Synthesis of a spacer-containing disaccharide fragment of Bordetella pertussis lipopolysaccharide

Article abstract of DOI:10.1016/S0008-6215(99)00318-3

The disaccharide 2-(p-aminophenyl)ethyl 4-O-(2-acetamido-2-deoxy-α-D-glucopyranosyl)-2,3-diacetamido-2,3-dideoxy-α-D-mannopyranoside uronate, which is assumed to be a partial structure of the Bordetella pertussis polysaccharide, was synthesized starting from D-glucose and D-glucosamine, respectively. The major synthetic transformations were conversion of D-glucosamine into the donor ethyl 3,4,6-tri-O-acetyl-2-azido-2-deoxy-1-thio-β-D-glucopyranoside and conversion of glucose, by a sequence involving 2,3-epoxide formation/opening, nucleophilic triflate displacement in the 3-position, and necessary protecting group manipulations, into the acceptor 2-(p-trifluoroacetamidophenyl)ethyl 6-O-benzyl-2,3-diazido-2,3-dideoxy-α-D-mannopyranoside. Coupling of the donor and acceptor units promoted by dimethyl(methylthio)sulfonium triflate followed by selective oxidation of the 6'-position and deprotection gave the target disaccharide. Copyright (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0008-6215(99)00318-3

www.elsevier.nl/locate/carres
Carbohydrate Research 327 (2000) 261–267
Synthesis of a spacer-containing disaccharide fragment
of Bordetella pertussis lipopolysaccharide
Magnus Nilsson, Thomas Norberg *
Department of Chemistry, Swedish Uni6ersity of Agricultural Sciences, PO Box 7015, S-750 07 Uppsala, Sweden
Received 30 July 1999; accepted 22 November 1999
Abstract
The disaccharide 2-(p-aminophenyl)ethyl 4-O-(2-acetamido-2-deoxy-a-
dideoxy-a- -mannopyranoside uronate, which is assumed to be a partial structure of the Bordetella pertussis
polysaccharide, was synthesized starting from -glucose and -glucosamine, respectively. The major synthetic
transformations were conversion of -glucosamine into the donor ethyl 3,4,6-tri-O-acetyl-2-azido-2-deoxy-1-thio-b-
-glucopyranoside and conversion of glucose, by a sequence involving 2,3-epoxide formation/opening, nucleophilic
triflate displacement in the 3-position, and necessary protecting group manipulations, into the acceptor 2-(p-trifl-
-mannopyranoside. Coupling of the donor and
D-glucopyranosyl)-2,3-diacetamido-2,3-
D
D
D
D
D
uoroacetamidophenyl)ethyl 6-O-benzyl-2,3-diazido-2,3-dideoxy-a-
D
acceptor units promoted by dimethyl(methylthio)sulfonium triflate followed by selective oxidation of the 6%-position
and deprotection gave the target disaccharide. © 2000 Elsevier Science Ltd. All rights reserved.
Keywords: Bordetella pertussis; Glycoconjugate; Vaccine
1. Introduction
charide–protein conjugate vaccines is
a
proven fact, and commercial vaccines already
exist, e.g., against childhood meningitis caused
by Haemophilus influenzae type B bacteria.
Even short synthetic fragments [3] of the natu-
ral polysaccharide have been shown to be
highly immunogenic when conjugated to a
protein.
To aid in the development of conjugate
vaccines, we have previously synthesized
penta- and decameric fragments [4,5] as well
as a pentameric analog [6] of H. influenzae
type B capsular polysaccharide, and a
nonasaccharide fragment [7] of the capsular
polysaccharide from Streptococcus pneumoniae
19F. The present article describes synthesis
work on a Bordetella pertussis lipopolysaccha-
ride fragment. The entire lipopolysaccharide
structure of B. pertussis has not yet been
published, but the partial structure shown
Bacterial lipopolysaccharides are important
bacterial surface components, that have been
studied for nearly a century by immunologists
and chemists. Chemical structures are now
known for lipopolysaccharides of a great
number of bacterial species [1]. Immunologi-
cally, the carbohydrate part of bacterial lipo-
polysaccharides give rise to highly specific
immune responses. Because of this, oligosac-
charides derived from bacterial lipopolysac-
charides should be efficient components of
glycoconjugate vaccines, in analogy with what
has been shown [2] with bacterial capsular
polysaccharides. Here, the efficacy of polysac-
* Corresponding author. Tel.: +46-18-671578; fax: +46-
18-673477.
E-mail address: thomas.norberg@kemi.slu.se (T. Norberg).
0008-6215/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved.
PII: S0008-6215(99)00318-3

262
M. Nilsson, T. Norberg / Carbohydrate Research 327 (2000) 261–267
below has been proposed [8,9]. Our own pre-
liminary NMR investigations [10] of a pen-
tasaccharide obtained from partial degrada-
tion of B. pertussis LPS confirmed the Glc-
NAc-(1“4)-ManpA2,3-(NAc)2- structural el-
ement, and indicated that both the glycosidic
linkages were alpha. The absolute configura-
tions of the component monosaccharides are,
to our knowledge, unknown, but our working
hypothesis is that the GlcNAc and Man-
NMR data of 2. Treatment of 2 with sodium
hydroxide in toluene–dimethyl sulfoxide–
butanol in the presence of tetrabutylammon-
ium hydrogensulfate and potassium carbonate
gave the allo-epoxide 3, which was directly
reacted with sodium azide and ammonium
chloride in dimethyl sulfoxide. The expected
2-azide 4 (diaxial epoxide opening) was iso-
lated in a total yield of 82% (from 2). In the
literature, similar preparations of 2-azido-altro
compounds carrying other glycosidic sub-
stituents have been reported [16]. To introduce
a second azido function, compound 4 was
treated with triflic anhydride–pyridine to give
an intermediate 3-triflate 5, which was directly
reacted with sodium azide in dimethyl sulfox-
ide. The resulting SN2 displacement product,
crystalline 2,3-diazido compound 6 (manno
configuration) was isolated in 52% yield. Next,
the temporary thioglycoside function in 6 was
replaced by a 2-(p-trifluoroacetamidophenyl)-
ethyl glycosidic function. Reaction of the ethyl
thioglycoside 6 with 2-(p-trifluoroacetami-
dophenyl)ethanol and dimethyl(methylthio)-
sulfonium triflate (DMTST) [17,18] resulted in
the formation of the two possible anomeric
glycosides 7 and 8, which were separated by
chromatography. The desired a glycoside 7
was isolated in 44% yield. The anomeric
configuration of 7 was ascertained by the
3JC-1,H-1 value (171 Hz for 7, as compared with
158 Hz for 8), as well as by observed strong
nOe contacts between H-1 and H-3/H-5 in 8
but not in 7. Variations of the glycosylation
conditions (including use of glycosyl bromide
and silver triflate or tetraethylammonium bro-
mide) in order to obtain a more favorable a/b
ratio were unsuccessful.
A(NAc)2 units are in the
D
form. Based on
this tentative structure and indications that
this part of the lipopolysaccharide is highly
immunogenic [8–10], we undertook the syn-
thesis of the disaccharide 16, the details of
which is hereby reported. The synthesized dis-
accharide carries an amino group for later
simple and specific conjugation to proteins.
The proposed partial structure [8,9] of B. per-
tussis lipopolysaccharide is shown in (1):
GlcNAcp-(1“4)-ManpA2,3-(NAc)2-(1“3)-
FucpN(Ac)Me-(1 − − − “
(1)
2. Results and discussion
The major challenge in the synthesis of
disaccharide 16 is the construction of the 2,3-
diacetamidomannuronate unit, a monosaccha-
ride, which to our knowledge, has not been
synthesized before. It has been detected as a
constituent of both B. pertussis [8,9] and Pseu-
domonas aureginosa [11] lipopolysaccharides,
in a glycoprotein from Bacillus stearother-
mophilus [12,13], and in teichuronic acids [14].
It was decided to synthesize a 2,3-diac-
etamido-
D
-mannuronic acid derivative from
Opening of the benzylidene ring in 7 with
sodium cyanoborohydride–fluoroboric acid
yielded compound 9 (73% yield from 7), which
was used as an acceptor in a DMTST-pro-
moted disaccharide coupling with donor 10
D
-glucose, and to introduce first the acet-
amido functions via azide opening of a 2,3-
epoxide (to give a 2-azido function) followed
by azide displacement of a 3-triflate (to give a
3-azido function), and then to introduce the
carboxylate function at the disaccharide stage
of the synthesis. The following steps were
carried out to synthesize disaccharide 16
(Scheme 1).
(prepared as described [19] from
D-glu-
cosamine). As expected, the a-disaccharide
derivative 11 was formed as a major product
together with minor amounts of the corre-
sponding b isomer. However, the a/b mixture
(86% total glycosylation yield, a/b ratio 83:17)
proved difficult to resolve by chromatography
at this stage, so the crude mixture was instead
Compound 1 [15] was monotosylated in the
3-position with p-toluenesulfonyl chloride in
pyridine to give crystalline 2 in 50% yield. The
1
position of tosylation was evident from the H

M. Nilsson, T. Norberg / Carbohydrate Research 327 (2000) 261–267
263
subjected to treatment with hydrogen sulfide
in pyridine–triethylamine–water (to reduce
the azido functions), followed by acetylation
of the formed triamino products with acetic
anhydride in pyridine. The resulting mix-
ture of triacetamido derivatives could now be
Scheme 1.

264
M. Nilsson, T. Norberg / Carbohydrate Research 327 (2000) 261–267
using a matrix of glycerol or m-nitrobenzyl
alcohol. For HRMS, PEG was used as an
internal standard. TLC was performed on Sil-
ica Gel F254 (E. Merck, Darmstadt, Germany)
with detection by UV light and by staining
with 5% sulfuric acid in ethanol or 0.5% nin-
hydrin in butanol. Column chromatography
resolved by column chromatography to give
the pure a anomer 13 in 40% yield (calculated
from 10). Removal of the benzyl group of 13
by catalytic hydrogenation gave the 6%-OH
compound 14, which was then treated with
dimethyl sulfoxide–acetic anhydride. The re-
sulting oxidation product (aldehyde 15) was
stable to further oxidation by the reagent, and
was therefore isolated (58% yield) and treated
with iodine in dioxane–water to complete the
oxidation to a carboxyl group (several other
attempts to carry out the hydroxyl to carboxyl
oxidation, i.e., with 2,2,6,6-tetramethylpipe-
ridinyloxy radical or pyridinium dichromate,
were made but gave inferior results). The
crude carboxylic acid was treated in situ with
aqueous potassium hydroxide to remove O-
acetyl groups. The conditions required for re-
moval of the acetate groups resulted in loss
also of the N-trifluoroacetyl group, so the
final product disaccharide 16 was isolated as a
2-(p-aminophenyl)ethyl glycoside in 67% yield
(from 15). Purification by gel filtration and
analysis by NMR and mass spectroscopy ver-
ified the structure of 16 in all respects. Conju-
gation of 16 to proteins and immunological
studies with the obtained neoglycoconjugate
will be reported elsewhere.
,
was performed on Matrex silica gel 60 A
(35–70 mm, Amicon). Molecular sieves (pow-
,
,
dered 3 A and 4 A) were dried at 280 °C in
vacuo overnight. Dichloromethane was dis-
tilled from P2O5 when necessary. Dimethyl-
(thiomethyl)sulfonium triflate (DMTST) was
prepared by mixing equimolar amounts of
methyl triflate and dimethyl disulfide in dry
dichloromethane at 0 °C, precipitating with
dry ether, and filtering off (the hygroscopic)
solid, essentially as previously described
[17,18]. The material was stored under dry
nitrogen at −20 °C.
Ethyl 4,6-O-benzylidene-3-O-p-toluenesul-
fonyl-1-thio-i- -glucopyranoside (2).—To a
D
stirred and cooled (0 °C) solution of ethyl
4,6-O-benzylidene-1-thio-b- -glucopyranoside
D
(1) [15] (5.0 g, 16.0 mmol) in pyridine was
added p-toluenesulfonyl chloride (9.2 g, 48.2
mmol) and the mixture was slowly allowed to
reach rt. After stirring for 70 h the mixture
was cooled (0 °C) and water (0.9 mL) was
added. The mixture was stirred for another 40
min, then diluted with toluene, washed with
water, aq 2 M sulfuric acid, aq. 1 M sodium
hydrogen carbonate, dried (Na2SO4), filtered
and concentrated to give a liquid residue (:
20 mL). This residue was applied to a short,
wide column (100 g silica, packed and eluted
with 6:1 toluene–ethyl acetate), pure fractions
were concentrated and crystallized from
ethanol. More crystals were obtained by re-
peating the chromatographic and crystalliza-
tion procedure with the contaminated
chromatographic fractions and the concen-
trated mother liquor, giving a total yield of
compound 2 of 3.76 g (50%) as needles, mp
115–116 °C, [h]D −83°; NMR data (CDCl3):
13C, l 15.2 (SCH2CH3), 21.7 (CH3Ar), 24.9
(SCH2CH3) 68.5 (C-6), 70.7 (C-5), 71.9 (C-2),
77.3 (C-4), 83.1 (C-3), 86.8 (C-1), 101.7
3. Experimental
General methods.—Concentrations were
performed at reduced pressure (bath tempera-
ture B40 °C, unless otherwise stated).
Weighed yields are calculated from residues
dried in vacuum overnight. Optical rotations
were measured at 27 °C (c 1.0, chloroform,
unless otherwise stated), using a Perkin–
Elmer 341 polarimeter. NMR spectra were
recorded for solutions in CDCl3 (internal
1
Me4Si, l=0.00) or D2O (internal acetone, H
13
l=2.225, C l=30.7) at 303 K unless other-
wise stated with Bruker DRX 400 or DRX
600 spectrometers. Only selected NMR data
are reported. Assignments were corroborated
by appropriate 2D experiments. Coupling
constants (in Hz) are given within brackets.
The FABMS spectra were recorded with a
Jeol JMS-SX/SX-102A instrument. Ions were
produced by a beam of Xe-atoms (6 keV),
1
(CHPh), H, l 1.32 (t, 3H, SCH2CH3), 2.31 (s,
3H, CH3Ar), 2.76 (m, 2H, SCH2CH3), 3.03 (d,
1 H, OH, JOH,2=2.4), 3.48 (m, 1 H, H-5), 3.63

M. Nilsson, T. Norberg / Carbohydrate Research 327 (2000) 261–267
265
another
1.5
h,
then
diluted
with
(t, 1 H, H-4, J4,5=9.5), 3.67–3.74 (m, 2 H,
H-2/H-6a), 4.34 (dd, 1 H, H-6b, J6b,5 =5.0,
dichloromethane, washed with aq 1 M sulfuric
acid, aq 1 M sodium hydrogen carbonate,
water, dried (Na2SO4), filtered and concen-
trated. The residue (containing crude 5) was
dissolved in dimethyl sulfoxide (20 mL) and
added to a mixture of sodium azide (2.72 g,
41.7 mmol) and dimethyl sulfoxide (50 mL).
After 30 min at rt the reaction mixture was
partitioned between toluene and water, and
the toluene layer was washed with water, dried
(Na2SO4), filtered and concentrated. Column
chromatography of the residue with ethyl ac-
etate in petroleum ether (stepwise gradient
elution, 17–50%) gave 6 (1.57 g, 52%) as a
solid which could be crystallized from ethanol
giving needles, mp 144–145 °C; [h]D −5°;
NMR data (CDCl3): 13C, l 15.1 (SCH2CH3),
26.0 (SCH2CH3), 63.1 (C-3), 65.1 (C-2), 68.4
(C-6), 72.1 (C-5), 77.4 (C-4), 84.5 (C-1), 101.7
J6b,6a =10.6), 4.51 (d, 1 H, H-1, J1,2=9.7),
4.70 (dd, 1 H, H-3, J3,2=8.6, J3,4 =9.5), 5.37
(s, 1 H, CHPh). (HRMS: Calc. for C22H27O7S:
467.1198. Found: 467.1219 [M+H+].
Ethyl 2-azido-4,6-O-benzylidene-2-deoxy-1-
thio-i- -altropyranoside (4).—To a stirred
D
solution of 2 (4.38 g, 9.38 mmol) in toluene–
dimethyl sulfoxide–n-butanol (80/4/1.6 mL)
was first added tetrabutylammonium hydro-
gensulfate (0.64 g, 1.89 mmol), then a pow-
dered mixture of sodium hydroxide (0.98 g,
24.5 mmol) and potassium carbonate (3.0 g,
21.7 mmol). The mixture was stirred for 2 h at
rt, then diluted with toluene, washed with
water, dried (Na2SO4), filtered and concen-
trated. A mixture of the residue (containing
crude 3), sodium azide (2.45 g, 37.7 mmol)
and ammonium chloride (2.52 g, 47.1 mmol)
in dimethyl sulfoxide (40 mL) was stirred at
80 °C for 20 h. The reaction mixture was then
diluted with ethyl acetate, washed with water
and aq 1 M sodium hydrogen carbonate, dried
(Na2SO4), filtered and concentrated. The
residue was crystallized from ethanol giving
needles of 4 (1.36 g, 43%). Column chro-
matography of the mother liquor with ethyl
acetate in petroleum ether (stepwise gradient
elution, 20–25%) gave more 4 (1.24 g, 39%),
mp 105–106 °C, [h]D −28°; NMR data
(CDCl3): 13C, l 15.1 (SCH2CH3), 26.1
(SCH2CH3), 65.6 (C-2), 67.1 (C-5), 68.6 (C-3),
69.1 (C-6), 76.4 (C-4), 82.1 (C-1), 102.3
1
(CHPh), H, l 1.31 (t, 3 H, SCH2CH3), 2.75
(q, 2 H, SCH2CH3), 3.46 (m, 1 H, H-5, J5,6b
=
5.0), 3.86 (t, 1 H, H-6a, J6a,6b =10.4), 3.92 (dd,
1 H, H-3, J3,4=10.1), 3.97 (dd, 1 H, H-2,
J2,3=3.7), 4.07 (t, 1 H, H-4, J4,5=9.3), 4.32
(dd, 1 H, H-6b), 4.73 (d, 1 H, H-1, J1,2 =1.5),
5.64 (CHPh). HRMS: Calc. for C15H19N6O3S:
363.1239, Found: 363.1284 [M+H+].
2-(p-Trifluoroacetamidophenyl)ethyl 2,3-di-
azido-6-O-benzyl-2,3-dideoxy-h- -mannopyra-
D
noside (9).—To a stirred mixture of 6 (0.65 g,
1.79 mmol), 2-(trifluoroacetamidophenyl)-
,
ethanol (0.50 g, 2.14 mmol) and 4 A molecu-
lar sieves in dichloromethane (30 mL) was
added DMTST (1.38 g, 5.36 mmol), and the
reaction mixture was stirred at rt for 70 h,
after which pyridine (1.5 mL) was added, and
stirring was continued for 30 min. The mix-
ture was then filtered through Celite, diluted
with dichloromethane, washed with aq 2 M
sulfuric acid, aq 1 M sodium hydrogen car-
bonate, water, dried (Na2SO4), filtered and
concentrated. Column chromatography of the
residue with ethyl acetate in petroleum ether
(stepwise gradient elution, 25–33%) gave first
7 (0.42 g, 44%, NMR data (CDCl3): 4.74
(1.6)/98.2 (171) (H-1/C-1) and then 8 (0.34 g,
36%, NMR data (CDCl3): 4.62 (nd)/100.8
(158) (H-1/C-1)). To a stirred mixture of the
a-glycoside 7 (0.15 g, 0.28 mmol), sodium
cyanoborohydride (71 mg, 1.12 mmol) and 3
1
(CHPh), H, l 1.32 (t, 3 H, SCH2CH3), 2.77
(q, 2 H, SCH2CH3), 3.82 (t, 1 H, H-6a,
J6a,6b =10.2), 3.86 (dd, 1 H, H-2, J2,3=3.5),
3.91 (dd, 1 H, H-4, J4,5 =9.6), 3.97 (m, 1 H,
H-5), 4.27 (t, 1 H, H-3, J3,4=3.0), 4.33 (dd, 1
H, H-6b, J6b,5 =4.8), 5.13 (d, 1 H, H-1, J1,2=
1.7), 5.63 (CHPh). HRMS: Calc. for
C15H20N3O4S: 338.1174. Found: 338.1183
[M+H+].
Ethyl
dideoxy-1-thio-i-
2,3-diazido-4,6-O-benzylidene-2,3-
-mannopyranoside (6).—A
D
solution of 4 (2.82 g, 8.36 mmol) in 2:1
dichloromethane–pyridine (30 mL) was
stirred and cooled (0 °C) while triflic anhy-
dride (3.5 mL, 20.9 mmol) was added drop-
wise during 15 min. After 1 h, cooling was
removed and the reaction mixture was stirred

266
M. Nilsson, T. Norberg / Carbohydrate Research 327 (2000) 261–267
,
The resulting mixture was concentrated and
co-concentrated twice with pyridine. The
residue, containing crude tri-amine 12, was
stirred with 2:1 pyridine–acetic anhydride (9
mL) for 90 min, then concentrated and co-
concentrated twice with toluene. Flash column
chromatography of the residue with methanol
in ethyl acetate (stepwise gradient elution, 10–
20%) gave the title compound 13 as a syrup
(0.154 g, 40%, calculated from 10), [h]D +70°.
NMR data (CDCl3) are given in the table
below.
A molecular sieves in THF (4 mL) at 0 °C,
was added dropwise a solution of fluoroboric
acid (54% in diethyl ether; 0.15 mL, 1.12
mmol) in THF (1 mL). Additional fluoroboric
acid (0.05 mL, 0.37 mmol) was added after 90
min, and after 4 h the reaction mixture was
filtered through Celite, diluted with ethyl ac-
etate, washed with aq 1 M sodium hydrogen
carbonate, brine, dried (Na2SO4), filtered and
concentrated. Column chromatography of the
residue with ethyl acetate in toluene (stepwise
gradient elution, 17–25%) gave the title com-
pound 9 as a solid (0.11 g, 73%), [h]D +62°.
NMR data (CDCl3): 13C, l 35.4 (CH2Ar), 61.9
(C-2), 62.1 (C-3), 68.4 (OCH2CH2Ar), 69.2
(C-4), 70.2 (C-5), 70.6 (C-6), 73.8 (CH2Ph),
1
2
3
4
5
6a
Glc 5.07 4.28 5.02 5.17 3.64 3.86 4.01
(3.5) (10.7) (10.6) (9.9) (12.6)
6b
1
97.8 51.4 71.7 67.4 69.2 61.4
(175)
Man 4.60 4.15 4.73 3.82 2.89 3.31 3.43
(1.9) (2.9) (9.7) (nd) (nd) (11.1)
97.2 53.6 50.5 71.4 71.2 69.2
(170)
97.4 (JH,C=171, C-1), H, l 2.88 (t, 2 H,
CH2Ar), 3.40 (m, 1 H, H-5), 3.59 (dd, 1 H,
H-6a, J6a,6b =9.9, J6a,5 =5.8), 3.64–3.68 (m,
2H, H-6b, OCHHCH2Ar), 3.74 (dd, 1 H, H-2,
J2,3 =3.6), 3.79 (dd, 1 H, H-3, J3,4=9.7),
3.83–3.92 (m, 2 H, H-4, OCHHCH2Ar), 4.50
and 4.59 (2 d, 2 H, CH2Ph), 4.74 (d, 1 H, H-1,
J1,2 =1.3). HRMS: Calc. for C23H25F3N7O5:
536.1869, Found: 536.1851 [M+H+].
1
Further H/13C chemical shifts are 1.91/23.7
(s, 6 H, 2 CH3CO), 2.00/20.7, 2.04/20.9, 2.08/
20.9, 2.10/23.3 (4 s, 12 H, 4 CH3CO), 2.85,
2.98/35.8 (CH2Ar), 3.86, 4.01/61.4 (CH2CH2-
Ar), 5.87, 5.94, 6.30 (3 d, 3H, 3NHAc).
HRMS: Calc. for C41H52F3N4O15: 897.3381.
Found: 897.3428 [M+H+].
2-(p-Trifluoroacetamidophenyl)ethyl 4-O-(2-
acetamido-3,4,6-tri-O-acetyl-2-deoxy-h- -glu-
D
copyranosyl)-2,3-diacetamido-6-O-benzyl-2,3-
dideoxy-h- -mannopyranoside (13).—A mix-
ture of ethyl 3,4,6-tri-O-acetyl-2-azido-2-de-
-glucopyranoside (10 [19], 0.169
D
2-(p-Aminophenyl)ethyl 4-O-(2-acetamido-
oxy-1-thio-a-
D
2-deoxy-h-
2,3-dideoxy-h-
D
-glucopyranosyl)-2,3-diacetamido-
-mannopyranoside uronate
g, 0.451 mmol), 9 (0.254 g, 0.47 mmol), 2,6-di-
tert-butyl-4-methylpyridine (0.049 g, 0.237
D
(16).—A mixture of 13 (50 mg, 0.056 mmol)
and Pd/C (25 mg; 10%) in 1:1 ethanol–ethyl
acetate (6 mL) was hydrogenated at atmo-
spheric pressure for 2 h, then additional Pd/C
(50 mg; 10%) was added and the mixture was
hydrogenated for another 3 h. The mixture
was filtered through Celite, diluted with
ethanol, concentrated and dried in vacuum for
12 h. The residue (containing crude 14) was
dissolved in a mixture of 2:1 dimethyl sulfox-
ide–acetic anhydride (3 mL) and stirred at rt.
for 6 h, then diluted with ethyl acetate,
washed with water, dried (Na2SO4), filtered
and concentrated. Column chromatography of
the residue with methanol in 9:1 ethyl acetate
gave the aldehyde 15 as a syrup (26.2 mg,
58%). To a stirred solution of 15 (22 mg, 0.015
mmol) and iodine (8 mg, 0.0185 mmol) in 6:4
,
mmol) and
4
A
molecular sieves in
dichloromethane (8 mL), was stirred at rt for
45 min. DMTST (0.349 g, 1.35 mmol) was
added and the reaction mixture was stirred at
rt for 14 h. Then pyridine was added, the
mixture was filtered through Celite, diluted
with dichloromethane, washed with aq. 2 M
sulfuric acid, aq 1 M sodium hydrogen car-
bonate, dried (Na2SO4), filtered and concen-
trated. Column chromatography of the
residue with ethyl acetate in toluene (20%)
gave the a-disaccharide 11 in admixture with
its b isomer (0.328 g, 86%, a/b ratio: 83:17).
To a solution of this syrupy a/b-mixture (0.32
g, 0.38 mmol) in 2:1.5:1 pyridine–triethyl-
amine–water (19.5 mL) was slowly bubbled
hydrogen sulfide for 55 min. The flask was
then sealed and stirring was continued for 1 h.

M. Nilsson, T. Norberg / Carbohydrate Research 327 (2000) 261–267
267
References
dioxane–water (1.5 mL) was added dropwise
aq 4% (w/v) potassium hydroxide. The pH
was kept between 8–9 for 1 h until 15 was
consumed, then an additional aq 4% (w/v)
potassium hydroxide was added until the pH
was 11. The reaction mixture was heated to
60 °C and stirred for another 36 h. Then dry
ice was added until neutral pH, and the mix-
ture was concentrated. Column chromatog-
raphy of the residue (12:3:3:2–4:3:3:2 ethyl
acetate–methanol–acetic acid–water) fol-
lowed by gel filtration (Sephadex G-15
column) using 95:5 water–butan-1-ol as elu-
ant gave the title compound 16 as a white
powder (11 mg, 67% from 15), [h]D +19° (c
0.5, water). NMR data (CDCl3) are given in
the table below.
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1
2
3
4
5
6a
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Glc 5.07 3.82 3.69 3.47 3.72 3.81 3.81
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96.6 53.9 70.7 69.8 72.1 60.3
Man 4.69 4.17 4.37 3.95 3.96
(3.2) (9.1)
98.1 51.1 50.7 70.8 73.9 176.1
Further H/13C chemical shifts are 1.88/22.1,
1
2.01/22.2, 2.03/22.1 (3 s, 9 H, 3 CH3CO),
2.95/34.6 (CH2Ar), 3.76, 3.96/68.5 (CH2-
CH2Ar), 7.10/120.1, 7.34/130.4 (Ar), 174.5,
174.8, 174.9 (3 CꢀO). HRMS: Calc. for
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.
.