Synthesis of a tetrasaccharide substrate of heparanase

Article abstract of DOI:10.1016/j.carres.2008.06.011

A tetrasaccharide, corresponding to the heparan sulfate heparanase substrate, namely β-d-GlcA(2S)-(1→4)-α-d-GlcN(NS,6S)-(1→4)-β-d-GlcA-(1→4)-α-d-GlcN(NS,6S)-OMe, was synthesized in a convergent manner via coupling of a pair of the disaccharide building blocks as a key step.

Full text of DOI:10.1016/j.carres.2008.06.011

Carbohydrate Research 343 (2008) 2853–2862
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Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Synthesis of a tetrasaccharide substrate of heparanase
*
Jianfang Chen, Ying Zhou, Chen Chen, Weichang Xu, Biao Yu
State Key Laboratory of Bio-organic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai 200032, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 12 May 2008
Received in revised form 8 June 2008
Accepted 11 June 2008
Available online 18 June 2008
A tetrasaccharide, corresponding to the heparan sulfate heparanase substrate, namely b-D-GlcA(2S)-
(1?4)-
a-D-GlcN(NS,6S)-(1?4)-b-D-GlcA-(1?4)-a-D-GlcN(NS,6S)-OMe, was synthesized in a convergent
manner via coupling of a pair of the disaccharide building blocks as a key step.
Ó 2008 Elsevier Ltd. All rights reserved.
Dedicated to Professor Yongzheng Hui on
the occasion of his 70th birthday
Keywords:
Heparan sulfate
Synthesis
Heparanase
Glycosylation
Thioglycoside
Trifluoroacetimidate
O-
1. Introduction
-
OSO3
O
O
O
-
O-
Heparan sulfate proteoglycans (HSPGs), consisting of a central
protein core covalently linked to heparan sulfate (HS) polysaccha-
ride chains, make up a significant proportion of the cell basement
membranes and the extracellular matrix (ECM), which is com-
posed of a network of macromolecules that serve to maintain tis-
sue and cellular architecture.1,2 Heparanase is a mammalian
O
OSO3
O
HO
-O3SHN
HO
O
-O3SO
O
O
O
OH
HO
HO
-O3SHN
OH
ΔHexA(2S)-GlcN(NS,6S)-GlcA-GlcN(NS,6S)
endo-b-D-glucuronidase that specifically cleaves HS of the HSPGs
-
O-
in the ECM and basal membrane, and is preferentially expressed
in many tumor cells.3 Digestion of the ECM or basal membrane
by heparanase releases HS-bound growth factors that promote
angiogenesis, tumor growth, progression, invasion, and metastasis.
Therefore, heparanase has become an emerging target for the
development of novel anti-cancer drugs.4,5 Nevertheless, the sub-
strate specificity of heparanase has only recently been started to
be understood,3b,6 by employing purified recombinant human
heparanase and a series of the structurally defined HS fragments.7
OSO3
O
O
O
HO
-
O-
O
OSO3
O
HO
-O3SHN
HO
O
-O3SO
O
O
O
HO
HO
OH
-O3SHN
1
OMe
Figure 1. The minimum substrate of heparanase and its analog 1.
port the synthesis of a tetrasaccharide, GlcA(2S)-GlcN(NS,6S)-
GlcA-GlcN(NS,6S)-OMe (1, Fig. 1), where the replacement of the
DHexUA with GlcA, and the installation of an
anomeric -OMe at the reducing end, would facilitate the synthe-
The tetrasaccharide fragment
DHexUA(2S)-GlcN(NS,6S)-GlcA-
GlcN(NS,6S) (Fig. 1) turns out to be the minimum sequence well
recognized by heparanase. The availability of a good heparanase
substrate would lead to the establishment of a quantitative assay
of the enzyme activity thus facilitating the development of thera-
peutic strategies targeting this important enzyme. Herein, we re-
non-reducing end
a
sis as well as the stability of the substrate.
2. Results and discussion
Referring to the previous protecting-group strategies for hepa-
rin synthesis,8 we envisioned the fully protected tetrasaccharide
* Corresponding author.
E-mail address: byu@mail.sioc.ac.cn (B. Yu).
0008-6215/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2008.06.011

2854
J. Chen et al. / Carbohydrate Research 343 (2008) 2853–2862
1
OPMB
O
OMe
O
O
AcO
O
OPMB
O
OBn
O
BnO
O
BnO
N
AZMBO
3 O
O
BnO
BnO
2
N
OBz
3 OMe
OPMB
O
OMe
O
OBn
O
OPMB
O
O
O
AcO
HO
O
O
O
CF3
+
BnO
BnO
BnO
BnO
N3
AZMBO
OBz
N
3 OMe
NPh
3
4
O
OPMB
O
OPMB
O
MP
O
O
S
HO
HO
BnO
OTBS
+
+
BnO
BnO
OR
N3
5
N
3 OMe
6 R = AZMB
8
7 R = Bz
Scheme 1. Retrosynthetic plan for tetrasaccharide 1.
2 as an advanced precursor to the target molecule 1 (Scheme 1).
Benzyl, acetyl, and benzoyl groups were used as permanent pro-
tecting groups for the hydroxyl groups; the carboxylic acid groups
were blocked with methyl and/or benzyl groups; p-methoxybenzyl
(PMB) and 2-(azidomethyl)benzoyl (AZMB)9 groups were used for
the temporary protection of the hydroxyl groups destined for sul-
fonation; and an azide was employed as the precursor to the sulfo-
(68% from GlcN). Diol 10 was subjected to selective protection with
TBSCl in the presence of imidazole at low temperature (ꢀ15 °C,
CH2Cl2) followed with BnBr and NaH in THF, providing the 3-O-
Bn-1-b-O-TBS ether 11 regio- and stereoselectively in a good 69%
yield. Regioselective opening of the 4,6-O-p-methoxybenzylidene
acetal in 11 with NaBH3CN and TFA in a mixed solvent of CH2Cl2
and THF led to the desired 6-O-PMB derivative 5 (88% yield).11
Treatment of the crude 9 with HCl-containing methanol gave the
methyl glycoside 15 (70% for two steps),12 which was subjected
to the 4,6-O-p-methoxybenzylidene acetal formation (p-MePh-
CH(OMe)2, p-TsOHꢁH2O, DMF, 40 °C) to provide 16 (75%). Protection
of the remaining 3-OH with a benzyl group (BnBr, NaH, Bu4N+Iꢀ,
THF) led to 17 (76%). Regioselective opening of the 4,6-O-p-
methoxybenzylidene acetal in 17 (NaBH3CN, TFA, CH2Cl2, THF) fur-
nished the desired 6-O-PMB derivative 8; at this stage, the two ano-
mers (43% and 36% yields, respectively) were able to be separated
by column chromatography on silica gel in nearly equal amount.
The o-methylphenyl thioglycosides 6 and 7 were synthesized
nated 2-amino groups of the
D-GlcN residues. Considering the
pseudo-symmetry of tetrasaccharide 2, we planned to adopt a con-
vergent 2+2 coupling of the disaccharide donor 3 and acceptor 4.
For the preparation of the disaccharides (3 and 4), GlcN and Glc
derivatives 5–8 were required.
The synthesis of the GlcN and Glc derivatives 5–8 is depicted in
Scheme 2. The 4-OH free GlcN derivatives 5 and 8 were both pre-
pared from
D
-glucosamine hydrochloride in five steps (16% and
-glucopyranose
-glucosamine
17% yields, respectively). Thus, 2-azido-2-deoxy-
D
9, resulting from the diazotransfer reaction of
D
hydrochloride with triflyl azide,10 was treated with p-MeOP-
hCH(OMe)2 in the presence of p-TsOH in DMF to give 1,3-diol 10
from 1,2,4,6-tetra-O-acetyl-3-O-benzyl-
D
-glucopyranoside 12,13
OH
O
O
O
MP
MP
ref.10
ref.13
a
O
O
d
b, c
HO
HO
O
O
D-GlcN
D-Glc
OH
OH
OTBS
O
5
HO
BnO
N3
N3
N3
10
9
11
OAc
O
OAc
O
O
MP
i
e
f, g
O
AcO
BnO
AcO
S
OAc
S
BnO
BnO
OR
OAc
OAc
12
13
14 R = H
6 R = AZMB
7 R = Bz
h
OH
O
O
ref.12
MP
k
j
O
l
HO
HO
O
D-GlcN
OMe
OMe
8 + 8β
RO
N3
N3
15
16 R = H
17 R = Bn
Scheme 2. Synthesis of the monosaccharide building blocks 5–8. Reagents and conditions: (a) p-MeOPhCH(OMe)2, p-TsOH, DMF, 40 °C, 68%; (b) TBSCl, imidazole, CH2Cl2,
ꢀ15 °C; (c) NaH, BnBr, THF, 0 °C to rt, 69% (for 2 steps); (d) NaBH3CN, CF3CO2H, 4 Å MS, THF, CH2Cl2, rt, 88%; (e) o-methylbenzenethiol, BF3ꢁEt2O, CH2Cl2, 0 °C to rt, 44%; (f)
NaOMe, MeOH, rt, 99%; (g) p-MeOPhCH(OMe)2, p-TsOHꢁH2O, CH2Cl2, 40 °C, 90%; (h) AZMBCl, DMAP, CH2Cl2, rt, 93%; (i) BzCl, Et3N, DMAP, CH2Cl2, rt, 88%; (j) p-
MeOPhCH(OMe)2, p-TsOHꢁH2O, DMF, 40 °C, 75%; (k) BnBr, NaH, Bu4N+Iꢀ, THF, 0 °C to rt, 76%; (l) NaBH3CN, CF3CO2H, 4 Å MS, THF, CH2Cl2, rt, 43% (for 8), 36% (for 8b).

J. Chen et al. / Carbohydrate Research 343 (2008) 2853–2862
2855
OH
O
BnO
OAZMB
OPMB
O
OPMB
O
O
MP
O
a
b
HO
BnO
O
O
O
OTBS
OTBS
e
6
5
+
BnO
BnO
OAZMB
N3
N3
18
19
O
O
OMe
OMe
O
OPMB
O
OPMB
O
c
O
f
HO
AcO
O
O
OTBS
OH
3
BnO
BnO
BnO
OAZMB
BnO
N3
OAZMB
N3
22
20
21
R = H
R = Ac
d
OPMB
O
OPMB
O
OH
O
O
MP
O
g
h
i
O
HO
BnO
O
O
7
8
4
+
BnO
BnO
23
BnO
OBz
N
OBz
N
3OMe
3OMe
24
Scheme 3. Synthesis of the disaccharide building blocks 3 and 4. Reagents and conditions: (a) BSP, 3 Å MS, TTBP, Tf2O, CH2Cl2, ꢀ60 to ꢀ40 °C to rt, 87%; (b) AcOH, H2O (70%),
70 °C, 93%; (c) (i) TEMPO, BAIB, CH2Cl2, H2O, rt; (ii) CH2N2, Et2O, rt, 88% (for 2 steps); (d) Ac2O, Et3N, DMAP, CH2Cl2, rt, 95%; (e) TBAF, AcOH, THF, ꢀ15 °C to rt, 86%; (f)
ClC(@NPh)CF3, K2CO3, acetone, rt, 97%; (g) BSP, 3 Å MS, TTBP, Tf2O, CH2Cl2, ꢀ60 to ꢀ40 °C to rt, 90%; (h) HOAc–H2O (70%), 70 °C, 89%; (i) TEMPO, BAIB, CH2Cl2, H2O, rt; (ii)
BnBr, KHCO3, DMF, rt, 88% (for 2 steps).
which was readily prepared from
D
-glucose in four steps and
ꢀ20 °C. However, the reaction was found to be sluggish and when
51% overall yield (Scheme 3). Treatment of 12 with o-methyl-
benzenethiol in the presence of BF3ꢁEt2O provided the b-thiogly-
coside 13 in a moderate 44% yield. Removal of the three O-acetyl
groups in 13 (NaOMe, MeOH), followed by protection of the 4,6-
di-OH with p-methoxybenzylidene provided 14 in 90% yield.
Installation of an AZMB or a Bz group (AZMBCl or BzCl, DMAP,
CH2Cl2) at the remaining 2-OH furnished the desired thioglyco-
sides 6 and 7.
the donor was completely hydrolyzed, the desired tetrasaccharide
2a/b was isolated in only trace amounts, with the acceptor being
partly recovered (Scheme 4, Table 1, entry 1). Changing the pro-
moter to TBSOTf, we were able to obtain the tetrasaccharide in a
moderate 36% yield in favor of the
a anomer (a:b = 9:1) (Table 1,
entry 2). Toluene has been found to be the better solvent in some
condensations between a glucosyl donor and oligosaccharide
acceptors bearing a 4-OH uronate moiety.18 Indeed, the present
The desired GlcA-(1?4)-GlcN disaccharide donor 3 and accep-
tor 4 were synthesized as shown in Scheme 3. Glycosidic coupling
of thioglucoside 6 with tert-butyldimethylsilyl 2-azido-3-O-ben-
condensation in toluene gave the tetrasaccharide 2
58% yield, albeit the :b selectivity was only moderate (4:6
(Table 1, entry 3). Lowering the temperature from ꢀ20 °C to
ꢀ40 °C, we were pleased to find that the /b ratio was improved
while the yield remained good (Table 1, entry 4). We also tried
homogeneous - and b-glycosyl trifluoroacetimidates 3 and 3b
(and the corresponding disaccharide trichloroacetimidate as well
(results not shown)) as donors in the glycosylation reaction with
acceptor 4 under similar conditions as for entry 3. Similar results
both in yield and stereoselectivity were attained (Table 1, entries
5 and 6). It has long been known that under SN1 conditions the ef-
fect of ethers on the oxycarbenium intermediate generally results
in the formation of the thermodynamically more stable glycoside
anomer, due to stabilization of the epimeric oxonium anomer by
the reverse anomeric effect.19 We thus explored the reactions with
ether or THF as solvent, but the coupling yields were much lower
(Table 1, entries 7 and 8).
a
/b in a better
a
a
/b)
zyl-2-deoxy-6-O-p-methoxybenzyl-b-
D-glucopyranoside 5 under
a
the action of BSP (1-benzenesulfinyl-piperidine)–Tf2O in the pres-
ence of 2,4,6-tri-tert-butylpyrimidine (TTBP) in CH2Cl2 provided
the b-linked disaccharide 18 in a satisfactory 87% yield (H-10,
4.55 ppm, J = 8.4 Hz).14 Removal of the 4,6-O-benzylidene group
in 18 with 70% HOAc gave diol 19 (93%). The resulting primary hy-
droxyl group was then selectively oxidized with the combination
of a catalytic amount of 2,2,6,6-tetramethyl-1-piperidinyloxy free
radical (TEMPO) and a slight excess of [bis(acetoxy)iodo]benzene
(BAIB) in a biphasic dichloromethane–water solvent system,15
leading to the corresponding glucuronic acid derivative, which
was subsequently treated with CH2N2 to provide the methyl uro-
nate 20 in an excellent 88% yield. The remaining 40-OH was pro-
tected with an acetate to give 21. Cleavage of the 1-O-TBS group
was achieved with tetrabutylammonium fluoride (TBAF) in the
presence of acetic acid, providing lactol 22, which was readily con-
a
a
The PMB groups in 2
rodicyanobenzoquinone (DDQ)20 to afford cleanly the desired
anomer 25 and the b-anomer 25b via column chromatography.
Subsequent reduction of the azido groups in 25 was realized with
a/b were selectively removed with dichlo-
a
-
verted into the desired glycosyl trifluoroacetimidate
3
a
(ClC(@NPh)CF3, K2CO3, acetone, 97%).16
a
Under similar conditions for the coupling of 6 and 5 (BSP, TTBP,
Tf2O, CH2Cl2), glycosylation of methyl 2-azido-3-O-benzyl-2-
deoxy-6-O-p-methoxybenzyl-a-D-glucopyranoside 8 with thiogly-
PPh3 in the presence of silica gel in a mixed solvent of tetrahydro-
furan and water,21 the AZMB group was simultaneously removed,
furnishing tetrasaccharide 26 in 86% yield. The unprotected hydro-
xyl groups and amino groups by simultaneous O- and N-sulfona-
tion afforded a mixture of partially sulfonated tetrasaccharides.
On the basis of this finding, the deprotection–sulfonation sequence
was altered. Thus, O-sulfonation of 26 was achieved by treatment
with SO3ꢁEt3N in DMF at 55 °C, followed by N-sulfonation using
SO3ꢁpyridine in triethylamine–pyridine,22 affording the completely
sulfonated tetrasaccharide, which was unambiguously confirmed
by ESI-MS analyses. The sulfonated tetrasaccharide was directly
subjected to saponification to remove the Ac, Bz, methyl, and ben-
zyl groups, followed by hydrogenolytic cleavage of the benzyl
groups. The target tetrasaccharide 1 was successfully obtained
coside 7 provided the desired b-linked disaccharide 23 in an
excellent 90% yield (H-10, 4.42 ppm, J = 7.8 Hz). Similar transfor-
mations as for 18?20, that is, removal of the 40,60-O-benzylidene
group, selective oxidation of the resulting 60-OH with TEMPO–
BAIB, and benzyl ester formation (BnBr, KHCO3, DMF) furnished
the desired disaccharide acceptor 4 in good yield (78%, three
steps).
With the disaccharide trifluoroacetimidate 3 and disaccharide
acceptor 4 available, we then investigated the glycosylation reac-
tion between donor 3 and acceptor 4 under conventional condi-
tions,17 using CH2Cl2 as solvent and TMSOTf as the promoter at

2856
J. Chen et al. / Carbohydrate Research 343 (2008) 2853–2862
O
OMe
O
OPMB
O
a
b
AcO
BnO
O
O
OBn
O
OPMB
O
3 + 4
BnO
OAZMB
N3
O
O
BnO
BnO
/β
2α
OBz
N
3OMe
O
OMe
O
OH
O
O
OMe
O
BnO
OAZMB
O
OBn
O
OH
O
OH
O
AcO
BnO
O
O
BnO
OH
O
OBn
O
AcO
BnO
+
O
O
O
N3
BnO
OAZMB
BnO
O
N3
O
OBz
N
3OMe
BnO
BnO
β
25
25α
OBz
N
3OMe
O
OMe
OH
O
O
AcO
c
O
d, e, f
O
BnO
BnO
OBn
O
OH
O
1
H2N
OH
O
O
BnO
BnO
26
OBz
H2N
OMe
Scheme 4. Synthesis of the target tetrasaccharide 1. Reagents and conditions: (a) TBSOTf (0.2 equiv), 4 Å MS, toluene, ꢀ40 °C, 71%; (b) DDQ, CH2Cl2, H2O, rt, 43% (for 25
a),
39% (for 25b); (c) PPh3, THF, silica gel, H2O, rt, 86%; (d) (i) SO3ꢁEt3N, DMF, 55 °C; (ii) SO3ꢁpyridine, Et3N, pyridine, rt; (e) 2 N NaOH, MeOH, H2O, rt; (f) H2, Pd–C (10%), EtOH, rt,
36 h, 22% (for 3 steps).
Table 1
Glycosylation of disaccharide 4 with disaccharide trifluoroacetimidate 3a
Entry
Donor
Promoter
Solvent
Temperature (°C)
Yieldb (%)
a
/bc
1
2
3
4
5
6
7
8
3
3
3
3
TMSOTf
TBSOTf
TBSOTf
TBSOTf
TBSOTf
TBSOTf
TBSOTf
TBSOTf
CH2Cl2
CH2Cl2
Toluene
Toluene
Toluene
Toluene
Et2O
ꢀ20
ꢀ20
ꢀ20
ꢀ40
ꢀ40
ꢀ40
ꢀ20
ꢀ20
Trace
36
58
71
68
65
11
Trace
9:1
4:6
6:4
6:4
6:4
3a
3b
3
3
THF
a
Compound 3 (1.0 equiv), 4 (1.0 equiv), promoter (0.2 equiv), 4 Å molecular sieves.
b
c
Isolated yields.
a
/b ratio was determined by 1H NMR spectrometry.
after ion-exchange with Dowex 50WX4-Na+ resin, desalting with
Sephadex G-15, and lyophilization in 22% yield (for three steps).
In summary, tetrasaccharide 1, corresponding to the heparan
3.1.1. 2-Azido-2-deoxy-4,6-O-(4-methoxybenzylidene)-a/b-D-
glucopyranose (10)
Crude 9, prepared from D-glucosamine hydrochloride (50.0 g,
sulfate sequence heparanase substrate, namely b-
D-GlcA(2S)-
0.23 mol),10 was dissolved in DMF (150 mL), and p-TsOH (2.0 g)
and p-methoxybenzaldehyde dimethyl acetal (47.0 mL, 0.28 mol)
were added. After stirring at 40 °C under reduced pressure for
8 h, the mixture was concentrated in vacuo. The obtained anomers
were purified by silica gel column chromatography (2:1 petroleum
ether–EtOAc) to afford 10 (19.8 g, 68%) as a white solid. Rf = 0.52
(1:1 petroleum ether–EtOAc); 1H NMR (300 MHz, CD3OD): d
7.43–7.38 (m, 2H), 6.90–6.86 (m, 2H), 5.52 (s, 1H), 5.20 (d, 0.5H,
J = 3.3 Hz), 4.88 (s, 2H), 4.62 (d, 0.5H, J = 8.1 Hz), 4.22 (dd, 0.5H,
J = 10.8, 5.4 Hz), 4.15 (dd, 0.5H, J = 9.9, 5.1 Hz), 4.07 (t, 0.5H,
J = 9.9 Hz), 4.01–3.93 (m, 0.5H), 3.78 (s, 3H), 3.71 (t, 0.5H,
J = 10.2 Hz), 3.58 (t, 0.5H, J = 9.3 Hz), 3.47 (t, 1H, J = 9.0 Hz), 3.30
(br, 1H), 3.23–3.16 (m, 1H); 13C NMR (75 MHz, CD3OD): d 161.6,
131.5, 131.4, 128.9, 128.9, 114.4, 103.1, 103.0, 97.9, 94.1, 83.5,
82.3, 73.2, 70.3, 70.0, 69.6, 67.7, 65.8, 63.8, 55.7; ESI-MS: 324.2
[M+H]+; Anal. Calcd for C14H17N3O6: C, 52.01; H, 5.30; N, 13.00.
Found: C, 57.76; H, 5.30; N, 12.77; IR (thin film in NaCl)
(1?4)- -GlcN(NS,6S)-(1?4)-b- -GlcA-(1?4)- -GlcN(NS,6S)-
a-
D
D
a-D
OMe, has been synthesized in a convergent manner via coupling of
a pair of the disaccharide building blocks as the key step. The pres-
ent synthesis requires a total of 43 steps, with the longest linear se-
quence of 21 steps and in 0.5% overall yield from
D
-glucose.
However, the 2+2 glycosylation in building the GlcN-a-(1?4)-GlcA
linkage demands further improvement.
3. Experimental
3.1. General methods23
Solvents were purified in the usual way. Thin layer chromatog-
raphy (TLC) was performed on precoated plates of Silica Gel HF254
(0.5 mm, Yantai, China). Flash column chromatography was per-
formed on Silica Gel H (10–40
lm, Yantai, China). Optical rotations
m
max = 2124, 1246, 1089, 985 cmꢀ1
.
were determined with a Perkin–Elmer Model 241 MC polarimeter.
NMR spectra were recorded on a Bruker AM 300 spectrometer with
Me4Si as the internal standard. J values were given in Hertz. Mass
spectra were obtained using HP5989A or a VG Quatro mass spec-
trometer. Elemental analyses were performed on a Perkin–Elmer
Model 2400 instrument.
3.1.2. tert-Butyldimethylsilyl 2-azido-3-O-benzyl-2-deoxy-4,6-
O-(4-methoxybenzylidene)-b- -glucopyranoside (11)
D
To a cooled solution of compound 10 (13.0 g, 0.040 mol) and
imidazole (6.8 g, 0.10 mol) in dry CH2Cl2 (40.0 mL) at ꢀ15 °C, TBSCl

J. Chen et al. / Carbohydrate Research 343 (2008) 2853–2862
2857
(6.7 g, 0.044 mol) was added. After stirring for 2.5 h, the reaction
mixture was quenched by the addition of H2O (40.0 mL). The or-
ganic phase was separated and the remaining aqueous phase was
extracted with CH2Cl2 (3 ꢂ 100 mL). The combined organic phase
was washed with brine, dried with Na2SO4, and then filtered and
concentrated in vacuo. The residue was purified by silica gel col-
umn chromatography (8:1 petroleum ether–EtOAc) to give tert-
ether–EtOAc) to give 13 (2.56 g, 44%) as a yellow solid. Rf = 0.60
(5:2 petroleum ether–EtOAc); ½a D28
ꢃ
ꢀ12.6 (c 1.0, CHCl3); 1H NMR
(300 MHz, CDCl3): d 7.52 (d, 1H, J = 6.9 Hz), 7.32–7.13 (m, 8H),
5.16 (d, 1H, J = 9.6 Hz), 5.09 (d, 1H, J = 9.6 Hz), 4.65–4.57 (m, 3H),
4.21–4.09 (m, 2H), 3.72 (t, 1H, J = 9.3 Hz), 3.63–3.58 (m, 1H), 2.39
(s, 3H), 2.06 (s, 3H), 2.05 (s, 3H), 1.96 (s, 3H); 13C NMR (75 MHz,
CDCl3): d 170.6, 169.3, 169.2, 139.8, 137.6, 132.8, 132.2, 130.2,
128.4, 128.0, 127.8, 127.7, 126.5, 86.7, 81.4, 75.9, 74.1, 71.3, 69.6,
62.5, 20.9, 20.8, 20.7; Anal. Calcd for C26H30O8S: C, 62.14; H, 6.02.
butyldimethylsilyl
2-azido-2-deoxy-4,6-O-(4-methoxybenzyli-
dene)-b-
D
-glucopyranoside24 (14.2 g, 81%) as a white solid. To a
cooled solution of the above product (11.6 g, 0.027 mol) in dry
THF (100.0 mL) at 0 °C, NaH (60% dispersion in mineral oil,
2.13 g, 0.053 mol) was added. After the mixture had been stirred
for 1 h, benzyl bromide (4.8 mL, 0.040 mol) was added. Stirring
continued at room temperature for 24 h, the reaction mixture
was quenched by the addition of CH3OH (2.5 mL). The resulting
mixture was filtered through a pad of Celite and the filtrate con-
centrated in vacuo. The residue was purified by silica gel column
chromatography (25:1 petroleum ether–EtOAc) to afford 11
(12.1 g, 85%) as a white solid. Rf = 0.82 (2:1 petroleum ether–
Found: C, 61.79; H, 5.91; IR (thin film in NaCl)
mmax = 1741, 1234,
1040 cmꢀ1
.
3.1.5. 2-Methylphenyl 3-O-benzyl-4,6-O-(4-
methoxybenzylidene)-1-thio-b- -glucopyranoside (14)
D
To a solution of 13 (1.0 g, 2.0 mmol) in CH3OH (20 mL) at room
temperature was added CH3ONa (46 mg, 2.0 mmol). After stirring
at room temperature for 5 h, the reaction mixture was quenched
by the addition of H+-ion-exchange resin (1.5 g). The mixture
was filtered and concentrated in vacuo. The residue was purified
by silica gel column chromatography (2:1 petroleum ether–EtOAc)
EtOAc); ½a 2D8
ꢃ
ꢀ89.7 (c 1.0, CHCl3); 1H NMR (300 MHz, CDCl3): d
7.41–7.25 (m, 7H), 6.90 (d, 2H, J = 8.7 Hz), 5.52 (s, 1H), 4.89 (d,
1H, J = 11.1 Hz), 4.78 (d, 1H, J = 11.3 Hz), 4.58 (d, 1H, J = 7.5 Hz),
4.27 (dd, 1H, J = 11.8, 5.1 Hz), 3.81 (s, 3H), 3.77–3.66 (m, 2H,
J = 9.9, 5.1 Hz), 3.50 (t, 1H, J = 9.3 Hz), 3.41–3.33 (m, 2H), 0.93 (s,
9H), 0.16 (s, 3H), 0.15 (s, 3H); 13C NMR (75 MHz, CDCl3): d 160.0,
138.0, 129.6, 128.3, 128.1, 127.8, 127.3, 113.6, 101.3, 97.4, 81.53
78.8, 74.8, 68.7, 68.5, 66.3, 55.3, 25.5, 17.9, ꢀ4.4, ꢀ5.2; Anal. Calcd
for C27H37N3O6Si: C, 61.46; H, 7.07; N, 7.96. Found: C, 61.71; H,
to give 2-methylphenyl 3-O-benzyl-1-thio-b-D-glucopyranoside
(750 mg, 99%) as a white solid. Rf = 0.40 (1:1 petroleum ether–
EtOAc); ½a 2D5
ꢃ
ꢀ85.3 (c 1.0, CHCl3); 1H NMR (300 MHz, CDCl3): d
7.62–7.12 (m, 9H), 4.96–4.84 (m, 5H), 4.65 (d, 1H, J = 8.7 Hz),
3.88–3.83 (m, 1H), 3.69–3.64 (m, 1H), 3.50–3.39 (m, 2H), 3.37–
3.30 (m, 2H), 2.41 (s, 3H); 13C NMR (75 MHz, CD3OD): d 140.4,
139.9, 135.2, 132.2, 131.1, 129.2, 129.1, 128.5, 128.1, 127.7, 89.5,
88.0, 82.0, 76.4, 74.3, 71.2, 62.8, 21.0; Anal. Calcd for C20H24O5S:
C, 63.81; H, 6.43. Found: C, 63.61; H, 6.51; IR (thin film in NaCl)
7.15; N, 7.66; IR (thin film in NaCl)
mmax = 2111, 1614, 1515,
1252, 1078 cmꢀ1
.
m
max = 3306, 1123, 1037, 745 cmꢀ1. The same procedure described
for the preparation of compound 10 (from compound 9) was em-
3.1.3. tert-Butyldimethylsilyl 2-azido-3-O-benzyl-2-deoxy-6-O-
(4-methoxybenzyl)-b- -glucopyranoside (5)
ployed for the preparation of 14 from 2-methylphenyl 3-O-ben-
zyl-1-thio-b-D-glucopyranoside. Purification by silica gel column
D
A mixture of compound 11 (200 mg, 0.38 mmol), NaBH3CN
(238 mg, 3.8 mmol), and freshly activated 4 Å molecular sieves
(500 mg) in dry THF (3.8 mL) and CH2Cl2 (3.8 mL) was stirred at
room temperature under argon for 40 min. Then CF3CO2H
(0.55 mL, 7.6 mmol) was added dropwise. After stirring 9 h at room
temperature, the reaction mixture was quenched by addition of a
satd aq NaHCO3 (50.0 mL). The mixture was filtered through a
pad of Celite. The organic phase was separated and the remaining
aqueous phase was extracted with CH2Cl2 (3 ꢂ 100 mL). The com-
bined organic phase was washed with brine, dried with Na2SO4,
and then filtered and concentrated in vacuo. The residue was puri-
fied by silica gel column chromatography (8:1 petroleum ether–
EtOAc) to give monosaccharide 5 (176 mg, 88%) as a colorless syr-
chromatography (6:1:1 petroleum ether–EtOAc–CH2Cl2) gave
monosaccharide 14 (1.0 g, 90%) as a white solid. Rf = 0.80 (3:1
petroleum ether–EtOAc); ½a D28
ꢃ
ꢀ40.9 (c 1.0, CHCl3); 1H NMR
(300 MHz, CDCl3): d 7.52 (d, 1H, J = 7.2 Hz), 7.38–7.16 (m, 10H),
6.90 (d, 1H, J = 8.7 Hz), 5.52 (s, 1H), 4.94 (d, 1H, J = 11.7 Hz), 4.78
(d, 1H, J = 11.7 Hz), 4.63 (d, 1H, J = 9.3 Hz), 4.33 (dd, 2H, J = 10.5,
5.1 Hz), 3.80 (s, 3H), 3.75 (t, 1H, J = 10.2 Hz), 3.68–3.45 (m, 4H),
2.64 (br, 1H), 2.45 (s, 3H); 13C NMR (75 MHz, CD3OD): d 160.0,
140.3, 138.1, 133.1, 131.3, 130.4, 129.6, 128.4, 128.2, 128.1,
127.8, 127.3, 126.6, 113.5, 101.2, 88.5, 81.6, 80.9, 74.7, 72.5, 70.5,
68.5, 55.2, 21.018; Anal. Calcd for C28H30O6S: C, 67.99; H, 6.11.
Found: C, 68.24; H, 6.16; IR (thin film in NaCl)
mmax = 3294, 1517,
1251, 1070 cmꢀ1
.
up. Rf = 0.56 (3:1 petroleum ether–EtOAc); ½a D28
ꢀ31.1 (c 1.2,
ꢃ
CHCl3); 1H NMR (300 MHz, CDCl3): d 7.38–7.30 (m, 5H), 7.23 (d,
2H, J = 8.7 Hz), 6.87 (d, 2H, J = 8.4 Hz), 4.90 (d, 1H, J = 11.1 Hz),
4.77 (d, 1H, J = 11.1 Hz), 4.53–4.49 (m, 3H), 3.79 (s, 3H), 3.68–
3.59 (m, 3H), 3.42–3.37 (m, 1H), 3.33–3.18 (m, 2H), 2.72 (br, 1H),
0.93 (d, 9H, J = 7.8 Hz), 0.17 (s, 3H), 0.16 (s, 3H); 13C NMR
(75 MHz, CDCl3): d 159.2, 138.2, 129.7, 129.3, 128.5, 128.0, 127.9,
113.8, 97.2, 82.2, 74.9, 73.8, 73.3, 72.1, 70.1, 68.0, 55.2, 25.6,
17.9, ꢀ4.3, ꢀ5.3; HRMS (MALDI/DHB) m/z: calcd for C27H39N3O6Si-
3.1.6. 2-Methylphenyl 2-O-(2-(azidomethyl)benzoyl)-3-O-
benzyl-4,6-O-(4-methoxybenzylidene)-1-thio-b-D-
glucopyranoside (6)
2-(Azidomethyl)benzoic acid (286 mg, 1.6 mmol) was dissolved
in CHCl3 (2.0 mL), and thionyl chloride (0.36 mL, 4.8 mmol) was
added. The reaction mixture was heated to reflux for 5 h, cooled,
and concentrated in vacuo using a base trap. The resulting residue
was coevaporated with toluene (3 ꢂ 15 mL) to yield the crude 2-
Na, 552.2506; found, 552.2504; IR (thin film in NaCl)
1614, 1515, 1251, 1075 cmꢀ1
m
max = 2111,
(azidomethyl)benzoyl chloride. A solution of compound 14
.
(266 mg, 0.54 mmol) in dry CH2Cl2 (2.5 mL) was stirred at room
temperature, and DMAP (330 mg, 2.7 mmol) was added, followed
by a solution of the crude 2-(methylazido)benzoyl (AZMB) chloride
in CH2Cl2 (1.0 mL). After stirring at room temperature under argon
for 14 h, the reaction mixture was diluted with CH2Cl2 (100 mL)
and washed twice with satd aq NaHCO3 and once with water.
The organic layer was dried with Na2SO4, and then filtered and
concentrated in vacuo. The residue was purified by silica gel col-
umn chromatography (12:1:2 petroleum ether–EtOAc–CH2Cl2) to
give compound 6 (326 mg, 93%) as a white solid. Rf = 0.45 (4:1
3.1.4. 2-Methylphenyl 2,4,6-tri-O-acetyl-3-O-benzyl-1-thio-b-D-
glucopyranoside (13)
To a solution of 12 (5.1 g, 12.0 mmol) and 2-methylbenzeneth-
iol (2.8 mL, 23.0 mmol) in anhydrous CH2Cl2 (30 mL) at 0 °C was
added BF3ꢁEt2O (1.7 mL, 13.0 mmol). After stirring at rt for 24 h,
the reaction mixture was quenched by the addition of Et3N
(1.5 mL). The mixture was concentrated in vacuo. The residue
was purified by silica gel column chromatography (5:1 petroleum

2858
J. Chen et al. / Carbohydrate Research 343 (2008) 2853–2862
petroleum ether–EtOAc); ½a D25
ꢃ
+44.6 (c 1.0, CHCl3); 1H NMR
103.3, 101.2, 81.5, 79.0, 74.9, 68.4, 66.1, 66.0, 57.4, 55.2; HRMS
(MALDI/DHB) m/z: calcd for C22H25N3O6Na, 452.1743; found,
450.1637.
(300 MHz, CDCl3): d 7.87 (d, 1H, J = 7.8 Hz), 7.59–7.37 (m, 6H),
7.16–7.11(m, 8H), 6.91 (d, 2H, J = 9.0 Hz), 5.58 (s, 1H), 5.36 (t, 1H,
J = 10.2 Hz), 4.88–4.82 (m, 2H), 4.76 (d, 2H, J = 6.3 Hz), 4.65 (d,
1H, J = 12.0 Hz), 4.37 (dd, 1H, J = 10.5, 5.1 Hz), 3.90–3.83 (m, 3H),
3.80 (s, 3H), 3.56–3.50 (m, 1H), 2.25 (s, 3H); 13C NMR (75 MHz,
CDCl3): d 164.9, 160.0, 139.9, 137.8, 137.7, 132.9, 132.3, 132.3,
130.9, 130.3, 129.5, 129.3, 128.1, 128.1, 127.9, 127.8, 127.6,
127.3, 126.6, 113.6, 101.2, 87.2, 81.4, 79.4, 74.2, 71.9, 70.5, 68.4,
55.2, 52.8, 20.8; Anal. Calcd for C35H34O7S: C, 66.14; H, 5.40; N,
6.43. Found: C, 66.15; H, 5.52; N, 6.44; IR (thin film in NaCl)
3.1.10. Methyl 2-azido-3-O-benzyl-2-deoxy-6-O-
(4-methoxybenzyl)-
methyl 2-azido-3-O-benzyl-2-deoxy-6-O-
(4-methoxybenzyl)-b- -glucopyranoside (8b)
a-D-glucopyranoside (8) and
D
The same procedure described for the preparation of compound
5 (from compound 11) was employed for the preparation of 8 and
8b from 17. Purification by silica gel column chromatography (7:1
m
max = 2104, 1726, 1517, 1249 cmꢀ1
.
petroleum ether–EtOAc) gave methyl glycoside acceptor
8
(203 mg, 43%) and compound 8b (170 mg, 36%) as colorless syrups.
3.1.7. 2-Methylphenyl 2-O-benzoyl-3-O-benzyl-4,6-O-(4-
methoxybenzylidene)-1-thio-b- -glucopyranoside (7)
Compound 8: Rf = 0.35 (3:1 petroleum ether–EtOAc); ½a D22
ꢃ
+51.1 (c
D
0.9, CHCl3); 1H NMR (300 MHz, CDCl3): 7.39–7.23 (m, 7H), 6.87 (d,
2H, J = 9.0 Hz), 4.91 (d, 1H, J = 8.1 Hz), 4.82–4.78 (m, 2H), 4.50 (AB,
d, 2H, J = 11.7 Hz), 3.80 (s, 3H), 3.83–3.62 (m, 5H), 3.42 (s, 3H), 3.37
(dd, 1H, J = 9.9, 3.6 Hz), 2.51 (d, 1H, J = 1.5 Hz); 13C NMR (75 MHz,
CDCl3): d 159.3, 139.0, 129.7, 129.3, 128.5, 128.1, 127.9, 113.8,
98.7, 79.9, 75.0, 73.3, 72.2, 69.8, 69.4, 62.9, 55.2, 55.2; HRMS (MAL-
DI/DHB) m/z: calcd for C22H27N3O6Na, 452.1798; found, 452.1791;
A solution of compound 14 (1.0 g, 2.0 mmol) and DMAP (50 mg)
in dry CH2Cl2 (10.0 mL) was stirred at room temperature, and Et3N
(1.5 mL, 10.2 mmol) was added, followed by addition of BzCl
(0.5 mL, 4.1 mmol). After stirring under argon for 12 h, the reaction
mixture was diluted with CH2Cl2 (100 mL), and washed twice with
satd aq NaHCO3 and once with water. The organic layer was dried
with Na2SO4, and then filtered and concentrated in vacuo. The res-
idue was purified by silica gel column chromatography (10:1
petroleum ether–EtOAc) to give compound 7 (1.1 g, 88%) as a white
IR (cmꢀ1
) mmax = 2112, 1613, 1515, 1250, 1075. Compound 8b:
Rf = 0.33 (3:1 petroleum ether–EtOAc); ½a D28
ꢀ42.5 (c 1.0, CHCl3);
ꢃ
1H NMR (300 MHz, CDCl3): 7.41–7.24 (m, 7H), 6.89 (d, 2H,
J = 8.4 Hz), 4.91 (d, 1H, J = 11.4 Hz), 4.80 (d, 1H, J = 11.4 Hz), 4.51
(AB, d, 2H, J = 11.4 Hz), 4.18 (d, 1H, J = 7.8 Hz), 3.80 (s, 3H), 3.70
(d, 2H, J = 4.5 Hz), 3.61 (t, 1H, J = 9.3 Hz), 3.56 (s, 3H), 3.43–3.24
(m, 3H), 2.77 (br, 1H); 13C NMR (75 MHz, CDCl3): d 159.3, 138.0,
129.6, 129.4, 128.5, 128.1, 128.0, 113.8, 102.9, 82.5, 75.1, 73.7,
73.4, 72.3, 69.8, 65.6, 57.1, 55.2; HRMS (MALDI/DHB) m/z: calcd
for C22H27N3O6Na, 452.1798; found, 452.1781; IR (thin film in
solid. Rf = 0.35 (5:1 petroleum ether–EtOAc); ½a D28
ꢃ
+51.3 (c 1.0,
CHCl3); 1H NMR (300 MHz, CDCl3): d 8.01 (d, 2H, J = 7.5 Hz), 7.60
(t, 1H, J = 7.2 Hz), 7.48–7.43 (m, 5H), 7.23–7.08 (m, 8H), 6.91 (d,
2H, J = 9.0 Hz), 5.57 (s, 1H), 5.41–5.35 (m, 1H), 4.81 (d, 1H,
J = 7.5 Hz), 4.80 (d, 1H, J = 11.7 Hz), 4.67 (d, 1H, J = 11.7 Hz), 4.36
(dd, 1H, J = 10.5, 4.8 Hz), 3.88–3.82 (m, 3H), 3.80 (s, 3H), 3.56–
3.50 (m, 1H), 2.23 (s, 3H); 13C NMR (75 MHz, CDCl3): d 165.0,
160.0, 140.1, 137.7, 133.2, 132.7, 132.5, 130.3, 129.9, 129.6,
128.3, 128.1, 128.0, 127.5, 127.3, 126.5, 113.6, 110.2, 87.5, 81.3,
79.2, 74.1, 72.0, 70.5, 68.5, 55.2, 20.8; Anal. Calcd for C35H34O7S:
C, 70.21; H, 5.72. Found: C, 69.78; H, 5.65; IR (thin film in NaCl)
NaCl) m .
max = 2112, 1613, 1515, 1250, 1075 cmꢀ1
3.1.11. tert-Butyldimethylsilyl (2-O-(2-(azidomethyl)benzoyl)-
3-O-benzyl-4,6-O-(4-methoxybenzylidene)-b-
glucopyranosyl)-(1?4)-2-azido-3-O-benzyl-2-deoxy-
6-O-(4-methoxybenzyl)-b- -glucopyranoside (18)
m
max = 1723, 1294, 1097 cmꢀ1
.
D
-
D
3.1.8. Methyl 2-azido-2-deoxy-4,6-O-(4-methoxybenzylidene)-
/b- -gluco-pyranose (16)
The same procedure described for the preparation of com-
A solution of compound 6 (200 mg, 0.31 mmol), BSP (70 mg,
0.34 mmol), and TTBP (152 mg, 0.61 mmol) in dry CH2Cl2
(3.0 mL) was stirred for 30 min under argon over activated 3 Å
molecular sieves (300 mg). The mixture was cooled to ꢀ60 °C,
a
D
pound 10 (from compound 9) was employed for the preparation
of 16 from 15. Purification by silica gel column chromatography
(4:1 petroleum ether–EtOAc) gave compound 16 (2.6 g, 75%) as
a white solid. Rf = 0.40 (3:1 petroleum ether–EtOAc); 1H NMR
(300 MHz, CDCl3): 7.43–7.39 (m, 2H), 6.90 (d, 2H, J = 7.2 Hz),
5.50 (s, 1H), 4.80 (d, 0.4H, J = 3.0 Hz), 4.36–425 (m, 1.7H), 4.18
(t, 0.5H, J = 9.3 Hz), 3.92–3.63 (m, 1.8H), 3.81 (s, 3.0H), 3.60 (s,
1.8H), 3.51 (d, 0.9H, J = 9.0 Hz), 3.45 (s, 1.5H), 3.43–3.31 (m,
1.6H), 2.78 (br s, 0.9H); 13C NMR (75 MHz, CDCl3): d 160.35,
129.2, 127.5, 127.5, 103.4, 102.0, 101.8, 99.3, 81.7, 80.5, 72.0,
69.0, 68.7, 68.4, 66.3, 66.1, 63.2, 62.2, 57.4, 55.4, 55.2; HRMS
(MALDI/DHB) m/z: calcd for C15H20N3O6, 338.1352; found,
338.1354.
and Tf2O (5 lL, 0.037 mmol) was added. The temperature was
raised to ꢀ40 °C and stirring continued for 15 min. Then a solution
of the compound 5 (103 mg, 0.19 mmol) in dry CH2Cl2 (0.5 mL)
was added, and the resulting mixture was allowed to warm slowly
to room temperature. The reaction mixture was quenched by the
addition of Et3N (0.5 mL) and the mixture filtered through a pad
of Celite. The filtrate was concentrated and purified by silica gel
column chromatography (10:1 petroleum ether–EtOAc) to afford
disaccharide 18 (180 mg, 87%) as a colorless syrup. Rf = 0.55 (3:1
petroleum ether–EtOAc);
½
a 2D5
ꢃ
ꢀ4.2 (c 1.0, CHCl3); 1H NMR
(300 MHz, CDCl3): d 7.77 (d, 1H, J = 8.4 Hz), 7.63–7.52 (m, 2H),
7.44–7.30 (m, 8H), 7.20–7.11 (m, 7H), 6.93–6.87 (m, 4H), 5.49 (s,
1H), 5.18 (t, 1H, J = 8.1 Hz), 4.94 (d, 1H, J = 3.0 Hz), 4.90 (s, 1H),
4.84 (d, 1H, J = 12.0 Hz), 4.75 (d, 1H, J = 10.8 Hz), 4.68–4.53 (m,
4H), 4.38 (d, 1H, J = 6.0 Hz), 4.38 (d, 1H, J = 12.0 Hz), 4.17 (dd, 1H,
J = 10.2, 4.2 Hz), 4.00–3.94 (m, 1H), 3.96 (s, 3H), 3.83 (s, 3H), 3.70
(d, 1H, J = 9.0 Hz), 3.66 (d, 1H, J = 9.6 Hz), 3.55 (dd, 1H, J = 11.7,
2.7 Hz), 3.48 (t, 1H, J = 10.2 Hz), 3.37 (d, 1H, J = 11.4 Hz), 3.30–
3.25 (m, 3H), 3.15 (br d, 1H, J = 9.3 Hz), 0.89 (s, 9H), 0.10 (s, 3H),
0.09 (s, 3H); 13C NMR (75 MHz, CDCl3): d 164.5, 156.0, 159.3,
138.5, 138.2, 137.9, 133.0, 130.6, 130.0, 129.8, 129.6, 128.2,
128.1, 127.9, 127.8, 127.7, 127.5, 127.3, 113.8, 113.5, 101.1,
100.4, 96.9, 81.7, 80.6, 78.4, 76.1, 75.1, 74.6, 74.1, 73.7, 73.0,
68.4, 68.1, 67.1, 66.0, 55.2, 55.2, 52.8, 25.5, 17.9, ꢀ4.4, ꢀ5.3; Anal.
3.1.9. Methyl 2-azido-3-O-benzyl-2-deoxy-4,6-O-(4-
methoxybenzylidene)-a/b-D-glucopyranose (17)
The same procedure described for the preparation of compound
11 (from compound 10) was employed for the preparation of 17
from 16. Purification by silica gel column chromatography (10:1
petroleum ether–EtOAc) gave compound 17 (881 mg, 76%) as a
white solid. Rf = 0.50 (5:1 petroleum ether–EtOAc); 1H NMR
(300 MHz, CDCl3): 7.43–7.27 (m, 7H), 6.90 (d, 2H, J = 8.7 Hz), 5.54
(s, 1H), 4.95–4.89 (m, 1H), 4.80–4.77 (m, 1.5H), 4.36–4.24 (m,
1.5H), 4.05 (t, 0.5H, J = 10.2 Hz), 3.82 (s, 3H), 3.87–3.66 (m, 2.5H),
3.59–3.53 (m, 1.8H), 3.48–3.37 (m, 2.2H); 13C NMR (75 MHz,
CDCl3): d 160.0, 137.7, 129.5, 128.3, 128.2, 127.9, 127.2, 113.6,

J. Chen et al. / Carbohydrate Research 343 (2008) 2853–2862
2859
Calcd for C56H66N6O13Si: C, 63.50; H, 6.28; N, 7.93. Found: C, 63.52;
H, 6.41; N, 7.73; IR (thin film in NaCl) max = 2932, 2860, 2110,
1733, 1614, 1516, 1251, 1072 cmꢀ1
3.1.14. tert-Butyldimethylsilyl (methyl 4-O-acetyl-2-O-(2-
(azidomethyl)benzoyl)-3-O-benzyl-b-
glucopyranosyluronate)-(1?4)-2-azido-3-O-benzyl-2-deoxy-6-
O-(4-methoxybenzyl)-b- -glucopyranoside (21)
m
D-
.
D
To a solution of compound 20 (126 mg, 0.13 mmol) and DMAP
(4 mg) in dry CH2Cl2 (2.0 mL), Et3N (0.4 mL) was added followed
by addition of Ac2O (0.2 mL). The reaction mixture was stirred at
room temperature under argon for 2 h and then concentrated.
The residue was purified by silica gel column chromatography
(4:1 petroleum ether–EtOAc) providing 21 (125 mg, 95%) as a col-
3.1.12. tert-Butyldimethylsilyl (2-O-(2-(azidomethyl)benzoyl)-
3-O-benzyl-b- -glucopyranosyl)-(1?4)-2-azido-3-O-benzyl-2-
deoxy-6-O-(4-methoxybenzyl)-b- -glucopyranoside (19)
A solution of compound 18 (120 mg, 0.11 mmol) in 70% aque-
ous HOAc (6.0 mL) was heated at 70 °C for 30 min and then con-
centrated. The residue was purified by silica gel column
chromatography (2:1 petroleum ether–EtOAc) to provide 19
D
D
orless syrup. Rf = 0.75 (2:1 petroleum ether–EtOAc); ½a D28
ꢀ7.3 (c
ꢃ
1.0, CHCl3); 1H NMR (300 MHz, CDCl3): d 7.81 (d, 1H, J = 8.1 Hz),
7.61–7.55 (m, 2H), 7.43–6.93 (m, 13H), 6.91 (d, 2H, J = 8.1 Hz),
5.28–5.20 (m, 2H), 5.06 (d, 1H, J = 11.7 Hz), 4.90 (d, 1H,
J = 14.7 Hz), 4.74 (d, 1H, J = 11.7 Hz), 4.69 (d, 1H, J = 8.1 Hz), 4.65–
4.49 (m, 4H), 4.38 (br d, 1H, J = 7.2 Hz), 4.19 (d, 1H, J = 11.4 Hz),
3.96 (t, 1H, J = 9.0 Hz), 3.82 (s, 3H), 3.74 (d, 1H, J = 10.2 Hz), 3.67–
3.52 (m, 2H), 3.56 (s, 3H), 3.40–3.25 (m, 3H), 3.16 (br d, 1H,
J = 9.6 Hz), 2.00 (s, 3H), 0.90 (s, 9H), 0.09 (s, 3H), 0.08 (s, 3H); 13C
NMR (75 MHz, CDCl3): d 169.3, 167.3, 164.1, 159.3, 138.6, 138.4,
137.4, 133.2, 130.5, 130.1, 129.8, 129.7, 128.2, 128.2, 127.9,
127.8, 127.7, 127.5, 127.5, 127.3, 113.9, 100.0, 97.0, 80.7, 79.7,
76.7, 75.0, 74.4, 74.1, 73.0, 72.8, 72.8, 71.2, 68.3, 67.2, 55.2, 52.8,
52.7, 25.5, 20.6, 17.9, ꢀ4.4, ꢀ5.3; HRMS (MALDI/DHB) m/z: calcd
for C51H62N6O14SiNa, 1033.3991; found, 1033.4021; IR (thin film
(99 mg, 93%) as
a
colorless syrup. Rf = 0.50 (1:1 petroleum
ether–EtOAc); ½a D26
ꢃ
ꢀ0.36 (c 1.0, CHCl3); 1H NMR (300 MHz,
CDCl3): d 7.82 (d, 1H, J = 8.4 Hz), 7.63–7.52 (m, 2H), 7.42–7.30
(m, 6H), 7.26–7.16 (m, 7H), 6.93 (d, 2H, J = 8.4 Hz), 5.14 (t, 1H,
J = 8.4 Hz), 4.94 (d, 1H, J = 9.0 Hz), 4.90 (d, 1H, J = 5.4 Hz), 4.76
(d, 1H, J = 10.8 Hz), 4.69–4.61 (m, 4H), 4.55 (d, 1H, J = 7.8 Hz),
4.40–4.38 (m, 1H), 4.25 (d, 1H, J = 11.7 Hz), 3.94 (br t, 1H,
J = 9.3 Hz), 3.80 (s, 3H), 3.68–3.55 (m, 3H), 3.48–3.36 (m, 3H),
3.30–3.27 (m, 2H), 3.24–3.13 (m, 2H), 2.60 (br s, 1H), 1.87 (br s,
1H), 0.90 (s, 9H), 0.11 (s, 3H), 0.10 (s, 3H); 13C NMR (75 MHz,
CDCl3): d 164.4, 159.3, 138.4, 137.9, 133.2, 130.5, 129.9, 129.9,
129.8, 128.4, 128.4, 127.9, 127.8, 127.7, 127.7, 127.6, 127.5,
113.9, 99.8, 96.9, 83.0, 80.6, 75.8, 75.2, 75.1, 74.8, 74.5, 73.7,
73.1, 70.8, 68.2, 67.1, 62.0, 55.2, 52.9, 25.5, 17.9, ꢀ4.4, ꢀ5.3; HRMS
(MALDI/DHB) m/z: calcd for C48H60N6O12SiNa, 963.3936; found,
in NaCl) m .
max = 2950, 2932, 2111, 1760, 1251, 1075 cmꢀ1
963.3938; IR (thin film in NaCl)
mmax = 2931, 2860, 2111, 1731,
3.1.15. (Methyl 4-O-acetyl-2-O-(2-(azidomethyl)benzoyl)-3-O-
benzyl-b- -glucopyranosyluronate)-(1?4)-2-azido-3-O-benzyl-
2-deoxy-6-O-(4-methoxybenzyl)- /b- -glucopyranose (22)
To a cooled solution of compound 21 (125 mg, 0.12 mmol) in
L, 0.15 mmol) was added fol-
lowed by addition of TBAF (1 M, 0.15 mL). The temperature was
raised to room temperature and stirring continued for 3 h. The
reaction mixture was diluted with CH2Cl2 (50 mL) and washed
twice with satd aq NaHCO3, dried with Na2SO4, and then filtered
and concentrated in vacuo. The residue was purified by silica gel
column chromatography (1.8:1 petroleum ether–EtOAc) to provide
1252, 1068 cmꢀ1
.
D
a
D
3.1.13. tert-Butyldimethylsilyl (methyl 2-O-(2-
(azidomethyl)benzoyl)-3-O-benzyl-b-
glucopyranosyluronate)-(1?4)-2-azido-3-O-benzyl-2-deoxy-6-
O-(4-methoxybenzyl)-b- -glucopyranoside (20)
D
-
THF (3.0 mL) at ꢀ15 °C, HOAc (9
l
D
Compound 19 (140 mg, 0.15 mmol) was dissolved in CH2Cl2–
water (2:1 v/v, 2.1 mL), and TEMPO (5 mg, 0.03 mmol) and BAIB
(120 mg, 0.37 mmol) were added subsequently. After the biphasic
mixture was stirred vigorously for 1 h, the reaction mixture was
quenched by the addition of satd aq NaHSO3 (5.0 mL). The layers
were separated and the aqueous layer was acidified with 1 M aque-
ous HCl, and extracted three times with CH2Cl2 (100 mL). The com-
bined organic layer was washed with brine, dried with Na2SO4, and
then filtered and concentrated in vacuo to yield the corresponding
crude glucuronic acid. The crude glucuronic acid was dissolved in
ether (10 mL) and treated with a solution of diazomethane in ether
(ꢄ0.3 M, 2.0 mL). After stirring for 10 min, the yellow solution was
treated with a few drops of acetic acid until the mixture turned col-
orless. The solvents were evaporated and the residue was purified
by silica gel column chromatography (3:1 petroleum ether–EtOAc)
to afford disaccharide 20 (126 mg, 88%) as a white solid. Rf = 0.40
22 (95 mg, 86%) as a white solid (a/b, 3:2). Rf = 0.50 (1:1 petroleum
ether–EtOAc); 1H NMR (300 MHz, CDCl3): 7.78–7.74 (m, 1H), 7.61
(t, 1H, J = 7.8 Hz), 7.53 (br d, 1H, J = 7.5 Hz), 7.46–7.41 (m, 3H),
7.39–7.32 (m, 2H), 7.30–7.22 (m, 3H), 7.18–7.09 (m, 5H), 6.96 (t,
2H, J = 8.1 Hz), 5.26–5.18 (m, 3H), 5.09 (d, 0.4H, J = 11.1 Hz), 4.90
(dd, 1H, J = 14.7, 3.9 Hz), 4.74–4.67 (m, 2H), 4.65 (br s, 1H), 4.58
(br s, 1H), 4.55–4.37 (m, 2H), 4.13 (dd, 1H, J = 12.0, 2.4 Hz), 3.95
(AB, d, 1H, J = 9.6 Hz), 3.89–3.82 (m, 3.6H), 3.76 (br d, 1H,
J = 9.6 Hz), 3.69 (dd, 1H, J = 9.9, 0.9 Hz), 3.65–3.48 (m, 5H), 3.41–
3.25 (m, 2H), 3.20–3.13 (m, 1H), 2.01 (d, 3H, 4.90 J = 1.8 Hz); HRMS
(ESI) m/z calcd: for C45H48N6O14Na, 919.3126; found, 919.3115; IR
(thin film in NaCl)
mmax = 3447, 2955, 2110, 1760, 1734, 1514,
(2:1 petroleum ether–EtOAc); ½a D25
ꢃ
+1.1 (c 1.0, CHCl3); 1H NMR
1250, 1076 cmꢀ1
.
(300 MHz, CDCl3): d 7.79 (d, 1H, J = 7.8 Hz), 7.63–7.52 (m, 2H),
7.41–7.20 (m, 8H), 7.14 (br s, 5H), 6.91 (d, 2H, J = 8.4 Hz), 5.14 (t,
1H, J = 9.0 Hz), 4.99 (d, 1H, J = 11.4 Hz), 4.91 (d, 1H, J = 14.7 Hz),
4.83 (d, 1H, J = 11.4 Hz), 4.72 (d, 1H, J = 11.1 Hz), 4.66–4.56 (m,
4H), 4.39 (br d, 1H, J = 6.6 Hz), 4.19 (d, 1H, J = 12.0 Hz), 4.03–3.95
(m, 2H), 3.81 (s, 3H), 3.68–3.55 (m, 2H), 3.60 (s, 3H), 3.46 (t, 1H,
J = 8.7 Hz), 3.41 (br d, 1H, J = 6.6 Hz), 3.31–3.29 (m, 2H), 3.15 (br
s, 2H), 0.90 (s, 9H), 0.10 (s, 3H), 0.09 (s, 3H); 13C NMR (75 MHz,
CDCl3): d 169.6, 164.4, 159.3, 138.7, 138.3, 137.9, 133.1, 130.6,
129.9, 129.7, 128.2, 128.1, 127.9, 127.7, 127.6, 127.3, 113.9,
100.1, 97.0, 81.2, 80.7, 76.2, 74.8, 74.6, 74.4, 73.8, 73.0, 72.9, 72.4,
68.1, 67.1, 55.2, 52.8, 52.7, 25.5, 17.9, ꢀ4.4, ꢀ5.3; Anal. Calcd for
C49H60N6O13Si: C, 60.73; H, 6.24; N, 8.67. Found: C, 60.35; H,
3.1.16. (Methyl 4-O-acetyl-2-O-(2-(azidomethyl)benzoyl)-3-O-
benzyl-b- -glucopyranosyluronate)-(1?4)-2-azido-3-O-benzyl-
2-deoxy-6-O-(4-methoxybenzyl)- -glucopyranosyl N-
phenyltrifluoroacetimidate (3 ) and (Methyl 4-O-acetyl-O-(2-
(azidomethyl)-benzoyl)-3-O-benzyl-b-
glucopyranosyluronate)-(1?4)-2-azido-3-O-benzyl-2-deoxy-6-
O-(4-methoxybenzyl)-b- -glucopyranosyl N-
D
a
-D
a
D
-
D
phenyltrifluoroacetimidate (3b)
To a solution of compound 22 (163 mg, 0.18 mmol) and K2CO3
(30 mg, 0.22 mmol) in acetone (2.0 mL), ClCNPhCF3 (57 mg,
0.27 mmol) was added. The reaction mixture was stirred at room
temperature under argon for 8 h and then filtered and concen-
trated in vacuo. The residue was purified by silica gel column chro-
matography (3:1 petroleum ether–EtOAc, 1% Et3N, v/v) to provide
6.32; N, 8.45; IR (thin film in NaCl)
mmax = 2955, 2932, 2860,
2111, 1733, 1252, 1071 cmꢀ1
.

2860
J. Chen et al. / Carbohydrate Research 343 (2008) 2853–2862
disaccharide trifluoroacetimidate 3 (188 mg, 97%) as a colorless
7.22–7.15 (m, 5H), 6.99 (d, 2H, J = 8.7 Hz), 5.15 (t, 1H, J = 9.3 Hz),
4.99 (d, 1H, J = 11.1 Hz), 4.78–4.72 (m, 3H), 4.59 (AB, d, 2H,
J = 11.4 Hz), 4.39 (d, 1H, J = 7.8 Hz), 4.25 (d, 1H, J = 12.0 Hz), 3.94
(t, 1H, J = 9.3 Hz), 3.82 (t, 1H, J = 9.9 Hz), 3.80 (s, 3H), 3.64 (t, 2H,
J = 11.1 Hz), 3.50 (t, 1H, J = 9.3 Hz), 3.42–3.26 (m, 5H), 3.30 (s,
3H), 3.21–3.15 (m, 1H), 3.29 (d, 1H, J = 3.3 Hz); 13C NMR
(75 MHz, CDCl3): d 164.8, 159.5, 138.1, 137.8, 133.3, 130.3, 129.6,
129.4, 128.4, 127.7, 127.6, 127.5, 126.9, 114.1, 99.8, 98.5, 82.9,
78.0, 75.4, 75.1, 74.9, 73.8, 73.2, 70.7, 69.9, 66.5, 62.9, 62.0, 55.3,
55.2; HRMS (MALDI/DHB) m/z: calcd for C42H47N3O12Na,
syrup (
a
/b, 3:2). Compound 3
ꢃ
a
: Rf = 0.80 (1:1 petroleum ether–
EtOAc); ½a 2D6
+35.5 (c 1.0, CHCl3); 1H NMR (300 MHz, CDCl3): d
7.75 (d, 1H, J = 8.1 Hz), 7.62 (dt, 1H, J = 8.4, 0.9 Hz), 7.53 (br d,
1H, J = 7.2 Hz), 7.48–7.34 (m, 5H), 7.30–7.23 (m, 5H), 7.19–7.05
(m, 6H), 6.98 (br d, 2H, J = 8.7 Hz), 6.76 (br d, 2H, J = 7.5 Hz),
5.29–5.21 (m, 3H), 4.90 (d, 1H, J = 14.1 Hz), 4.73–4.64 (m, 3H),
4.90–4.60 (m, 3H), 4.18 (d, 1H, J = 12.0 Hz), 4.10 (t, 1H,
J = 9.0 Hz), 3.88 (t, 1H, J = 10.2 Hz), 3.84 (s, 3H), 3.72 (d, 1H,
J = 9.9 Hz), 3.66–3.51 (m, 4H), 3.54 (s, 3H), 3.36 (br d, 1H,
J = 9.6 Hz), 2.02 (s, 3H); 13C NMR (75 MHz, CDCl3): d 169.2, 167.2,
164.3, 159.9, 143.2, 138.4, 138.2, 137.5, 133.2, 130.4, 130.1,
129.8, 128.7, 128.3, 128.2, 128.1, 127.9, 127.8, 127.5, 127.4,
124.4, 119.3, 114.2, 100.0, 93.7, 79.8, 78.0, 76.2, 75.6, 74.2, 73.2,
73.0, 72.8, 72.8, 71.2, 66.5, 62.3, 55.2, 52.8, 52.6, 20.6. Compound
808.3057; found, 808.3041; IR (thin film in NaCl)
mmax = 2919,
2110, 1731, 1515, 1267, 1044 cmꢀ1
.
3.1.19. Methyl (benzyl 2-O-benzoyl-3-O-benzyl-b-
glucopyranosyluronate)-(1?4)-2-azido-3-O-benzyl-2-deoxy-6-
O-(4-methoxybenzyl)- -glucopyranoside (4)
D-
3b: Rf = +0.85 (1:1 petroleum ether–EtOAc); ½a D26
ꢃ
+25.1 (c 1.0,
a-D
CHCl3); 1H NMR (300 MHz, CDCl3): d 7.76 (d, 1H, J = 7.5 Hz), 7.61
(t, 1H, J = 7.2 Hz), 7.52 (d, 1H, J = 7.2 Hz), 7.47–7.35 (m, 5H),
7.32–7.20 (m, 5H), 7.17–7.00 (m, 6H), 6.96 (d, 2H, J = 8.7 Hz),
6.75 (d, 2H, J = 7.5 Hz), 5.25–5.12 (m, 3H), 4.90 (d, 1H,
J = 15.0 Hz), 4.73 (br d, 2H, J = 11.4 Hz), 4.64 (t, 1H, J = 6.9 Hz),
4.59–4.48 (m, 3H), 4.21 (d, 1H, J = 12.0 Hz), 4.06 (t, 1H,
J = 9.6 Hz), 3.83 (s, 3H), 3.71 (d, 1H, J = 10.2 Hz), 3.58–3.52 (m,
3H), 3.57 (s, 3H), 3.42 (br d, 2H, J = 10.2 Hz), 2.02 (s, 3H); 13C
NMR (75 MHz, CDCl3): d 169.2, 167.6, 167.2, 164.4, 164.1, 161.7,
159.6, 147.4, 143.1, 138.8, 138.6, 138.2, 137.5, 134.2, 133.3,
130.4, 130.0, 129.8, 128.7, 128.2, 127.9, 127.7, 127.5, 127.4,
124.4, 119.2, 115.6, 114.1, 99.8, 95.6, 80.7, 79.7, 75.8, 75.4, 75.2,
74.2, 73.1, 72. 9, 72.8, 71.3, 70.7, 66.5, 64.8, 55.2, 52.9, 52.6, 20.6,
16.4.
Compound 24 (203 mg, 0.26 mmol) was dissolved in CH2Cl2–
water (2:1 v/v, 4.5 mL), subsequently TEMPO (8 mg, 0.05 mmol)
and BAIB (209 mg, 0.65 mmol) were added. After the biphasic mix-
ture was stirred vigorously for 1 h, the reaction mixture was
quenched by addition of saturated aqueous NaHSO3 (5.0 mL). The
layers were separated and the aqueous layer was acidified with
1 M aqueous HCl, and extracted thrice with CH2Cl2 (100 mL). The
combined organic layer was washed with brine, dried with Na2SO4,
and then filtered and concentrated in vacuo, yielding the corre-
sponding crude glucuronic acid. The crude glucuronic acid was dis-
solved in dry DMF (5.0 mL), and KHCO3 (100 mg, 1.0 mmol) was
added, followed by addition of benzyl bromide (59 lL, 0.5 mmol).
After stirring vigorously for 10 h, the reaction mixture was treated
with a few drops of acetic acid. The solvents were evaporated and
the residue was purified by silica gel column chromatography (3:1
petroleum ether–EtOAc) to afford disaccharide 4 (202 mg, 88%) as
3.1.17. Methyl (2-O-benzoyl-3-O-benzyl-4,6-O-(4-
methoxybenzylidene)-b-
D
-glucopyranosyl)-(1?4)-2-azido-3-O-
a colorless syrup. Rf = 0.40 (2:1 petroleum ether–EtOAc); ½a D26
ꢃ
benzyl-2-deoxy-6-O-(4-methoxybenzyl)-
a-D
-glucopyranoside
+55.1 (c 1.0, CHCl3); 1H NMR (300 MHz, CDCl3): d 7.94 (d, 2H,
J = 7.8 Hz), 7.62 (t, 1H, J = 6.9 Hz), 7.49 (t, 2H, J = 7.5 Hz), 7.40–
7.26 (m, 7H), 7.15 (s, 5H), 6.98 (d, 2H, J = 8.4 Hz), 5.17 (t, 1H,
J = 9.0 Hz), 5.10 (d, 1H, J = 11.1 Hz), 4.78–4.70 (m, 3H), 4.63 (t,
2H, J = 9.6 Hz), 4.43 (d, 1H, J = 8.1 Hz), 4.21 (d, 1H, J = 11.7 Hz),
4.02 (t, 1H, J = 9.3 Hz), 3.95 (t, 1H, J = 9.3 Hz), 3.84–3.78 (m, 1H),
3.82 (s, 3H), 3.67 (t, 2H, J = 9.9 Hz), 3.56 (s, 3H), 3.44–3.31 (m,
4H), 3.28 (s, 3H), 3.07 (br, 1H); 13C NMR (75 MHz, CDCl3): d
169.5, 164.7, 159.6, 138.6, 138.0, 133.3, 130.2, 129.7, 129.6,
128.5, 128.2, 128.1, 128.0, 127.7, 127.6, 127.3, 114.2, 100.3, 98.8,
81.3, 78.0, 75.1, 74.7, 74.0, 73.2, 73.0, 72.3, 69.9, 66.8, 62.8, 55.3,
55.2, 52.6, 29.7; HRMS (MALDI/DHB) m/z: calcd for C43H47N3O13Na,
(23)
The same procedure described for the preparation of compound
18 (from compounds 6 and 5) was employed for the preparation of
23 from 7 and 8. Purification by silica gel column chromatography
(4:1 petroleum ether–EtOAc) gave disaccharide 23 (190 mg, 90%)
as a white solid. Rf = 0.40 (3:1 petroleum ether–EtOAc); ½a D26
ꢃ
+34.0 (c 1.1, CHCl3); 1H NMR (300 MHz, CDCl3): d 7.94 (d, 2H,
J = 8.4 Hz), 7.63 (t, 1H, J = 7.5 Hz), 7.51–7.25 (m, 11H), 7.10 (br s,
5H), 6.92 (d, 4H, J = 8.7 Hz), 5.46 (s, 1H), 5.20 (t, 1H, J = 9.0 Hz),
5.00 (d, 1H, J = 10.5 Hz), 4.79 (d, 1H, J = 12.3 Hz), 4.73–4.69 (m,
3H), 4.60 (d, 1H, J = 12.0 Hz), 4.42 (d, 1H, J = 7.8 Hz), 4.20 (d, 1H,
J = 13.2 Hz), 4.15 (dd, 1H, J = 10.5, 4.8 Hz), 3.98 (t, 1H, J = 9.6 Hz),
3.85–3.79 (m, 1H), 3.83 (s, 3H), 3.70 (s, 3H), 3.69–3.65 (m, 2H),
3.53 (t, 1H, J = 9.3 Hz), 3.44–3.31 (m, 4H), 3.29 (s, 3H), 3.16–3.14
(m, 1H); 13C NMR (75 MHz, CDCl3): d 164.7, 160.0, 159.5, 138.4,
137.9, 133.3, 130.0, 129.7, 129.7, 129.6, 129.5, 128.4, 128.1,
128.2, 127.9, 127.7, 127.5, 127.5, 127.3, 114.0, 113.5, 101.1,
100.5, 98.5, 81.6, 78.3, 78.0, 76.4, 75.1, 74.2, 73.7, 73.1, 70.0, 68.4,
66.6, 66.0, 62.8, 55.3, 55.2, 55.2; Anal. Calcd for C50H53N3O13: C,
66.43; H, 5.91; N, 4.65. Found: C, 66.37; H, 6.06; N, 4.47; IR (thin
836.3007; found, 836.3019; IR (thin film in NaCl)
mmax = 3497,
2918, 2109, 1720, 1515, 1139, 1073 cmꢀ1
.
3.1.20. Methyl (methyl 4-O-acetyl-2-O-(2-
(azidomethyl)benzoyl)-3-O-benzyl-b-
glucopyranosyluronate)-(1?4)-(2-azido-3-O-benzyl-2-deoxy-6-
O-(4-methoxybenzyl)- /b- -glucopyranosyl)-(1?4)-(benzyl 2-
O-benzoyl-3-O-benzyl-b- -glucopyranosyluronate)-(1?4)-2-
azido-3-O-benzyl-2-deoxy-6-O-(4-methoxybenzyl)-
glucopyranoside (2 /b)
D-
a
D
D
a-D-
film in NaCl)
m
max = 2110, 1720, 1517, 1251, 1098 cmꢀ1
.
a
A mixture of the disaccharide trifluoroacetimidate 3 (60 mg,
0.056 mmol) and disaccharide acceptor 4 (50 mg, 0.056 mmol) in
dry toluene (2.0 mL) was stirred in the presence of freshly acti-
vated powdered 4 Å molecular sieves (150 mg) for 30 min at room
temperature under argon. After cooling to ꢀ40 °C, TBSOTf (0.05 M,
0.24 mL) was added dropwise. The reaction mixture was stirred at
ꢀ40 °C until TLC indicated complete conversion of the donor 3 and
then was quenched by the addition of Et3N (0.2 mL). The resulting
mixture was filtered through a pad of Celite and concentrated. The
residue was purified by silica gel column chromatography (5:2
3.1.18. Methyl (2-O-benzoyl-3-O-benzyl-b-
D
-glucopyranosyl)-
(1?4)-2-azido-3-O-benzyl-2-deoxy-6-O-(4-methoxybenzyl)-
-glucopyranoside (24)
The same procedure described for the preparation of compound
a-
D
19 (from compound 18) was employed for the preparation of 24
from 23. Purification by silica gel column chromatography (3:2
petroleum ether–EtOAc) gave disaccharide 25 (122 mg, 89%) as a
white solid. Rf = 0.40 (1:1 petroleum ether–EtOAc); ½a D26
ꢃ
+48.7 (c
1.0, CHCl3); 1H NMR (300 MHz, CDCl3): d 7.95 (d, 2H, J = 8.7 Hz),
7.62 (t, 1H, J = 7.5 Hz), 7.49 (t, 2H, J = 7.8 Hz), 7.41–7.29 (m, 7H),
petroleum ether–EtOAc) to provide tetrasaccharide 2a/b (70 mg,

J. Chen et al. / Carbohydrate Research 343 (2008) 2853–2862
2861
71%) as a colorless syrup (
a
/b, 3:2). Rf = 0.35 (2:1 petroleum ether–
127.1, 100.8, 100.7, 98.7, 97.5, 82.3, 79.7, 77.8, 77.6, 75.5, 75.3,
74.9, 74.6, 74.2, 73.8, 73.1, 73.0, 71.4, 70.7, 67.3, 63.2, 63.0, 60.3,
60.0, 55.3, 52.9, 52.6, 29.7, 20.6; HRMS (MALDI/DHB): calcd for
C78H81N9O24Na, 1550.5292; found, 1550.5267; IR (thin film in
EtOAc); 1H NMR (300 MHz, CDCl3): d 7.87–7.71 (m, 6.1H), 7.65–
7.57 (m, 5.5H), 7.50–7.36 (m, 19.1H), 7.32–7.24 (m, 18.4H), 7.22–
7.11 (m, 16.5H), 7.08–6.88 (m, 17.4H), 5.47 (d, 0.8H, J = 4.2 Hz),
5.27–5.19 (m, 5.7H) 5.13–5.03 (m, 4.4H), 4.95 (t, 2.5H, J = 8.7 Hz),
4.89–4.80 (m, 2.4H), 4.76 (s, 2.2H), 4.73–4.65 (m, 6.0H), 4.61–
4.49 (m, 9.4H), 4.47–4.34 (m, 5.9H), 4.24–4.14 (6.5H), 4.11–3.85
(m, 7.7H), 3.78–3.69 (m, 17.6H), 3.66–3.63 (m, 2.4H), 3.56 (s,
7.0H), 3.63–3.26 (m, 20.8H), 3.11 (t, 1.1H, J = 4.8 Hz), 2.82 (d,
1.0H, J = 9.6 Hz), 2.03 (s, 2.5H), 2.01 (s, 3.0H); HRMS (MALDI/
DHB) m/z: calcd for C94H97N9O26Na, 1790.6442; found,
NaCl) m .
max = 2927, 2110, 1736, 1264, 1071 cmꢀ1
3.1.22. Methyl (methyl 4-O-acetyl-3-O-benzyl-b-
glucopyranosyluronate)-(1?4)-(2-amino-3-O-benzyl-2-deoxy-
-glucopyranosyl)-(1?4)-(benzyl 2-O-benzoyl-3-O-benzyl-b-
-glucopyranosyluronate)-(1?4)-2-amino-3-O-benzyl-2-deoxy-
-glucopyranoside (26)
Compound 25 (19 mg, 0.012 mmol), PPh3 (36 mg,
D-
a-D
D
a-D
1790.6429; IR (thin film in NaCl)
m
max = 2937, 2111, 1735, 1611,
a
1515, 1456, 1364, 1251, 1073, 1037 cmꢀ1
.
0.120 mmol), and silica gel (5 mg) were dissolved in THF–water
(9:1, 2.0 mL). After stirring at room temperature for 37 h, the reac-
tion mixture was concentrated. The residue was eluted from a
Sephadex LH-20 chromatography with MeOH–CH2Cl2 1:1 to afford
26 (14 mg, 86%) as a colorless syrup. Rf = 0.40 (10:1, MeOH–
3.1.21. Methyl (methyl 4-O-acetyl-2-O-(2-
(azidomethyl)benzoyl)-3-O-benzyl-b-
glucopyranosyluronate)-(1?4)-(2-azido-3-O-benzyl-2-deoxy-b-
-glucopyranosyl)-(1?4)-(benzyl 2-O-benzoyl-3-O-benzyl-b-
glucopyranosyluronate)-(1?4)-2-azido-3-O-benzyl-2-deoxy-6-
O-(4-methoxybenzyl)- -glucopyranoside (25b) and methyl
(methyl 4-O-acetyl-2-O-(2-(azidomethyl)benzoyl)-3-O-benzyl-
b- -glucopyranosyluronate)-(1?4)-(2-azido-3-O-benzyl-2-
deoxy- -glucopyranosyl)-(1?4)-(benzyl 2-O-benzoyl-3-O-
benzyl-b- -glucopyranosyluronate)-(1?4)-2-azido-3-O-benzyl-
2-deoxy- -glucopyranoside (25
To a solution of the tetrasaccharide 2
D
-
D
D-
CH2Cl2); ½a 2D5
ꢃ
+41.9 (c1.0, CHCl3); 1H NMR (300 MHz, CDCl3): d
8.08 (d, 2H, J = 7.2 Hz), 7.60 (t, 1H, J = 7.5 Hz), 7.47 (t, 2H,
J = 7.8 Hz), 7.34–7.24 (m, 20H), 7.14 (s, 5H), 5.42 (t, 1H,
J = 8.4 Hz), 5.24 (d, 1H, J = 3.6 Hz), 5.12–4.96 (m, 6H), 4.80–4.72
(m, 3H), 4.68–4.51 (m, 5H), 4.34 (t, 1H, J = 7.8 Hz), 4.18 (d, 1H,
J = 8.4 Hz), 3.96–3.70 (m, 7H), 3.63–3.58 (m, 4H), 3.55 (s, 3H),
3.44–3.32 (m, 3H), 3.21 (s, 3H), 2.68–2.62 (m, 2H), 1.94 (s, 3H);
13C NMR (75 MHz, CDCl3): d 169.7, 167.9, 167.6, 164.8, 139.1,
138.9, 138.2, 137.3, 134.9, 133.5, 129.7, 129.4, 128.7, 128.6,
128.6, 128.5, 128.4, 128.3, 127.9, 127.9, 127.7, 127.6, 127.5,
103.4, 100.8, 99.9, 98.6, 82.2, 81.9, 81.3, 77.9, 77.5, 75.5, 75.4,
75.4, 75.3, 74.8, 74.4, 74.2, 73.9, 73.0, 72.2, 71.0, 67.7, 61.0, 60.5,
56.0, 55.7, 55.2, 52.5, 29.7, 20.6; HRMS (MALDI/DHB) m/z: calcd
for C37H43NO13Na, 1339.5050; found, 1339.5027; IR (thin film in
a-D
D
a-D
D
a
-D
a)
a/b (51 mg, 0.029 mmol) in
CH2Cl2 (2.0 mL), H2O (0.2 mL) was added followed by addition of
DDQ (33 mg, 0.14 mmol). After stirring at room temperature for
4 h, the reaction mixture was diluted with CH2Cl2 (80 mL), and
washed twice with satd aq NaHCO3 and once with water. The or-
ganic layer was dried with Na2SO4, and then filtered and concen-
trated in vacuo. The residue was purified by silica gel column
chromatography (5:2 petroleum ether–EtOAc) to give compound
NaCl)
m
max = 2927, 1744, 1265, 1027, 699 cmꢀ1
.
25b (17 mg, 39%) and compound 25
a
(19 mg, 43%) as colorless syr-
3.1.23. Methyl (2-O-sulfo-b-
D
-glucopyranosyluronate)-(1?4)-
ups. Compound 25b: Rf = 0.15 (1:1 petroleum ether–EtOAc); ½a D26
ꢃ
(2-deoxy-2-sulfamido-6-O-sulfo-
-glucopyranosyluronate)-(1?4)-2-deoxy-2-sulfamido-6-O-
sulfo- -glucopyranoside (1)
a-D-glucopyranosyl)-(1?4)-(b-
+3.2 (c 1.0, CHCl3); 1H NMR (500 MHz, CDCl3): d 8.00 (d, 2H,
J = 7.5 Hz), 7.84 (d, 1H, J = 7.8 Hz), 7.61 (t, 1H, J = 7.6 Hz), 7.56–
7.54 (m, 2H), 7.45–7.35 (m, 10H), 7.32–7.19 (m, 11H), 7.14–7.08
(m, 11H), 5.31–5.27 (m, 3H), 5.08–5.04 (m, 3H), 4.88–4.83 (m,
3H), 4.76–4.59 (m, 8H), 4.55–4.52 (m, 2H), 4.17 (t, 1H, J = 7.8 Hz),
4.08 (d, 1H, J = 7.2 Hz), 4.04 (d, 1H, J = 9.6 Hz), 3.92–3.83 (m, 4H),
3.75–3.71 (m, 3H), 3.75–3.53 (m, 1H), 3.56 (s, 3H), 3.41 (d, 1H,
J = 7.8 Hz), 3.36–3.24 (m, 3H), 3.27 (s, 3H), 3.19–3.11 (m, 2H),
2.69 (d, 1H, J = 10.0 Hz), 1.97 (s, 3H); 13C NMR (75 MHz, CDCl3): d
169.3, 167.3, 164.8, 164.2, 138,6, 138.5, 138.4, 133.4, 137.3,
134.7, 133.4, 130.6, 129.9, 129.7, 129.4, 129.0, 128.8, 128.6,
128.4, 128.3, 128.3, 128.2, 128.1, 127.8, 127.7, 127.6, 127.5,
127.5, 127.3, 126.8, 101.2, 101.0, 100.3, 98.8, 80.4, 79.8, 79.4,
78.0, 77.8, 76.1, 75.3, 75.2, 74.7, 74.6, 74.6, 74.1, 73.2, 73.1, 72.9,
71.4, 70.7, 67.7, 66.3, 63.3, 60.4, 60.1, 55.3, 53.0, 52.7, 20.6; HRMS
(MALDI/DHB) m/z: calcd for C78H81N9O24Na, 1550.5292; found,
D
a-D
SO3ꢁNEt3 (30 mg, 0.165 mmol) was added to a solution of 26
(14 mg, 0011 mmol) in anhydrous DMF (1.0 mL). After stirring for
24 h at 55 °C under an argon atmosphere, the reaction mixture
was quenched with Et3N (0.2 mL) and diluted with MeOH
(1.0 mL) and CH2Cl2 (1.0 mL). The solution was layered on the
top of a Sephadex LH-20 chromatography column that was eluted
with MeOH–CH2Cl2 (1:1). The fraction that contained the sulfo-
nated tetrasaccharide was pooled and evaporated to dryness. The
residue was dissolved in anhydrous pyridine (1 mL), after which
Et3N (0.2 mL) was added followed by addition of SO3ꢁ Py (18 mg,
0.11 mmol). After stirring for 2 h at room temperature under an ar-
gon atmosphere, the reaction mixture was diluted with MeOH
(1.0 mL) and CH2Cl2 (1.0 mL). The solution was layered on the
top of a Sephadex LH-20 chromatography column that was eluted
with MeOH–CH2Cl2 1:1. The fractions that contained the sulfo-
nated tetrasaccharide were pooled and evaporated to dryness to
yield the crude sulfonated tetrasaccharide. ESI-MS: (m/z): calcd
for C70H77N2O35S4, 544.4; found, 544.7 [MꢀSO3ꢀ3H+]3ꢀ; m/z calcd
for C70H77N2O38S5, 571.1; found, 571.5 [Mꢀ3H+]3ꢀ; m/z calcd for
C70H78N2O35S4, 817.2; found, 817.2 [MꢀSO3ꢀ2H+]2ꢀ; m/z calcd
1550.5256; IR (thin film in NaCl)
m
max = 2928, 2112, 1734, 1456,
Rf = +0.25 (1:1 petroleum
31.0 (c 1.0, CHCl3); 1H NMR (500 MHz, CDCl3):
1265, 1072 cmꢀ1
. Compound 25a:
ether–EtOAc); ½a D26
ꢃ
d 8.04 (d, 2H, J = 7.3 Hz), 7.94 (d, 1H, J = 7.8 Hz), 7.58 (t, 1H,
J = 7.5 Hz), 7.54 (t, 1H, J = 7.4 Hz), 7.47–7.41 (m, 6H), 7.36–7.33
(m, 4H), 7.29–7.04 (m, 8H), 7.15–7.06 (m, 12H), 5.37–5.29 (m,
4H), 5.20 (d, 1H, J = 11.2 Hz), 4.97 (d, 1H, J = 9.9 Hz), 4.90–4.88
(m, 2H), 4.72 (d, 1H, J = 10.5 Hz), 4.64–4.58 (m, 7H), 4.58 (s, 1H),
4.52–4.46 (m, 2H), 4.24 (t, 1H, J = 7.8 Hz), 3.97 (t, 2H, J = 9.5 Hz),
3.94–3.76 (m, 6H), 3.73–3.63 (m, 3H), 3.58 (br d, 1H, J = 6.6 Hz),
3.53 (s, 3H), 3.38 (dd, 2H, J = 11.1, 4.8 Hz), 3.26 (s, 3H), 3.21–3.19
(m, 2H), 1.98 (s, 3H); 13C NMR (75 MHz, CDCl3): d 169.3, 167.5,
167.3, 164.8, 164.3, 138.8, 138.4, 138.2, 137.3, 137.2, 134.4,
133.5, 133.4, 130.6, 129.7, 129.4, 129.3, 128.7, 128.7, 128.6,
128.5, 128.3, 128.2, 128.2, 127.9, 127.8, 127.8, 127.7, 127.4,
for C70H78N2O38S5, 857.1; found, 857.3 [Mꢀ2H+]2ꢀ
.
The crude sulfonated tetrasaccharide was dissolved in MeOH–
water (9:1 v/v, 2.0 mL) and treated with a 2 M aqueous solution
of NaOH (0.5 mL). After stirring at room temperature for 48 h,
the reaction mixture was quenched by addition of H+-ion-ex-
change resin (1.5 g). The filtrate that contained sulfonated tetrasac-
charide was hydrogenated in the presence of 10% Pd–C. After 36 h,
the suspension was filtered, concentrated, and eluted from a col-
umn of Dowex 50WX4-Na+ with H2O. The fractions that contained

2862
J. Chen et al. / Carbohydrate Research 343 (2008) 2853–2862
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sulfonated tetrasaccharide were pooled and concentrated. The res-
idue was diluted with H2O. The solution was layered on the top of a
Sephadex G-15 chromatography column that was eluted with
0.5 M aqueous solution of NaCl. The fractions that contained the
sulfonated tetrasaccharide were pooled and concentrated. The res-
idue was diluted with H2O. The solution was layered on the top of a
Sephadex G-15 chromatography column that was eluted with H2O.
The fractions that contained the sulfonated tetrasaccharide were
pooled and evaporated to dryness to yield tetrasaccharide 1
(3 mg, 22%) as a white solid. 1H NMR (400 MHz, D2O): d 5.74 (d,
1H, J = 3.5 Hz), 5.04 (d, 1H, J = 3.0 Hz), 4.62 (dd, 2H, J = 8.1,
2.6 Hz), 4.49 (d, 1H, J = 10.7 Hz), 4.42 (br s, 2H), 4.15–4.05 (m,
2H), 4.04–3.96 (m, 3H), 3.94–3.84 (m, 8H), 3.82–3.77 (m, 2H),
3.76—3.70 (m, 3H), 3.69–3.56 (m, 5H), 3.51 (s, 3H), 3.47–3.35 (m,
5H); 13C NMR (75 MHz, D2O): d 173.8, 165.3, 104.9, 104.7, 99.0,
98.0, 80.7, 79.5, 78.7, 78.6, 78.3, 77.8, 76.0, 75.7, 74.7, 74.5, 71.9,
71.5, 71.3, 70.8, 68.8, 68.3, 65.2, 58.1, 56.3; ESI-MS: m/z calcd for
C25H40N2O36S5, 552.0; found, 551.9 [Mꢀ2H+]2ꢀ, m/z calcd for
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Acknowledgements
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This work is supported by the National Natural Science Founda-
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