2 3.6 Hz,
H-1), 4.63 (ddd, 1 H, J2,3 10.5 Hz, H-2), 4.13 (ddt, 1 H,
OCH2), 4.00 (dd, 1 H, J5,6a=J5,6b 6.8 Hz, H-5), 3.93
(ddt, 1 H, OCH2), 3.54 (ddd, 1 H, J6a,6b 11.7, J6a,OH 6.9
Hz, H-6a), 3.35 (ddd, 1 H, J6b,OH 6.9 Hz, H-6b), 2.31
(dd, 1 H, OH), 1.87 (s, 3 H, Ac), 1.22 and 1.08 (2 s, 18
H, 2 t-Bu). 13C NMR (CDCl3): l 178.71 and 178.39 (2
(CO), 169.70 (NHAc), 133.23 (ꢀCHꢁ), 118.18 (ꢁCH2),
97.4 (C-1), 69.77 (C-5), 68.64 (OCH2), 68.39 (C-3),
68.09 (C-4), 60.74 (C-6), 48.10 (C-2), 39.21 and 38.83 [2
Me3CCO], 27.27 and 26.95 [2 Me3CCO], 23.27
(CH3CO). Anal. Calcd for C21H35NO8: C, 58.73; H,
8.21; N, 3.26. Found: C, 58.68; H, 8.06; N, 3.23.
3.4. Allyl 2-acetamido-2-deoxy-3,6-di-O-pivaloyl-a-D-
glucopyranoside (2b)
Compound 2b was prepared from 1b (1.5 g, 5.74 mmol)
in the same fashion as described for 2a to afford 2b as
crystals. Yield: 2.1 g (85%), mp 75 °C (n-hexane–
3.6. Allyl 2-acetamido-3-O-acetyl-2-deoxy-4,6-di-O-pi-
valoyl-a-D-galactopyranoside (4)
1
EtOAc); [h]2D0 +60° (c 1.0, CHCl3). H NMR (CDCl3):
l 5.86 (m, 1 H, ꢀCHꢁ), 5.74 (d, 1 H, JNH,2 9.7 Hz, NH),
5.27 (dq, 1 H, ꢁCH2trans), 5.21 (dq, 1 H, ꢁCH2cis), 5.08
(dd, 1 H, J2,3 10.8, J3,4 9.2 Hz, H-3), 4.82 (d, 1 H, J1,2
3.7 Hz, H-1), 4.41 (dd, 1 H, J6a,5 4.7, J6a,6b 12.0 Hz,
H-6a), 4.29 (dd, 1 H, J6b,5 2.3 Hz, H-6b), 4.26 (ddd, 1
H, H-2), 4.18 (ddt,1 H, OCH2), 3.97 (ddt, 1 H, OCH2),
3.82 (ddd, 1 H, J4,5 10.5 Hz, H-5), 3.52 (ddd, 1 H, J4,OH
5.0 Hz, H-4), 2.94 (d, 1 H, OH), 1.92 (s, 3 H, Ac), 1.22
and 1.18 (2 s, 18 H, 2 t-Bu). Anal. Calcd for
C21H35NO8: C, 58.73; H, 8.21; N, 3.26. Found: C,
58.52; H, 8.28; N, 3.19.
A solution of 3b (10 mg) and a catalytic amount of
4-N,N-dimethylaminopyridine in dry pyridine (2 mL)
was stirred with Ac2O (200 mL) overnight at room
temperature (rt). Methanol (0.5 mL) was added, the
solution was coevaporated three times with addition of
toluene and concentrated. Purification of the residue on
silica gel (2:1 toluene–EtOAc) furnished 4 as syrup.
Yield: 10 mg (91%); [h]2D0 +72° (c 0.8, CHCl3). 1H
NMR (CDCl3): l 5.92 (m, 1 H, ꢀCHꢁ), 5.59 (d, 1 H,
JNH,2 9.8 Hz, NH), 5.38 (d, 1 H, J3,4 3.3 Hz, H-4), 5.29
(dq, 1 H, ꢁCH2trans), 5.25 (dq, 1 H, ꢁCH2cis), 5.18 (dd,

40
H. Amer et al. / Carbohydrate Research 338 (2003) 35–45
1 H, J2,3 11.3 Hz, H-3), 4.94 (d, 1 H, J1,2 3.6 Hz, H-1),
4.61 (ddd, 1 H, H-2), 4.24–4.12 (m, 2 H, H-5, OCH2),
4.09–3.96 (m, 3 H, H-6a, H-6b, OCH2), 1.96 (s, 3 H,
Ac), 1.27 and 1.19 (2 s, 18 H, 2 t-Bu).
H, H-2), 2.14, 2.07, 2.05, 1.99 and 1.98 (5 s, 15 H, 5
Ac), 1.26 and 1.22 (2 s, 18 H, 2 t-Bu). Anal. Calcd for
C35H53NO17: C, 55.33; H, 7.03; N, 1.84. Found: C,
55.23; H, 7.22; N, 1.87.
Further elution afforded 8a as syrup. Yield: 60 mg
(10%).
3.7. 3,4,6-Tri-O-acetyl-1,2-O-[1-exo-(allyl 2-acetamido-
2-deoxy-4,6-di-O-pivaloyl-b-
D-galactopyranoside)] ethyl-
idene-a- -galactopyranose (7)
D
3.9. Allyl 2,3,4,6-tetra-O-acetyl-b-D-galactopyranosyl-
(1“3)-2-acetamido-2-deoxy-4,6-di-O-pivaloyl-a-D-
galactopyranoside (8b)
Boron trifluoride etherate (125 mL, 0.98 mmol) was
added dropwise for 2 h to a stirred mixture of 3a (350
mg, 0.82 mmol), 6 (430 mg, 0.87 mmol) and powdered
The coupling of donor 6 (320 mg, 0.65 mmol) and
acceptor 3b (250 mg, 0.58 mmol) was performed in the
same way as described for 8a to give 8b as syrup.Yield:
,
molecular sieves 4 A (1.5 g) in CH2Cl2 (20 mL) under
Ar at −25 °C. The suspension was diluted with
CH2Cl2 (50 mL), neutralized with Et3N (0.4 mL),
filtered over Celite and the filtrate was concentrated.
The residue was purified on a column of silica gel (2:3
toluene–EtOAc) to give 7 (exo-isomer, 190 mg, 31%)
1
200 mg (45%); [h]D20 +56° (c 0.9, CHCl3). H NMR
(CDCl3): l 5.90 (m, 1 H, ꢀCHꢁ), 5.85 (d, 1 H, JNH,2 9.0
Hz, NH), 5.37 (d, 1 H, J3,4 3.4 Hz, H-4), 5.34 (d, 1 H,
J3%,4% 3.4 Hz, H-4%), 5.29 (dq, 1 H, ꢁCH2trans), 5.25 (dq, 1
H, ꢁCH2cis), 5.10 (dd, 1 H, J1%,2% 7.9, J2%,3% 10.5 Hz, H-2%),
4.96 (d, 1 H, J1,2 3.7 Hz, H-1), 4.94 (dd, 1 H, H-3%), 4.59
(d, 1 H, H-1%), 4.56 (ddd, 1 H, J2,3 10.9 Hz, H-2),
4.25–4.05 (m, 5 H, H-5, H-6a, H-6a%, H-6b%, OCH2),
4.05–3.80 (m, 4 H, H-3, H-6b, H-5%, OCH2), 2.16, 2.06,
2.05, 1.99 and 1.96 (5 s, 15 H, 5 Ac), 1.25 and 1.21 (2
s, 18 H, 2 t-Bu). Anal. Calcd for C35H53NO17: C, 55.33;
H, 7.03; N, 1.84. Found: C, 55.16; H, 7.00; N, 1.88.
1
and the endo-isomer (30 mg, 5%). H NMR (CDCl3): l
5.90 (m, 1 H, ꢀCHꢁ), 5.79 (d, 1 H, J1%,2% 4.4 Hz, H-1%),
5.71 (d, 1 H, JNH,2 7.0 Hz, NH), 5.37 (dd, 1 H, J3%,4% J4%,5%
3.4 Hz, H-4%), 5.36 (d, 1 H, J3,4 3.3 Hz, H-4), 5.30 (d, 1
H, J1,2 8.3 Hz, H-1), 5.28 (dq, 1 H, ꢁCH2trans), 5.21 (dq,
1 H, ꢁCH2cis), 5.03 (dd, 1 H, J2%,3% 6.1 Hz, H-3%), 4.57
(dd, 1 H, J2,3 10.8, J3,4 3.4 Hz, H-3), 4.33 (ddt, 1 H,
OCH2), 4.31 (dd, 1 H, H-2%), 4.27 (ddd, 1 H, J5%,6a% 6.7
Hz, H-5%), 4.18 (dd, 1 H, J6a%,6b% 11.4 Hz, H-6%a), 4.14–
4.02 (m, 4 H, H-6a, H-6b, H-6b%, OCH2), 3.93 (dd, 1 H,
J5,6a 6.6, J5,6b 7.5 Hz, H-5), 3.13 (ddd, H-2), 2.13, 2.06,
2.05 and 1.97 (4 s, 12 H, 4 Ac), 1.63 (s, 3 H, Me), 1.23
and 1.20 (2 s, 18 H, 2 t-Bu).
3.10. Allyl b-
D-galactopyranosyl-(1“3)-2-acetamido-2-
deoxy-4,6-di-O-pivaloyl-b-D-galactopyranoside (9a)
A solution of 8a (140 mg) in dry MeOH (20 mL) was
stirred with 0.1 M methanolic NaOMe (0.35 mL) for 3
h at rt. The pH of the solution was adjusted to 7.0 by
addition of Dowex 50 (H+) resin. The resin was filtered
off and the filtrate was taken to dryness to give 9a as a
syrup. Yield: 95 mg (87%); [h]2D0 +19° (c 0.7, MeOH).
1H NMR (D2O): l 5.90 (m, 1 H, ꢀCHꢁ), 5.49 (d, 1 H,
J3,4 3.1 Hz, H-4), 5.31 (dq, 1 H, ꢁCH2trans), 5.26 (dq, 1
H, ꢁCH2cis), 4.66 (d, 1 H, J1,2 7.9 Hz, H-1), 4.36 (d, 1
H, J1%,2% 7.7 Hz, H-1%), 4.33 (ddt, 1 H, OCH2), 4.20–4.00
(m, 6 H, H-5, H-6a, H-6b, H-2, H-3, OCH2), 3.88 (d, 1
H, J3%,4% 3.5 Hz, H-4%), 3.76 (dd, 1 H, J6a%,5% 7.5, J6a%,6b%
11.5 Hz, H-6a%), 3.71 (dd, 1 H, J6b%,5% 4.6 Hz, H-6b%),
3.62 (dd, 1 H, H-5%), 3.57 (dd, 1 H, J2%,3% 10.0 Hz, H-3%),
3.38 (dd, 1 H, H-2%), 2.02 (s, 3 H, Ac), 1.26 and 1.19 (2
s, 18 H, t-Bu). Anal. Calcd for C27H45NO13·2 H2O: C,
51.95; H, 7.44; N, 2.26. Found: C, 51.67; H, 7.87; N,
2.23.
3.8. Allyl 2,3,4,6-tetra-O-acetyl-b-D-galactopyranosyl-
(1“3)-2-acetamido-2-deoxy-4,6-di-O-pivaloyl-b-D-
galactopyranoside (8a)
A solution of 6 (240 mg, 0.49 mmol) in CH2Cl2 (1 mL)
was added dropwise for 1 h to a suspension of com-
pound 3a (200 mg, 0.47 mmol), BF3·Et2O (64 mL, 0.51
,
mmol) and powdered molecular sieves 4 A (1 g) in
CH2Cl2 (10 mL) at −25° under Ar. The mixture was
diluted with CH2Cl2 (50 mL), and Et3N (0.5 mL) was
added. The suspension was filtered over Celite, the
filtrate was concentrated and the residue was purified
on a column of silica gel (40:1 CH2Cl2–EtOH) to give
8a as syrup. Yield: 140 mg (40%); [h]2D0 +20° (c 0.7,
1
CHCl3). H NMR (CDCl3): l 5.89 (m, 1 H, ꢀCHꢁ),
5.66 (d, 1 H, JNH,2 6.9 Hz, NH), 5.42 (d, 1 H, J3,4 3.6
Hz, H-4), 5.35 (d, 1 H, J3%,4% 3.3 Hz, H-4%), 5.29 (dq, 1
H, ꢁCH2trans), 5.22 (dq, 1 H, ꢁCH2cis), 5.09 (dd, 1 H,
J1%,2% 7.6, J2%,3% 10.4 Hz, H-2%), 5.07 (d, 1 H, J1,2 8.5 Hz,
H-1), 4.98 (dd, 1 H, H-3%), 4.75 (dd, 1 H, J2,3 10.8 Hz,
H-3), 4.61 (d, 1 H, H-1%), 4.34 (ddt, 1 H, OCH2), 4.16
(dd, 1 H, J5%,6a% 4.8, J6a%,6b% 11.4 Hz, H-6a%), 4.13–4.04
(m, 3 H, H-6a, H-6b, OCH2), 3.99 (dd, 1 H, J5%,6b% 7.9
Hz, H-6b%), 3.92–3.38 (m, 2 H, H-5, H-5%), 3.25 (ddd, 1
3.11. Allyl b-
D-galactopyranosyl-(1“3)-2-acetamido-2-
deoxy-4,6-di-O-pivaloyl-a-D-galactopyranoside (9b)
The de-O-acetylation of 8b (180 mg) was carried out in
the same fashion as for 8a to give 9b as syrup. Yield:
1
135 mg (97%); [h]2D0 +88° (c 0.5, MeOH). H NMR
(D2O): l 5.97 (m, 1 H, ꢀCHꢁ), 5.55 (d, 1 H, J3,4 3.0 Hz,

H. Amer et al. / Carbohydrate Research 338 (2003) 35–45
41
H-4), 5.35 (dq, 1 H, ꢁCH2trans), 5.27 (dq, 1 H, ꢁCH2cis),
3.14. Allyl 4,6-O-p-methoxybenzylidene-b-
ranosyl-(1“3)-2-acetamido-2-deoxy-4,6-di-O-
pivaloyl-b- -galactopyranoside (11a)
D-galactopy-
5.00 (d, 1 H, J1,2 3.5 Hz, H-1), 4.48 (dd, 1 H, J2,3 11.1
Hz, H-2), 4.40 (d, 1 H, J1%,2% 7.8 Hz, H-1%), 4.24 (dd, 1
H, J3,4 3.0 Hz, H-3), 4.25–4.11 (m, 3 H, H-5, H-6a,
OCH2), 4.06 (ddt, 1 H, OCH2), 4.00 (dd, 1 H, J6b,5 8.1,
J6a,6b 11.3 Hz, H-6b), 3.88 (d, 1 H, J3%,4% 3.6 Hz, H-4%),
3.77 (dd, 1 H, J6a%,5% 7.5 Hz, H-6a%), 3.71 (dd, 1 H, J6b%,5%
4.3 Hz, H-6b%), 3.62 (dd, 1 H, H-5%), 3.58 (dd, 1 H, J2%,3%
10.0 Hz, H-3%), 3.38 (dd, 1 H, H-2%), 2.02 (s, 3 H, Ac),
1.26 and 1.19 (2 s, 18 H, 2 t-Bu). Anal. Calcd for
C27H45NO13·H2O: C, 53.44; H, 7.55; N, 2.34. Found: C,
53.19; H, 7.77; N, 2.30.
D
A mixture of 9a (140 mg, 0.24 mmol), anisaldehyde
dimethylacetal (170 mL, 0.93 mmol) and p-toluenesul-
fonic acid monohydrate (15 mg) in dry DMF (3 mL)
was stirred overnight at rt. Triethylamine (0.1 mL) was
added and the solution was concentrated. The residue
was purified on silica gel (20:1 CH2Cl2–EtOH) to fur-
nish 11a as colourless syrup. Yield: 120 mg (72%); [h]D20
1
+63° (c 0.7, CHCl3). H NMR (CDCl3): l 7.50–6.80
(m, 4 H, arom. H), 6.05 (d, 1 H, JNH,2 7.4 Hz, NH),
5.88 (m, 1 H, ꢀCHꢁ), 5.47 (s, 1 H, PhCHO), 5.37 (d, 1
H, J3,4 3.4 Hz, H-4), 5.27 (dq, 1 H, ꢁCH2trans), 5.19 (dq,
1 H, ꢁCH2cis), 4.75 (d, 1 H, J1,2 8.2 Hz, H-1), 4.48 (dd,
1 H, J2,3 10.7, J3,4 3.4 Hz, H-3), 4.40 (d, 1 H, J1%,2% 7.4
Hz, H-1%), 4.35 (ddt, 1 H, OCH2), 4.24 (dd, 1 H, J5%,6a%
1.2, J6a%,6b% 12.3 Hz, H-6a%), 4.18 (dd, 1 H, J5,6a 5.3, J6a,6b
11.4 Hz, H-6a), 4.15–4.02 (m, 2 H, H-6b, OCH2), 3.99
(dd, 1 H, J5%,6b% 1.6 Hz, H-6b%), 3.86 (dd, 1 H, J5,6b 7.7
Hz, H-5), 3.81 (s, 3 H, OMe), 3.75–3.55 (m, 4 H, H-2,
H-2%, H-3%, OH), 3.44 (m, 1 H, H-5%), 2.69 (bs, 1 H,
OH), 1.99 (s, 3 H, Ac), 1.24 and 1.20 (2 s, 18 H, 2
t-Bu). Anal. Calcd for C35H51NO14·0.5 H2O: C, 58.28;
H, 7.16; N, 1.90. Found: C, 58.48; H, 7.29; N, 1.95.
3.12. Allyl b-
D-galactopyranosyl-(1“3)-2-acetamido-2-
deoxy-b- -galactopyranoside (10a)
D
Compound 9a (15 mg, 0.03 mmol) was dissolved in
90% aq dioxane (3 mL), and then 40% aq tetra-n-butyl-
ammonium hydroxide (40 mL, 0.22 mmol) was added.
After stirring overnight at ambient temperature, the
solution was neutralized by addition of Dowex 50 (H+)
resin. The resin was filtered off, the filtrate was concen-
trated and the residue was purified on a column of
silica gel (1:1 EtOH–EtOAc) to give 10a as an amor-
phous powder. Yield: 8 mg (75%); [h]2D0 −11° (c 0.5,
1
MeOH). H NMR (D2O): l 5.91 (m, 1 H, ꢀCHꢁ), 5.31
3.15. Allyl 4,6-O-p-methoxybenzylidene-b-
ranosyl-(1“3)-2-acetamido-2-deoxy-4,6-di-O-
pivaloyl-a- -galactopyranoside (11b)
D-galactopy-
(dq, 1 H, ꢁCH2trans), 5.26 (dq, 1 H, ꢁCH2cis), 4.55 (d, 1
H, J1,2 8.5 Hz, H-1), 4.43 (d, 1 H, J1%,2% 7.6 Hz, H-1%),
4.34 (ddt, 1 H, OCH2), 4.25–4.10 (m, 2 H, H-4, OCH2),
4.01 (dd, 1 H, J1,2 8.5, J2,3 10.9 Hz, H-2), 3.9 (d, 1 H,
J3%,4% 3.3 Hz, H-4%), 3.89–3.64 (m, 7 H, H-3, H-5, H-6a,
H-6b, H-5%, H-6a%, H-6b%), 3.65 (dd, 1 H, J2%,3% 9.9 Hz,
H-3%), 3.52 (dd, 1 H, H-2%) and 2.01 (s, 3 H, Ac). Anal.
Calcd for C17H29NO11: C, 48.22; H, 6.90; N, 3.31.
Found: C, 44.26; H, 6.66; N, 2.97.
D
Compound 11b was prepared from 9b (100 mg, 0.17
mmol) and anisalde hydedimethylacetal (125 mL, 0.69
mmol) in the same way as described for the preparation
of 11a. A colorless syrup was obtained after chro-
matography on silica gel (20:1 CH2Cl2–EtOH). Yield:
90 mg (75%); [h]2D0 +55° (c 0.9, CHCl3). 1H NMR
(CDCl3): l 7.45–6.80 (m, 4 H, arom. H), 6.21 (d, 1 H,
JNH,2 6.8 Hz, NH), 5.87 (m, 1 H, ꢀCHꢁ), 5.50 (s, 1 H,
3.13. Allyl b-
D-galactopyranosyl-(1“3)-2-acetamido-2-
PhCHO), 5.35–5.51 (m, 4 H, H-1, H-4, ꢁCH2trans,
deoxy-a- -galactopyranoside (10b)
D
ꢁCH2cis), 4.46 (d, 1 H, J1%,2% 7.9 Hz, H-1%), 4.38–4.00 (m,
11 H, H-2, H-3, H-5, OH, H-6a, H-6b, H-4%, H-6a%,
H-6b%, 2 OCH2), 3.85–3.75 (m, 4 H, OMe, H-2%), 3.66
(ddd, 1 H, J2%,3% 9.5, J3%,4% 3.5, J3%,OH 6.4 Hz, H-3%), 3.50
(m, 1 H, H-5%), 2.59 (d, 1 H, OH), 1.99 (s, 3 H, Ac),
1.26 and 1.19 (2 s, 18 H, 2 t-Bu). Anal. Calcd for
C35H51NO14: C, 59.23; H, 7.24; N, 1.97. Found: C,
58.88; H, 7.29; N, 1.74.
The deprotection of 9b (15 mg) was done as described
for 9a to give 10b as syrup. Yield: 9.5 mg (89%); [h]D20
+132° (c 0.7, MeOH). H NMR (D2O): l 5.97 (m, 1
1
H, ꢀCHꢁ), 5.35 (dq, 1 H, ꢁCH2trans), 5.25 (dq, 1 H,
ꢁCH2cis), 4.94 (d, 1 H, J1,2 3.8 Hz, H-1), 4.46 (d, 1 H,
J1%,2% 7.7 Hz, H-1%), 4.34 (dd, 1 H, J2,3 11.1 Hz, H-2),
4.24 (d, 1 H, J3,4 3.2 Hz, H-4), 4.21 (ddt, 1 H, OCH2),
4.10–3.95 (m, 3 H, H-3, H-5, OCH2), 3.90 (d, 1 H, J3%,4%
3.4, Hz, H-4%), 3.85–3.68 (m, 4 H, H-6a, H-6b, H-6a%,
H-6b%), 3.64 (dd, 1 H, J6a%,5% 6.9, J6b%,5% 4.4 Hz, H-5%), 3.62
(dd, 1 H, J2%,3% 10.0 Hz, H-3%), 3.51 (dd, 1 H, H-2%) and
2.01 (s, 3 H, Ac). Anal. Calcd for C17H29NO11·H2O: C,
46.25; H, 7.08; N, 3.17. Found C, 46.34; H, 7.02; N,
3.02.
3.16. Allyl 3-O-benzoyl-4,6-O-p-methoxybenzylidene-b-
D-galactopyranosyl-(1“3)-2-acetamido-2-deoxy-4,6-di-
O-pivaloyl-b- -galactopyranoside (12a)
D
A mixture of 11a (115 mg, 0.16 mmol) and benzoyl
imidazole (60 mg, 0.35 mmol) in dry CHCl3 (10 mL)
was refluxed for 48 h at 80 °C. The mixture was diluted

42
H. Amer et al. / Carbohydrate Research 338 (2003) 35–45
with CH2Cl2 (50 mL) and MeOH (0.2 mL) was added.
Concentration of the solution gave a residue, which was
purified on silica gel (20:1 CH2Cl2–EtOH) to furnish
12a as syrup. Yield: 95 mg (72%); [h]2D0 +63° (c 0.7,
CHCl3). 1H NMR (CDCl3): l 8.50–6.80 (m, 9 H, arom.
H), 6.10 (d, 1 H, JNH,2 7.5 Hz, NH), 5.87 (m, 1 H,
ꢀCHꢁ), 5.45 (s, 1 H, PhCHO), 5.37 (d, 1 H, J3,4 3.5 Hz,
H-4), 5.27 (dq, 1 H, ꢁCH2trans), 5.18 (dq, 1 H, ꢁCH2cis),
5.09 (dd, 1 H, J2%,3% 10.1, J3%,4% 3.4 Hz, H-3%), 4.62 (d, 1 H,
J1%,2% 8.3 Hz, H-1%), 4.58 (d, 1 H, J1,2 7.8 Hz, H-1),
4.45–4.38 (m, 2 H, H-3, H-4%), 4.34 (ddt, 1 H, OCH2),
4.25 (dd, 1 H, J5%,6a% 1.1, J6a%,6b% 12.2 Hz, H-6a%), 4.18 (dd,
1 H, J5,6a 5.4, J6a,6b 11.4 Hz, H-6a), 4.13–4.04 (m, 3 H,
H-6b, H-2%, OCH2), 4.02 (dd, 1 H, J5%,6b% 1.3 Hz, H-6b%),
3.88–3.82 (m, 2 H, H-5, OH), 3.79 (s, 3 H, OMe), 3.76
(ddd, 1 H, J2,3 10.3 Hz, H-2), 3.59 (m, 1 H, H-5%), 1.98
(s, 3 H, Ac), 1.23 and 1.21 (2 s, 18 H, 2 t-Bu). Anal.
Calcd for C42H55NO15: C, 61.98; H, 6.81; N, 1.72.
Found: C, 61.76; H, 6.64; N, 1.64.
Hz, NH), 5.87 (m, 1 H, ꢀCHꢁ), 5.49 (s, 1 H, PhCHO),
5.40 (m, 1 H, H-4), 5.28 (dq, 1 H, ꢁCH2trans), 5.23–5.15
(m, 2 H, H-1, ꢁCH2cis), 5.11 (dd, 1 H, J2%,3% 10.0, J3%,4% 3.4
Hz, H-3%), 4.65 (d, 1 H, J1%,2% 7.9 Hz, H-1%), 4.47 (d, 1 H,
H-4%), 4.38–4.13 (m, 8 H, H-2, H-3, H-5, H-6a, H-2%,
H-6a%, OH, OCH2), 4.13–4.00 (m, 3 H, H-6b, H-6b%,
OCH2), 3.83 (s, 3 H, OMe), 3.66 (m, 1 H, H-5%), 2.03 (s,
3 H, Ac), 1.28 and 1.23 (2 s, 18 H, 2 t-Bu). Anal. Calcd.
for C42H55NO15: C, 61.98; H, 6.81; N, 1.72. Found: C,
61.68; H, 6.43; N, 1.59.
3.19. Allyl 3,4-di-O-acetyl-2-O-methyl-a-
osyl-(1“2)-3-O-benzoyl-4,6-O-p-methoxybenzylidene-
b- -galactopyranosyl-(1“3)-2-acetamido-2-deoxy-4,6-
di-O-pivaloyl-b- -galactopyranoside (15a) and allyl 3,4-
di-O-acetyl-2-O-methyl-b- -fucopyranosyl-(1“2)-3-O-
benzoyl-4,6-O-p-methoxybenzylidene-b- -galactopyran-
L-fucopyran-
D
D
L
D
osyl-(1“3)-2-acetamido-2-deoxy-4,6-di-O-pivaloyl-b-D-
galactopyranoside (16a)
3.17. Allyl 2-O-acetyl-3-O-benzoyl-4,6-O-p-methoxy-
A solution of NIS (16 mg, 0.7 mmol) and TfOH (4 mL)
in 1:1 CH2Cl2–Et2O (2 mL) was added to a suspension
of 12a (40 mg, 0.05 mmol), 14 (20 mg, 0.07 mmol) and
benzylidene-b-D-galactopyranosyl-(1“3)-2-acetamido-
2-deoxy-4,6-di-O-pivaloyl-b-
D-galactopyranoside (13a)
,
powdered molecular sieves 4 A (0.4 g) in 1:1 CH2Cl2–
A solution of 12a (40 mg) and a catalytic amount of
4-N,N-dimethylaminopyridine in dry pyridine (2 mL)
was stirred with Ac2O (200 mL) overnight at rt.
Methanol (1 mL) was added and the solution was
coevaporated three times with addition of toluene. The
solution was concentrated and purified on silica gel (1:1
n-hexane–EtOAc) to furnish 13a as a syrup. Yield: 40
mg (95%); [h]2D0 +90° (c 1.0, CHCl3). 1H NMR
(CDCl3): l 8.20–6.80 (m, 9 H, arom. H), 5.96–5.75 (m,
2 H, NH, CHꢁ), 5.50–5.40 (m, 3 H, H-2%, H-4, Ph-
CHO), 5.26 (dq, 1 H, ꢁCH2trans), 5.20 (dq, 1 H,
ꢁCH2cis), 5.09 (dd, 1 H, J2%,3% 10.4, J3%,4% 3.5 Hz, H-3%),
5.05 (d, 1 H, J1,2 8.8 Hz, H-1), 4.71 (dd, 1 H, J2,3 10.5,
J3,4 3.5 Hz, H-3), 4.69 (d, 1 H, J1%,2% 7.9 Hz, H-1%), 4.44
(d, 1 H, H-4%), 4.37–4.26 (m, 2 H, H-6a%, OCH2), 4.21
(dd, 1 H, J5,6a 4.1, J6a,6b 11.2 Hz, H-6a), 4.07 (ddt, 1 H,
OCH2), 4.03–3.94 (m, 2 H, H-6b, H-6b%), 3.88 (dd, 1 H,
J5,6a 4.1, J5,6b 7.1 Hz, H-5), 3.79 (s, 3 H, OMe), 3.54 (m,
1 H, H-5%), 3.37 (ddd, 1 H, J2,NH 7.0 Hz, H-2), 1.97,
1.96 (2 s, 6 H, 2 Ac), 1.23 and 1.21 (2 s, 18 H, 2 t-Bu).
Et2O (2 mL) at −20° under Ar. The suspension was
stirred overnight at 5°, diluted with CH2Cl2 (50 mL)
and filtered over Celite. The filtrate was washed with
5% aq Na2S2O3, satd aq NaHCO3 and dried (Na2SO4).
The solution was concentrated and purified on silica gel
(1:1 toluene–EtOAc) to afford 15a as syrup. Yield: 22
mg (42%); [h]2D0 +37° (c 0.5, CHCl3). 1H NMR
(CDCl3): l 8.10–6.80 (m, 9 H, arom. H), 6.57 (d, 1 H,
JNH,2 6.6 Hz, NH), 5.92 (m, 1 H, ꢀCHꢁ), 5.57 (d, 1 H,
J3,4 3.7 Hz, H-4), 5.36–5.42 (m, 2 H, H-1¦, PhCHO),
5.30 (dq, 1 H, ꢁCH2trans), 5.26–5.15 (m, 3 H, H-3%,
H-4¦, ꢁCH2cis), 5.12 (dd, 1 H, J2¦,3¦ 10.5, J3¦,4¦ 3.2 Hz,
H-3¦), 4.96 (d, 1 H, J1,2 8.2 Hz, H-1), 4.94 (dd, 1 H, J2,3
9.9 Hz, H-3), 4.66 (d, 1 H, J1%,2% 7.7 Hz, H-1%), 4.45–4.28
(m, 4 H, H-4%, H-6a%, H-5¦, OCH2), 4.25 (dd, 1 H, J5,6a
3.8, J6a,6b 11.2 Hz, H-6a), 4.19 (dd, 1 H, J2%,3% 9.8 Hz,
H-2%), 4.10 (ddt, 1 H, OCH2), 4.05–3.90 (m, 3 H, H-5,
H-6b, H-6b%), 3.82 (s, 3 H, MeOPh), 3.55 (m, 1 H,
H-5%), 3.45 (dd, 1 H, J1¦,2¦ 3.5 Hz, H-2¦), 3.34 (ddd, 1 H,
J2,3 9.9 Hz, H-2), 3.03 (s, 3 H, OMe), 2.14, 2.00, 1.97 (3
s, 9 H, 3 Ac), 1.26, 1.22 (2 s, 18 H, 2 t-Bu) and 1.09 (d,
3 H, J5¦,6¦ 6.5 Hz, H-6¦). Anal. Calcd for C53H71NO21:
C, 60.16; H, 6.76; N, 1.32. Found: C, 60.00; H, 7.00; N,
1.39.
3.18. Allyl 3-O-benzoyl-4,6-O-p-methoxybenzylidene-b-
D-galactopyranosyl-(1“3)-2-acetamido-2-deoxy-4,6-di-
O-pivaloyl-a- -galactopyranoside (12b)
D
Further elution afforded 16a as syrup. Yield: 8 mg
1
Compound 12b was prepared from 11b (85 mg, 0.12
mmol) and benzoyl imidazole (45 mg, 0.26 mmol) in
the same way as described for the preparation of 12a.
Chromatography on silica gel (20:1 CH2Cl2–EtOH)
furnished 12b as a colorless syrup. Yield: 85 mg (87%);
[h]2D0 +112° (c 0.9, CHCl3). 1H NMR (CDCl3): l
8.20–6.80 (m, 9 H, arom. H), 6.28 (d, 1 H, JNH,2 6.7
(19%). H NMR (CDCl3): l 8.10–6.80 (m, 9 H, arom.
H), 6.43 (d, 1 H, JNH,2 7.0 Hz, NH), 5.91 (m, 1 H,
ꢀCHꢁ), 5.45 (d, 1 H, J3,4 3.4 Hz, H-4), 5.41 (s, 1 H,
PhCHO), 5.27 (dq, 1 H, ꢁCH2trans), 5.18 (dq, 1 H,
ꢁCH2cis), 5.08 (dd, 1 H, J2%,3% 10.8, J3%,4% 3.4 Hz, H-3%),
5.06 (d, 1 H, J3¦,4¦ 3.5 Hz, H-4¦), 5.01 (d, 1 H, J1,2 8.4
Hz, H-1), 4.83 (dd, 1 H, J2¦,3¦ 10.3 Hz, H-3¦), 4.66 (d, 1

H. Amer et al. / Carbohydrate Research 338 (2003) 35–45
43
3.21. Allyl 3,4-di-O-acetyl-2-O-methyl-a-
osyl-(1“2)-4,6-di-O-acetyl-3-O-benzoyl-b-
ranosyl-(1“3)-2-acetamido-2-deoxy-4,6-di-O-pivaloyl-
b- -galactopyranoside (17a)
L
-fucopyran-
H, J1¦,2¦ 7.6 Hz, H-1¦), 4.55 (d, 1 H, J1%,2% 7.9 Hz, H-1%),
4.50–3.85 (m, 10 H, H-3, H-5, H-6a, H-6b, H-2%, H-4%,
H-6a%, H-6b%, 2 OCH2), 3.79 (s, 3 H, MeOPh), 3.65–
3.50 (m, 5 H, H-2, H-5¦, OMe), 3.48 (m, 1 H, H-5%),
3.08 (dd, 1 H, H-2¦), 2.03, 2.00 and 1.84 (3 s, 9 H, 3
Ac), 1.28, 1.21 (2 s, 18 H, 2 t-Bu) and 1.00 (d, 3 H,
J5¦,6¦ 6.4 Hz, H-6¦).
D-galactopy-
D
A solution of 15a (20 mg, 0.02 mmol) in CH2Cl2 (3 mL)
was treated with 90% aq CF3CO2H (20 mL) at rt.
Triethylamine (0.1 mL) was added and the solution was
concentrated. The residue was dried, dissolved in pyri-
dine (2 mL) and stirred with a catalytic amount of
4-N,N-dimethylaminopyridine and Ac2O (200 mL)
overnight at rt. Methanol (0.5 mL) was added and the
solution was concentrated. Purification of the residue
on silica gel (1:2 toluene–EtOAc) afforded 17a as
syrup. Yield: 18 mg (94%); [h]2D0 −11° (c 0.9, CHCl3).
1H NMR (CDCl3): l 8.00–7.40 (m, 5 H, arom. H), 6.50
(d, 1 H, J2,NH 6.5 Hz, NH), 5.93 (m, 1 H, ꢀCHꢁ), 5.51
(d, 1 H, J3%,4% 3.3 Hz, H-4%), 5.49 (d, 1 H, J3,4 4.0 Hz,
H-4), 5.39 (d, 1 H, J1¦,2¦ 3.5 Hz, H-1¦), 5.36–5.27 (m, 2
H, H-3%, ꢁCH2trans), 5.26–5.18 (m, 2 H, H-4¦, ꢁCH2cis),
5.13 (dd, 1 H, J2¦,3¦ 10.6, J3¦,4¦ 3.3 Hz, H-3¦), 5.05 (dd,
1 H, J2,3 10.0 Hz, H-3), 5.02 (d, 1 H, J1,2 8.3 Hz, H-1),
4.69 (d, 1 H, J1%,2% 7.7 Hz, H-1%), 4.45–4.30 (m, 2 H,
H-5¦, OCH2), 4.20–3.88 (m, 7 H, H-6a, H-6b, H-2%,
H-5%, H-6a%, H-6b%, H-4¦, OCH2), 3.47 (dd, 1 H, H-2¦),
3.21 (ddd, 1 H, H-2), 3.07 (s, 3 H, OMe), 2.15, 2.08 and
2.00 (3 s, 15 H, 5 Ac), 1.29 and 1.23 (2 s, 18 H, 2 t-Bu),
1.16 (d, 3 H, J5¦,6¦ 6.5 Hz, H-6¦). Anal. Calcd for
C49H69NO22: C, 57.47; H, 6.79; N, 1.37. Found: C,
57.64; H, 6.77; N, 1.45.
3.20. Allyl 3,4-di-O-acetyl-2-O-methyl-a-
osyl-(1“2)-3-O-benzoyl-4,6-O-p-methoxybenzylidene-
b- -galactopyranosyl-(1“3)-2-acetamido-2-deoxy-4,6-
di-O-pivaloyl-a- -galactopyranoside (15b) and allyl 3,4-
di-O-acetyl-2-O-methyl-b- -fucopyranosyl-(1“2)-3-O-
benzoyl-4,6-O-p-methoxybenzylidene-b- -galactopyran-
L-fucopyran-
D
D
L
D
osyl-(1“3)-2-acetamido-2-deoxy-4,6-di-O-pivaloyl-a-D-
galactopyranoside (16b)
Compound 12b (32 mg, 0.04 mmol) was reacted with 14
(15 mg, 0.05 mmol) in the same way as described for
the preparation of 15a. Purification of the residue on
silica gel (1:1 toluene–EtOAc) afforded 15b as syrup.
1
Yield: 20 mg (48%); [h]2D0 +103° (c 0.7, CHCl3). H
NMR (CDCl3): l 8.10–6.80 (m, 10 H, arom. H, NH),
5.81 (m, 1 H, ꢀCHꢁ), 5.43 (s, 1 H, PhCHO), 5.40 (d, 1
H, J3,4 3.1 Hz, H-4), 5.27 (dd, 1 H, J2%,3% 10.1, J3%,4% 4.0
Hz, H-3%), 5.26–5.20 (m, 3 H, H-1¦, H-4¦, ꢁCH2trans),
5.20–5.12 (m, 2 H, H-1, ꢁCH2cis), 4.98 (dd, 1 H, J2¦,3¦
10.5, J3¦,4¦ 3.2 Hz, H-3¦), 4.50 (d, 1 H, J1%,2% 7.6 Hz,
H-1%), 4.48 (ddd, 1 H, J1,2 3.7, J2,3 10.8, J2,NH 8.3 Hz,
H-2), 4.40–4.28 (m, 4 H, H-6a, H-4%, H-6a%, H-5¦),
4.17–4.04 (m, 3 H, H-3, H-6b, OCH2), 4.01 (dd, 1 H,
J2%,3% 10.1 Hz, H-2%), 3.96–3.78 (m, 6 H, H-6b%, H-5,
MeOPh, OCH2), 3.45 (m, 1 H, H-5%), 3.36 (dd, 1 H,
J1¦,2¦ 3.5 Hz, H-2¦), 3.02 (s, 3 H, OMe), 2.08, 1.98 and
1.85 (3 s, 9 H, 3 Ac), 1.26 and 1.21 (2 s, 18 H, 2 t-Bu),
1.07 (d, 3 H, J5¦,6¦ 6.5 Hz, H-6¦). Anal. Calcd for
C53H71NO21: C, 60.16; H, 6.76; N, 1.32. Found: C,
59.64; H, 6.87; N, 1.25.
3.22. Allyl 3,4-di-O-acetyl-2-O-methyl-a-
osyl-(1“2)-4,6-di-O-acetyl-3-O-benzoyl-b-
ranosyl-(1“3)-2-acetamido-2-deoxy-4,6-di-O-pivaloyl-
a- -galactopyranoside (17b)
L-fucopyran-
D-galactopy-
D
A solution of 16b (22 mg, 0.02 mmol) in CH2Cl2 (3 mL)
was treated with 90% aq CF3CO2H (20 mL) at rt for 1
h. Treatment as described for 17a gave a residue which
was dissolved in pyridine (2 mL) and stirred with a
catalytic amount of 4-N,N-dimethylaminopyridine and
Ac2O (200 mL) overnight at rt. Methanol (0.5 mL) was
added and the solution was concentrated. Purification
of the residue on silica gel (1:2 toluene–EtOAc) af-
forded 17b as syrup. Yield: 19 mg (90%); [h]2D0 +45° (c
Further elution afforded 16b as syrup. Yield: 9 mg
1
(20%). H NMR (CDCl3): l 8.25–6.75 (m, 10 H, arom.
H, NH), 5.84 (m, 1 H, ꢀCHꢁ), 5.42 (s, 1 H, PhCHO),
5.38 (d, 1 H, J3,4 2.8 Hz, H-4), 5.22 (dq, 1 H, ꢁCH2trans),
5.20–5.10 (m, 3 H, H-1, H-3%, ꢁCH2cis), 5.01 (d, 1 H,
J3¦,4¦ 3.4 Hz, H-4¦), 4.88 (dd, 1 H, J2¦,3¦ 10.3 Hz, H-3¦),
4.62 (ddd, 1 H, J1,2 3.4, J2,3 10.8, J2,NH 7.7 Hz, H-2),
4.58 (d, 1 H, J1¦,2¦ 7.7 Hz, H-1¦), 4.50 (d, 1 H, J1%,2% 7.9
Hz, H-1%), 4.42–4.30 (m, 3 H, H-6a, H-4%, H-6a%),
4.20–4.05 (m, 4 H, H-3, H-6b, H-2%, OCH2), 3.95 (ddt,
1 H, OCH2), 3.92–3.87 (m, 5 H, H-5, H-6b%, MeOPh),
3.61–3.54 (m, 4 H, H-5¦, OMe), 3.46 (m, 1 H, H-5%),
3.27 (dd, 1 H, H-2¦), 2.00 and 1.88 (2 s, 9 H, 3 Ac),
1.28, 1.21 (2 s, 18 H, 2 t-Bu), and 0.85 (d, 3 H, J5¦,6¦ 6.4
Hz, H-6¦).
1
0.9, CHCl3). H NMR (CDCl3): l 7.97–7.35 (m, 5 H,
arom. H), 6.97 (d, 1 H, JNH,2 7.5 Hz, NH), 5.83 (m, 1
H, ꢀCHꢁ), 5.50–5.45 (m, 2 H, H-4, H-4%), 5.38 (dd, 1
H, J2%,3% 9.9, J3%,4% 3.5 Hz, H-3%), 5.30–5.15 (m, 4 H, H-1¦,
H-4¦, ꢁCH2trans, ꢁCH2cis), 5.14 (d, 1 H, J1,2 3.5 Hz,
H-1), 5.04 (dd, 1 H, J2¦,3¦ 10.5, J3¦,4¦ 3.2 Hz, H-3¦), 4.62
(d, 1 H, J1%,2% 7.4 Hz, H-1%), 4.50 (ddd, 1 H, J1,2 3.4, J2,3
11.0 Hz, H-2), 4.40 (dd, 1 H, J5¦,6¦ 6.5 Hz, H-5¦), 4.38
(dd, 1 H, J5,6a 3.2, J6a,6b 11.7 Hz, H-6a), 4.19–4.00 (m,
5 H, H-3, H-5, H-6a%, H-6b%, OCH2), 4.00–3.80 (m, 4
H, H-6b, H-2%, H-5%, OCH2), 3.39 (dd, 1 H, J1¦,2¦ 3.6

44
H. Amer et al. / Carbohydrate Research 338 (2003) 35–45
Hz, H-2¦), 3.00 (s, 3 H, OMe), 2.10, 2.09, 2.07, 2.01 and
1.90 (5 s, 15 H, 5 Ac), 1.29 and 1.22 (2 s, 18 H, 2 t-Bu),
1.14 (d, 3 H, H-6¦). Anal. Calcd for C49H69NO22: C,
57.47; H, 6.79; N, 1.37. Found: C, 57.32; H, 7.04; N,
1.47.
49.39; H, 7.08; N, 2.40: Found: C, 49.01; H, 7.20; N,
2.42.
3.25. 3-(2-Aminoethylthiopropyl) 2-O-methyl-a-
fucopyranosyl-(1“2)-b- -galactopyranosyl-(1“3)-2-
-galactopyranoside hydrochloride
L-
D
acetamido-2-deoxy-b-
D
3.23. Allyl 2-O-methyl-a-
L-fucopyranosyl-(1“2)-b-
D-
(19a)
galactopyranosyl-(1“3)-2-acetamido-2-deoxy-b-D-
galactopyranoside (18a)
A solution of 18a (3.9 mg, 0.007 mmol), cysteamine
hydrochloride (4.2 mg, 0.037 mmol) in water (0.4 mL)
was stirred under UV light for 4 h at rt. The solution
was then purified on Dowex AG WX8 (NH+4 form)
using a gradient 0“0.1 M aq NH3. Carbohydrate-con-
taining fractions were pooled and lyophilized to afford
19a as an amorphous powder. Yield: 3.1 mg (70%);
A solution of 17a (16 mg, 0.016 mmol) in dry MeOH
(10 mL) was stirred with 0.1 M methanolic NaOMe
(0.5 mL) overnight at rt. The pH of the solution was
adjusted to 7.0 by addition of Dowex 50 (H+) resin, the
resin was filtered off and the filtrate was taken to
dryness. The residue was dissolved in aq 90% 1,4-diox-
ane (2.5 mL) and aq 40% tetra-n-butylammonium hy-
droxide (50 mL, 0.077 mmol) was added. After stirring
overnight at ambient temperature, the solution was
neutralized by addition of Dowex 50 (H+) resin and the
resin was filtered off. The filtrate was concentrated and
the residue was first purified on column of silica gel (C,
3:2 EtOH–EtOAc), then on a column of Sephadex
LH-20 (MeOH) to give 18a as syrup. Yield: 7.5 mg
(82%); [h]2D0 −55° (c 0.6, H2O). 1H NMR (D2O): l 5.88
(m, 1 H, ꢀCHꢁ), 5.45 (d, 1 H, J1¦,2¦ 3.9 Hz, H-1¦), 5.27
(dq, 1 H, ꢁCH2trans), 5.23 (dq, 1 H, ꢁCH2cis), 4.58 (d, 1
H, J1%,2% 7.6 Hz, H-1%), 4.35 (d, 1 H, J1,2 8.1 Hz, H-1),
4.33 (m, 1 H, OCH2), 4.22 (dd, 1 H, J5¦,6¦ 6.6 Hz, H-5¦),
4.15–4.05 (m, 2 H, H-4, OCH2), 3.98 (dd, 1 H, J2,3 10.9
Hz, H-2), 3.89 (dd, 1 H, J3,4 3.1 Hz, H-3), 3.86 (d, 1 H,
J3%,4% 3.3 Hz, H-4%), 3.84–3.69 (m, 5 H, H-6a, H-6b,
H-3%, H-6a%, H-6b%), 3.69–3.60 (m, 5 H, H-2%, H-5%, H-5,
H-3¦, H-4¦), 3.49 (s, 3 H, OMe), 3.44 (dd, 1 H, J2¦,3¦
10.3 Hz, H-2¦), 2.02 (s, 3 H, Ac), 1.20 (d, 3 H, H-6¦).
Anal. Calcd for C24H41NO15: C, 49.39; H, 7.08; N, 2.40:
Found: C, 49.69; H, 6.77; N, 2.41.
1
[h]2D0 −26° (c 0.3, H2O). H NMR (D2O): l 5.40 (d, 1
H, J1¦, 2¦ 3.9 Hz, H-1¦), 4.54 (d, 1 H, J1%,2% 7.6 Hz, H-1%),
4.25 (d, 1 H, J1,2 7.6 Hz, H-1), 4.16 (dd, 1 H, J5¦,6¦ 6.6
Hz, H-5¦), 4.05 (d, 1 H, J3,4 2.1 Hz, H-4), 4.00–3.85 (m,
3 H, H-2, H-3, OCH2), 3.82 (d, 1 H, J3%,4% 3.5 Hz, H-4%),
3.78–3.68 (m, 5 H, H-6a, H-6b, H-3, H-6a%, H-6b%),
3.68–3.53 (m, 6 H, H-5, H-5%, H-2%, H-3¦, H-4¦, OCH2),
3.44 (s, 1 H, OMe), 3.40 (dd, 1 H, J2¦,3¦ 10.3 Hz, H-2¦),
3.03 (t, 2 H, CH2N), 2.73 (t, 2 H, SCH2), 2.54 (t, 2 H,
CH2S), 1.99 (s, 3 H, Ac), 1.77 (m, 2 H, CH2), 1.20 (d,
3 H, H-6¦). MALDI-TOF MS: m/z 662.03 [MH]+,
684.45 [M+Na]+.
3.26. 3-(2-Aminoethylthiopropyl) 2-O-methyl-a-
fucopyranosyl-(1“2)-b- -galactopyranosyl-(1“3)-2-
-galactopyranoside hydrochloride
L-
D
acetamido-2-deoxy-a-
D
(19b)
A solution of 18b (5.6 mg, 0.01 mmol) and cysteamine
hydrochloride (5.9 mg, 0.052 mmol) in water (0.6 mL)
was stirred under UV light for 3 h at rt. The solution
was then purified on Dowex AG WX8 (NH+4 form)
using a gradient 0“0.1 M aq NH3. Carbohydrate-con-
taining fractions were pooled and lyophilized to give
19b as an amorphous powder. Yield: 4.5 mg (71%);
3.24. Allyl 2-O-methyl-a-L-fucopyranosyl-(1“2)-b-D-
galactopyranosyl-(1“3)-2-acetamido-2-deoxy-a-D-
galactopyranoside (18b)
1
[h]2D0 +18° (c 0.5, H2O). H NMR (D2O): l 5.41 (d, 1
H, J1¦,2¦ 3.9 Hz, H-1¦), 4.82 (d, 1 H, J1,2 3.1 Hz, H-1),
4.56 (d, 1 H, J1%,2% 7.5 Hz, H-1%), 4.22–4.90 (m, 5 H,
H-2, H-3, H-4, H-5, H-5¦), 3.82 (d, 1 H, J3%,4% 3.4 Hz,
H-4%), 3.80–3.65 (m, 6 H, H-6a, H-6b, H-3%, H-6a%,
H-6b%, OCH2), 3.65–3.54 (m, 4 H, H-2%, H-5%, H-3¦,
H-4¦), 3.52–3.37 (m, 5 H, H-2¦, OCH2, OMe), 3.09 (t,
2 H, CH2N), 2.76 (t, 2 H, SCH2), 2.64 (t, 2 H, CH2S),
1.99 (s, 3 H, Ac), 1.84 (m, 2 H, CH2), 1.14 (d, 3 H, J5¦,6¦
6.6 Hz, H-6¦). MALDI-TOF MS: m/z 684.45 [M+
Na]+.
The deprotection of 17b (12 mg) was done as described
for 17a. The residue was purified on a column of silica
gel (3:2 EtOH–EtOAc) and then on a column of Sep-
hadex LH-20 to give 18b as syrup. Yield: 5.0 mg (73%);
1
[h]2D0 +14° (c 0.6, H2O). H NMR (D2O): l 5.95 (m, 1
H, ꢀCHꢁ), 5.45 (d, 1 H, J1¦,2¦ 3.9 Hz, H-1¦), 5.32 (dq, 1
H, ꢁCH2trans), 5.24 (dq, 1 H, ꢁCH2cis), 4.90 (d, 1 H, J1,2
2.7 Hz, H-1), 4.62 (d, 1 H, J1%,2% 7.7 Hz, H-1%), 4.27–4.10
(m, 5 H, H-2, H-3, H-4, H-5¦, OCH2), 4.07–3.93 (m, 2
H, H-5, OCH2), 3.87 (d, 1 H, J3%,4% 3.5 Hz, H-4%),
3.85–3.70 (m, 5 H, H-6a, H-6b, H-3%, H-6a%, H-6b%),
3.70–3.60 (m, 4 H, H-2%, H-5%, H-3¦, H-4¦), 3.52–3.40
(m, 4 H, H-2¦, OMe), 2.03 (s, 3 H, Ac), 1.18 (d, 3 H,
J5¦,6¦ 6.6 Hz, H-6¦). Anal. Calcd for C24H41NO15: C,
3.27. Synthesis of the BSA-conjugate 20a
A solution of thiophosgene (2 mL) in CHCl3 (2 mL) was
added to 19a (3.1 mg, 0.0047 mmol) dissolved in 0.1 M

H. Amer et al. / Carbohydrate Research 338 (2003) 35–45
45
NaHCO3 (1.5 mL). The solution was kept under high
References
speed stirring for 2 h at rt. The organic phase was
removed and the aqueous phase was extracted 3 times
with CHCl3 (3 mL). Traces of CHCl3 in the aq phase
were removed by evaporation under a stream of N2.
The aqueous phase was then added to a solution of
BSA (3.1 mg) in a 1:1 mixture of 0.3 M NaCl and 0.1
M NaHCO3 (1.5 mL). The solution was slowly stirred
for 48 h at rt and was then separated by gel permeation
chromatography on a Sephadex G-25 column (0.01 M
NaHCO3). The BSA-glycoconjugate fractions (ninhy-
drine-positive) were pooled and dialyzed against water
for 48 h. Lyophilization gave the BSA-conjugate 20a.
Yield: 2.3 mg. The carbohydrate–BSA ratio was deter-
mined via MALDI-TOF-MS: [M]+ 71,227 which corre-
sponds to 6.8 mol ligand/mol BSA.
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3.28. Synthesis of the BSA-conjugate 20b
A solution of thiophosgene (2 mL) in CHCl3 (2 mL) was
added to 19b (4.5 mg, 0.0068 mmol) dissolved in 0.1 M
NaHCO3 (1.5 mL). The solution was kept under high
speed stirring at rt for 2 h. Work-up was done as
described for 20a. The aqueous phase was added to a
solution of BSA (3.0 mg) in a 1:1 mixture of 0.3 M
NaCl and 0.1 M NaHCO3 (1.5 mL). The reaction
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was separated by gel permeation chromatography on a
Sephadex G-25 column. The ninhydrine-positive frac-
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3.1 mol ligand/mol BSA.
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8
Financial support by a scholarship from OAD (Aus-
trian Academic Exchange Office) to Hassan Amer is
gratefully acknowledged. The authors also thank Dr
Michael Puchberger for recording NMR spectra, Dr
Fritz Altmann for providing MALDI measurements
and Maria Hobel for technical assistance.
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55–59.