Synthesis of hyaluronic-acid-related oligosaccharides and analogues, as their 4-methoxyphenyl glycosides, having N-acetyl-β-D-glucosamine at the reducing end

Article abstract of DOI:10.1016/S0008-6215(98)00116-5

To contribute to the possibilities to study the ability of oligosaccharide fragments of hyaluronic acid to induce angiogenesis, several hyaluronic-acid-related oligosaccharides and their 6-O-sulfated analogues were synthesised as their 4-methoxyphenyl glycosides having 2-acetamido-2-deoxy-D-glucopyranose at the reducing end. In all syntheses described, the D-glucopyranosyluronic acid residue was obtained by oxidation at C-6 of a corresponding D-glucopyranosyl residue after construction of the oligosaccharide backbone, using pyridinium dichromate and acetic anhydride.

Full text of DOI:10.1016/S0008-6215(98)00116-5

Carbohydrate Research 309 (1998) 161±174
Synthesis of hyaluronic-acid-related
oligosaccharides and analogues, as their
4-methoxyphenyl glycosides, having
N-acetyl-ꢀ-d-glucosamine at the reducing end
Koen M. Halkes a, Ted M. Slaghek b, Teija K. Hypponen a,
Peter H. Kruiskamp a, Tomoya Ogawa c, 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
c TheInstituteofPhysicalandChemicalResearch(RIKEN),Wako-shi,Hirosawa2-1,Saitama,350-01,
Japan
Received 19 January 1998; accepted 3 March 1998
Abstract
To contribute to the possibilities to study the ability of oligosaccharide fragments of hyaluronic
acid to induce angiogenesis, several hyaluronic-acid-related oligosaccharides and their 6-O-sul-
fated analogues were synthesised as their 4-methoxyphenyl glycosides having 2-acetamido-2-
deoxy-d-glucopyranose at the reducing end. In all syntheses described, the d-glucopyr-
anosyluronic acid residue was obtained by oxidation at C-6 of a corresponding d-glucopyranosyl
residue after construction of the oligosaccharide backbone, using pyridinium dichromate and
acetic anhydride. # 1998 Elsevier Science Ltd. All rights reserved
Keywords: Oxidation; Hyaluronic acid oligosaccharides; Hyaluronic acid sulfated analogues
1. Introduction
(1!3)-ꢀ-d-GlcpNAc-(1!]n. It is a major compo-
nent of several soft connective tissues and has also
been found in certain bacterial strains [2]. HA is
biosynthesised at the inner side of plasma mem-
branes by a membrane-bound HA synthetase, and
is then extruded to the cell surface [3].
HA obtained from di€erent tissue sources exhi-
bits considerable variation in size, and biological
activity has been shown to be critically dependent
Hyaluronic acid (HA) is a linear extracellular
polysaccharide [1], consisting of disaccharide
repeating units of 2-acetamido-2-deoxy-d-glucose
and d-glucuronic acid, namely, [!4)-ꢀ-d-GlcpA-
* 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)00116-5

162
K.M. Halkes et al./Carbohydrate Research 309 (1998) 161±174
on molecular mass in a number of experimental
systems [4]. Native high-molecular-mass HA is
anti-angiogenic [5], whereas HA degradation pro-
ducts, containing 3±10 disaccharide units, stimu-
late endothelial cell proliferation and migration [6],
and induce angiogenesis in the chick chor-
ioallantoic membrane (CAM) assay [6,7]. How-
ever, biological studies performed so far always
used a mixture of oligosaccharides obtained by
digestion of HA from biological sources. There-
fore, it is not known which oligosaccharides are
responsible for the observed stimulating e€ects on
angiogenesis. To circumvent the drawbacks con-
nected to the use of oligosaccharides isolated from
biological sources, a synthetic program was initi-
ated in our laboratory to obtain a wide range of
medium-sized, well-de®ned oligosaccharide ele-
ments of HA.
In this paper we demonstrate, by synthesis of
several HA oligosaccharide 4-methoxyphenyl gly-
cosides (Table 1, compounds 1±3), that oxidation
of d-glucose into d-glucuronic acid of the larger
oligosaccharide fragments in the ®nal stage of the
synthetic route is possible, using pyridinium
dichromate and acetic anhydride [16,17]. Since our
synthetic approach o€ers also the possibility to
introduce other groups instead of the normal car-
boxylic acid function, two sulfated analogues of HA
fragments are synthesised (Table 1, compounds 4
and 5) because it is known that other sulfated
oligosaccharides [18] decrease the angiogenesis in
CAM experiments. Therefore, it is interesting
to study the behaviour of these sulfated analogues
of HA oligosaccharides in the above mentioned
biological assays.
As a result of this research program, several oli-
gosaccharide fragments constituted of even or odd
numbers of monosaccharides with either 2-acet-
amido-2-deoxy-d-glucose or d-glucuronic acid at
the reducing end have been synthesised. In our
synthetic strategy, the d-glucuronic acid residue
was obtained by oxidation of the primary hydroxyl
function of a corresponding d-glucose residue.
Using the above mentioned approach, so far ꢀ-d-
GlcpA-(1!3)-ꢀ-d-GlcpNAc, ꢀ-d-GlcpNAc-(1!4)-
ꢀ-d-GlcpA-(1!3)-ꢀ-d-GlcpNAc, ꢀ-d-GlcpA-(1!3)-
ꢀ-d-GlcpNAc-(1!4)-ꢀ-d-GlcpA-(1!3)-ꢀ-d-
GlcpNAc [8,9], ꢀ-d-GlcpNAc-(1!4)-ꢀ-d-GlcpA
and ꢀ-d-GlcpNAc-(1!4)-ꢀ-d-GlcpA-(1!3)-ꢀ-d-
GlcpNAc-(1!4)-ꢀ-d-GlcpA [10,11] have been
synthesised as their 4-methoxyphenyl glycosides.
Using a similar approach, the disaccharide ꢀ-d-
GlcpA-(1!3)-ꢀ-d-GlcpNAc as its methyl glycoside
was synthesised [12]. Recently, the expeditious and
stereocontrolled syntheses of HA di-, tri- [13],
tetra-, hexa-, and octasaccharides [14] fragments
having a 1-O-methyl ꢀ-d-glucuronic acid residue at
the reducing end, by direct coupling of d-glu-
curonic acid derivatives with suitably protected
2-deoxy-2-trichloroacetamido-d-glucose residues
[15] were described.
2. Results and discussion
To establish a convenient and systematic synth-
esis of the target oligosaccharides, several key
intermediates (Scheme 1; 6±9) were synthesised as
described previously by Slaghek et al. [8±11].
Starting from these intermediates, the backbone of
larger oligosaccharides can be synthesised. The
unique properties of the protecting group of the
primary hydroxyl function of the glucose residues,
the levulinoyl group, allows its selective removal.
The obtained hydroxyl functions can be oxidised,
and after deprotection the target structures will be
obtained.
For the synthesis of pentasaccharide 1, having a
GlcNAc residue at the reducing end, disaccharide
acceptor 6 was coupled with donor 7 in dichloro-
methane in the presence of boron tri¯uoride ethyl
etherate (0.3 equiv based on 6) which gave tri-
saccharide derivative 10 in 69% yield (Scheme 2).
Removal of the allyloxycarbonyl group, applying
tetrakis(triphenylphosphine)palladium and mor-
pholine [19,20], a€orded 11 in 89% yield. Di-
saccharide donor 13 was prepared by oxidative
removal [21] of the 4-methoxyphenyl group from 8
Table 1
Synthesised oligosaccharide fragments of hyaluronic acid and analogues (MP=4-methoxyphenyl)
ꢀ-d-GlcpNAc-(1!4)-ꢀ-d-GlcpA-(1!3)-ꢀ-d-GlcpNAc-(1!4)-ꢀ-d-GlcpA-(1!3)-ꢀ-d-GlcpNAc-(1!O)MP (1)
ꢀ-d-GlcpA-(1!3)-ꢀ-d-GlcpNAc-(1!4)-ꢀ-d-GlcpA-(1!3)-ꢀ-d-GlcpNAc-(1!4)-ꢀ-d-GlcpA-(1!3)-ꢀ-d-GlcpNAc-(1!O)MP (2)
ꢀ-Á4,5GlcpA-(1!3)-ꢀ-d-GlcpNAc-(1!4)-ꢀ-d-GlcpA-(1!3)-ꢀ-d-GlcpNAc-(1!4)-ꢀ-d-GlcpA-(1!3)-ꢀ-d-GlcpNAc-(1!O)MP (3)
ꢀ-d-GlcpNAc-(1!4)-ꢀ-d-Glcp6S-(1!3)-ꢀ-d-GlcpNAc-(1!4)-ꢀ-d-Glcp6S-(1!3)-ꢀ-d-GlcpNAc-(1!O)MP (4)
ꢀ-d-Glcp6S-(1!3)-ꢀ-d-GlcpNAc-(1!4)-ꢀ-d-Glcp6S-(1!3)-ꢀ-d-GlcpNAc-(1!4)-ꢀ-d-Glcp6S-(1!3)-ꢀ-d-GlcpNAc-(1!O)MP (5)

K.M. Halkes et al./Carbohydrate Research 309 (1998) 161±174
163
with ammonium cerium(IV) nitrate (! 12), fol-
lowed by imidoylation [22] (trichloroacetonitrile
and 1,8-diazabicyclo[5.4.0]undec-7-ene in dichloro-
methane), in an overall yield of 67%.
Condensation of 11 with 13 in dichloromethane,
using trimethylsilyl tri¯ate (0.05 equiv based on
11), gave the pentasaccharide derivative 14 in 81%
yield (Scheme 3). The allyloxycarbonyl group was
split o€ using tetrakis(triphenylphosphine)palladium
and morpholine to yield the pentasaccharide
derivative 15 (89%). Acidic removal of the isopropyl-
idene functions of 15, followed by conventional
acetylation of the hydroxyl functions, provided 16
(overall yield 90%), which was delevulinoylated
Scheme 1. Key intermediates as synthesised by Slaghek et al. [8±11]
Scheme 2. Synthetic intermediates towards pentasaccharide derivative 14
Scheme 3. Synthetic intermediates towards pentasaccharide 1

164
K.M. Halkes et al./Carbohydrate Research 309 (1998) 161±174
using hydrazinium acetate [23,24] to a€ord 17
(74%). Slaghek et al. [8±11] described good results
for the oxidation of primary hydroxyl to carboxyl
functions using a Swern oxidation with oxalyl
chloride and methyl sulfoxide [25] followed by
oxidation with NaClO2 [26]. However, using this
method in the present case gave unsatisfactory
results for the oxidation of 17. Application of pyr-
idinium dichromate [27] in combination with
molecular sieves [28] generated the desired product
18 (61%), but the extremely long reaction time
(72 h) made this procedure less attractive. Pre-
viously, Garegg et al. [16] described that addition
of acetic anhydride accelerated a pyridinium
9 (Scheme 1) in dichloromethane in the presence of
trimethylsilyl tri¯ate (0.05 equiv based on 15) to
a€ord the hexasaccharide derivative 19 in 62%
yield (Scheme 4). Removal of the isopropylidene
functions under acidic conditions, followed by
conventional acetylation of the hydroxyl functions
gave 20 in an overall yield of 84% based on 19.
Delevulinoylation using hydrazinium acetate pro-
vided triol 21 (75%), and subsequent oxidation of
the primary hydroxyl functions, as described for
the preparation of 18, yielded 22 (58%), as veri®ed
1
by H NMR analysis of the methyl-esteri®ed deri-
vative. Dephthaloylation/deacylation of compound
22 using methylamine in ethanol (10 days), fol-
lowed by re-N-acetylation, gave a complex mixture.
Treatment of this mixture with sodium methoxide
in methanol (pH 11), Dowex-50 (H+), and acetic
anhydrideÐmethanol resulted in the formation of
the elimination product 3, containing a terminal
unsaturated uronic acid residue which could be
isolated in 40% yield. Although being an undesired
side product, 3 could be of interest to test in bio-
logical assays since it re¯ects the nonreducing ter-
minus of enzymatic degradation products of native
hyaluronic acid when incubated with testicular
hyaluronidase. To avoid this side reaction, the
deprotection strategy was adapted and 22 was
chlorochromate-mediated
oxidation
reaction,
whereas Corey et al. [17] published that addition of
acetic anhydride and tert-butanol to a pyridinium
dichromate-mediated oxidation reaction a€orded
the tert-butyl ester of the glucuronic acid residues.
We found that oxidation of diol 17 using pyr-
idinium dichromate and acetic anhydride without
the addition of tert-butanol in dichloromethane
gave, after a reaction period of 3 h, 18 in 70%
yield. In order to demonstrate the presence of two
d-glucuronic acid residues in 18, a small amount of
the product was esteri®ed with diazomethane in
1
ether and analysed by H NMR spectroscopy. The
ꢀ
detection of two singlets at 3.499 and 3.442 ppm
(COOCH3) and two doublets at 3.706 and
3.543 ppm (H-50 and H-5000) showed unambiguously
the presence of two glucuronic acid residues.
Dephthaloylation/deacylation of 18 using methyl-
amine [29] in ethanol (10 days), and re-N-acetyla-
heated with ethylenediamine in 1-butanol at 90 C
[30], followed by re-N,O-acetylation using acetic
anhydride and pyridine in the presence of a cata-
lytic amount of 4-dimethylaminopyridine. Finally,
de-O-acetylation using aq 2 M sodium hydroxide in
ꢀ
tetrahydrofuran at 0 C [31] and subsequent neu-
ꢀ
tion with acetic anhydride in methanol at 0 C,
a€orded the target compound 1 (64%).
tralisation yielded the desired compound 2 (73%).
For the preparation of 4 and 5, representing the
sulfated hyaluronic acid analogues of 1 and 2, the
pentasaccharide derivative 17 (Scheme 3) and hex-
asaccharide derivative 21 (Scheme 4) were each
The starting compound for the synthesis of the
target structure 2 was the pentasaccharide deriva-
tive 15 (Scheme 3). Acceptor 15 was condensed with
Scheme 4. Synthetic intermediates towards hexasaccharides 2 and 3

K.M. Halkes et al./Carbohydrate Research 309 (1998) 161±174
165
treated with the sulfur trioxide-trimethylamine
complex (5 equiv/OH-group) to a€ord the penta-
saccharide disulfate analogue 23 (88%) and the hexa-
saccharide trisulfate analogue 24 (78%), respectively
(Scheme 5). Deblocking of both compounds, using
the procedure as described for compound 2 includ-
ing conversion into the sodiated form, rendered the
sulfated pentasaccharide 4 and hexasaccharide 5 in
yields of 68 and 88%, respectively.
for internal Me4Si (ꢁ 0) for solutions in CDCl3, or
by reference to acetone (ꢁ 2.225) for solutions in
D2O. 13C (APT, 75 MHz) NMR spectra were
recorded at 27 ꢀC with a Bruker AC 300 or a Varian
Gemini-300 instrument; indicated ppm values for
ꢁC are relative to the signal of CDCl3 (ꢁ 76.9) for
solutions in CDCl3. Two-dimensional double-
1
quantum ®ltered H±1H correlation spectra (2D
DQF 1H±1H COSY) were recorded usingꢀa 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-
sector 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 Xe atoms. Ele-
mental analyses were carried out by H. Kolbe
Mikroanalytisches Laboratorium (Mulheim an der
Ruhr, Germany).
1
The H NMR spectra of 1±5 are in full agree-
ment with the expected structures, and in accor-
dance with those reported for the smaller synthetic
oligosaccharide 4-methoxyphenyl glycosides [8±
11]. The synthesised 4-methoxyphenyl glycosides
1±5 will be tested in biological assays for their
ability to in¯uence the angiogenesis.
3. Experimental
4-Methoxyphenyl (3-O-allyloxycarbonyl-2-deoxy-
4,6-O-isopropylidene-2-phthalimido-b-d-glucopyrano-
syl)-(1!4)-(6-O-levulinoyl-2,3-di-O-p-toluoyl-b-d-
glucopyranosyl)-(1!3)-2-deoxy-4,6-O-isopropyl-
idene-2-phthalimido-b-d-glucopyranoside (10).ÐTo
a solution of 4-methoxyphenyl (6-O-levulinoyl-2,3-
di-O-p-toluoyl-ꢀ-d-glucopyranosyl)-(1!3)-2-deoxy-
4,6-O-isopropylidene-2-phthalimido-ꢀ-d-glucopyr-
anoside [9] (6; 0.87 g, 0.91mmol) and 3-O-allyloxy-
carbonyl-2-deoxy-4,6-O-isopropylidene-2-phthalimido-
ꢀ-d-glucopyranosyl trichloroacetimidate [8] (7;
0.95 g, 1.64 mmol) in dry CH2Cl2 (10 mL), con-
taining 3 A molecular sieves (0.8 g), was added
General methods.ÐReactions were monitored by
TLC on Kieselgel 60 F254 (E. Merck); compounds
were visualised by charring with aq 50% H2SO4,
after examination under UV light. In the work-up
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
.
BF3 OEt2 (34 ꢂL). After stirring for 30 min, TLC
1
polarimeter, using a 10 cm 1 mL cell. H NMR
(95:5 CH2Cl2±acetone) showed the disappearance
of 7 and the formation of 10 (Rf 0.21). Then the
mixture was neutralised with Et3N, diluted with
CH2Cl2 (150 mL), ®ltered through Celite, and
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
Scheme 5. Synthetic intermediates towards the sulfated hyaluronic acid analogues 4 and 5

166
K.M. Halkes et al./Carbohydrate Research 309 (1998) 161±174
washed with aq 5% NaCl, and the organic layer
was dried, ®ltered, and concentrated. Column
chromatography (9:1 CH2Cl2±acetone) of the residue
gave 10, ꢀisolated as a white foam (0.86 g, 69%);
[ꢃ]d +38 (c 1); NMR (CDCl3): 1H, ꢁ 7.725, 7.302,
7.134, and 6.977 (4 d, each 2 H, 2 COC6H4CH3),
6.68±6.60 (m, 4 H, C6H4OCH3), 5.61±5.51 (m, 1 H,
COOCH2CHˆCH2), 5.537 (d, 1 H, J1,2 8.3 Hz, H-
1.198 (4 s, each 3 H, 2 C(CH3)2); 13C, ꢁ 171.7
(COCH2CH2COCH3), 164.8 and 164.5 (2
COC6H4CH3), 99.4 and 99.3 (2 C(CH3)2), 99.8,
98.5, and 97.7 (C-1,10,100), 61.7, 61.6, and 60.8 (C-
6,60,600), 56.8, 55.4, and 55.1 (C-2,200 and
C6H4OCH3), 37.9, 29.8, and 27.5 (COCH2CH2-
COCH3), 21.4 (COC6H4CH3), 29.0, 28.6, 18.9, and
18.5 (2 C(CH3)2). Anal. Calcd for C68H70N2O23: C,
63.64; H, 5.50. Found: C, 63.49; H, 5.59.
1), 5.367 (dd, 1 H, J2 ,3 10.2, J3 ,4 9.3 Hz, H-300),
00 00
00 00
5.337 (d, 1 H, J1 ,2 8.2 Hz, H-100), 5.307 (dd, 1 H,
(3-O-Allyloxycarbonyl-2-deoxy-4,6-O-isopropyl-
idene-2-phthalimido-b-d-glucopyranosyl)-(1!4)-6-
O-levulinoyl-2,3-di-O-p-toluoyl-a=b-d-glucopyranose
(12).ÐTo a solution of 4-methoxyphenyl (3-O-
allyloxycarbonyl-2-deoxy-4,6-O-isopropylidene-2-
phthalimido-ꢀ-d-glucopyranosyl)-(1!4)-6-O-levu-
linoyl-2,3-di-O-p-toluoyl-ꢀ-d-glucopyranoside [10]
(8; 1.29 g, 1.24 mmol) in toluene (50 mL) and
acetonitrile (75 mL) was added water and ammo-
nium cerium(IV) nitrate (6.8 g, 12.4 mmol). After
stirring for 2.5 h, TLC (6:1 CH2Cl2±acetone)
showed the disappearance of 8 (Rf 0.83) and the
formation of 12 (Rf 0.58). The mixture was diluted
with EtOAc (300 mL), washed with aq 10%
NaHCO3 (2Â) and aq 5% NaCl (2Â), dried, ®l-
tered, and concentrated. Column chromatography
(9:1 CH2Cl2±acetone) of the residue gave 12, iso-
lated as a yellow syrup (0.85 g, 73%); [ꢃ]d +50ꢀ (c
1); 13C NMR (CDCl3): ꢁ 171.8 (COCH2CH2-
COCH3), 165.7 and 164.9 (2 COC6H4CH3), 154.1
(COOCH2CHˆCH2), 131.0 (COOCH2CHˆCH2),
118.2 (COOCH2CHˆCH2), 99.3 [C(CH3)2], 98.4
00 00
J2 ,3 9.6, J3 ,4 10.0 Hz, H-30), 5.07±4.93 (m, 2 H,
COOCH2CHˆCH2), 5.035 (dd, 1 H, H-20), 4.811
0
0
0
0
(d, 1 H, J1 ,2 8.0 Hz, H-10), 4.398 (dd, 1 H, J2,3
10.4 Hz, H-2), 4.153 (dd, 1 H, H-200), 3.657 (s, 3 H,
C6H4OCH3), 2.352 and 2.336 (2 s, each 3 H, 2
COC6H4CH3), 2.249 (s, 3 H, COCH2CH2COCH3),
1.485, 1.336, 1.221, and 1.174 (4 s, each 3 H, 2
C(CH3)2); 13C, ꢁ 171.7 (COCH2CH2COCH3), 164.8
and 164.5 (2 COC6H4CH3), 154.0 (COOCH2CHˆ
0
0
CH2),
131.1
(COOCH2CHˆCH2),
118.2
(COOCH2CHˆCH2), 99.3 (2 C(CH3)2), 99.8, 98.3,
and 97.7 (C-1,10,100), 55.3 (2 C) and 55.0 (C-2,200
and C6H4OCH3), 37.8, 29.7, and 27.5
(COCH2CH2COCH3), 21.4 (COC6H4CH3), 28.9,
28.5, 18.9, and 18.4 (2 C(CH3)2). Anal. Calcd for
C72H75N2O25: C, 63.24; H, 5.46. Found: C, 63.18;
H, 5.49.
4-Methoxyphenyl (2-deoxy-4,6-O-isopropylidene-
2-phthalimido-b-d-glucopyranosyl)-(1!4)-(6-O-
levulinoyl-2,3-di-O-p-toluoyl-b-d-glucopyranosyl)-
(1!3)-2-deoxy-4,6-O-isopropylidene-2-phthalimido-
b-d-glucopyranoside (11).ÐTo a solution of 10
(0.86 g, 0.63 mmol) in THF (12 mL) and morpho-
line (0.47 mL) was added tetrakis(triphenylphos-
phine)palladium (142 mg). The mixture was stirred
and boiled under re¯ux until the de-allyloxy-
carbonylation was complete (11; Rf 0.45, TLC (9:1
CH2Cl2±acetone)). Then the mixture was diluted
with EtOAc (100 mL) and washed with aq 5%
NaCl, and the organic layer was dried, ®ltered, and
concentrated. Column chromatography (9:1
CH2Cl2±acetone) of the residue gave 11, isolated as
a syrup (0.72 g, 89%); [ꢃ]d +45.5ꢀ (c 1); NMR
(C-10),
95.4
(C-1ꢀ),
89.7
(C-1ꢃ),
68.2
(COOCH2CHˆCH2), 61.8 and 60.8 (C-6,60), 55.3
(C-20), 37.7, 29.6, and 27.4 (COCH2CH2COCH3),
21.3 (COC6H4CH3), 28.5 and 18.4 [C(CH3)2].
Anal. Calcd for C48H51NO18: C, 61.99; H, 5.53.
Found: C, 61.67; H, 5.64.
(3-O-Allyloxycarbonyl-2-deoxy-4,6-O-isopropyl-
idene-2-phthalimido-b-d-glucopyranosyl)-(1!4)-6-
O-levulinoyl-2,3-di-O-p-toluoyl-a-d-glucopyranosyl
trichloroacetimidate (13).ÐTo a solution of 12
(0.85 g, 0.79 mmol) in dry CH2Cl2 (12 mL) and tri-
chloroacetonitrile (1.0 mL) was added 1,8-diazabi-
cyclo[5.4.0]undec-7-ene (30 ꢂL). After stirring
overnight, TLC (9:1 CH2Cl2±acetone) showed a
complete conversion of 12 into 13 (Rf 0.89), and
the mixture was puri®ed by column chromato-
graphy (93:7 CH2Cl2±acetone) to yield 13, isolated
as a colorless syrup (0.87 g, 91%); [ꢃ]d +23ꢀ (c 1);
1H NMR (CDCl3): ꢁ 7.919, 7.817, 7.236, and 7.118
(4 d, each 2 H, 2 COC6H4CH3), 6.585 (d, 1 H, J1,2
3.7 Hz, H-1), 5.64±5.51 (m, 1 H, COOCH2CHˆCH2),
1
(CDCl3): H, ꢁ 7.715, 7.294, 7.117, and 6.971 (4 d,
each 2 H, 2 COC6H4CH3), 6.68±6.60 (m, 4 H,
C6H4OCH3), 5.536 (d, 1 H, J1,2 8.4 Hz, H-1), 5.228
(d, 1 H, J1 ,2 8.2 Hz, H-100), 5.032 (dd, 1 H, J1 ,2
00 00
0
0
8.0, J2 ,3 9.5 Hz, H-20), 4.813 (d, 1 H, H-10), 4.394
0
0
00 00
(dd, 1 H, J2,3 10.4 Hz, H-2), 4.033 (dd, 1 H, J2 ,3
10.4 Hz, H-200), 3.644 (s, 3 H, C6H4OCH3), 2.344
and 2.325 (2 s, each 3 H, 2 COC6H4CH3), 2.236 (s,
3 H, COCH2CH2COCH3), 1.484, 1.332, 1.234, and

K.M. Halkes et al./Carbohydrate Research 309 (1998) 161±174
167
5.496 (d, 1 H, J1 ,2 8.2 Hz, H-10), 5.342 (dd, 1 H,
J2,3 10.2 Hz, H-2), 5.338 (dd, 1 H, J3,4 9.5 Hz, H-3),
5.09±4.95 (m, 2 H, COOCH2CHˆCH2), 4.254 (dd,
28.8, 28.6, 18.9, 18.7, and 18.4 (3 C(CH3)2).
FABMS (positive-ion mode; C116H119N3O40): m/z
2216 [M+Na]+.
0
0
1 H, J2 ,3 10.2 Hz, H-20), 2.386 and 2.326 (2 s, each
3 H, 2 COC6H4CH3), 2.211 (s, 3 H, COCH2CH2-
COCH3), 1.253 (s, 6 H, C(CH3)2). Anal. Calcd for
C50H51N2O18Cl3: C, 55.90; H, 4.78. Found: C,
55.73; H, 4.71.
4-Methoxyphenyl (2-deoxy-4,6-O-isopropylidene-
2-phthalimido-b-d-glucopyranosyl)-(1!4)-(6-O-
levulinoyl-2,3-di-O-p-toluoyl-b-d-glucopyranosyl)-
(1!3)-(2-deoxy-4,6-O-isopropylidene-2-phthalim-
ido-b-d-glucopyranosyl)-(1!4)-(6-O-levulinoyl-
2,3-di-O-p-toluoyl-b-d-glucopyranosyl)-(1!3)-2-
deoxy-4,6-O-isopropylidene-2-phthalimido-b-d -
glucopyranoside (15).ÐTo a solution of 14 (0.30 g,
0.14 mmol) in THF (4 mL) and morpholine (91 ꢂL)
was added tetrakis(triphenylphosphine)palladium
(30 mg). The mixture was stirred and boiled under
re¯ux until the de-allyloxycarbonylation was com-
plete (15; Rf 0.17, TLC (85:15 CH2Cl2±acetone)).
Then the mixture was diluted with EtOAc (100 mL)
and washed with aq 5% NaCl, and the organic
layer was dried, ®ltered, and concentrated. Column
chromatography (85:15 CH2Cl2±acetone) of the
residue gave 15, isolated as a glass (0.26 g, 89%);
[ꢃ]d +80ꢀ (c 1); NMR (CDCl3): 1H, ꢁ 7.679, 7.666,
7.264, 7.213, 7.103, 7.093, 6.958, and 6.952 (8 d,
each 2 H, 4 COC6H4CH3), 6.66±6.59 (m, 4 H,
C6H4OCH3), 5.282 and 5.272 (2 dd, each 1 H,
0
0
4-Methoxyphenyl (3-O-allyloxycarbonyl-2-deoxy-
4,6-O-isopropylidene-2-phthalimido-b-d-glucopyrano-
syl)-(1!4)-(6-O-levulinoyl-2,3-di-O-p-toluoyl-b-d-
glucopyranosyl)-(1!3)-(2-deoxy-4,6-O-isopropyl-
idene-2-phthalimido-b-d-glucopyranosyl)-(1!4)-
(6-O-levulinoyl-2,3-di-O-p-toluoyl-b-d-glucopyrano-
syl)-(1!3)-2-deoxy-4,6-O-isopropylidene-2-phthal-
imido-b-d-glucopyranoside (14).ÐTo a solution of
13 (0.58 g, 0.54 mmol) and 11 (0.23 g, 0.18 mmol) in
dry CH2Cl2 (7.5 mL), containing 3 A molecular
sieves (0.5 g), was added Me3SiOTf (17 ꢂL). After
stirring for 10 min, TLC (9:1 CH2Cl2±acetone)
showed the disappearance of 11, and the formation
of 14 (Rf 0.44). Then Et3N was added, and the
mixture was diluted with EtOAc (150 mL), ®ltered
through Celite, and washed with aq 5% NaCl. The
organic layer was dried, ®ltered, and concentrated.
Column chromatography (9:1 CH2Cl2±acetone) of
the residue gave 14, isolated as a white foam
0
0
000 000
0
0
000 000
J2 ,3 =J2 ,3 =9.5 Hz, J3 ,4 =J3 ,4 =9.3 Hz, H-
30,3000), 5.503, 5.192, and 5.019 (3 d, each 1 H,
J1,2/1 ,2 /1
(0.32 g, 81%); [ꢃ]d +78ꢀ (c 1); NMR (CDCl3): H,
8.4, 8.2, and 8.3 Hz, H-1,100,10000),
0000,20000
1
00 00
0
0
000 000
ꢁ 7.687, 7.665, 7.265, 7.220, 7.114, 7.089, 6.959, and
6.952 (8 d, each 2 H, 4 COC6H4CH3), 6.66±6.59
(m, 4 H, C6H4OCH3), 5.63±5.49 (m, 1 H,
4.963 and 4.913 (2 dd, each 1 H, J1 ,2 /1 ,2 7.9 and
8.0 Hz, H-20,2000), 4.718 and 4.659 (2 d, each 1 H, H-
10,1000), 4.493, 4.352, and 4.021 (3 dd, each 1 H,
00 00
COOCH2CHˆCH2), 5.07±4.92 (m,
2
H,
J2,3/2 ,3 /2
10.4, 10.4, and 10.3 Hz, H-2,200,20000),
0000,30000
COOCH2CHˆCH2), 5.502, 5.258, and 5.018 (3 d,
3.642 (s, 3 H, C6H4OCH3), 2.358, 2.333, 2.319, and
2.311 (4 s, each 3 H, 4 COC6H4CH3), 2.234 and
2.209 (2 s, each 3 H, 2 COCH2CH2COCH3), 1.419,
1.262, 1.230, 1.183, 1.143, and 1.136 (6 s, each 3 H, 3
C(CH3)2); 13C, ꢁ 171.7 and 171.6 (2 COCH2CH2-
COCH3), 164.8 and 164.5 (COC6H4CH3), 99.4 (2
C) and 99.3 (3 C(CH3)2), 99.7, 99.6, 98.5, 98.3, and
97.8 (C-1,10,100,1000,10000), 61.8, 61.5 (2 C), and 60.9 (2
C) (C-6,60,600,6000,60000), 56.8, 55.4, 55.3, and 55.2 (C-
2,200,20000 and C6H4OCH3), 37.9, 29.7, and 27.6
(COCH2CH2COCH3), 21.4 (COC6H4CH3), 28.9,
28.8, 28.7, 18.9, 18.7, and 18.6 (3 C(CH3)2). Anal.
Calcd for C112H115N3O38: C, 63.72; H, 5.49.
Found: C, 63.28; H, 5.36.
4-Methoxyphenyl (3,4,6-tri-O-acetyl-2-deoxy-2-
phthalimido-b-d-glucopyranosyl)-(1!4)-(6-O-
levulinoyl-2,3-di-O-p-toluoyl-b-d-glucopyranosyl)-
(1!3)-(4,6-di-O-acetyl-2-deoxy-2-phthalimido-b-
d-glucopyranosyl)-(1!4)-(6-O-levulinoyl-2,3-di-
O-p-toluoyl-b-d-glucopyranosyl)-(1!3)-4,6-di-O-
0000,20000
00 00
each 1 H, J1,2/1 ,2 /1
8.4, 8.4, and 8.3 Hz, H-
0
1,100,10000), 4.962 and 4.917 (2 dd, each 1 H, J1 ,2 /
0
0
0
000 000
1000,2000
7.9 and 8.0 Hz, J2 ,3 /2 ,3 9.8 and 9.6 Hz, H-
20,2000), 4.719 and 4.658 (2 d, each 1 H, H-10,1000),
00 00
4.496, 4.354, and 4.024 (3 dd, each 1 H, J2,3/2 ,3 /
20000,30000
10.4, 10.4, and 10.3 Hz, H-2,200,20000), 3.637 (s,
3 H, C6H4OCH3), 2.356, 2.329, 2.321, and 2.307 (4
s, each 3 H, 4 COC6H4CH3), 2.236 and 2.207 (2 s,
each 3 H, 2 COCH2CH2COCH3), 1.420, 1.263,
1.204, 1.153, 1.144, and 1.134 (6 s, each 3 H, 3
C(CH3)2); 13C, ꢁ 171.7 and 171.5 (2 COCH2CH2-
COCH3), 164.8, 164.7, 164.5, and 164.4 (4
COC6H4CH3), 154.1 (COOCH2CHˆCH2), 130.9
(COOCH2CHˆCH2), 99.7, 99.6, 98.3 (2 C), and
97.8 (C-1,10,100,100010000), 99.4 (2 C) and 98.9
(3C(CH3)2), 61.8, 61.5 (2 C), and 60.8 (2 C) (C-
6,60,600,6000,60000), 55.4 (2 C), 55.3, and 55.2 (C-
2,200,20000 and C6H4OCH3), 37.9, 29.7, and 27.5
(COCH2CH2COCH3), 21.4 (COC6H4CH3), 28.9,

168
K.M. Halkes et al./Carbohydrate Research 309 (1998) 161±174
acetyl-2-deoxy-2-phthalimido-b-d-glucopyranoside
(16).ÐTo a solution of 15 (0.23 g, 0.11 mmol) in
CH2Cl2 (5 mL) and H2O (12.5 ꢂL) was added
CF3CO2H (125 ꢂL). The mixture was stirred for
45 min, when TLC (9:1 CH2Cl2±MeOH) showed
the disappearance of 15 (Rf 0.91) and the forma-
tion of a slower moving spot (Rf 0.63). Then the
mixture was diluted with EtOAc (100 mL) and
washed with aq 10% NaHCO3 and aq 5% NaCl.
The aqueous layers were extracted twice with
EtOAc. The combined organic layers were dried,
®ltered, and concentrated. To a solution of the
residue in pyridine (5 mL) was added Ac2O (5 mL)
and a catalytic amount of 4-dimethylaminopyr-
idine. After stirring overnight at room tempera-
ture, TLC (4:1 CH2Cl2±acetone) showed the
acetylation to be complete (16; Rf 0.68). The mix-
ture was concentrated and co-concentrated with
toluene, EtOH, and CH2Cl2 (3Â20 mL). Column
chromatography (85:15 CH2Cl2±acetone) of the
residue gave 16, isolated as a glass (0.22 g, 90%);
[ꢃ]d +48ꢀ (c 1); NMR (CDCl3): 1H, ꢁ 7.680, 7.639,
7.349, 7.338, 7.045, 7.017, 6.998, and 6.973 (8 d,
each 2 H, 4 COC6H4CH3), 6.67±6.59 (m, 4 H,
(1!3)-4,6-di-O-acetyl-2-deoxy-2-phthalimido-b-d-
glucopyranoside (17).ÐTo solution of 16
(200 mg, 88 ꢂmol) in EtOH (12 mL) and toluene
a
.
(6 mL) was added NH2NH2 HOAc (76 mg,
0.88 mmol). The mixture was stirred for 40 min,
when TLC (4:1 CH2Cl2±acetone) showed the con-
version of 16 into 17 (Rf 0.57), then concentrated.
Column chromatography (4:1 CH2Cl2±acetone) of
the residue yielded 17, isolated as a glass (136 mg,
74%); [ꢃ]d +45ꢀ (c 1); H NMR (CDCl3): ꢁ 7.716,
1
7.659, 7.436, 7.420, 7.115, 7.062, 7.027, and 7.006
(8 d, each 2 H, 4 COC6H4CH3), 6.67±6.61 (m, 4 H,
C6H4OCH3), 5.588 (dd, 1 H, J3
9.1 Hz, H-30000),
0000,40000
0
5.369 and 5.318 (2 t, each 1 H, J2 ,3 =J2 ,3 =
0
000 000
8.8 Hz, J3 ,4 /3 ,4 8.7 and 8.8 Hz, H-30,3000), 5.452,
0
0
000 000
00 00
0000,20000
5.362, and 5.024 (3 d, each 1 H, J1,2/1 ,2 /1
8.4,
8.5, and 8.4 Hz, H-1,100,10000), 4.925 and 4.872 (2 dd,
each 1 H, H-20,2000), 4.545 and 4.488 (2 d, each 1 H,
J1 ,2 /1 ,2 7.2 and 6.9 Hz, H-10,1000), 3.661 (s, 3 H,
C6H4OCH3), 2.365, 2.352, 2.336, and 2.311 (4 s,
each 3 H, 4 COC6H4CH3), 2.053, 1.910, 1.905,
1.891, 1.880, 1.819, and 1.775 (7 s, each 3 H, 7 Ac).
FABMS (positive-ion mode; C107H105N3O41): m/z
2110 [M+Na]+, 2088 [M+H]+.
0
0
000 000
C6H4OCH3), 5.596 (dd, 1 H, J3
5.335, 5.272, and 4.849 (3 d, each 1 H, J1,2/1 ,2 /
9.1 Hz, H-30000),
4-Methoxyphenyl (3,4,6-tri-O-acetyl-2-deoxy-2-
phthalimido-b-d-glucopyranosyl)-(1!4)-(2,3-di-O-
p-toluoyl-b-d-glucopyranosyluronic acid)-(1!3)-
(4,6-di-O-acetyl-2-deoxy-2-phthalimido-b-d-gluco-
pyranosyl)-(1!4)-(2,3-di-O-p-toluoyl-b-d-gluco-
pyranosyluronic acid)-(1!3)-4,6-di-O-acetyl-2-
deoxy-2-phthalimido-b-d-glucopyranoside (18).Ð
To a solution of 17 (55 mg, 26 ꢂmol) in dry
CH2Cl2 (4 mL) containing Ac2O (26 ꢂL) was
added pyridinium dichromate (39 mg, 104 ꢂmol).
After stirring for 1 h, the brown suspension became
a brown solution, and after 3 h TLC (10:5:0.5
CH2Cl2±acetone±HOAc) showed the conversion of
17 (Rf 0.87) into 18 (Rf 0.13). After addition of
EtOAc (25 mL), the suspension was applied to col-
umn chromatography (98:2 EtOAc±HOAc) to
yield 18, isolated as a glass (38 mg, 70%); [ꢃ]d
+30ꢀ (c 1); NMR (CDCl3): 1H, ꢁ 5.347, 5.231, and
00 00
0000,40000
00 00
8.3, 8.2, and 8.4 Hz, H-1,100,10000), 4.995 and
10000,20000
0
0
000 000
4.983 (2 dd, each 1 H, J1 ,2 /1 ,2 7.5 and 7.6 Hz,
0
000
0
0
000 000
J2 ,3 =J2 ,3 =9.4 Hz, H-2 ,2 ), 4.518, 4.391, and
00 00
0000,30000
4.004 (3 dd, each 1 H, J2,3/2 ,3 /2
10.7, 10.7, and
10.8 Hz, H-2,200,20000), 4.322 and 4.252 (2 d, each 1
H, H-10,1000), 3.655 (s, 3 H, C6H4OCH3), 2.383,
2.359, 2.295, and 2.285 (4 s, each 3 H, 4
COC6H4CH3), 2.214 and 2.199 (2 s, each 3 H, 2
COCH2CH2COCH3), 2.056, 1.896, 1.888, 1.869,
1.859, 1.838, and 1.759 (7 s, each 3 H, 7 Ac); 13C, ꢁ
171.7 and 171.5 (2 COCH2CH2COCH3), 170.5,
170.3, 170.2, 169.8, 169.1, 169.0, and 168.9 (7
COCH3), 164.9 and 164.7 (3 C) (4 COC6H4CH3),
100.8, 100.6, and 97.5 (3 C) (C-1,10,100,1000,10000),
62.4 (2 C), 62.2, 61.8, and 61.4 (C-6,60,600,6000,60000),
55.4, 55.2 (2 C), and 54.6 (C-2,200,20000 and
C6H4OCH3), 37.6, 29.7, and 27.5 (COCH2CH2-
COCH3), 21.4 (COC6H4CH3), 21.5, 20.5 (3 C),
20.4 (2 C), and 20.1 (7 COCH3). Anal. Calcd for
C117H117N3O45: C, 61.50; H, 5.16. Found: C,
61.48; H, 5.26.
0000,20000
4.881 (3 d, each 1 H, J1,2/1 ,2 /1
8.6, 8.5, and
8.3 Hz, H-1,100,10000), 4.392 and 4.365 (2 d, each 1 H,
J1 ,2 /1 ,2 8.1 and 7.8 Hz, H-10,1000), 3.658 (s, 3 H,
C6H4OCH3), 2.389, 2.367, 2.295, and 2.277 (4 s,
each 3 H, 4 COC6H4CH3), 2.061, 1.886, 1.862,
1.855, 1.828, 1.797, and 1.762 (7 s, each 3 H, 7 Ac);
13C, ꢁ 170.7 (2 C), 170.4 (2 C), 169.7 (2 C), 169.5,
and 169.2 (2 C) (7 COCH3 and 2 COOH), 164.7
and 164.2 (COC6H4CH3), 101.5, 100.8, and 97.5 (3
C) (C-1,10,100,1000,10000), 62.3 (2 C) and 61.7 (C-
0
0
000 000
4-Methoxyphenyl (3,4,6-tri-O-acetyl-2-deoxy-2-
phthalimido-b-d-glucopyranosyl)-(1!4)-(2,3-di-O-
p-toluoyl-b-d-glucopyranosyl)-(1!3)-(4,6-di-O-
acetyl-2-deoxy-2-phthalimido-b-d-glucopyranosyl)-
(1!4)-(2,3-di-O-p-toluoyl-b-d-glucopyranosyl)-

K.M. Halkes et al./Carbohydrate Research 309 (1998) 161±174
169
6,600,60000), 55.4, 55.2 (2 C), and 54.5 (C-2,200,20000 and
C6H4OCH3), 21.4 (COC6H4CH3), 21.5, 20.6 (2 C),
20.5, 20.4 (2 C), and 20.1 (7 COCH3).
H, C6H4OCH3), 2.049, 2.023, and 2.019 (3 s, each 3
H, 3 NHCOCH3). FABMS (negative-ion mode;
C43H63N3O29): m/z 1084 [M H] .
A small amount of 18 was esteri®ed with diazo-
1
4-Methoxyphenyl (6-O-levulinoyl-2,3,4-tri-O-p-
toluoyl-b-d-glucopyranosyl)-(1!3)-(2-deoxy-4,6-
O-isopropylidene-2-phthalimido-b-d-glucopyranosyl)-
(1!4)-(6-O-levulinoyl-2,3-di-O-p-toluoyl-b-d-
glucopyranosyl)-(1!3)-(2-deoxy-4,6-O-isopropyl-
idene-2-phthalimido-b-d-glucopyranosyl)-(1!4)-
(6-O-levulinoyl-2,3-di-O-p-toluoyl-b-d-glucopyrano-
syl)-(1!3)-2-deoxy-4,6-O-isopropylidene-2-phthal-
imido-b-d-glucopyranoside (19).ÐTo a solution of
15 (141 mg, 67 ꢂmol) and 6-O-levulinoyl-2,3,4-tri-
O-p-toluoyl-ꢀ-d-glucopyranosyl trichloroacetimid-
ate [8] (9; 129 mg, 167 ꢂmol) in dry CH2Cl2 (5 mL),
containing 3 A molecular sieves (0.2 g), was added
at 0 ꢀC Me3SiOTf (6.5 ꢂL). After stirring for
45 min, TLC (93:7 CH2Cl2±acetone) showed the
disappearance of 15 and the formation of 19 (Rf
0.27). Then the mixture was neutralised with Et3N,
diluted with CH2Cl2 (150 mL), ®ltered through
Celite, and washed with aq 5% NaCl, and the
organic layer was dried, ®ltered, and concentrated.
Column chromatography (9:1 CH2Cl2±acetone) of
the residue gave 19, isolated as a glass (112 mg,
62%); [ꢃ]d +32ꢀ (c 1); NMR (CDCl3): 1H, ꢁ 7.534,
7.528, 7.490, 7.460, 7.255, 7.246, 7.204, 7.108,
7.093, 7.080, 6.970, 6.960, 6.952, and 6.949 (14 d,
each 2 H, 7 COC6H4CH3), 6.68±6.58 (m, 4 H,
C6H4OCH3), 5.502, 5.040, and 5.001 (3 d, each 1
00 00
methane in ether, and analysed by H NMR: ꢁ
7.689, 7.651, 7.337, 7.328, 7.071, 7.025, 6.992, and
6.967 (8 d, each 2 H, 4 COC6H4CH3), 6.625 (bs, 4
0000,40000
H, C6H4OCH3), 5.606 (dd, 1 H, J3
9.1 Hz,
H-30000), 5.336, 5.279, and 4.801 (3 d, each 1 H,
8.5, 8.4, and 8.6 Hz, H-1,100,10000),
0000,20000
00 00
J1,2/1 ,2 /1
5.334 and 5.244 (2 dd, each 1 H, J3 ,4 /3 ,4 9.0
0
0
000 000
and 9.2 Hz, H-30,3000), 5.024 and 4.969 (2 dd, each
0
0
000 000
0
0
000 000
1 H, J1 ,2 /1 ,2 7.4 and 7.6 Hz, J2 ,3 /2 ,3 9.2 and
9.3 Hz, H-20,2000), 4.629, 4.519, and 4.388 (3 dd,
00 00
0000,30000
each 1 H, J2,3/2 ,3 /2
10.9, 10.7, and 10.9 Hz,
H-2,200,20000), 4.363 and 4.326 (2 d, each 1 H, H-
10,1000), 3.706 and 3.543 (2 d, each 1 H, J4 ,5 /4 ,5 9.6
and 9.3 Hz, H-50,5000), 3.657 (s, 3 H, C6H4OCH3),
3.499 and 3.442 (2 s, each 3 H, 2 COOCH3), 2.391,
2.362, 2.306, and 2.281 (4 s, each 3 H, 4
COC6H4CH3), 2.057, 1.890, 1.881, 1.853, 1.837,
1.764, and 1.743 (7 s, each 3 H, 7 Ac). FABMS
(positive-ion mode; C109H105N3O43): m/z 2166
[M+Na]+.
0
0
000 000
4-Methoxyphenyl (2-acetamido-2-deoxy-b-d-
glucopyranosyl)-(1!4)-(b-d-glucopyranosyluronic
acid)-(1!3)-(2-acetamido-2-deoxy-b-d-glucopyrano-
syl)-(1!4)-(b-d-glucopyranosyluronic acid)-(1!3)-
2-acetamido-2-deoxy-b-d-glucopyranoside (1).ÐA
solution of 18 (30 mg, 14 ꢂmol) in ethanolic 33%
MeNH2 (7.5 mL) was stirred for 10 days at room
temperature, during which the mixture was con-
centrated repeatedly and new reagent (5Â7.5 mL)
added. After concentration, the residue was dis-
solved in dꢀry MeOH (5 mL), Ac2O (100 ꢂL) was
added at 0 C, and the mixture was stirred for 2 h.
Then, TLC (4:2:2:0.5 1-BuOH±EtOH±H2O±
HOAc) showed the complete disappearance of 18
and the formation of 1 (Rf 0.45), and the solution
was concentrated and co-concentrated with 1:1
toluene±MeOH (3Â10 mL). Gel ®ltration on
Sephadex G-10 (water) of the residue yielded 1,
isolated after lyophilisation as a white, amorphous
0000,20000
H, J1,2/1 ,2 /1
8.4, 8.8, and 8.6 Hz, H-1,100,10000),
0
5.095, 4.956, and 4.872 (3 dd, each 1 H, J1 ,2 /1 ,2 /
0
000 000
0
0
000 000
100000,200000
00000,300000
9.0, 9.5,
7.8, 7.9, and 8.0 Hz, J2 ,3 /2 ,3 /2
and 9.6 Hz, H-20,2000,200000), 4.879, 4.713, and 4.586 (3
d, each 1 H, H-10,1000,100000), 4.491, 4.366, and 4.352
00 00
0000,30000
(3 dd, each 1 H, J2,3/2 ,3 /2
10.4, 10.4, and
10.2 Hz, H-2,200,20000), 3.636 (s, 3 H, C6H4OCH3),
2.357, 2.351, 2.326, 2.301, and 2.240 (5 s, 3,3,6,6,3
H, 7 COC6H4CH3), 2.219, 2.203, and 2.146 (3 s,
each 3 H, 3 COCH2CH2COCH3), 1.416, 1.278,
1.258, 1.253, 1.248, and 1.087 (6 s, each 3 H, 3
C(CH3)2); 13C, ꢁ 172.1 and 171.5 (2 C) (3
COCH2CH2COCH3), 165.4, 164.7 (2 C), 164.5 (2
C), and 164.3 (2 C) (7 COC6H4CH3), 99.4, 99.1,
and 98.9 (3 C(CH3)2), 99.7 (3 C), 98.3 (2 C), and
97.8 (C-1,10,100,1000,10000,100000), 62.5, 61.8, 61.5 (2 C),
and 60.8 (2 C) (C-6,60,600,6000,60000,600000), 55.4 (C-
2,200,20000 and C6H4OCH3), 37.7, 29.7, and 27.6
(COCH2CH2COCH3), 21.4 (COC6H4CH3), 28.9 (3
C), 18.9, and 18.7 (2 C) (3 C(CH3)2). FABMS
(positive-ion mode; C147H149N3O48): m/z 2748
[M+Na]+.
powder (10 mg, 64%); [ꢃ]d +12ꢀ (c 0.5, H2O); H
1
NMR (D2O): ꢁ 7.06±6.96 (m, 4 H, C6H4OCH3),
5.054 (d, 1 H, J1,2 8.6 Hz, H-1), 4.564 and 4.533 (2
8.5 and 8.4 Hz, H-100,10000),
0000,20000
00 00
d, each 1 H, J1 ,2 /1
4.511 and 4.466 (2 d, each 1 H, J1 ,2 /1 ,2 7.8 and
0
0
000 000
7.9 Hz, H-10,1000), 4.089 (dd, 1 H, J2,3 10.9 Hz, H-2),
00 00
0000,30000
3.851 and 3.704 (2 dd, each 1 H, J2 ,3 /2
10.7
and 10.9 Hz, H-200,20000), 3.387 and 3.347 (2 dd, each
1 H, J2 ,3 /2 ,3 9.4 and 9.5 Hz, H-20,2000), 3.808 (s, 3
0
0
000 000

170
K.M. Halkes et al./Carbohydrate Research 309 (1998) 161±174
4-Methoxyphenyl (6-O-levulinoyl-2,3,4-tri-O-p-
COCH3).
FABMS
(positive-ion
mode;
toluoyl-b-d-glucopyranosyl)-(1!3)-(4,6-di-O-
acetyl-2-deoxy-2-phthalimido-b-d-glucopyranosyl)-
(1!4)-(6-O-levulinoyl-2,3-di-O-p-toluoyl-b-d-
glucopyranosyl)-(1!3)-(4,6-di-O-acetyl-2-deoxy-
2-phthalimido-b-d-glucopyranosyl)-(1!4)-(6-O-
levulinoyl-2,3-di-O-p-toluoyl-b-d-glucopyranosyl)-
(1!3)-4,6-di-O-acetyl-2-deoxy-2-phthalimido-b-d-
glucopyranoside (20).ÐTo a solution of 19 (90 mg,
33 ꢂmol) in CH2Cl2 (5 mL) and H2O (5 ꢂL) was
added CF3CO2H (50 ꢂL). The mixture was stirred
for 30 min, when TLC (9:1 CH2Cl2±MeOH)
showed the disappearance of 19 (Rf 0.93) and the
formation of a slower moving spot (Rf 0.70). Then
the mixture was diluted with EtOAc (100 mL), and
washed with aq 10% NaHCO3 and aq 5% NaCl.
The aqueous layers were extracted twice with
EtOAc. The combined organic layers were dried,
®ltered, and concentrated. To a solution of the
mixture in pyridine (5 mL) were added Ac2O
(5 mL) and a catalytic amount of 4-dimethylami-
nopyridine. After stirring overnight at room tem-
perature, TLC (95:5 CH2Cl2±MeOH) showed the
acetylation to be complete (20; Rf 0.37). The
mixture was concentrated and co-concentrated
with toluene, EtOH, and CH2Cl2 (3Â20 mL).
Column chromatography (95:5 CH2Cl2±MeOH) of
the residue gave 20, isolated as a glass (80 mg,
C150H149N3O54): m/z 2880 [M+Na]+, 2858
[M+H]+.
4-Methoxyphenyl
(2,3,4-tri-O-p-toluoyl-b-d-
glucopyranosyl)-(1!3)-(4,6-di-O-acetyl-2-deoxy-
2-phthalimido-b-d-glucopyranosyl)-(1!4)-(2,3-di-
O-p-toluoyl-b-d-glucopyranosyl)-(1!3)-(4,6-di-O-
acetyl-2-deoxy-2-phthalimido-b-d-glucopyranosyl)-
(1!4)-(2,3-di-O-p-toluoyl-b-d-glucopyranosyl)-(1
!3)-4,6-di-O-acetyl-2-deoxy-2-phthalimido-b-d-
glucopyranoside (21).ÐTo a solution of 20 (65 mg,
23 ꢂmol) in EtOH (6 mL) and toluene (3 mL) was
.
added NH2NH2 HOAc (30 mg, 0.34 mmol). The
mixture was stirred for 2 h, when TLC (1:1
CH2Cl2±EtOAc) showed the conversion of 20 into
21 (Rf 0.49). Then, the mixture was concentrated,
and column chromatography (3:1 CH2Cl2±acet-
one) of the residue yielded 21, isolated as a glass
(44 mg, 75%); [ꢃ]d +37ꢀ (c 1); NMR (CDCl3):
1H, ꢁ 7.741, 7.671, 7.652, 7.543, 7.445, 7.416,
7.403, 7.125, 7.082, 7.055, 7.029, 7.000, 6.983,
and 6.970 (14 d, each 2 H, 7 COC6H4CH3), 6.70±
6.60 (m, 4 H, C6H4OCH3), 5.621 (t, 1 H,
J3
=J4
and 5.012 (3 d, each 1 H, J1,2/1 ,2 /1
=9.6 Hz, H-400000), 5.360, 5.045,
00000,500000
00000,400000
00 00
0000,20000
8.5, 8.5,
and 8.4 Hz, H-1,100,10000), 5.151, 4.834, and 4.723 (3
0
0
000 000
dd, each 1 H, J1 ,2 /1 ,2 /1
00000,200000
7.7, 7.3, and 7.1 Hz,
9.6, 8.6, and 9.1 Hz, H-20,2000,200000),
4.588, 4.535, and 4.396 (3 d, each 1 H, H-
0
J2 ,3 /2 ,3 /2
0
000 000
00000,300000
ꢀ
1
84%); [ꢃ]d +24 (c 1); NMR (CDCl3): H, ꢁ 6.68±
6.59 (m, 4 H, C6H4OCH3), 5.510 (t, 1 H,
10,1000,100000), 4.482, 4.148, and 4.095 (3 dd, each 1 H,
00000,400000
00000,500000
00 00
J2,3/2 ,3 /2
J3
=J4
=9.6 Hz, H-400000), 5.337, 4.885,
11.1, 11.0, and 11.3 Hz, H-2,200,20000),
0000,30000
00 00
0000,20000
and 4.843 (3 d, each 1 H, J1,2/1 ,2 /1
8.5, 8.5,
3.656 (s, 3 H, C6H4OCH3), 2.379, 2.355, 2.348,
2.325, 2.305, and 2.217 (6 s, 3,3,3,6,3,3 H, 7
COC6H4CH3), 2.052, 2.010, 1.920, 1.903, 1.877,
and 1.781 (6 s, each 3 H, 6 Ac); 13C, ꢁ 170.5, 170.4,
170.3, 169.5, 169.4, and 169.2 (6 COCH3), 165.9,
165.5, 164.7 (2 C), 164.6, and 164.5 (2 C) (7
COC6H4CH3), 99.9 and 99.5 (2 C) (C-10,1000,100000),
97.9, 97.8, and 97.5 (C-1,100,10000), 62.0, 61.4 (2 C),
60.9, and 60.3 (2 C) (C-6,60,600,6000,60000,600000), 55.4,
55.5, 55.3, and 55.2 (C-2,200,20000 and C6H4OCH3),
21.5, 21.4, and 21.3 (COC6H4CH3), 20.6, 20.5, and
20.4 (COCH3). FABMS (positive-ion mode;
C135H131N3O48): m/z 2584 [M+Na]+, 2562
[M+H]+.
4-Methoxyphenyl (2,3,4-tri-O-p-toluoyl-b-d-
glucopyranosyluronic acid)-(1!3)-(4,6-di-O-acetyl-
2-deoxy-2-phthalimido-b-d-glucopyranosyl)-(1!4)-
(2,3-di-O-p-toluoyl-b-d-glucopyranosyluronic acid)-
(1!3)-(4,6-di-O-acetyl-2-deoxy-2-phthalimido-b-
d-glucopyranosyl)-(1!4)-(2,3-di-O-p-toluoyl-b-d-
glucopyranosyluronic acid)-(1!3)-4,6-di-O-acetyl-
and 8.4 Hz, H-1,100,10000), 5.141, 4.992, and 4.927 (3
0
0
000 000
dd, each 1 H, J1 ,2 /1 ,2 /1
00000,200000
7.8, 7.5, and 7.5 Hz,
10.3, 9.1, and 9.2 Hz, H-
0
0
000 000
00000,300000
J2 ,3 /2 ,3 /2
20,2000,200000), 4.486, 4.324, and 4.162 (3 d, each 1 H,
H-10,1000,100000), 4.391, 4.061, and 3.982 (3 dd, each 1
00 00
0000,30000
H, J2,3/2 ,3 /2
10.8, 10.7, and 10.8 Hz, H-
2,200,20000), 3.647 (s, 3 H, C6H4OCH3), 2.394, 2.374,
2.352, 2.308, 2.279, and 2.220 (6 s, 3,3,3,3,6,3 H, 7
COC6H4CH3), 2.215, 2.192, and 2.136 (3 s, each 3
H, 3 COCH2CH2COCH3), 2.053, 1.994, 1.897,
1.872, 1.851, 1.786 (6 s, each 3 H, 6 Ac); 13C, ꢁ
171.9, 171.5, and 171.4 (3 COCH2CH2COCH3),
170.5, 170.3 (2 C), 169.0, 168.9, and 168.7 (6
COCH3), 165.4, 164.7 (2 C), 164.6 (2 C), and 164.5
(2 C) (7 COC6H4CH3), 100.7 (2 C), 100.5, 97.3 (3
C) (C-1,10,100,1000,10000,100000), 62.1 (3 C), 62.0, and 61.6
(2 C) (C-6,60,600,6000,60000,600000), 55.3, 55.2, and 55.1 (2
C) (C-2,200,20000 and C6H4OCH3), 37.5, 29.5, and
27.4 (COCH2CH2COCH3), 21.4 and 21.3
(COC6H4CH3), 20.5 (3 C), 20.4, 20.3, and 20.2 (6

K.M. Halkes et al./Carbohydrate Research 309 (1998) 161±174
171
2-deoxy-2-phthalimido-b-d-glucopyranoside (22).Ð
To a solution of 21 (40 mg, 16 ꢂmol) in dry
CH2Cl2 (2 mL), containing Ac2O (22 ꢂL), was
added pyridinium dichromate (35 mg, 93 ꢂmol).
After stirring for 1 h the brown suspension became
a brown solution, and after 6 h TLC (5:5:1
CH2Cl2±EtOAc±HOAc) showed the conversion of
21 (Rf 0.70) into 22 (Rf 0.19). After the addition of
EtOAc (25 mL), the suspension was applied to col-
umn chromatography (98:2 EtOAc±HOAc) to
yield 22, isolated as a glass (24 mg, 58%); [ꢃ]d 2ꢀ
(c 1).
mixture was stirred for 2 h. Then, TLC (4:2:3:2 1-
BuOH±EtOH±H2O±HOAc) showed the formation
of a major compound (Rf 0.23). After concentra-
tion, gel ®ltration on Sephadex G-10 (water) of the
residue yielded 3, isolated after lyophilisation as a
white, amorphous powder (3 mg, 40%); [ꢃ]d +108ꢀ
(c 0.25, H2O); 1H NMR (D2O): ꢁ 7.11±6.95 (m, 4 H,
C6H4OCH3), 5.849 (d, 1 H, J3
00000,200000
4.0 Hz, H-400000),
00000,400000
5.151 (d, 1 H, J1
4.9 Hz, H-100000), 5.053 (d, 1
H, J1,2 8.6 Hz, H-1), 4.588 and 4.563 (2 d, each 1
8.0 and 8.5 Hz, H-100,10000), 4.510 and
0000,20000
00 00
H, J1 ,2 /1
0
0
4.465 (2 d, each 1 H, J1 ,2 /1 ,2 7.7 and 7.8 Hz, H-
000 000
10,1000), 4.136 (t, 1 H, J2
(dd, 1 H, J2,3 10.4 Hz, H-2), 3.845 (dd, 2 H, J2 ,3 /
4.3 Hz, H-300000), 4.089
00000,300000
A small amount of 22 was esteri®ed with diazo-
1
00 00
methane in ether, and analysed by H NMR: ꢁ
10.2 Hz, H-200,20000), 3.750 (dd, 1 H, H-200000),
3.725 (s, 3 H, C6H4OCH3), 3.386 and 3.347 (2 dd,
20000,30000
7.692, 7.645, 7.559, 7.407, 7.324, 7.299, 7.115,
7.021, 6.993, and 6.966 (10 d, 2,4,2,2,2,2,2,4,4,4 H,
7 COC6H4CH3), 6.624 (bs, 4 H, C6H4OCH3), 5.577
0
000
0
0
000 000
each 1 H, J2 ,3 /2 ,3 9.5 and 9.4 Hz, H-2 ,2 ), 2.064,
2.032, and 2.021 (3 s, each 3 H, 3 NHCOCH3).
FABMS (negative-ion mode; C49H69N3O34): m/z
1264 [M+Na-H] , 1242 [M-H] .
(t, 1 H, J3
=J4
4.831, and 4.796 (3 d, each 1 H, J1,2/1 ,2 /1
=9.5 Hz, H-400000), 5.335,
00000,500000
00000,400000
0000,20000
00 00
8.5,
8.4, and 8.4 Hz, H-1,100,10000), 4.513, 4.354, and
0
0
000 000
00000,200000
4.212 (3 d, each 1 H, J1 ,2 /1 ,2 /1
7.8, 7.4, and
4-Methoxyphenyl (b-d-glucopyranosyluronic acid)-
(1!3)-(2-acetamido-2-deoxy-b-d-glucopyranosyl)-
(1!4)-(b-d-glucopyranosyluronic acid)-(1!3)-
(2-acetamido-2-deoxy-b-d-glucopyranosyl)-(1!4)-
(b-d-glucopyranosyluronic acid)-(1!3)-2-acetamido-
2-deoxy-b-d-glucopyranoside (2).ÐTo a solution
of 22 (16 mg, 6 ꢂmol) in 1-BuOH (5 mL) was added
ethylenediamineꢀ (1 mL). The mixture was stirred
overnight at 90 C under Ar, when TLC (4:2:2:0.5
1-BuOH±EtOH±H2O±HOAc) showed the dis-
appearance of 22. The mixture was concentrated
and co-concentrated with toluene (5Â10 mL). The
residue was dissolved in dry pyridine (5 mL), and
Ac2O (5 mL) and a catalytic amount of 4-dimethyl-
aminopyridine were added. The mixture was stir-
red overnight at room temperature, then
concentrated and co-concentrated with toluene and
EtOH (2Â20 mL). The yellow, amorphous solid
7.6 Hz, H-10,1000,100000), 3.657 (s, 3 H, C6H4OCH3),
3.591, 3.427, and 3.418 (3 s, each 3 H, 3 COOCH3),
2.402, 2.383, 2.359, 2.327, 2.286, 2.277, and 2.238
(7 s, each 3 H, 7 COC6H4CH3), 2.079, 1.874, 1.850,
1.844, 1.833, and 1.710 (6 s, each 3 H, 6 Ac).
FABMS (positive-ion mode; C138H131N3O51): m/z
2668 [M+Na]+, 2646 [M+H]+.
4-Methoxyphenyl
(4,5Á-glucopyranosyluronic
acid)-(1!3)-(2-acetamido-2-deoxy-b-d-glucopyrano-
syl)-(1!4)-(b-d-glucopyranosyluronic acid)-(1!3)-
(2-acetamido-2-deoxy-b-d-glucopyranosyl)-(1!4)-
(b-d-glucopyranosyluronic acid)-(1!3)-2-acetamido-
2-deoxy-b-d-glucopyranoside (3).ÐA solution of 22
(12 mg, 4.5 ꢂmol) in ethanolic 33% MeNH2
(7.5 mL) was stirred for 10 days at room tempera-
ture, during which the mixture was concentrated
and repeatedly new reagent (5Â7.5 mL) added.
After ®nal concentration, the residue was dissolved
in dry MeꢀOH (5 mL) and Ac2O (100 ꢂL) was
added at 0 C, and the mixture was stirred for 2 h.
Then, TLC (4:2:2:0.5 1-BuOH±EtOH±H2O±
HOAc) showed a complex mixture. The solution
was concentrated and co-concentrated with 1:1
toluene±MeOH (3Â10 mL). To a solution of the
residue in MeOH (5 mL) was added NaOMe (pH
11), and the mixture was stirred overnight. After
concentration a solution of the residue in MeOH
(10 mL) was stirred with Dowex-50 (H+) for 1 h,
then the mixture was ®ltered and concentrated.
The residue was dissolved in dry MeOH (5 mL)
ꢀ
was dissolved in THF (5 mL), and at 0 C was
added aq 2 M NaOH (1 mL) under vigorous stir-
ring. After stirring for 6 h at 0 ꢀC, TLC (4:2:2:0.5 1-
BuOH±EtOH±H2O±HOAc) showed the conver-
sion into 2 (Rf 0.35), and the solution was neu-
tralised with aq 1 M HCl. After concentration and
co-concentration
with
1:1
toluene-MeOH
(3Â10 mL), gel ®ltration on Sephadex G-10 (water)
of the residue yielded 2, isolated after lyophilisation
as a ꢀwhite, amorphous powder (5 mg, 73%); [ꢃ]d
1
+44 (c 0.25, H2O); H NMR (D2O): ꢁ 7.05±6.97
(m, 4 H, C6H4OCH3), 5.053 (d, 1 H, J1,2 8.5 Hz, H-
8.5 Hz, H-100,10000),
00 00
1), 4.563 (d, 2 H, J1 ,2 /1
0000,20000
ꢀ
0
4.509 and 4.464 (2 d, 1,2 H, J1 ,2 /1 ,2 /1
0
000 000
00000,200000
7.7
and Ac2O (100 ꢂL) was added at 0 C, and the

172
K.M. Halkes et al./Carbohydrate Research 309 (1998) 161±174
and 8.1 Hz, H-10,1000,100000), 4.087 (dd, 1 H, J2,3
pyranoside (4).ÐTo a solution of 23 (49 mg,
21 ꢂmol) in 1-BuOH (5 mL) was added ethylene-
diamine (1 mL). The mixture was stirred overnight
00 00
0000,30000
10.3 Hz, H-2), 3.842 (dd, 2 H, J2 ,3 /2
10.2 Hz,
H-200,20000), 3.501, 3.380, and 3.375 (3 dd, each 1 H,
9.4, 9.4, and 9.5 Hz, H-20,2000,200000),
at 90 C under Ar, when TLC (4:2:2:0.5 1-BuOH±
ꢀ
0
J2 ,3 /2 ,3 /2
0
000 000
00000,300000
3.806 (s, 3 H, C6H4OCH3), 2.030, 2.020, and 2.018
(3 s, each 3 H, 3 NHCOCH3). FABMS (negative-
ion mode; C49H71N3O35): m/z 1282 [M+Na H] ,
1260 [M H] .
EtOH±H2O±HOAc) showed the disappearance of
23. The mixture was concentrated and co-con-
centrated with toluene (5Â10 mL). The residue was
dissolved in dry pyridine (5 mL), and Ac2O (5 mL)
and a catalytic amount of 4-dimethylaminopyr-
idine were added. The mixture was stirred over-
night at room temperature, then concentrated and
co-concentrated with toluene and EtOH
(2Â20 mL). The yellow, amorphous solid was dis-
4-Methoxyphenyl (3,4,6-tri-O-acetyl-2-deoxy-2-
phthalimido-b-d-glucopyranosyl)-(1!4)-(sodium
2,3-di-O-p-toluoyl-b-d-glucopyranosyl 6-sulfate)-(1
!3)-(4,6-di-O-acetyl-2-deoxy-2-phthalimido-b-d-
glucopyranosyl)-(1!4)-(sodium 2,3-di-O-p-toluoyl-
b-d-glucopyranosyl 6-sulfate)-(1!3)-4,6-di-O-
acetyl-2-deoxy-2-phthalimido-b-d-glucopyranoside
(23).ÐTo a solution of 17 (50 mg, 24 ꢂmol) in dry
DMF (4 mL) was added sulfur trioxide-trimethyl-
amine complex (50 mg, 360 ꢂmol), and the mixture
ꢀ
solved in THF (5 mL), and at 0 C under vigorous
stirring, aq 2 M NaꢀOH (1 mL) was added. After
stirring for 6 h at 0 C, TLC analysis (4:2:2:0.5 1-
BuOH±EtOH±H2O±HOAc) showed the conver-
sion into a new spot with Rf 0.32, then aq 1 M HCl
was added to neutralise the solution. The mixture
was concentrated and co-concentrated with 1:1
toluene±MeOH (3Â10 mL), and a solution of the
residue in MeOH (10 mL) was stirred with Dowex-
50 (Na+) for 1 h, then ®ltered and concentrated.
Gel ®ltration on Sephadex G-10 (water) of the
residue yielded 4, isolated after lyophilisation as a
slightly yellow, amorphous powder (17 mg, 68%);
ꢀ
was stirred overnight at 50 C under Ar. Then,
TLC (85:15 CH2Cl2±MeOH) showed the dis-
appearance of 17 and the formation of a new spot
(Rf 0.57). After the addition of MeOH (1 mL),
stirring was continued for 15 min. The mixture was
concentrated and a solution of the residue in
MeOH (10 mL) was stirred with Dowex-50 (Na+)
for 1 h, then the mixture was ®ltered and con-
centrated. Column chromatography (9:1 CH2Cl2-
MeOH) of the residue a€orded 23 as a white,
amorphous powder (49 mg, 88%); [ꢃ]d 5ꢀ (c 1);
ꢀ
1
[ꢃ]d 32 (c 1, H2O); H NMR (D2O): ꢁ 7.06±6.97
(m, 4 H, C6H4OCH3), 5.073 (d, 1 H, J1,2 8.4 Hz, H-
00 00
0000,20000
1), 4.634 and 4.601 (2 d, each 1 H, J1 ,2 /1
9.0
NMR (CDCl3): H, ꢁ 7.666, 7.640, 7.306, 7.301,
7.106, 7.085, 7.036, and 7.019 (8 d, each 2 H, 4
and 8.4 Hz, H-100,10000), 4.537 and 4.515 (2 d, each 1
H, J1 ,2 /1 ,2 7.8 and 8.4 Hz, H-10,1000), 4.269 (dd, 2
1
0 0 000 000
0 0 000 000 0 0 000 000
H, J5 ,6a /5 ,6a 6.0 Hz, J6a ,6b /6a ,6b 10.8 Hz, H-
COC6H4CH3), 6.67±6.59 (m, 4 H, C6H4OCH3),
9.0 Hz, H-30000), 5.599, 5.421,
0
0
000
000
0000,40000
5.628 (dd, 1 H, J3
and 5.164 (3 d, each 1 H, J1,2/1 ,2 /1
6a0,6a000), 4.112 (dd, 2 H, J5 ,6b /5 ,6b < 1 Hz, H-
6b0,6b000), 4.074 (dd, 1 H, J2,3 10.3 Hz, H-2), 3.810
(s, 3 H, C6H4OCH3), 3.364 and 3.327 (2 dd, each 1
00 00
0000,20000
8.2, 8.5,
and 8.3 Hz, H-1,100,10000), 4.444 and 4.420 (2 d, each
1 H, J1 ,2 /1 ,2 6.8 and 7.3 Hz, H-10,1000), 3.632 (s, 3
H, C6H4OCH3), 2.397, 2.386, and 2.294 (3 s, 3,3,6
H, 4 COC6H4CH3), 2.124, 2.103, 2.035, 1.911,
1.852, 1.884, and 1.716 (7 s, each 3 H, 7 Ac); 13C, ꢁ
170.8, 170.7 (2 C), 170.6, 170.4, 169.9, and 169.2 (7
COCH3), 164.9 (2 C), 164.8, and 164.7 (4
COC6H4CH3), 100.2, 99.4, 96.9, 95.5, and 95.4 (C-
1,10,100,1000,10000), 65.4 and 63.8 (C-60,6000), 61.7 (2 C)
and 61.0 (C-6,600,60000), 55.2, 55.2, 54.7, and 54.2 (C-
2,200,20000 and C6H4OCH3), 20.7, 20.6, 20.5, and 20.4
(4 COC6H4CH3), 20.1, 19.9, 19.7, 19.6 (2 C), 19.5,
and 19.3 (7 COCH3).
H, J2 ,3 /2 ,3 9.0 and 8.4 Hz, H-20,2000), 2.086, 2.064,
and 2.019 (3 s, each 3 H, 3 NHCOCH3). FABMS
(positive-ion mode; C43H65N3O33S2Na2): m/z 1284
[M+Na]+, 1262 [M+H]+.
0
0
000 000
0
0
000 000
4-Methoxyphenyl (sodium 2,3,4-tri-O-p-toluoyl-
b-d-glucopyranosyl
6-sulfate)-(1!3)-(4,6-di-O-
acetyl-2-deoxy-2-phthalimido-b-d-glucopyranosyl)-
(1!4)-(sodium 2,3-di-O-p-toluoyl-b-d-glucopyrano-
syl 6-sulfate)-(1!3)-(4,6-di-O-acetyl-2-deoxy-2-
phthalimido-b-d-glucopyranosyl)-(1!4)-(sodium
2,3-di-O-p-toluoyl-b-d-glucopyranosyl 6-sulfate)-(1
!3)-4,6-di-O-acetyl-2-deoxy-2-phthalimido-b-d-
glucopyranoside (24).ÐTo a solution of 21 (33 mg,
13 ꢂmol) in dry DMF (5 mL) was added sulfur tri-
oxide±trimethylamine complex (37 mg, 268 ꢂmol),
4-Methoxyphenyl
(2-acetamido-2-deoxy-b-d-
glucopyranosyl)-(1!4)-(sodium b-d-glucopyranosyl
6-sulfate)-(1!3)-(2-acetamido-2-deoxy-b-d-gluco-
pyranosyl)-(1!4)-(sodium b-d-glucopyranosyl 6-
sulfate)-(1!3)-2-acetamido-2-deoxy-b-d-gluco-
ꢀ
and the mixture was stirred overnight at 50 C
under Ar. Then, TLC (4:1 CH2Cl2-MeOH) showed

K.M. Halkes et al./Carbohydrate Research 309 (1998) 161±174
173
the disappearance of 21 and the formation of 24
(Rf 0.40). After the addition of MeOH (1 mL),
stirring was continued for 15 min. The mixture was
concentrated and a solution of the residue in
MeOH (10 mL) was stirred with Dowex-50 (Na+)
for 1 h, then the mixture was ®ltered, and con-
centrated. Column chromatography (85:15
CH2Cl2±MeOH) of the residue a€orded 24 as a
white, amorphous powder (28 mg, 78%); [ꢃ]d +64ꢀ
EtOH±H2O±HOAc) showed the conversion into a
new spot with Rf 0.55. The solution was neutralised
with aq 1 M HCl, concentrated and co-con-
centrated with 1:1 toluene±MeOH (3Â10 mL), and
a solution of the residue in MeOH (10 mL) was
stirred with Dowex-50 (Na+) for 1 h, then ®ltered
and concentrated. Gel ®ltration on Sephadex G-10
(water) of the residue yielded 5, isolated after lyo-
philisation as a white, amorphous powder (11 mg,
88%); [ꢃ]d 112ꢀ (c 0.5, H2O); H NMR (D2O): ꢁ
1
1
(c 0.5); NMR (CDCl3): H, ꢁ 7.579, 7.531, 7.493,
7.409, 7.311, 7.209, 7.202, 6.993, 6.948, 6.928,
6.889, 6.881, 6.873, and 6.853 (14 d, each 2 H, 7
COC6H4CH3), 6.508 (bs, 4 H, C6H4OCH3), 5.422
7.06±6.96 (m, 4 H, C6H4OCH3), 5.066 (d, 1 H, J1,2
00 00
8.8 Hz, H-1), 4.638 and 4.632 (2 d, each 1 H, J1 ,2 /
8.4 and 8.5 Hz, H-100,10000), 4.532, 4.516, and
10000,20000
(t, 1 H, J3
5.008, and 4.980 (3 d, each 1 H, J1,2/1 ,2 /1
=J4
=9.5 Hz, H-400000), 5.214,
00000,400000
00000,500000
00000,200000
8.1, 8.1, and
0
4.505 (3 d, each 1 H, J1 ,2 /1 ,2 /1
0
000 000
8.5,
7.7 Hz, H-10,1000,100000), 4.330 and 4.273 (2 dd, 1,2 H,
00 00
0000,20000
8.2, and 7.9 Hz, H-1,100,10000), 4.842, 4.751, and
0
0
J5 ,6a /5 ,6a /5
000
000
0 0 000 000
00000,6a00000
2.2 and 2.9 Hz, J6a ,6b /6a ,6b /
7.8, 7.5, and
11.4 and 12.1 Hz, H-6a0,6a000,6a00000), 4.208
0
0
000 000
4.678 (3 dd, each 1 H, J1 ,2 /1 ,2 /1
00000,200000
6a00000,6b00000
0
0
000 000
0
0
000
000
00000,300000
00000,6b00000
5.2 and
7.5 Hz, J2 ,3 /2 ,3 /2
9.5, 9.5, and 9.1 Hz, H-
and 4.109 (2 dd, 1,2 H, J5 ,6b /5 ,6b /5
20,2000,200000), 4.696, 4.183, and 4.105 (3 d, each 1 H,
H-10,1000,100000), 3.543 (s, 3 H, C6H4OCH3), 2.288,
2.262, 2.253, 2.198, 2.186, 2.159, and 2.116 (7 s,
each 3 H, 7 COC6H4CH3), 2.026, 1.985, 1.947,
1.895, 1.796, and 1.756 (6 s, each 3 H, 6 Ac); 13C, ꢁ
171.2 (2 C), 170.9 (2 C), 170.7, and 170.4 (6
COCH3), 165.5, 165.4, 165.0, 164.9 (2 C), and
164.8 (2 C) (7 COC6H4CH3), 100.5, 99.7 (2 C),
97.2, 95.7, and 95.4 (C-1,10,100,1000,10000,100000), 66.5 (2
C), 64.6, and 62.0 (3 C) (C-6,60,600,6000,60000,600000), 55.3
(C-2,200,20000 and C6H4OCH3), 21.2, 21.1, and 21.0
(COC6H4CH3), 20.4, 20.2, and 20.1 (COCH3).
4-Methoxyphenyl (sodium b-d-glucopyranosyl 6-
sulfate)-(1!3)-(2-acetamido-2-deoxy-b-d-gluco-
pyranosyl)-(1!4)-(sodium b-d-glucopyranosyl 6-
sulfate)-(1!3)-(2-acetamido-2-deoxy-b-d-gluco-
pyranosyl)-(1!4)-(sodium b-d-glucopyranosyl 6-
sulfate)-(1!3)-2-acetamido-2-deoxy-b-d-gluco-
pyranoside (5).ÐTo a solution of 24 (25 mg,
8.9 ꢂmol) in 1-BuOH (5 mL) was added ethylene-
diamine (1 mL). The mixture was stirred overnight
4.1 Hz, H-6b0,6b000,6b00000), 4.083 (dd, 1 H, J2,3
10.3 Hz, H-2), 3.809 (s, 3 H, C6H4OCH3), 3.384,
0
0
000 000
00000,300000
3.330, and 3.304 (3 dd, each 1 H, J2 ,3 /2 ,3 /2
9.1, 9.4, and 8.9 Hz, H-20,2000,200000), 2.074, 2.067, and
2.021 (3 s, each 3 H, 3 NHCOCH3). FABMS
(positive-ion mode; C49H74N3O41S3Na3): m/z 1526
[M+H]+.
Acknowledgements
The authors wish to thank Mrs. A.C.H.T.M.
van der Kerk-van Hoof for recording FAB mass
spectra.
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