An expeditious preparation of various sulfoforms of the disaccharide β-D-Galp-(1→3)-D-Galp, a partial structure of the linkage region of proteoglycans, as their 4-methoxyphenyl β-D-glycosides

Article abstract of DOI:10.1016/j.carres.2003.10.012

An expeditious preparation of various sulfoforms of the disaccharide 4-methoxyphenyl O-(β-D-galactopyranosyl)-(1→3)-β-D- galactopyranoside, namely the 4^I- and 6^I-sulfate, the 4^II- and 6^II-sulfate, and the 6^I,6 ^II-disulfate derivatives, is reported for the first time. These molecules will be useful for the study of the early steps of the biosynthesis and sorting of proteoglycans. All target compounds were readily obtained from the common key intermediate 4-methoxyphenyl O-(2,3-di-O-benzoyl-4,6-di-O- levulinoyl-β-D-galactopyranosyl)-(1→3)-2-O-benzoyl-4, 6-O-benzylidene-β-D-galactopyranoside, easily prepared from the common starting material 4-methoxyphenyl 4,6-O-benzylidene-β-D-galactopyranoside. Noticeable is the possible preparation of the different 6-O-sulfonated species through a one-pot procedure starting from a tetrol precursor.

Full text of DOI:10.1016/j.carres.2003.10.012

Carbohydrate
RESEARCH
Carbohydrate Research 339 (2004) 349–359
An expeditious preparation of various sulfoforms of the disaccharide
b- -Galp, a partial structure of the linkage region of
D
-Galp-(1 fi 3)-
D
proteoglycans, as their 4-methoxyphenyl b- -glycosides
D
Jean-Claude Jacquinet*
Institut de Chimie Organique et Analytique, UMR CNRS 6005, UFR Faculteꢀ des Sciences, Universiteꢀ d’Orleꢀans,
BP 6759, F-45067 Orleꢀans Cedex, France
Received 16 September 2003; accepted 24 October 2003
Abstract—An expeditious preparation of various sulfoforms of the disaccharide 4-methoxyphenyl O-(b-
D-galactopyranosyl)-
(1 fi 3)-b-
D
-galactopyranoside, namely the 4I- and 6I-sulfate, the 4II- and 6II-sulfate, and the 6I,6II-disulfate derivatives, is reported
for the first time. These molecules will be useful for the study of the early steps of the biosynthesis and sorting of proteoglycans. All
target compounds were readily obtained from the common key intermediate 4-methoxyphenyl O-(2,3-di-O-benzoyl-4,6-di-O-lev-
ulinoyl-b-
D
-galactopyranosyl)-(1 fi 3)-2-O-benzoyl-4,6-O-benzylidene-b-
D-galactopyranoside, easily prepared from the common
-galactopyranoside. Noticeable is the possible preparation of the different
starting material 4-methoxyphenyl 4,6-O-benzylidene-b-
D
6-O-sulfonated species through a one-pot procedure starting from a tetrol precursor.
ꢀ 2003 Elsevier Ltd. All rights reserved.
Keywords: Proteoglycan; Linkage region; Digalactoside; Sulfoforms
1. Introduction
introduction of the D-GlcA residue. Thus, the presence
of this phosphate group should be related to the very
early steps of the biosynthesis of GAGs. The biological
significance of the possible sulfations is however still
unknown, but these modifications are possibly related to
GAG chain elongation and sorting. The question is
what determines whether the GAG chain becomes a
glucosaminoglycan (heparin, heparan sulfate) or a
galactosaminoglycan (chondroitin sulfates, dermatan
sulfate) starting from a common precursor. Investiga-
tions of the structural variations in the GAG–protein
linkage region may clarify the biological functions of
these unique modifying groups. Since the human
galactose b-1,3-glucuronyltransferase (GlcAT-1), the
key enzyme in the biosynthesis of GAGs, is now avail-
able,8;9 it should be relevant to gain insight into its
specificity, particularly toward variously sulfated oligo-
saccharides of the linkage region. Several syntheses of
glycopeptides of various length containing a sulfate
Proteoglycans (PGs) are complex macromolecular gly-
coproteins that contain a core protein to which side
chains of glycosaminoglycans (GAGs) are covalently
attached to
L-serine residues. Although GAGs have
different repetitive disaccharide units, they are, with the
exception of keratan sulfate, covalently linked to the
protein core through a common tetrasaccharide se-
quence,1 namely b-
b- -Galp-(1 fi 4)-b-
been demonstrated that this linkage region may be
D
-GlcpA-(1 fi 3)-b-
D
-Galp-(1 fi 3)-
D
D-Xylp (Fig. 1). However, it has
occasionally modified by sulfation at C-4 and/or C-6 of
D
-Gal units2–4 as well as by phosphorylation at C-2
the
of the
D
-Xyl residue.5;6 Recently, it has been established
that phosphorylation should be a transient phenome-
non,7 and that the phosphate content decreases after
group on one or the other D-Gal units have been re-
ported10–12 in the last decade, but no preparation of all
the possible sulfoforms has been described.
* Tel.: +33-238-417072; fax: +33-238-417281; e-mail: jean-claude.jac-
quinet@univ-orleans.fr
0008-6215/$ - see front matter ꢀ 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2003.10.012

350
J.-C. Jacquinet / Carbohydrate Research 339 (2004) 349–359
a
a
a
a
b
O
HO
O
OH
HO
HN
OH
HO
O
COONa
O
HO
O
O
O
O
O
O
HO
O
HN
HO
HO
HO
Figure 1. The carbohydrate–protein linkage region of proteoglycans. The arrows indicate possible substitution with a sulfate (a) or a phosphate (b)
group.
As a preliminary study devoted to the elucidation of
the role of these sulfate groups in the biosynthesis of
chondroitin sulfates in relation with osteoarthritis, we
now report on for the first time an expeditious prep-
aration of various sulfoforms of the disaccharide
examined. This later should be useful for the prepara-
tion of a symmetrically protected disaccharide deriva-
tive, potential precursor of tetrol 12. Oxidative removal
of the anomeric 4-methoxyphenyl group in 4 with ceric
ammonium nitrate (CAN) was troublesome and gave
the corresponding hemiacetal in low yield with extensive
degradation. This intermediate was treated with tri-
chloroacetonitrile and 1,8-diazabicyclo-[5,4,0]-undec-7-
ene (DBU) in dichloromethane to afford the a-imidate 6
in 48% overall yield, the structure of which was deduced
from its 1H NMR spectrum (Table 1). However, similar
treatment of 5 gave readily the crystalline a-imidate 7 in
69% overall yield.
4-methoxyphenyl O-(b-
D
D
-galactopyranosyl)-(1 fi 3)-b-
-galactopyranoside, namely its 6I-, 6II-, 4I-, and 4II-
monosulfate and 6I,6II-disulfate derivatives. The phenyl
group will be useful for the detection of the oligosac-
charides and the methoxy group will serve as a marker to
check the purity of the products by NMR spectroscopy.
2. Results and discussion
Coupling reactions that involved acceptor 3 and do-
nors 6 and 7 was next studied (Scheme 2). Condensation
of imidate 6 (1.3 equiv) and alcohol 3 in dichlorome-
thane, in the presence of trimethylsilyl triflate, afforded a
ꢀ1:1 mixture of disaccharides 8a and 8b. Changes in the
temperature, the nature of the solvent or the catalyst
(not described in Section 3) did not significantly modify
the product distribution. Such a lack of stereoselectivity
For the synthesis of the target sulfoforms 16, 19, 22, 24,
and 27, a common disaccharide intermediate (9) was
designed, which possesses at C-4 and C-6 a pair of
orthogonal protecting groups, that is, benzylidene acetal
and 4-oxopentanoyl (levulinoyl) esters, thus allowing
selective removal and regioselective introduction of the
sulfate groups. This later may, in turn, be prepared from
a glycosyl acceptor (3) and a glycosyl donor (7), both
readily available from a common starting material (1).
Preparation of the glycosyl acceptor 3 and donor 7
was achieved as follows (Scheme 1). Treatment of
in glycosylation reactions with activated
D-galacto
structures bearing 4,6-acetal protection (4,6-O-benzyl-
idene15 or 4,6-O-di-tert-butylsilylene16), despite the
presence of a C-2 participating group, has still been
observed. However, coupling of imidate 7 and acceptor
3 under the same conditions afforded the crystalline key
disaccharide intermediate 9 in 70% yield, the structure of
known 4-methoxyphenyl 4,6-O-benzylidene-b-D-galac-
topyranoside (1)13 with benzoyl cyanide and triethyl-
amine in acetonitrile gave the crystalline 3-O-benzoyl
derivative 2 in 75% yield. This later was submitted to
3 fi 2 O-acyl migration by treatment14 with dilute so-
dium hydroxide in acetone at 0 ꢁC to afford crystalline 3
in 82% yield, the structure of which was evident from its
1H NMR spectrum (Table 1). For the preparation of the
glycosyl donor 7, diol 1 was benzoylated to give crys-
talline 4 in 92% yield. This provides a stereocontrolling
auxiliary at C-2, which would induce 1,2-trans linkage
formation after activation at the anomeric center. Acid
hydrolysis of 4 with 90% trifluoroacetic acid gave the
corresponding diol, which was directly treated with
4-oxopentanoic acid (levulinic acid), 1,3-dicyclohexyl-
carbodiimide, and 4-dimethylaminopyridine in dichlo-
romethane to give crystalline 5 in 87% yield. At this
point, transformation of 4 into the a-imidate 6 was first
1
which was easily deduced from its H NMR spectrum
(Table 2).
Transformation of 9 into the intermediate diols 11
and 14 and tetrol 12 was then achieved as follows
(Scheme 3). Acid hydrolysis of 9 with 90% trifluoro-
acetic acid followed by conventional benzoylation gave
10 in 87% yield. Treatment of 10 with in situ prepared
hydrazine acetate17 afforded crystalline 11 in 90% yield.
Selective O-delevulinoylation of 9 followed by acid
hydrolysis as described above gave the crystalline tetrol
12 in 81% overall yield, whereas O-delevulinoylation of
9 followed by benzoylation gave crystalline 13 in 88%
yield. Acid hydrolysis of 13 afforded the crystalline diol
14 in 92% yield. The structure of these intermediates
were easily assigned through their 1H NMR spectra
(Table 2).

J.-C. Jacquinet / Carbohydrate Research 339 (2004) 349–359
Ph
351
Ph
Ph
O
O
O
O
O
O
O
O
O
b
a
d
d
BzO
OMP
OMP
OMP
OMP
HO
OMP
HO
HO
BzO
HO
2
1
3
c
Ph
Ph
O
O
O
O
O
O
BzO
BzO
BzO
BzO
O
CCl3
6
4
HN
e
LevO OLev
LevO
BzO
OLev
O
O
BzO
BzO
BzO
O
CCl3
7
5
HN
Scheme 1. Reagents and conditions: (a) PhCOCN, Et3N, MeCN, rt, 30 min; (b) 0.05 M NaOH, acetone, 0 ꢁC, 3 min; (c) PhCOCl, pyridine, 0 ꢁC, 1 h;
(d) CAN, toluene–MeCN–water, rt, 15 min; then CCl3CN, DBU, CH2Cl2, rt, 30 min; (e) 90% TFA, rt, 15 min; then levulinic acid, DCC, DMAP,
CH2Cl2, rt, 2 h.
Table 1. 1H NMR data: carbohydrate ring protons for monosaccharide derivatives 2–7a
2
3
4
5
6
7
H-1
J1;2
4.90
7.8
5.05
8.0
5.19
8.0
5.17
8.0
6.89
3.6
6.79
3.8
H-2
J2;3
4.40
5.60
6.09
5.89
6.07
5.90
10.2
10.0
10.0
10.2
10.0
10.0
H-3
J3;4
5.20
3.8
3.95
3.9
5.42
3.8
5.47
3.8
5.88
3.8
5.81
3.8
H-4
J4;5
4.50
1.0
4.35
0.9
4.65
0.9
5.68
1.0
4.77
0.8
5.78
1.0
H-5
J5;6a
J5;6b
H-6a
J6a;6b
H-6b
3.65
2.5
3.65
2.8
3.77
2.5
4.17
5.0
4.15
4.60
6.0
2.5
4.35
2.8
4.45
2.5
4.46
6.5
4.35
6.5
4.20
4.42
)12.0
4.15
)12.4
4.05
)11.0
4.10
)12.0
4.18
)11.0
4.24
)11.0
4.20
aChemical shifts (d, in ppm) and coupling constants (J, in Hz) for solns in CDCl3.
Having in hand the intermediates 11, 12, and 14, their
transformation into the target sulfoforms 16, 19, 22, 24,
and 27 was readily achieved as follows (Scheme 4).
Treatment of diols 11 and 14 and tetrol 12 with sulfur
trioxide–trimethylamine complex (1.5 equiv per primary
hydroxyl group) in N,N-dimethylformamide at 40 ꢁC for
90 min followed by ion-exchange chromatography (Naþ
resin) afforded the sodium salts 15, 21, and 23, respec-
tively, in 82%, 80%, and 83% yields, respectively.
recorded in the same mixture of solvents (3:1 CD3OD–
CDCl3, for solubility reason) showed the expected18
downfield shifts (Dd ꢀ 0:3–0.5 ppm) of the signals for H-
6a and H-6b in the 6-O-sulfonated-
D
-galacto species.
-galacto
For the preparation of the 4-O-sulfonated-
D
derivatives, protection at O-6 was first required.19 Thus,
treatment of diols 11 and 14 with benzoyl cyanide in
pyridine afforded alcohols 17 and 25 in 87% and 88%
yields, respectively. These later were O-sulfonated by
treatment with an excess (10 equiv) of sulfur trioxide–
trimethylamine complex in N,N-dimethylformamide at
1
Comparison of the H NMR spectra of 15, 21, and 23
with those of their nonsulfated precursors (Table 2), all

352
J.-C. Jacquinet / Carbohydrate Research 339 (2004) 349–359
easily assigned through spin-decoupling and 2D NMR
1
Ph
Ph
O
experiments, and their H NMR spectra (Table 2) also
showed the expected downfields shifts of the signals for
H-6a and H-6b in 28 and 29.
O
O
O
O
O
a
BzO
6
3
+
O
OMP
BzO
BzO
In conclusion, we have reported a stereocontrolled
and high-yielding approach for the preparation of var-
ious sulfoforms of the digalactoside 20. The use of
common precursors and the possible one-pot prepara-
tion of the 6-O-sulfonated derivatives considerably
reduce the number of transformations, and render this
route attractive for the preparation of larger molecules
from the glycosaminoglycan–protein linkage region of
GAGs. These sulfoforms are currently being evaluated
in biological assays, and the results of these studies will
be reported elsewhere in due course.
8a, 8b
Ph
O
LevO
BzO
OLev
O
O
O
O
a
7
3
+
OMP
BzO
BzO
9
Scheme 2. Reagents and conditions: (a) donor (1.3 equiv), TMSOTf,
ꢁ
mol. sieves 4 A, CH2Cl2, rt, 30 min.
60 ꢁC for 3 days followed by ion-exchange to give the
sodium salts 18 and 26 in 93% and 90% yields, respec-
tively. Comparison of the 1H NMR spectra of 18 and 26
(Table 2) with those of their precursors also showed the
expected19 downfield shifts (Dd ꢀ 0:8–0.9 ppm) of the
signals for H-4 in the 4-O-sulfonated derivatives. Final
saponification of 15, 18, 21, 23, and 26 with sodium
hydroxide in methanol gave the 6II-sulfate 16, the 4II-
sulfate 19, the 6I,6II-sulfate 22, the 6I-sulfate 24, and the
4I-sulfate 27, respectively, in 89–91% yields. Similarly,
the crystalline nonsulfated derivative 20 was prepared
by saponification of tetrol 12 in 91% yield. The 1H
NMR spectra of the five sulfoforms were compared with
those of their nonsulfated congener 20 (Table 2). Par-
ticularly relevant were the downfield shifts (Dd ꢀ 0:3–
0.6 ppm) of the signals for H-6a and H-6b in the
6-sulfated species, as well as the downfield shifts (Dd
0.7–1.0 ppm) of the signals for H-4 in the 4-sulfated
derivatives. Comparison of the 13C NMR spectra (Table
3) also showed the expected19 downfield shifts (Dd ꢀ 6–
7 ppm) of the signals for C-6 and the downfield shifts
(Dd ꢀ 7–8 ppm) of the signals for C-4 in the sulfated
species. These NMR data are in complete agreement
with the expected structures, and accord well with those
3. Experimental
3.1. General methods
Melting points were determined in capillary tubes with a
€
Buchi apparatus and are uncorrected. Optical rotations
were measured with a Perkin–Elmer 241 polarimeter. 1H
NMR spectra were recorded at 25 ꢁC with Bruker DPX
250 Advance and Varian Unity 500 spectrometers
operating at 250 and 500 MHz, respectively, with Me4Si
as internal standard, unless otherwise stated. 13C NMR
spectra were recorded with a Bruker Advance DPX 250
spectrometer operating at 62.8 MHz. Assignments were
based on homo- and heteronuclear correlations using
the supplierꢀs sofware. Low resolution mass spectra were
obtained on a Perkin–Elmer Sciex API 3000 spectro-
meter operating in the ion-spray (IS) mode. Flash-
column chromatography was performed on silica gel (E.
Merck, 40–63 lm). Elemental analyses were performed
by the Service Central de Microanalyse du CNRS
(Vernaison, France).
3.2. 4-Methoxyphenyl 3-O-benzoyl-4,6-O-benzylidene-
b-D-galactopyranoside (2)
reported for synthetic derivatives having D-Gal units O-
sulfonated at C-4 or C-6,10–12 as well as for fragments
isolated from natural sources.2–4
Triethylamine (0.5 mL) was added to a suspension of
4-methoxyphenyl 4,6-O-benzylidene-b- -galactopyran-
oside (1) (3.0 g, 8 mmol) and benzoyl cyanide (1.38 g,
10.4 mmol) in anhyd MeCN (40 mL), and the mixture
was stirred for 30 min at rt. Methanol (1 mL) was added,
and the mixture was concentrated. The solid residue was
Noteworthy is the possibility to prepare the 6I-, 6II-
sulfated and the 6I,6II-disulfated derivatives through a
one-pot procedure starting from tetrol 12 (Scheme 5).
Careful O-sulfonation of 12 (1equiv) with the sulfur tri-
oxide–trimethylamine complex (2.5 equiv) in N,N-di-
methylformamide at 40 ꢁC for 40 min (optimal
formation of the monosubstituted derivatives deter-
mined by TLC analysis), followed by ion-exchange,
afforded, besides traces of tetrol 12 (5%), the 6I-sulfate
28, the 6II-sulfate 29, and the 6I,6II-disulfate 21,
respectively, as their sodium salts, in 26%, 24%, and 40%
yields, respectively. The structures of 28 and 29 were
D
recrystallized from hot EtOH to give 2 (2.88 g, 75%); mp
22
D
202–203 ꢁC; ½aꢁ
+55ꢁ (c 1, CHCl3); 1H NMR
(250 MHz, CDCl3): carbohydrate ring protons (see Ta-
ble 1); d 8.10–6.70 (m, 14H, aromatic H), 5.50 (s, 1H,
PhCH), 3.75 (s, 3H, OCH3), 2.45 (d, 1H, J 3.0 Hz, HO-
2); ISMS: m=z 501 [M+Na]þ. Anal. Calcd for C27H26O8:
C, 67.77; H, 5.48. Found: C, 67.82; H, 5.41.

J.-C. Jacquinet / Carbohydrate Research 339 (2004) 349–359
Table 2. 1H NMR data: carbohydrate ring protons for disaccharide derivatives 8–29a
353
H-1I
H-2I
H-3I
H-4I
H-1II
H-2II
H-3II
H4II
H-5I
H-6aI
H-6bI
H-5II
H-6aII
H-6bII
J1;2
J2;3
J3;4
J4;5
J1;2
J2;3
J3;4
J4;5
8a
5.07
7.5
5.90
10.0
5.85
10.1
5.76
10.0
5.85
10.0
5.83
10.2
5.73
8.0
4.10
3.8
4.12
0.8
3.52
3.57
3.66
4.30
4.28
3.85
3.39
3.65
4.51
3.70
4.01
4.21
3.80
3.65
4.10
4.01
4.07
4.00
3.92
4.05
3.85
4.10
3.71
4.29
4.37
4.15
4.68
4.61
3.91
4.30
3.90
4.48
3.75
4.65
4.85
3.80
3.80
4.30
4.18
4.28
4.14
4.60
4.70
3.70
4.34
3.80
3.65
4.08
4.15
4.45
4.40
3.81
3.90
3.80
4.48
3.54
4.55
4.60
3.52
3.52
4.30
4.10
4.28
4.11
4.60
4.70
3.52
4.18
3.70
5.71
3.8
5.70
10.2
5.78
10.0
5.64
10.1
5.43
10.1
5.61
10.0
5.64
10.0
5.78
10.2
5.32
10.1
5.50
10.2
3.59
10.2
5.56
10.1
5.48
10.2
3.56
10.2
3.55
10.1
5.67
10.1
3.55
10.2
5.69
10.2
3.55
10.1
5.81
10.2
5.77
10.2
3.56
10.1
5.65
10.2
5.58
10.2
5.59
3.8
4.38
1.0
3.72
3.58
4.05
4.02
3.70
3.85
4.34
4.39
4.04
3.87
4.14
3.92
3.80
3.65
4.10
3.87
4.46
3.67
4.38
4.43
3.79
3.73
4.00
3.94
4.19
4.40
4.27
3.90
3.91
4.74
4.64
4.37d
4.11
4.65
4.30
3.80
3.78
4.30
4.18
4.64
3.80
4.60
4.70
3.80
3.93
4.25
3.40
4.04
4.30
4.08
3.90
3.81
4.37
4.54
4.21
4.09
4.55
4.30
3.52
3.54
4.30
4.10
4.51
3.53
4.60
4.70
3.52
3.82
4.18
8b
5.03
8.0
4.41
3.8
4.47
0.8
5.15
8.0
5.17
3.8
4.53
0.8
9
5.02
8.0
4.28
3.9
4.53
0.8
5.05
8.0
5.23
3.8
5.57
0.8
10
4.93
8.0
4.49
3.8
5.95
0.9
5.07
8.0
5.17
3.9
5.49
0.8
11b
12b
13
4.95
8.0
4.24
3.9
6.10
0.8
5.05
8.0
5.12
3.8
4.28
0.8
4.96
8.0
4.10
3.8
4.28
0.8
4.99
8.0
5.20
3.8
4.38
0.8
4.94
8.0
5.82
10.1
5.79
10.0
5.74
10.0
3.58
10.1
5.78
10.0
5.71
10.1
3.56
10.1
3.58
10.2
5.55
10.0
3.59
10.2
5.62
10.0
3.57
10.0
5.74
10.0
5.55
10.1
3.56
10.1
5.55
10.0
5.47
10.0
4.17
3.9
4.47
0.8
5.15
8.0
5.47
3.9
5.94
0.9
14b
15b
16c
17b
18b
19c
20c
21b
22c
23b
24c
25b
26b
27b
28b
29b
4.89
8.0
4.01
3.8
4.24
0.9
5.03
8.0
5.54
3.9
5.94
0.9
4.94
8.0
4.60
3.9
5.99
0.8
5.14
8.0
5.07
3.8
4.23
0.9
4.53
8.0
3.86
3.8
4.21
1.0
4.91
8.0
3.60
3.7
3.87
0.9
4.92
8.0
4.32
3.8
5.95
0.8
4.95
8.0
5.11
3.8
4.25
0.9
4.86
8.0
4.32
3.8
5.78
0.8
4.95
8.0
5.21
3.9
5.04
0.9
4.60
8.0
4.01
3.9
4.13
1.0
4.92
8.0
3.69
3.8
4.82
0.8
4.53
8.0
3.82
3.8
4.15
0.8
4.88
8.0
3.66
3.6
3.80
0.9
4.96
8.0
4.13
3.8
4.41
0.8
4.97
8.0
5.21
3.9
4.28
0.8
4.57
8.0
3.84
3.7
4.23
0.8
4.89
8.0
3.61
3.6
3.88
0.9
4.91
8.0
4.11
3.9
4.37
0.9
5.13
8.0
5.55
3.8
5.93
0.8
4.53
8.0
3.83
3.6
4.21
0.9
4.89
8.0
3.48
3.8
3.83
0.8
4.87
8.0
4.05
3.8
4.29
0.8
5.03
8.0
5.51
3.9
5.96
0.9
4.93
8.0
4.25
3.9
5.26
0.9
5.15
8.0
5.54
3.8
5.91
0.8
4.61
8.0
4.01
3.8
4.82
0.8
4.91
8.0
3.68
3.6
4.14
0.9
4.93
8.0
4.05
3.8
4.47
0.9
4.95
8.0
5.19
3.7
4.26
0.8
4.85
8.0
4.02
3.8
4.32
0.9
4.88
8.0
5.07
3.8
4.15
0.9
aFor solns in CDCl3, unless otherwise stated.
b3:1 CD3OD–CDCl3.
cD2O.
dValues in bold type reflect the locations of the sulfate groups.
3.3. 4-Methoxyphenyl 2-O-benzoyl-4,6-O-benzylidene-b-
from MeOH–pyridine to give 3 (3.14 g, 82%); mp 254–
1
22
D
D
-galactopyranoside (3)
256 ꢁC; ½aꢁ )3ꢁ (c 1, pyridine); H NMR (250 MHz,
CDCl3): carbohydrate ring protons (see Table 1);
d 8.10–6.70 (m, 14H, aromatic H), 5.65 (s, 1H, PhCH),
3.75 (s, 3H, OCH3), 2.70 (d, 1H, J 10.0 Hz, HO-3);
ISMS: m=z 501 [M+Na]þ, 479 [M+H]þ. Anal. Calcd
for C27H26O8: C, 67.77; H, 5.48. Found: C, 67.70; H,
5.39.
A soln of 2 (3.83 g, 8 mmol) in acetone (80 mL) was
treated at 0 ꢁC with NaOH (0.05 M, 80 mL), and the
mixture was vigorously stirred for 3 min at this tem-
perature. The copious precipitate was collected by fil-
tration, washed well with cold water, and recrystallized

354
J.-C. Jacquinet / Carbohydrate Research 339 (2004) 349–359
RO
BzO
OBz
BzO
HO
RO
OSO3Na
O
OR
OR
O
O
O
RO
O
O
OMP
OMP
OMP
a
c
BzO
BzO
RO
RO
15 R = Bz
16 R = H
11
b
10 R = Lev
11 R = H
a, b
R1O
RO
OR2
OR
O
O
RO
O
RO
RO
HO
BzO
OH
OH
HO
c, a
17 R = R2 = Bz, R1 = H
18 R = R2 = Bz, R1 = SO3Na
19 R = R2 = H, R1 = SO3Na
O
O
d
b
9
O
OMP
BzO
BzO
12
HO
HO
OH
OH
HO
O
O
c, b
OR1
BzO
BzO
O
OBz
RO
OMP
OMP
b
a
HO
20
HO
O
O
O
OMP
BzO
12
BzO
HO
RO
HO OSO3Na
O
OSO3Na
O
13 R, R1 = PhCH
14 R = R1 = H
O
RO
RO
Scheme 3. Reagents and conditions: (a) 90% TFA, rt, 15 min; (b)
PhCOCl, pyridine, rt, 16 h; (c) hydrazine acetate, pyridine, rt, 8 min.
21 R = Bz
22 R = H
b
RO
RO
OR
HO
OSO3Na
O
O
3.4. 4-Methoxyphenyl 2,3-di-O-benzoyl-4,6-O-benzyl-
idene-b-D-galactopyranoside (4)
O
a
c
OMP
RO
RO
14
23 R = Bz
24 R = H
A mixture of 1 (1.82 g, 4.9 mmol) and benzoyl chloride
(1.82 mL, 15.7 mmol) in anhyd pyridine (20 mL) was
stirred for 1 h at 0 ꢁC. Methanol (2 mL) was added, and
the mixture was diluted with CH2Cl2 (100 mL), washed
with water, satd aq NaHCO3, and water, dried (MgSO4)
and concentrated. The residue was crystallized from
b
R1O
OR2
O
RO
RO
OR
O
O
OMP
RO
RO
EtOAc–petroleum ether to afford 4 (2.64 g, 92%); mp
25 R = R2 = Bz, R1 = H
26 R = R2 = Bz, R1 = SO3Na
27 R = R2 = H, R1 = SO3Na
22
D
196–197 ꢁC; ½aꢁ
+121ꢁ (c 1, CHCl3); 1H NMR
d
b
(250 MHz, CDCl3): carbohydrate ring protons (see Ta-
ble 1); d 8.10–6.70 (m, 19H, aromatic H), 5.58 (s, 1H,
PhCH), 3.75 (s, 3H, OCH3); ISMS: m=z 605 [M+Na]þ.
Anal. Calcd for C34H30O9: C, 70.09; H, 5.08. Found: C,
70.15; H, 5.08.
Scheme 4. Reagents and conditions: (a) Me3NÆSO3 (1.5 equiv), DMF,
40 ꢁC, 90 min; then ion-exchange resin [Na]þ; (b) 4 M NaOH, MeOH,
rt, 2 h; (c) PhCOCN, pyridine, rt, 16 h; (d) Me3NÆSO3 (10 equiv),
DMF, 60 ꢁC, 72 h.
3.5. 4-Methoxyphenyl 2,3-di-O-benzoyl-4,6-di-O-levuli-
noyl-b-D-galactopyranoside (5)
tered off, washed with CH2Cl2, and the filtrate was
washed with cold 0.1 M HCl, satd aq NaHCO3, and
water, dried (MgSO4) and concentrated. A soln of the
residue in 1:1 toluene–EtOAc was filtered through a pad
of Celite, and the clear filtrate was concentrated. The
A soln of 4 (2.65 g, 4.55 mmol) in 9:1 TFA–water
(15 mL) was stirred for 15 min at rt, then was concen-
trated, evaporated with water (3 · 10 mL), and dried
under diminished pressure. A soln of the residue, levu-
linic acid (1.31 g, 11.2 mmol) and DMAP (122 mg,
1 mmol) in anhyd CH2Cl2 (40 mL) was treated por-
tionwise with DCC (2.32 g, 11.2 mmol), and the mixture
was stirred for 2 h at rt. The precipitated DCU was fil-
residue was crystallized from EtOAc–petroleum ether to
22
D
give 5 (2.74 g, 87% from 4); mp 128–129 ꢁC; ½aꢁ +72ꢁ (c
1
1, CHCl3); H NMR (250 MHz, CDCl3): carbohydrate
ring protons (see Table 1); d 8.0–6.70 (m, 14H, aromatic

J.-C. Jacquinet / Carbohydrate Research 339 (2004) 349–359
355
Table 3. 13C NMR data (62.8 MHz, D2O, 25 ꢁC) for sulfoforms 16, 19, 22, 24, 27, and their nonsulfated congener 20a
16
19
20
22
24
27
C-1I
C-2I
C-3I
C-4I
C-5I
C-6I
C-1II
C-2II
C-3II
C-4II
C-5II
C-6II
ArC
101.62
69.77
82.76
68.46
75.35
61.19
104.38
71.09
72.60
68.55
72.83
67.65
154.82
151.14
118.38
115.21
55.96
101.69
71.31
82.39
68.45
75.16
60.88
104.47
71.74
69.96
76.92
74.55
61.13
154.85
151.17
118.42
115.21
55.96
101.66
69.97
82.30
68.50
75.21
60.91
104.58
71.28
72.75
68.80
75.32
61.13
154.91
151.16
118.42
115.25
55.96
101.53
69.74
82.32
68.42
72.73
67.29
104.39
71.10
72.55
68.42
73.05
67.93
154.81
151.24
118.31
115.22
55.96
101.62
69.82
82.15
68.13
72.77
67.19
104.59
71.25
71.73
68.78
75.24
61.12
154.84
151.21
118.34
115.22
55.96
101.78
72.81
78.46
75.57
74.64
60.96
104.29
70.42
71.24
69.10
75.35
61.30
154.93
151.11
118.50
115.21
55.96
OCH3
aBold type values reflect the positions of sulfations.
OR1
HO
HO
BzO
OR
Anal. Calcd for C29H24Cl3NO8: C, 56.10; H, 3.90; N,
2.26. Found: C, 56.01; H, 3.98; N, 2.15.
O
O
a
O
OMP
21
12
+
BzO
BzO
28 R = SO3Na, R1 = H
29 R = H, R1 = SO3Na
3.7. 2,3-Di-O-benzoyl-4,6-di-O-levulinoyl-1-O-trichloro-
acetimidoyl-a-D-galactopyranose (7)
Scheme 5. Reagents and conditions: (a) Me3NÆSO3 (2.5 equiv), DMF,
40 ꢁC, 40 min; then ion-exchange resin [Na]þ.
A mixture of 5 (1.38 g, 2 mmol) and CAN (5.48 g,
10 mmol) was treated for 15 min at rt as described for
the preparation of 6. Flash chromatography of the res-
idue on a column of silica gel with 1:1 toluene–EtOAc
gave the corresponding free hemiacetal (950 mg).
The above isolated hemiacetal was treated as de-
scribed for the preparation of 5. Flash chromatography
of the residue on a column of silica gel with 2:1 toluene–
EtOAc containing 0.2% of Et3N followed by crystalli-
H), 3.75 (s, 3H, OCH3), 2.60 (m, 8H, CH2CO), 2.20,
2.10 (2s, 6H, COCH3); ISMS: m=z 714 [M+Na]þ. Anal.
Calcd for C37H38O13: C, 64.34; H, 5.54. Found: C, 64.31;
H, 5.52.
3.6. 2,3-Di-O-benzoyl-4,6-O-benzylidene-1-O-trichloro-
-galactopyranose (6)
acetimidoyl-a-
D
zation of the residue from diethyl ether afforded 7
22
(1.01 g, 69% from 6); mp 106–107 ꢁC; ½aꢁ +110ꢁ (c 1,
D
A mixture of 4 (0.58 g, 1 mmol) and CAN (1.65 g,
3 mmol) in 1:1.5:1 toluene–MeCN–water (17.5 mL) was
stirred for 15 min at 0 ꢁC, then was diluted with EtOAc
(50 mL), washed with water, satd aq NaHCO3, and
water, dried (MgSO4) and concentrated. Flash chro-
matography of the residue on a column of silica gel with
9:1 CH2Cl2–EtOAc gave the corresponding free hemi-
acetal (250 mg).
CHCl3); 1H NMR (250 MHz, CDCl3): carbohydrate
ring protons (see Table 1); d 8.60 (s, 1H, C ¼ NH), 8.0–
7.30 (m, 10H, Ph), 2.60 (m, 8 H, CH2CO), 2.20, 2.10 (2s,
6H, COCH3); ISMS: m=z 752 [M+Na]þ for 35Cl. Anal.
Calcd for C32H32Cl3NO12: C, 52.72; H, 4.42; N, 1.92.
Found: C, 52.63; H, 4.51; N, 1.78.
A mixture of the above isolated hemiacetal, CCl3CN
(0.5 mL, 5 mmol), and DBU (20 lL, 0.13 mmol) in an-
hyd CH2Cl2 (3 mL) was stirred for 30 min at rt, then was
concentrated. Flash chromatography of the residue on a
column of silica gel with 2:1 petroleum ether–EtOAc
containing 0.2% of Et3N gave 6 (303 mg, 48% from 4) as
3.8. 4-Methoxyphenyl O-(2,3-di-O-benzoyl-4,6-O-benzyl-
idene-a- (8a) and b-
D
-galactopyranosyl)-(1 ! 3)-2-O-
benzoyl-4,6-O-benzylidene-b-
D
-galactopyranoside (8b)
A mixture of imidate 5 (250 mg, 0.4 mmol), alcohol 3
ꢁ
(143 mg, 0.3 mmol), and 4 A powdered molecular sieves
(200 mg) in anhyd CH2Cl2 (4 mL) was stirred for 30 min
under dry Ar. A soln of Me3SiOTf in toluene (1 M,
64 lL) was added, and the mixture was stirred for
30 min. Triethylamine (0.1 mL) was added, and the
22
1
a colorless syrup; ½aꢁ +165ꢁ (c 1, CHCl3); H NMR
D
(250 MHz, CDCl3): carbohydrate ring protons (see Ta-
ble 1); d 8.60 (s, 1H, C@NH), 8.0–7.20 (m, 15H, Ph),
5.60 (s, 1H, PhCH); ISMS: m=z 644 [M+Na]þ for 35Cl.

356
J.-C. Jacquinet / Carbohydrate Research 339 (2004) 349–359
mixture was filtered and concentrated. Flash chroma-
tography of the residue on a column of silica gel with
20:1 CH2Cl2–EtOAc containing 0.1% of Et3N gave first
2.70 (m, 8H, CH2CO), 2.20, 2.05 (2s, 6H, COCH3);
ISMS: m=z 1188 [M+Na]þ. Anal. Calcd for C71H64O22:
C, 65.97; H, 5.19. Found: C, 65.91; H, 5.17.
8a (87 mg, 31%); mp 255–257 ꢁC (from EtOAc–petro-
22
leum ether); ½aꢁ +200ꢁ (c 1, CHCl3); 1H NMR
3.11. 4-Methoxyphenyl O-(2,3-di-O-benzoyl-b-
D
-galacto-
D-galactopyran-
D
(250 MHz, CDCl3): carbohydrate ring protons (see Ta-
ble 2); d 8.10–6.70 (m, 29H, aromatic H), 5.33, 5.03 (2s,
2H, PhCH), 3.72 (s, 3H, OCH3); ISMS: m=z 960
[M+Na]þ. Anal. Calcd for C54H48O15: C, 69.22; H, 5.16.
Found: C, 69.12; H, 5.26.
pyranosyl)-(1 ! 3)-2,4,6-tri-O-benzoyl-b-
oside (11)
A soln of 10 (1.07 g, 0.92 mmol) in pyridine (10 mL) was
treated for 8 min at rt with a freshly prepared mixture of
pyridine (12 mL), HOAc (8 mL), and hydrazine hydrate
(1 mL), then was diluted with CH2Cl2 (100 mL), washed
with water, brine, and water, dried (MgSO4) and con-
Next eluted was 8b (90 mg, 32%); mp 310–312 ꢁC
22
(from MeOH–CH2Cl2); ½aꢁ +94ꢁ (c 1, CHCl3); 1H
D
NMR (250 MHz, CDCl3): carbohydrate ring protons
(see Table 2); d 8.10–6.70 (m, 29H, aromatic H), 5.50,
5.44 (2s, 2H, PhCH), 3.72 (s, 3H, OCH3); ISMS: m=z
960 [M+Na]þ. Anal. Calcd for C54H48O15: C, 69.22; H,
5.16. Found: C, 69.28; H, 5.21.
centrated. Crystallization of the residue from hot EtOH
22
gave 11 (802 mg, 90%); mp 218–219 ꢁC; ½aꢁ +91ꢁ (c 1,
D
CHCl3); 1H NMR (250 MHz, 3:1 CD3OD–CDCl3):
carbohydrate ring protons (see Table 2); d 8.10–6.70 (m,
29H, aromatic H), 3.67 (s, 3H, OCH3); ISMS: m=z 992
[M+Na]þ. Anal. Calcd for C54H48O17: C, 66.94; H, 4.99.
Found: C, 66.81; H, 5.04.
3.9. 4-Methoxyphenyl O-(2,3-di-O-benzoyl-4,6-di-O-lev-
-galactopyranosyl)-(1 ! 3)-2-O-benzoyl-4,6-
ulinoyl-b-
D
O-benzylidene- b-
D
-galactopyranoside (9)
3.12. 4-Methoxyphenyl O-(2,3-di-O-benzoyl-b-
D
-galacto-
pyranosyl)-(1 ! 3)-2-O-benzoyl-b-
D-galactopyranoside
A mixture of imidate 7 (520 mg, 0.7 mmol) and alcohol 3
(263 mg, 0.55 mmol) was treated as described for the
preparation of 8a,b. Flash chromatography of the resi-
due on a column of silica gel with 1:1 toluene–EtOAc
containing 0.1% of Et3N and crystallization from
EtOAc–petroleum ether gave 9 (403 mg, 70%); mp 177–
(12)
Compound 9 (1.05 g, 1 mmol) was treated as described
for the preparation of 11 to give the intermediate diol as
a sparingly soluble solid. A suspension of this solid in
9:1 TFA–water (15 mL) was stirred for 15 min at rt, and
the resulting clear soln was then concentrated, and
evaporated with water (3 · 10 mL). The solid residue was
22
1
178 ꢁC; ½aꢁ +54ꢁ (c 1, CHCl3); H NMR (250 MHz,
D
CDCl3): carbohydrate ring protons (see Table 2); d
8.10–6.70 (m, 24H, aromatic H), 5.62 (s, 1H, PhCH),
3.70 (s, 3H, OCH3), 2.70 (m, 8H, CH2CO), 2.20, 2.05
(2s, 6H, COCH3); ISMS: m=z 1063 [M+Na]þ. Anal.
Calcd for C57H56O19: C, 65.51; H, 5.40. Found: C, 65.62;
H, 5.31.
recrystallized from MeOH–pyridine to give 12 (617 mg,
22
D
81%); mp 244–246 ꢁC; ½aꢁ +40ꢁ (c 1, pyridine); 1H
NMR (250 MHz, 3:1 CD3OD–CDCl3): carbohydrate
ring protons (see Table 2); d 8.10–6.70 (m, 19H, aro-
matic H), 3.70 (s, 3H, OCH3); ISMS: m=z 783 [M+Na]þ.
Anal. Calcd for C40H40O15: C, 63.15; H, 5.30. Found: C,
62.98; H, 5.41.
3.10. 4-Methoxyphenyl O-(2,3-di-O-benzoyl-4,6-di-O-
levulinoyl-b-
D
-galactopyranosyl)-(1 ! 3)-2,4,6-tri-O-
benzoyl-b- -galactopyranoside (10)
D
3.13. 4-Methoxyphenyl O-(2,3,4,6-tetra-O-benzoyl-b-
galactopyranosyl)-(1 ! 3)-2-O-benzoyl-4,6-O-benzyl-
D-
A soln of 9 (1.40 g, 1.34 mmol) in 9:1 TFA–water
(15 mL) was stirred for 15 min at rt, then was concen-
trated, evaporated with water (3 · 10 mL), and dried
under diminished pressure. A mixture of the residue and
benzoyl chloride (0.6 mL, 5 mmol) in anhyd pyridine
(10 mL) was stirred overnight at rt. Methanol (1 mL)
was added, and the mixture was diluted with CH2Cl2
(100 mL), washed with water, satd aq NaHCO3, and
water, dried (MgSO4) and concentrated. Flash chro-
matography of the residue on a column of silica gel with
idene-b-D-galactopyranoside (13)
Compound 9 (1.05 g, 1 mmol) was treated as described
for the preparation of 11. Conventional benzoylation
(benzoyl chloride in pyridine) of the resulting diol fol-
lowed by flash chromatography on a column of silica gel
with 5:1 toluene–EtOAc and crystallization of the resi-
due from hot EtOH afforded 13 (930 mg, 88%); mp 158–
22
D
1
160 ꢁC; ½aꢁ +92ꢁ (c 1, CHCl3); H NMR (250 MHz,
CDCl3): carbohydrate ring protons (see Table 2);
d 8.10–6.70 (m, 34H, aromatic H), 5.53 (s, 1H, PhCH),
3.71 (s, 3H, OCH3); ISMS: m=z 1070 [M+Na]þ. Anal.
Calcd for C61H52O17: C, 69.31; H, 4.96. Found: C, 69.40;
H, 5.02.
3:2 toluene–EtOAc gave 10 (1.36 g, 87%) as a white
22
D
foam; ½aꢁ +81ꢁ (c 1, CHCl3); 1H NMR (250 MHz,
CDCl3): carbohydrate ring protons (see Table 2);
d 8.10–6.70 (m, 29H, aromatic H), 3.67 (s, 3H, OCH3),

J.-C. Jacquinet / Carbohydrate Research 339 (2004) 349–359
357
3.14. 4-Methoxyphenyl O-(2,3,4,6-tetra-O-benzoyl-b-
D
-
3.17. 4-Methoxyphenyl O-(2,3,6-tri-O-benzoyl-b-
D
-
galactopyranosyl)-(1 ! 3)-2-O-benzoyl-b-
D
-galacto-
galactopyranosyl)-(1 ! 3)-2,4,6-tri-O-benzoyl-b-
D
-
pyranoside (14)
galactopyranoside (17)
A soln of 13 (2.11 g, 2 mmol) in 9:1 TFA–water (20 mL)
was stirred for 20 min at rt, then was concentrated, and
evaporated with water (2 · 10 mL). Recrystallization of
A mixture of 11 (388 mg, 0.4 mmol) and benzoyl cyanide
(106 mg, 0.8 mmol) in anhyd pyridine (8 mL) was stirred
overnight at rt. Methanol (0.5 mL) was added, and the
mixture was concentrated. Flash chromatography of the
the residue from hot EtOH gave 14 (1.77 g, 92%); mp
22
D
230–232 ꢁC; ½aꢁ
+13ꢁ (c 1, CHCl3); 1H NMR
residue on a column of silica gel with 6:1 toluene–EtOAc
22
(250 MHz, 3:1 CD3OD–CDCl3): carbohydrate ring
protons (see Table 2); d 8.10–6.70 (m, 29H, aromatic H),
3.68 (s, 3H, OCH3); ISMS: m=z 992 [M+Na]þ. Anal.
Calcd for C54H48O17: C, 66.94; H, 4.99. Found: C, 66.82,
H, 5.06.
gave 17 (374 mg, 87%) as a white foam; ½aꢁ +74ꢁ (c 1,
D
CHCl3); 1H NMR (250 MHz, 3:1 CD3OD–CDCl3):
carbohydrate ring protons (see Table 2); d 8.10–6.70 (m,
34H, aromatic H), 3.67 (s, 3H, OCH3); ISMS: m=z 1096
[M+Na]þ. Anal. Calcd for C61H52O18: C, 68.28; H, 4.88.
Found: C, 68.32; H, 4.80.
3.15. 4-Methoxyphenyl O-(2,3-di-O-benzoyl-6-O-sodium
-galactopyranosyl)-(1 ! 3)-2,4,6-tri-O-
3.18. 4-Methoxyphenyl O-(2,3,6-tri-O-benzoyl-4-O-
sodium sulfonato-b-
sulfonato-b-
D
D
-galactopyranosyl)-(1 ! 3)-2,4,6-
benzoyl-b- -galactopyranoside (15)
D
tri-O-benzoyl-b- -galactopyranoside (18)
D
A mixture of 11 (388 mg, 0.4 mmol) and sulfur trioxide–
trimethylamine complex (84 mg, 0.6 mmol) in anhyd
DMF (10 mL) was stirred for 90 min at 40 ꢁC, then was
cooled. Methanol (0.5 mL) was added, and the mixture
was concentrated. Flash chromatography of the residue
on a column of silica gel with 9:1 CH2Cl2–MeOH con-
taining 2% of Et3N followed by elution from a column
(1.5 · 25 cm) of Sephadex SP C25 [Naþ] with 9:5:1
A mixture of 17 (280 mg, 0.26 mmol) and sulfur triox-
ide–trimethylamine complex (350 mg, 2.6 mmol) in
anhyd DMF (5 mL) was stirred for 72 h at 60 ꢁC, then was
cooled and treated as described for the preparation of 15
22
D
to give 18 (284 mg, 93%) as a white powder; ½aꢁ +78ꢁ (c
1
1, MeOH); H NMR (250 MHz, 3:1 CD3OD–CDCl3):
carbohydrate ring protons (see Table 2); d 8.10–6.70 (m,
34H, aromatic H), 3.63 (s, 3H, OCH3); ISMS: m=z 1198
[M+Na]þ. Anal. Calcd for C61H51NaO21S: C, 62.35; H,
4.37; S, 2.73. Found: C, 62.12; H, 4.51; S, 2.54.
CH2Cl2–MeOH–water afforded 15 (352 mg, 82%) as a
22
D
white powder; ½aꢁ +80ꢁ (c 1, MeOH); 1H NMR
(250 MHz, 3:1 CD3OD–CDCl3): carbohydrate ring
protons (see Table 2); d 8.10–6.70 (m, 29H, aromatic H),
3.63 (s, 3H, OCH3); ISMS: m=z 1093 [M+Na]þ. Anal.
Calcd for C54H47NaO20S: C, 60.56; H, 4.05; S, 2.99.
Found: C, 60.30; H, 4.21; S, 2.80.
3.19. 4-Methoxyphenyl O-(4-O-sodium sulfonato-b-
D-
galactopyranosyl)-(1 ! 3)-b- -galactopyranoside (19)
D
Compound 18 (240 mg, 0.2 mmol) was treated as de-
scribed for the preparation of 16 to give 19 (100 mg,
3.16. 4-Methoxyphenyl O-(6-O-sodium sulfonato-b-
D-
22
D
1
89%) as a white powder; ½aꢁ )5.5ꢁ (c 1, water); H
galactopyranosyl)-(1 ! 3)-b- -galactopyranoside (16)
D
NMR (500 MHz, D2O, internal acetone, dH 2.225):
carbohydrate ring protons (see Table 2); d 6.95 (m, 4H,
aromatic H), 3.70 (s, 3H, OCH3); 13C NMR (D2O,
internal acetone, dC 30.45): see Table 3. Anal. Calcd for
C19H27NaO15S: C, 41.46; H, 4.94; S, 5.82. Found: C,
41.18; H, 5.15; S, 5.61.
A soln of 15 (320 mg, 0.3 mmol) in MeOH (8 mL)
was treated for 2 h at rt with NaOH (4 M, 1.5 mL), then
was diluted with water (8 mL). The pH of the soln was
brought to 3.5 (pH meter control) with Amberlite IR-
120 [Hþ] resin, and the mixture was filtered, concen-
trated and dried under diminished pressure. Benzoic
acid was extracted with cold abs EtOH (2 · 5 mL), and
the residue was dissolved in water (5 mL). The soln was
brought to pH 7 with 0.1 M NaOH, then was eluted
from a column (1.5 · 80 cm) of Sephadex LH-20 with
3.20. 4-Methoxyphenyl O-(b-
D-galactopyranosyl)-
(1 ! 3)-b- -galactopyranoside (20)
D
A soln of 12 (228 mg, 0.3 mmol) in MeOH (10 mL) was
treated for 2 h with methanolic MeONa (1 M, 1 mL),
then was deionized with Amberlite IR-120 [Hþ] resin,
filtered, and concentrated. Crystallization of the residue
water to give 16 (148 mg, 90%) as a white hygroscopic
22
D
powder; ½aꢁ )7ꢁ (c 1, water); 1H NMR (500 MHz, D2O,
internal acetone, dH 2.225): carbohydrate ring protons
(see Table 2); d 6.95 (m, 4H, aromatic H), 3.70 (s, 3H,
OCH3); 13C NMR (D2O, internal acetone, dC 30.45): see
Table 3. Anal. Calcd for C19H27NaO15S: C, 41.46; H,
4.94; S, 5.82. Found: C, 41.21; H, 5.11; S, 5.70.
from MeOH gave 20 (122 mg, 91%); mp 192–193 ꢁC;
22
½aꢁ )6ꢁ (c 1, water); 1H NMR (500 MHz, D2O, internal
D
acetone, dH 2.225): carbohydrate ring protons (see Table
2); d 6.95 (m, 4H, aromatic H), 3,69 (s, 3H, OCH3); 13C

358
J.-C. Jacquinet / Carbohydrate Research 339 (2004) 349–359
NMR (D2O, internal acetone, dC 30.45): see Table 3.
Anal. Calcd for C19H28O12: C, 50.89; H, 6.29. Found: C,
50.77; H, 6.33.
(see Table 2); d 6.95 (m, 4H, aromatic H), 3.70 (s, 3H,
OCH3); 13C NMR (D2O, internal acetone, dC 30.45): see
Table 3. Anal. Calcd for C19H27NaO15S: C, 41.46; H,
4.94; S, 5.82. Found: C, 41.25; H, 5.07; S, 9.60.
3.21. 4-Methoxyphenyl O-(2,3-di-O-benzoyl-6-O-sodium
sulfonato-b-
D
-galactopyranosyl)-(1 ! 3)-2-O-benzoyl-6-
3.25. 4-Methoxyphenyl O-(2,3,4,6-tetra-O-benzoyl-b-D-
O-sodium sulfonato-b-
D
-galactopyranoside (21)
galactopyranosyl)-(1 ! 3)-2,6-di-O-benzoyl-b-
D-galacto-
pyranoside (25)
A mixture of 12 (228 mg, 0.3 mmol) and sulfur trioxide–
trimethylamine complex (120 mg, 0.9 mmol) in anhyd
DMF (8 mL) was stirred for 90 min at 40 ꢁC, then was
treated as described for the preparation of 15. Recrys-
Compound 14 (436 mg, 0.45 mmol) was treated as de-
scribed for the preparation of 17. Crystallization of the
residue from EtOAc–petroleum ether gave 25 (426 mg,
22
tallization of the solid residue from aq MeOH gave 21
88%); mp 240–242 ꢁC; ½aꢁ +119ꢁ (c 1, CHCl3); 1H
D
22
(232 mg, 80%); mp 232–234 ꢁC; ½aꢁ +70ꢁ (c 0.5,
NMR (250 MHz, 3:1 CD3OD–CDCl3): carbohydrate
ring protons (see Table 2); d 8.10–6.70 (m, 34H, aro-
matic H), 3.67 (s, 3H, OCH3); ISMS: m=z 1096
[M+Na]þ. Anal. Calcd for C62H52O18: C, 68.28; H, 4.88.
Found: C, 68.21; H, 4.82.
D
MeOH); 1H NMR (250 MHz, 3:1 CD3OD–CDCl3):
carbohydrate ring protons (see Table 2); d 8.10–6.70 (m,
19H, aromatic H), 3.63 (s, 3H, OCH3); ISMS: m=z 988
[M+Na]þ. Anal. Calcd for C40H38Na2O21S2: C, 49.80;
H, 3.57; S, 6.65. Found: C, 49.52; H, 3.74; S, 6.41.
3.26. 4-Methoxyphenyl O-(2,3,4,6-tetra-O-benzoyl-b-
galactopyranosyl)-(1 ! 3)-2,6-di-O-benzoyl-4-O-sodium
sulfonato-b- -galactopyranoside (26)
D-
3.22. 4-Methoxyphenyl O-(6-O-sodium sulfonato-b-
D
-
galactopyranosyl)-(1 ! 3)-6-O-sodium sulfonato-b-
D
-
D
galactopyranoside (22)
Compound 25 (322 mg, 0.3 mmol) was treated as de-
scribed for the preparation of 18 to give 26 (317 mg,
Compound 21 (410 mg, 0.42 mmol) was treated as
described for the preparation of 16 to give 22 (252 mg,
22
D
1
90%) as a white powder; ½aꢁ +93ꢁ (c 1, MeOH); H
22
D
91%) as a white hygroscopic powder; ½aꢁ )11ꢁ (c 1,
NMR (250 MHz, 3:1 CD3OD–CDCl3): carbohydrate
ring protons (see Table 2); d 8.10–6.70 (m, 34H, aro-
matic H), 3.66 (s, 3H, OCH3); ISMS: m=z 1198
[M+Na]þ. Anal. Calcd for C61H51NaO21S: C, 62.35; H,
4.37; S, 2.73. Found: C, 62.15; H, 4.51; S, 2.52.
1
water); H NMR (500 MHz, D2O, internal acetone, dH
2.225): carbohydrate ring protons (see Table 2); d 6.95
(m, 4H, aromatic H), 3.71 (s, 3H, OCH3); 13C NMR
(D2O, internal acetone, dC 30.45): see Table 3. Anal.
Calcd for C19H26Na2O18S2: C, 34.97; H, 4.02; S, 9.83.
Found: C, 34.70; H, 4.21; S, 9.58.
3.27. 4-Methoxyphenyl O-(b-
(1 ! 3)-4-O-sodium sulfonato-b-
D
-galactopyranosyl)-
-galactopyranoside (27)
D
3.23. 4-Methoxyphenyl O-(2,3,4,6-tetra-O-benzoyl-b-
galactopyranosyl)-(1 ! 3)-2-O-benzoyl-6-O-sodium sul-
fonato-b- -galactopyranoside (23)
D-
Compound 26 (258 mg, 0.22 mmol) was treated as de-
scribed for the preparation of 16 to give 27 (109 mg,
D
22
D
90%) as a white hygroscopic powder; ½aꢁ )5ꢁ (c 1,
1
Compound 14 (388 mg, 0.4 mmol) was treated as de-
scribed for the preparation of 16. Crystallization of the
residue from EtOH gave 23 (356 mg, 83%); mp 190–
water); H NMR (500 MHz, D2O, internal acetone, dH
2.225): carbohydrate ring protons (see Table 2); d 6.95
(m, 4H, aromatic H), 3.70 (s, 3H, OCH3); 13C NMR
(D2O, internal acetone, dC 30.45): see Table 3. Anal.
Calcd for C19H27NaO15S: C, 41.46; H, 4.94; S, 5.82.
Found: C, 41.21; H, 5.12; S, 5.59.
22
D
1
192 ꢁC; ½aꢁ +108ꢁ (c 1, MeOH); H NMR (250 MHz,
3:1 CD3OD–CDCl3): carbohydrate ring protons (see
Table 2); d 8.10–6.70 (m, 29H, aromatic H), 3.66 (s, 3H,
OCH3); ISMS: m=z 1093 [M+Na]þ. Anal. Calcd for
C54H47NaO20S: C, 60.56; H, 4.05; S, 2.99. Found: C,
60.48; H, 4.12; S, 2.79.
3.28. Controlled O-sulfonation of tetrol 12
A mixture of 12 (152 mg, 0.2 mmol) and sulfur trioxide–
trimethylamine complex (66 mg, 0.5 mmol) in anhyd
DMF (6 mL) was stirred at 40 ꢁC. After 40 min, TLC
(5:1 CH2Cl2–MeOH) showed optimal formation of
intermediate monosulfates. The mixture was then
cooled, quenched with MeOH (0.5 mL), and concen-
trated. Flash chromatography of the residue on a col-
umn of silica gel with 7:1 CH2Cl2–MeOH containing 2%
of Et3N gave first the starting material (8 mg, 5%).
3.24. 4-Methoxyphenyl O-(b-
(1 ! 3)-6-O-sodium sulfonato-b-
D
-galactopyranosyl)-
-galactopyranoside (24)
D
Compound 23 (321 mg, 0.3 mmol) was treated as de-
scribed for the preparation of 16. Crystallization of the
residue from aq MeOH gave 24 (150 mg, 91%); mp 226–
22
228 ꢁC; ½aꢁ )9ꢁ (c 1, water); 1H NMR (500 MHz, D2O,
D
internal acetone, dH 2.225): carbohydrate ring protons

J.-C. Jacquinet / Carbohydrate Research 339 (2004) 349–359
359
2. Sugahara, K.; Yamashina, I.; De Waard, P.; Van Halbeek,
H.; Vliegenthart, J. F. G. J. Biol. Chem. 1988, 263, 10168–
10174.
3. Yamada, S.; Oyama, M.; Yuki, Y.; Kato, K.; Sugahara,
K. Eur. J. Biochem. 1995, 233, 687–693.
Next eluted was a second fraction that was submitted
to ion-exchange as described previously. Crystallization
of the residue from MeOH gave the 6I-sulfate 28 (45 mg,
22
D
26%); mp 225–228 ꢁC; ½aꢁ +91ꢁ (c 1, MeOH); 1H NMR
(250 MHz, 3:1 CD3OD–CDCl3): carbohydrate ring
protons (see Table 2); d 8.10–6.70 (m, 19H, aromatic H),
3.66 (s, 3H, OCH3); ISMS: m=z 886 [M+Na]þ. Anal.
Calcd for C40H39NaO18S: C, 55.68; H, 4.56. Found: C,
55.39; H, 4.72.
4. Kitagawa, H.; Oyama, M.; Masayama, K.; Yamaguchi,
Y.; Sugahara, K. Glycobiology 1997, 7, 1175–1180.
5. Oegama, T. R.; Kraft, E. L.; Jourdian, G. W.; Van Valen,
T. R. J. Biol. Chem. 1984, 259, 1720–1726.
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6. Fransson, L.-A.; Silverberg, I.; Carlstedt, J. J. Biol. Chem.
1985, 260, 14722–14726.
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7. Moses, J.; Oldberg, A.; Cheng, F.; Fransson, L.-A. Eur. J.
Biochem. 1997, 248, 521–526.
Further elution gave a third fraction that was treated
similarly to give the 6II-sulfate 29 (41 mg, 24%) as a
22
D
white powder; ½aꢁ +78ꢁ (c 1, MeOH); 1H NMR
8. Ouzzine, M.; Gulberti, S.; Netter, P.; Magdalou, J.;
Fournel-Gigleux, S. J. Biol. Chem. 2000, 275, 28254–
28260.
9. Pedersen, L. C.; Tsuchida, K.; Kitagawa, H.; Sugahara,
K.; Darden, T. A.; Negishi, M. J. Biol. Chem. 2000, 275,
34580–34585.
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(250 MHz, 3:1 CD3OD–CDCl3): carbohydrate ring
protons (see Table 2); d 8.10–6.80 (m, 19H, aromatic H),
3.67 (s, 3H, OCH3); ISMS: m=z 886 [M+Na]þ. Anal.
Calcd for C40H39NaO18S: C, 55.68; H, 4.56. Found: C,
55.42; H, 4.75.
Elution with 4:1 CH2Cl2–MeOH gave a fourth frac-
tion that was submitted to the same treatment to give
22
D
the disulfate 21 (78 mg, 40%), mp 231–233 ꢁC; ½aꢁ +69ꢁ
12. Tamura, J.-I.; Nishihara, J. J. Org. Chem. 2001, 66, 3074–
3083.
13. Ohlsson, J.; Magnusson, G. Carbohydr. Res. 2000, 329,
49–55.
(c 0.8, MeOH).
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Acknowledgements
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15. Belot, F.; Jacquinet, J.-C. Carbohydr. Res. 2000, 325, 93–
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The author thanks Mr. J. Blu for valuable technical
assistance. This work was supported by a grant-in-aid
from CNRS–INSERM–MRT through the ACI 2001
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ꢀ
ꢀ
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