Synthesis of various sulfoforms of the trisaccharide β-D-GlcpA-(1→3)-β-D-Galp-(1→3)-β-D-Galp-(1→OMP) as probes for the study of the biosynthesis and sorting of proteoglycans

Article abstract of DOI:10.1039/b314244b

A straightforward preparation of various sulfoforms of the trisaccharide 4-methoxyphenyl O-(sodium β-D-glucopyranosyluronate)-(1 → 3)-(β-D-galactopyranosyl)-(1 → 3)-β-D-galactopyranoside (1), namely its 6a- and 4a-monosulfate, 6b- and 4b-monosulfate and 6a,6b-disulfate derivatives, is reported/or the first time. These compounds, which are partial structures of the linkage region of proteoglycans, will serve as probes for the study of the biosynthesis and sorting of these macromolecules. A key trisaccharide derivative, in which the two similar D-Gal units were differentiated at C-4,6 with 4,6-benzylidene and 4,6-di-tert-butylsilylene acetals, respectively, was used as a common intermediate. Both acetal groups showed excellent orthogonality, and allowed the preparation of all target compounds in high yield. Noteworthy is the possibility to prepare the 6a- and 6b-monosulfated and the 6a,6b-disulfated species through a one-pot regioselective procedure starting from a tetrol precursor.

Full text of DOI:10.1039/b314244b

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Synthesis of various sulfoforms of the trisaccharide
ꢀ-D-GlcpA-(1 3)-ꢀ-D-Galp-(1 3)-ꢀ-D-Galp-(1 OMP) as probes
for the study of the biosynthesis and sorting of proteoglycans
Bertrand Thollas and Jean-Claude Jacquinet
Institut de Chimie Organique et Analytique – UMR CNRS 6005, UFR Faculté des Sciences,
Université d’Orléans, B.P. 6759, 45067 Orleans Cedex, France.
E-mail: Jean-Claude.Jacquinet@univ-orleans.fr
Received 7th November 2003, Accepted 4th December 2003
First published as an Advance Article on the web 14th January 2004
A straightforward preparation of various sulfoforms of the trisaccharide 4-methoxyphenyl O-(sodium β--gluco-
pyranosyluronate)-(1 3)-(β--galactopyranosyl)-(1 3)-β--galactopyranoside (1), namely its 6a- and 4a-mono-
sulfate, 6b- and 4b-monosulfate and 6a,6b-disulfate derivatives, is reported for the first time. These compounds,
which are partial structures of the linkage region of proteoglycans, will serve as probes for the study of the
biosynthesis and sorting of these macromolecules. A key trisaccharide derivative, in which the two similar -Gal
units were differentiated at C-4,6 with 4,6-benzylidene and 4,6-di-tert-butylsilylene acetals, respectively, was used as a
common intermediate. Both acetal groups showed excellent orthogonality, and allowed the preparation of all target
compounds in high yield. Noteworthy is the possibility to prepare the 6a- and 6b-monosulfated and the 6a,6b-
disulfated species through a one-pot regioselective procedure starting from a tetrol precursor.
C-6 of the -Gal units4–6 as well as by phosphorylation at C-2
Introduction
of the -Xyl unit.7,8 Although the biological significance of
Proteoglycans (PGs) are macromolecules composed of glycos-
these modifications is still not yet deciphered, it has been
aminoglycan chains (GAGs) covalently bound to a protein
suggested2 that they could act as biosynthetic signals. Recently,
core.1 They are ubiquitously distributed on the cell surface and
it has been demonstrated9 that phosphorylation at C-2 of the
in the extracellular matrix. GAGs are increasingly implicated as
regulators of many biological processes such as cell growth,
-Xyl unit should be a transient phenomenon, involved only in
the very early steps of the biosynthesis of GAGs. But what is
adhesion and recognition, blood-coagulation, viral and bac-
the exact function of the sulfate groups? Investigations of the
terial infections, and cytokine action, owing to their capacity to
possible structural variations in this linkage region may help to
interact with protein ligands through specific oligosaccharide
clarify the biological function of these unique modifying
groups.
To address this issue, several syntheses of glycopeptides of
sequences.2 GAG assembly starts with the attachment of
-xylose to one or more -serine residues within the protein
core. The sequential addition of two -galactose and one
-glucuronic acid units give rise to a tetrasaccharide inter-
various lengths of the linkage region, containing a sulfate group
on one or the other -Gal unit, have been reported,10–12 but no
mediate that lies at a bifurcation in the biosynthetic pathway
systematic preparation of all possible sulfoforms has been des-
(Fig. 1). Attachment of an α--GlcNAc residue to this linkage
region initiates the formation of glucosaminoglycans (heparin,
heparan sulfate) whereas those of a β--GalNAc unit lead to
the assembly of galactosaminoglycans (chondroitins sulfate,
dermatan sulfate). GAG chains mostly consist of hexosamine
and hexuronic acid arranged in alternating sequences, and these
repeating units contain various sulfate substituents which create
a great degree of structural and functional diversity. The fact
cribed. Within the frame of a programme devoted to the study
of the biosynthesis of chondroitins sulfate in relation with
osteoarthritis, we now report on for the first time a straight-
forward preparation of various sulfoforms of the trisaccharide
4-methoxyphenyl O-(sodium β--glucopyranosyluronate)-
(1 3)-(β--galactopyranosyl)-(1 3)-β--galactopyranoside
(1), namely its 6a- and 4a-monosulfated, 6b- and 4b-mono-
sulfated, and 6a,6b-disulfated derivatives (Fig. 2), in which the
that the linkage region should be common to all GAG species
phenyl group will be useful for the detection of the transfer
contrasts sharply with the structural heterogeneity of the
products and the methoxy group will serve as a marker for
repeating disaccharide region. Hence, the question arises how
NMR studies.
these different GAGs can be synthesized on this common
structure since chain elongation proceeds in a stepwise fashion
and is governed by the high substrate specificity of the glyco-
Results and discussion
syltransferases involved.3
Interestingly, it has been reported that this common linkage
region may be occasionally modified by sulfation at C-4 and/or
For the synthesis of the target sulfoforms 2–6, a common key
trisaccharide intermediate (16) was designed, in which the two
Fig. 1 The linkage region of proteoglycans. The arrows indicate possible substitutions with sulfate and phosphate groups.
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 4 3 4 – 4 4 2
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 4
434

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(J1,2 3.5 Hz). Condensation of the imidate 9 (1.4 mol equiv.)
with the alcohol 7 (1 mol equiv.), in dichloromethane at room
temperature, and in the presence of trimethylsilyl triflate
(TMSOTf, 15% based on 9) afforded the disaccharide 10 in 55%
yield, this moderate yield being essentially due to difficulties
encountered in chromatographic separation of the mixture. The
anomeric configuration of the newly established interglycosidic
1
linkage was deduced from its H NMR spectrum (δ 5.01, J1,2
8.0 Hz, H-1b). Treatment of 10 with hydrazine acetate16 in
pyridine gave the crystalline triol 11 in 90% yield.
Transformation of 11 into the key trisaccharide derivative 16
was next achieved as follows (Scheme 2). First of all, the triol 11
was treated with benzaldehyde and TFA (5%, v/v) to give the
crystalline bis-benzylidene acetal 12, a symmetrically protected
derivative which could serve as a precursor of tetrol 19, in 88%
yield. Treatment of 11 with di-tert-butylsilyl ditriflate 17,18 and
2,6-lutidine in dichloromethane gave smoothly the crystalline
silylene acetal 13 in 86% yield, the position of which was
deduced from its 1H NMR spectrum (δ 2.60, d, J 10.8 Hz, HO-
3b). The convenience of the 4,6-di-tert-butylsilylene acetal
(DTBS) in glycoconjugate synthesis was recently highlighted.19
Coupling reactions that involved the alcohols 12 and 13 and the
donor methyl 2,3,4-tri-O-benzoyl-1-O-trichloroacetimidoyl-α-
-glucopyranuronate 1420 were achieved as described for the
preparation of 10 to afford the crystalline trisaccharides 15 and
16 in 25 and 70% yields, respectively, the structures of which
were easily deduced from their 1H NMR spectra (δ 4.98,
J1,2 7.0 Hz, and δ 4.96, J1,2 7.0 Hz, H-1c, respectively). The low
yield obtained in the coupling reaction with 12 was rather
unexpected, and changes in the reaction conditions (solvent,
catalyst, temperature, details not presented in the Experimental
section) did not significantly improve the yield, but this result
was in agreement with those reported for glucuronylation reac-
tion of 4,6-O-benzylidene--galacto acceptors.21 However, the
acceptor 13, in which the bulky silylene acetal was expected to
cause steric hindrance at O-3b, gave a satisfactory result prob-
ably due to subtle electronic factors, possibly originating from
the strong electron-donating effect of the tert-butyl groups
which should increase the nucleophilic character of the oxygen
atom at C-3b.
The protective group pattern in 16 allowed access to the tar-
get sulfoforms depending on the partial deprotection sequence
(Scheme 3). Attempted deprotection of the DTBS group in 16
under standard conditions (tetra-n-butylammonium fluoride–
acetic acid in oxolane) failed, but treatment of 16 with triethyl-
amine–trihydrofluoride complex22 (Et3Nؒ3HF) in tetrahydro-
furan at 0 ЊC gave smoothly the crystalline diol 17 in nearly
quantitative yield. Careful treatment of 16 with 80% TFA in
dichloromethane at 0 ЊC allowed isolation of the crystalline diol
18 in 80% yield. Acid hydrolysis of 17 with 90% TFA gave
the crystalline tetrol 19 in 85% yield, whereas conventional
benzoylation (benzoyl chloride in pyridine) of 17 followed by
hydrolysis as described above afforded the crystalline diol 20 in
83% overall yield. Benzoylation of 18 followed by desilylation,
as described above, provided the diol 21 in 81% overall yield.
The 1H NMR spectra for 19–21 are in complete agreement with
the expected structures and showed high purity for these
crystalline intermediates.
Fig. 2 The trisaccharide derivative 1 and its sulfoforms 2–6.
-Gal units were differentiated at C-4 and C-6 by the use of 4,6-
benzylidene and 4,6-di-tert-butylsilylene acetals, respectively.
The latter may be prepared by stereoselective assembly of a
glucuronyl donor (14) and a digalactosyl acceptor (13) easily
available from a digalactosyl precursor (11), which may, in turn,
be conveniently prepared from a single starting material (7).
The stereoselective coupling of each unit, ensured by the pres-
ence of a stereocontrolling auxiliary (benzoyl group) at C-2,
was designed to be performed using the trichloroacetimidate
glycosylation procedure.13
Preparation of the intermediate disaccharide derivative 11
was achieved as follows (Scheme 1). The common starting
material was the known14 4-methoxyphenyl 2-O-benzoyl-4,6-
O-benzylidene-β--galactopyranoside 7, easily obtained from
-galactose in multigram quantities. Acid hydrolysis of 7 with
90% trifluoroacetic acid (TFA) followed by treatment of
the resulting triol with 4-oxopentanoic acid (levulinic acid),
1,3-dicyclohexylcarbodiimide and 4-dimethylaminopyridine
(DMAP) in dichloromethane afforded the crystalline tri-
levulinate 8 in 80% overall yield. Introduction of the tri-
chloroacetimidoyl group at C-1 was then achieved through
oxidative removal of the anomeric 4-methoxyphenyl group with
cerium() ammonium nitrate (CAN),15 followed by imidoyl-
ation of the intermediate free hemiacetal with trichloro-
acetonitrile and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) to
give the α-imidate 9 in 70% overall yield, the anomeric con-
1
figuration of which was deduced from its H NMR spectrum
Transformation of 19–21 into the target molecules 1–6 was
next achieved as follows (Scheme 4). To avoid an elimination
reaction under basic conditions on the sensitive methyl uronate
moiety,20 saponification of the ester groups in 19 was performed
first by treatment with lithium hydroperoxide16 in THF, fol-
lowed by methanolic sodium hydroxide to afford the target tri-
saccharide derivative 1, as its sodium salt, in 92% yield. Tetrol
19 and diols 20 and 21 were regioselectively O-sulfonated at C-6
by treatment with the sulfur trioxide–trimethylamine complex
(Me3NؒSO3, 2 mol equiv. per primary hydroxy group) in DMF
at 40 ЊC, followed by ion-exchange chromatography (Naϩ resin)
to afford the sodium salts 22, 23 and 26, respectively, in 83, 86
Scheme 1 Reagents and conditions: a) 90% TFA, 15 min; then LevOH,
DCC, DMAP, CH2Cl2, 2 h, 80%; b) CAN, toluene–MeCN–water,
15 min; then CCl3CN, DBU, CH2Cl2, 30 min, 70%; c) TMSOTf, mol.
sieves 4 Å, CH2Cl2, 30 min, 55%; d) hydrazine acetate, pyridine, 8 min,
90%. MP = 4-methoxyphenyl; Lev = 4-oxopentanoyl (levulinoyl).
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 4 3 4 – 4 4 2
435

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Scheme 2 Reagents and conditions: a) PhCHO, TFA, 30 min, 88%; b) DTBS ditriflate, 2,6-lutidine, CH2Cl2, 2 h, 86%; c) TMSOTf, mol. sieves 4 Å,
CH2Cl2, 30 min, 15 : 25%; 16 : 70%.
Scheme 3 Reagents and conditions: a) Et3Nؒ3HF, THF, 0 ЊC, 4 h, 95%; b) 80% TFA, CH2Cl2, 0 ЊC, 15 min, 80%; c) 90% TFA, 15 min, 85%;
d) PhCOCl, pyridine, 0 ЊC, 4 h, quantitative.
and 87% yields, respectively. Very small amounts (2–3%) of the
corresponding 4,6-disulfate derivatives were also obtained, but
easily removed by simple silica chromatography. It is worth not-
ing that tin-mediated regioselective sulfation23–25 proceeded in
lower yields, mainly due to the difficulty in removing completely
the tin salts. Comparison of the 1H NMR spectra of 22, 23 and
26 with those of their non-sulfated precursors, all recorded in
3 : 1 CD3OD–CDCl3 (a mixture which provided good solubility
and resolution for all compounds), showed the expected26,27
downfield shifts (∆δ ∼ 0.4 ppm) of the signals for Gal H-6 in
sulfates 22, 23 and 26, and no change of the signals for Gal H-4,
demonstrating that sulfation occurred exclusively at C-6. For
the preparation of the 4-sulfated derivatives, the low reactivity
of the axial 4-hydroxy groups in -galacto structures requires
first temporary protection at O-6.28 Thus, treatment of diols 20
and 21 with benzoyl cyanide in pyridine gave the alcohols 24
and 27 in 91 and 90% yields, respectively. These later were sul-
fated by treatment with a large excess (10 mol equiv.) of Me3Nؒ
SO3 in DMF at 60 ЊC for 4 days, followed by ion-exchange, to
give the sodium salts 25 and 28 in 88 and 89% yields, respect-
those of their precursors, recorded as described above, also
showed the expected26 significant downfield shifts (∆δ
∼ 0.9 ppm) of the signals for Gal H-4 in sulfates 25 and 28.
Saponification of the esters 22, 23, 25, 26 and 28, as described
for the preparation of 1, afforded the target sulfoforms 6 and
2–5, respectively, in ∼90% yields. The 1H NMR data of the five
sulfoforms were compared with those of their non sulfated
congener 1 (Table 1). Particularly relevant were the expected26
downfield shifts (∆δ ∼ 0.4 ppm) of the signals for Gal H-6 in the
6-sulfated species, and those (∆δ ∼ 0.6 ppm) of the signals for
Gal H-4 in the 4-sulfated derivatives. Comparison of the 13C
NMR data (Table 2) also showed the expected26 downfield
shifts (∆δ ∼ 7 ppm) of the signals for Gal C-6 in the 6-sulfated
derivatives and those (∆δ ∼ 9 ppm) of the signals for Gal C-4 in
the 4-sulfated species. These NMR data are in complete agree-
ment with the expected structures, and in accord with those
reported for synthetic derivatives containing -Gal units bear-
ing sulfate groups at C-4 or C-610–12 as well as for fragments
isolated from natural proteoglycans.4–6
In a continuous effort to try to reduce the number of
transformations in a multi-step preparation, we attempted the
1
ively. Comparison of the H NMR spectra of 25 and 28 with
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 4 3 4 – 4 4 2
436

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Table 1 1H NMR data (500 MHz, D2O, 25 ЊC, internal acetone,
δH 2.225 ppm) for trisaccharide 1 and its sulfoforms 2–6a
1
2
3
4
5
6
H-1a
H-2a
H-3a
H-4a
H-5a
H-6a
4.70
3.70
3.94
4.27
3.80
3.80
4.72
3.74
3.97
4.34
3.97
4.14
4.24
4.75
3.74
4.16
4.94
3.98
3.80
4.71
3.76
3.94
4.36
3.82
3.82
4.79
3.72
3.96
4.27
3.82
3.82
4.74
3.76
3.97
4.37
3.88
4.24
4.30
H-1b
H-2b
H-3b
H-4b
H-5b
H-6b
5.05
3.72
3.94
4.20
3.80
3.80
5.12
3.74
3.76
4.22
3.78
3.78
5.22
3.74
3.98
4.09
3.86
3.80
5.18
3.76
3.94
4.24
3.95
4.22
4.30
5.15
3.72
4.08
4.82
3.96
3.82
5.11
3.76
3.97
4.28
3.95
4.24
4.30
H-1c
H-2c
H-3c
H-4c
H-5c
4.68
3.43
3.52
3.52
3.75
4.70
3.44
3.54
3.54
3.76
4.70
3.44
3.54
3.54
3.80
4.70
3.44
3.53
3.53
3.80
4.74
3.44
3.54
3.54
3.80
4.71
3.45
3.54
3.54
3.82
a Bold-type values reflect the positions of sulfation.
Table 2 13C NMR data (62.8 MHz, D2O, 25 ЊC, internal acetone,
δC 30.35 ppm) for trisaccharide 1 and its sulfoforms 2–6a
1
2
3
4
5
6
C-1a
C-2a
C-3a
C-4a
C-5a
C-6a
101.67
69.94
82.26
68.13
75.01
60.90
101.56
69.84
82.06
68.24
75.82
67.30
101.74
70.37
78.40
77.51
74.67
60.95
101.57
69.78
82.48
68.13
75.42
61.24
101.65
69.98
82.39
68.45
75.21
60.91
101.49
69.77
82.28
68.02
72.64
67.80
C-1b
C-2b
C-3b
C-4b
C-5b
C-6b
104.20
70.38
82.61
68.51
75.18
61.17
104.25
70.37
82.84
68.32
74.99
61.12
104.17
70.44
82.54
68.60
75.13
61.29
104.04
70.23
82.75
68.57
72.75
68.03
104.35
71.24
78.42
76.87
74.69
61.15
104.01
70.14
82.42
68.59
73.09
68.02
C-1c
C-2c
C-3c
C-4c
C-5c
C-6c
103.79
73.33
75.49
71.92
76.21
175.79
103.80
73.35
75.51
71.97
76.37
176.11
103.89
73.24
75.20
71.98
76.41
176.12
103.84
73.35
75.54
71.97
76.39
176.14
103.89
73.31
75.37
72.06
76.50
176.07
103.81
73.34
75.50
71.96
76.38
176.09
Scheme 4 Reagents and conditions: a) LiOH–H2O2, THF; then NaOH,
MeOH, 1: 92%, 2–6: 90%; b) Me3NؒSO3, DMF, 40 ЊC, 90 min; then ion-
exchange (Naϩ resin), 22: 83%, 23: 86%; 26: 87%; c) PhCOCN, pyridine,
16 h, 24: 91%, 27: 90%; d) Me3NؒSO3 (10 mol equiv.), DMF, 60 ЊC, 4 d,
25: 88%, 28: 89%.
a Bold-type values reflect the positions of sulfation.
regioselective monosulfation of tetrol 19 (Scheme 5). Thus,
carefully controlled treatment of 19 (1 mol equiv.) with Me3Nؒ
SO3 (1.5 mol equiv. per primary hydroxy group) in DMF at
40 ЊC for 40 min (optimal reaction time for the major formation
of the monosubstituted species determined by TLC control),
followed by silica chromatography and ion-exchange, afforded,
beside traces of the starting tetrol, the 6a-sulfate 29, the 6b-
sulfate 30 and the 6a,6b-disulfate 22, respectively, in 27, 20 and
38% yields, respectively. Since mass spectrometry showed that
29 and 30 are monosulfated derivatives, the problem was to
determine on which of the two similar -Gal units was located
the sulfate group. Their 1H NMR spectra were recorded under
the conditions reported above, and the use of spin decoupling
and 2D experiments allowed unambiguous assignment of the
Gal-6 protons. The expected downfield shifts (∆δ ∼ 0.4 ppm) of
the signals for Gal H-6 in the sulfated moiety were observed. In
addition, saponification of 29 and 30, as described, afforded the
sulfoforms 2 and 4, respectively, with physical data identical to
those of the independently prepared derivatives.
Scheme 5 Regioselective O-sulfonation of tetrol 19. Reagents and
conditions: a) Me3NؒSO3, DMF, 40 ЊC, 40 min, 29: 27%, 30: 20%, 22:
38%.
the trisaccharide 1. The use of common precursors and the
possible regioselective preparation of the 6-sulfated species
render this route attractive for the further preparation of more
In conclusion, we have reported a stereocontrolled and high-
yielding approach for the preparation of a set of sulfoforms of
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 4 3 4 – 4 4 2
437

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complex structures of the linkage region of proteoglycans.
These molecules will be useful to assess the role of the various
sulfate groups in the biosynthesis of GAGs. These com-
pounds are currently being evaluated in biological assays, and
the results of these studies will be reported elsewhere in due
course.
afforded the α-imidate 9 (1.31 g, 70% from 8) as a colourless
glass; [α]D ϩ96 (c 1 in CHCl3); (Found: C, 49.7; H, 4.8; N, 1.8.
C30H34Cl3NO13 requires C, 49.8; H, 4.7; N, 1.9%); δH(250 MHz,
CDCl3) 2.05, 2.15, 2.18 (9 H, 3 s, 3 × COCH3), 2.62 (12 H,
m, 6 × CH2CO), 4.15 (2 H, m, 2 × H-6), 4.50 (1 H, m, H-5), 5.62
(3 H, m, H-2, H-3, H-4), 6.72 (1 H, d, J1,2 3.5, H-1), 7.20–8.0
(5 H, m, Ph) and 8.60 (1 H, s, C᎐NH ); m/z 795 [M ϩ Na]ϩ, 611
᎐
[M Ϫ CCl3CONH]ϩ for 35Cl.
Experimental
4-Methoxyphenyl O-(2-O-benzoyl-3,4,6-tri-O-levulinoyl-ꢀ-D-
galactopyranosyl)-(1 3)-2-O-benzoyl-4,6-O-benzylidene-ꢀ-D-
galactopyranoside 10
General procedures
Melting points were determined with a Büchi apparatus and
are uncorrected. Optical rotations were measured at room
temperature (22 ЊC) in a 1 dm cell with a Perkin-Elmer
241 polarimeter and [α]D values are given in units of 10Ϫ1
deg cm2 gϪ1. NMR spectra were recorded at 25 ЊC on Bruker
DPX 250 Advance and Varian Unity 500 spectrometers
with Me4Si as internal reference, unless otherwise stated,
and J values are quoted in Hz. Assignments were based on
homo- and heteronuclear correlations using the manufacturers
software. In the description of the NMR spectra, a, b, and c
refer to the monosaccharide units (a: reducing end) in the
oligosaccharides. Low-resolution mass spectra were recorded
with a Perkin-Elmer Sciex API 3000 spectrometer in the
ion-spray (IS) mode. TLC was performed on Merck 60 F254
precoated plates, and compounds were detected by spraying
the plates with 5% H2SO4 in EtOH, and heating. Flash silica
chromatography was performed using Merck silica gel C60
(0.040–0.063 mm). Elemental analyses were carried out at
the Service Central de Microanalyse du CNRS (Vernaison,
France).
A mixture of imidate 9 (4.92 g, 6.8 mmol), alcohol 7 (2.35 g,
4.9 mmol) and 4 Å powdered molecular sieves (3.0 g) in
dry dichloromethane (60 cm3) was stirred for 1 h at rt under
dry argon. A solution of Me3SiOTf in dry toluene (1 mol
dmϪ3, 1 cm3) was added, and the mixture was stirred for
30 min, then was quenched with Et3N (0.4 cm3), filtered and
concentrated. Flash silica chromatography (5 : 2 EtOAc–
toluene, containing 0.1% of Et3N) gave the disaccharide 10
(2.80 g, 55%) as a white foam; [α]D ϩ38 (c 1 in CHCl3);
(Found: C, 63.4; H, 5.7. C55H58O20 requires C, 63.6; H, 5.6%);
δH(250 MHz, CDCl3) 2.10, 2.15, 2.20 (9 H, 3 s, 3 × COCH3),
2.55 (12 H, m, 6 × CH2CO), 3.63 (1 H, m, H-5a), 3.69
(3 H, s, OCH3), 3.95 (1 H, m, H-5b), 4.10 (2 H, m, 2 × H-6a),
4.22 (1 H, dd, J2,3 10.0, J3,4 3.6, H-3a), 4.30 (2 H, m, 2 × H-6b),
4.48 (1 H, dd, J4,5 0.8, H-4a), 4.91 (1 H, d, J1,2 8.0, H-1a),
5.0 (1 H, d, J1,2 8.0, H-1b), 5.02 (1 H, dd, J2,3 10.4, J3,4 3.6,
H-3b), 5.40 (2 H, m, H-2b, H-4b), 5.60 (1 H, s, PhCH ), 5.76
(1 H, dd, H-2a) and 6.70–8.0 (19 H, m, ArH); m/z 1062
[M ϩ Na]ϩ.
4-Methoxyphenyl 2-O-benzoyl-3,4,6-tri-O-levulinoyl-ꢀ-D-
galactopyranoside 8
4-Methoxyphenyl O-(2-O-benzoyl-ꢀ-D-galactopyranosyl)-
(1 3)-2-O-benzoyl-4,6-O-benzylidene-ꢀ-D-galactopyranoside
A solution of 4-methoxyphenyl 2-O-benzoyl-4,6-O-benzyl-
idene-β--galactopyranoside 714 (4.78 g, 10 mmol) in 90% aq
CF3COOH (40 cm3) was stirred for 15 min at rt, then was
concentrated, evaporated with water (3 × 20 cm3) and dried
in vacuo. A mixture of the solid residue, levulinic acid (4.20 g,
36 mmol) and DMAP (0.36 g, 3 mmol) in dry dichloromethane
(80 cm3) was treated portionwise with DCC (7.50 g, 36 mmol),
and the mixture was stirred for 2 h. The solids were filtered off,
washed with dichloromethane (100 cm3), and the filtrate was
washed with cold 0.1 M hydrochloric acid, saturated aq
NaHCO3 and water, dried (MgSO4) and concentrated. Crystal-
lization of the residue from EtOAc–petroleum ether gave the
tri-levulinate 8 (5.48 g, 80% from 7); mp 94–95 ЊC; [α]D ϩ22
(c 1 in CHCl3); (Found: C, 61.3; H, 5.9. C35H40O14 requires C,
61.4; H, 5.9%); δH(250 MHz, CDCl3) 2.04, 2.16, 2.20 ( 9 H, 3 s,
3 × COCH3), 2.60 (12 H, m, 6 × CH2CO), 3.74 (3 H, s, OCH3),
4.08 (1 H, m, H-5), 4.22 (2 H, m, 2 × H-6), 5.06 (1 H, d, J1,2 8.0,
H-1), 5.23 (1 H, dd, J2,3 10.2, J3,4 3.6, H-3), 5.52 (1 H, dd, J4,5
0.8, H-4), 5.69 (1 H, dd, H-2) and 6.70–8.0 (9 H, m, ArH); m/z
708 [M ϩ Na]ϩ.
11
A freshly prepared mixture of pyridine–acetic acid–hydrazine
hydrate (6 : 4 : 0.5, 42 cm3) was added to a solution of 10
(2.08 g, 2 mmol) in pyridine (10 cm3), and the mixture was
stirred for 8 min at rt, then was diluted with dichloromethane
(250 cm3), washed with water, saturated aq NaHCO3 and water,
dried (MgSO4) and concentrated. Crystallization of the residue
from MeOH gave the triol 11 (1.34 g, 90%); mp 256–258 ЊC;
[α]D ϩ18 (c 1 in CHCl3); (Found: C, 64.4; H, 5.5. C40H40O14
requires C, 64.5; H, 5.4%); δH(250 MHz, CD3OD) 3.60 (2 H, m,
H-5a, H-3b), 3.68 (3 H, s, OCH3), 3.75 (1 H, m, H-5b), 3.80
(2 H, m, 2 × H-6b), 3.84 (1 H, dd, J3,4 3.6, J4,5 0.8, H-4b), 4.18
(2 H, m, 2 × H-6a), 4.22 (1 H, dd, J2,3 10.2, J3,4 3.6, H-3a), 4.54
(1 H, dd, J4,5 0.8, H-4a), 4.78 (1 H, d, J1,2 8.0, H-1a), 5.01 (1 H,
d, J1,2 8.0, H-1b), 5.24 (1 H, dd, J2,3 10.2, H-2b), 5.37 (1 H, s,
PhCH ), 5.64 (1 H, dd, H-2a) and 6.70–8.0 (19 H, m, ArH);
m/z 768 [M ϩ Na]ϩ.
4-Methoxyphenyl O-(2-O-benzoyl-4,6-O-benzylidene-ꢀ-D-
galactopyranosyl)-(1 3)-2-O-benzoyl-4,6-O-benzylidene-ꢀ-D-
galactopyranoside 12
2-O-Benzoyl-3,4,6-tri-O-levulinoyl-1-O-trichloroacetimidoyl-
ꢁ-D-galactopyranose 9
A mixture of triol 11 (372 mg, 0.5 mmol), benzaldehyde (5 cm3)
and CF3COOH (0.2 cm3) was stirred for 30 min at rt, then was
cooled to 0 ЊC. Triethylamine (0.6 cm3) was added, and the mix-
ture was diluted with dichloromethane (50 cm3), washed with
saturated aq NaHCO3 and water, dried (MgSO4) and concen-
trated. Recrystallization of the jelly-like residue from EtOH
afforded the alcohol 12 (369 mg, 88%); mp 310–313 ЊC; [α]D ϩ50
(c 1 in pyridine); (Found: C, 67.7; H, 5.25. C47H44O14 requires C,
67.8; H, 5.3%); δH(250 MHz, CDCl3) 2.55 (1 H, d, J 10.0,
HO-3b), 3.45 (1 H, m, H-5a), 3.55 (1 H, m, H-5b), 3.70 (1 H,
dd, J2,3 10.3, J3,4 3.6, H-3b), 3.72 (3 H, s, OCH3), 4.10 (4 H, m,
2 × H-6, H-3a, H-4b), 4.35 (3 H, m, 2 × H-6, H-4a), 5.0 (1 H, d,
J1,2 8.0, H-1a), 5.02 (1 H, d, J1,2 8.0, H-1b), 5.35 (1 H, dd, H-2b),
A mixture of 8 (1.77 g, 2.6 mmol) and CAN (7.12 g, 13 mmol)
in toluene–acetonitrile–water (1 : 1.5 : 1, 70 cm3) was stirred for
15 min at rt, then was poured into ice-cold water and extracted
with EtOAc (3 × 50 cm3). The combined extracts were washed
with saturated aq NaHCO3 and water, dried (MgSO4) and con-
centrated. Flash silica chromatography (6 : 1 EtOAc–petroleum
ether) gave the corresponding hemiacetal (1.35 g, 90%) as a
yellow foam. Trichloroacetonitrile (2 cm3, 20 mmol) was added
to a solution of the hemiacetal and DBU (0.06 cm3, 0.4 mmol)
in dry dichloromethane (10 cm3), and the mixture was stirred
for 30 min at rt, then was concentrated. Flash silica chromato-
graphy (4 : 1 EtOAc–petroleum ether, containing 0.1% of Et3N)
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 4 3 4 – 4 4 2
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5.43, 5.53 (2 H, 2 s, 2 × PhCH ), 5.81 (1 H, dd, J2,3 10.2, H-2a)
and 6.70–8.10 (24 H, m, ArH); m/z 856 [M ϩ Na]ϩ.
4-Methoxyphenyl O-(methyl 2,3,4-tri-O-benzoyl-ꢀ-D-gluco-
pyranosyluronate)-(1 3)-(2-O-benzoyl-ꢀ-D-galactopyranosyl)-
(1 3)-2-O-benzoyl-4,6-O-benzylidene-ꢀ-D-galactopyranoside
17
4-Methoxyphenyl O-(2-O-benzoyl-4,6-O-di-tert-butylsilylene-ꢀ-
D-galactopyranosyl)-(1 3)-2-O-benzoyl-4,6-O-benzylidene-ꢀ-
D-galactopyranoside 13
To a cooled (0 ЊC) solution of 16 (1.0 g, 0.72 mmol) in dry THF
(15 cm3) was added Et3Nؒ3HF (0.25 cm3, 1.5 mmol), and the
mixture was stirred for 4 h at 0 ЊC, then was diluted with di-
chloromethane (100 cm3) and pyridine (10 cm3, to avoid pre-
cipitation of the diol), washed with saturated aq NaHCO3 and
water, dried (MgSO4) and concentrated. Recrystallization of
the solid residue from MeOH–pyridine gave the diol 17 (856
mg, 95%); mp 320–323 ЊC; [α]D ϩ28 (c 0.5 in pyridine); (Found:
C, 65.3; H, 5.15. C68H62O23 requires C, 65.5; H, 5.0%); δH(250
MHz, (CD3)2SO ϩ D2O) 3.50 (1 H, m, H-5a), 3.65 (3 H, s,
OCH3), 3.70 (3 H, s, COOCH3), 3.90 (1 H, dd, J2,3 10.0, J3,4 3.8,
H-3b), 4.0 (4 H, m, H-3a, H-5b, H-6a, H-6b), 4.15 (1 H, d, J4,5
10.0, H-5c), 4.25 (4 H, m, H-4a, H-4b, H-6a, H-6b), 4.80 (1 H,
d, J1,2 8.0, H-1a), 4.90 (1 H, d, J1,2 8.0, H-1b), 5.0 (1 H, d, J1,2
7.5, H-1c), 5.45 (3 H, m, H-2b, H-2c, H-4c), 5.65 (2 H, m, H-2a,
H-3c) and 6.70–8.10 (24 H, m, ArH); m/z 1270 [M ϩ Na]ϩ.
To a cooled (0 ЊC) solution of triol 11 (745 mg, 1 mmol)
and 2,6-lutidine (0.29 cm3, 2.5 mmol) in dry dichloromethane
(10 cm3) was added di-tert-butylsilyl ditriflate (0.39 cm3,
1.15 mmol), and the mixture was stirred for 1 h at 0 ЊC, then
for 1 h at rt. The mixture was diluted with dichloromethane
(50 cm3), washed with water, cold 0.1 M hydrochloric acid,
saturated aq NaHCO3 and water, dried (MgSO4) and concen-
trated. Flash silica chromatography (5 : 2 toluene–EtOAc) and
crystallization of the residue from EtOAc–petroleum ether gave
the silylene acetal 13 (765 mg, 86%); mp 285–287 ЊC; [α]D ϩ17
(c 1 in CHCl3); (Found: C, 65.1; H, 6.4. C48H56O14Si requires C,
65.1; H, 6.4%); δH(250 MHz, CDCl3) 1.01, 1.05 (18 H, 2 s,
2 × C(CH3)3), 2.60 (1 H, d, J 10.8, HO-3b), 3.55 (3 H, m, H-5a,
H-5b, H-3b), 4.20 (5 H, m, 2 × H-6a, 2 × H-6b, H-3a), 4.40
(2 H, m, H-4a, H-4b), 4.92 (1 H, d, J1,2 8.0, H-1a), 4.96 (1 H, d,
J1,2 8.0, H-1b), 5.34 (1 H, dd, J2,3 10.3, H-2b), 5.43 (1 H, s,
PhCH ), 5.75 (1 H, dd, J2,3 10.4, H-2a) and 6.70–8.0 (19 H, m,
ArH); m/z 908 [M ϩ Na]ϩ.
4-Methoxyphenyl O-(methyl 2,3,4-tri-O-benzoyl-ꢀ-D-gluco-
pyranosyluronate)-(1 3)-(2-O-benzoyl-4,6-O-di-tert-butyl-
silylene-ꢀ-D-galactopyranosyl)-(1 3)-2-O-benzoyl-ꢀ-D-galacto-
pyranoside 18
To a cooled (0 ЊC) solution of 16 (0.5 g, 0.36 mmol) in di-
chloromethane (12 cm3) was added dropwise 80% aq CF3-
COOH (1.2 cm3), and the mixture was stirred for 15 min at
0 ЊC, then was diluted with dichloromethane (50 cm3), washed
with water, saturated aq NaHCO3 and water, dried (MgSO4)
and concentrated. Flash silica chromatography (8 : 1 dichloro-
methane–acetone) and recrystallization of the residue from
EtOH afforded the diol 18 (0.42 g, 80%); mp 265–268 ЊC; [α]D
ϩ33 (c 1 in CHCl3); (Found: C, 63.6; H, 5.8. C69H74O23Si
requires C, 63.8; H, 5.75%); δH(250 MHz, CDCl3) 1.01, 1.10
(18 H, 2 s, 2 × C(CH3)3), 2.28, 2.84 (2 H, 2 br s, 2 × OH ), 3.49
(1 H, m, H-5b), 3.64 (3 H, s, OCH3), 3.68 (3 H, s, COOCH3),
3.85 (2 H, m, 2 × H-6a), 3.92 (1 H, dd, J2,3 10.2, J3,4 3.6, H-3b),
4.01 (1 H, dd, J2,3 10.0, J3,4 3.6, H-3a), 4.16 (1 H, d, J4,5 10.0,
H-5c), 4.25 (2 H, m, 2 × H-6b), 4.69 (1 H, dd, J4,5 0.8, H-4b),
4.75 (1 H, d, J1,2 8.0, H-1a), 4.88 (1 H, d, J1,2 8.0, H-1b), 5.0
(1 H, d, J1,2 7.0, H-1c), 5.43 (1 H, dd, J2,3 9.0, H-2c), 5.50 (2 H,
m, H-2b, H-4c), 5.63 (1 H, dd, H-2a), 5.67 (1 H, t, J3,4 9.0, H-3c)
and 6.70–8.0 (29 H, m, ArH); m/z 1323 [M ϩ Na]ϩ.
4-Methoxyphenyl O-(methyl 2,3,4-tri-O-benzoyl-ꢀ-D-gluco-
pyranosyluronate)-(1 3)-(2-O-benzoyl-4,6-O-benzylidene-ꢀ-D-
galactopyranosyl)-(1 3)-2-O-benzoyl-4,6-O-benzylidene-ꢀ-D-
galactopyranoside 15
A mixture of alcohol 12 (166 mg, 0.2 mmol) and methyl 2,3,4-
tri-O-benzoyl-1-O-trichloroacetimidoyl-α--glucopyranuron-
ate 1420 (200 mg, 0.3 mmol) was treated as described for the
preparation of 10. Flash silica chromatography (15 : 1 di-
chloromethane–EtOAc, containing 0.1% of Et3N) and crystal-
lization of the residue from EtOH afforded the trisaccharide
15 (67 mg, 25%); mp 305–308 ЊC; [α]D ϩ30 (c 1 in CHCl3);
(Found: C, 67.3; H, 5.1. C75H66O23 requires C, 67.5; H,
5.0%); δH(250 MHz, CDCl3) 3.33 (1 H, m, H-5a), 3.46 (1 H,
m, H-5b), 3.71 (3 H, s, OCH3), 3.95 (1 H, dd, J2,3 10.2, J3,4 3.6,
H-3a), 4.01 (2 H, m, 2 × H-6), 4.30 (1 H, d, J4,5 10.0, H-5c), 4.32
(3 H, m, 2 × H-6, H-3b), 4.40 (2 H, m, H-4a, H-4b), 4.95 (1 H,
d, J1,2 8.0, H-1a), 4.96 (1 H, d, J1,2 8.0, H-1b), 4.98 (1 H, d,
J1,2 7.0 Hz, H-1c), 5.29, 5.44 (2 H, 2 s, 2 × PhCH ), 5.45 (1 H,
dd, J2,3 9 0, H-2c), 5.53 (1 H, dd, J2,3 10.3, H-2b), 5.70 (3 H, m,
H-2a, H-3c, H-4c) and 6.70–8.0 (39 H, m, ArH); m/z 1358
[M ϩ Na]ϩ.
4-Methoxyphenyl O-(methyl 2,3,4-tri-O-benzoyl-ꢀ-D-gluco-
pyranosyluronate)-(1 3)-(2-O-benzoyl-ꢀ-D-galactopyranosyl)-
(1 3)-2-O-benzoyl-ꢀ-D-galactopyranoside 19
4-Methoxyphenyl O-(methyl 2,3,4-tri-O-benzoyl-ꢀ-D-gluco-
pyranosyluronate)-(1 3)-(2-O-benzoyl-4,6-O-di-tert-butyl-
silylene-ꢀ-D-galactopyranosyl)-(1 3)-2-O-benzoyl-4,6-O-
benzylidene-ꢀ-D-galactopyranoside 16
A solution of 17 (0.44 g, 0.35 mmol) in 90% aq CF3COOH (10
cm3) was stirred for 15 min at rt, then was concentrated, evap-
orated with water (3 × 10 cm3) and dried in vacuo. Recrystalliz-
ation of the solid residue from MeOH gave the tetrol 19 (345
mg, 85%); mp 205–207 ЊC; [α]D ϩ20 (c 1 in CHCl3); (Found: C,
63.1; H, 5.2. C61H58O23 requires C, 63.2; H, 5.05%); δH(250
MHz, 3 : 1 CD3OD–CDCl3) 3.63 (3 H, s, OCH3), 3.65 (3 H, s,
COOCH3), 3.66 (1 H, m, H-5a), 3.80 (1 H, dd, J2,3 10.3, J3,4 3.6,
H-3a), 3.85 (5 H, m, H-5b, 2 × H-6a, 2 × H-6b), 3.92 (1 H, dd,
J2,3 10.2, J3,4 3.6, H-3b), 4.17 (1 H, dd, J4,5 0.8, H-4b), 4.22 (1 H,
dd, J4,5 0.8, H-4a), 4.32 (1 H, d, J4,5 9.5, H-5c), 4.69 (1 H, d, J1,2
8.0, H-1a), 4.82 (1 H, d, J1,2 8.0, H-1b), 4.96 (1H, d, J1,2 7.5,
H-1c), 5.35 (2 H, m, H-2b, H-2c), 5.46 (1 H, dd, H-2a), 5.48
(1 H, t, J3,4 9.5, H-4c), 5.71 (1 H, t, J2,3 9.5, H-3c) and 6.70–8.0
(29 H, m, ArH); m/z 1160 [M ϩ H]ϩ, 1182 [M ϩ Na]ϩ.
A mixture of alcohol 13 (885 mg, 1 mmol) and imidate 14
(930 mg, 1.4 mmol) was treated as described for the preparation
of 10. Flash silica chromatography (18 : 1 dichloromethane–
EtOAc, containing 0.1% of Et3N) and crystallization of the
residue from EtOH gave the trisaccharide 16 (972 mg, 70%); mp
263–265 ЊC; [α]D ϩ16 (c 1 in CHCl3); (Found: C, 65.7; H, 5.7.
C76H78O23Si requires C, 65.8; H, 5.7%); δH(250 MHz, CDCl3)
1.01, 1.15 (18 H, 2 s, 2 × C(CH3)3), 3.45 (1 H, m, H-5b), 3.50
(1 H, m, H-5a), 3.65 (3 H, s, OCH3), 3.69 (3 H, s, COOCH3),
3.93 (1 H, dd, J2,3 10.2, J3,4 3.6, H-3b), 4.10 (2 H, m, H-6a,
H-6b), 4.17 (1 H, dd, J4,5 10.0, H-5c), 4.21 (1 H, dd, J2,3 10.3,
J3,4 3.6, H-3a), 4.30 (3 H, m, H-4a, H-6a, H-6b), 4.70 (1 H, dd,
J4,5 0.8, H-4b), 4.92 (1 H, d, J1,2 8.0, H-1a), 4.93 (1 H, d, J1,2 8.0,
H-1b), 4.96 (1 H, d, J1,2 7.0, H-1c), 5.34 (1 H, s, PhCH ), 5.45
(2 H, m, H-2c, H-2b), 5.59 (1 H, dd, J3,4 9.0, H-4c), 5.65 (1 H,
dd, H-2a), 5.66 (1 H, t, J2,3 9.0, H-3c) and 6.70–8.10 (34 H, m,
ArH); m/z 1411 [M ϩ Na]ϩ.
4-Methoxyphenyl O-(methyl 2,3,4-tri-O-benzoyl-ꢀ-D-gluco-
pyranosyluronate)-(1 3)-(2,4,6-tri-O-benzoyl-ꢀ-D-galacto-
pyranosyl)-(1 3)-2-O-benzoyl-ꢀ-D-galactopyranoside 20
A mixture of diol 17 (0.4 g, 0.32 mmol) and benzoyl chloride
(0.15 cm3, 1.3 mmol) in dry pyridine (6 cm3) was stirred for
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 4 3 4 – 4 4 2
439

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4 h at 0 ЊC, then was quenched with MeOH (0.5 cm3), diluted
with dichloromethane (50 cm3), washed with water, saturated
aq NaHCO3 and water, dried (MgSO4) and concentrated. A
solution of the crude solid residue in 90% aq CF3COOH (10
cm3) was stirred for 15 min at rt, then was concentrated,
evaporated with water (3 × 10 cm3) and dried in vacuo. Flash
silica chromatography (8 : 1 dichloromethane–acetone) and
crystallization of the residue from MeOH afforded the diol 20
(363 mg, 83% from 17); mp 233–235 ЊC; [α]D ϩ42 (c 1 in
CHCl3); (Found: C, 65.75; H, 5.0. C75H66O25 requires C, 65.9;
H, 4.85%); δH(250 MHz, 3 : 1 CD3OD–CDCl3) 3.49 (1 H, m,
H-5a), 3.60 (3 H, s, OCH3), 3.61 (1 H, dd, J 5,6 and 12.0, H-6a),
3.64 (3 H, s, COOCH3), 3.79 (1 H, dd, J5,6 7.0, H-6a), 3.88 (1 H,
dd, J2,3 10.2, J3,4 3.6, H-3a), 4.12 (1 H, dd, J4,5 0.8, H-4a), 4.24
(1 H, m, H-5b), 4.28 (1 H, d, J4,5 9.5, H-5c), 4.36 (1 H, dd,
J2,3 10.2, J3,4 3.6, H-3b), 4.50 (2 H, m, 2 × H-6b), 4.76 (1 H, d,
J1,2 8.0, H-1a), 4.82 (1 H, d, J1,2 8.0, H-1b), 4.94 (1 H, d, J1,2 7.5,
H-1c), 5.21 (1 H, dd, J2,3 9.0, H-2c), 5.50 (3 H, m, H-2a, H-2b,
H-4c), 5.58 (1 H, dd, J3,4 9.5, H-3c), 5.88 (1 H, dd, J4,5 0.8,
H-4b) and 6.70–8.10 (39 H, m, ArH); m/z 1368 [M ϩ H]ϩ, 1390
[M ϩ Na]ϩ.
4-Methoxyphenyl O-(methyl 2,3,4-tri-O-benzoyl-ꢀ-D-gluco-
pyranosyluronate)-(1 3)-(2-O-benzoyl-6-O-sodium sulfonato-ꢀ-
D-galactopyranosyl)-(1 3)-2-O-benzoyl-6-O-sodium sulfonato-
ꢀ-D-galactopyranoside 22
A solution of the tetrol 19 (116 mg, 0.16 mmol) and the sulfur
trioxide–trimethylamine complex (84 mg, 0.64 mmol) in dry
DMF (4 cm3) was stirred for 90 min at 40 ЊC under dry argon,
then was cooled, quenched with MeOH (1 cm3) and concen-
trated. Flash silica chromatography (4 : 1 dichloromethane–
MeOH, containing 2% of Et3N) gave a fraction that was eluted
from a column (1.5 × 20 cm) of Sephadex SP C25 [Naϩ]
resin with 9 : 5 : 1 dichloromethane–MeOH–water to give the
disodium salt 22 (184 mg, 83%) as a white powder; [α]D ϩ37
(c 1 in MeOH); (Found: C, 53.45; H, 4.3. C61H56Na2O29S2
requires C, 53.75; H, 4.15%); δH(250 MHz, 3 : 1 CD3OD–
CDCl3) 3.58 (3 H, s, OCH3), 3.64 (3 H, s, COOCH3), 3.83 (2 H,
m, H-5a, H-5b), 3.92 (1 H, dd, J2,3 10.0, J3,4 3.6, H-3a), 3.93
(1 H, dd, J2,3 10.2, J3,4 3.6, H-3b), 4.20 (2 H, m, H-4a, H-4b),
4.25 (4 H, m, 2 × H-6a, 2 × H-6b), 4.34 (1 H, d, J4,5 9.5, H-5c),
4.74 (1 H, d, J1,2 8.0, H-1a), 4.82 (1 H, d, J1,2 8.0, H-1b), 5.01
(1 H, d, J1,2 7.0, H-1c), 5.25 (1 H, dd, J2,3 9.0, H-2c), 5.48 (2 H,
m, H-2b, H-4c), 5.59 (2 H, m, H-2a, H-4c) and 6.70–8.0 (29 H,
m, ArH); m/z 1386 [M ϩ Na]ϩ.
4-Methoxyphenyl O-(methyl 2,3,4-tri-O-benzoyl-ꢀ-D-gluco-
pyranosyluronate)-(1 3)-(2-O-benzoyl-ꢀ-D-galactopyranosyl)-
(1 3)-2,4,6-tri-O-benzoyl-ꢀ-D-galactopyranoside 21
4-Methoxyphenyl O-(sodium ꢀ-D-glucopyranosyluronate)-
(1 3)-(6-O-sodium sulfonato-ꢀ-D-galactopyranosyl)-(1 3)-
6-O-sodium sulfonato-ꢀ-D-galactopyranoside 6
Compound 18 (585 mg, 0.45 mmol) was benzoylated as
described for the preparation of 20, then was desilylated as
described for the preparation of 17. Crystallization of the solid
residue from EtOH gave the diol 21 (0.50 g, 81% from 18); mp
255–257 ЊC; [α]D ϩ23 (c 1 in CHCl3); (Found: C, 65.8; H,
5.0. C75H66O25 requires C, 65.9; H, 4.85%); δH(250 MHz,
3 : 1 CD3OD–CDCl3) 3.59 (1 H, m, H-5b), 3.60 (3 H, s, OCH3),
3.63 (3 H, s, COOCH3), 3.68 (1 H, dd, J 5.0 and 12.0,
H-6b), 3.81 (1 H, dd, J2,3 10.2, J3,4 3.6, H-3b), 3.86 (1 H, dd,
J5,6 7.0, H-6b), 4.08 (1 H, dd, J4,5 0.8, H-4b), 4.20 (1 H, m,
H-5a), 4.31 (1 H, d, J4,5 9.5, H-5c), 4.39 (1 H, dd, J2,3 10.3,
J3,4 3.6, H-3a), 4.50 (2 H, m, 2 × H-6a), 4.73 (1 H, d, J1,2 8.0,
H-1a), 4.91 (1 H, d, J1,2 8.0, H-1b), 4.98 (1 H, d, J1,2 7.5,
H-1c), 5.23 (1 H, dd, H-2b), 5.29 (1 H, dd, J2,3 9.0, H-2c),
5.47 (1 H, t, J3,4 9.5, H-4c), 5.66 (2 H, m, H-2a, H-3c), 5.84 (1 H,
dd, J4,5 0.8, H-4a) and 6.70–8.10 (39 H, m, ArH); m/z 1390
[M ϩ Na]ϩ.
The ester 22 (177 mg, 0.13 mmol) was saponified as described
for the preparation of 1 to give the target 6a,6b-disulfate 6
(100 mg, 91%) as a white hygroscopic powder; [α]D Ϫ13 (c 1 in
water); (Found: C, 35.0; H, 4.15. C25H33Na3O24S2 requires C,
35.3; H, 3.9%); δH(500 MHz, D2O, internal acetone) data for
ring protons are reported in Table 1, 3.72 (3 H, s, OCH3) and
6.90 (4 H, m, ArH); δC(62.8 MHz, D2O, internal acetone) data
for ring carbons are reported in Table 2, 55.99 (OCH3), 115.24,
118.31, 151.24, and 154.82 (6 C, ArC).
4-Methoxyphenyl O-(methyl 2,3,4-tri-O-benzoyl-ꢀ-D-gluco-
pyranosyluronate)-(1 3)-(2,4,6-tri-O-benzoyl-ꢀ-D-galacto-
pyranosyl)-(1 3)-2-O-benzoyl-6-O-sodium sulfonato-ꢀ-D-
galactopyranoside 23
A solution of the diol 20 (205 mg, 0.15 mmol) and the sulfur
trioxide–trimethylamine complex (40 mg, 0.3 mmol) in dry
DMF (4 cm3) was stirred for 7 h at 40 ЊC, then was cooled,
quenched with MeOH (0.5 cm3) and concentrated. Flash silica
chromatography (10 : 1 dichloromethane–MeOH, containing
2% of Et3N) gave a fraction that was submitted to ion-exchange
as described for the preparation of 22 to afford the 6a-sulfate 23
(190 mg, 86%) as a white powder; [α]D ϩ36 (c 1 in CHCl3);
(Found: C, 60.8; H, 4.7. C75H65NaO28S requires C, 61.0; H,
4.45%); δH(250 MHz, 3 : 1 CD3OD–CDCl3) 3.57 (3 H, s,
OCH3), 3.64 (3 H, s, COOCH3), 3.84 (1 H, m, H-5a), 3.93 (1 H,
dd, J2,3 10.2, J3,4 3.6, H-3a), 4.20 (3 H, m, 2 × H-6a, H-4a), 4.30
(1 H, m, H-5b), 4.34 (1 H, d, J4,5 9.5, H-5c), 4.39 (1 H, dd, J2,3
10.3, J3,4 3.6, H-3b), 4.50 (2 H, m, 2 × H-6b), 4.77 (1 H, d, J1,2
8.0, H-1a), 4.89 (1 H, d, J1,2 8.0, H-1b), 5.04 (1 H, d, J1,2 7.5,
H-1c), 5.21 (1 H, dd, J2,3 9.0, H-2c), 5.50 (3 H, m, H-2a, H-2b,
H-4c), 5.61 (1 H, dd, J3,4 9.5, H-3c), 5.92 (1 H, dd, J4,5 0.8,
H-4b) and 6.70–8.10 (39 H, m, ArH); m/z 1499 [M ϩ Na]ϩ.
4-Methoxyphenyl O-(sodium ꢀ-D-glucopyranosyluronate)-
(1 3)-(ꢀ-D-galactopyranosyl)-(1 3)-ꢀ-D-galactopyranoside 1
A solution of the ester 19 (208 mg, 0.18 mmol) in THF (5 cm3)
was treated at 0 ЊC with a freshly prepared solution of hydrogen
peroxide (30 wt% solution in water, 0.6 cm3) and lithium
hydroxide (1 mol dmϪ3, 1 cm3), and the mixture was stirred for
1 h at 0 ЊC and 5 h at rt, then was cooled to 0 ЊC. Methanol
(2 cm3) and sodium hydroxide (4 mol dmϪ3, 2 cm3) were
added, and the mixture was stirred for 4 h at rt. Water (10 cm3)
was added, and the mixture was treated with Amberlite IR-120
[Hϩ] resin to pH 3 (pHmeter control), then was filtered, concen-
trated and dried in vacuo. Benzoic acid was extracted from
the glassy residue with cold abs EtOH (2 × 5 cm3). The remain-
ing solid was dissolved in water (5 cm3), and the pH of
the solution was brought to 7 (pHmeter control) with diluted
aq sodium hydroxide. The solution was eluted from a column
(2 × 80 cm) of Sephadex LH-20 with water and freeze-dried
to give the target trisaccharide 1 (107 mg, 92%) as a white
powder; [α]D Ϫ18 (c 1 in water); (Found: C; 46.25; H, 5.6.
C25H35NaO18 requires C, 46.45; H, 5.45%); δH(500 MHz,
D2O, internal acetone) data for ring protons are reported in
Table 1, 3.70 (3 H, s, OCH3) and 6.90 (4 H, m, ArH); δC(62.8
MHz, D2O, internal acetone) data for ring carbons are reported
in Table 2, 55.98 (OCH3), 115.22, 118.42, 151.15, and 154.87
(6 C, ArC).
4-Methoxyphenyl O-(sodium ꢀ-D-glucopyranosyluronate)-
(1 3)-(ꢀ-D-galactopyranosyl)-(1 3)-6-O-sodium sulfonato-
ꢀ-D-galactopyranoside 2
The ester 23 (177 mg, 0.12 mmol) was saponified as described
for the preparation of 1 to give the target 6a-sulfate 2 (80 mg,
89%) as a white hygroscopic powder; [α]D Ϫ16 (c 1 in water);
(Found: C, 39.9; H, 4.8. C25H34Na2O21S requires C, 40.1; H,
4.6%); δH(500 MHz, D2O, internal acetone) data for ring
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 4 3 4 – 4 4 2
440

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protons are reported in Table 1, 3.71 (3 H, s, OCH3) and 6.90
(4 H, m, ArH); δC(62.8 MHz, D2O, internal acetone) data for
ring carbons are reported in Table 2, 55.99 (OCH3), 115.24,
118.34, 151.20, and 154.87 (6 C, ArC).
m, H-5b), 4.21 (1 H, dd, J4,5 0.8, H-4b), 4.28 (3 H, m, H-5b,
2 × H-6b), 4.37 (1 H, d, J4,5 9.5, H-5c), 4.41 (1 H, dd, J2,3 10.3,
J3,4 3.6, H-3a), 4.50 (2 H, m, 2 × H-6a), 4.74 (1 H, d, J1,2 8.0,
H-1a), 4.94 (1 H, d, J1,2 8.0, H-1b), 5.05 (1 H, d, J1,2 7.5, H-1c),
5.17 (1 H, dd, J2,3 9.5, H-2c), 5.31 (1 H, dd, H-2b), 5.45 (1 H, t,
J3,4 9.5, H-4c), 5.59 (1 H, dd, H-2a), 5.71 (1 H, dd, H-3c), 5.94
(1 H, dd, J4,5 0.8, H-4a) and 6.70–8.10 (39 H, m, ArH); m/z 1496
[M ϩ Na]ϩ.
4-Methoxyphenyl O-(methyl 2,3,4-tri-O-benzoyl-ꢀ-D-gluco-
pyranosyluronate)-(1 3)-(2,4,6-tri-O-benzoyl-ꢀ-D-galacto-
pyranosyl)-(1 3)-2,6-di-O-benzoyl-ꢀ-D-galactopyranoside 24
A mixture of diol 20 (178 mg, 0.13 mmol) and benzoyl cyanide
(35 mg, 0.26 mmol) in dry pyridine (4 cm3) was stirred overnight
at rt, then was quenched with MeOH (0.5 cm3) and concen-
trated. Flash silica chromatography (9 : 1 dichloromethane–
EtOAc) and crystallization of the residue from MeOH gave the
alcohol 24 (174 mg, 91%); mp 222–224 ЊC; [α]D ϩ55 (c 1 in
CHCl3); (Found: C, 66.8; H, 4.95. C82H70O26 requires C, 66.9;
H, 4.8%); δH(250 MHz, 3 : 1 CD3OD–CDCl3) 3.63 (3 H, s,
OCH3), 3.67 (3 H, s, COOCH3), 3.80 (2 H, m, H-5a, H-5b), 3.91
(1 H, dd, J2,3 10.2, J3,4 3.6, H-3a), 4.17 (1 H, d, J4,5 9.5, H-5c),
4.19 (1 H, dd, J4,5 0.8, H-4a), 4.32 (1 H, dd, J2,3 10.2, J3,4 3.6,
H-3b), 4.50 (4 H, m, 2 × H-6a, 2 × H-6b), 4.74 (1 H, d, J1,2 8.0,
H-1a), 4.83 (1 H, d, J1,2 8.0, H-1b), 4.94 (1 H, d, J1,2 7.5, H-1c),
5.25 (1 H, dd, J2,3 9.0, H-2c), 5.58 (4 H, m, H-2a, H-2b, H-3c,
H-4c), 5.88 (1 H, dd, J4,5 0.8, H-4b) and 6.70–8.10 (44 H, m,
ArH); m/z 1494 [M ϩ Na]ϩ.
4-Methoxyphenyl O-(sodium ꢀ-D-glucopyranosyluronate)-
(1 3)-(6-O-sodium sulfonato-ꢀ-D-galactopyranosyl)-(1 3)-
ꢀ-D-galactopyranoside 4
The ester 26 (160 mg, 108 µmol) was saponified as described for
the preparation of 1 to give the target 6b-sulfate 4 (73 mg, 90%)
as a white powder; [α]D Ϫ11 (c 1 in water); (Found: C, 39.8; H,
4.85. C25H34 Na2O21S requires C, 40.1; H, 4.6%); δH(500 MHz,
D2O, internal acetone) data for ring protons are reported in
Table 1, 3.72 (3 H, s, OCH3) and 6.90 (4 H, m, ArH); δC(62.8
MHz, D2O, internal acetone) data for ring carbons are reported
in Table 2, 56.00 (OCH3), 115.24, 118.38, 151.13, and 154.86
(6 C, ArC).
4-Methoxyphenyl O-(methyl 2,3,4-tri-O-benzoyl-ꢀ-D-gluco-
pyranosyluronate)-(1 3)-(2,6-di-O-benzoyl-ꢀ-D-galactopyrano-
syl)-(1 3)-2,4,6-tri-O-benzoyl-ꢀ-D-galactopyranoside 27
4-Methoxyphenyl O-(methyl 2,3,4-tri-O-benzoyl-ꢀ-D-gluco-
pyranosyluronate)-(1 3)-(2,4,6-tri-O-benzoyl-ꢀ-D-galacto-
pyranosyl)-(1 3)-2,6-di-O-benzoyl-4-O-sodium sulfonato-ꢀ-D-
galactopyranoside 25
The diol 21 (205 mg, 0.15 mmol) was treated as described for
the preparation of 24. Crystallization of the residue from EtOH
gave the alcohol 27 (198 mg, 90%); mp 196–198 ЊC; [α]D ϩ33
(c 1 in CHCl3); (Found: C, 66.8; H, 4.9. C82H70O26 requires C,
66.9; H, 4.8%); δH(250 MHz, 3 : 1 CD3OD–CDCl3) 3.57 (3 H, s,
OCH3), 3.65 (3 H, s, COOCH3), 3.86 (2 H, m, H-3b, H-5a), 4.09
(1 H, m, H-5b), 4.22 (1 H, d, J4,5 9.5, H-5c), 4.25 (1 H, dd, J3,4
3.6, J4,5 0.8, H-4b), 4.27 (1 H, dd, J2,3 10.2, J3,4 3.6, H-3a), 4.55
(4 H, m, 2 × H-6a, 2 × H-6b), 4.74 (1 H, d, J1,2 8.0, H-1a), 4.85
(1 H, d, J1,2 8.0, H-1b), 4.91 (1 H, d, J1,2 7.0, H-1c), 5.32 (2 H, m,
H-2b, H-2c), 5.58 (1 H, t, J3,4 9.5, H-4c), 5.68 (2 H, m, H-2a,
H-3c), 5.85 (1 H, dd, J4,5 0.8, H-4a) and 6.70–8.10 (44 H, m,
ArH): m/z 1494 [M ϩ Na]ϩ.
A mixture of alcohol 24 (147 mg, 0.1 mmol) and the sulfur tri-
oxide–trimethylamine complex (132 mg, 1 mmol) in dry DMF
(3 cm3) was stirred for 4 d at 60 ЊC under dry argon, then was
treated as described for the preparation of 23 to give the sodium
salt 25 (138 mg, 88%) as a white powder; [α]D ϩ44 (c 1 in
CHCl3); (Found: C, 62.4; H, 4.6. C82H69NaO29S requires C,
62.6; H, 4.4%); δH(250 MHz, 3 : 1 CD3OD–CDCl3) 3.59 (3 H, s,
OCH3), 3.64 (3 H, s, COOCH3), 3.91 (1 H, m, H-5a), 4.11 (1 H,
dd, J2,3 10.2, J3,4 3.6, H-3a), 4.21 (1 H, m, H-5b), 4.36 (1 H, d,
J4,5 9.5, H-5c), 4.38 (1H, dd, J2,3 10.1, J3,4 3.6, H-3b), 4.60 (4 H,
m, 2 × H-6a, 2 × H-6b), 4.81 (1 H, d, J1,2 8.0, H-1a), 4.91 (1 H,
d, J1,2 8.0, H-1b), 5.04 (1 H, d, J1,2 7.5, H-1c), 5.14 (1 H, dd,
J4,5 0.8, H-4a), 5.23 (1 H, dd, J2,3 9.5, H-2c), 5.43 (1 H, dd,
H-2b), 5.56 (2 H, m, H-2a, H-4c), 5.64 (1 H, t, J3,4 9.5, H-3c),
5.97 (1 H, dd, J4,5 0.8, H-4b) and 6.70–8.10 (44 H, m, ArH); m/z
1597 [M ϩ Na]ϩ.
4-Methoxyphenyl O-(methyl 2,3,4-tri-O-benzoyl-ꢀ-D-gluco-
pyranosyluronate)-(1 3)-(2,6-di-O-benzoyl-4-O-sodium
sulfonato-ꢀ-D-galactopyranosyl)-(1 3)-2,4,6-tri-O-benzoyl-
ꢀ-D-galactopyranoside 28
The alcohol 27 (147 mg, 0.1 mmol) was sulfated as described for
the preparation of 24 to give the sodium salt 28 (140 mg, 89%)
as a white powder; [α]D ϩ34 (c 1 in CHCl3); (Found: C, 62.4; H,
4.65. C82H69NaO29S requires C, 62.6; H, 4.4%); δH(250 MHz,
3 : 1 CD3OD–CDCl3) 3.57 (3 H, s, OCH3), 3.64 (3 H, s, CO-
OCH3), 4.0 (3 H, m, H-3b, H-5a, H-5b), 4.27 (1 H, dd, J2,3 10.2,
J3,4 3.6, H-3a), 4.34 (1 H, d, J4,5 9.5, H-5c), 4.50 (4 H, m, 2 ×
H-6a, 2 × H-6b), 4.74 (1 H, d, J1,2 8.0, H-1a), 4.78 (1 H, d, J1,2
8.0, H-1b), 4.90 (1 H, d, J1,2 7.5, H-1c), 5.11 (1 H, dd, J3,4 3.6,
J 4,5 0.8, H-4b), 5.14 (1 H, dd, J2,3 9.0, H-2c), 5.45 (1 H, dd, J2,3
10.2, H-2b), 5.62 (1 H, dd, H-2a), 5.58 (2 H, m, H-3c, H-4c),
5.78 (1 H, dd, J4,5 0.8, H-4a) and 6.70–8.10 (44 H, m, ArH); m/z
1597 [M ϩ Na]ϩ.
4-Methoxyphenyl O-(sodium ꢀ-D-glucopyranosyluronate)-
(1 3)-(ꢀ-D-galactopyranosyl)-(1 3)-4-O-sodium sulfonato-
ꢀ-D-galactopyranoside 3
The ester 25 (127 mg, 80 µmol) was saponified as described for
the preparation of 1 to give the target 4a-sulfate 3 (54 mg, 90%)
as a white powder; [α]D Ϫ15 (c 1 in water); (Found: C, 39.9; H,
4.7. C25H34Na2O21S requires C, 40.1; H, 4.6%); δH(500 MHz,
D2O, internal acetone) data for ring protons are reported in
Table 1, 3.71 (3 H, s, OCH3) and 6.90 (4 H, m, ArH); δC(62.8
MHz, D2O, internal acetone) data for ring carbons are reported
in Table 2, 55.99 (OCH3), 115.24, 118.49, 151.11, and 154.95
(6 C, ArC).
4-Methoxyphenyl O-(sodium ꢀ-D-glucopyranosyluronate)-
(1 3)-(4-O-sodium sulfonato-ꢀ-D-galactopyranosyl)-(1 3)-
ꢀ-D-galactopyranoside 5
4-Methoxyphenyl O-(methyl 2,3,4-tri-O-benzoyl-ꢀ-D-gluco-
pyranosyluronate)-(1 3)-(2-O-benzoyl-6-O-sodium sulfonato-
ꢀ-D-galactopyranosyl)-(1 3)-2,4,6-tri-O-benzoyl-ꢀ-D-galacto-
pyranoside 26
The ester 28 (110 mg, 70 µmol) was saponified as described for
the preparation of 1 to give the target 4b-sulfate 5 (48 mg, 90%)
as a white powder; [α]D Ϫ17 (c 1 in water); (Found: C, 39.8; H,
4.75. C25H34Na2O21S requires C, 40.1; H, 4.6%); δH(500 MHz,
D2O, internal acetone) data for ring protons are reported in
Table 1, 3.72 (3 H, s, OCH3) and 6.90 (4 H, m, ArH); δC(62.8
MHz, D2O, internal acetone) data for ring carbons are reported
in Table 2, 56.00 (OCH3), 115.24, 118.41, 151.15, and 154.89
(6 C, ArC).
The diol 21 (205 mg, 0.15 mmol) was sulfated as described
for the preparation of 23 to give the sodium salt 26 (192 mg,
87%) as a white powder; [α]D ϩ28 (c 1 in CHCl3); (Found: C,
60.8; H, 4.6. C75H65NaO28S requires C, 61.0; H, 4.45%); δH(250
MHz, 3 : 1 CD3OD–CDCl3) 3.61 (3 H, s, OCH3), 3.65 (3 H, s,
COOCH3), 3.85 (1 H, dd, J2,3 10.2, J3,4 3.6, H-3b), 3.91 (1 H,
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 4 3 4 – 4 4 2
441

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4-Methoxyphenyl O-(methyl 2,3,4-tri-O-benzoyl-ꢀ-D-gluco-
pyranosyluronate)-(1 3)-(2-O-benzoyl-ꢀ-D-galactopyranosyl)-
(1 3)-2-O-benzoyl-6-O-sodium sulfonato-ꢀ-D-galactopyrano-
side 29 and 4-methoxyphenyl O-(methyl 2,3,4-tri-O-benzoyl-ꢀ-D-
glucopyranosyluronate)-(1 3)-( 2-O-benzoyl-6-O-sodium
sulfonato-ꢀ-D-galactopyranosyl)-(1 3)-2-O-benzoyl-ꢀ-D-
galactopyranoside 30
References
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103–124.
A mixture of tetrol 19 (116 mg, 0.1 mmol) and the sulfur tri-
oxide–trimethylamine complex (40 mg, 0.3 mmol) in dry DMF
(4 cm3) was stirred for 40 min at 40 ЊC under dry argon, then
was directly quenched with MeOH (0.5 cm3) and concentrated.
Flash silica chromatography (8 : 1 dichloromethane–MeOH,
containing 2% of Et3N) gave first the starting tetrol 19 (6 mg,
5%). Next eluted was a fraction that was submitted to ion-
exchange as described previously to give the 6a-sulfated deriv-
ative 29 (34 mg, 27%) as a white powder; [α]D ϩ32 (c 1 in
MeOH); δH(500 MHz, 3 : 1 CD3OD–CDCl3) 3.57 (3 H, s,
OCH3), 3.64 (3 H, s, COOCH3), 3.74 (1 H, m, H-5a), 3.84 (1 H,
m, H-5b), 3.87 (2 H, m, 2 × H-6b), 3.93 (1 H, dd, J2,3 10.2, J3,4
3.6, H-3a), 3.99 (1 H, dd, J2,3 10.3, J3,4 3.6, H-3b), 4.15 (1 H,
dd, J4,5 0.8, H-4b), 4.17 (1 H, dd, J4,5 0.8, H-4a), 4.28 (2 H, m,
2 × H-6a), 4.32 (1 H d, J4,5 9.5, H-5c), 4.71 (1 H, d, J1,2 8.0,
H-1a), 4.80 (1 H, d, J1,2 8.0, H-1b), 4.95 (1 H, d, J1,2 7.5, H-1c),
5.21 (1 H, dd, J2,3 9.0, H-2c), 5.45 (3 H, m, H-2b, H-3c, H-4c),
5.51 (1 H, dd, H-2a) and 6.70–8.0 (29 H, m, ArH); m/z 1284
[M ϩ Na]ϩ.
Further elution gave a second fraction that was treated simi-
larly to give the 6b-sulfated derivative 30 (26 mg, 20%) as a
colourless glass; [α]D ϩ26 (c 1 in MeOH); δH(500 MHz, 3 : 1
CD3OD–CDCl3) 3.55 (3 H, s, OCH3), 3.63 (3 H, s, COOCH3),
3.68 (1 H, m, H-5a), 3.82 (1 H, m, H-5b), 3.85 (2 H, m, 2 ×
H-6a), 3.94 (1 H, dd, J2,3 10.2, J3,4 3.6, H-3b), 3.99 (1 H, dd,
J2,3 10.1, J3,4 3.6, H-3a), 4.16 (1 H, dd, J4,5 0.8, H-4a), 4.18
(1 H, dd, J4,5 0.8, H-4b), 4.24 (2 H, m, 2 × H-6b), 4.34 (1 H,
d, J4,5 9.5, H-5c), 4.69 (1 H, d, J1,2 8.0, H-1a), 4.76 (1 H, d,
J1,2 8.0, H-1b), 4.99 (1 H, d, J1,2 7.5, H-1c), 5.17 (1 H, dd,
J2,3 9.0, H-2c), 5.45 (1 H, dd, H-2b), 5.51 (2 H, m, H-3c, H-4c),
5.56 (1 H, dd, H-2a) and 6.70–8.0 (29 H, m, ArH); m/z 1284
[M ϩ Na]ϩ.
12 J.-I. Tamura and J. Nishihara, J. Org. Chem., 2001, 66, 3074–
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Acknowledgements
This work was supported by a grant-in-aid from CNRS–
INSERM–MRT through the ACI 2001 “Molécules et Cibles
Thérapeutiques”. B.T. thanks the MRT for a studentship.
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Res., 1998, 314, 283–288.
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 4 3 4 – 4 4 2
442