Synthesis of α-galactosylated fragments related to the core-structure of the GPI anchor of Trypanosoma brucei

Article abstract of DOI:10.1016/S0008-6215(00)00071-9

A series of octyl glycosides di- to tetrasaccharides related to the GPI anchor of Trypanosoma brucei was prepared. Treatment of octyl 2-O-benzoyl-4,6-O-(1,1,3,3-tetraisopropyl-1,3-disiloxane-1,3-diyl)-α-D-mannopyranoside with ethyl 2,3,4,6-tetra-O-benzyl-1-thio-β-D-galactopyranoside under activation with bromine and silver trifluoromethanesulfonate afforded the α-linked disaccharide octyl 2-O-benzoyl-3-O-(2,3,4,6-tetra-O-benzyl-α-D-galactopyranosyl)-4,6-O-(1,1,3,3-tetraisopropyl-1,3-disiloxane-1,3-diyl)-α-D-mannospyranoside, the siloxane ring of which was regioselectively opened with a HF-pyridine complex to give the disaccharide acceptor octyl 3-O-(2,3,4,6-tetra-O-benzyl-α-D-galactopyranosyl)-2-O-benzoyl-4-O-(3-fluoro-1,1,3,3-tetraisopropyl-1,3-disiloxane-3-yl)-α-D-mannopyranoside (4). Mannosylation of 4 with benzobromomannose (7), followed by fluoride catalyzed desilylation gave the trisaccharide octyl 2-O-benzoyl-6-O-(2,3,4,6-tetra-O-benzoyl-α-D-mannopyranosyl)-3-O-(2,3,4,6-tetra-O-benzyl-α-D-galactopyranosyl)-α-D-mannospyranoside, which was deblocked via the deacylated intermediate octyl 3-O-(2,3,4,6-tetra-O-benzyl-α-D-galactopyranosyl)-6-O-(α-D-mannopyranosyl)-α-D-mannospyranoside to afford the octyl glycoside trisaccharide octyl 3-O-(α-D-galactopyranosyl)-6-O-(α-D-mannopyranosyl)-α-D-mannospyranoside. Glycosylation of 4 with 3,4,6-tri-O-acetyl-2-O-(2,3,4,6-tetra-O-benzoyl-α-D-mannopyranosyl)-α-D-mannopyranosyl trichloroacetimidate resulted in the tetrasaccharide octyl 2-O-benzoyl-4-O-(1-fluoro-1,1,3,3-tetraisopropyl-1,3-disiloxane-3-yl)-3-O-(2,3,4,6-tetra-O-benzyl-α-D-galactopyranosyl)-6-O-[2-O-(2,3,4,6-tetra-O-benzoyl-α-D-mannopyranosyl)-3,4,6-tri-O-acetyl-α-D-mannopyranosyl]-α-D-mannospyranoside, sequential desilylation, deacylation and debenzylation, respectively, of which via the intermediate octyl 2-O-benzoyl-3-O-(2,3,4,6-tetra-O-benzyl-α-D-galactopyranosyl)-6-O-[2-O-(2,3,4,6-tetra-O-benzoyl-α-D-mannopyranosyl)-3,4,6-tri-O-acetyl-α-D-mannopyranosyl]-α-D-mannospyranoside afforded the octyl glycoside tetrasaccharide octyl 3-O-(α-D-galactopyranosyl)-6-O-[2-O-(α-D-mannopyranosyl)-α-D-mannopyranosyl]-α-D-mannospyranoside. Copyright (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0008-6215(00)00071-9

www.elsevier.nl/locate/carres
Carbohydrate Research 327 (2000) 367–375
Synthesis of a-galactosylated fragments related to the
core-structure of the GPI anchor of Trypanosoma brucei
Thomas Ziegler a,*, Ralf Dettmann a, Michael Duszenko b
a Institute of Organic Chemistry, Uni6ersity of Cologne, Greinstraße 4, D-50939 Cologne, Germany
b Institute of Physiological Chemistry, Uni6ersity Tu¨bingen, Hoppe-Seyler-Straße 4, D-72076 Tu¨bingen, Germany
Received 24 September 1999; accepted 24 February 2000
Abstract
A series of octyl glycosides di- to tetrasaccharides related to the GPI anchor of Trypanosoma brucei was prepared.
Treatment of octyl 2-O-benzoyl-4,6-O-(1,1,3,3-tetraisopropyl-1,3-disiloxane-1,3-diyl)-a-
2,3,4,6-tetra-O-benzyl-1-thio-b- -galactopyranoside under activation with bromine and silver trifluoromethanesul-
fonate afforded the a-linked disaccharide octyl 2-O-benzoyl-3-O-(2,3,4,6-tetra-O-benzyl-a- -galactopyranosyl)-4,6-O-
(1,1,3,3-tetraisopropyl-1,3-disiloxane-1,3-diyl)-a- -mannospyranoside, the siloxane ring of which was regioselectively
opened with a HF–pyridine complex to give the disaccharide acceptor octyl 3-O-(2,3,4,6-tetra-O-benzyl-a- -galactopy-
ranosyl)-2-O-benzoyl-4-O-(3-fluoro-1,1,3,3-tetraisopropyl-1,3-disiloxane-3-yl)-a- -mannopyranoside (4). Mannosyla-
tion of 4 with benzobromomannose (7), followed by fluoride catalyzed desilylation gave the trisaccharide octyl
2-O-benzoyl-6-O-(2,3,4,6-tetra-O-benzoyl-a- -mannopyranosyl)-3-O-(2,3,4,6-tetra-O-benzyl-a- -galactopyranosyl)-
a-
galactopyranosyl)-6-O-(a-
3-O-(a- -galactopyranosyl)-6-O-(a-
acetyl-2-O-(2,3,4,6-tetra-O-benzoyl-a-
D-mannopyranoside with ethyl
D
D
D
D
D
D
D
D
-mannospyranoside, which was deblocked via the deacylated intermediate octyl 3-O-(2,3,4,6-tetra-O-benzyl-a-
D
-
D
-mannopyranosyl)-a-
D
-mannospyranoside to afford the octyl glycoside trisaccharide octyl
D
D
-mannopyranosyl)-a-
D
-mannospyranoside. Glycosylation of 4 with 3,4,6-tri-O-
-mannopyranosyl trichloroacetimidate resulted in the
D
-mannopyranosyl)-a-
D
tetrasaccharide octyl 2-O-benzoyl-4-O-(1-fluoro-1,1,3,3-tetraisopropyl-1,3-disiloxane-3-yl)-3-O-(2,3,4,6-tetra-O-ben-
zyl-a- -galactopyranosyl)-6-O-[2-O-(2,3,4,6-tetra-O-benzoyl-a- -mannopyranosyl)-3,4,6-tri-O-acetyl-a-
mannopyranosyl] - a - - mannospyranoside, sequential desilylation, deacylation and debenzylation, respectively, of
which via the intermediate octyl 2-O-benzoyl-3-O-(2,3,4,6-tetra-O-benzyl-a- -galactopyranosyl)-6-O-[2-O-(2,3,4,6-
tetra-O-benzoyl-a- -mannopyranosyl)-3,4,6-tri-O-acetyl-a- -mannopyranosyl]-a- -mannospyranoside afforded the
octyl glycoside tetrasaccharide octyl 3-O-(a- -galactopyranosyl)-6-O-[2-O-(a- -mannopyranosyl)-a- -mannopyra-
nosyl]-a- -mannospyranoside. © 2000 Elsevier Science Ltd. All rights reserved.
D
D
D -
D
D
D
D
D
D
D
D
D
Keywords: GPI anchor; Trypanosoma brucei; Glycodesilylation
glycosyl-phosphatidylinositol (GPI) anchor,
which is covalently linked to the C-terminus
of the protein. The latter is a membrane an-
chor motif of variant surface glycoprotein
(VSG), a surface protein, which forms a pro-
tective barrier around the bloodstream forms
of the protozoan parasite Trypanosoma brucei
and accounts for about 10% of the total try-
panosomal proteins [1]. Furthermore, GPI-
1. Introduction
Eukaryotic membrane proteins are attached
to the membrane either by membrane-span-
ning hydrophobic peptide domains or by a
* Corresponding author. Tel.: +49-221-4703078; fax: +
49-221-4705057.
E-mail address: aco10@rrz.uni-koeln.de (T. Ziegler).
0008-6215/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved.
PII: S0008-6215(00)00071-9

368
T. Ziegler et al. / Carbohydrate Research 327 (2000) 367–375
anchored proteins are found in virtually all
classes of eukaryotes [2] and VSG was the first
protein of which the GPI structure was com-
pletely determined. However, a clear and
defined biological function of the GPI anchor
has still to be elucidated, but it is noteworthy
that GPI-anchored proteins are involved in
important processes of cell–cell interaction,
cell adhesion, and immunoresponse [3].
animals and man, the respective galactosyl-
transferases are of fundamental interest as
possible targets for a new anti-trypanosomal
strategy. Thus, synthetic fragments related to
trypanosomal GPI anchors can serve as
probes for revealing the biosynthesis and bio-
logical properties of these structures and sev-
eral chemical syntheses of GPI anchors and
fragments thereof are described already in the
literature [12–19].
Studies of GPI structures obtained from
several sources [2–8] revealed that all GPI
anchors contain a highly conserved pentasac-
charide core structure ABCDE (Fig. 1). In the
case of the trypanosomal VSG GPI anchor,
this core structure is differently galactosylated.
These variable galactose side-chains may be
important for the integrity of the VSG coat in
order to serve as a molecular diffusion barrier
and could perform space-filling functions in
order to compensate for differences in the
three-dimensional VSG structures [9]. For ex-
ample, in T. brucei variant MITat 1.4 strain
427 [10], the core structure of the GPI anchor
is modified by a diantennary galactan tetra-
saccharide, which is a-(1“3)-linked to the
mannosyl residue C (Fig. 1). Although the
distinct enzymes involved in the biosynthetic
pathway of trypanosomal GPI anchors are
still unknown, it was reported recently that
octyl glycosides and octyl 1-thio-glycosides
may serve as substrates for trypanosomal a-
galactosyltransferases [11–13]. Since try-
panosomes can cause severe infections in
In continuation of our previous study on
synthetic substrates for trypanosomal UDP-
gal:glycosylphosphatidylinositol anchor a-
(1“3)-galactosyltransferases [12], we describe
the chemical synthesis of a-galactosylated
fragments related to the GPI anchor of T.
brucei. In particular, syntheses of a-galactosy-
lated mono- to trisaccharide octyl glycopyrano-
sides of the core region CDE (Fig. 1) are
described that are planned to serve as sub-
strates
transferases.
for
trypanosomal
a-galactosyl-
2. Results and discussion
Originally, it was planned to construct the
a-(1“3)-galactosylated key fragment C (Fig.
1) by intramolecular glycosylation [20] of a
4,6-tetraisopropyl-1,3-disiloxane-1,3-diyl pro-
tected mannopyranoside related to compound
1. However, preliminary studies revealed that
problems arose with this approach since rear-
rangement of the siloxane protecting group
resulted in exclusive a-(1“6)-galactosylation
of that mannosyl acceptor [21]. Therefore, a
classical approach using siloxane protected
mannoside 1 [12] as the acceptor was chosen
here. Previously, Kovac and Edgar [22]
showed for a-(1“3)-selective galactosylations
of a series of 4,6-O-protected glucosamine
derivatives with tetra-O-benzyl-a- -galacto-
D
pyranosyl chloride that 4,6-siloxane blocked
acceptors reacted rather sluggishly or with low
diastereoselectivity. Therefore, we chose ethyl
2,3,4,6-tetra-O-benzyl-1-thio-a- -galacto-
D
pyranoside (2) [23] as the donor in combination
with Bundles [24] activation procedure. Thus,
under optimized reaction conditions (see Sec-
tion 3), the a-(1“3)-linked disaccharide 3 was
obtained in 48% yield and no b-galactosylated
Fig. 1.

T. Ziegler et al. / Carbohydrate Research 327 (2000) 367–375
369
glycosylation. Thus, tetrasaccharide 12 could
be isolated in 52% yield besides unreacted 4
(45%). This may be due to a steric hindrance
of the substituents at the 3- and 4-position of
4 since previously, similar (1“6)-selective
condensations of 11 with octyl 2-O-benzoyl-4-
O-(1-fluoro-1,1,3,3-tetraisopropyl-1,3-disilox-
disaccharide could be detected in the crude
reaction mixture. Other activation procedures
of 2 gave lower yields or anomeric mixtures of
the corresponding (1“3)-linked disaccharide.
The a-configuration of the galactosyl residue
in compound 3 was evident from the NMR
spectra, which showed characteristic coupling
constants of J1,2 =3.4 Hz and JC-1,H-1 =165
Hz [25]. The siloxane ring of compound 3 was
regioselectively opened with HF–pyridine
complex [26] to afford first octyl disaccharide
4 (90%). The regioselective ring opening of
compound 3 instead of a direct glycodesilyla-
tion methodology with mannosyl fluorides, as
previously described for similar siloxane pro-
tected monosaccharide octyl glycosides [12],
was chosen here because compound 4 was
planned to be condensed with bromide 7 and
imidate 11 (see below). Although compound 4
was only obtained in medium overall yield, its
preparation from easily accessible intermedi-
ates 1 and 2 appears to be straightforward
since it can be used in a flexible strategy for
the construction of various GPI anchor frag-
ments as follows. Conventional sequential de-
blocking of the siloxane moiety (Bu4NF)
afforded first 5 debenzoylation and debenzyla-
tion of which gave disaccharide 6 in 70%
overall yield. Also, disaccharide 4 served as
the glycosyl acceptor for further elongation of
the sugar chain. First, silver trifluoromethane-
sulfonate promoted condensation of 4 with
benzobromomannose 7, followed by fluoride
catalyzed removal of the siloxane moiety, gave
partially blocked trisaccharide 8 (78%). Next,
conventional removal of the benzoyl groups
(Zemplen) resulted in intermediate 9 (69%),
which was finally debenzylated to give octyl
glycoside trisaccharide 10 in 64% yield. Re-
cently, compound 10 has also been prepared
by a classical approach via galactosylation of
a partially benzoylated octyl mannodisaccha-
ride and has been used as substrate for enzy-
matic glycosylations with trypanosomal
membrane preparations [27]. For the con-
struction of the tetrasaccharide compound,
disaccharide 4 was coupled with disaccharide
imidate 11, which has been previously used for
the preparation of GPI anchor-related frag-
ments [12]. However, TMSOTf promoted re-
action of 4 and 11 resulted in incomplete
ane-1-yl)-a- -mannopyranoside proceeded in
D
high yield [12]. Final sequential deblocking of
12 via saccharide 13 (89%) afforded tetrasac-
charide octyl glycoside 14.
Previously, enzymatic a-galactosylations of
the
trisaccharide
a-
D
-Manp-(1“2)-a- -
D
Manp-(1“6)-a- -Manp-1-O-(CH2)7CH3 us-
D
ing trypanosomal membrane fractions and
radiolabelled UDP-Gal have been shown to
result in the formation of three different tetra-
saccharides in which the a-linked galactosyl
residues were shown by glycosidase digestion
to be attached to mannose residues 1, 2, and
3, respectively [12]. No conclusion, however,
could be deduced from these galactosylation
experiments to which position of the respec-
tive mannosyl residues the galactosyl residues
were linked. Preliminary enzymatic galactosy-
lations of compounds 6, 10, and 14, respec-
tively, with radiolabeled UDP-galactose and
trypanosomal membrane fractions, as previ-
ously described [12], revealed that saccharides
10 and 14 were significantly galactosylated at
a faster rate than the corresponding non-
galactosylatedcounterparts.Forexample,tetra-
saccharide 14 reacted about 1000-fold faster
than the aforementioned mannosyl trisaccha-
ride octyl glycoside, as was judged from the
relative rate of incorporation of radiolabeled
galactose. Similarly, compound 10 was re-
cently described to be most probably a-galac-
tosylated at position 2 of the galactosyl
residue [27]. Thus, it must be concluded that
the three tetrasaccharides obtained previously
by enzymatic galactosylation of a-
D
-Manp-
(1“2)-a- -Manp-(1“6)-a- -Manp-1-O-(C-
D
D
H2)7CH3 do not contain tetrasaccharide 14.
This finding could be the result of the presence
of different a-galactosyltransferases in the try-
panosomal membrane fraction used here. Al-
ternatively, significantly different substrate
selectivities of a single galactosyltransferase
could account for this finding. Therefore, fur-
ther investigations toward enzymatic galacto-

370
T. Ziegler et al. / Carbohydrate Research 327 (2000) 367–375
1
sylations of synthetic GPI anchor fragments
are now under investigation.
CHCl3); H NMR (CDCl3): l 8.05–7.00 (m,
25 H, ArH), 5.40 (dd, 1 H, J1,2 1.6, J2,3 2.7 Hz,
H-2Man), 5.02 (d, 1 H, H-1Man), 4.97 (d, 1 H,
J1,2 3.4 Hz, H-1Gal), 4.83 (d, 1 H, J −11.3 Hz,
PhCH2), 4.79–4.57 (m, 5 H, PhCH2), 4.44 (d,
1 H, J −11.3 Hz, PhCH2), 4.17 (dd, 1 H, J5,6a
1.2 Hz, J6a,6b −12.5 Hz, H-6aMan), 4.08–3.84
(m, 8 H, PhCH2, H-2Gal, H-3Man, H-4Man, H-
3. Experimental
Mps were determined with a Bu¨chi appara-
tus and are uncorrected. Optical rotations
were measured with a Perkin–Elmer 241 po-
4
Gal, H-5Man, H-5Gal), 3.68–3.39 (m, 5 H,
OCH2, H-3Gal, H-6bMan, H-6aGal), 3.18 (dd, 1
H, J5,6b 4.7 Hz, J6a,6b -8.5 Hz, H-6bGal), 1.50–
0.81 (m, 45 H, CH, CH2, CH3); 13C NMR
(CDCl3): l 166.0 (CO), 139.1, 139.0, 138.9,
137.9, 133.2, 129.9, 129.7, 128.4, 128.3, 128.1,
128.0, 127.9, 127.6, 127.5, 127.3, 127.2 (ArC),
100.9 (JC-1,H-1 165 Hz, C-1Gal), 97.2 (JC-1,H-1
171 Hz, C-1Man), 80.6, 78.9, 76.7, 74.8
(PhCH2), 74.7, 73.7 (C-5Man), 73.6 (C-3Man),
73.1 (2 C, PhCH2), 72.9 (PhCH2), 69.7 (C-
1
larimeter at 20 °C. H and 13C NMR spectra
were recorded with a Bruker AC 300F spec-
trometer at 300 and 75.46 MHz, respectively.
For trisaccharides 8–10 and tetrasaccharides
12–14, protons and carbons of the 1st, 2nd
and 3rd mannosyl residue, where the 1st
residue is that one bearing the aglycone, was
assigned with non-acentuated, single acentu-
ated and double acentuated numbers. Thin-
layer chromatography (TLC) was performed
on precoated silica gel plates (E. Merck,
60F254) using appropriately adjusted mixtures
of toluene–acetone or CCl4–acetone. Detec-
tion of the products was achieved by charring
with 5% H2SO4 in EtOH. Column chromatog-
raphy was carried out on silica gel using
columns of different length. Solvents were des-
tilled prior to use. Solutions in organic sol-
vents were dried with anhyd Na2SO4, and
concentrated at 2 kPa, B40 °C. Compounds
1 [12], 2 [23], 7 [28] and 11 [12] were prepared
as described in the literature.
4
2
Man), 68.2 (C-6Gal), 67.6 (OCH2), 65.9 (C-
Man), 61.0 (C-6Man), 31.9, 29.4, 29.3, 26.1, 22.7
(CH2), 17.9, 17.8, 17.5, 17.4, 17.3, 17.1, 14.1,
13.3, 13.0, 12.7 (CH, CH3).
Octyl
tetraisopropyl-1,3-disiloxane-3-yl)-3-O-(2,3,4,
6-tetra-O-benzyl-h- -galactopyranosyl)-h- -
2-O-benzoyl-4-O-(1-fluoro-1,1,3,3-
D
D
mannospyranoside (4).—HF-pyridine (70%,
0.4 mL) was added to a stirred solution of 3
(1.7 g, 1.46 mmol) in CH2Cl2 at room temper-
ature (rt). After 45 min, the solution was
washed with an aq NaHCO3 solution, dried,
and concentrated. Chromatography (30:1
CCl4–acetone) of the residue afforded 4 (1.54
g, 90%) as a colorless oil; [h]D +9.2° (c 0.9,
Octyl 2-O-benzoyl-3-O-(2,3,4,6-tetra-O-ben-
zyl - h -
D
- galactopyranosyl) - 4,6 - O - (1,1,3,3-
tetraisopropyl - 1,3 - disiloxane - 1,3 - diyl) - h - -
D
1
CHCl3); H NMR (CDCl3): l 8.06–6.99 (m,
mannospyranoside (3).—Tetramethyl urea
(580 mL, 4.84 mmol) and silver trifl-
uoromethanesulfonate (1.26 g, 4.88 mmol)
were added to a stirred solution of 1 (1.55 g,
25 H, ArH), 5.36 (t, 1 H, J1,2 2.95 Hz, H-
2
Man), 5.02 (d, 1 H, H-1Man), 4.89 (d, 1 H, J1,2
3.4 Hz, H-1Gal), 4.83–4.64 (m, 5 H, PhCH2),
4.47 (d, 1 H, J 7.8 Hz, OH), 4.40 (dd, 1 H, J
−11.3 Hz, PhCH2), 4.73 (dd, 1 H, J5,6a 2.6
Hz, J6a,6b −8.1 Hz, H-6aMan), 4.00 (dd, 1 H,
J2,3 3.2, J3,4 10.8 Hz, H-3Man), 3.92–3.67 (m, 9
,
2.43 mmol), 2 (1.72 g, 2.94 mmol) and 3 A
molecular sieves in toluene (30 mL). After 20
min, bromine (195 mL, 3.7 mmol) was added
and the mixture was stirred until TLC indi-
cated complete conversion of 1 and 2 (4.5 h).
The reaction was quenched with triethylamine
(1.9 mL) and diluted with toluene. The mix-
ture was filtered through a layer of Celite
washed with water, aq Na2S2O3 solution, wa-
ter, aq NaHCO3 solution, dried and concen-
trated. Chromatography (1:3 n-hexane–
CH2Cl2) of the residue afforded 3 (1.36 g,
48%) as a colorless oil; [h]D 90° (c 1.5,
H, H-2Gal, H-3Gal, H-4Man, H-4Gal,, H-5Man
,
H-5Gal, H-6bMan, H-6aGal, PhCH2), 3.49–3.40
(m, 2 H, OCH2), 2.98 (dd, 1 H, J5,6b 4.4, J6a,6b
7.9 Hz, H-6bGal), 1.63–0.85 (m, 45 H, CH,
13
CH2, CH3); C NMR (CDCl3): l 165.6 (CO),
138.9, 138.8, 138.6, 137.9, 133.2, 130.0, 129.6,
128.6, 128.3, 128.26, 128.2, 128.0, 127.8, 127.7,
127.6, 127.5, 127.4, 127.3, 127.2 (ArC), 99.8
(C-1Gal), 96.3 (C-1Man), 79.4, 79.0, 75.9 (C-2Gal
,

T. Ziegler et al. / Carbohydrate Research 327 (2000) 367–375
371
with a catalytic amount of 1 M NaOMe in
MeOH for 4 days. The solution was neutral-
ized with ion-exchange resin (Dowex 1X8, H+)
and concentrated. Chromatography (3:1
CCl4–acetone) of the residue afforded a color-
less oil, which was dissolved in aq 90% MeOH
(8 mL) and AcOH (0.5 mL) and hy-
drogenolyzed with H2 (1 atm) over a catalytic
amount of Pd–C. The mixture was stirred
until TLC (9:4:2 EtOAc–i-propanol–water)
indicated the end of the reaction. Filtration
and concentration of the mixture, followed by
chromatography of the residue with water on
Bio-Gel P2 afforded 6 (118 mg, 85%); [h]D
+68.4° (c 0.3, H2O); 13C NMR (D2O): l
101.9 (C-1Gal), 101.1 (C-1Man), 80.8 (C-3Man),
73.2 (C-5Man), 72.2 (C-2Man), 71.0 (C-2Gal),
70.6 (C-5Gal), 70.2 (C-3Gal), 70.0 (C-4Gal), 68.9
(OCH2), 66.5 (C-4Man), 62.1 (C-6Gal), 61.7 (C-
C-3Gal, C-4Gal, C-5Gal), 74.8 (2 C, C-3Man
,
CH2), 73.6, 73.0, 72.9 (CH2), 72.5 (C-5Man),
69.8 (C-4Man), 68.5 (C-6Gal), 67.7, 67.6
(OCH2), 61.9 (C-6Man), 31.8, 29.5, 29.4, 29.3,
26.1, 22.7 (CH2), 17.6, 17.3, 17.1, 16.8, 16.7,
14.1, 12.8, 12.5, 12.4 (CH, CH3); Anal. Calcd
for C67H93FO13Si2: C, 68.21; H, 7.98. Found:
C, 68.10; H, 7.93.
Octyl
benzyl-h-
2-O-benzoyl-3-O-(2,3,4,6-tetra-O-
-galactopyranosyl)-h- -mannospyra-
D
D
noside (5).—A solution of 4 (500 mg, 0.42
mmol) in THF (10 mL) was treated with a
catalytic amount of Bu4NF at rt for 1 h. The
mixture was concentrated, the residue dis-
solved in CH2Cl2, washed with aq NaHCO3
solution, dried and concentrated. Chromatog-
raphy (7:1 CCl4–acetone) afforded 5 (317 mg,
82%) as a colorless oil; [h]D +21.7° (c 1.2,
1
CHCl3); H NMR (CDCl3): l 8.05–7.17 (m,
6
Man), 32.9, 30.5, 30.3 (2 C), 27.1, 23.7 (CH2),
25 H, PhH), 5.42 (dd, 1 H, J1,2 1.6, J2,3 3.1 Hz,
H-2Man), 5.03 (d, 1 H, J1,2 3.8 Hz, H-1Gal), 4.88
(d, 1 H, H-1Man), 4.85 (d, 1 H, J −11.5 Hz,
PhCH2), 4.82 (d, 1 H, J −11.3 Hz, PhCH2),
4.68 (d, 1 H, J −11.6 Hz, PhCH2), 4.61 (d, 1
H, J −11.8 Hz, PhCH2), 4.58 (d, 1 H, J
−11.7 Hz, PhCH2), 4.49 (d, 1 H, J −11.4
Hz, PhCH2), 4.38 (d, 1 H, J −11.8 Hz,
PhCH2), 4.31 (d, 1 H, J −11.8 Hz, PhCH2),
4.11 (t, 1 H, J3,4 =J4,5 9.5 Hz, H-4Man), 4.04
(dd, 1 H, J2,3 10 Hz, H-2Gal), 3.97 (dd, 1 H,
15.0 (CH3); Anal. Calcd for C20H38NaO11:
477.23118 [M+Na]+. Found: m/z 477.23123
[M+Na]+ (HRFABMS).
Octyl 2-O-benzoyl-6-O-(2,3,4,6-tetra-O-ben-
zoyl-h-
D
-mannopyranosyl)-3-O-(2,3,4,6-tetra-
-galactopyranosyl)-h- -mann-
O-benzyl-h-
D
D
ospyranoside (8).—Silver triflate (121 mg, 0.47
mmol) was added at −10 °C under Ar to a
stirred mixture of 4 (0.5 g, 0.42 mmol) and 3
,
A molecular sieves (0.5 g) in CH2Cl2 (3 mL).
After 0.5 h, a solution of 7 (307 mg, 0.47
mmol) and sym-collidine (45 mL, 0.34 mmol)
in CH2Cl2 (1 mL) was added dropwise and the
mixture was stirred for 1 h, filtered through a
layer of Celite, washed with an aq Na2S2O3
solution, 1 M HCl, an aq NaHCO3 solution,
dried and concentrated. Chromatography
(30:1 CCl4 –acetone) of the residue afforded
an oil, which was dissolved in THF (5 mL)
and treated with a catalytic amount of
Bu4NF, as described for the preparation of 5.
Chromatography (30:1 CCl4 –acetone) of the
residue afforded 8 (483 mg, 78%); [h]D +2.1°
H-3Man), 3.92–3.64 (m, 8 H, H-3Gal, H-4Man
H-5Man, H-5Gal, H-6aMan, H-6bMan, H-6aGal
,
,
H-6bGal), 3.53 (t, 1 H, J3,4 =J4,5 8.7 Hz, H-
Gal), 3.46–3.37 (m, 2 H, OCH2), 2.21 (brs, 1
4
H, OH), 1.77 (brs, 1 H, OH), 1.62–1.55 (m, 2
H, CH2), 1.37–1.04 (m, 10 H, CH2), 0.91–
0.87 (m, 3 H, CH3); 13C NMR (CDCl3): l
166.1 (PhCO), 139.1, 138.8, 138.5, 138.1,
133.7, 130.2, 128.9, 128.8, 128.7, 128.6, 128.4,
128.1, 127.9, 127.8 (PhC), 101.6 (C-1Gal), 97.8
(C-1Man), 81.0 (C-3Man), 79.5 (C-5Gal), 76.4 (C-
2Gal), 74.7, 74.4 (PhCH2), 74.3 (C-3Gal), 73.2,
72.4 (PhCH2), 72.2 (C-4Gal), 71.7 (C-5Man),
69.7 (C-2Man), 68.3 (C-6Gal), 68.0 (OCH2), 68.0
(C-4Man), 62.9 (C-6Man), 31.8, 29.4, 29.2, 26.1,
22.7 (CH2), 14.1 (CH3); Anal. Calcd for
C55H66O12: C, 71.87; H, 7.24. Found: C, 71.67;
H, 7.26.
1
(c 1.0, CHCl3); H NMR (CDCl3): l 8.01–
7.07 (m, 25 H, PhH), 6.07 (t, 1 H, J3,4 =J4,5
10.0 Hz, H-4%), 5.89 (dd, 1 H, J2,3 3.1 Hz,
H-3%), 5.71 (dd, 1 H, J1,2 1.8 Hz, H-2%), 5.42
(dd, 1 H, J1,2 1.5 Hz, J2,3 3.0 Hz, H-2), 5.15 (d,
1 H, H-1%), 4.95 (d, 1 H, J1,2 3.8 Hz, H-1Gal),
4.85 (d, 1 H, H-1), 4.79 (d, 1 H, J −11.4 Hz,
PhCH2), 4.75 (d 1 H, J −11.2 Hz, PhCH2),
Octyl 3-O-(h-
D
-galactopyranosyl)-h- -man-
D
nospyranoside (6).—A solution of 5 (299 mg,
0.33 mmol) in MeOH (20 mL) was treated

372
T. Ziegler et al. / Carbohydrate Research 327 (2000) 367–375
71.62 (C-5%), 71.1 (C-3), 70.8 (OCH2), 70.7
(C-2), 70.13 (C-2%), 68.1 (C-6Gal), 67.1 (C-4%),
66.1 (C-6), 65.4 (C-4), 61.7 (C-6%), 32.3, 29.9
(2C), 29.7, 26.6, 23.1 (CH2), 14.5 (CH3); Anal.
Calcd for C54H72O16: 976.5. Found m/z 999.4
[M+Na]+ (FABMS).
4.63 (d, 1 H, J −11.7 Hz, PhCH2), 4.60–4.34
(m, 4 H, H-5%, PhCH2), 4.33 (d, 1 H, J −11.7
Hz, PhCH2), 4.25 (d, 1 H, J 11.8 Hz, PhCH2),
4.10 (t, 1 H, J3,4=J4,5 9.6 Hz, H-4), 4.08–3.65
(m, 11 H, H-2Gal, H-3, H-3Gal, H-5, H-5Gal
,
H-6a,b, H-6a,b%,H-6a,bGal), 3.51 (t, 1 H, J3,4=
J4,5 8.7 Hz, H-4Gal), 3.44–3.36 (m, 2 H,
OCH2), 1.67–1.53 (m, 3 H, OH, CH2), 1.32–
1.16 (m, 10 H, CH2), 0.79–0.74 (m, 3 H,
CH3); 13C NMR (CDCl3): l 166.3, 165.9,
165.5, 165.4, 165.2 (PhꢀCO), 138.8, 138.5,
138.3, 137.8, 133.4, 133.3, 133.2, 133.1, 130.1,
129.9 (2 C), 129.5, 129.4, 129.2, 128.7, 128.6,
128.5, 128.4, 128.3 (2 C), 128.1 (2 C), 127.8,
127.6, 127.4 (ArC), 102.0 (C-1Gal), 98.0 (C-1%),
97.6 (C-1), 81.8 (C-3), 79.6 (C-5Gal), 76.5 (C-
2Gal), 74.9, 74.5 (PhCH2), 74.4 (C-3Gal), 73.3,
72.5 (PhCH2), 72.2 (C-4Gal), 71.5 (C-5), 70.5
(C-5%), 70.3 (C-3%), 69.8 (C-2), 68.9 (C-2%), 68.4
(C-6Gal), 67.0 (C-4), 66.9 (C-4%), 66.6 (C-6),
62.8 (C-6%), 31.9, 29.6 (2C), 29.4, 26.3,
22.6 (CH2), 14.2 (CH3); Anal. Calcd for
C89H92O21: 1496.6. Found: m/z 1496.4 [M]+
(FABMS).
Octyl 3-O-(h-
- mannopyranosyl) - h -
D
-galactopyranosyl)-6-O-(h-
- mannospyranoside
D
D
(10).—Compound 9 (184 mg, 0.19 mmol) was
dissolved in 90% aq MeOH (8 mL) and AcOH
(0.5 mL) and treated with H2 (1 atm) and a
catalytical amount of Pd–C until all starting
material was consumed and the mixture was
filtered and concentrated. Chromatography of
the residue with water on Bio-Gel P2 afforded
10 (73 mg, 64%); [h]D +70.5° (c 0.2, water);
13C NMR (D2O): l 102.1 (C-1Gal), 101.2 (C-1),
100.7 (C-1%), 81.3 (C-3), 73.8 (C-5), 72.4 (C-2),
72.2 (C-3%), 71.9 (C-2%), 71.2 (C-5%), 71.0 (C-
2Gal), 70.6 (C-5Gal), 70.2(C-3Gal), 70.0 (C-4Gal),
69.2 (OCH2), 67.7 (C-4%), 66.3 (C-4,6), 62.2
(C-6Gal), 61.9 (C-6%), 32.7, 30.0 (2C), 26.9 and
23.5 (CH2), 14.9 (CH3); Anal. Calcd for
C26H48O16Na+:
639.2840.
639.28332 [M+Na]+ (HRFABMS).
Octyl 2-O-benzoyl-4-O-(1-fluoro-1,1,3,3-
tetraisopropyl-1,3-disiloxane-3-yl)-3-O-(2,3,4,
6-tetra-O-benzyl-h- -galactopyranosyl)-6-O-
[2-O-(2,3,4,6-tetra-O-benzoyl-h- -mannopyra-
nosyl)-3,4,6-tri-O-acetyl-h- -mannopyranosyl]-
h- -mannospyranoside (12).—A catalytic
Found:
m/z
Octyl3-O-(2,3,4,6-tetra-O-benzyl-h-
D
-galac-
topyranosyl)-6-O-(h- -mannopyranosyl)-h- -
D
D
mannospyranoside (9).—A catalytic amount of
NaOMe was added to a solution of 8 (462 mg,
0.31 mmol) in MeOH (30 mL). After 5 days,
the mixture was neutralized with ion-exchange
resin (Dowex 5w80) and filtered. Chromatog-
raphy (10:1 CH2Cl2–MeOH) of the residue
afforded 9 (209 mg, 69%) as a white foam;
[h]D +64.6° (c 1.0, CHCl3); 1H NMR
(CDCl3): l 7.40–7.19 (m, 20 H, PhH), 4.95 (d,
1 H, J1%,2% 2.3 Hz, H-1%), 4.90–4.79 (m, 4 H,
PhCH2), 4.75 (d, 1 H, J1,2 4.0 Hz, H-1Gal), 4.48
(d, 1 H, J −11.8 Hz, PhCH2), 4.42 (d, 1 H, J
−12.0 Hz, PhCH2), 4.40 (d, 1 H, J1,2 2.2 Hz,
H-1), 4.20 (dd, 1 H, J2,3 3.5, J3,4 7.6 Hz, H-3),
D
D
D
D
amount of Me3SiOTf (10 mL, 0.05 mmol) was
added at −30 °C to a stirred solution of 4
,
(420 mg, 0.355 mmol) and 4 A molecular
sieves (0.5 g) in CH2Cl2 (4.5 mL), followed by
dropwise addition of a solution of 11 (468 mg,
0.455 mmol) in CH2Cl2 (4.5 mL). After 20 min
an additional amount of 11 (192 mg, 0.187
mmol) was added, the mixture was stirred for
0.5 h, and the reaction was quenched with
pyridine. Concentration of the mixture and
chromatography (10:1 CCl4–acetone) of the
residue afforded first unchanged 4 (197 mg,
47%). Eluted next was 12 (375 mg, 52%); [h]D
4.08–3.11 (m, 28 H, H-2, 2%, 2Gal, H-3%, 3Gal
,
H-4, 4%, 4Gal, H-5, 5%, 5Gal, H-6a,b, 6a,b%,
6a,bGal, OH, PhCH2, OCH2), 1.47 (brs, 2 H,
CH2), 1.27 (brs, 10 H, CH2), 0.88 (brt, 3 H,
CH3); 13C NMR (CDCl3): l 138.7, 138.5,
137.8, 137.3, 128.9, 128.8, 128.6, 128.5, 128.4
(2C), 128.3, 128.0, 127.9 (ArC), 100.8 (C-1Gal),
100.5 (C-1%), 100.2 (C-1), 83.5 (C-3), 79.6 (C-
1
−5° (c 0.8, CHCl3); H NMR (CDCl3): l
8.15–6.89 (m, 5 H, ArH), 6.15 (t, 1 H, J3¦,4¦
=
J4¦,5¦ 10.3 Hz, H-4¦), 5.89 (dd, 1 H, J2¦,3¦ 3.1
Hz, H-3¦), 5.53 (dd, 1 H, J1,2 1.9 Hz, H-2),
5.44 (t, 1 H, J3%,4%=J4%,5% 9.8 Hz, H-4%), 5.33
(dd, 1 H, J2%,3% 3.7 Hz, H-3%), 5.32 (dd, 1 H,
5Gal), 76.9 (C-2Gal), 75.3 (C-3Gal), 74.8, 74.1
(2C), 73.4 (PhCH2), 72.6 (C-5), 72.0 (C-4Gal),

T. Ziegler et al. / Carbohydrate Research 327 (2000) 367–375
373
J1¦,2¦ 1.6 Hz, H-2¦), 5.21 (s, 1 H, H-1%), 5.11
(d, 1 H, H-1¦), 4.89 (d, 1 H, J1,2 2.8 Hz,
H-1Gal), 4.82–4.64 (m, 6 H, H-1%, PhCH2),
4.58 (t, 1 H, J3,4 =J4,5 9.2 Hz, H-4), 4.48–
4.19 (m, 6 H, H-2%, H-2Gal, H-6a¦, PhCH2),
4.13–3.89 (m, 9 H, H-3, H-3Gal, H-5, H-5%,
H-5¦, H-5Gal, H-6b¦, H-6a,b%), 3.78–3.68 (m,
4 H, H-6a,b, H-6aGal, OCH2), 3.55–3.49 (m,
2 H, 1 H, OCH2), 3.42 (t, 1 H, J3,4l =J4,5 9.0
Hz, H-4Gal), 3.82 (dd, 1 H, J5,6b 4.6, J6a,6b 8.3
Hz, H-6bGal), 2.23, 2.11, 2.08 (s, 3 H, CH3),
1.75–1.63 (m, 2 H, CH2), 1.55–0.88 (m, 41
H, CH, CH2, CH3); 13C NMR (CDCl3): l
171.1, 170.3, 169.6 (CO), 166.0, 165.5, 165.3,
165.1, 165.0 (PhCO), 139.0 (3 C), 138.0,
133.6, 133.4, 133.1, 132.9, 129.9, 129.8, 129.7,
128.8, 128.4, 128.3, 128.1, 127.8 (2 C), 127.6,
127.4, 127.3 (ArC), 100.1 (C-1Gal), 99.5 (C-
1¦), 98.7 (C-1%), 96.3 (C-1), 79.7 (C-3), 79.3
(C-5Gal), 77.4 (C-2%), 76.1 (C-2Gal), 75.0 (C-
H-5¦, H-6a,b%, H-6a,b¦, PhCH2), 4.27–3.57
(m, 10 H, H-2Gal, H-3, H-3Gal,, H-4, H-5,
H-5Gal, H-6a,b, H-6a,bGal), 3.55 (t, 1 H,
J3,4=J4,5 8.8 Hz, H-4Gal), 3.38–3.28 (m, 2 H,
OCH2), 2.11 (s, 3 H, CH3), 2.05 (s, 3 H,
CH3), 1.96 (s, 3 H, CH3), 1.74 (brs, 1 H,
OH), 1.52–1.50 (brm, 2 H, CH2), 1.19–1.13
(brs, 10 H, CH2), 0.82–0.77 (brm, 3 H, CH3);
13C NMR (CDCl3): l 171.0, 170.4, 169.5
(CO), 165.9, 165.5 (2 C), 165.1, 165.0
(PhCO), 138.7, 138.4, 138.1, 137.8, 133.5,
133.4, 133.1, 133.0, 132.9, 129.8, 129.7, 129.3,
129.1, 128.9, 128.6, 128.5, 128.4, 128.3 (2 C),
128.2, 128.1, 127.9, 127.6, 127.4, 127.3 (ArC),
101.9 (C-1Gal), 99.6 (C-1¦), 98.4 (C1%), 97.7
(C-1), 81.6 (C-3), 79.6 (C-5Gal), 77.6 (C-2%),
76.4 (C-2Gal), 74.8, 74.4 (PhCH2), 74.3 (C-
3
Gal), 73.2, 72.5 (PhCH2), 72.3 (C-4Gal), 71.7
(C-5), 70.7 (C-5%), 70.5 (C-5¦), 69.7 (C-2),
69.5 (2 C, C-3%, C-3¦), 68.6 (C-2¦), 68.4 (C-
4Gal), 74.8 (2 C, C-3Gal, PhCH2), 73.6, 73.2
6Gal), 68.0 (OCH2), 67.0 (C-4), 66.6 (C-4%),
(PhCH2), 73.0 (2 C, C-5, PhCH2), 70.5 (C-5%),
70.4 (C-5¦), 70.0 (C-2), 69.5 (C-3%), 69.2 (C-
3¦), 68.6 (C-2¦), 68.5 (C-6Gal), 67.9 (C-4), 67.6
(OCH2), 66.8 (C-4%), 66.3 (C-4¦), 65.5 (C-6),
62.4 (2 C, C-6%, C-6¦), 31.9, 29.5, 29.4 (2 C),
26.3, 22.8 (CH2), 21.0, 20.7, 17.8, 17.4, 17.3,
17.2, 16.8, 14.2, 12.5, 12.2 (CH, CH3); Anal.
Calcd for C113H135FO30Si2: C, 66.26; H, 6.64.
Found: C, 66.15; H, 6.64.
66.4 (C-4¦), 65.8 (C-6), 62.8 (C-6%), 62.3 (C-
6¦), 31.9, 29.5 (2 C), 29.3, 26.3, 22.8 (CH2),
21.0, 20.9 (2 C), 14.2 (CH3); Anal. Calcd for
C101H108O29: C, 67.93; H, 6.10. Found: C,
67.82; H, 6.11.
Octyl 3-O-(h-
O-(h- -mannopyranosyl)-h-
syl]-h- -mannospyranoside (14).—A solution
D-galactopyranosyl)-6-O-[2-
D
D
-mannopyrano-
D
Octyl 2-O-benzoyl-3-O-(2,3,4,6-tetra-O-ben-
of 13 (230 mg, 0.129 mmol) in MeOH (30
mL) was treated with a catalytic amount of
NaOMe as described for the preparation of
compound 8. The crude deacylated interme-
diate was dissolved in 90% aq MeOH (90%)
and hydrogenolyzed as described for the
preparation of compound 10. Chromatogra-
phy of the residue with water on Bio-Gel P-2
afforded 14 (48 mg, 78%); [h]D +63° (c 0.2,
H2O); 13C NMR (D2O): l 103.5 (C-1¦), 102.0
(C-1Gal), 101.0 (C-1), 99.2 (C-1%), 80.5 (C-3),
79.9 (C-2%), 74.4 (C-5¦), 73.9 (C-5), 72.5 (C-
2), 72.3 (C-2¦), 71.5 (C-5%), 71.4 (C-2Gal), 71.1
(C-5Gal), 71.0 (C-3Gal), 70.5 (C-3%), 70.4 (C-
3¦), 69.9 (C-4Gal), 69.3 (OCH2), 68.00 (2 C,
C-4%,4¦), 66.74 (C-6), 66.68 (C-4), 62.4 (C-
6¦),62.3 (C-6Gal), 62.0 (C-6%), 32.3, 29.6 (2 C),
29.53, 29.52 and 23.2 (CH2), 14.6 (CH3);
Anal. Calcd for C32H58O21Na+: 801.336828.
Found: m/z 801.33607 [M+Na]+ (HR-
FABMS) (Scheme 1).
zyl-h-
tetra-O-benzoyl-h-
tri-O-acetyl-h- -mannopyranosyl]-h-
D
-galactopyranosyl)-6-O-[2-O-(2,3,4,6-
D
-mannopyranosyl)-3,4,6-
-mannos-
D
D
pyranoside (13).—A solution of 12 (320 mg,
0.156 mmol) in THF (5 mL) was treated with
a catalytic amount of Bu4NF as described for
the preparation of compound 8. Chromatog-
raphy (7:1 CCl4–acetone) of the residue af-
forded 13 (247 mg, 89%) as a white foam;
[h]D −2.8° (c 0.7, CHCl3); 1H NMR
(CDCl3): l 8.01–7.04 (m, 45 H, PhH), 6.02
(t, 1 H, J3¦,4¦=J4¦,5¦ 10.0 Hz, H-4¦), 5.86 (dd,
1 H, J2¦,3¦ 3.1 Hz, H-3¦), 5.59 (dd, 1 H, J1,2
1.8, J2,3 2.8 Hz, H-2), 5.40, (t, 1 H, J3%,4%
=
J4%,5% 9.6 Hz, H-4%), 5.35–5.32 (m, 2 H, H-3%,
H-2¦), 5.10 (d, 1 H, J1%,2% 0.6 Hz, H-1%), 4.93
(d, 1 H, J1,2 3.4 Hz, H-1Gal), 4.85 (d, 1 H,
H-1), 4.83 (d, 1 H, J1¦,2¦ 1.6 Hz, H-1¦), 4.79
(d, 1 H, J −11.8 Hz, PhCH2), 4.73 (d, 1 H,
J −11.4 Hz, PhCH2), 4.63 (d, 1 H, J 11.6
Hz, PhCH2), 4.58–4.30 (m, 12 H, H-2%, H-5%,

374
T. Ziegler et al. / Carbohydrate Research 327 (2000) 367–375
Scheme 1.
[8] M.A. Deeg, D.R. Humphrey, S.M. Yang, T.R. Fergu-
Acknowledgements
son, V.N. Reinhold, T.L. Rosenberry, J. Biol. Chem.,
267 (1992) 18573–18580.
This work was financially supported by the
Deutsche Forschungsgemeinschaft and the
Fonds der Chemischen Industrie. We thank
Dr H. Schmickler, C. Schmitz for performing
the NMR spectra and the elemental analyses.
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