Preparation of spacer-containing di-, tri-, and tetrasaccharide fragments of the circulating anodic antigen of Schistosoma mansoni for diagnostic purposes

Article abstract of DOI:10.1016/S0008-6215(98)00125-6

The chemical synthesis of β-D-GlcpA-(1→3)-β-D-GalpNAc-(1→O)CH₂CH=CH₂, β-D-GalpNAc-(1→6)-[β-D-GlcpA-(1→3)]-β-D-GalpNAc-(1→O)CH₂CH =CH₂, and β-D-GlcpA-(1→3)-β-D-GalpNAc-(1→6)-[β-D-GlcpA- (1→3)]-β-D-GalpNAc-(1→O)CH

Full text of DOI:10.1016/S0008-6215(98)00125-6

Carbohydrate Research 309 (1998) 175±188
Preparation of spacer-containing di-, tri-, and
tetrasaccharide fragments of the circulating
anodic antigen of Schistosoma mansoni for
diagnostic purposes
Koen M. Halkes a, Henricus J. Vermeer a, Ted M. Slaghek b,
Peter A.V. van Hooft a, Arnoud Loof a, Johannis P. Kamerling a,*,
Johannes F.G. Vliegenthart a
a Bijvoet Center, Department of Bio-Organic Chemistry, Utrecht University, PO Box 80.075, NL-3508
TB Utrecht, The Netherlands
b Agrotechnological Research Institute (ATO-DLO), PO Box 17, NL-6700 AA Wageningen, The
Netherlands
Received 19 January 1998; accepted 20 April 1998
Abstract
The chemical synthesis of ꢀ-d-GlcpA-(1!3)-ꢀ-d-GalpNAc-(1!O)CH2CH=CH2, ꢀ-d-Galp-
NAc-(1!6)-[ꢀ-d-GlcpA-(1!3)]-ꢀ-d-GalpNAc-(1!O)CH2CH=CH2, and ꢀ-d-GlcpA-(1!3)-ꢀ-d-
GalpNAc-(1!6)-[ꢀ-d-GlcpA-(1!3)]-ꢀ-d-GalpNAc-(1!O)CH2CH=CH2 is described. These oli-
gosaccharides represent fragments of the circulating anodic antigen, secreted by the parasite
Schistosoma mansoni in the circulatory system of the host. The applied synthesis strategy includes
the preparation of a non-oxidised backbone oligosaccharide, with a levulinoyl group at O-6 of the
ꢀ-d-glucose residue. After the selective removal of the levulinoyl group, the obtained hydroxyl
functions were converted into carboxyl groups, using pyridinium dichromate and acetic anhydride
in dichloromethane, to a€ord the desired glucuronic-acid-containing oligosaccharides. Subse-
quently, the allyl glycosides have been elongated with cysteamine to give the corresponding amine-
spacer-containing oligosaccharides. # 1998 Elsevier Science Ltd. All rights reserved
Keywords: Oxidation; Schistosoma mansoni; Circulating anodic antigen
1. Introduction
million people predominantly in third world coun-
tries. Schistosomiasis is caused by the presence of
the blood-¯uke Schistosoma (Trematode) [2] in the
blood-vessels of the human host (®nal host).
Because for cure of the infection chemotherapy [3±5]
is available, early diagnosis is important. At present,
diagnosis relies mainly on the parasitological
Human schistosomiasis is one of the major
parasitic diseases in the world [1], a€ecting 200
* Corresponding author. Fax: +31 30 2540980; e-mail:
kame@boc.chem.uu.nl
0008-6215/98/$Ðsee front matter # 1998 Elsevier Science Ltd. All rights reserved
PII S0008-6215(98)00125-6

176
K.M. Halkes et al./Carbohydrate Research 309 (1998) 175±188
examination of urine and faeces for the presence of
Schistosoma eggs. In addition, for diagnosis, ser-
ological methods are increasingly used, aiming at
detection of speci®c antibodies or of speci®c anti-
gens. Diagnosis based on the presence of Schisto-
soma antigens in the circulatory system or the urine
of the host is increasingly used [6±9]. The gut of the
parasite is an important source of these antigens
since many gut-associated antigens are excreted
into the circulation of the host following digestion
of food (e.g., blood cells, proteins) by the parasite.
One of the major gut-associated antigens is the
circulating anodic antigen (CAA). CAA is a glyco-
protein in which the major threonine-linked car-
bohydrate part consists of disaccharide repeating
units, namely {!6)-[ꢀ-d-GlcpA-(1!3)]-ꢀ-d-Galp-
NAc-(1!}n, probably connected to the protein via
a, yet unknown, core saccharide with GlcNAc at
the reducing end [10]. As CAA is an important
circulating antigen and the glycan part strongly
immunogenic, it would be an interesting target
antigen for antibody assays. However, this
approach is limited by the availability of CAA
from biological sources in sucient amounts. In
order to replace isolated CAA in diagnostic meth-
ods, a synthetic program for the preparation of a
wide range of medium-sized oligosaccharide frag-
ments of CAA was initiated, to determine the
optimal epitope of CAA that can act as an immu-
nologic determinant.
In this report, the stereoselective synthesis of the
CAA fragments 1, 3, and 5 and their cysteamine
elongated derivatives 2, 4, and 6 is described
(Fig. 1). A preliminary report on the synthesis of 5
has appeared [11].
The compounds 2, 4, and 6 are suitable for con-
jugation [12±14] with protein carriers, to be tested
in immunological assays with the aim to develop a
new diagnostic method for schistosomiasis.
2. Results and discussion
To establish a convenient and systematic route
for the synthesis of the target structures 1±6, the
disaccharide building block allyl (6-O-levulinoyl-
2,3,4-tri-O-p-toluoyl-ꢀ-d-glucopyranosyl)-(1!3)-4-
O-acetyl-6-O-tert-butyldimethylsilyl-2-deoxy-2-
phthalimido-ꢀ-d-galactopyranoside (16) (Scheme 1)
was developed which can easily be transformed
into an acceptor (17) or a donor (18), both suitable
for the synthesis of the larger oligosaccharides.
Fig. 1. Target structures for the synthetic route.

K.M. Halkes et al./Carbohydrate Research 309 (1998) 175±188
177
Scheme 1. Synthesis of the mono- and disaccharide building blocks; OMP, OC6H4OCH3; TBDMS, ((CH3)3C)(CH3)Si; Lev,
COCH2CH2COCH; T, COC6H4CH3
After the construction of the backbone oligo-
saccharide, the unique protecting group at the C-6
position of the ꢀ-d-glucose residue, the levulinoyl
group, can be selectively removed to obtain a
hydroxyl function which can be oxidised to prepare
the desired ꢀ-d-glucuronic acid residue.
for the synthesis of monosaccharide 9 another
strategy than reported here. Although the overall
yield of the latter synthetic route is slightly higher,
our present strategy is less time-consuming and
involves less puri®cation steps. Oxidative removal
[20] of the 4-methoxyphenyl group at C-1 in a two-
phase system using ammonium cerium(IV) nitrate
in acetonitrile±toluene±water (!10), followed by
imidoylation [21] of the anomeric center in the
presence of 1,8-diazabicyclo[5.4.0]undec-7-ene yiel-
ded donor 11 (overall yield 78%). Condensation of
allyl 4,6-O-benzylidene-2-deoxy-2-phthalimido-ꢀ-
d-glucopyranoside (12) [22,23] with 11 in dichloro-
methane in the presence of trimethylsilyl tri¯ate
(0.05 equiv based on 11), a€orded disaccharide 13
(89%). After removal of the benzylidene function in
tri¯uoroacetic acid±dichloromethane-water (!14,
98%), the primary hydroxyl function of the gluco-
samine unit was selectively protected with a silyl
ether group using tert-butyldimethylsilyl chloride
in pyridine at 0 ꢀC (!15, 96%).
The monosaccharide building blocks used for
the synthesis of 16 are depicted in Scheme 1.
Starting from ꢀ-d-glucose pentaacetate, the 4-
methoxyphenyl function was introduced at the
anomeric center using trimethylsilyl tri¯ate [15] as
a catalyst (!7, 77%). Subsequent Zemplen deace-
tylation and selective silylation [16] of the primary
hydroxyl function, using tert-butyldimethylsilyl
ꢀ
chloride in pyridine at 0 C, followed by toluoyla-
tion of the remaining hydroxyl groups with p-
toluoyl chloride in pyridine, a€orded 8 in 63%
overall yield. After removal of the tert-butyldi-
methylsilyl ether group, using p-toluenesulfonic
acid in acetonitrile±water, levulinoylation of the
HO-6 function with levulinic acid in the presence
of 2-chloro-1-methylpyridinium iodide and 1,4-
diazabicyclo[2.2.2]octane in 1,2-dichloroethane
[17,18] gave 9 (80%). Slaghek et al. [19] followed
At this stage of the synthetic route, the ꢀ-d-
glucosamine moiety was transformed into the
desired ꢀ-d-galactosamine residue by epimerisation

178
K.M. Halkes et al./Carbohydrate Research 309 (1998) 175±188
of the hydroxyl function at C-4 [24,25]. To this
end, a tri¯ate group was introduced applying
tri¯uoromethanesulfoꢀnic anhydride in dichloro-
methane±pyridine (0 C), and via a SN2 displace-
ment using tetrabutylammonium acetate in N,N-
dimethylformamide, the desired 4-O-acetyl-pro-
tected building block 16 was obtained in 78% yield.
Removal of the silyl ether group under acidic
conditions (p-toluenesulfonic acid in acetonitrile±
water) gave disaccharide derivative 17 (91%).
Compound 17 is a suitable acceptor for the synth-
esis of the larger oligosaccharides (see below).
However, compound 17 can also be used as an
intermediate in the synthesis of disaccharide target
structure 1. For the last mentioned application, the
primary hydroxyl function of the galactosamine
unit in 17 was acetylated with acetic anhydride in
pyridine (!19, 88%). Then the levulinoyl group at
HO-60 was removed using hydrazinium acetate
[26,27] (!20, 94%), and subsequently the ꢀ-d-glu-
cose residue could be converted into the desired ꢀ-
d-glucuronic acid moiety through oxidation. In the
case of the disaccharide derivative the best result
was obtained using a Swern oxidation with oxalyl
chloride and methyl sulfoxide [28] yielding the
aldehyde intermediate, which was further oxidised
with NaClO2 [29] to a€ord disaccharide derivative
21 (83%). Dephthaloylation/deacylation of 21
using methylamine [30] in ethanol (2 days), and
subsequent re-N-acetylation with acetic anhydride in
methanol at 0 ꢀC, a€orded target compound 1 (88%).
For the synthesis of the target trisaccharide 3,
acceptor disaccharide 17 was coupled with ethyl
3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-1-thio-ꢀ-d-
galactopyranoside (22) in dichloromethane, using
N-iodosuccinimide and tri¯ic acid [31] as catalytic
system, to a€ord 23 (80%) (Scheme 2). Removal of
the levulinoyl function of the glucose residue (see
19), gave trisaccharide derivative 24 (91%). Con-
trary to the oxidation of 20, no satisfactory results
were obtained for the oxidation of 24 via the Swern
method. However, the mild oxidation of 24 with
pyridinium dichromate in dichloromethane in the
presence of pulverised 4 A molecular sieves [32]
resulted in the formation of 25 in 86% yield.
Dephthaloylation/deacylation of 25 using methyl-
amine [30] in ethanol (5 days), and subsequent re-
N-acetylation with acetic anhydride in methanol at
ꢀ
0 C, a€orded the target compound 3 (74%).
In order to prepare the tetrasaccharide target
structure 5, disaccharide donor 18 was synthesised
from 16 in a two reaction step sequence (Scheme
1). Isomerisation of the double bond of the allyl
function at C-1 [33], followed by removal of the 1-
propenyl function using N-iodosuccinimide in tet-
rahydrofuran and water [34] a€orded the hemi-
acetal derivative of 16. Activation of the anomeric
center was achieved by imidoylation [21] using tri-
chloroacetonitrile and 1,8-diazabicyclo[5.4.0]undec-
7-ene in dichloromethane resulting in the di-
saccharide imidate 18 (overall yield 71%, based on
16). Condensation of 17 with 18 in dichloro-
methane in the presence of trimethylsilyl tri¯ate
(0.05 eq based on 18) a€orded the tetrasaccharide
derivative 26 (73%) (Scheme 3). The silyl ether
group could be removed selectively under acidic
conditions (p-toluenesulfonic acid in acetonitrile±
water), and the product was acetylated with acetic
anhydride in pyridine to a€ord 27 (90% over two
steps). Note that the desilylated intermediate in the
last reaction step sequence can be used as a tetra-
saccharide acceptor, suitable for the synthesis of
Scheme 2. Intermediates towards trisaccharide 3

K.M. Halkes et al./Carbohydrate Research 309 (1998) 175±188
179
Scheme 3. Intermediates towards tetrasaccharide 5
larger oligosaccharide structures. Compound 27
was delevulinoylated (see 19), to a€ord diol 28 in
98% yield. Then, oxidation of the two primary
hydroxyl functions (see 24) resulted in the forma-
tion of 29 (60%). However, the extremely long
reaction time necessary (up to 96 h) to complete the
reaction, made this method less attractive. There-
fore, pyridinium dichromate in dichloromethane in
the presence of acetic anhydride [35] was attemp-
ted, since it was claimed that acetic anhydride
facilitates the reduction of chromium(VI) from the
intermediate ester, thereby accelerating the reac-
tion [36]. Indeed, by using the latter method, the
reaction time for the oxidation of tetrasaccharide
28 into 29 was decreased to 4.5 h and the yield
slightly increased to 64%. Dephthaloylation/deacyl-
ation of 29 using methylamine [30] in ethanol (7
days), and re-N-acetylation with acetic anhydride in
procedures of reaction mixtures, organic solutions
were washed with appropriate amounts of the
indicated aq solutions, then dried (MgSO4), aꢀnd
concentrated under reduced pressure at 20±40 C
(water-bath). Column chromatography was per-
formed on Kieselgel 60 F254 (E. Merck, 70±230
mesh).
ꢀ
Optical rotations were measured at 20 C for
solutions in CHCl3 with a Perkin±Elmer 241
1
polarimeter, using a 10 cm 1 mL cell. H NMR
spectra were recorded with Bruker AC 300, Bruker
AMX 500 or Bruker AMX 600 spectrometers; the
values of ꢁH are given in ppm relative to the signal
for internal Me4Si (ꢁ 0) for solutions in CDCl3, or
by reference to acetone (ꢁ 2.225) for solutions in
D2O. 13C (APꢀT, 75 MHz) NMR spectra were
recorded at 27 C with a Bruker AC 300 spectro-
meter or a Varian Gemini-300 instrument; indi-
cated ppm values for ꢁC are relative to the signal of
CDCl3 (ꢁ 76.9) for solutions in CDCl3. Two-
ꢀ
methanol at 0 C, a€orded the target compound 5
(63%).
1
To facilitate the conjugation of 1, 3, and 5 to
bovine serum albumin, their allyl functions were
elongated with cysteamine using cysteamine hydro-
chloride (2 equiv) in water under UV light [37] for
initiation of the reaction, to obtain the amine-
spacer-containing epitopes 2, 4, and 6. The synthesis
of the neoglycoconjugates and subsequent immu-
nological studies are currently under investigation.
dimensional double-quantum ®ltered H±1H cor-
1
relation spectra (2D DQF H±1H COSY) of the
products 1±6 were recorded using a Bruker AMX
500 apparatus (500 MHz) at 27 ꢀC. Fast-atom-
bombardment mass spectrometry (FABMS) was
performed on a JEOL JMS SX/SX 102A four-sec-
tor mass spectrometer, operated at 10 kV accel-
erating voltage, equipped with a JEOL MS-FAB
10 D FAB gun, operated at 10 mA emission cur-
rent, producing a beam of 6 keV Xenon atoms.
Elemental analyses were carried out by H. Kolbe
Mikroanalytisches Laboratorium (Mulheim an der
Ruhr, Germany).
3. Experimental
General methods.ÐReactions were monitored by
TLC on Kieselgel 60 F254 (E. Merck); compounds
were visualised, after examination under UV light,
by charring with aq 50% H2SO4. In the work-up
4-Methoxyphenyl
2,3,4,6-tetra-O-acetyl-b-d-
glucopyranoside (7).ÐTo a solution of 1,2,3,4,6-
penta-O-acetyl-ꢀ-d-glucopyranose (5.0 g, 12.8 mmol),

180
K.M. Halkes et al./Carbohydrate Research 309 (1998) 175±188
4-methoxyphenol (2.4 g, 14.7 mmol) in dry CH2Cl2
(40 mL), containing 4 A molecular sieves (5 g), was
added at 0 C Me3SiOTf (0.25 mL, 1.35 mmol).
4-Methoxyphenyl
6-O-levulinoyl-2,3,4-tri-O-p-
toluoyl-b-d-glucopyranoside (9).ÐTo a solution of
8 (4.4 g, 5.83 mmol) in acetonitrile (45 mL) con-
taining water (5 mL), was added p-toluenesulfonic
acid (pH 3). After 5 h, TLC (Rf 0.74; 9:1 CH2Cl2±
EtOAc) showed a complete removal of the silyl
function. The mixture was diluted with CH2Cl2
(250 mL), washed with aq 5% NaHCO3 and aq 5%
NaCl, and the organic layer was dried, ®ltered, and
concentrated. To a solution of the residue in 1,2-
dichloroethane (100 mL) was added levulinic acid
(1.25 mL, 11.7 mmol) and 2-chloro-1-methylpyr-
idinium iodide (5.96 g, 23.4 mmol). The mixture
was stirred for 15 min, then 1,4-diazabicy-
clo[2.2.2]octane (3.27 g, 29.0 mmol) was added and
the stirring was continued for another 20 min,
when TLC (Rf 0.86; 6:1 CH2Cl2-acetone) revealed
the reaction to be complete. The mixture was ®l-
tered through Celite, diluted with EtOAc (200 mL),
washed with aq 5% NaCl, and the organic phase
was dried, ®ltered, and concentrated. Column
chromatography (9:1 CH2Cl2±acetone) of the resi-
due yielded 9, isolated as a syrup (3.4 g, 80%);
ꢀ
After stirring for 2.5 h, TLC (2:1 toluene±EtOAc)
showed the disappearance of the starting material
and the formation of a new spot (Rf 0.66). Then
the mixture was neutralised with Et3N, diluted with
CH2Cl2 (150 mL), washed with aq 5% NaHCO3
and aq 5% NaCl, and the organic layer was dried,
®ltered, and concentrated. Crystallisation from i-
PrOH a€orded 7, isolated as white crystals (4.6 g,
77%); [ꢂ]d 21ꢀ (c 1); mp 102 ꢀC; 1H NMR
(CDCl3): ꢁ 6.946 and 6.817 (2 d, each 2 H,
C6H4OCH3), 4.953 (d, 1 H, J1,2 7.3 Hz, H-1), 4.288
(dd, 1 H, J5,6b 5.1, J6a,6b 12.1 Hz, H-6b), 4.168 (dd,
1 H, J5,6a 2.2 Hz, H-6a), 3.775 (s, 3 H,
C6H4OCH3), 2.083, 2.076, 2.041, and 2.032 (4 s,
each 3 H, 4 Ac). Anal. Calcd for C21H26O11: C,
55.50; H, 5.77. Found: C, 55.48; H, 5.76.
4-Methoxyphenyl
6-O-tert-butyldimethylsilyl-
2,3,4-tri-O-p-toluoyl-b-d-glucopyranoside (8).ÐTo
a solution of 7 (4.3 g, 9.25 mmol) in MeOH (40 mL)
was added NaOMe (pH 10). The solution was
stirred for 8 h, then neutralised by addition of
Dowex-50 (H+). The mixture was ®ltered and
concentrated, after which the residue was dissolved
in pyridine (20 mL), and tert-butyldimethylsꢀilyl
chloride (1.81 g, 12.0 mmol) was added at 0 C.
TLC analysis (Rf 0.52; 9:1 CH2Cl2±MeOH) after
4.5 h showed the silylation of the primary hydroxyl
function to be complete. The mixture was allowed
to warm to room temperature, and p-toluoyl
chloride (6.5 mL, 55.5 mmol) was added. The mix-
ture was stirred overnight when TLC (Rf 0.87; 97:3
CH2Cl2±acetone) showed the reaction to be com-
plete. The mixture was diluted with EtOAc
(250 mL), washed with aq 5% NaHCO3 and aq 5%
NaCl, and the organic layer was dried, ®ltered, and
concentrated. Crystallisation from EtOH gave
compound 8, isolated as light yellow crystals (4.4 g,
63%); [ꢂ]d 38ꢀ (c 1); mp 86 ꢀC; 1H NMR
(CDCl3): ꢁ 7.850, 7.733, 7.298, 7.165, 7.152, and
7.063 (6 d, each 2 H, 3 COC6H4CH3), 6.984 and
6.755 (2 d, each 2 H, C6H4OCH3), 5.889 (t, 1 H,
J2,3 9.8, J3,4 9.6 Hz, H-3), 5.680 (dd, 1 H, J1,2
7.9 Hz, H-2), 5.501 (dd, 1 H, J4,5 9.6 Hz, H-4),
5.212 (d, 1 H, H-1), 3.775 (s, 3 H, C6H4OCH3),
2.342, 2.333, and 2.273 (3 s, each 3 H, 3
COC6H4CH3), 0.913 (s, 9 H, Si(CH3)2C(CH3)3),
0.055 and 0.026 (2 s, each 3 H, Si(CH3)2C(CH3)3).
Anal. Calcd for C43H50O10Si: C, 68.41; H, 6.68.
Found: C, 68.17; H, 6.79.
[ꢂ]d+22ꢀ (c 1); NMR (CDCl3): H, ꢁ 7.853, 7.809,
1
7.740, 7.162, and 7.080 (5 d, 2,2,2,4,2 H, 3
COC6H4CH3), 6.961 and 6.779 (2 d, each 2 H,
C6H4OCH3), 5.704 (dd, 1 H, J1,2 7.7, J2,3 9.5 Hz,
H-2), 5.231 (d, 1 H, H-1), 3.744 (s, 3 H,
C6H4OCH3), 2.676 (m, 4 H, COCH2CH2COCH3),
2.348 and 2.289 (2 s, 6,3 H, 3 COC6H4CH3),
2.160 (s, 3 H, COCH2CH2COCH3); 13C, d 205.8
(COCH2CH2COCH3), 171.8 (COCH2CH2COCH3),
165.4, 164.9, and 164.8 (3 COC6H4CH3), 100.5 (C-
1), 63.0 (C-6), 55.2 (C6H4OCH3), 37.5, 29.3, and
27.5 (COCH2CH2COCH3), 21.2 (COC6H4CH3).
Anal. Calcd for C42H42O12: C, 68.28; H, 5.73.
Found: C, 68.35; H, 5.82.
6-O-Levulinoyl-2,3,4-tri-O-p-toluoyl-a,b-d-gluco-
pyranose (10).ÐTo a solution of 9 (5.47 g,
7.40 mmol) in 1:1:1 toluene±acetonitrile±water
(600 mL) was added ammonium cerium(IV) nitrate
(40.7 g, 74.0 mmol). After 30 min, TLC (Rf 0.19; 9:1
CH2Cl2±acetone) showed the conversion of 9 into
10 to be complete. The mixture was diluted with
EtOAc (500 mL), washed with aq saturated
NaHCO3 and water, and the organic layer was
dried, ®ltered, and concentrated. Column chroma-
tography (9:1 CH2Cl2±acetone) of the residue gave
10, isolated as a syrup (4.11 g, 88%); [ꢂ]d +24ꢀ (c
1
1) (ꢂ:ꢀ 2.7:1); H NMR (CDCl3): ꢁ 5.733 (d, 0.73
H, J1,2 3.3 Hz, H-1ꢂ), 4.664 (d, 0.27 H, J1,2 8.1 Hz,
H-1ꢀ), 2.67±2.62 (m, 4 H, COCH2CH2-COCH3),

K.M. Halkes et al./Carbohydrate Research 309 (1998) 175±188
181
2.334, 2.325, and 2.266 (3 s, each 3 H, 3 COC6H4CH3),
2.170 (s, 3 H, COCH2CH2COCH3). Anal. Calcd
for C35H36O11: C, 66.45; H, 5.74. Found: C, 66.16;
H, 5.82.
27.8 (COCH2CH2COCH3), 21.5 (2 C) and 21.3 (3
COC6H4CH3).
Allyl (6-O-levulinoyl-2,3,4-tri-O-p-toluoyl-b-d-
glucopyranosyl)-(1!3)-2-deoxy-2-phthalimido-b-
d-glucopyranoside (14).ÐTo a solution of 13
(0.69 g, 0.66 mmol) in CH2Cl2 (20 mL) and water
(63 ꢃL) was added CF3CO2H (0.54 mL). The mix-
ture was stirred for 30 min, when TLC (4:1
CH2Cl2±acetone) showed the formation of a slower
moving spot (Rf 0.45). The mixture was diluted
with CH2Cl2 (150 mL), washed with aq 10%
NaHCO3 and aq 10% NaCl, and the aqueous
phases were twice extracted with CH2Cl2 (50 mL).
The combined organic layers were dried, ®ltered,
and concentrated. Column chromatography (4:1
CH2Cl2±acetone) of the residue gave 14, isolated as
a glass (0.62 g, 98%); [ꢂ]d +4ꢀ (c 1); NMR
6-O-Levulinoyl-2,3,4-tri-O-p-toluoyl-a-d-gluco-
pyranosyl trichloroacetimidate (11).ÐTo a solution
of 10 (2.45 g, 3.88 mmol) in dry CH2Cl2 (12 mL)
and trichloroacetonitrile (4.1 mL, 40.8 mmol) was
added 1,8-diazabicyclo[5.4.0]undec-7-ene (140 ꢃL).
The mixture was stirred overnight and applied to
column chromatography (95:5 CH2Cl2±acetone) to
yield 11, isolated as a white foam (2.77 g, 89%);
[ꢂ]d +38ꢀ (c 1); 1H NMR (CDCl3): ꢁ 8.631 (s, 1 H,
C(NH)CCl3), 7.832, 7.748, 7.168, 7.147, and 7.086
(5 d, 4,2,2,2,2 H, 3 COC6H4CH3), 6.961 (d, 1 H,
J1,2 3.6 Hz, H-1), 5.545 (dd, 1 H, J2,3 10.3 Hz, H-2),
2.74±2.61 (m, 4 H, COCH2CH2COCH3), 2.356,
2.341, and 2.292 (3 s, each 3 H, 3 COC6H4CH3),
2.179 (s, 3 H, COCH2CH2COCH3). Anal. Calcd
for C37H36Cl3NO11: C, 57.19; H, 4.67. Found: C,
56.76; H, 4.68.
1
(CDCl3): H, ꢁ 7.742, 7.521, 7.323, 7.137, 6.964,
and 6.823 (6 d, each 2 H, 3 COC6H4CH3), 5.60±
5.48 (m, 1 H, OCH2CH=CH2), 5.390 (dd, 1 H,
J1 ,2 7.9, J2 ,3 9.9 Hz, H-20), 4.997 (d, 1 H, J1,2
8.5 Hz, H-1), 5.03±4.89 (m, 2 H, OCH2CH=CH2),
4.816 (d, 1 H, H-10), 4.629 (dd, 1 H, J2,3 10.8 Hz,
H-2), 2.82±2.61 (m, 4 H, COCH2CH2COCH3),
2.353, 2.313, and 2.224 (3 s, each 3 H, 3
COC6H4CH3), 2.215 (s, 3 H, COCH2CH2COCH3);
0
0
0
0
Allyl (6-O-levulinoyl-2,3,4-tri-O-p-toluoyl-b-d-
glucopyranosyl)-(1!3)-4,6-O-benzylidene-2-deoxy-
2-phthalimido-b-d-glucopyranoside (13).ÐTo
a
solution of 11 (0.77 g, 0.99 mmol) and allyl 4,6-O-
benzylidene-2-deoxy-2-phthalimido-ꢀ-d-glucopyran-
oside [24,25] (12; 0.29 g, 0.66 mmol) in CH2Cl2
(8 mL), containing 4 A molecular sieves (0.8 g), was
13C,
ꢁ
204.8 (COCH2CH2COCH3), 172.1
(COCH2CH2COCH3), 165.4, 165.0, and 164.2 (3
COC6H4CH3), 130.7 (OCH2CH=CH2), 117.1
(OCH2CH=CH2), 101.2 and 97.3 (C-1,10), 62.8
and 62.6 (C-6,60), 54.6 (C-2), 37.7, 29.6, and 27.6
(COCH2CH2COCH3), 21.5 (2 C) and 21.3 (3
COC6H4CH3). Anal. Calcd for C52H53NO17: C,
64.79; H, 5.54. Found: C, 64.79; H, 5.49.
ꢀ
added at 0 C Me3SiOTf (6.3 ꢃL, 33 ꢃmol). After
stirring for 10 min, TLC (9:1 CH2Cl2±acetone)
showed the disappearance of 12 and the formation
of a new product (Rf 0.77). The mixture was
neutralised with Et3N, diluted with CH2Cl2
(150 mL), washed with aq 5% NaCl, and the
organic layer was dried, ®ltered, and concentrated.
Column chromatography (95:5 CH2Cl2±acetone)
of the residue gave 13, isolated as a glass (0.62 g,
89%); [ꢂ]d +31ꢀ (c 1); NMR (CDCl3): 1H, ꢁ 7.662,
7.532, 7.249, 7.110, 6.969, and 6.941 (6 d, each 2 H,
3 COC6H4CH3), 5.674 (s, 1 H, CHC6H5), 5.63±
5.51 (m, 1 H, OCH2CH=CH2), 5.230 (dd, 1 H,
Allyl (6-O-levulinoyl-2,3,4-tri-O-p-toluoyl-b-d-
glucopyranosyl)-(1!3)-6-O-tert-butyldimethylsilyl-
2-deoxy-2-phthalimido-b-d-glucopyranoside (15).Ð
To a solution of 14 (0.57 g, 0.59 mmol) in pyridine
(18 mL) was added tert-butyldimethylsilyl chloride
(0.27 g, 1.77 mmol). The mixture was stirred for 6 h
at 0 ꢀC when TLC (8:1 CH2Cl2±acetone) showed a
complete conversion of 14 into 15 (Rf 0.84). The
mixture was diluted with EtOAc (150 mL), washed
with aq 10% NaHCO3 and aq 10% NaCl, and the
organic layer was dried, ®ltered, and concentrated.
Column chromatography (9:1 CH2Cl2±acetone) of
the residue yielded 15, isolated as a glass (0.60 g,
J1 ,2 8.0, J2 ,3 9.6 Hz, H-20), 5.166 (d, 1 H, J1,2
8.5 Hz, H-1), 5.07±4.93 (m, 2 H, OCH2CH=CH2),
4.916 (d, 1 H, H-10), 4.788 (dd, 1 H, J2,3 10.4 Hz,
H-2), 2.72±2.50 (m, 4 H, COCH2CH2COCH3),
2.350, 2.323, and 2.234 (3 s, each 3 H, 3
COC6H4CH3), 2.191 (s, 3 H, COCH2CH2COCH3);
0
0
0
0
13C,
ꢁ
206.2 (COCH2CH2COCH3), 172.1
96%); [ꢂ]d +5ꢀ (c 1); NMR (CDCl3): H, ꢁ 7.741,
1
(COCH2CH2COCH3), 165.5, 164.8, and 164.3 (3
COC6H4CH3), 133.1 (OCH2CH=CH2), 117.4
(OCH2CH=CH2), 101.6, 100.2, and 97.5
(CHC6H5 and C-1,10), 55.0 (C-2), 37.8, 29.7, and
7.520, 7.327, 7.135, 6.961, and 6.823 (6 d, each 2 H,
3 COC6H4CH3), 5.61±5.48 (m, 1 H, OCH2CH=
CH2), 5.395 (dd, 1 H, J1 ,2 8.0, J2 ,3 9.9 Hz, H-20),
4.940 (d, 1 H, J1,2 8.4 Hz, H-1), 5.01±4.89 (m, 2 H,
0
0
0
0

182
K.M. Halkes et al./Carbohydrate Research 309 (1998) 175±188
OCH2CH=CH2), 4.801 (d, 1 H, H-10), 4.601
(dd, 1 H, J2,3 10.8 Hz, H-2), 2.82±2.62 (m, 4 H,
COCH2CH2COCH3), 2.333, 2.313, and 2.220 (3 s,
each 3 H, 3 COC6H4CH3), 2.210 (s, 3 H,
COCH2CH2COCH3), 0.909 (s, 9 H, Si(CH3)2-
C(CH3)3), 0.090 and 0.081 (2 s, each 3 H,
Si(CH3)2C(CH3)3); 13C, ꢁ 206.3 (COCH2CH2-
COCH3), 172.1 (COCH2CH2COCH3), 165.5,
165.0, and 164.3 (3 COC6H4CH3), 130.8
(OCH2CH=CH2), 117.0 (OCH2CH=CH2), 101.3
and 96.8 (C-1,10), 63.0 and 62.5 (C-6,60), 54.7 (C-2),
37.8, 29.7, and 27.6 (COCH2CH2COCH3), 25.8
[Si(CH3)2C(CH3)3], 21.5 (2 C) and 21.4 (3
COC6H4CH3), 18.3 [Si(CH3)2C(CH3)3], 5.3
[Si(CH3)2C(CH3)3]. Anal. Calcd for C58H67-
NO17Si: C, 64.61; H, 6.26. Found: C, 65.08; H,
6.37.
169.9 (COCH3), 165.5, 164.9, and 164.2 (3
COC6H4CH3), 130.9 (OCH2CH=CH2), 117.3
(OCH2CH=CH2), 101.1 and 97.3 (C-1,10), 62.2
and 62.1 (C-6,60), 52.4 (C-2), 37.9, 29.7, and 27.8
(COCH2CH2COCH3), 25.6 [Si(CH3)2C(CH3)3],
21.5 (2 C) and 21.4 (3 COC6H4CH3), 20.8
(COCH3), 18.1 [Si(CH3)2C(CH3)3], 5.6 and 5.5
[Si(CH3)2C(CH3)3]. FABMS (positive-ion mode;
C60H69NO18Si): m/z 1142 [M+Na]+, 1120
[M+H]+.
Allyl (6-O-levulinoyl-2,3,4-tri-O-p-toluoyl-b-d-
glucopyranosyl)-(1!3)-4-O-acetyl-2-deoxy-2-
phthalimido-b-d-galactopyranoside (17).ÐTo
a
solution of 16 (0.32 g, 0.28 mmol) in acetonitrile
(27 mL), containing water (2.7 mL), was added p-
toluenesulfonic acid (0.17 g, 1.0 mmol). The mix-
ture was stirred for 2 h, when TLC (Rf 0.33; 8:1
CH2Cl2±acetone) showed the reaction to be com-
plete. The mixture was concentrated, diluted with
EtOAc (150 mL), washed with aq 10% NaHCO3
and aq 10% NaCl, and the organic layer was dried,
®ltered, and concentrated. Column chromato-
graphy (9:1 CH2Cl2±acetone) of the residue ren-
dered 17, isolated as a syrup (0.26 g, 91%); [ꢂ]d
+3ꢀ (c 1); NMR (CDCl3): 1H, ꢁ 7.739, 7.548,
7.322, 7.124, 6.975, and 6.834 (6 d, each 2 H, 3
COC6H4CH3), 5.599 (d, 1 H, J3,4 3.2, J4,5 < 1 Hz,
Allyl (6-O-levulinoyl-2,3,4-tri-O-p-toluoyl-b-d-
glucopyranosyl)-(1!3)-4-O-acetyl-6-O-tert-butyl-
dimethylsilyl-2-deoxy-2-phthalimido-b-d-galacto-
pyranoside (16).ÐTo a solution of 15 (0.61 g,
0.57 mmol) in CH2Cl2 (27 mL), containing pyrꢀidine
(0.5 mL, 5.7 mmol), was added slowly at 0 C a
solution of tri¯uoromethanesulfonic anhydride
(0.38 mL, 2.26 mmol) in CH2Cl2 (5 mL). After stir-
ꢀ
ring for 6 h at 0 C, TLC (Rf 0.75; 95:5 CH2Cl2±
acetone) showed the tri¯ation to be complete. The
mixture was diluted with EtOAc (150 mL), washed
with aq 10% NaHCO3 and aq 10% NaCl, and the
organic layer was dried, ®ltered, concentrated and
co-concentrated with toluene. The residue was dis-
solved in DMF (16 mL) and tetrabutylammonium
acetate (0.85 g, 2.8 mmol) was added. The mixture
was stirred overnight at room temperature, diluted
with EtOAc (150 mL), washed with aq 10% NaCl,
dried, ®ltered, concentrated and co-concentrated
with toluene. Column chromatography (97:3
CH2Cl2±acetone) of the residue a€orded 16, iso-
lated as a syrup (0.49 g, 78%); [ꢂ]d +8ꢀ (c 1);
H-4), 5.64±5.51 (m, 1 H, OCH2CH=CH2), 5.314
0
0
0
0
(dd, 1 H, J1 ,2 7.9, J2 ,3 9.7 Hz, H-2 ), 5.062 (d, 1 H,
0
J1,2 8.6 Hz, H-1), 5.06±4.92 (m,
2
H,
OCH2CH=CH2), 4.903 (dd, 1 H, J2,3 11.3 Hz, H-
3), 4.871 (d, 1 H, H-10), 4.571 (dd, 1 H, H-2), 2.88±
2.53 (m, 4 H, COCH2CH2COCH3), 2.322, 2.309,
and 2.225 (3 s, each 3 H, 3 COC6H4CH3), 2.296 (s,
3 H, COCH2CH2COCH3), 2.203 (s, 3 H, Ac); 13C,
ꢁ 206.5 (COCH2CH2COCH3), 172.3 (COCH2CH2-
COCH3), 168.6 (COCH3), 165.3, 164.8, and 163.9
(3 COC6H4CH3), 130.7 (OCH2CH=CH2), 117.2
(OCH2CH=CH2), 101.4 and 97.5 (C-1,10), 62.1
and 59.8 (C-6,60), 52.1 (C-2), 37.7, 29.5, and 27.5
(COCH2CH2COCH3), 21.3 (2 C) and 21.1 (3
COC6H4CH3), 20.8 (COCH3). Anal. Calcd for
C54H55NO18: C, 64.47; H, 5.51. Found: C, 64.42;
H, 5.62.
1
NMR (CDCl3): H, ꢁ 7.732, 7.557, 7.359, 7.120,
6.979, and 6.879 (6 d, each 2 H, 3 COC6H4CH3),
5.601 (d, 1 H, J3,4 3.3, J4,5 < 1 Hz, H-4), 5.66±5.53
0
0
(m, 1 H, OCH2CH=CH2), 5.395 (dd, 1 H, J1 ,2
7.8, J2 ,3 9.8 Hz, H-20), 5.042 (d, 1 H, J1,2 8.6 Hz,
H-1), 5.07±4.93 (m, 2 H, OCH2CH=CH2), 4.833
(dd, 1 H, J2,3 11.2 Hz, H-3), 4.799 (d, 1 H, H-10),
4.526 (dd, 1 H, H-2), 2.83±2.53 (m, 4 H,
COCH2CH2COCH3), 2.326 and 2.228 (2 s, 6,3 H, 3
COC6H4CH3), 2.215 (s, 3 H, COCH2CH2COCH3),
2.193 (s, 3 H, Ac), 0.893 (s, 9 H, Si(CH3)2C(CH3)3),
0.064 (s, 6 H, Si(CH3)2C(CH3)3); 13C, ꢁ 206.4
(COCH2CH2COCH3), 172.2 (COCH2CH2COCH3),
0
0
(6-O-Levulinoyl-2,3,4-tri-O-p-toluoyl-b-d-gluco-
pyranosyl)-(1!3)-4-O-acetyl-6-O-tert-butyldimethyl-
silyl-2-deoxy-2-phthalimido-b-d-galactopyranosyl
trichloroacetimidate(18).ÐA solution of 16 (0.21 g,
0.19 mmol),
tris(triphenylphosphine)rhodium(I)
chloride (64 mg, 74 ꢃmol), and a catalytic amount
of 1,4-diazabicyclo[2.2.2]octane in EtOH (27 mL)
was boiled under re¯ux for 45 min. The mixture

K.M. Halkes et al./Carbohydrate Research 309 (1998) 175±188
183
was cooled to room temperature, diluted with
CH2Cl2 (100 mL), washed with aq 10% NaCl,
dried, ®ltered, and concentrated. To a solution of
the residue in THF (15 mL), containing water
(1.5 mL), was added N-iodosuccinimide (112 mg,
0.50 mmol). After stirring for 30 min, the mixture
was diluted with CH2Cl2 (150 mL), washed with aq
10% NaHSO3, aq 10% NaHCO3, and aq 10%
NaCl, and the organic layer was dried, ®ltered, and
concentrated. Column chromatography (95:5
CH2Cl2±acetone) of the residue gave the deal-
lylated intermediate, isolated as a syrup. To a
solution of the residue and trichloroacetonitrile
(0.14ꢀmL, 1.34 mmol) in CH2Cl2 (7 mL) was added
at 0 C 1,8-diazabicyclo[5.4.0]undec-7-ene (4.7 ꢃL,
31 ꢃmol). The mixture was stirred overnight and
applied to column chromatography (Rf 0.55; 95:5
CH2Cl2±acetone) to yield 18, isolated as a glass
2.090 (4 s, 6,6,3,3 H, 3 COC6H4CH3, COCH2CH2-
COCH3, and 2 Ac); 13C, ꢁ 172.2 (COCH2CH2-
COCH3), 170.4 and 169.8 (2 COCH3), 164.8
(2 C) and 164.1 (3 COC6H4CH3), 133.1
(OCH2CH=CH2), 117.4 (OCH2CH=CH2), 101.1
and 97.9 (C-1,10), 62.5 (C-6,60), 52.1 (C-2), 37.8,
29.5, and 27.7 (COCH2CH2COCH3), 21.4
(COC6H4CH3), 20.8 and 20.1 (2 COCH3).
Allyl (2,3,4-tri-O-p-toluoyl-b-d-glucopyranosyl)-
(1!3)-4,6-di-O-acetyl-2-deoxy-2-phthalimido-b-d-
galactopyranoside (20).ÐTo a solution of 19
(0.16 g, 0.15 mmol) in EtOH (20 mL) and toluene
.
(6 mL) was added NH2NH2 HOAc (70 mg,
0.76 mmol). The mixture was stirred for 90 min,
when TLC (9:1 CH2Cl2±acetone) showed the con-
version of 19 into 20 (Rf 0.64), then concentrated.
Column chromatography (9:1 CH2Cl2±acetone) of
the residue yielded 20, isolated as a glass (136 mg,
(0.14 g, overall yield 71%); [ꢂ]d +19ꢀ (c 1); H
94%); [ꢂ]d +7ꢀ (c 1); NMR (CDCl3): H, ꢁ 7.752,
1
1
NMR (CDCl3): ꢁ 7.755, 7.572, 7.377, 7.126, 6.986,
and 6.882 (6 d, each 2 H, 3 COC6H4CH3), 6.334 (d,
1 H, J1,2 8.9 Hz, H-1), 5.705 (d, 1 H, J3,4 3.3, J4,5 <
7.585, 7.345, 7.137, 7.006, and 6.901 (6 d, each 2 H,
3 COC6H4CH3), 5.737 (d, 1 H, J3,4 3.4, J4,5 <
1 Hz, H-4), 5.69±5.61 (m, 1 H, OCH2CH=CH2),
1 Hz, H-4), 5.274 (dd, 1 H, J1 ,2 7.8, J2 ,3 9.8 Hz,
H-20), 4.988 (dd, 1 H, J2,3 11.2 Hz, H-3), 4.795 (d, 1
H, H-10), 4.748 (dd, 1 H, H-2), 2.79±2.60 (m, 4 H,
COCH2CH2COCH3), 2.327 and 2.233 (2 s, 6,3 H, 3
COC6H4CH3), 2.258 (s, 3 H, COCH2CH2COCH3),
2.209 (s, 3 H, Ac), 0.870 (s, 9 H, Si(CH3)2C(CH3)3),
0.043 and 0.028 (2 s, each 3 H, Si(CH3)2C(CH3)3).
Anal. Calcd for C58H65Cl3N2O18Si: C, 57.45; H,
5.40. Found: C, 57.83; H, 5.29.
5.287 (dd, 1 H, J1 ,2 7.8, J2 ,3 9.8 Hz, H-20), 5.038
(d, 1 H, J1,2 8.5 Hz, H-1), 5.01±4.94 (m, 2 H,
OCH2CH=CH2), 4.885 (d, 1 H, H-10), 4.559 (dd,
1 H, J2,3 11.2 Hz, H-2), 2.339, 2.292, and 2.229
(3 s, each 3 H, 3 COC6H4CH3), 2.036 (s, 6 H,
2 Ac); 13C, ꢁ 170.4 and 169.8 (2 COCH3), 165.4,
165.0, and 163.9 (3 COC6H4CH3), 133.0
(OCH2CH=CH2), 117.4 (OCH2CH=CH2), 101.9
and 97.4 (C-1,10), 52.1 (C-2), 21.4 (COC6H4CH3),
20.3 and 20.1 (2 COCH3). Anal. Calcd for
C51H51NO17: C, 64.48; H, 5.41. Found: C, 64.34;
H, 5.50.
0
0
0
0
0
0
0
0
Allyl (6-O-levulinoyl-2,3,4-tri-O-p-toluoyl-b-d-
glucopyranosyl)-(1!3)-4,6-di-O-acetyl-2-deoxy-2-
phthalimido-b-d-galactopyranoside (19).ÐTo
a
solution of 17 (0.28 g, 0.28 mmol) in pyridine
(5 mL) were added Ac2O (5 mL) and a catalytic
amount of 4-dimethylaminopyridine. After stirring
overnight at room temperature, TLC (95:5
CH2Cl2±acetone) showed the acetylation to be
complete (19; Rf 0.64). The mixture was con-
centrated and co-concentrated with toluene, EtOH,
and CH2Cl2 (3Â20 mL). Column chromatography
(95:5 CH2Cl2±acetone) of the residue a€orded 19,
isolated as a glass (0.26 g, 88%); [ꢂ]d +8ꢀ (c 1);
Allyl (2,3,4-tri-O-p-toluoyl-b-d-glucopyranosyl-
uronic acid)-(1!3)-4,6-di-O-acetyl-2-deoxy-2-
phthalimido-b-d-galactopyranoside (21).ÐTo
a
cold ( 78 ꢀC) 2 M solution of oxalyl chloride
(0.24 mL, 0.48 mmol) in CH2Cl2 (2 mL) was added
Me2SO (73.2 ꢃL, 1.03 mmol), and the mixture was
stirred for 10 min. A solution of 20 (47 mg,
50 ꢃmol) in CH2Cl2 (2 mL) was added, and the
mixture was stirred for 1 h, whereby in 20 min a
white precipitate was formed. Diisopropylethyla-
mine (0.36 mL) was added, and after 10 min the
mixture was diluted with EtOAc (150 mL), washed
with aq 1 M HCl, and the organic layer was dried,
®ltered, and concentrated. To a solution of the
residue in tert-BuOH (2 mL) and 2-methyl-2-
butene (0.77 mL), containing water (1.27 mL) and
NaH2PO4 (126 mg), was added NaClO2 (126 mg).
The mixture was stirred overnight, when TLC
1
NMR (CDCl3): H, ꢁ 7.738, 7.562, 7.353, 7.132,
6.990, and 6.871 (6 d, each 2 H, 3 COC6H4CH3),
5.60±5.51 (m, 1 H, OCH2CH=CH2), 5.266 (dd, 1
H, J1 ,2 7.6, J2 ,3 9.8 Hz, H-20), 5.052 (d, 1 H, J1,2
8.7 Hz, H-1), 5.06±4.94 (m, 2 H, OCH2CH=CH2),
4.862 (dd, 1 H, J2,3 11.3, J3,4 3.3 Hz, H-3), 4.788 (d,
1 H, H-10), 4.535 (dd, 1 H, H-2), 2.88±2.53 (m, 4 H,
COCH2CH2COCH3), 2.331, 2.238, 2.208, and
0
0
0
0

184
K.M. Halkes et al./Carbohydrate Research 309 (1998) 175±188
(10:9:1 CH2Cl2±EtOAc±HOAc) showed a complete
conversion of 20 into 21 (Rf 0.64). The mixture was
concentrated, and a solution of the residue in water
was washed with hexane, acidi®ed with aq 1 M
HCl, and extracted with EtOAc (3Â20 mL), and
the organic layer was dried, ®ltered, and con-
centrated. Column chromatography (10:9:1
CH2Cl2±EtOAc±HOAc) of the residue yielded 21,
isolated as a white solid (37 mg, 83%); [ꢂ]d +3ꢀ
4-O-acetyl-2-deoxy-2-phthalimido-b-d-galacto-
pyranoside (23).ÐTo a solution of 17 (85 mg,
84 ꢃmol) and ethyl 3,4,6-tri-O-acetyl-2-deoxy-2-
phthalimido-1-thio-ꢀ-d-galactopyranoside
(22;
85 mg, 0.17 mmol) in CH2Cl2 (5.5 mL), containing
4 A molecular sieves (0.2 g), was added dropwise at
ꢀ
20 C a solution of N-iodosuccinimide (24 mg,
0.11 mmol) and HOTf (106 ꢃL, 11 ꢃmol) in dry
CH2Cl2 (2 mL). After stirring for 30 min, TLC (9:1
CH2Cl2±acetone) showed the disappearance of 17
and the formation of a new product (Rf 0.61). The
mixture was neutralised with Et3N, diluted with
CH2Cl2 (100 mL), washed with aq 5% NaHSO3,
aq 5% NaHCO3, and aq 5% NaCl, and the
organic layer was dried, ®ltered, and concentrated.
Column chromatography (94:6 CH2Cl2±acetone)
of the residue gave 23, isolated as a white solid (96
1
(c 1); NMR (CDCl3): H, ꢁ 7.802, 7.590, 7.301,
7.151, 7.007, and 6.777 (6 d, each 2 H, 3
COC6H4CH3), 5.715 (d, 1 H, J3,4 3.4, J4,5 < 1 Hz,
H-4), 5.73±5.66 (m, 1 H, OCH2CH=CH2), 5.287
(dd, 1 H, J1 ,2 7.7, J2 ,3 9.1 Hz, H-20), 4.996 (d, 1 H,
J1,2 8.6 Hz, H-1), 4.986 (d, 1 H, H-10), 4.486 (dd, 1
H, J2,3 11.3 Hz, H-2), 2.099 and 2.072 (2 s, each 3
H, 2 Ac); 13C, ꢁ 165.2, 164.9, and 163.9 (3
COC6H4CH3), 133.0 (OCH2CH=CH2), 117.1
(OCH2CH=CH2), 101.3 and 97.4 (C-1,10), 52.1
(C-2), 21.4 and 21.3 (COC6H4CH3), 21.0 and 20.5
(2 COCH3). Anal. Calcd for C51H49NO18: C,
63.61; H, 5.04. Found: C, 63.28; H, 5.28.
0
0
0
0
ꢀ
1
mg, 80%); [ꢂ]d +13 (c 1); NMR (CDCl3): H, ꢁ
7.724, 7.536, 7.314, 7.120, 6.971, and 6.851 (6 d,
0
each 2 H, 3 COC6H4CH3), 5.807 (dd, 1 H, J2 ,3
0
11.5, J3 ,4 3.4 Hz, H-30), 5.487 (d, 1 H, J4 ,5
<
0
0
0
0
1 Hz, H-40), 5.453 (d, 1 H, J3,4 3.6, J4,5 < 1 Hz, H-
4), 5.39±5.28 (m, 1 H, OCH2CH=CH2), 5.353 and
Allyl (b-d-glucopyranosyluronic acid)-(1!3)-2-
acetamido-2-deoxy-b-d-galactopyranoside (1).ÐA
solution of 21 (17 mg, 18 ꢃmol) in ethanolic 33%
MeNH2 (7.5 mL) was stirred for 2 days at room
temperature, during which the mixture was once
concentrated and new reagent (7.5 mL) added.
After concentration, the residue was dissolved in
dry MeOH (5 mL), and Ac2O (100 ꢃL) was added
at 0 ꢀC. The mixture was stirred for 2 h, when TLC
(4:2:2:0.5 1-BuOH±EtOH±H2O±HOAc) showed a
complete conversion of 21 into 1 (Rf 0.53). The
solution was concentrated and co-concentrated
with 1:1 toluene±MeOH (3Â10 mL). Gel ®ltration
over Sephadex G-10 (water) of the residue yielded
1, isolated after lyophilisation as a white, amor-
0
0
4.863 (2 d, each 1 H, J1,2/1 ,2 8.4 and 8.5 Hz, H-
1,10), 5.218 (dd, 1 H, J1 ,2 7.8, J2 ,3 9.8 Hz, H-200),
4.98±4.86 (m, 2 H, OCH2CH=CH2), 4.746 (dd, 1
H, J2,3 11.2 Hz, H-3), 4.726 (d, 1 H, H-100), 4.526
and 4.409 (2 dd, each 1 H, H-2,20), 2.335, 2.325,
and 2.237 (3 s, each 3 H, 3 COC6H4CH3), 2.206 (s,
3 H, COCH2CH2COCH3), 2.203, 2.179, 2.099, and
1.835 (4 s, each 3 H, 4 Ac); 13C, ꢁ 172.1
(COCH2CH2COCH3), 170.2, 170.0 (2 C), and
169.5 (4 COCH3), 165.3, 164.8, and 164.0 (3
COC6H4CH3), 134.0 (OCH2CH=CH2), 117.4
(OCH2CH=CH2), 101.0, 98.1, and 96.7 (C-
1,10,100), 52.2 and 51.2 (C-2,20), 37.7, 29.6, and 27.7
(COCH2CH2COCH3), 21.4 and 21.3 (2 C) (3
COC6H4CH3), 20.7, 20.5 (2 C), and 20.3 (4
COCH3). Anal. Calcd for C74H74N2O27: C, 62.44;
H, 5.24. Found: C, 62.28; H, 5.36.
00 00
00 00
1
phous powder (7 mg, 88%); NMR (D2O): H, ꢁ
5.912 (m, 1 H, OCH2CH=CH2), 5.36±5.23 (m, 2
H, OCH2CH=CH2), 4.544 (d, 1 H, J1,2 8.6 Hz, H-
1), 4.501 (d, 1 H, J1 ,2 7.9 Hz, H-10), 4.027 (dd, 1 H,
J2,3 10.9 Hz, H-2), 3.831 (dd, 1 H, J3,4 3.2 Hz, H-3),
Allyl (3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-
b-d-galactopyranosyl)-(1!6)-[(2,3,4-tri-O-p-
toluoyl-b-d-glucopyranosyl)-(1!3)]-4-O-acetyl-2-
deoxy-2-phthalimido-b-d-galactopyranoside (24).Ð
To a solution of 23 (96 mg, 67 ꢃmol) in EtOH
(11 mL) and toluene (5 mL) was added
0
0
3.330 (t, 1 H, J2 ,3 8.0 Hz, H-20), 2.116 (s, 3 H,
0
0
NHCOCH3); 13C,
ꢁ
175.4 (COOH), 174.6
(NHCOCH3), 133.4 (OCH2CH=CH2), 118.3
(OCH2CH=CH2), 103.8 and 100.2 (C-1,10), 60.9
(C-6), 51.1 (C-2), 22.1 (NHCOCH3). FABMS
(positive-ion mode; C17H27NO12): m/z 460
[M+Na]+.
Allyl (3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-
b-d-galactopyranosyl)-(1!6)-[(6-O-levulinoyl-
2,3,4-tri-O-p-toluoyl-b-d-glucopyranosyl)-(1!3)]-
.
NH2NH2 HOAc (61 mg, 67 ꢃmol). The mixture
was stirred for 2 h, when TLC (9:1 CH2Cl2±acet-
one) showed the conversion of 23 into 24 (Rf 0.61),
then concentrated. Column chromatography (95:5
CH2Cl2±acetone) of the residue a€orded 24, iso-
lated as a glass (84 mg, 91%); [ꢂ]d +1ꢀ (c 1); NMR

K.M. Halkes et al./Carbohydrate Research 309 (1998) 175±188
185
1
(CDCl3): H, ꢁ 7.734, 7.516, 7.279, 7.131, 6.956,
and 6.777 (6 d, each 2 H, 3 COC6H4CH3), 5.803
uct (Rf 0.38). The solution was concentrated and
co-concentrated with 1:1 toluene±MeOH
(dd, 1 H, J2 ,3 11.5, J3 ,4 3.3 Hz, H-30), 5.586 (d, 1
(3Â10 mL). Gel ®ltration over Sephadex G-10
(water) of the residue yielded 3, isolated after lyo-
philisation as a white, amorphous powder (7 mg,
74%); NMR (D2O): 1H, ꢁ 5.914 (m, 1 H,
OCH2CH=CH2), 5.34±5.27 (m, 2 H, OCH2CH=
0
0
0
0
0
0
H, J3,4 3.5, J4,5 < 1 Hz, H-4), 5.478 (d, 1 H, J4 ,5
< 1 Hz, H-40), 5.346 and 4.867 (2 d, each 1 H, J1,2/
8.6 and 8.5 Hz, H-1,10), 5.237 (dd, 1 H, J1 ,2
00 00
10,20
7.8, J2 ,3 9.8 Hz, H-200), 4.96±4.82 (m, 2 H,
00 00
OCH2CH=CH2), 4.812 (d, 1 H, H-100), 4.733 (dd,
1 H, J2,3 11.1 Hz, H-3), 4.534 and 4.421 (2 dd, each
1 H, H-2,200), 2.332, 2.276, and 2.248 (3 s, each 3 H,
3 COC6H4CH3), 2.213, 2.087, and 1.840 (3 s, 6,3,3
H, 4 Ac); 13C, ꢁ 170.2, 170.1, 169.5, and 168.5 (4
COCH3), 165.3, 165.0, and 163.9 (3 COC6H4CH3),
130.6 (OCH2CH=CH2), 117.5 (OCH2CH=CH2),
101.7, 97.8, and 96.8 (C-1,10,100), 52.1 and 51.1 (C-
2,20), 21.4, 21.2, 20.9, 20.5, and 20.3 (COC6H4CH3
and COCH3). Anal. Calcd for C69H68N2O25: C,
62.53; H, 5.17. Found: C, 62.39; H, 5.31.
CH2), 4.553 (d, 1 H, J1 ,2 7.9 Hz, H-100), 4.542 and
00 00
0
4.475 (2 d, each 1 H, J1,2/1 ,2 8.6 and 8.5 Hz,
0
H-1,10), 4.110 and 3.939 (2 d, each 1 H, J3,4/3 ,4 2.8
0
0
and 2.7 Hz, J4,5/4 ,5 <1 Hz, H-4,40), 4.019 and
0
0
0
0
3.899 (2 dd, each 1 H, J2,3/2 ,3 11.2 and 10.7 Hz,
00 00
H-2,20), 3.356 (dd, 1 H, J2 ,3 8.4 Hz, H-200), 2.025
and 2.012 (2 s, each 3 H, 2 NHCOCH3); 13C, ꢁ
177.6 and 177.4 (2 NHCOCH3), 175.4 (COOH),
135.9 (OCH2CH=CH2), 121.2 (OCH2CH=CH2),
107.0, 104.6, and 102.8 (C-1,10,100), 55.2 and 53.8
(C-2,20), 25.1 and 25.0 (NHCOCH3). FABMS
(positive-ion mode; C25H40N2O17): m/z 663
[M+Na]+.
Allyl (3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-
b-d-galactopyranosyl)-(1!6)-[(2,3,4-tri-O-p-
toluoyl-b-d-glucopyranosyluronic acid)-(1!3)]-4-
O-acetyl-2-deoxy-2-phthalimido-b-d-galactopyrano-
side (25).ÐTo a solution of 24 (45 mg, 34 ꢃmol) in
dry CH2Cl2 (5 mL), containing 4 A powdered
molecular sieves (300 mg), was added pyridinium
dichromate (62 mg, 161 ꢃmol). After stirring for
48 h, TLC (8:4:1 CH2Cl2±EtOAc±HOAc) showed
the conversion of 24 into 25 (Rf 0.68). After the
addition of EtOAc (25 mL), the suspension was
®ltered through Celite and applied to column
chromatography (8:4:1 CH2Cl2±EtOAc±HOAc) to
yield 25, isolated as a glass (39 mg, 86%); [ꢂ]d +4ꢀ
(c 1); 13C NMR (CDCl3): ꢁ 170.2, 170.1, 169.5, and
168.5 (4 COCH3), 165.3, 165.0, and 163.9 (3
COC6H4CH3), 130.6 (OCH2CH=CH2), 117.5
(OCH2CH=CH2), 101.7, 97.8, and 96.8 (C-
1,10,100), 52.1 and 51.1 (C-2,20), 21.4, 21.2, 20.9,
20.5, and 20.3 (COC6H4CH3 and COCH3). Anal.
Calcd for C69H66N2O26: C, 61.88; H, 4.97. Found:
C, 61.66; H, 5.12.
Allyl (2-acetamido-2-deoxy-b-d-galactopyranosyl)-
(1!6)-[(b-d-glucopyranosyluronic acid)-(1!3)]-
2-acetamido-2-deoxy-b-d-galactopyranoside (3).Ð
A solution of 25 (21 mg, 15 ꢃmol) in ethanolic
33% MeNH2 (7.5 mL) was stirred for 5 days at
room temperature, during which the mixture was
concentrated repeatedly and new reagent
(5Â7.5 mL) added. After concentration, the residue
was dissolved in dry MeOH (5 mL), and Ac2O
Allyl (6-O-levulinoyl-2,3,4-tri-O-p-toluoyl-b-d-
glucopyranosyl)-(1!3)-(4-O-acetyl-6-O-tert-butyl-
dimethylsilyl-2-deoxy-2-phthalimido-b-d-galacto-
pyranosyl)-(1!6)-[(6-O-levulinoyl-2,3,4-tri-O-p-
toluoyl-b-d-glucopyranosyl)-(1!3)]-4-O-acetyl-2-
deoxy-2-phthalimido-b-d-galactopyranoside (26).Ð
To a solution of 17 (94 mg, 94 ꢃmol) and 18
(187 mg, 150 ꢃmol) in CH2Cl2 (5 mL), containing
4 A molecular sieves (0.3 g), was added Me3SiOTf
(0.8 ꢃL, 4.3 ꢃmol). After stirring for 10 min, TLC
(9:1 CH2Cl2±acetone) showed the disappearance of
17 and the formation of a new product (Rf 0.66).
The mixture was neutralised with Et3N, diluted
with CH2Cl2 (100 mL), washed with aq 5% NaCl,
and the organic layer was dried, ®ltered, and con-
centrated. Column chromatography (95:5 CH2Cl2±
acetone) of the residue a€orded 26, isolated as a
glass (141 mg, 73%); [ꢂ]d 1ꢀ (c 1); NMR (CDCl3):
1H, ꢁ 7.736, 7.708, 7.549, 7.521, 7.472, 7.331, 7.278,
7.114, 6.980, 6.952, 6.870, and 6.843 (12 d, each 2
H, 6 COC6H4CH3), 5.659 and 5.403 (2 d, each 1 H,
J3,4/3 ,4 3.2 and 3.1 Hz, J4,5/4 ,5 < 1 Hz, H-4,40),
5.29±5.21 (m, 1 H, OCH2CH=CH2), 5.237 and
0
0
0
0
00 00 000 000
5.193 (2 dd, each 1 H, J1 ,2 /1 ,2 7.8 and 7.9 Hz,
00 00 000 000
J2 ,3 /2 ,3 9.8 and 9.9 Hz, H-200,2000), 5.031 and
0
0
4.731 (2 d, each 1 H, J1,2/1 ,2 8.4 and 8.5 Hz, H-
1,10), 4.752 and 4.682 (2 d, each 1 H, H-100,1000),
0
0
4.465 and 4.347 (2 dd, each 1 H, J2,3/2 ,3 11.2 and
11.3 Hz, H-2,20), 2.336, 2.309, 2.222, 2.198, and
2.138 (5 s, each 6 H, 6 COC6H4CH3, 2 COCH2CH2-
COCH3, and 2 Ac), 0.907 (s, 9 H, Si(CH3)2C-
(CH3)3), 0.770 (s, 6 H, Si(CH3)2C(CH3)3); 13C, ꢁ
ꢀ
(100 ꢃL) was added at 0 C. The mixture was stir-
red for 2 h, when TLC (4:2:2:0.5 1-BuOH±EtOH-
H2O±HOAc) showed the formation of a new prod-

186
K.M. Halkes et al./Carbohydrate Research 309 (1998) 175±188
172.0 (COCH2CH2COCH3), 169.7 and 169.4
(2 COCH3), 166.5, 165.3 (2 C), 164.8, and 164.0
(2 C) (6 COC6H4CH3), 133.2 (OCH2CH=CH2),
100.9 (2 C), 96.6, and 95.1 (C-1,10,100,1000), 52.3 and
52.0 (C-2,20), 37.7, 29.5, and 27.7 (COCH2CH2-
COCH3), 25.6 [Si(CH3)2C(CH3)3], 21.3 and 21.2
(COC6H4CH3), 20.6 (COCH3), 17.9 [Si(CH3)2-
C(CH3)3], 5.3 and 5.1 [Si(CH3)2C(CH3)3].
FABMS (positive-ion mode; C107H106N2O36): m/z
2017 [M+Na]+, 1995 [M+H]+.
Allyl (2,3,4-tri-O-p-toluoyl-b-d-glucopyranosyl)-
(1!3)-(4,6-di-O-acetyl-2-deoxy-2-phthalimido-b-
d-galactopyranosyl)-(1!6)-[(2,3,4-tri-O-p-toluoyl-
b-d-glucopyranosyl)-(1!3)]-4-O-acetyl-2-deoxy-
2-phthalimido-b-d-galactopyranoside (28).ÐTo a
solution of 27 (90 mg, 45 ꢃmol) in EtOH (7 mL)
.
Allyl (6-O-levulinoyl-2,3,4-tri-O-p-toluoyl-b-d-
glucopyranosyl)-(1!3)-(4,6-di-O-acetyl-2-deoxy-
2-phthalimido-b-d-galactopyranosyl)-(1!6)-[(6-
O-levulinoyl-2,3,4-tri-O-p-toluoyl-b-d-glucopyranosyl)-
(1!3)]-4-O-acetyl-2-deoxy-2-phthalimido-b-d-
galactopyranoside (27).ÐTo a solution of 26
(69 mg, 33 ꢃmol) in acetonitrile (3 mL), containing
water (0.3 mL), was added p-toluenesulfonic acid
(21 mg, 0.12 mmol). The mixture was stirred for
30 min, when TLC (Rf 0.07; 8:1 CH2Cl2±acetone)
showed the desilylation to be complete. The mix-
ture was concentrated, diluted with EtOAc
(100 mL), washed with aq 10% NaHCO3 and aq
10% NaCl, and the organic layer was dried, ®l-
tered, and concentrated. To a solution of the resi-
due in pyridine (5 mL) were added Ac2O (5 mL)
and a catalytic amount of 4-dimethylaminopyr-
idine. The solution was stirred overnight when
TLC (Rf 0.69; 9:1 CH2Cl2±acetone) showed a
complete conversion. The mixture was con-
centrated and co-concentrated with toluene, EtOH,
and CH2Cl2 (3Â20 mL). Column chromatography
(93:7 CH2Cl2±acetone) of the residue gave 27, iso-
lated as a glass (57 mg, 90%); [ꢂ]d +4ꢀ (c 1); NMR
and toluene (3 mL) was added NH2NH2 HOAc
(39 mg, 0.45 mmol). The mixture was stirred for
2 h, then concentrated. The residue was applied to
column chromatography (9:1 CH2Cl2±acetone) to
yield ꢀ28, isolated as a glass (79 mg, 98%); [ꢂ]d
1
+1.4 (c 1); NMR (CDCl3): H, ꢁ 7.750, 7.745,
7.508, 7.270, 7.264, 7.128, 6.952, 6.777, and 6.764
(9 d, 2,2,4,2,2,4,4,2,2 H, 6 COC6H4CH3), 5.730
0
0
and 5.531 (2 d, each 1 H, J3,4/3 ,4 3.4 and 3.6 Hz,
0
J4,5/4 ,5 < 1 Hz, H-4,40), 5.263 and 5.217 (2 dd,
0
00 00 000
H, J1 ,2 /1 ,2
000 000
000 00 000
each
00 00
1
J2 ,3 =J2 ,3 =10.0 Hz, H-2 ,2 ), 5.027 and 4.775
7.8 and 7.9 Hz,
(2 d, each 1 H, J1,2/1 ,2 8.5 and 8.6 Hz, H-1,10),
0
0
4.866 and 4.793 (2 d, each 1 H, H-100,1000), 4.515 and
0
0
4.361 (2 dd, each 1 H, J2,3/2 ,3 11.0 and 11.1 Hz, H-
2,20), 2.329, 2.313, 2.269, and 2.213 (4 s, 6,3,6,3 H,
6 COC6H4CH3), 2.207 and 2.107 (2 s, 6,3 H, 3 Ac);
13C, ꢁ 171.5, 171.4, and 170.4 (3 COCH3), 166.6,
166.5, 165.3 (2 C), 164.9, and 163.9 (6
COC6H4CH3), 132.5 (OCH2CH=CH2), 117.6
(OCH2CH=CH2), 101.8, 101.6, 98.0, and 96.6 (C-
1,10,100,1000), 51.9 (C-2,20), 21.3 and 21.2
(COC6H4CH3), 20.9 (2 C) and 20.4 (3 COCH3).
FABMS (positive-ion mode; C97H94N2O32): m/z
1821 [M+Na]+, 1799 [M+H]+.
1
(CDCl3): H, ꢁ 7.733, 7.718, 7.541, 7.526, 7.323,
7.296, 7.113, 7.107, 6.961, 6.952, 6.845, and 6.832
Allyl (2,3,4-tri-O-p-toluoyl-b-d-glucopyranosyl-
(12 d, each 2 H, 6 COC6H4CH3), 5.635 and 5.429
0
1 Hz, H-4,40), 5.33±5.22 (m, 1 H, OCH2CH=CH2),
uronic
acid)-(1!3)-(4,6-di-O-acetyl-2-deoxy-2-
0
0
0
(2 d, each 1 H, J3,4/3 ,4 3.6 and 3.5 Hz, J4,5/4 ,5 <
phthalimido-b-d-galactopyranosyl)-(1!6)-[(2,3,4-
tri-O-p-toluoyl-b-d-glucopyranosyluronic acid)-(1
!3)]-4-O-acetyl-2-deoxy-2-phthalimido-b-d -
galactopyranoside (29).ÐTo a solution of 28
(23 mg, 13 ꢃmol) in dry CH2Cl2 (3 mL), containing
Ac2O (129 ꢃL, 130 ꢃmol), was added pyridinium
dichromate (48 mg, 130 ꢃmol). After stirring for
4.5 h, TLC (8:2:1 CH2Cl2±EtOAc±HOAc) showed
the conversion of 28 into 29 (Rf 0.13). After the
addition of EtOAc (25 mL), the suspension was
applied to column chromatography (8:4:1 CH2Cl2±
EtOAc±HOAc) to a€ord 29, isolated as a glass
00 00 000 000
5.246 and 5.213 (2 dd, each 1 H, J1 ,2 /1 ,2 7.8 and
00 000
00 00
000 000
7.9 Hz, J2 ,3 =J2 ,3 =9.7 Hz, H-2 ,2 ), 5.040 and
0
0
4.777 (2 d, each 1 H, J1,2/1 ,2 8.5 and 8.8 Hz, H-
1,10), 4.779 and 4.722 (2 d, each 1 H, H-100,1000),
0
0
4.487 and 4.364 (2 dd, each 1 H, J2,3/2 ,3 11.2 and
11.1 Hz, H-2,20), 2.309, 2.295, 2.241, 2.208, 2.203,
2.198, 2.144, and 2.121 (8 s, 6,6,3,6,3,3,3,3 H, 6
COC6H4CH3, 2 COCH2CH2COCH3, and 3 Ac);
13C, ꢁ 172.0 and 171.9 (2 COCH2CH2COCH3),
170.4, 169.8, and 169.7 (3 COCH3), 165.2, 164.7,
and 163.9 (COC6H4CH3), 130.6 (OCH2CH=CH2),
117.3 (OCH2CH=CH2), 101.0 (2 C), 98.2, and
96.6 (C-1,10,100,1000), 52.0 (C-2,20), 37.7, 29.5, and
27.6 (COCH2CH2COCH3), 21.3 and 21.1
(COC6H4CH3), 20.6 (2 C) and 20.4 (3 COCH3).
(14 mg, 64%); [ꢂ]d +6ꢀ (c 0.5); NMR (CDCl3): H,
1
ꢁ 7.750, 7.744, 7.508, 7.269, 7.262, 7.129, 6.936,
6.777, and 6.764 (9 d, 2,2,4,2,2,4,4,2,2 H, 6
COC6H4CH3), 5.728 and 5.530 (2 d, each 1 H,
J3,4/3 ,4 2.9 and 3.8 Hz, J4,5/4 ,5 < 1 Hz, H-4,40),
0
0
0
0

K.M. Halkes et al./Carbohydrate Research 309 (1998) 175±188
187
00 00 000 000
5.261 and 5.214 (2 dd, each 1 H, J1 ,2 /1 ,2 7.9 and
00 00 000 000
temperature, during which the mixture was con-
centrated repeatedly and new reagent (7Â7.5 mL)
added. After concentration, the residue was dis-
solved in dry MeOH (5 mL), and Ac2O (100 ꢃL)
was added at 0 ꢀC. The mixture was stirred for 2 h,
when TLC (4:2:2:0.5 1-BuOH±EtOH±H2O±HOAc)
showed the formation of a major spot (Rf 0.21).
The solution was concentrated and co-con-
centrated with 1:1 toluene±MeOH (3Â10 mL). Gel
®ltration over Sephadex G-10 (water) of the
residue yielded 5, isolated after lyophilisation as
a white, amorphous powder (4.5 mg, 63%); [ꢂ]d
7.8 Hz, J2 ,3 /2 ,3 9.9 and 10.0 Hz, H-200,2000), 5.025
and 4.791 (2 d, each 1 H, J1,2/1 ,2 8.5 and 8.6 Hz, H-
1,10), 4.868 and 4.774 (2 d, each 1 H, H-100,1000),
0
0
0
0
4.513 and 4.358 (2 dd, each 1 H, J2,3/2 ,3 11.3 and
11.4 Hz, H-2,20), 2.333, 2.272, and 2.218 (3 s, each
6 H, 6 COC6H4CH3), 2.315, 2.207, and 2.108 (3 s,
each 3 H, 3 Ac); 13C, ꢁ 171.5, 171.2, and 170.6 (3
COCH3), 168.4, 166.5, 165.1, 164.8, and 163.8
(COC6H4CH3), 132.7 (OCH2CH=CH2), 117.5
(OCH2CH=CH2), 101.3 (2 C), 97.9, and 96.9 (C-
1,10,100,1000), 52.1 (C-2,20), 21.4, 21.3, and 20.8
(COC6H4CH3), 20.6, 20.5, and 19.2 (3 COCH3).
A small amount of 29 was esteri®ed with diazo-
methane in ether, and analysed by 1H NMR
(CDCl3): ꢁ 7.746, 7.735, 7.565, 7.553, 7.309, 7.286,
7.130, 6.992, 6.984, 6.883, and 6.858 (11 d,
2,2,2,2,2,2,4,2,2,2,2 H, 6 COC6H4CH3), 5.583 and
10ꢀ (c 0.4, H2O); NMR (D2O): H, ꢁ 5.914 (m,
1
1 H, OCH2CH=CH2), 5.34±5.27 (m, 2 H,
OCH2CH=CH2), 4.528 and 4.511 (2 d, each 1 H,
J1,2/1 ,2 8.4 and 8.2 Hz, H-1,10), 4.506 and 4.490 (2
0
0
d, each 1 H, J1 ,2 /1 ,2 8.0 and 7.9 Hz, H-100,1000),
00 00 000 000
0
0
4.182 and 4.154 (2 d, 0each 1 H, J3,4=J3 ,4 =3.3 Hz,
0
0
0
0
5.365 (2 d, each 1 H, J3,4/3 ,4 3.1 and 3.9 Hz, J4,5/
40,50
J4,5/4 ,5 <1 Hz, H-4,4 ), 2.012 and 2.006 (2 s, each 3
H, 2 NHCOCH3). FABMS (positive-ion mode;
C31H48N2O23): m/z 839 [M+Na]+.
< 1 Hz, H-4,40), 5.282 and 5.244 (2 dd, each 1
00 00 000 000
00 00 000 000
H, J1 ,2 /1 ,2 7.7 and 7.8 Hz, J2 ,3 /2 ,3 9.5 and
9.6 Hz, H-200,2000), 5.009 and 4.767 (2 d, each 1 H,
General protocol for the preparation of the 3-(2-
aminoethylthio)propyl adducts of 1, 3, and 5.ÐThe
allyl glycoside and cysteamine hydrochloride (3 eq
based on the allyl glycoside) were dissolved in
water (50 ꢃL/ꢃmol allyl glycoside) and the solution
was irradiated for 8±16 h in quartz with an UV
lamp. Excess of cysteamine hydrochloride was
removed by HiTrap gel ®ltration (aq 5%
NH4HCO3) to a€ord a mixture of the desired 3-(2-
J1,2/1 ,2 8.5 and 8.4 Hz, H-1,10), 4.817 and 4.742 (2
0
0
d, each 1 H, H-100,1000), 4.521 and 4.382 (2 dd, each
0
0
0
1 H, J2,3=J2 ,3 =11.2 Hz, H-2,2 ), 4.216 and 4.157
00 00 000 000
(2 d, each 1 H, J4 ,5 /4 ,5 9.6 and 9.7 Hz, H-500,5000),
3.708 and 3.676 (2 s, each 3 H, 2 COOCH3), 2.333,
2.314, and 2.237 (3 s, each 6 H, 6 COC6H4CH3),
2.221, 2.132, and 2.123 (3 s, each 3 H, 3 Ac).
FABMS (positive-ion mode; C99H94N2O34): m/z
1877 [M+Na]+, 1855 [M+H]+.
aminoethylthio)propyl adducts and
a
minor
Allyl (b-d-glucopyranosyluronic acid)-(1!3)-
(2-acetamido-2-deoxy-b-d-galactopyranosyl)-(1!6)-
[(b-d-glucopyranosyluronic acid)-(1!3)]-2-acet-
amido-2-deoxy-b-d-galactopyranoside (5).ÐA solu-
tion of 29 (15 mg, 8.5 ꢃmol) in ethanolic 33%
MeNH2 (7.5 mL) was stirred for 7 days at room
amount of the starting material. Separation was
achieved by column chromatography (1:1:0.2
CH2Cl2±MeOH±HOAc) to yield the pure 3-(2-
aminoethylthio)propyl glycosides. The products 2
{[ꢂ]d 17ꢀ (c 0.9, H2O)}, 4 {[ꢂ]d +3ꢀ (c 0.3, H2O)},
and 6 {[ꢂ]d 10ꢀ (c 0.5, H2O)} were obtained in a
Table 1
1H Chemical shifts (ꢁ, ppm) and coupling constants (Hz; in brackets) of the 3-(2-aminoethylthio)propyl adducts 2, 4, and 6
Protons
Compound
2
4
6 b
GalNAc
GlcA0
GalNAc
GalNAc0
GlcA00
GalNAc
GalNAc0
GlcA00
GlcA000
H-1
H-2
4.485 (8.5)
3.98 (10.9)
3.833 (2.9)
4.175 (<1)
1.86
4.506 (7.8) 4.454 (8.6) 4.482 (9.3) 4.499 (8.0) 4.454 (8.5)
3.331
3.470
4.542 (8.4)
4.00
3.85 (2.8)
4.507 (7.8) 4.498 (7.6)
3.188
3.47
3.336 (8.3) 3.98
3.90
3.470 (9.1) 3.841 (2.8) 3.75 (2.7)
3.98
3.82 (2.8)
3.188
3.47
H-3
H-4
n.da
4.154
1.87
2.63
3.70
4.04
3.942
n.da
4.153 (<1) 4.179 (<1)
n.d.a
n.d.a
O(CH2)3S(CH2)2NH2
1.88
2.63
3.68
3.96
2.63
3.71
3.95
an.d., not determined.
bGlcA00 is terminal ꢀ-d-glucuronic acid residue; GlcA000 is branching ꢀ-d-glucuronic acid residue.

188
K.M. Halkes et al./Carbohydrate Research 309 (1998) 175±188
1
yield of 85, 63, and 78%, respectively. For H
NMR data of the products, see Table 1.
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The authors wish to thank Dr. P.H. Kruiskamp
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