Synthesis of chondroitin sulfate E octasaccharide in a repeating region involving an acetamide auxiliary

Article abstract of DOI:10.1016/j.carres.2007.09.009

Chondroitin sulfate E repeating octasaccharide was effectively synthesized in a stereocontrolled manner by adopting an acetamide-type disaccharide unit. In the tetrasaccharide synthesis we isolated a characteristic glycosyl imidate as a reactive intermedi

Full text of DOI:10.1016/j.carres.2007.09.009

Available online at www.sciencedirect.com
Carbohydrate Research 343 (2008) 39–47
Synthesis of chondroitin sulfate E octasaccharide in a
repeating region involving an acetamide auxiliary
Jun-ichi Tamura,* Yuka Nakada, Kayo Taniguchi and Manami Yamane
Department of Regional Environment, Faculty of Regional Sciences, Tottori University,
Koyamacho-minami 4-101, Tottori 680-8551, Japan
Received 18 July 2007; received in revised form 25 September 2007; accepted 26 September 2007
Available online 1 October 2007
Abstract—Chondroitin sulfate E repeating octasaccharide was effectively synthesized in a stereocontrolled manner by adopting an
acetamide-type disaccharide unit. In the tetrasaccharide synthesis we isolated a characteristic glycosyl imidate as a reactive interme-
diate. An acetamide auxiliary involves the glycosylation mechanism.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Keywords: Glycosylation; Oligosaccharide; Chondroitin sulfate
1. Introduction
2. Results and discussion
Chondroitin sulfate (CS) is one of the several classes of
glycosaminoglycans (GAGs) that widely exists in animal
tissues. The nonreducing part of CS contains a repetitive
disaccharide region composed of b-D-glucuronic acid
(GlcA) and N-acetylgalactosamine (GalNAc). Recent
reports have described the biological properties of CSs
in the repeating disaccharide region, especially at the
oligosaccharide level.1–3 However, little is known about
the optimum structures of the CS oligosaccharides, such
as the length of the glycans for respective biological
activities. These facts prompted us to work toward
achieving the systematic and effective syntheses of CS
oligosaccharides. In the synthesis of CS repeating
oligosaccharides, we optimized the glycan-elongation
reaction by adopting the trichloroacetimidate of 2-O-
(4-methylbenzoyl)GlcA and the 3-OH of GalNAc, and
finally succeeded in obtaining the CS repeating octasac-
charide (1).
We used an acetamide-type (b-GalNAc-GlcA) of disac-
charide units 34 by the reduction of azide of 25 for the
synthesis of the CS oligosaccharides (Scheme 1). The
azide group was hydrogenolyzed in the presence of
Pd/C and AcOH, then acetylated with Ac2O to give 3
in 91% yield (in two steps). Byproduct 4 was generated
in the absence of AcOH. In fact, Lindlar-catalyzed
reduction of 2 for 13 days resulted in a complete migra-
tion of the luvulinoyl group. Subsequent acetylation
afforded 4 in 75% yield (in two steps). The 4-methoxy-
phenyl (MP) group of 3 was removed, and the resulting
hemiacetal was converted to the corresponding trichlo-
roacetimidate 5.6 As described previously,6 glycosyl-
ation with the acetamide-type substrates 5 + 85 as well
as 5 + 126 in the presence of trimethylsilyl trifluoro-
methanesulfonate (TMSOTf, 0.7 equiv of donor) and
AW300 molecular sieves in CH2Cl2 at ꢀ20 ꢁC to room
temperature yielded 11 and 22 in 71% and 66%,
respectively.
In the synthesis of tetrasaccharide 11, we observed
dynamic changes on TLC analysis during the coupling
reaction as follows: (1) 8 was trimethylsilylated at the
beginning; (2) the silylated product decreased and a
new spot appeared within a few hours; then (3) the
*
Corresponding author. Tel./fax: +81 857 31 5108; e-mail:
jtamura@rstu.jp
0008-6215/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2007.09.009

40
J. Tamura et al. / Carbohydrate Research 343 (2008) 39–47
Scheme 1. Synthetic route to the tetrasacharides. Reagents and conditions: (a) Lindlar catalyst, H2, AcOH/EtOAc; (b) Lindlar catalyst, H2/EtOAc;
(c) CAN/CH3CN–H2O; (d) CCl3CN, DBU/CH2Cl2; (e) H2NNH2ÆAcOH/toluene–EtOH; (f) PivCl, DMAP/CH2Cl2; (g) TMSOTf, MS AW300/
CH2Cl2, ꢀ20 ꢁC to room temperature, overnight. Abbreviations: Lev, MeCO(CH2)2CO–; MBz, p-MeC6H4CO–; MP, p-MeOC6H4–, Piv, Me3CCO–.
TLC pattern became complicated, but (4) the spots
finally focused on the desired product 11. On the other
hand, the coupling of the corresponding azide-type
substrate did not show such complicated TLC patterns.
A spot that transiently appeared in the acetamide-type
system may indicate a rather pivotal intermediate that
certainly involved the acetamide.
spectrum showed two characteristic peaks at 161.41
and 15.99 ppm that can be assigned as N@C and
N@C–Me, respectively. These values were in good
agreement with those previously reported by Liao and
Auzanneau.7 We determined the structure of temporary
product 10 as depicted in Scheme 1. The MALDITOF
MS data also supported the structure.
We were able to isolate the product of the coupling
reaction of 5 + 8 after 3 h in 50% yield by silica gel col-
umn chromatography. This compound was stable only
for a few days as a solution in chloroform. The fully
characterized 1H NMR spectrum showed H-1III at
6.12 ppm with J1,2 = 7.8 Hz. The chemical shift of H-
2III was not affected, which means the newly formed gly-
coside did not link through an orthoester-type linkage.
The newly generated stereochemistry at C-1III of GlcA
was determined as b (no a-isomer was detected), per-
haps due to the neighboring-group-effect of GlcA. In
addition, the NHII peak disappeared. The 13C NMR
Imidate 10, which is of limited stability, seems kineti-
cally formed by a nucleophilic attack of the acetamide
oxygen that competes with the vicinal hydroxyl group.
Similar glycosyl imidates with an acetamido group have
already been reported. In 1976 Pougny and Sinay first
¨
isolated the glycosyl imidate formed with fucosyl chlo-
ride and fully protected GlcNAc in a Koenigs–Knorr-
type condensation.8 In 1982 Hindsgaul et al. proposed
a similar glycosyl imidate formation by circumstantial
evidence.9 In 2003 Liao and Auzanneau isolated the
1,2-trans glycosyl imidate by the coupling of rhamosyl
trichloroacetimidate with the 4-OH of LacNAc.7 The

J. Tamura et al. / Carbohydrate Research 343 (2008) 39–47
41
authors reported that the glycosyl imidate independently
rearranged to the desired (1!4)-linkage. However, a
very recent report by Martin-Lomas and co-workers
suggested that the isolated 1,2-cis glycosyl imidate was
not converted to the final glycoside.10 Our results
showed, at least, that the 3-OH of GalNAc had low
reactivity in the glycosylation comparable to that of 4-
OH of GlcNAc, which was notoriously difficult to
glycosylate.
To investigate the glycosylation mechanism and the
role of 10 in detail, we mixed 10 and azide-type acceptor
74,11 in the presence of TMSOTf and AW300 molecular
sieves in CH2Cl2 at 0 ꢁC to room temperature. The cross
reaction gradually proceeded and was completed after
6.5 h to give 11 and 13 in 36% and 18% yields, respec-
tively (Table 1, entry1). The decreased concentration
of the reaction mixture gave little change in the ratio
of 11/13 (entry 2), while the concentration of 7 affected
the ratio (entry 3). These facts suggest that 10 was not
converted to 11 via an intramolecular attack including
anomerization of 10, but the reaction proceeded via an
SN1 type reaction (Scheme 2).
In a previous report6 on the synthesis of chondroitin
sulfate E hexasaccharide, we also described the success-
ful pivaloylation at the nonreducing terminal for the
hexasaccharide in a final step. However, the pivaloyl-
ation for the hexasaccharide proved to be tricky and
sometimes generated overacylated products containing
–NAcPiv or –N@C(OPiv)Me type structure in moderate
yields (data not shown). This unfavorable reaction
happened due to the high reactivity of the acetamides
of 19. In the synthesis of the chondroitin sulfate E
repeating oligosaccharide, the protection of the hydro-
xyl group at the nonreducing end is critical. We realized
that the levulinoyl group was not suitable for the
reaction by the following reaction. Deprotection of the
benzylidene acetal of 3 with camphorsulfonic acid for
5 d generated 14 together with a triol 15 in 54% and
24% yield, respectively. In contrast, the pivaloyl group
was not affected under the same reaction conditions
Table 1. Cross reaction of 10 and 7
Entry
10 (lmol)
7 (lmol)
Ratio 10/7
Solvent (mL)
Yield 11 (%)
Yield 13 (%)
Ratio 11/13
1
2
3
17
22
20
21
27
121
0.81
0.81
0.17
2
20
2
36
29
13
18
12
31
2.0
2.4
0.4
Ph
O
Ph
O
MBzO
O
MBzO
O
N
OMP
O
O
MBzO
COOMe
O
HO
NHAc
O
MBzO
OMP
LevO
COOMe
O
O
O
O
O
HO
H+/TMS+
O
MBzO
COOMe
N3
O
O
O
Ph
CH3
10
7
O
Ph
O
O
MBzO
MBzO
O
OMP
O
O
HO
COOMe
NHAc
Ph
O
AcHN
O
MeOOC
O
MBzO
LevO
O
O
MBzO
O
O
OMP
O
MBzO
O
HO
O
COOMe
O
N3
O
Ph
+
11
13
Scheme 2. Postulated reaction pathway via interglycosidic imidate 10.

42
J. Tamura et al. / Carbohydrate Research 343 (2008) 39–47
two steps). Surprisingly, the coupling of 6 and the tetra-
saccharide acceptor 12 in the same manner as above
afforded hexasaccharide 18 in 95% yield (Scheme 4).
Corresponding levulinated donor 5 afforded 17 in 66%
yield.6 Further elongation on 196 with 6 gave octasac-
charide 20 in 58% yield. The four benzylidene acetals
of 20 were completely removed with camphorsulfonic
acid to afford 21 in 61% yield without affecting the pival-
oate, then 21 was sulfated with SO3ÆMe3N to give 22 in
95% yield. Finally, all the protecting groups of 22 were
removed under basic conditions to give 1 in 70% yield.
The 1H NMR and ESI mass spectra showed satisfactory
results.
Scheme 3. Removal of the benzylidene acetal in an acidic condition.
(Scheme 3). The corresponding pivaloate 9 afforded the
4,6-diol 16 quantitatively.5 These facts encouraged us to
use a pivaloated donor for end-capping.
We examined the glycan elongation with the pivaloat-
ed donor 6, which was obtained from 9 in 71% yield (in
Scheme 4. Synthesis of targeting chondroitin sulfate E repeating octasaccharide 1. Reagents and conditions: (a) TMSOTf, MS AW300/CH2Cl2,
ꢀ20 ꢁC to room temperature, overnight; (b) camphorsulfonic acid/CH2Cl2–MeOH; (c) SO3ÆMe3N/DMF; (d) LiOH/aq THF, then NaOH/aq MeOH.

J. Tamura et al. / Carbohydrate Research 343 (2008) 39–47
43
In summary, we optimized the superior glycan-elon-
gation system of the glycosyl donor equipped with
2-O-(4-methylbenzoyl)GlcA with the 3-OH of the
GalNAc acceptor in the presence of TMSOTf. On the
way to the tetrasaccharide we isolated a pivotal tetraosyl
intermediate composed of an interglycosidic imidate.
Prolonged reaction converted the intermediate to the
desired glycoside. Finally we attained a synthesis of
chondroitin sulfate E octasaccharide. The reactive and
b-selective coupling reaction provided an effective
strategy for the synthesis of longer CS oligosaccharides.
pressure. The residue was diluted with pyridine (3 mL).
Ac2O (3 mL) was added to the solution. Two hours later
a portion of ice was added to the reaction mixture with
stirring overnight. After usual workup the crude materi-
als obtained were passed through a silica gel column
chromatography eluted with 5:1!1:1 toluene–EtOAc
to give 3 (279.0 mg, 91%) as a syrup. 1H NMR data were
in good agreement with those already reported.4
3.3. Methyl 3-O-acetyl-4,6-O-benzylidene-2-deoxy-2-
levulinamido-b-D-galactopyranosyl-(1!4)-O-{4-methoxy-
phenyl 2,3-di-O-(4-methylbenzoyl)-b-D-glucopyran-
osid}uronate (4)
3. Experimental
To a solution of 2 (186.0 mg, 201.0 lmol) in EtOAC
(10 mL) was added Lindlar catalyst (514 mg). The mix-
ture was vigorously stirred under a hydrogen atmosphere
for 3 days. The reaction mixture was passed through a
Celite, and the volatiles were removed under diminished
pressure. To a solution of the residue in EtOAC (10 mL)
was again added Lindlar catalyst (503 mg) and the mix-
ture was vigorously stirred for 10 days under a hydrogen
atmosphere. Insoluble materials were removed as above,
and the volatiles were removed under diminished pres-
sure. The residue was diluted with MeOH (3 mL), and
Ac2O (3 mL) was added to the solution with stirring
overnight. A portion of ice was then added to the reac-
tion mixture. After usual workup the crude materials
obtained were passed through a silica gel column and
3.1. General methods
Optical rotation values were obtained at 22 3 ꢁC with a
Horiba SEPA-200 polarimeter. 1H and 13C NMR assign-
ments were confirmed by 2D HH COSY and HMQC
experiments, respectively, with a JEOL ECP 500 MHz
spectrometer. Mass spectra were recorded on an Auto-
flex III MALDITOF mass spectrometer (Bruker Dalton-
ics) and a Q-TOF2 ESI mass spectrometer (Micromass).
Signal assignments such as H-1III stand for a proton at
C-1 of sugar residue 3. Silica gel chromatography and
analytical TLC were, respectively, conducted in a
column of Silica Gel 60 (E. Merck), a column of Silica
Gel 60N (spherical neutral; Kanto Kagaku), and on glass
plates coated with Silica Gel F254 (E. Merck). The gel for
size-exclusion chromatography (Sephadex LH-20) was
purchased from GE Healthcare Bio-Sciences. Molecular
sieves (MS) 4A and AW300 were purchased from GL
Science and activated at 200 ꢁC under reduced pressure
prior to use. All reactions in organic solvents were per-
formed under a dry Ar atmosphere. The usual workup
involved the organic phase of the reaction mixture being
successively washed with aq NaHCO3 and brine, and
dried over anhyd MgSO4.
eluted with 10:1!1:10 toluene–EtOAc to give
4
(142.3 mg, 75%) as a syrup: Rf 0.45 (40:1 EtOAc–
1
MeOH); [a]D +98 (c 0.33, CHCl3); H NMR (CDCl3):
d 7.86–7.79 (m, 4H, Ph), 7.49–7.48 (m, 2H, Ph), 7.35–
7.31 (m, 3H, Ph), 7.18–7.15 (m, 4H, Ph), 6.92–6.89 (m,
2H, PhOMe), 6.79–6.76 (m, 2H, PhOMe), 5.77 (t, 1H,
J2,3 = J3,4 = 9.4 Hz, H-3I), 5.71 (dd, 1H, J1,2 = 8.7 Hz,
H-2I), 5.48 (s, 1H, PhCH), 5.36 (d, 1H, JNH,2 = 10.1 Hz,
NHII), 5.21 (d, 1H, H-1I), 5.20 (m, 1H, H-1II), 5.06 (dd,
1H, J2,3 = 11.5 Hz, J3,4 = 1.8 Hz, H-3II), 4.65 (m, 1H,
H-2II), 4.60 (t, 1H, H-4I), 4.24 (d, 1H, H-5I), 4.24 (m,
1H, H-6bII), 4.23 (d, 1H, H-4II), 3.99 (d, 1H,
J6a,6b = 12.6 Hz, H-6aII), 3.75 (2s, 3H · 2, 2OMe), 3.60
(s, 1H, H-5II), 2.76–2.50 (m, 4H, 2CH2), 2.35 (s,
3H · 2, 2PhMe), 2.05 (s, 3H, COCH3), 1.60 (s, 3H,
NAc). Anal. Calcd for C50H53NO17Æ0.5H2O: C, 63.27;
H, 5.75; N, 1.48. Found: C, 63.42; H, 5.71; N, 1.13.
3.2. Preparation of methyl 2-acetamido-4,6-O-benzyl-
idene-2-deoxy-3-O-levulinoyl-b-D-galactopyranosyl-
(1!4)-O-{4-methoxyphenyl 2,3-di-O-(4-methylbenzoyl)-
b-D-glucopyranosid}uronate (3)4
To a solution of 24 (301.6 mg, 331.4 lmol) in EtOAC
(13 mL) were added Lindlar catalyst (618 mg) and HOAc
(250 lL). The mixture was vigorously stirred under a
hydrogen atmosphere for 1 day. The reaction mixture
was passed through Celite, and the volatiles of the filtrate
were removed under diminished pressure. To the solution
of the residue in EtOAC (13 mL) were added Lindlar
catalyst (606 mg) and HOAc (180 lL) again, and the
mixture was vigorously stirred overnight under a hydro-
gen atmosphere. The insoluble materials were removed as
above, and the volatiles were removed under diminished
3.4. Methyl 2-N-[(2-acetamido-4,6-O-benzylidene-2-
deoxy-3-O-levulinoyl-b-D-galactopyranosyl)-(1!4)-O-
{methyl 2,3-di-O-(4-methylbenzoyl)-b-D-glucopyranuron-
osyl}]acetimido-4,6-O-benzylidene-2-deoxy-b-D-galacto-
pyranosyl-(1!4)-O-{4-methoxyphenyl 2,3-di-O-(4-
methylbenzoyl)-b-D-glucopyranosid}uronate (10)
To a solution of 56 (87.8 mg, 87.9 lmol, 1.46 equiv) and
85 (51.7 mg, 61.4 lmol) in CH2Cl2 (3 mL) was added

44
J. Tamura et al. / Carbohydrate Research 343 (2008) 39–47
MS AW300 (500 mg). The mixture was stirred for 1 h at
room temperature and then cooled to ꢀ20 ꢁC. TMSOTf
(11 lL, 61 lmol) was added while continuing the stirring
for 3 h, gradually increasing the temperature to 3 ꢁC.
Et3N (18 lL, 0.13 mmol) was added, the mixture was
filtered through cotton, and the volatiles were removed
under diminished pressure. The crude materials
obtained were passed through a column of silica gel
(2:1!1:3 toluene–EtOAc containing 0.1% Et3N) to give
10 (51.3 mg, 50%) as a syrup: Rf 0.55 (40:1 EtOAc–
mixture was stirred for 1 h at room temperature and
then cooled to ꢀ20 ꢁC. TMSOTf (22 lL, 0.12 mmol,
0.9 equiv) was added while continuing the stirring over-
night, gradually increasing the temperature to room
temperature. After similar workup the crude materials
obtained were passed through a gel-permeation column
(LH-20, 1:1 CHCl3–MeOH) to give tetrasaccharide 13
(154.9 mg, 72%) as a syrup: Rf 0.46 (40:1 EtOAc–
1
MeOH); [a]D +23 (c 0.86, CHCl3); H NMR (CDCl3):
d 7.96 (d, 2H, J = 8.0 Hz, Ph), 7.90–7.78 (m, 6H, Ph),
7.58 (d, 2H, J = 6.7 Hz, Ph), 7.41–7.28 (m, 9H, Ph),
7.23–7.09 (m, 5H, Ph), 7.03 (d, 2H, J = 8.0 Hz, Ph),
6.92–6.89 (m, 2H, PhOMe), 6.77–6.74 (m, 2H, PhOMe),
5.75 (t, 1H, J2,3 = J3,4 = 8.9 Hz, H-3I), 5.67 (s, 1H,
PhCH), 5.61 (dd, 1H, J2,3 = 3.2 Hz, J3,4 = 7.6 Hz,
H-3III), 5.56 (dd, 1H, J1,2 = 7.3 Hz, H-2I), 5.34–5.28
(m, 3H, H-1III, 2III, NHIV), 5.26 (s, 1H, PhCH), 5.21
(d, 1H, H-1I), 4.81 (dd, 1H, J4,5 = 10.5 Hz, H-4III),
4.50 (t, 1H, H-4I), 4.48 (d, 1H, J3,4 = 3.4 Hz, H-4II),
4.43 (m, 1H, H-3IV), 4.38 (d, 1H, H-5III), 4,35 (d, 1H,
J1,2 = 8.3 Hz, H-1II), 4.30 (d, 1H, H-5I), 4.21 (d, 1H,
J1,2 = 8.3 Hz, H-1IV), 4.05 (m, 1H, H-2IV), 3.90 (d, 1H,
J3,4 = 3.2 Hz, H-4IV), 3.86 (s, 3H, OMe), 3.85 (m, 1H,
H-6aIV), 3.75–3.74 (2s, 3H · 2, 2OMe), 3.69 (m, 2H,
H-2II,6aII), 3.56 (d, 1H, J6a,6b = 12.8 Hz, H-6bII), 3.49
(d, 1H, J6a,6b = 12.2 Hz, H-6bIV), 3.47 (dd, 1H,
J2,3 = 10.3 Hz, H-3II), 3.06 (s, 1H, H-5II), 2.75–2.40
(m, 4H, 2CH2), 2.59 (s, 1H, H-5IV), 2.45, 2.39, 2.34,
2.30 (4s, 3H · 4, 4PhMe), 2.05 (s, 3H, COCH3), 1.78
(s, 3H, NAc). Anal. Calcd for C86H88N4O29Æ3.3H2O:
C, 60.71; H, 5.62; N, 3.29. Found: C, 61.02; H, 5.32;
N, 2.89.
1
MeOH); H NMR (CDCl3): d 7.89–7.78 (m, 8H, Ph),
7.33–7.22 (m, 10H, Ph), 7.16–7.07 (m, 8H, Ph), 6.96–
6.95 (m, 2H, PhOMe), 6.81–6.79 (m, 2H, PhOMe),
6.12 (d, 1H, J1,2 = 7.8 Hz, H-1III), 5.76 (t, 1H,
J2,3 = J3,4 = 8.9 Hz, H-3III), 5.61 (t, 1H, J2,3 = J3,4
=
7.8 Hz, H-3I), 5.48 (m, 2H, H-2III, NHIV), 5.47 (m,
1H, H-2I), 5.34 (s, 1H, PhCH), 5.26 (s, 1H, PhCH),
5.17 (d, 1H, J1,2 = 6.2 Hz, H-1I), 5.12 (dd, 1H, J2,3
11.2 Hz, J3,4 = 3.4 Hz, H-3IV), 4.87 (d, 1H, J1,2
8.3 Hz, H-1IV), 4.60 (t, 1H, H-4I), 4,54 (d, 1H, J1,2
=
=
=
7.8 Hz, H-1II), 4.44 (br t, 1H, J = 9.2 Hz, H-4III), 4.30
(d, 1H, J4,5 = 9.6 Hz, H-5III), 4.20 (d, 1H, H-5I), 4.14
(d, 1H, J6a,6b = 11.5 Hz, H-6aII), 3.97 (d, 1H, H-4IV),
3.88 (m, 1H, H-2IV), 3.85 (m, 1H, H-6bII), 3.84 (m,
1H, H-4II), 3.78 (m, 1H, H-6aIV), 3.78, 3.73, 3.72 (3s,
3H · 3, 3OMe), 3.57 (dd, 1H, J2,3 = 9.2 Hz, J3,4
=
3.2 Hz, H-3II), 3.54 (d, J6a,6b = 12.4 Hz, 1H, H-6bIV),
3.41 (dd, 1H, H-2II), 3.24 (s, 1H, H-5II), 2.94 (s, 1H,
H-5IV), 2.74–2.35 (m, 4H, 2CH2), 2.30, 2.29, 2.26, 2.24
(4s, 3H · 4, 4PhMe), 2.04 (s, 3H, COCH3), 1.91 (s,
3H, NAc), 1.66 (s, 3H, N@CMe); 13C NMR
(125 MHz, CDCl3): d 206.62 (C@O, ketone), 172.11,
170.53, 168.57, 168.36, 165.47, 165.28, 165.18, 164.96
(OC@O, NHC@O), 161.41 (N@CMe), 155.99–126.42
(aromatic), 118.56, 114.48 (o-, m-C of MP), 103.00
(C-1II), 100.95, 100.60 (PhCH · 2), 100.22 (C-1IV), 100.05
(C-1I), 91.92 (C-1III), 77.00 (C-4III), 75.38 (C-4II), 74.66
(C-5I), 74.48 (C-4I), 74.31 (C-5III), 73.09 (C-3II), 73.06
(C-3III), 72.72 (C-4IV), 71.54 (C-2I), 71.46 (C-3I), 71.04
(C-2III), 70.72 (C-3IV), 68.95 (C-6II), 68.19 (C-6IV),
66.82 (C-5II), 66.17 (C-5IV), 61.04 (C-2II), 55.54, 52.88,
52.62 (OMe · 3), 51.47 (C-2IV), 37.75 (CH2), 29.60
(Me of Lev), 28.13 (CH2), 23.46 (NCOMe), 21.56
(PhMe), 15.99 (N@CMe); MALDITOF MS: [M+Na]+
calcd for C88H92N2O30, 1679.56; found, 1679.56.
3.6. Methyl 2-acetamido-2-deoxy-3-O-levulinoyl-b-D-
galactopyranosyl-(1!4)-O-{4-methoxyphenyl 2,3-di-O-
(4-methylbenzoyl)-b-D-glucopyranosid}uronate (14) and
methyl 2-acetamido-2-deoxy-b-D-galactopyranosyl-
(1!4)-O-{4-methoxyphenyl 2,3-di-O-(4-methylbenzoyl)-
b-D-glucopyranosid}uronate (15)
To a solution of 3 (75.5 mg, 80.3 lmol) in CH2Cl2
(4.4 mL) and MeOH (4.4 mL) was added camphor-
sulfonic acid (95.4 mg, 411 lmol) with stirring at room
temperature. After 5 days, an excess amount of Et3N
was added to the reaction mixture, and the volatiles were
removed under diminished pressure. The crude materials
obtained were passed through a gel-permeation column
(LH-20, 1:1 CHCl3–MeOH) and a silica gel column
eluting with 1:3!1:5 toluene–EtOAc followed by
100:1!10:1 EtOAc–MeOH to give 14 (37.0 mg) and 15
(14.8 mg) in 54 and 24% yield, respectively.
3.5. Methyl (2-acetamido-4,6-O-benzylidene-2-deoxy-3-
O-levulinoyl-b-D-galactopyranosyl)-(1!4)-O-{methyl
2,3-di-O-(4-methylbenzoyl)-b-D-glucopyranosyluronate}-
(1!3)-O-(2-azido-4,6-O-benzylidene-2-deoxy-b-D-galac-
topyranosyl)-(1!4)-O-{4-methoxyphenyl 2,3-di-O-(4-
methylbenzoyl)-b-D-glucopyranosid}uronate (13)
3.6.1. Data for 14. Rf 0.55 (5:1 EtOAc–MeOH); [a]D
1
To a solution of disaccharide 5 (165.8 mg, 169.5 lmol,
1.3 equiv) and disaccharide 74,11 (107.6 mg, 130.3 lmol)
in CH2Cl2 (6 mL) was added MS AW300 (800 mg). The
+48.0 (c 0.50, CHCl3); H NMR (CDCl3): d 7.91 (d,
2H, J = 8.0 Hz, Ph), 7.85 (d, 2H, J = 8.0 Hz, Ph), 7.22
(d, 2H, J = 8.3 Hz, Ph), 7.17 (d, 2H, J = 8.3 Hz, Ph),

J. Tamura et al. / Carbohydrate Research 343 (2008) 39–47
45
6.93–6.91 (m, 2H, Ph), 6.78–6.76 (m, 2H, Ph), 5.73 (t,
1H, J2,3 = J3,4 = 8.7 Hz, H-3I), 5.58 (m, 2H, H-2II,
NH), 5.25 (d, 1H, J1,2 = 7.1 Hz, H-1I), 4.98 (dd, 1H,
J2,3 = 8.0 Hz, J3,4 = 3.0 Hz, H-3II), 4.85 (d, 1H,
J1,2 = 8.3 Hz, H-1II), 4.47 (dd, 1H, J4,5 = 8.9 Hz,
H-4I), 4.33 (d, 1H, H-5I), 3.98 (m, 1H, H-2II), 3.89 (br
s, 1H, H-4II), 3.78, 3.75 (2s, 3H · 2, 2OMe), 3.35 (br s,
1H, H-5II), 3.32 (m, 2H, H-6IIab), 2.89 (d, 1H,
J4,OH = 4.4 Hz, OH-4II), 2.82–2.48 (m, 4H, 2CH2),
2.38, 2.36 (2s, 3H · 2, PhMe), 2.17 (s, 3H, COCH3),
1.95 (m, 1H, OH-6II), 1.86 (s, 3H, NAc). Anal. Calcd
for C43H49NO17ÆH2O: C, 59.36; H, 5.92; N, 1.61. Found:
C, 59.53; H, 5.61; N, 1.38.
toluene–EtOAc); 1H NMR (CDCl3): d 8.64 (s, 1H,
NH), 7.89 (d, 2H, J = 8.3 Hz, Ph), 7.81 (d, 2H,
J = 8.3 Hz, Ph), 7.72–7.16 (m, 5H, Ph), 7.14–7.12 (m,
4H, Ph), 6.73 (d, 1H, J1,2 = 3.7 Hz, H-1I), 6.13 (t, 1H,
J2,3 = J3,4 = 9.9 Hz, H-3I), 5.37 (dd, 1H, H-2I), 5.36 (d,
1H, J2,NH = 7.3 Hz, NHII), 5.31 (s, 1H, PhCH), 5.16
(dd, 1H, J2,3 = 11.5 Hz, J3,4 = 3.4 Hz, H-3II), 4.96 (d,
1H, J1,2 = 8.5 Hz, H-1II), 4.60 (d, 1H, J4,5 = 10.1 Hz,
H-5I), 4.46 (t, 1H, H-4I), 4.11 (d, 1H, H-4II), 3.88 (m,
2H, H-6aII, 2II), 3.82 (s, 3H, OMe), 3.56 (dd,
1H, J5,6a = 1.6 Hz, Jgem = 12.4 Hz, H-6bII), 2.88 (s,
1H, H-5II), 2.33, 2.32 (2s, 3H · 2, 2PhMe), 1.94 (s, 3H,
NAc), 1.13 (s, 9H, tert-Bu).
3.6.2. Data for 15. Rf 0.25 (5:1 EtOAc–MeOH); [a]D
+23.8 (c 1.06, CHCl3); H NMR (CDCl3): d 7.88 (d,
3.7.2. Methyl (2-acetamido-4,6-O-benzylidene-2-deoxy-3-
O-pivaloyl-b-D-galactopyranosyl)-(1!{4)-[methyl 2,3-di-
O-(4-methylbenzoyl)-b-D-glucopyranosyluronate]-(1!3)-
(2-acetamido-4,6-O-benzylidene-2-deoxy-b-D-galactopyr-
anosyl)-(1!}24)-[4-methoxyphenyl 2,3-di-O-(4-methyl-
1
2H, J = 8.0 Hz, Ph), 7.84 (d, 2H, J = 8.3 Hz, Ph), 7.22
(d, 2H, J = 8.3 Hz, Ph), 7.18 (d, 2H, J = 8.3 Hz, Ph),
6.95–6.93 (m, 2H, Ph), 6.80–6.78 (m, 2H, Ph), 6.23 (s,
1H, NH), 5.69 (br t, 1H, J = 8.3 Hz, H-3I), 5.63 (dd,
1H, J1,2 = 6.9 Hz, J2,3 = 9.2 Hz, H-2I), 5.20 (d, 1H,
H-1I), 4.39 (br d, 1H, J = 9.6 Hz, H-5I), 4.39 (br d,
1H, J = 7.1 Hz, H-1II), 4.37 (br t, 1H, J = 8.0 Hz,
H-4I), 3.87, 3.76 (2s, 3H · 2, 2OMe), 3.67 (m, 1H,
H-2II), 3.66 (m, 1H, H-4II), 3.57 (m, 1H, H-3II), 3.31
(m, 1H, H-6aII), 3.18 (m, 1H, H-6bII), 2.79 (s, 1H,
H-5II), 2.38, 2.36 (2s, 3H · 2, 2PhMe), 2.03 (s, 3H,
NAc). Anal. Calcd for C38H43NO15ÆH2O: C, 59.13;
H, 5.89; N, 1.82. Found: C, 59.03; H, 5.51; N, 1.56.
benzoyl)-b-D-glucopyranosid]uronate
(18).6 To
a
solution of 6 (6.28 g, 6.51 mmol) and 12 (5.08 g,
3.26 mmol) in CH2Cl2 (255 mL) was added MS
AW300 (15.4 g). The mixture was stirred for 1 h at room
temperature and then cooled to ꢀ20 ꢁC. TMSOTf
(0.88 mL, 4.9 mmol) was added while continuing the
stirring for 4 h until a temperature of 0 ꢁC was reached.
Additional TMSOTf (0.38 mL, 2.1 mmol) was then
added with a gradual increase to room temperature
and stirring overnight. The reaction was worked up by
the addition of excess amount of Et3N (1.8 mL) and
aq NaHCO3. CHCl3 was added to the mixture, and
the insoluble materials were removed by filtering
through Celite. The organic phase was washed with aq
NaHCO3 and brine, and dried over anhyd MgSO4.
The crude materials thus obtained were passed through
a gel-permeation column (LH-20, 1:1 CHCl3–MeOH)
and a silica gel column (2:1!1:15 toluene–EtOAc
followed by 50:1!5:1 EtOAc–MeOH) to give 18
(7.32 g) in 95% yield. Physical data were in good agree-
ment with those already reported.6
3.7. Preparation of methyl (2-acetamido-4,6-O-benzyl-
idene-2-deoxy-3-O-pivaloyl-b-D-galactopyranosyl)-(1!
{4)-[methyl 2,3-di-O-(4-methylbenzoyl)-b-D-glucopyrano-
syluronate]-(1!3)-(2-acetamido-4,6-O-benzylidene-2-
deoxy-b-D-galactopyranosyl)-(1!}24)-[4-methoxyphenyl
2,3-di-O-(4-methylbenzoyl)-b-D-glucopyranosid]uronate
(18)6
3.7.1. Methyl (2-acetamido-4,6-O-benzylidene-2-deoxy-3-
O-pivaloyl-b-D-galactopyranosyl)-(1!4)-2,3-di-O-(4-meth-
ylbenzoyl)-l-O-trichloroacetimidoyl-a-D-glucopyranuro-
nate (6). To a solution of 95 (1.72 g, 1.86 mmol) in
CH3CN (80 mL) and H2O (20 mL) was added ceriu-
m(IV) ammonium nitrate (5.08 g, 9.27 mmol) with stir-
ring. After 1 h the reaction mixture was extracted with
CHCl3. The organic phase was washed with brine and
dried over anhyd MgSO4. The crude materials were
eluted through a silica gel column (5:1!1:5 toluene–
EtOAc). The hemiacetal thus obtained (1.27 g,
1.55 mmol, 84% yield) was diluted with CH2Cl2
(40 mL) and CCl3CN (6.0 mL, 60 mmol). To the solu-
tion was added DBU (60 lL, 0.60 lmol) at 0 ꢁC with
stirring for 1 h. The reaction mixture was directly eluted
through a silica gel column (5:1!2:1 toluene–EtOAc) to
give 6 quantitatively as a syrup that was used for the
next reaction without further purification. Rf 0.60 (1:3
3.8. Methyl (2-acetamido-4,6-O-benzylidene-2-deoxy-3-
O-pivaloyl-b-D-galactopyranosyl)-(1!{4)-[methyl 2,3-di-
O-(4-methylbenzoyl)-b-D-glucopyranosyluronate]-(1!3)-
(2-acetamido-4,6-O-benzylidene-2-deoxy-b-D-galactopyr-
anosyl)-(1!}34)-[4-methoxyphenyl 2,3-di-O-(4-methyl-
benzoyl)-b-D-glucopyranosid]uronate (20)
To a solution of 6 (233.2 mg, 0.242 mmol) and 19
(277.2 mg, 0.122 mmol) in CH2Cl2 (10 mL) was added
MS AW300 (580 mg). The mixture was stirred for 1 h
at room temperature and then cooled to ꢀ20 ꢁC.
TMSOTf (44 lL, 0.24 mmol) was added while contin-
uing the stirring for 1 day and gradually increasing the
temperature to room temperature. The reaction was
worked up by the addition of an excess amount of

46
J. Tamura et al. / Carbohydrate Research 343 (2008) 39–47
Et3N (67 lL) and aq NaHCO3. CHCl3 was added to the
mixture, and the insoluble materials were removed by
filtering through Celite. The organic phase was washed
with aq NaHCO3 and brine, and dried over anhyd
MgSO4. The crude materials obtained were passed
through a gel-permeation column (LH-20, 1:1 CHCl3–
MeOH) to give 20 (219.0 mg) in 58% yield: Rf 0.73
(246 mg, 1.77 mmol) while stirring at 60 ꢁC for 3 days.
The reaction mixture was then cooled to room tempera-
ture and subjected directly to gel-permeation chromato-
graphy (LH-20, 1:1 CHCl3–MeOH) and an ion-
exchange resin column [Dowex AG50W-X8 (Na+), 8:1
MeOH–H2O] to give 22 (37.4 mg) in 95% yield. This
compound was used for the next reaction without
further purification. To a solution of 22 (37.4 mg,
10.5 lmol) in THF (2.2 mL) and H2O (0.3 mL) was
added 1.25 M LiOH (0.6 mL) at 0 ꢁC. After stirring
overnight, all the volatiles were removed in vacuo. The
residue was dissolved in MeOH (1.6 mL) and CH2Cl2
(0.3 mL), to which was added 0.5 M NaOH (1 mL)
dropwise while stirring at room temperature. After
18 h, the reaction was quenched with 50% AcOH. All
the volatiles were removed under reduced pressure, and
the residue was subjected to gel-permeation chromato-
graphy (LH-20, 1% AcOH) to afford 1 (18.0 mg) in
70% yield: Rf 0.32 (RP-TLC, 82:18 CH3CN–H2O);
1
(40:1 EtOAc–MeOH); [a]D +11.6 (c 0.88, CHCl3); H
NMR (CDCl3): d 7.97 (d, 2H, J = 8.2 Hz, Ph), 7.92
(m, 4H, Ph), 7.87–7.79 (m, 12H, Ph), 7.66 (d, 2H,
J = 7.6 Hz, Ph), 7.56 (m, 2H, Ph), 7.45 (m, 2H, Ph),
7.40–7.35 (m, 3H, Ph), 7.32–7.24 (m, 11H, Ph), 7.21–
7.12 (m, 12H, Ph), 7.06 (d, 2H, J = 8.3 Hz, Ph), 6.89–
6.87 (m, 2H, Ph), 6.76–6.73 (m, 2H, Ph), 5.72 (t, 1H,
J2,3 = J3,4 = 8.9 Hz, H-3I), 5.70 (s, 1H, PhCH), 5.58
(m, 4H, H-3III, 3V, 3VII, PhCH), 5.47 dd, (1H,
J1,2 = 7.1 Hz, H-2I), 5.45 (s, 1H, PhCH), 5.42 (d, 1H,
J2,NH = 6.9 Hz, NHVIII), 5.29 (s, 1H, PhCH), 5.17 (m,
3H, H-1I, 2III/V/VII, 1VIII), 5.04 (br t, 1H, J = 4.0 Hz,
H-2III/V/VII), 4.96 (m, 4H, H-1III, 1V, 1VII, 2III/V/VII),
4.86 (dd, 1H, J3,4 = 7.3 Hz, J4,5 = 10.8 Hz, H-4VII),
4.73 (m, 2H, H-4III/V, NH2/4/6), 4.67–4.58 (m, 3H,
H-1II/IV/VI, 4V/III, 3VIII), 4.50 (t, 1H, H-4I), 4.49 (d, 1H,
J3,4 = 4.1 Hz, H-4II/IV/VI), 4.40 (d, 1H, J3,4 = 3.2 Hz,
H-4II/IV/VI), 4.38 (d, 1H, J2,NH = 8.7 Hz, NHII/IV/VI),
4.32 (d, 1H, H-5VII), 4.30 (d, 1H, J3,4 = 3.4 Hz, H-4VIII),
4.27 (d, 1H, J4,5 = 10.3 Hz, H-5III/V), 4.19 (br d, 3H,
J = 9.1 Hz, H-5I, 5V/III, 1II/IV/VI), 4.12 (br d, 3H,
J = 4.4 Hz, H-1II/IV/VI 4II/IV/VI, NHII/IV/VI), 3.99–3.78
(m, 7H, H-2II/IV/VI · 2, 3II/IV/VI · 2, 6abII/IV/VI, 6aVIII),
3.75–3.49 (m, 7H, H-2II/IV/VI, 3II/IV/VI, 6abII/IV/VI · 2,
6bVIII), 3.73, 3.70, 3.68, 3.64, 3.64 (5s, 3H · 5, 5OMe),
3.22 (m, 1H, H-2VIII), 2.97 (s, 1H, H-5VIII), 2.78, 2.68,
2.49 (3s, 1H · 3, H-5II, 5IV, 5VI), 2.44, 2.41, 2.40, 2.39,
2.36, 2.34, 2.30 (7s, 24H, 8MePh), 1.75, 1.68, 1.67,
1.63 (4s, 3H · 4, 4NAc), 1.07 (s, 9H, tert-Bu). Anal.
Calcd for C164H172N4O55Æ4H2O: C, 62.50; H, 5.77; N,
1.77. Found: C, 62.42; H, 5.48; N, 1.44.
1
[a]D +4.9 (c 0.61, H2O); H NMR (D2O): d 7.13–7.09
(m, 2H, Ph), 6.99–6.97 (m, 2H, Ph), 5.09 (d, 1H,
J1,2 = 8.0 Hz, H-1I), 4.79 [3s, 3H, H-4 (GalNAc · 3)],
4.72 [s, 1H, H-4 (GalNAc)], 4.68 [d, 1H, J1,2 = 7.1 Hz,
H-1 (GalNAc)], 4.66 [d, 1H, J1,2 = 6.9 Hz, H-1(Gal-
NAc)], 4.62–6.55 [m, 5H, H-1 (GlcA · 3, GalNAc · 2)],
4.30–4.19 [m, 8H, H-6ab (GalNAc · 4)], 4.16 (d, 1H,
J4,5 = 9.6 Hz, H-5I), 4.11–4.07 [m, 4H, H-5 (Gal-
NAc · 4)], 4.09–4.06 [m, 3H, H-3 (GalNAc · 3)], 4.03–
3.97 [m, 6H, H-5 (GlcA · 3), H-2 (GalNAc · 3)], 3.94
(t, 1H, H-4I), 3.89 [m, 2H, H-2, 3 (GalNAc)], 3.89–
3.83 [m, 3H, H-4 (GlcA · 3)], 3.81 (s, 3H, OMe), 3.79
(m, 1H, H-3I), 3.68–3.64 [m, 3H, H-3 (GlcA · 3)], 3.64
(m, 1H, H-2I), 3.44–3.40 [m, 3H, H-2 (GlcA · 3)],
2.02, 2.00, 1.99, 1.99 (4s, 4H · 3, 4NAc). ESIMS: calcd
for C63H81N4O70S8Na8 (Mꢀ3H)3ꢀ, 817.66; found,
817.65; calcd for C63H82N4O70S8Na7 (MꢀNaꢀ2H)3ꢀ
,
810.33; found, 810.32; calcd for C63H83N4O70S8Na6
(Mꢀ2NaꢀH)3ꢀ
, 803.01; found, 803.00; calcd for
C63H84N4O70S8Na5 (Mꢀ3Na)3ꢀ, 795.68; found, 795.67.
3.9. Disodium 2-acetamido-2-deoxy-4,6-di-O-sulfonate-b-
D-galactopyranosyl-(1![4)-b-D-glucopyranosyluronic
acid-(1!3)-(disodium 2-acetamido-2-deoxy-4,6-di-O-sul-
fonate-b-D-galactopyranosyl)-(1!]34)-b-D-glucopyrano-
siduronic acid (1)
Acknowledgments
This research was financially supported by Taisho Phar-
maceutical Co., Ltd. The authors are grateful to Ms. M.
Tanmatsu at the Center for Instrumental Analysis of
Tottori University for elemental analyses. We also thank
Mr. Akira Higuchi (Taisho Pharmaceutical Co., Ltd)
and Venture Business Laboratory of Tottori University
for the measurement of ESIMS and MALDITOF MS,
respectively.
Camphorsulfonic acid (28.2 mg, 121 lmol) was added to
a solution of 20 (55.6 mg, 18.0 lmol) in CH2Cl2
(4.2 mL) and MeOH (4.2 mL) while stirring. After stir-
ring for 22 h an excess amount of Et3N was added to
the reaction mixture, and the volatiles were removed un-
der reduced pressure. The residue was subjected to gel-
permeation chromatography (LH-20, 1:1 CHCl3–
Supplementary data
1
MeOH) to give 21 (30.3 mg) in 61% yield. H NMR
measurement indicated complete disappearance of the
four benzylidene acetals. To a solution of 21 (30.3 mg,
11.1 lmol) in DMF (2 mL) was added SO3ÆMe3N
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.carres.
2007.09.009.

J. Tamura et al. / Carbohydrate Research 343 (2008) 39–47
47
5. Tamura, J.; Neumann, K. W.; Kurono, S.; Ogawa, T.
Carbohydr. Res. 1998, 305, 43–63.
References
6. Tamura, J.; Tokuyoshi, M. Biosci. Biotechnol. Biochem.
2004, 68, 2436–2443.
7. Liao, L.; Auzanneau, F.-I. Org. Lett. 2003, 5, 2607–2610.
1. Takagaki, K.; Munakata, H.; Kakizaki, I.; Iwafune, M.;
Itabashi, T.; Endo, M. J. Biol. Chem. 2002, 277, 8882–
8889.
2. Kawashima, H.; Atarashi, K.; Hirose, M.; Hirose, J.;
Yamada, S.; Sugahara, K.; Miyasaka, M. J. Biol. Chem.
2002, 277, 12921–12930.
3. Tully, S. E.; Mabon, R.; Gama, C. I.; Tsai, S. M.; Liu, X.;
Hsieh-Wilson, L. C. J. Am. Chem. Soc. 2004, 126, 7736–
7737.
4. Tamura, J.; Neumann, K. W.; Ogawa, T. Bioorg. Med.
Chem. Lett. 1995, 5, 1351–1354.
8. Pougny, J.-R.; Sinay, P. Carbohydr. Res. 1976, 47, 69–79.
¨
9. Hindsgaul, O.; Norberg, T.; Le Pendu, J.; Lemieux, R. U.
Carbohydr. Res. 1982, 109, 109–142.
´
´
10. de Paz, J.-L.; Ojeda, R.; Barrientos, A. G.; Penades, S.;
´
Martın-Lomas, M. Tetrahedron: Asymmetry 2005, 16,
149–158.
11. Tamura, J.; Neumann, K. W.; Ogawa, T. Justus Liebigs
Ann. Chem. 1996, 1239–1257.