Syntheses of chondroitin 4- and 6-sulfate pentasaccharide derivatives having a methyl β-D-glucopyranosiduronic acid at the reducing end

Article abstract of DOI:10.1016/S0008-6215(00)00032-X

The syntheses are reported of β-D-GlcpA-(1→3)-β-D-GalpNAc-(1→4)-β-D-GlcpA-(1→3)-β-D-GalpNAc-(1→4)-β-D-GlcpA-(1→OMe), O-sulfonated at C-4 or C-6 of the aminosugar moieties, which represent structural elements of chondroitin 4- and 6-sulfate proteoglycans. Starting from a synthetic disaccharide glycosyl acceptor, the stepwise or blockwise construction of the sugar backbone with appropriate synthons led to a pentasaccharide tetraol, which was used as a common intermediate. Selective 6-O-sulfonation of this tetraol, followed by saponification, gave the 6-sulfate derivative, whereas selective 6-O-benzoylation, followed by O-sulfonation and saponification, afforded the 4-sulfate derivative as their sodium salts. Copyright (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0008-6215(00)00032-X

www.elsevier.nl/locate/carres
Carbohydrate Research 326 (2000) 88–97
Syntheses of chondroitin 4- and 6-sulfate
pentasaccharide derivatives having a methyl
-glucopyranosiduronic acid at the reducing end
b-D
Fre´de´ric Be´lot, Jean-Claude Jacquinet *
Institut de Chimie Organique et Analytique, UPRES-A CNRS 6005, UFR Faculte´ des Sciences,
Uni6ersite´ d’Orle´ans, BP 6759, F-45067 Orle´ans, France
Received 25 November 1999; accepted 13 January 2000
Abstract
The syntheses are reported of b-
D
-GlcpA-(1“3)-b-
D
-GalpNAc-(1“4)-b-
D
-GlcpA-(1“3)-b-
D
-GalpNAc-(1“4)-
b-D
-GlcpA-(1“OMe), O-sulfonated at C-4 or C-6 of the aminosugar moieties, which represent structural elements
of chondroitin 4- and 6-sulfate proteoglycans. Starting from a synthetic disaccharide glycosyl acceptor, the stepwise
or blockwise construction of the sugar backbone with appropriate synthons led to a pentasaccharide tetraol, which
was used as a common intermediate. Selective 6-O-sulfonation of this tetraol, followed by saponification, gave the
6-sulfate derivative, whereas selective 6-O-benzoylation, followed by O-sulfonation and saponification, afforded the
4-sulfate derivative as their sodium salts. © 2000 Elsevier Science Ltd. All rights reserved.
Keywords: Pentasaccharide; Chondroitin 4- and 6-sulfate; Glycosaminoglycans
1. Introduction
biological roles are highly diversified, ranging
from simple mechanical support functions to
more intricate effects such as cell recognition
[3], development of osteoarthritis [4], inhibi-
tion of human C1q factor [5], AT-III-medi-
ated anticoagulant activity [6], but many
other, still poorly understood, effects have
been reported. Determination of the precise
structure of the sequences involved in such
events is highly complicated by the micro-
heterogeneity of the polymers. Chemical or
enzymatic controlled degradation affords
complex mixtures of products from which
pure fragments could be isolated with great
difficulty and in low yields. Thus, chemical
synthesis of molecules of definite size and
structure remains one of the most efficient
ways to answer these questions.
Chondroitin sulfates occur in many tissues
as side chains of proteoglycans. They are
found at the cell surface or intracellularly in
secretory granules, as well as in various body
fluids [1,2]. They are linear copolymers built
essentially from dimeric units composed of
D
-glucuronic acid (GlcA) and 2-acetamido-2-
deoxy- -galactose (GalNAc). In the major
D
variants, the 4- and 6-O-positions of the Gal-
NAc residues are found sulfonated, but sev-
eral types having one or more sulfate groups
at various positions are also known. Their
* Corresponding author. Tel.: +33-238-417072; fax: +33-
238-417281.
E-mail address: jean-claude.jacquinet@univ-orleans.fr (J.-
C. Jacquinet)
Several syntheses of chondroitin 4- and 6-
sulfate fragments have been reported in the
0008-6215/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved.
PII: S0008-6215(00)00032-X

F. Be´lot, J.-C. Jacquinet / Carbohydrate Research 326 (2000) 88–97
89
last decade. The basic reducing disaccharides
2. Results and discussion
[7], their methyl [8,9] or 4-methoxyphenyl [10]
glycosides have been prepared, as well as
structures of higher molecular size such as
trisaccharide [10–12] or tetrasaccharide [10]
derivatives. We now report for the first time
the syntheses of chondroitin 4- and 6-sulfate
pentasaccharide derivatives 1 and 2 having a
For the syntheses of the target pentasacchar-
ides 1 and 2, the tetraol 15, which may be
obtained from a glycosyl donor (5) and a
tetrasaccharide glycosyl acceptor (12), was de-
signed as a common intermediate. This latter
may, in turn, be prepared from a disaccharide
glycosyl acceptor (3) by stepwise addition of
appropriate monosaccharide glycosyl donors
methyl b-
D
-glucopyranosiduronic acid at the
reducing end.
Fig. 1. Structures 1–10.

90
F. Be´lot, J.-C. Jacquinet / Carbohydrate Research 326 (2000) 88–97
the acetyl groups in 11 with guanidine [15] led
to important degradation, whereas the use of a
catalytic amount of sodium methoxide at vari-
ous temperatures and in various solvents led to
extensive b-elimination reaction at the GlcA
moieties. This problem was overcome by the
use of acid-catalysed methanolysis [16]. Thus,
careful treatment of 11 with methanolic hydro-
gen chloride in dichloromethane, followed by
benzylidenation of the crude intermediate with
benzaldehyde and trifluoroacetic acid, af-
forded the crystalline tetrasaccharide acceptor
12 in 60% overall yield. Under these condi-
tions, the benzylidene group on the first amino
sugar residue was partially removed, but easily
reintroduced in the benzylidenation reaction
(Fig. 2).
(4, then 10), or blockwise addition of a disac-
charide glycosyl donor (7) (Fig. 1).
Preparation of the target glycosyl acceptor
12 was achieved as follows. In the first route,
methyl 2,3-di-O-benzoyl-4-O-chloroacetyl-1-O
- trichloroacetimidoyl - a -
D
- glucopyranuron-
ate (4) [13] (1.2 equivalents) was reacted with
methyl (4,6 - O - benzylidene - 2 - deoxy - 2 - tri-
chloroacetamido-b-
D
-galactopyranosyl)-(1“4)-
(methyl 2,3 - di - O - benzoyl - b -
D
- glucopyran-
osid)uronate (3) [12] in toluene at room temper-
ature, in the presence of trimethylsilyl triflate,
to give the crystalline trisaccharide derivative 8
in 84% yield. When the same reaction was
performed in dichloromethane (details not pre-
sented in Section 3), a major product was
isolated, the 1H NMR spectrum of which indi-
cated it to be an orthoester derivative [l 5.95
(d, 1 H, J1,2 5.0 Hz, H-1III), 4.62 (m, 1 H, J2,3
4.0, J2,4 1.0 Hz, H-2III)]. This derivative could
not be rearranged cleanly into 8 by treatment
with trimethylsilyl triflate in toluene or
dichloromethane. Selective removal of the
monochloroacetyl group in 8 with thiourea
afforded the crystalline acceptor 9 in 87%
yield. Condensation of 3,4,6-tri-O-acetyl-2-de-
oxy-2-trichloroacetamido-1-O-trichloroace-
Condensation of methyl 2,3,4-tri-O-benzo-
yl-1-O-trichloroacetimidoyl-a- -glucopyran-
D
uronate (5) [11] (2 equivalents) with alcohol
12 in toluene, as described above for the
preparation of 8, afforded the crystalline pen-
tasaccharide derivative 13 in 55% yield, and no
orthoester formation was observed. This result
contrasts with those reported [12] for a similar
coupling reaction of 5 with the disaccharide
acceptor 3 (85%), and the use of a larger excess
of 5 (3–4 equivalents) did not significantly
increase the yield. The N-trichloroacetyl group
in 13 was transformed into N-acetyl by treat-
ment [17] with tributylstannane and azobi-
sisobutyronitrile (AIBN) in benzene at 80 °C
to afford the crystalline diacetamide 14 in 79%
yield. Treatment of 14 with aqueous acetic acid
at 100 °C gave the crystalline tetraol 15, the
common intermediate, in 79% yield.
timidoyl-a- -galactopyranose (10) [12,14] (1.4
D
equivalents) with 9 in dichloromethane, as
described above for the preparation of 8, gave
the crystalline tetrasaccharide derivative 11 in
78% yield. In the second route, the crystalline
disaccharide imidate 7 was prepared in 74%
yield from methyl (3,4,6-tri-O-acetyl-2-deoxy-
2-trichloroacetamido-b-
(1“4)-(4-methoxyphenyl 2,3-di-O-benzoyl-b-
-glucopyranosid)uronate (6) [12] by oxidative
D
-galactopyranosyl)-
D
Preparation of the target molecules 1 and 2
was then achieved as follows. Treatment of 15
with the sulfur trioxide–trimethylamine com-
plex (3.5 equivalents for each hydroxyl group)
in N,N-dimethylformamide at 50 °C for 3 h,
followed by ion-exchange chromatography, af-
forded the crystalline sodium salt 16 in 87%
removal of the 4-methoxyphenyl group with
ceric ammonium nitrate, followed by imidoy-
lation with trichloroacetonitrile and 1,8-diaz-
abicyclo[4,5,0]undec-7-ene (DBU). Coupling
of imidate 7 (1.2 equivalents) with alcohol 3 in
dichloromethane, as described above for the
preparation of 8, afforded crystalline 11 in
50% yield. Both routes, achieved with moder-
ate excess (1.2–1.4 equivalents) of the corre-
sponding glycosyl donors, compare well (57
and 50% yield, respectively), and no formation
of a-linked species was observed. Transforma-
tion of 11 into a suitable glycosyl acceptor was
then studied. Attempted selective removal of
1
yield. Since comparison of the H NMR spec-
tra of 16 and 15 (see Table 2) did not clearly
give evidence of the 6-O-sulfonation, a fraction
of 16 (details not presented in Section 3) was
O-acetylated (acetic anhydride in pyridine).
1
The H NMR spectrum of this derivative
showed, inter alia, two telling signals at l 5.27

F. Be´lot, J.-C. Jacquinet / Carbohydrate Research 326 (2000) 88–97
91
Fig. 2. Structures 11–18.
and 5.20 (2 dd, 2 H, J3,4 3.5, J4,5 1.0 Hz,
H-4II,IV), and two signals at l 1.80 and 1.78
(2 s, 3 H each, Ac), characteristic of a 4-O-
and 17 showed (Table 2) the expected [9]
downfield shifts (ꢀ1 ppm) of the signals for
H-4II,IV in 18. Saponification of the ester
groups was then achieved by treatment of 16
and 18 with lithium hydroperoxide [18] in
aqueous tetrahydrofuran (THF) at −5 °C,
followed by methanolic sodium hydroxide,
to afford the target molecules 2 and 1 in 80
acetylated- -galacto derivative, thus ensuring
D
that O-sulfonation occurred exclusively at O-
6. Treatment of the tetraol 15 with benzoyl
cyanide (2 molar equivalents) in pyridine at
room temperature afforded the 6-O-benzoyl-
ated derivative 17 in 77% yield. Comparison
1
and 78% yields, respectively. The H (Table
1
of the H NMR spectra (see Table 2) of 17
2) and 13C NMR spectra of 1 and 2 are in
full agreement with the expected structures,
and in accord with those reported for syn-
thetic di-, tri-, and tetrasaccharide derivatives
[9–11] and for polymeric chondroitin 4- and
6-sulfates [19].
In conclusion, stereocontrolled syntheses
of chondroitin 4- and 6-sulfate pentasaccha-
ride derivatives 1 and 2 are reported. These
molecules are currently being evaluated in
conformational studies and in biological
assays.
(chloroform-d) and 15 (methanol-d4) showed
no significant downfield shift of the signals
for H-4II,IV, thus excluding any O-benzoyla-
tion at C-4. Sulfation of 17, as described for
the preparation of 16, was somewhat trou-
blesome, and required a large excess of
reagent (15 equivalents for each hydroxyl
group) and prolonged reaction time to go to
completion, but the crystalline sodium salt
18 was nevertheless isolated in 88% yield.
1
Comparison of the H NMR spectra of 18

92
F. Be´lot, J.-C. Jacquinet / Carbohydrate Research 326 (2000) 88–97
3. Experimental
methoxyphenyl 2,3-di-O-benzoyl-b- -gluco-
D
pyranosid)uronate (6) [12] (0.3 g, 0.31 mmol)
and ceric ammonium nitrate (1.8 g, 3.3 mmol)
in 1:1.5:1 toluene–MeCN–water (15 mL) was
vigorously stirred for 45 min at room tempera-
ture (rt). The mixture was then diluted with
EtOAc (50 mL), washed with water, dried
(MgSO4), and concentrated. The residue was
eluted from a column (25 g) of silica gel with
1:1 EtOAc–petroleum ether to give the corre-
sponding free hemiacetal (0.23 g, 86%).
General methods.—Melting points were de-
termined in capillary tubes with a Bu¨chi appara-
tus and are uncorrected. Optical rotations were
measured at 20–25 °C with a Perkin–Elmer 241
1
polarimeter. H and 13C NMR spectra were
recorded at 25 °C with a Bruker DPX-250
spectrometer operating at 250 and 63 MHz,
respectively, with Me4Si as internal standard,
unlessotherwisestated. Assignmentswerebased
on homo- and heteronuclear correlations using
the supplier’s software. Mass spectra were ob-
tained on a Perkin–Elmer SCIEX API 300
spectrometer operating in the ion-spray (IS)
mode. Flash-column chromatography was per-
formed on Silica Gel (E. Merck, 40–63 mm).
Elemental analyses were performed by the Ser-
vice Central de Microanalyse du CNRS (Ver-
naison, France).
A mixture of the above isolated hemiacetal,
CCl3CN (0.32 mL, 0.32 mmol), and DBU (8 mL,
50 mmol) in anhyd CH2Cl2 (4 mL) was stirred
for 1 h at rt, then concentrated. The residue was
eluted from a column (20 g) of silica gel with
1:1 EtOAc–petroleum ether containing 0.5% of
Et3N to give 7 (232 mg, 74% from 6); mp
129–130 °C (from EtOAc–petroleum ether);
1
[h]D +32° (c 1, CHCl3); H NMR (CDCl3):
Methyl (3,4,6-tri-O-acetyl-2-deoxy-2-trichl-
oroacetamido-i- -galactopyranosyl)-(1“4)-
carbohydrate ring protons (see Table 1); 8.65 (s,
1 H, CꢀNH), 8.0–7.20 (m, 10 H, Ph), 3.82, (s,
3 H, COOCH3), 2.10, 2.0, 1.95 (3 s, 9 H, Ac).
Anal. Calcd for C37H36Cl6N2O17: C, 44.73; H,
3.65; N, 2.82. Found: C, 44.64; H, 3.85; N, 2.85.
D
2,3-di-O-benzoyl-1-O-trichloroacetimidoyl-h-
-glucopyranuronate (7).—A mixture of methyl
D
(3,4,6 - tri - O - acetyl - 2 - deoxy - 2 - trichloro-
acetamido-b- -galactopyranosyl)-(1“4)-(4-
D
Table 1
1H NMR data a: carbohydrate ring protons for compounds 7–9, 11 and 12
Proton
7
8
9
11
12
H-1I
6.69 (3.5)
5.47 (9.0)
5.98 (9.0)
4.38 (9.5)
4.52
4.68 (7.0)
5.30 (9.0)
5.61 (9.0)
4.57 (9.0)
4.14
4.72 (7.0)
5.33 (9.0)
5.46 (9.0)
4.58 (9.0)
4.10
4.67 (7.0)
5.33 (9.0)
5.52 (9.0)
4.57 (9.0)
4.12
4.67 (7.0)
5.31 (9.0)
5.55 (9.0)
4.57 (9.0)
4.12
H-2I
H-3I
H-4I
H-5I
H-1II
H-2II
H-3II
H-4II
H-5II
H-6a,bII
NHII
H-1III
H-2III
H-3III
H-4III
H-5III
H-1IV
H-2IV
H-3IV
H-4IV
H-5IV
H-6a,bIV
NHIV
4.94 (8.0)
3.91 (11.0)
5.14 (3.5)
5.12 (1.0)
3.70
5.25 (8.0)
3.64 (11.0)
4.59 (3.5)
4.31 (1.0)
3.28
3.85, 3.80
7.03 (7.0)
5.14 (7.0)
5.44 (9.0)
5.68 (9.0)
5.48 (9.5)
4.20
5.20 (8.0)
3.62 (11.0)
4.57 (3.5)
4.28 (1.0)
3.53
3.95, 3.80
7.07 (7.0)
5.12 (7.0)
5.44 (9.0)
5.72 (9.0)
4.10 (9.5)
4.15
5.23 (8.0)
3.62 (11.0)
4.53 (3.5)
4.26 (1.0)
3.89
3.95, 3.90
6.86 (7.0)
5.09 (7.0)
5.35 (9.0)
5.68 (9.0)
4.35 (9.5)
4.13
4.78 (8.0)
3.65 (11.0)
5.04 (3.5)
5.10 (1.0)
3.72
5.23 (8.0)
3.62 (11.0)
4.51 (3.5)
4.30 (1.0)
3.30
3.90, 3.80
6.78 (7.0)
5.11 (7.0)
5.32 (9.0)
5.67 (9.0)
4.48 (9.5)
4.17
4.59 (8.0)
3.65 (11.0)
3.92 (3.5)
3.95 (1.0)
3.19
3.35
6.74 (8.5)
3.31, 3.25
6.64 (8.5)
3.90, 3.80
6.75 (8.0)
a Chemical shifts (l, ppm) and coupling constants (J, Hz) in CDCl3.

F. Be´lot, J.-C. Jacquinet / Carbohydrate Research 326 (2000) 88–97
93
Methyl
chloroacetamido-i-
(methyl 2,3-di-O-benzoyl-i-
luronate)-(1“3)-(4,6-O-benzylidene-2-deoxy-
2 - trichloroacetamido - i - - galactopyranosyl)-
(1“4)-(methyl 2,3-di-O-benzoyl-i- -glucopy-
ranosid)uronate (11).—(a) A mixture of 9 (0.3
g, 0.245 mmol), 3,4,6-tri-O-acetyl-2-deoxy-2-
trichloroacetamido-1-O-trichloroacetimidoyl-
(3,4,6-tri-O-acetyl-2-deoxy-2-tri-
-galactopyranosyl)-(1“4)-
-glucopyranosy-
Methyl
chloroacetyl - i -
(1“3) - (4,6 - O - benzylidene - 2 - deoxy - 2 - tri-
chloroacetamido-i-
(methyl 2,3-di-O-benzoyl-i-
uronate (8).—A mixture of methyl (4,6-O-
benzylidene-2-deoxy-2-trichloroacetamido-b-
-galactopyranosyl)-(1“4)-(methyl 2,3-di-O-
(methyl
2,3-di-O-benzoyl-4-O-
D
- glucopyranosyluronate)-
D
D
D
-galactopyranosyl)-(1“4)-
D
-glucopyranosid)-
D
D
D
benzoyl-b-
(0.6 g, 0.73 mmol), methyl 2,3-di-O-benzoyl-4-
O-chloroacetyl-1-O-trichloroacetimidoyl-a-
D
-glucopyranosid)uronate (3) [12]
a- -galactopyranose (10) [12,14] (204 mg, 0.34
D
D-
mmol) in anhyd CH2Cl2 (8 mL) was treated as
described for the preparation of 8. The residue
was eluted from a column (40 g) of silica gel
with 2:1 toluene–EtOAc containing 0.2% of
Et3N to give 11 (320 mg, 78%); mp 151–
153 °C (from EtOAc–petroleum ether); [h]D
glucopyranuronate (4) [13] (0.56 g, 0.87
,
mmol), and powdered 4 A molecular sieves
(0.5 g) in anhyd toluene (20 mL) was stirred
for 1 h at rt under dry Ar. A solution of
Me3SiOTf in toluene (1 M, 0.13 mL) was
added, and the mixture was stirred for 1 h at
rt. Triethylamine (0.14 mL) was added, and
the mixture was filtered, and concentrated.
The residue was eluted from a column (100 g)
of silica gel with 4:1 toluene–EtOAc contain-
ing 0.2% of Et3N to give 8 (0.8 g, 84%); mp
249–250 °C (from EtOAc–petroleum ether);
1
+3° (c 1, CHCl3); H NMR (CDCl3): carbo-
hydrate ring protons (see Table 1); 8.0–7.20
(m, 25 H, Ph), 5.31 (s, 1 H, PhCH), 3.82, 3.78
(2 s, 6 H, COOCH3), 3.48 (s, 3 H, OCH3),
1.92, 1.90, 1.88 (3 s, 9 H, Ac). Anal. Calcd for
C72H70Cl6N2O30: C, 52.17; H, 4.22; N, 1.69.
Found: C, 51.92; H, 4.37; N, 1.71.
(b) A mixture of 3 (100 mg, 0.12 mmol) and
7 (144 mg, 0.14 mmol) was treated as de-
scribed in (a). The residue was eluted from a
column (25 g) of silica gel with 2:1 toluene–
EtOAc containing 0.2% of Et3N, and crystal-
lized from EtOAc–petroleum ether to give 11
(100 mg, 50%); mp 151–153 °C.
1
[h]D +37° (c 1, CHCl3); H NMR (CDCl3):
carbohydrate ring protons (see Table 1); 8.0–
7.20 (m, 25 H, Ph), 5.34 (s, 1 H, PhCH), 3.93
(ABq, 2 H, COCH2Cl), 3.83, 3.75 (2 s, 6 H,
COOCH3), 3.48 (s, 3 H, OCH3). Anal. Calcd
for C60H55Cl4NO23: C, 55.39; H, 4.23; N, 1.07.
Found: C, 55.25; H, 4.32; N, 1.12.
Methyl (methyl 2,3-di-O-benzoyl-i-
copyranosyluronate) - (1“3) - (4,6 - O - benzyli-
dene-2-deoxy-2-trichloroacetamido-i- -galac-
topyranosyl)-(1“4)-(methyl 2,3-di-O-benzoyl-
i- -glucopyranosid)uronate (9).—A mixture
D-glu-
Methyl (4,6-O-benzylidene-2-deoxy-2-tri-
chloroacetamido-i-
(methyl 2,3-di-O-benzoyl-i-
luronate)-(1“3) -(4,6-O-benzylidene-2-deoxy-
2 - trichloroacetamido - i - - galactopyranosyl)-
(1“4)-(methyl 2,3-di-O-benzoyl-i- -glucopy-
D
-galactopyranosyl)-(1“4)-
D
D
-glucopyranosy-
D
D
of 8 (585 mg, 0.45 mmol) and thiourea (0.1 g,
1.35 mmol) in pyridine (5 mL) and abs EtOH
(12 mL) was stirred for 2 h at 80 °C, then
cooled, diluted with CH2Cl2 (60 mL), washed
with water, dried (MgSO4), and concentrated.
The residue was eluted from a column (40 g)
of silica gel with 2:1 toluene–EtOAc to give 9
(0.48 g, 87%); mp 262–264 °C (from EtOAc–
D
ranosid)uronate (12).—A solution of AcCl
(0.2 mL) in anhyd MeOH (5 mL) was added
to a solution of 11 (0.39 g, 0.23 mmol) in
anhyd CH2Cl2 (5 mL), and the mixture was
stirred for 24 h at rt, then deionized with
Dowex 1X2-400 (OH−) resin, filtered, concen-
trated, and dried. A mixture of the residue,
benzaldehyde (6 mL), and CF3COOH (0.1
mL) was stirred for 1.5 h at rt, then cooled to
0 °C. Triethylamine (0.5 mL) was added, and
the mixture was diluted with CH2Cl2 (40 mL),
washed with water, dried (MgSO4), and con-
centrated. The residue was eluted from a
column (20 g) of silica gel with 1:1“3:1
1
petroleum ether); [h]D +32° (c 1, CHCl3); H
NMR (CDCl3): carbohydrate ring protons
(see Table 1); 8.0–7.20 (m, 25 H, Ph), 5.29 (s,
1 H, PhCH), 3.80, 3.75 (2 s, 6 H, COOCH3),
3.48 (s, 3 H, OCH3), 3.29 (bs, 1 H, HO-4III).
Anal. Calcd for C58H54Cl3NO22: C, 56.89; H,
4.41; N, 1.14. Found: C, 56.72; H, 4.54; N,
1.29.

94
F. Be´lot, J.-C. Jacquinet / Carbohydrate Research 362 (2000) 88–97
EtOAc–petroleum ether containing 0.5% of
Et3N, and crystallized from EtOAc–
petroleum ether to give 12 (228 mg, 60%); mp
(30 g) of silica gel with 1:1 EtOAc–CH2Cl2 to
give 14 (257 mg, 79%); mp 240–242 °C (from
EtOH–petroleum ether); [h]D +10° (c 1,
1
1
172–174 °C; [h]D −6° (c 1, CHCl3); H NMR
CHCl3); H NMR (CDCl3): carbohydrate ring
(CDCl3): carbohydrate ring protons (see Table
1); 8.0–7.20 (m, 30 H, Ph), 5.37, 5.23 (2 s, 2
H, PhCH), 3.82, 3.81 (2 s, 6 H, COOCH3),
3.49 (s, 3 H, OCH3), 2.95 (bs, 1 H, HO-3IV).
Anal. Calcd for C73H70Cl6N2O27: C, 54.07; H,
4.32; N, 1.73. Found: C, 54.05; H, 4.29; N,
1.78.
protons (see Table 2); 8.0–7.20 (m, 45 H, Ph),
5.28, 5.27 (2 s, 2 H, PhCH), 3.76, 3.67, 3.58 (3
s, 9 H, COOCH3), 3.47 (s, 3 H, OCH3), 1.57,
1.54 (2 s, 6 H, NAc); ISMS: m/z 1915, [M+
H]+. Anal. Calcd for C101H96N2O36: C, 63.38;
H, 5.06; N, 1.46. Found: C, 63.08; H, 5.20; N,
1.44.
Methyl (methyl 2,3,4-tri-O-benzoyl-i-
copyranosyluronate) - (1“3) - (4,6 - O - benzyli-
dene-2-deoxy-2-trichloroacetamido-i- -galac-
topyranosyl)-(1“4)-(methyl 2,3-di-O-benzoyl-
i - - glucopyranosyluronate) - (1“3) - (4,6 - O-
benzylidene-2-deoxy-2-trichloroacetamido-i-
galactopyranosyl)-(1“4)-(methyl 2,3-di-O-
benzoyl-i- -glucopyranosid)uronate (13).—A
D-glu-
Methyl (methyl 2,3,4-tri-O-benzoyl-i-
copyranosyluronate) - (1“3) - (2 - acetamido - 2-
deoxy-i- -galactopyranosyl) - (1“4) - (methyl
2,3-di-O-benzoyl-i- -glucopyranosyluronate)-
(1“3)-(2-acetamido-2-deoxy-i- -galactopyr-
anosyl)-(1“4)-(methyl 2,3-di-O-benzoyl-i- -
D-glu-
D
D
D
D
D
D-
D
glucopyranosid)uronate (15).—A suspension of
14 (192 mg, 0.1 mmol) in AcOH (8 mL) was
stirred at 100 °C. Water (4 mL) was added
dropwise, and the mixture was stirred for 30
min at 100 °C, then cooled, concentrated, and
evaporated with water (3×10 mL). The
residue was eluted from a column (15 g) of
silica gel with 20:1 EtOAc–EtOH to give 15
(138 mg, 79%); mp 197–198 °C (from CHCl3–
D
mixture of 12 (308 mg, 0.19 mmol) and methyl
2,3,4-tri-O-benzoyl-1-O-trichloroacetimidoyl-
a- -glucopyranuronate (5) [11] (255 mg, 0.38
D
mmol) in anhyd toluene (9 mL) was treated as
described for the preparation of 8. The residue
was eluted from a column (40 g) of silica gel
with 4:1 toluene–EtOAc containing 0.2% of
Et3N to give 13 (222 mg, 55%); mp 172–
174 °C (from EtOAc–petroleum ether); [h]D
1
petroleum ether); [h]D −3° (c 1, MeOH); H
NMR (CD3OD): carbohydrate ring protons
(see Table 2); 8.10–7.20 (m, 35 H, Ph), 3.77,
3.68, 3.58 (3 s, 9 H, COOCH3), 3.42 (s, 3 H,
OCH3), 1.35, 1.34 (2 s, 6 H, NAc); ISMS: m/z
1
+9.5° (c 1, CHCl3); H NMR (CDCl3): car-
bohydrate ring protons (see Table 2); 8.0–7.20
(m, 45 H, Ph), 5.34, 5.29 (2 s, 2 H, PhCH),
3.81, 3.77, 3.62 (3 s, 9 H, COOCH3), 3.48 (s, 3
H, OCH3). Anal. Calcd for C101H90Cl6N2O36:
C, 57.21; H, 4.28; N, 1.32. Found: C, 57.02;
H, 4.44; N, 1.40.
1741,
[M+H]+.
Anal.
Calcd
for
C87H90N2O36: C, 60.0; H, 5.21; N, 1.61.
Found: C, 59.80; H, 5.32; N, 1.52.
Methyl (methyl 2,3,4-tri-O-benzoyl-i-
copyranosyluronate)-(1“3)-(sodium 2-acetam-
ido-2-deoxy-6-O-sulfonato-i- -galactopyr-
anosyl)-(1“4)-(methyl 2,3-di-O-benzoyl-i-
glucopyranosyluronate)-(1“3)-(sodium 2-ace-
tamido-2-deoxy-6-O-sulfonato-i- -galactopyr-
anosyl)-(1“4)-(methyl 2,3-di-O-benzoyl-i-
D-glu-
Methyl (methyl 2,3,4-tri-O-benzoyl-i-
copyranosyluronate)-(1“3)-(2-acetamido-4,6-
O-benzylidene-2-deoxy-i- -galactopyranosyl)-
(1“4)-(methyl 2,3-di-O-benzoyl-i- -glucopyr-
anosyluronate) - (1“3) - (2 - acetamido - 4,6 - O-
benzylidene-2-deoxy-i- -galactopyranosyl)-
(1“4)-(methyl 2,3-di-O-benzoyl-i- -glucopyr-
D-glu-
D
D
D-
D
D
D
D-
D
glucopyranosid)uronate (16).—A mixture of 15
(261 mg, 0.15 mmol) and sulfur trioxide–
trimethylamine complex (132 mg, 1 mmol) in
anhyd DMF (8 mL) was stirred for 3 h at
50 °C under dry Ar, then cooled. Methanol
(0.5 mL) was added, and the mixture was
concentrated. The residue was eluted from a
column (30 g) of silica gel with 6:1 CH2Cl2–
MeOH, then from a column (1.5×25 cm) of
Sephadex SP C-25 (Na+) with 9:5:1 CH2Cl2–
anosid)uronate (14).—A mixture of 13 (360
mg, 0.17 mmol), Bu3SnH (0.46 mL, 1.7
mmol), and AIBN (20 mg) in dry benzene (10
mL) was stirred for 30 min at rt under dry Ar,
then heated at 80 °C for 2 h, cooled, and
concentrated. The residue was stirred for 1 h
at 0 °C with petroleum ether (30 mL), and the
precipitate was filtered off, washed with
petroleum ether, and eluted from a column

Table 2
1H NMR data a: carbohydrate ring protons for pentasaccharide derivatives 13–18, 1, and 2
b
Protons
13
14
15
16 b
17
18 b
1 c
2 c
GlcA H-1
H-2
5.18, 5.05, 4.67 5.10, 4.90, 4.63 5.20, 488, 4.78
5.17, 4.90, 4.78 4.93, 4.78, 4.60 5.29, 5.25, 5.24 4.35, 4.34, 4.27
4.37, 4.36, 4.25
3.23, 3.21, 3.18
3.50, 3.35, 3.34
3.56, 3.55, 3.43
3.63–3.58
/
5.49, 5.31, 5.29 5.47, 5.30, 5.28 5.51, 5.25, 5.20 5.49, 5.25, 5.18 5.39, 5.36, 5.35 5.66, 5.62, 5.35 3.25, 3.23, 3.22
5.77, 5.65, 5.64 5.83, 5.67, 5.64 6.00, 5.58, 5.55 5.95, 5.60, 5.59 5.87, 5.61, 5.53 6.13, 5.83, 5.75 3.55, 3.39, 3.37
5.55, 4.55, 4.52 5.59, 4.46, 4.39 5.61, 4.35, 4.34 5.60, 4.32, 4.31 5.50, 4.27, 4.25 5.66, 4.39, 4.37 3.57, 3.54, 3.45
4.27, 4.12, 4.11 4.27, 4.10, 4.08 4.59, 4.18, 4.16 4.59, 4.20, 4.15 4.27, 4.05, 4.01 4.69, 4.35, 4.33 3.70–3.65
H-3
H-4
H-5
GalN H-1
H-2
5.21, 5.12
3.68, 3.65
4.54, 4.48
4.32, 4.20
3.25, 3.10
5.11, 4.97
3.31, 3.17
4.62, 4.52
4.14, 4.10
2.92, 2.87
3.80–3.70
5.36, 5.28
5.18, 5.02
3.24, 3.15
4.57, 4.47
4.09, 3.95
3.15, 3.10
3.80–3.70
5.01, 4.91
3.64, 3.62
4.56, 4.43
4.11, 4.01
3.50, 3.45
3.85–3.75
4.90, 4.88
3.05, 3.03
4.67, 4.58
4.01, 3.86
3.51, 3.49
3.98, 3.70–3.60
5.45, 5.33
5.30, 5.22
3.64, 3.62
4.58, 4.57
5.10, 5.01
3.58, 3.57
4.10–3.90
4.47, 4.46
3.92, 3.91
3.97, 3.96
4.72, 4.64
3.75, 3.68
3.75–3.65
4.42, 4.41
3.89, 3.86
3.85, 3.84
4.08, 4.05
3.73, 3.69
4.12–4.08
H-3
H-4
H-5
326
(
H-6a,b 3.95–3.85
NH 6.92, 6.81
a Chemical shifts (l, ppm) in CDCl3, unless otherwise stated.
b CD3OD.
c D2O.

96
F. Be´lot, J.-C. Jacquinet / Carbohydrate Research 326 (2000) 88–97
mido-6-O-benzoyl-2-deoxy-4-O-sulfonato-i-
galactopyranosyl)-(1“4)-(methyl 2,3-di-O-
benzoyl-i- -glucopyranosyluronate)-(1 “ 3)-
(sodium 2-acetamido-6-O-benzoyl-2-deoxy-4-
O-sulfonato-i- -galactopyranosyl)-(1 “ 4)-
(methyl 2,3-di-O-benzoyl-i- -glucopyranosid)-
D
-
MeOH–water to give 16 (254 mg, 87%); mp
184–186 °C (dec., from CHCl3–petroleum
1
D
ether); [h]D −5° (c 1, MeOH); H NMR
(CD3OD): carbohydrate ring protons (see
Table 2); 8.10–7.20 (m, 35 H, Ph), 3.79, 3.77,
3.64 (3 S, 9 H, COOCH3), 3.44 (s, 3 H,
OCH3), 1.42, 1.34 (2 s, 6 H, NAc). Anal.
Calcd for C87H86N2Na2O42S2·2H2O: C, 52.89;
H, 4.59; N, 1.42. Found: C, 52.69; H, 4.71; N,
1.29.
D
D
uronate (18).—A mixture of 17 (125 mg, 64
mmol) and sulfur trioxide–trimethylamine
complex (170 mg, 1.28 mmol) in anhyd DMF
was stirred for 48 h at 50 °C under dry Ar.
More reagent (85 mg, 0.64 mmol) was then
added, and the mixture was stirred for further
48 h at 50 °C, then cooled. Methanol (1 mL)
was added, and the mixture was stirred for 1 h
at rt, then concentrated. The residue was
eluted twice from a column (25 g) of silica gel
with 10:1 CH2Cl2–MeOH, then from a
column (1.5×25 cm) of Sephadex SP C-25
(Na+) with 9:5:1 CH2Cl2–MeOH–water to
give 18 (120 mg, 88%); 202–204 °C (dec., from
CHCl3–petroleum ether); [h]D +9° (c 1,
O-Acetylated
derivative;
1H
NMR
(CD3OD): l 8.0–7.30 (m, 35 H, Ph), 5.89,
5.61, 5.48 (3 t, 3 H, J2,3 =J3,4=9.0 Hz, H-
3I,III,V), 5.58 (dd, 1 H, J4,5 9.5 Hz, H-4V), 5.34,
5.22, 5.13 (3 dd, 3 H, J1,2 7.0 Hz, H-2I,III,V),
5.27, 5.20 (2 dd, 2 H, J3,4 3.5, J4,5 1.0 Hz,
H-4II,IV), 5.13, 4.96, 4.78 (3 d, 3 H, H-1I,III,V),
4.97, 4.88 (2 d, 2 H, J1,2 8.0 Hz, H-1II,IV), 4.56,
4.51 (2 dd, 2 H, J2,3 11.0 Hz, H-3II,IV), 4.47,
4.17, 4.12 (3 d, 3 H, H-5I,III,V), 4.32, 4.23 (2
dd, 2 H, H-4I,III), 3.85–3.75 (m, 4 H, H-
6a,bII,IV), 3.79, 3.76, 3.64 (3 s, 9 H, COOCH3),
3.65–3.55 (m, 2 H, H-2II,IV), 3.50–3.45 (m, 2
H, H-5II,IV), 1.80, 1.78 (2 s, 6 H, OAc), 1.42,
1.36 (2 s, 6 H, NAc).
1
MeOH); H NMR (CD3OD): carbohydrate
ring protons (see Table 2); 8.20–7.20 (m, 45
H, Ph), 3.87, 3.68, 3.58 (3 s, 9 H, COOCH3),
3.42 (s, 3 H, OCH3), 1.35, 1.32 (2 s, 6 H,
NAc). Anal. Calcd for C101H94N2Na2O44S2·
H2O: C, 55.96; H, 4.46; N, 1.29. Found: C,
55.71; H, 4.62; N, 1.18.
Methyl (methyl 2,3,4-tri-O-benzoyl-i-
copyranosyluronate)-(1 “ 3)-(2-acetamido-6-
O - benzoyl - 2 - deoxy - i - - galactopyranosyl)-
(1“4)-(methyl 2,3-di-O-benzoyl-i- -glucopyr-
anosyluronate)-(1“3)-(2-acetamido-6-O-ben-
zoyl-2-deoxy-i- -galactopyranosyl)-(1 “ 4)-
(methyl 2,3-di-O-benzoyl-i- -glucopyran-
D-glu-
D
Sodium (sodium i-
ate)-(1“3)-(sodium 2-acetamido-2-deoxy-4-O
-sulfonato-i- -galactopyranosyl)-(1“4)-(sod-
ium i- -glucopyranosyluronate)-(1“3)-(sod-
ium 2-acetamido-2-deoxy-4-O-sulfonato-i-
galactopyranosyl)-(1“4)-(methyl i- -gluco-
D
-glucopyranosyluron-
D
D
D
D
D
D-
osid)uronate (17).—A mixture of 15 (244 mg,
0.14 mmol) and benzoyl cyanide (74 mg, 0.57
mmol) in anhyd pyridine (8 mL) was stirred
overnight at rt. Methanol (0.75 mL) was
added, and the mixture was concentrated, and
evaporated with toluene (3×10 mL). The
residue was eluted from a column (25 g) of
silica gel with 30:1 CH2Cl2–MeOH to give 17
(210 mg, 77%); mp 188–190 °C (from
EtOAc–petroleum ether); [h]D +19° (c 1,
D
pyranosid)uronate (1).—A solution of 18 (120
mg, 56 mmol) in 2:1 THF–water (4 mL) was
treated at −5 °C with 30% H2O2 (0.45 mL)
and LiOH (1 M, 0.84 mL), and the mixture
was stirred for 2 h at this temperature and 16
h at rt, then cooled to 0 °C. Methanol (4 mL)
and NaOH (4 M, 0.62 mL) were added, and
the mixture was stirred for 8 h at rt, then
treated with Amberlite IR-120 (H+) resin to
pH 3 (pH meter control), filtered, and concen-
trated. The residue was stirred for 1 h at 0 °C
with abs EtOH (10 mL), and the precipitate
was filtered off, washed with cold abs EtOH,
and eluted from a column (12 g) of silica gel
with 12:5:3“5:3:2 EtOAc–MeOH–water,
then taken up in water (5 mL). The pH of the
solution was brought to 6.5 (pH meter con-
trol) with diluted NaOH, and the solution was
1
CHCl3); H NMR (CDCl3): carbohydrate ring
protons (see Table 2); 8.20–7.20 (m, 45 H,
Ph), 3.78, 3.57, 3.54 (3 s, 9 H, COOCH3), 3.48
(s, 3 H, OCH3), 1.37, 1.35 (2 s, 6 H, NAc);
ISMS: m/z 1947, [M+H]+. Anal. Calcd for
C101H96N2O38: C, 62.34; H, 4.97; N, 1.44.
Found: C, 62.14; H, 4.95; N, 1.55.
Methyl (methyl 2,3,4-tri-O-benzoyl-i-
D-glu-
copyranosyluronate)-(1“3)-(sodium 2-aceta-

F. Be´lot, J.-C. Jacquinet / Carbohydrate Research 326 (2000) 88–97
97
filtered, and freeze-dried to give 1 as an amor-
phous hygroscopic powder (56 mg, 80%); [h]D
6II,IV), 57.56 (OCH3), 51.69, 51.42 (C-2II,IV),
22.77, 22.52 (COCH3). Anal. Calcd for
C35H49N2Na5O35S2·2H2O: C, 33.03; H, 4.20;
N, 1.30. Found: C, 32.80; H, 4.33; N, 1.17.
1
−46° (c 1, H2O); H NMR (D2O, internal
H2O): carbohydrate ring protons (see Table
2); 3.43 (s, 3 H, OCH3), 1.94, 1.92 (2 s, 6 H,
NAc); 13C (D2O, internal acetone): l 176.12,
175.24, 175.16, 174.69, 174.46 (CꢀO), 103.88,
103.72, 103.48, 103.27 (C-1II–V), 101.97 (C-1I),
80.55, 80.27, 80.07 (C-4I,III, C-3II,IV), 76.79,
76.67, 76.51, 75.58, 75.27, 74.79, 74.14, 73.71,
72.77, 72.41, 72.01 (C-2,3I,III,V, C-4V, C-
3,4,5II,IV), 61.24, 61.05 (C-6II,IV), 57.48
(OCH3), 51.80, 51.62 (C-2II,IV), 22.74, 22.69
(COCH3). Anal. Calcd for C35H49N2Na5O35S2·
3H2O: C, 32.56; H, 4.29; N, 2.17. Found: C,
32.34; H, 4.41; N, 2.07.
References
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Sodium (sodium i-
ate)-(1“3)-(sodium 2-acetamido-2-deoxy-6-
O - sulfonato - i - - galactopyranosyl) - (1 “ 4)-
(sodium i- -glucopyranosyluronate)-(1“3)-
(sodium 2-acetamido-2-deoxy-6-O-sulfonato-
i- -galactopyranosyl)-(1“4)-(methyl i- -
D-glucopyranosyluron-
D
D
D
D
glucopyranosid)uronate (2).—A solution of 16
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1
[h]D −24° (c 1, H2O); H NMR (D2O, inter-
nal H2O): carbohydrate ring protons (see
Table 2); 3.42 (s, 3 H, OCH3), 1.89, 1.88 (2 s,
6 H, NAc); 13C (D2O, internal acetone): l
176.23, 176.10, 175.81, 175.12, 174.24 (CꢀO),
103.81, 103.56, 103.36, 103.12 (C-1II–V),
101.75 (C-1I), 81.75, 81.55, 81.04, 80.35 (C-
3II,IV, C-4I,III), 75.86, 75.56, 75.37, 74.56, 74.28,
73.37, 73.08, 71.97, 71.86, 71.77, 71.60 (C-
2,3I,III,V, C-4V, C3,4,5II,IV), 67.78, 67.62 (C-
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.