Synthesis and serological characterization of L-glycero-α-D-manno-heptopyranose-containing di- and tri-saccharides of the non-reducing terminus of the Escherichia coli K-12 LPS core oligosaccharide

Article abstract of DOI:10.1016/S0008-6215(98)00292-4

Synthesis of the title trisaccharide was accomplished by sugar chain extension starting from the non-reducing terminus: coupling of GlcpNAc with LD-Hepp, then adding Glcp-OAll. An alternative route started from the reducing end: coupling of LD-Hepp with G

Full text of DOI:10.1016/S0008-6215(98)00292-4

Carbohydrate Research 314 (1998) 85–93
Synthesis and serological characterization of
L
-glycero-a-
D-manno-heptopyranose-containing di- and
tri-saccharides of the non-reducing terminus of the
Escherichia coli K-12 LPS core oligosaccharide
Konstantin V. Antonov a, Leon V. Backinowsky b, Barbara Grzeszczyk c, Lore Brade d,
Otto Holst d, Aleksander Zamojski c,*
a M.M. Shemyakin and Yu.A. O6chinniko6 Institute of Bioorganic Chemistry, Russian Academy of Sciences,
ul. Miklukho-Maklaya 16/10, 117871 Moscow, Russian Federation
b N.D. Zelinsky Institute of Organic Chemistry, Leninsky pr. 47, 117913 Moscow, Russian Federation
c Institute of Organic Chemistry, Polish Academy of Sciences, ul. Kasprzaka 44, PL-01-224 Warsaw, Poland
d Di6ision of Medical and Biochemical Microbiology, Research Center Borstel,
Center for Medicine and Biological Sciences, Parkallee 22, D-23845 Borstel, Germany
Received 9 October 1998; accepted 5 November 1998
Abstract
Synthesis of the title trisaccharide was accomplished by sugar chain extension starting from the non-reducing
terminus: coupling of GlcpNAc with LD-Hepp, then adding Glcp-OAll. An alternative route started from the
reducing end: coupling of LD-Hepp with Glcp-OAll, then addition of GlcpNAc. In the synthesis of the title
disaccharide a modification of the first approach was employed. The allyl glycosides were coupled with cysteamine,
activated with thiophosgene and conjugated to bovine serum albumin (BSA). The neoglycoconjugates obtained were
used in immunochemical studies of monoclonal and polyclonal antibodies directed against Escherichia coli K-12
lipopolysaccharide. © 1998 Elsevier Science Ltd. All rights reserved.
Keywords: Polysaccharides; Escherichia coli; Sugar chain extension
carrier. One of the popular aglycons utilised
to this end is the allyl group.
1. Introduction
Allyl glycosides of various oligosaccharides
have been used for the preparation of
oligosaccharide–acrylamide copolymers, both
as such or following the addition of cys-
teamine and N-acryloylation [1]. Alterna-
tively, the cysteamine adducts can be
converted into the respective isothiocyanates
and then coupled to a protein carrier, e.g.,
bovine serum albumin (BSA). It is the latter
approach that has been used in the prepara-
tion of synthetic neoglycoconjugates related to
A prerequisite for the synthesis of neoglyco-
conjugates that can be used in immunochemi-
cal studies as haptens, antigens, or immuno-
sorbents is the preparation of appropriate
oligosaccharides, often in the form of gly-
cosides that can be subject to subsequent poly-
merisation or attachment to a macromolecular
* Corresponding author. Tel.: +48-22-6327030; fax: +48-
22-6326681.
E-mail address: awzam@ichf.edu.pl (A. Zamojski)
0008-6215/98/$ - see front matter © 1998 Elsevier Science Ltd. All rights reserved.
PII: S0008-6215(98)00292-4

86
K.V. Antono6 et al. / Carbohydrate Research 314 (1998) 85–93
Scheme 1.
the K-12 core of Escherichia coli [2] (synthesis
of the corresponding oligosaccharide deriva-
tives, viz., L-a-D-Hepp-(1“6)-a-D-Glcp-O-
All, L-a-D-Hepp-(1“6)-a-D-Glcp-(1“2)-a-D-
Glcp-O-All, and L-a-D-Hepp-(1“6)-a-D-
Glcp-(1“2)-a-D-Glcp-(1“3)-a-D-Glcp-O-All
has been described in Ref. [3] rather than in
Ref. [4], erroneously cited in Ref. [2]).
These glycoconjugates proved to be reactive
as immunogens and antigens, although they
did not bind to three monoclonal antibodies
raised in mice by immunisation with heat-
killed E. coli K-12 bacteria, and the allyl
glycosides of the oligosaccharides did not
manifest inhibitory activities in ‘deacylated
lipopolysaccharide–monoclonal antibody’ sys-
tems [2].
In the K-12 core, the heptose linked to the
Glcp-(1“2)-Glcp-(1“3)-Glcp sequence car-
ries at O-7 a b-GlcpNAc substituent in a
non-stoichiometric amount [2,5]. For further
insight into the immunochemistry of K-12
strains of E. coli and in a continuation of our
synthetic studies on oligosaccharide fragments
of the E. coli K-12 core [3,4], here we report
the syntheses of allyl glycosides 1 and 2.
Trioside 1 was prepared using two synthetic
strategies, which differed in the direction of
the chain extension.
2. Results
Synthesis of 1 and 2.—The common feature
of both approaches to 1 is that the glu-
cosamine unit was introduced by glycosylation
with a thioglycoside donor mediated by
methyl trifluoromethanesulfonate (MeOTf)
[6], while 1-O-trichloroacetimidate methodol-
ogy [7] was applied to create a Hep–Glc
linkage.
According to the first reaction (Scheme 1),
benzyl
2,3,4,6-tetra-O-benzyl-L-glycero-a-D-
manno-heptopyranoside (3) [8] was converted
into the corresponding peracetylated glycosyl
trichloroacetimidate, which was coupled with
allyl 2,3,4-tri-O-benzoyl-a-D-glucopyranoside
(4) as described earlier [3] using trimethylsilyl
trifluoromethanesulfonate (Me3SiOTf) as a
catalyst to give the bioside (5). In the prepara-
tion of the glycosyl acceptor 4, selective 6-O-
formylation of allyl a- -glucopyranoside
D
followed by benzoylation and acid-catalyzed
deformylation [9] were used instead of the
tritylation–benzoylation–detritylation proce-
dure employed before [3].
Peracylated allyl bioside 5 was deacylated
with Et3N in MeOH, the product was selec-
tively 7%-O-silylated with tert-butyldimethylsil-
yl chloride in pyridine and acetylated. Sub-
sequent desilylation with 40% HF in acetoni-
trile afforded a disaccharide glycosyl acceptor
(6) isolated in a yield of 75% over four steps.
The relatively high-field position of H-7%a,b (l
3.79, m) in the 1H NMR spectrum of
b-D-GlcpNAc (1“7)-L-a-D-Hepp-(1“6)
-a-D-Glcp-OAll
(1)
(2)
b-D-GlcpNAc (1“7)-L-a-D-Hepp-OAll

K.V. Antono6 et al. / Carbohydrate Research 314 (1998) 85–93
87
this disaccharide derivative proved that desilyl-
ation was not accompanied by acyl migration.
On the contrary, conventional desilylation
with Bu4NF in THF gave a complex mixture.
Unsatisfactory results were also obtained on
Bu4NF-promoted desilylation of a perbenzoyl
analogue of the disaccharide under consider-
ation. It should also be noted that an attempt
to prepare 7-O-tert-butyldimethylsilyl-heptose
from benzyl heptoside 3 failed due to the
instability of the silyl group under hy-
drogenolysis conditions (Scheme 2).
The introduction of the glucosamine residue
into the disaccharide acceptor 6 was accom-
plished successfully using ethyl 3,4,6-tri-O-
acetyl-2-deoxy-2-phthalimido-1-thio-b-D-gluco-
pyranoside (7) [10] as the glycosyl donor. The
MeOTf-mediated glycosylation [6] resulted in
a fully protected allyl trioside (8) in 70% yield.
The newly formed glycosidic bond was b-(1“
7) as shown by the NMR spectral data: lH-1%%
5.39 (d, J 8.4 Hz); lC-7% 67.9.
Scheme 3.
tive 3 served as the first glycosyl acceptor. Its
MeOTf-mediated coupling [6] with the donor
7 yielded 74% of the benzyl bioside (9).
This trioside was N,O-deprotected in one
step by treatment with ethylenediamine in
BuOH [11]; this procedure has been especially
recommended for N-dephthaloylation of allyl
glycosides of oligosaccharides containing 2-de-
oxy-2-phthalimidosugars. Subsequent N,O-
acetylation and O-deacetylation afforded the
target trisaccharide 1.
This was debenzylated by catalytic hy-
drogenolysis on Pd/C and the product was
acetylated. The thus obtained per-O-acetyl
disaccharide derivative (10) (yield 83% over
two steps) was selectively 1-O-deacetylated
with hydrazinium acetate (cf. [3]) and the
product (11) was converted into the disaccha-
ride 1-O-trichloroacetimidate (12) isolated by
chromatography on silica gel in 71% yield.
The low-field position of the signal for H-1 (l
6.21, br.s) and the presence of an NH-proton
An alternative glycosylation sequence was
also explored. In this case, the heptose deriva-
1
(l 8.78, s) in the H NMR spectrum corrobo-
rated its structure (Scheme 3).
To complete the assembly of the trisaccha-
ride, the acceptor 4 was glycosylated with the
disaccharide donor 12 in the presence of
Me3SiOTf to yield 78% of the trisaccharide
1
derivative (13). H NMR chemical shifts for
the protons of the GlcNAc and Hep units of
this trisaccharide almost coincided with those
of the trisaccharide 8, while the signals for the
protons of the Glc unit were predictably
shifted downfield.
The transformation of this peracylated allyl
trioside into the target product 1 was accom-
plished by the same deprotection–protection
procedure as described for the transformation
8“1. The NMR and mass spectra of the both
samples of the trioside 1 were identical. As-
Scheme 2.

88
K.V. Antono6 et al. / Carbohydrate Research 314 (1998) 85–93
Scheme 4.
1
signments of the signals in the H and 13C
NMR spectra were made with the use of 1D-
and 2D- (TOCSY and HMQC) spectroscopy.
For the synthesis of 2, peracetylated disac-
charide 10 was employed. Its treatment with
allyl alcohol in the presence of tin tetrachlor-
ide [12] afforded 67% of a-glycoside (14).
Dephthaloylation and acetylation gave 15
(99%) which was next fully deacetylated to
yield 90% of 2.
cosamine was a part of the epitopes recog-
nized by these antibodies. The data obtained
with the glycoconjugates described herein, al-
lowed to answer this question. It is clearly
shown that the terminal part of the core
oligosaccharide with or without the glu-
cosamine does not constitute any of the epi-
topes recognized by these MAs. Obviously,
they recognize an epitope which requires the
complete core region. This is in accordance
with observations made with an antibody
cross-reacting with the different E. coli core
types [13]. However, the polyclonal antiserum
contains antibody specificities recognizing epi-
topes in which this terminal glucosamine is
included.
Conjugation and serological characterization
of 1 and 2.—In order to study the immunore-
active properties of these oligosaccharides,
they were derivatized into neoglycoconjugates
by cysteamine spacer extension, activation
with thiophosgene and subsequent coupling of
the resulting isothiocyanate to BSA [2]
(Scheme 4). These conjugates will be abbrevi-
ated as GlcNAc-Hep-Glc-BSA and as Glc-
NAc-Hep-BSA, respectively. The amount of
ligand present in the conjugates was deter-
mined by measuring the amount of protein
and glucosamine. For GlcNAc-Hep-Glc-BSA
and as GlcNAc-Hep-BSA the analytical val-
ues were 123.5 and 74 nmol of ligand per mg
BSA, respectively. Three monoclonal antibod-
ies, S31-8, S31-14, and S31-21, and one poly-
clonal antiserum, described previously [2] were
tested in EIA with GlcNAc-Hep-Glc-BSA or
GlcNAc-Hep-BSA as solid phase antigens.
Another glycoconjugate containing the com-
plete core E. coli K-12 LPS was included. The
MAbs did not bind to GlcNAc-Hep-Glc-BSA
or GlcNAc-Hep-BSA, whereas the polyclonal
rabbit antiserum reacted with a titer of 80,000
and 20,000 with GlcNAc-Hep-Glc-BSA and
GlcNAc-Hep-BSA, respectively (Table 1). The
titer of this antiserum with the conjugate con-
taining the complete core was 320,000.
3. Experimental
General and serological methods.—Column
chromatography was performed on Silica Gel
230–400 or 70–230 mesh (E. Merck). Optical
rotations were determined with a Jasco DIP-
360 automatic polarimeter (Japan) at 209
1
13
2 °C. H and C NMR spectra were recorded
on Varian Gemini AC-200 (200 MHz) or
Bruker AM-500 (500 MHz) spectrometers in
CDCl3 solutions with Me4Si as the internal
standard. The spectra of 1 and 2 were
recorded on solutions in D2O with Bruker
DRX 600 spectrometer (Research Center
Borstel), operating frequencies 600 MHz for
1H NMR and 125.77 MHz for 13C NMR at
27 °C. The resonances were measured relative
to internal acetone [(CH3)2CO, l1H 2.225, l13C
31.07]. Mass spectra (LSIMS, positive mode)
were obtained with an AMD-604 mass
spectrometer.
During the characterization of the epitope
specificities of the three K-12 MAs, it could
not be determined whether the terminal glu-
The glycoconjugates GlcpNAc-Hepp-Glcp-
BSA and GlcpNAc-Hepp-BSA were prepared
as described [2]. The conjugates were used as

K.V. Antono6 et al. / Carbohydrate Research 314 (1998) 85–93
89
Table 1
Reactivity in EIAa of monoclonal and polyclonal antibodies against Escherichia coli K-12 LPSdeac–BSAb and synthetic
glycoconjugates
Antibody
Reactivity against indicated antigen in EIAc
E.c.K-12 LPSdeac–BSA
GlcNAc-Hep-Glc–BSA
GlcNAc-Hep–BSA
MAb S31-8d
MAb S31-14
MAb S31-21
Rabbit serume
16
8
16
\10,000
\10,000
\10,000
80,000
\10,000
\10,000
\10,000
20,000
320,000
a EIA plates were coated at a concentration of 400 pmol of ligand per mL (50 mL/well).
b LPSdeac was covalently linked to BSA (LPSdeac–BSA) by the glutardialdehyde method [14].
c Final concentration (ng/mL) in the case of MAbs or dilution in the case of the rabbit antiserum yielding OD405\0.2.
d MAbs from Ref. [2] were obtained after immunization with heat-killed E. coli K-12 bacteria.
e The serum was obtained from a rabbit (K259 day 58 from Ref. [2]) immunized with heat-killed E. coli K-12 bacteria.
solid antigens in an EIA described previously
[2]. The monoclonal and polyclonal antibodies
were described in the same Ref.
stirred for 1 h at room temperature. It was
then diluted with CHCl3, washed with aq
NaHCO3 and water, dried (MgSO4), and con-
centrated. The residue, practically pure 6 (40
mg, 75% with respect to 5), [h]D +69.4° (c
0.5, CHCl3), was used in subsequent transfor-
mations without additional purification.
Allyl 2,3,4-tri-O-acetyl-6-O-(2,3,4,6-tetra-
O-acetyl-L- glycero-h-D- manno-heptopyran-
osyl)-h-D-glucopyranoside (6).—Allyl 2,3,4-tri-
O-benzoyl-6-O-(2,3,4,6,7-penta-O-acetyl-L-
glycero - a - D - manno - heptopyranosyl] - a - D-
glucopyranoside (5) [3] (70 mg, 0.075 mmol)
was dissolved in dry MeOH (5 mL) and tri-
ethylamine (0.5 mL) was added. The reaction
mixture was stirred for 4 days at room tem-
perature, concentrated, and traces of water
were removed by coevaporation with pyridine
(twice).
The thus obtained allyl 6-O-(L-glycero-a-D-
manno-heptopyranosyl)-a-D-glucopyranoside
was dissolved in pyridine (3 mL), and tert-
butyldimethylchlorosilane (30 mg, 0.2 mmol)
was added. The mixture was stirred for 3 h at
25 °C (TLC control, 4:1 CHCl3–MeOH),
acetic anhydride (1 mL) was added, and stir-
ring was continued for 24 h at room tempera-
ture. The mixture was concentrated and the
residue was chromatographed on silica gel
(9:1“2:3 hexane–EtOAc) to give allyl 2,3,4-
tri-O-acetyl-6-O-(2,3,4,6-tetra-O-acetyl-7-O-
tert-butyldimethylsilyl-L-glycero-a-D-manno-
heptopyranosyl)-a-D-glucopyranoside (60 mg,
0.073 mmol).
Allyl 2,3,4-tri-O-acetyl-6-O-[2,3,4,6-tetra-
O-acetyl-7-O-(3,4,6-tri-O-acetyl-2-deoxy-2-
phthalimido-i-D-glucopyranosyl)-L-glycero-h-
D-manno-heptopyranosyl]-h-D-glucopyranoside
(8).—A solution of acceptor 6 (40 mg, 0.056
mmol) and ethyl 3,4,6-tri-O-acetyl-2-deoxy-2-
phthalimido-1-thio-b-D-glucopyranoside 7 (35
mg, 0.073 mmol) in dry ether (3 mL) was
˚
stirred with 4 A molecular sieves under Ar for
1 h. Then methyl trifluoromethanesulfonate
(50 ml) was added and stirring was continued
for 24 h at room temperature. An additional 5
mg of the glycosyl donor and 60 mL of the
promoter were added and stirring was contin-
ued for another 48 h. One drop of pyridine
was added to destroy the promoter, and the
mixture was filtered and concentrated.
Column chromatography of the residue
(7:3“1:1 toluene–EtOAc) afforded 8 (45 mg,
1
70%); [h]D +77.5° (c 1, CHCl3). H NMR
data: l 7.9–7.1 (m, 4 H, Ph), 5.90 (m, 1 H,
–CHꢀ), 5.76 (dd, 1 H, J3,2 10.6, J3,4 9.1 Hz,
H-3¦), 5.55 (dd, 1 H, J4,3 9.3, J4,5 10 Hz,
H-4), 5.50 (dd, 1 H, J3,2 10.2, J3,4 9.3 Hz,
H-3), 5.39 (d, 1 H, J1,2 8.4 Hz, H-1¦), 5.36
(m, 1 H, ꢀCH2), 5.28 (dd, 1 H, J3,2 3.4, J3,4
10.4 Hz, H-3%), 5.23 (m, 2 H, H-2%,
A solution of HF in acetonitrile (350 mL, 50
mL of 40% aq HF in 1 mL of acetonitrile) was
added to a solution of the above silyl ether in
2 mL of acetonitrile and the mixture was

90
K.V. Antono6 et al. / Carbohydrate Research 314 (1998) 85–93
H-2,3,4,5,6,7a,7b,2%,5%,6%a,6%b,
5×CH2Ph),
ꢀCH2), 5.19 (t, 1 H, J4,3=J4,5=10.4 Hz, H-
4%), 5.17 (ddd, 1 H, J6,5 1.8, J6,7a 4.4, J6,7b 4.7
Hz, H-6%), 5.14 (d, 1 H, J1,2 3.8 Hz, H-1), 5.07
(dd, 1 H, J4,3 9.1, J4,5 10.2 Hz, H-4¦), 4.99 (dd,
1 H, J2,1 3.8, J2,3 10.2 Hz, H-2), 4.82 (d, 1 H,
J1,2 1.8 Hz, H-1%), 4.30 (dd, 1 H, J6b,5 4.3, J6a,6b
12.2 Hz, H-6¦b), 4.29 (dd, 1 H, J2,1 8.4, J2,3
10.6 Hz, H-2¦), 4.23 (m, 1 H, –OCH2), 4.19
(dd, 1 H, J6a,5 2.5, J6b,6a 12.2 Hz, H-6¦a), 4.10
(dd, 1 H, J5,4 10.4, J5,6 1.8 Hz, H-5%), 4.05 (m,
1 H, –OCH2), 4.02 (ddd, 1 H, J5,4 10, J5,6a 2,
J5,6b 7.4 Hz, H-5), 3.92 (ddd, 1 H, J5,4 10.2,
J5,6a 2.5, J5,6b 4.3 Hz, H-5¦), 3.82 (dd, 1 H, J6b,5
7.4, J6b,6a 11.5 Hz, H-6b), 3.76, 3.75 (2 dd, 2
H, J7a,6 4.4, J7a,7b 11, J7b,6 4.7 Hz, H-7%a,b),
3.44 (dd, 1 H, J6a,5 2, J6a,6b 11.5 Hz, H-6a),
2.13, 2.12, 2.07, 2.05, 2.03, 2.02, 1.99, 1.95,
1.91, 1.86 (10 s, 30 H, CH3CO). 13C NMR
data: l 134.1–129.5 (C, Ph, ꢀCH–), 117.95
(CH2ꢀ), 99.34 (C-1¦), 97.71 (C-1%), 94.52 (C-1),
71.80 (C-5¦), 71.71 (C-2), 70.43 (C-3¦), 69.38
(C-2%), 69.19 (C-3%), 69.09, 69.00 (C-3, 4), 68.77
(C-4¦), 68.54 (–OCH2), 67.98 (C-5%), 67.9 (C-
7%), 67.79 (C-6%), 66.27 (C-6), 64.80 (C-4%),
62.08 (C-6¦), 54.47 (C-2’’), 20.83–20.27 (7 C,
2.04, 2.03, 1.85 (3s, 9 H, 3×CH3CO). 13C
NMR data: l 98.0 (C-1%), 96.6 (C-1), 80.3
(C-3), 74.1, 72.1, 71.9, 70.7, 68.9 (C-
2,4,5,6,4%,5%), 68.8 (C-7), 62.0 (C-6%), 54.6 (C-
2%). LSIMS (+): Calcd for C62H63NO16+Na:
m/z 1100. Found: m/z 1100.
2,3,4,6-Tetra-O-acetyl-7-O-(3,4,6-tri-O-
acetyl-2-deoxy-2-phthalimido-i-D-glucopyra-
nosyl) - L - glycero - h - D - manno - heptopyranose
(11).—A solution of 9 (800 mg, 0.74 mmol) in
a 1:1 MeOH–EtOAc mixture (50 mL) was
hydrogenolyzed over 10% palladium-on-char-
coal for 64 h. The mixture was filtered
through Celite and concentrated. The residue
was conventionally acetylated with acetic an-
hydride in pyridine (1:1) for 16 h at ambient
temperature. The mixture was concentrated,
toluene was added, and the solution was con-
centrated to dryness. Column chromatogra-
phy of the residue (4:1“1:1 hexane–EtOAc)
afforded 480 mg of a mixture of 1,2,3,4,6-
penta-O-acetyl-7-O-(3,4,6-tri-O-acetyl-2-de-
oxy - 2 - phthalimido - b - D - glucopyranosyl) - L-
glycero-a,b-D-manno-heptopyranoses
(10).
CH3CO).
LSIMS
(+):
Calcd
for
Hydrazinium acetate (30 mg, 0.32 mmol) was
added to a solution of this product (200 mg,
0.23 mmol) in DMF (3 mL). The mixture was
stirred for 2 h at room temperature, diluted
with EtOAc, and washed twice with water.
The organic layer was dried (MgSO4) and
concentrated. Column chromatography of the
residue (7:3“2:3 hexane–EtOAc) afforded
the title compound as a syrup (108 mg, 52%);
[h]D +0.7° (c 0.5, CHCl3). LSIMS (+):
Calcd for C35H41O20N+Na: m/z 818. Found:
m/z 818.
C50H61NO28+Na: m/z 1146.2. Found: m/z
1146.0.
Benzyl 2,3,4,6-tetra-O-benzyl-7-O-(3,4,6-
tri-O-acetyl-2-deoxy-2-phthalimido-i-D-glu-
copyranosyl) - L - glycero - h - D - manno - hepto-
pyranoside (9).—A solution of benzyl 2,3,4,6-
tetra-O-benzyl-L-glycero-a-D-manno-heptopy-
ranoside 3 (688 mg, 1.0 mmol) and ethyl
3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-1-
thio-b-D-glucopyranoside 7 (600 mg, 1.25
mmol) in dry Et2O (30 ml) was stirred with 4
˚
A molecular sieves at room temperature for 1
Allyl 2,3,4-tri-O-benzoyl-6-O-[2,3,4,6-tetra-
O-acetyl-7-O-(3,4,6-tri-O-acetyl-2-deoxy-2-
phthalimido-i-D-glucopyranosyl)-L-glycero-h-
D-manno-heptopyranosyl]-h-D-glucopyranoside
(13).—Potassium carbonate (0.3 g) and
trichloroacetonitrile (400 mL, 4 mmol) were
added to a solution of 11 (130 mg, 0.14 mmol)
in CH2Cl2 (5 mL) and the mixture was stirred
for 16 h and filtered through a layer of silica
gel. The sorbent was washed with CH2Cl2 (20
mL), hexane (50 mL), and the syrupy 2,3,4,6-
tetra-O-acetyl-7-O-(3,4,6-tri-O-acetyl-2-de-
oxy - 2 - phthalimido - b - D - glucopyranosyl) - L-
glycero - a,b - D - manno - heptopyranosyl tri-
h under Ar and methyl trifluoromethanesul-
fonate (700 mL, 6.2 mmol) was added. Stirring
was continued for 24 h at room temperature.
One drop of pyridine was added to destroy
the catalyst, and the mixture was filtered and
concentrated. Column chromatography of the
residue (toluene“4:1 toluene–EtOAc) af-
forded 9 (800 mg, 74%) as a syrup, [h]D
1
+30.4° (c 1.46, CHCl3). H NMR data: l
7.8–7.0 (m, 29 H, Ph), 5.8 (dd, 1 H, J3,2 10,5,
J3,4 9.4 Hz, H-3%), 5.42 (d, 1 H, J1,2 8.4 Hz,
H-1%), 5.17 (t, 1 H, J4,3 =J4,5=9.5 Hz, H-4%),
4.9 (d, 1 H, J1,2 1.1 Hz, H-1), 4.8-3.6 (m, 21 H,

K.V. Antono6 et al. / Carbohydrate Research 314 (1998) 85–93
91
J6a,6b 12.3 Hz, H-6a%), 4.17 (dd, 1 H, J6b,5 2.4,
J6b,6a 12.3 Hz, H-6b¦), 4.11 (dd, 1 H, J5,4 10.1,
J5,6 1.8 Hz, H-5%), 3.88 (ddd, 1 H, J5,4 10.1,
J5,6a 4.8, J5,6b 2.4 Hz, H-5¦), 3.86 (dd, 1 H, J6b,5
chloroacetimidate (12) was eluted with 4:1“
1:1 toluene–EtOAc, yield 110 mg (71%); [h]D
1
+31° (c 0.5, CHCl3). H NMR data: l 8.78
(s, 1 H, NH), 7.84 and 7.73 (2 m, 4 H, Ph),
6.21 (br. s, 1 H, H-1), 5.71 (dd, 1 H, J3,2 10.7,
J3,4 9.2 Hz, H-3%), 5.43 (dd, 1 H, J2,1 1.8, J2,3
3.2 Hz, H-2), 5.40 (d, 1 H, J1,2 8.4 Hz, H-1%),
5.35 (dd, 1 H, J3,2 3.3, J3,4 10 Hz, H-3), 5.31 (t,
1 H, J4,3 =J4,5=10.1 Hz, H-4), 5.22 (m, 1 H,
H-6), 5.13 (dd, 1 H, J4,3 9.3, J4,5 10.1 Hz,
H-4%), 4.30 (m, 1H, H-5), 4.29 (m, 1 H-6%b),
4.22 (dd, 1 H, J2,1 8.4, J2,3 10.7 Hz, H-2%), 4.15
(dd, 1 H, J6a,5 2.3, J6a,6b 12.2 Hz, H-6%a), 3.87
(ddd, 1 H, J5,4 10.2, J5,6a 2.4, J5,6b 4.2 Hz,
H-5%), 3.80 (dd, 1 H, J7b,6 7.3, J7b,7a 11.4 Hz,
H-7b), 3.71 (dd, 1 H, J7a,6 6.8, J7a,7b 11.4 Hz,
H-7a), 2.18, 2.13, 2.02, 1.99, 1.98, 1.96, 1.84 (7
s, 21 H, CH3CO). 13C NMR data: l 199.26
(C-1%), 94.40 (C-1), 71.71 (C-5%), 70.62 (C-3%),
70.49 (C-5), 68.94 (C-3), 68.52 (C-4%), 67.96
(C-7), 67.83 (C-2), 67.40 (C-6), 64.21 (C-4),
61.84 (C-6%), 54.38 (C-2%).
6.6, J6b,6a 11.3 Hz, H-6b), 3.75 (d, 2 H, J7a,6
=
J7b,6=6.9 Hz, H-7%a,b), 3.54 (dd, 1 H, J6a,5
1.8, J6a,6b 11.3 Hz, H-6a), 2.12, 2.10, 2.03,
1.96, 1.95, 1.91, 1.86 (7s, 21 H, CH3CO). 13C
NMR data: l 134.0–123.4 (Ph, –CH2CHꢀ),
117.85 (–CHꢀCH2), 99.26 (C-1¦), 97.62 (C-1%),
94.94 (C-1), 71.82 (C-5¦), 71.78 (C-2), 70.69
(C-3), 70.56 (C-3¦), 69.43 (C-4), 69.38 (C-2%),
69.13 (C-3%), 68.90 (C-5), 68.84 (C-4¦), 68.82
(–OCH2–), 68.09 (C-5%), 68.02 (C-7%), 67.76
(C-6%), 66.48 (C-6), 64.88 (C-4%), 62.11 (C-6¦),
54.46 (C-2¦), 20.82–20.34 (7 C, CH3CO).
LSIMS (+): Calcd for C65H67NO28+Na: m/z
1332.35. Found: m/z 1331.9.
Allyl 6 - O - [7 - O - (2 - deoxy - 2 - acetamido-
i-D - glucopyranosyl)-L - glycero-h-D - manno-
heptopyranosyl] - h - D - glucopyranoside (1).—
Ethylenediamine (5 mL) was added to a solu-
tion of 13 (177 mg, 0.13 mmol) in n-butanol
(25 ml) under Ar and the mixture was stirred
for 20 h at 90 °C. After the reaction was
completed (TLC control in 1:1:1 n-BuOH–
EtOH–25% aq NH3, one ninhydrin-positive
spot, no UV absorption), the mixture was
concentrated, and coevaporated twice with a
mixture EtOH–toluene. The residue was dis-
solved in a 1:1 mixture of Py–Ac2O (50 mL),
and a catalytic amount of 4-N,N-dimethyl-
aminopyridine was added. After 4 h at room
temperature, the mixture was concentrated to
dryness. Toluene was added to and distilled
from the residue, which was then chro-
matographed on silica gel with a gradient
(0“3%) of MeOH in CHCl3 to give allyl
2,3,4-tri-O-acetyl-6-O-[2,3,4,6-tetra-O-acetyl-
7-O-(3,4,6-tri-O-acetyl-2-deoxy-2-acetamido-
b-D - glucopyranosyl)-L - glycero-a-D - manno-
The thus obtained biosyl trichloroacetimi-
date 12 (110 mg, 0.11 mmol) and allyl 2,3,4-
tri-O-benzoyl-a-D-glucopyranoside 4 (60 mg,
0.11 mmol) were stirred in dry CH2Cl2 (10 ml)
˚
with 4 A molecular sieves under Ar for 1 h. A
solution of trimethylsilyl trifluoromethanesul-
fonate (3 mL, 0.016 mmol) in CH2Cl2 was
added to a mixture at −30 °C and stirring
was continued for 2 h at −30 to −15 °C.
One drop of pyridine was added to destroy
the catalyst, and the mixture was filtered and
concentrated. Column chromatography of the
residue (4:1“2:5 toluene–EtOAc) afforded
the title compound 13 (120 mg, 78%); [h]D
+42.5° (c 1,CHCl3). 1H NMR data: l 8.0–7.2
(m, 19 H, Ph), 6.21 (t, 1 H, J2,3 =J3,4=9.6
Hz, H-3), 5.95–5.87 (m, 1 H, ꢀCH–), 5.78
(dd, 1 H, J3,2 10.6, J3,4 9.0 Hz, H-3¦), 5.56 (dd,
1 H, J4,3 9.6, J4,5 10.2 Hz, H-4), 5.44 (d, 1 H,
J1,2 3.6 Hz, H-1), 5.38 (d, 1 H, J1,2 8.4 Hz,
H-1¦), 5.38 (dd, 1 H, J2,1 3.6, J2,3 10 Hz, H-2),
5.34 (dd, 1 H, J3,2 3.5, J3,4 10.1 Hz, H-3%), 5.26
(dd, 1 H, J2,1 1.3, J2,3 3.5 Hz, H-2%), 5.18 (t, 1
H, J4,3 =J4,5=10.1 Hz, H-4%), 5.14 (t, 1 H, J4,3
=J4,5 =9.5 Hz, H-4¦), 5.14 (dt,1 H, J6,5 1.8,
heptopyranosyl]-a-D-glucopyranoside
(120
1
mg, 89%); [h]D +32.7° (c 1, CHCl3). H
NMR data: l 5.95 (d, 1 H, J 8.4 Hz, NH),
5.90 (m, 1 H, –CHꢀ), 5.52 (t, 1 H, J3,4 =
J3,2=9.9 Hz, H-3), 5.39 (dd, 1 H, J3,2 10.2, J3,4
9.6 Hz, H-3¦), 5.36 (m, 1 H, ꢀCH2), 5.31 (m, 1
H, H-6%), 5.30 (dd, 1 H, J 10.2, 8.9 Hz, H-4%),
5.26 (m, 1 H, H-3%), 5.25 (m, 2H, H-2%, ꢀCH2),
5.14 (d, 1 H, J1,2 3.8 Hz, H-1), 5.05 (t, 1 H,
J4,3=J4,5 =9.8 Hz, H-4¦), 4.96 (dd, 1 H, J4,5
10.2, J4,3 9.5 Hz, H-4), 4.91 (dd, 1 H, J2,1 3.8,
J
6,7a =J6,7b =6.9 Hz, H-6%), 4.82 (d, 1 H, J1,2
1.3 Hz, H-1%), 4.32 (ddd, 1 H, J5,4 10.2, J5,6a
1.8, J5,6b 6.6 Hz, H-5), 4.3 (dd, 1 H, J2,1 8.4,
J2,3 10.6 Hz, H-2¦), 4.27 (dd, 1 H, J6a,5 4.8,

92
K.V. Antono6 et al. / Carbohydrate Research 314 (1998) 85–93
(C-2%), 73.1 (C-4¦), 68.4 (C-6%), 68.6 (–CH2O–
), 67.2 (C-4%), 62.1 (C-6), 66.5 (C-6¦), 56.8
(C-2¦), 22.0 (CH3CO). LSIMS (+): Calcd for
C24H41NO17+Na: m/z 638.05. Found: m/z
638.0.
J2,3 10.1 Hz, H-2), 4.87 (d, 1 H, J1,2 1.4 Hz,
H-1%), 4.84 (d, 1 H, J1,2 8.2 Hz, H-1¦), 4.25 (m,
1 H, –CH2O–), 4.24 (m, 1 H, H-6¦b), 4.15 (m,
1 H, H-5%), 4.14 (dd, 1 H, J6a,5 2.4, J6a,6b 12.2
Hz, H-6¦a), 4.08 (m, 1 H, H-5), 4.06 (m, 1 H,
–CH2O–), 3.88 (dd, 1 H, J7b,6 6.7, J7b,7a 11.7
Hz, H-7%b), 3.84 (dd, 1 H, J7a,6 7.8, J7a,7b 12.2
Hz, H-7%a), 3.76 (m 2 H, H-6b, H-5¦), 3.68
(ddd, 1 H, J2,NH 8.4, J2,1 8.3, J2,3 10.5 Hz,
H-2¦), 3.51 (dd, 1 H, J6a,5 2.0, J6a,6b 11.3 Hz,
H-6a), 2.17, 2.11, 2.09, 2.07, 2.06, 2.04, 2.02,
2.01, 2.00, 1.97, 1.95 (11 s, 33 H, CH3CO). 13C
NMR data: l 133 (–CHꢀ), 118 (ꢀCH2), 100.5
(C-1¦), 98.0 (C-1%), 94.5 (C-1), 72.1 (C-3¦), 71.7
(C-5¦), 70.8 (C-2), 70.1 (C-3), 69.5 (C-2%), 69.4
(C-4), 69.1 (C-6%), 68.5 (C-4¦), 68.4 (–CH2O–,
C-5%), 68.1 (C-5), 67.3 (C-3%), 67.1 (C-7%), 66.6
(C-6), 64.8 (C-4%), 62.0 (C-6¦), 55.4 (C-2¦).
LSIMS (+): Calcd for C44H61NO27+Na: m/z
1058.15. Found: m/z 1057.9.
Deprotection of 8 (20 mg) as described for
13 afforded the target allyl trioside 1 (10 mg),
whose NMR spectra were identical with those
listed above.
Allyl 2,3,4,6-tetra-O-acetyl-7-O-(3,4,6-tri-
O-acetyl-2-deoxy-2-phthalimido-i- -glucopy-
D
ranosyl)-L-glycero-h-D-manno-heptopyranoside
(14).—To a solution of 10 (106 mg) in freshly
˚
distilled CH2Cl2 (3 mL) molecular sieves 3 A
(172 mg) were added and the mixture was
stirred under argon at room temperature. Af-
ter 30 min the mixture was cooled (ice–water)
and a solution of SnCl4 (0.3 mL of a solution
made of 0.5 mL SnCl4 in 5 mL CH2Cl2) was
added. After 1 h, allyl alcohol (0.017 mL) was
added and stirring was continued for 16 h.
Solid NaHCO3 and Na2SO4·10H2O were
added and the suspension was stirred
overnight, then filtered through a Celite pad.
The pad was washed with ethanol and ace-
tone, the filtrate was concentrated and the
residue was chromatographed with 1:1 hex-
ane–EtOAc 1:1 to yield 14, 70 mg (67%); [h]D
Triethylamine (1 mL) was added to a solu-
tion of the peracetylated trisaccharide (120
mg) in dry MeOH (9 mL). After 48 h at room
temperature, the mixture was concentrated,
dissolved in water, filtered through Celite and
concentrated to give 1 (70 mg, 98%); [h]D
+33.5° (c 1, H2O), Rf 0.3 (10:3:2, n-
BuOH:MeOH:H2O).1H NMR data: l 6.00 (m,
1 H, –CHꢀ), 5.38 (m, 1 H, ꢀCH2), 5.29 (m, 1
H, ꢀCH2), 4.97 (d, 1 H, J1,2 3.8 Hz, H-1), 4.88
(d, 1 H, J1,2 1.3 Hz, H-1%), 4.57 (d, 1 H, J1,2 8.6
Hz, H-1¦), 4.23 (ddt, 1 H, –CH2O–), 4.17
(ddd, J6,5 1.5, J6,7a 4.0, J6,7b 8.5 Hz, H-6%), 4.11
(ddt, 1 H, –CH2O–), 4.00 (dd, 1 H, J7b,6 8.3,
J7b,7a 10.9 Hz, H-7%b), 3.96 (dd, 1 H, J2,1 1.7,
1
+36.5° (c 1.43, CHCl3). H NMR data: l
5.85 (m, 1 H, –CHꢀ), 5.76 (dd, 1 H, J3,2 10.7,
J3,4 9.1Hz, H-3%), 5.42 (d, 1 H, J1,2 8.4 Hz,
H-1%), 5.32 (dd, 1 H, J3,2 3.5, J3,4 10.2 Hz,
H-3), 5.26–5.18 (m, 5 H, H-2,4,6, CH2ꢀ), 5.16
(dd, 1 H, J4,3 9.2, J4,5 10.1Hz, H-4%), 4.83 (d, 1
H, J1,2 1.4 Hz, H-1), 4.30 (dd, 1 H, J6a,5 4.8,
J6a,6b 12.2 Hz, H-6%a), 4.29 (dd, 1 H, J2,1 8.4,
J2,3 10.7 Hz, H-2%), 4.21 (dd, 1 H, J6b,5 2.4,
J6b,6a 12.2 Hz, 6%b), 4.17 (m, 2 H, H-5,
CHHO), 3.92 (ddd, 1 H, J5,4 10.1, J5,6a 4.8,
J5,6b 2.4 Hz, H-5%), 3.88 (dd, 1 H, J7a,6 8.3,
J7a,7b 11.1 Hz, H-7a), 3.89 (m, 1 H, CHHO),
3.73 (dd, 1 H, J7b,6 6.2, J7b,7a 11.1 Hz, H-7b),
2.13 (×2), 2.03, 2.01, 1.96 (×2), 1.85 (5 s, 21
H, 7CH3CO). 13C NMR data: l 99.2 (C-1%),
96.2 (C-1), 71.9, 70.6, 69.7, 69.4, 68.8, 67.9,
67.8 (C-2, 3, 4, 5, 6, 3%, 4%, 5%), 67.5 (C-7), 62.1
(C-6%), 54.6 (C-2%). LSIMS (+): Calcd for
C38H45NO20+Na: m/z 858. Found: m/z 858.
J2,3 3.4 Hz, H-2%), 3.93 (dd, 1 H, J% 2.0, J6b,6a
6b,5
8.3 Hz, H-6¦b), 3.90 (m, 1 H, H-6b), 3.87 (t, 1
H, J4,3=J4,5 =9.8 Hz, H-4%), 3.86 (m, 1 H,
H-5), 3.80 (dd, 1 H, J3,2 3.6, J3,4 9.8 Hz, H-3%),
3.80 (m, 1 H, H-7%a), 3.76 (dd, 1 H, J6a,5 1.5,
J6a,6b 8.3 Hz, H-6¦a), 3.74 (dd, J2,1 8.6, J2,3
10.4 Hz, H-2¦), 3.72 (t, 1 H, J3,2 =J3,4=9.8
Hz, H-3), 3.70 (dd, 1 H, J6a,5 2.3, J6a,6b 9.8 Hz,
H-6a), 3.58 (dd, 1 H, J2,1 3.8, J2,3 9.8 Hz, H-2),
3.58 (dd, 1 H, H-7%a), 3.56 (m, 1 H, H-3¦),
3.49 and 3.48 (m, 2 H, H-4,4¦), 3.46 (m, 1 H,
13
H-5¦), 2.07 (s, 3 H, CH3CO). C NMR data:
l 133.8 (–CHꢀ), 118.1 (ꢀCH2), 102.8 (C-1¦),
100.9 (C-1%), 98.9 (C-1), 77.2 (C-5¦), 75.1 (C-
3¦), 74.8 (C-3), 72.8 (C-5%), 72.8 (C-2), 70.1
(C-7%), 72.5 (C-3%), 71.4 (C-5), 71.1 (C-4), 71.1
Allyl 7-O-(2-deoxy-2-acetamido-i- -glu-
D
copyranosyl) - L - glycero - h - D - manno - hepto-
pyranoside (2).—De-phthaloylation and sub-

K.V. Antono6 et al. / Carbohydrate Research 314 (1998) 85–93
93
sequent acetylation of 14 (90 mg) was per-
formed as for 13. The peracetyl derivative 15
H.-P. Cordes for help with 600 MHz NMR
spectroscopy. This work was financed in part
by the Polish–Russian Scientific Cooperation
Programme and by the Deutsche Forschung-
sanstalt fu¨r Luft- und Raumfahrt e. V. (grant
POL 150-96).
13
was obtained (80 mg). C NMR data: l 133.3
(–CHꢀ), 119 (CH2ꢀ), 100.7 (C-1%), 97.1 (C-1),
73.3, 72.5, 70.1, 69.9, 68.9 (×2), 67.6, 65.6
(C-2, 3, 4, 5, 6, 3%, 4%, 5%), 69.0 (CH2O), 67.3
(C-7), 62.6 (C-6%), 54.6 (C-2%).
To a solution of 15 (80 mg) in MeOH (2
mL) NaHCO3 (220 mg) was added and the
suspension was stirred overnight, then filtered
and the filtrate was concentrated. The residue
was chromatographed on a Sephadex G-10
column with water. After concentration and
lyophilization 39 mg (90%) of 2 was obtained,
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1
[h]D +12.6° (c 0.56, H2O); H NMR data: l
4.89 (d, 1 H, H-1), 4.53 (d, 1 H, H-1%), 4.12 (t,
1 H, J6,7a 5.7, J6,7b 7.7 Hz, H-6), 3.92 (dd, 1 H,
J7a,6 7.7, J7a,7b 10.5 Hz, H-7a), 3.91 (m, 2 H,
H-2,4%), 3.88 (H-6a%), 3.82 (t, 1 H, J4,3 9.6, J4,5
9.8 Hz, H-4), 3.74 (H-3), 3.73 (H-7b), 3.71
(H-6b%), 3.69 (dd, 1 H, J2,1 8.7, J2,3 8.9 Hz,
13
H-2%), 3.55 (H-5), 3.53 (H-3%), 3.43 (H-5%). C
NMR data: l 102.3 (C-1%), 99.9 (C-1), 76.8
(C-5%), 74.8 (C-3%), 72.3 (C-5), 72.1 (C-3), 71.9
(C-7), 71.1 (C-2,4), 67.8 (C-6), 66.9 (C-4), 61.6
(C-6%), 56.3 (C-2%). LSIMS (+): Calcd for
C18H31NO12+Na: m/z 476.1744. Found: m/z
476.1757.
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Acknowledgements
We thank P. Behrens, V. Susott and R.
Engel for the expert technical assistance, and
.