Synthesis of heparan sulfate tetrasaccharide as a substrate for human heparanase

Article abstract of DOI:10.1016/j.carres.2012.03.014

Regiospecifically sulfated heparan sulfate tetrasaccharide, GlcAβ-GlcN(NS6S)α-GlcAβ-GlcN(NS6S)α was first synthesized as an octyl glycoside. Total synthesis was achieved effectively by coupling the corresponding disaccharide units in short steps.

Full text of DOI:10.1016/j.carres.2012.03.014

Carbohydrate Research 353 (2012) 13–21
Contents lists available at SciVerse ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Synthesis of heparan sulfate tetrasaccharide as a substrate for human
heparanase
Naoko Takeda, Remina Ikeda-Matsumi, Kaoru Ebara-Nagahara, Miyuki Otaki-Nanjo,
⇑
Kayo Taniguchi-Morita, Miwa Nanjo, Jun-ichi Tamura
Department of Regional Environment, Tottori University, Tottori 680-8551, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Regiospecifically sulfated heparan sulfate tetrasaccharide, GlcAb-GlcN(NS6S)a-GlcAb-GlcN(NS6S)a was
first synthesized as an octyl glycoside. Total synthesis was achieved effectively by coupling the corre-
sponding disaccharide units in short steps.
Received 20 September 2011
Received in revised form 12 March 2012
Accepted 12 March 2012
Ó 2012 Elsevier Ltd. All rights reserved.
Available online 20 March 2012
Keywords:
Heparan sulfate
Oligosaccharide
Heparanase
Glycosaminoglycan
1. Introduction
demonstrated heparanase detection.7 To our knowledge, GlcAb-
GlcN (NS6S)a-GlcAb-GlcN(NS6S)a-type tetrasaccharide has never
Some enzymes are secreted and decompose the basal mem-
brane in the first step of metastasis by cancer cells. Heparanase
is also secreted and digests heparan sulfate polysaccharides, which
compose the basal membrane. Heparanase recognizes a few spe-
cific sequences of heparan sulfate. Okada et al. reported substrate
specificity using different types of heparan sulfate oligosaccharides
derived from natural products.1 Among them, three representa-
tive sequences were better substrates for heparanase digestion:
been synthesized. It is obviously easier to synthesize the tetrasac-
charide composed of a dimer of the repeating disaccharide than the
2S analogue of the GlcA residue that the heparanase would simi-
larly recognize.1 In this paper, we report the synthesis of heparan
sulfate tetrasaccharide, GlcAb-GlcN(NS6S)a-GlcAb-GlcN(NS6S)a, as
an octyl glycoside.
2. Results and discussion
D
HexA(2S)-GlcN(NS6S)
(NS6S) -GlcAb-GlcN(NS6S)
(NS6S) -GlcAb-GlcN(NS6S)
a
-GlcAb-GlcN(NS6S) I,
-GlcAb-GlcN(NS6S) II and
-GlcAb-GlcN(NS6S)a III. Sensitive detec-
a
D
HexA(2S)-GlcN
a
a
a
a
a
DHexA-GlcN
The retrosynthesis of the target tetrasaccharide 23 was ana-
lyzed as depicted in Scheme 1. Glycosyl donor 10 and acceptor
12 derived from a common disaccharide were coupled to each
other. Following glycosylation with n-octanol, sulfation and depro-
tection would lead to the final compound.
tion of heparanase secreted by cancer cells could open the way
to the development of a new type of diagnosis for cancer, espe-
cially at an early stage. Recent techniques could detect heparanase
activity by using radioisotopes,2,3 homogeneous time-resolving
fluorescence4 and FRET on gold nanoparticles.5 These methods still
employ naturally occurring heparin and heparan sulfate polysac-
charide, the entire structure of which has not been clarified. Chem-
ically synthesized heparan sulfate oligosaccharides are useful tools
for this purpose. Yu’s group synthesized I as a methyl glycoside,
which required many steps for total synthesis.6 Very recently,
Based on this strategy we prepared a common disaccharide 1,
the synthesis of which was previously reported by the coupling
of methyl 2,3,4,6-tetra-O-benzoyl-1-thio-b-
1,6-anhydro-2-azido-3-O-benzyl-2-deoxy- -glucopyranose in 74%
yield.8 We synthesized the same disaccharide via: (1) the coupling
of 2,3,4,6-tetra-O-acetyl- -glucopyranosyl trichloroacetimidate and
D-glucopyranoside and
D
D
the same glycosyl acceptor in 92% yield, and (2) subsequent quanti-
tative saponification. Disaccharide 1 was regiospecifically benzylid-
enated at the 40 and 60 positions with benzaldehyde dimethylacetal
under acidic conditions to give 2 in 87% yield (Scheme 2). The resid-
ual hydroxyl groups were benzylated with BnBr and Ag2O in DMF to
give 3 in 93% yield. Conversion to the corresponding uronate was
von Itstein’s group synthesized GlcN(NS)a-GlcAb-R-type disaccha-
rides, which lacked the 6-O-sulfate on GlcN residue, and
⇑
Corresponding author. Tel./fax: +81 857 31 5108.
E-mail address: jtamura@rstu.jp (J. Tamura).
0008-6215/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.carres.2012.03.014

14
N. Takeda et al. / Carbohydrate Research 353 (2012) 13–21
Scheme 1. Retrosynthetic analysis.
performed as follows. Reductive ring opening of the benzylideneacetal
of 3 with BH3ꢀNMe3 and AlCl3 in CH2Cl2–Et2O afforded an inseparable
mixture of 20,30,40-tri-O-benzyl and 20,30,60-tri-O-benzyl compounds 4
and 5 in 75% yield. The primary hydroxyl group of 4 was regioselec-
tively oxidized with NaClO in the presence of TEMPO. Subsequent oxi-
dation with NaClO2 furnished carboxylic acid, which was immediately
esterified with allyl alcohol to give 6 (69% in 3 steps). Allyl ester, being
distinguished from methyl ester, was prepared to equip appropriate
functional groups (not used in this synthesis) at the carboxylic acid.
Compound 5 was obtained in 23% yield. Next, we performed acetolysis
of 6 and obtained 7 in 94% yield. Successive deacetylation at the ano-
meric position of 7 was performed with H2NNH2ꢀAcOH in 93% yield.
(ꢁ40 °C to rt) afforded 13
tively, with higher yield and
The -isomer 13 was subjected to acetolysis in the same man-
ner as above to give the triacetate 14 in 90% yield, which was con-
verted to the corresponding hemiacetal 15 and the subsequent
a
and 13b in 65% and 11% yield, respec-
a-selectivity.
a
a
a-trichloroacetimidate 16 in 92% and 98% yields, respectively
(Scheme 3). A hydrophobic group such as an aglycon, such as the
n-octyl group,10 is often used, and is useful to isolate the com-
pound in a later stage of the synthesis. The imidate 16 was coupled
with n-octanol in the presence of TMSOTf at a lower temperature
(ꢁ20 °C to rt) to give octyl
26% and 46% yields, respectively.
markedly when coupled at rt; 17 and 17b in 67% and 20% yields,
respectively. Octyl glycoside 17 was saponified to give 18, which
a- and b-glycosides 17
a and 17b in
a-Stereoselectivity increased
The obtained hemiacetal 8 was converted to the corresponding
a-iso-
a
mers of trichloroacetimidate 9 and chloride 10 in 87% and 89% yields,
respectively. On the other hand, the benzylidene acetal of 3 was
removed to obtain 119 in 92% yield. The primary alcohol of 11 was reg-
iospecifically oxidized in the similar manner as above, and the resul-
tant carboxylic acid was converted to methyl ester 12 in 78% yield
(in 2 steps).
The coupling of the glycosyl donor (9 and 10) and the acceptor
12 is summarized in Table 1. Coupling of the imidate 9 and the
acceptor 12 under standard conditions using TMSOTf in CH2Cl2 at
a
was sulfated with SO3ꢀNMe3 in DMF to afford di-O-sulfate 19 in
85% yield in 3 steps. Hydrogenolysis of 19 in the presence of Pd-
C under an H2 atmosphere furnished 20 in 76% yield. The complete
reduction of both azides was confirmed by forming diacetamide
analogue with Ac2O in aqueous condition to give 21 in 78% yield.
The structure was assigned by 1H NMR and TOFMS. The non-pro-
tected tetrasaccharide 20 was finally N-sulfated with SO3ꢀpyridine
in aq NaOH to give the inseparable mixture of the desired 23 and
the sulfated analogue at O-3III 24 in 75% and 25% yields, respec-
tively. Ley’s group reported sodium-mediated regioselective O-
silylation,11 and explained the regioselectivity due to the complex
formation of the sodium cation and three oxygen atoms of the sub-
strate. We re-investigated the final N-sulfation of 20 in aq Et3N,
avoiding the hypothesized formation of sodium complex 22. We
successfully obtained the final compound 23 in 78% yield without
the formation of 24, as expected. We rationalized the unexpected
ꢁ20 °C afforded the desired
isomer 13b in 23% and 12% yields, respectively. Toluene instead
of CH2Cl2 was slightly effective (13 29%; 13b, 14%). TBDMSOTf-
mediated coupling of 9 and 12 in toluene improved the results;
13 and 13b in 43% and 25% yields, respectively. On the other
hand, coupling of the chloride 10 and 12 in the presence of AgOTf
and collidine at ꢁ20 °C to rt yielded 13 and 13b at 48% and 9%
yields, respectively; however, lower reaction temperature
a-glycoside 13a together with the b-
a
a
a
a

N. Takeda et al. / Carbohydrate Research 353 (2012) 13–21
15
Scheme 2. Synthesis of the disaccharide donors (9 and 10) and acceptor 12, and their coupling to give tetrasaccharide moiety. Reagents and conditions: (a) PhCH(OMe)2, p-
TsOH, THF, 87%; (b) BnBr, Ag2O, KI, DMF, 93%; (c) BH3ꢀNMe3, AlCl3, MS4 Å, CH2Cl2–Et2O, 75%; (d) (i) TEMPO, NaClO, NaHCO3, n-Bu4NBr, NaBr, EtOAc, H2O; (ii) NaClO2,
NaH2PO4ꢀ2H2O, tert-BuOH, 2-methyl-2-butene, H2O; (iii) AllOH, WSCDꢀHCl, HOBt, i-Pr2EtN, CH2Cl2, ꢁ20 °C to rt, 69% (for 3 steps); (e) Ac2O, AcOH, TFA, 0 °C to rt, 94%; (f)
H2NNH2ꢀAcOH, DMF, 50 °C, 93%; (g) CCl3CN, DBU, CH2Cl2, 0 °C, 87%; (h) MeClC@CClNMe2, CH2Cl2, 89%; (i) camphorsulfonic acid, CH2Cl2–MeOH, 92%; (j) (i) TEMPO, NaClO,
NaHCO3, n-Bu4NBr, KBr, EtOAc, H2O, 0 °C; (ii) TMSCHN2, Et2O–MeOH–PhMe, 78% (for 2 steps); (k) AgOTf, collidine, MS4A, CH2Cl2, ꢁ40 °C, 13
a: 65%, 13b: 11%.
Table 1
Glycosylation of disaccharide acceptor 12 with disaccharide donor 9 or10
Entry
Donor
Promoter
Solvent
Temperature (°C)
Yield (%)
a/b
13
a
13b
1
2
3
4
5
9
9
9
10
10
TMSOTf
TMSOTf
TBDMSOTf
AgOTf, collidine
AgOTf, collidine
CH2Cl2
Toluene
Toluene
CH2Cl2
CH2Cl2
ꢁ20
ꢁ20
ꢁ20
ꢁ20
ꢁ40
23
29
43
48
65
12
14
25
9
1.9
2.1
1.7
5.3
5.9
11

16
N. Takeda et al. / Carbohydrate Research 353 (2012) 13–21
Scheme 3. Synthesis toward the target tetrasaccharide 23. Reagents and conditions: (a) Ac2O, AcOH, TFA, 0 °C to rt, 90%; (b) H2NNH2ꢀAcOH, DMF, 50 °C, 92%; (c) CCl3CN, DBU,
CH2Cl2, 0 °C to rt, 98%; (d) TMSOTf, MSAW300, CH2Cl2, rt, 17
a
: 67%, 17b: 20%; (e) (i) aq LiOH, THF; (ii) aq NaOH, CH2Cl2–MeOH; (f) SO3ꢀNMe3, DMF, 60 °C, 85% (for 3 steps); (g)
H2, Pd-C, aq EtOH, 76%; (h) Ac2O, cat Et3N, H2O, 78%; (i) SO3ꢀpyridine, aq NaOH (pH 9), 23: 75%, 24: 25%; (j) SO3ꢀpyridine, aq Et3N (pH 10), 78%.
sulfation at O-3III in an aqueous condition owing to chelation of the
4. Experimental
sodium cation in carboxylic acid and O-3III, such as 22.
4.1. General methods
3. Conclusion
Optical rotations were measured at 22 3 °C with a JASCO
polarimeter DIP-18-1. 1H NMR assignments were confirmed by
two-dimensional HH COSY experiments with JEOL ECP 500 MHz
and BRUKER ADVANCE II 600 MHz spectrometers. As an example
of signal assignments, 1III stands for a proton at C-1 of sugar resi-
due III. Silica gel chromatography, analytical TLC, and preparative
TLC (PTLC) were performed on a column of Silica Gel 60 (E. Merck),
Silica Gel 60N (spherical neutral) (Kanto Kagaku), and glass plates
Heparanase secreted by cancer cells recognizes and digests hep-
aran sulfate, having sulfate groups at the specific positions. In order
to develop a new type of diagnosis for cancer, we synthesized the
heparin sulfate tetrasaccharide, GlcAb-GlcN(NS6S)a-GlcAb-GlcN
(NS6S) , as an octyl glycoside. Coupling of the corresponding
a
disaccharide units was achieved in an effective manner and the to-
tal synthesis was accomplished in short steps.

N. Takeda et al. / Carbohydrate Research 353 (2012) 13–21
17
coated with Silica Gel F254 (E. Merck), respectively. The gel for size-
exclusion chromatographies (S-X1 and Sephadex LH-20) were
from BioꢀRad and GE Healthcare, respectively. Molecular sieves
(MS) were from GL Science, Inc. and activated at 200 °C under
diminished pressure prior to use. All reactions in organic solvents
were performed under a dry Ar-containing atmosphere. As a usual
work-up, the organic phase of the reaction mixture was washed
with aq NaHCO3 and brine, and dried over anhyd MgSO4.
and the insoluble materials were filtered on Celite. The organic
phase was treated as described in Section 4.1. The volatiles were
removed under diminished pressure and the residue was subjected
to a column of silica gel (10:1–1:1 n-hexane–EtOAc) to give a mix-
ture of 4 and 5 (1.72 g) in 75% yield as a syrup. The mixture was
used for the next reaction without separation.
One portion of the mixture obtained above (360.3 mg,
0.51 mmol) was diluted with EtOAc (12 mL) and H2O (2 mL). To
this were added 1 M NaBr (0.3 mL), 1 M n-Bu4NBr (0.5 mL), TEMPO
(24.0 mg, 0.154 mmol) and satd NaHCO3 (1.4 mL), then 4% NaClO
soln (1.7 mL) was added with stirring at 0 °C. After 1 h pH of the
reaction mixture was lowered with 1 M HCl (0.52 mL) (pH 6–7)
at rt. Then, tert-BuOH (13 mL), 2-methyl-2-butene (5.5 mL), H2O
(13 mL), NaH2PO4ꢀ2H2O (1.37 g) and NaClO2 (1.37 g) were added
with stirring for 3 h, and the reaction mixture was extracted with
EtOAc. Organic phase was washed with brine and dried over
MgSO4. The volatiles were removed under diminished pressure
and the residue was applied to a column of silica gel (15:1–1:3 tol-
uene–EtOAc). The resultant syrupy materials (381.0 mg) and allyl
alcohol (0.1 mL, 1.6 mmol) were diluted with CH2Cl2 (26 mL). To
4.1.1. (4,6-O-Benzylidene-b-
anhydro-2-azido-3-O-benzyl-2-deoxy-b-
To a solution of b- -glucopyranosyl-(1?4)-1,6-anhydro-2-azido-
3-O-benzyl-2-deoxy-b- -glucopyranose (1, 796.9 mg, 1.81 mmol) in
D-glucopyranosyl)-(1?4)-1,6-
D-glucopyranose (2)
D
D
THF (9 mL) was added to benzaldehyde dimethylacetal (1.4 mL,
9.1 mmol) and catalytic amount of p-TsOH (pH <3) with stirring
overnight. The reaction mixture was neutralized with Et3N. The
volatiles were removed under diminished pressure and the residue
was subjected to a column of silica gel (3:1–1:5 n-hexane–EtOAc)
to give 2 (829.5 mg) in 87% yield as a syrup. [
a
]
ꢁ9.0 (c 0.49,
D
CHCl3); 1H NMR (CDCl3): d 7.50–7.31 (m, 10H, Ar H), 5.53 (s, 1H,
PhCH), 5.50 (s, 1H, H-1I), 4.70 (m, 1H, H-5I), 4.65, 4.59 (ABq, 2H,
J = 12.00 Hz, PhCH2), 4.51 (d, 1H, J1,2 = 7.62 Hz, H-1II), 4.23 (dd,
this solution were added iPr2EtN (92 lL, 0.53 mmol) and HOBt
(216.3 mg, 1.59 mmol). The mixture was cooled at ꢁ20 °C, and
WSCDꢀHCl (203.2 mg, 1.06 mmol) was added with stirring over-
night gradually raising to rt. The reaction mixture was diluted with
CHCl3. The organic phase was treated as described in Section 4.1.
The volatiles were removed under diminished pressure and the
residue was subjected to a column of silica gel (15:1–1:1 n-hex-
ane–EtOAc) to give 6 (268.6 mg) in 69% yield (2 steps) as a syrup.
Compound 5 (82.4 mg) was obtained in 23% yield. Compound 5:
1H, J5,6b = 4.98 Hz, Jgem = 10.50 Hz, H-6aII), 4.16 (d, 1H, Jgem
=
7.23 Hz, H-6aI), 3.84 (br s, 1H, H-3I), 3.82 (br t, 1H, J = 9.23 Hz,
H-3II), 3.80 (m, 2H, H-4I, 6bI), 3.75 (br t, 1H, J = 10.26 Hz, H-6bII),
3.57 (dd, 1H, J2,3 = 8.94 Hz, H-2II), 3.56 (br t, 1H, J = 9.27 Hz,
H-4II), 3.39 (dt, 1H, H-5II), 3.23 (s, 1H, H-2I). Anal. Calcd for
C26H29N3O9ꢀH2O: C, 57.23; H, 5.74. Found: C, 57.28; H, 5.46.
4.1.2. (2,3-Di-O-benzyl-4,6-O-benzylidene-b- -glucopyranosyl)-
D
[a]
+9.4 (c 0.98, CHCl3); 1H NMR (CDCl3): d 7.39–7.21 (m, 20H,
D
(1?4)-1,6-anhydro-2-azido-3-O-benzyl-2-deoxy-b-
glucopyr anose (3)
D
-
Ar H), 5.50 (s, 1H, H-1I), 5.04, 4.73 (ABq, 2H, J = 10.87 Hz, PhCH2),
4.96, 4.73 (ABq, 2H, J = 11.53 Hz, PhCH2), 4.58 (m, 3H, H-5I, PhCH2),
4.53, 4.50 (ABq, 2H, J = 11.95 Hz, PhCH2), 4.52 (d, 1H, J1,2 = 7.68 Hz,
H-1II), 4.06 (dd, 1H, J5,6a = 0.60 Hz, Jgem = 7.38 Hz, H-6aI), 3.96 (br t,
1H, J = 1.57 Hz, H-3I), 3.74 (s, 1H, H-4I), 3.71 (dd, 1H, J5,6a = 6.84 Hz,
Jgem = 10.38 Hz, H-6aII), 3.70 (dd, 1H, J5,6b = 3.00 Hz, H-6bI), 3.66
(dd, 1H, J5,6b = 5.76 Hz, H-6bII), 3.57 (br t, 1H, J = 9.27 Hz, H-4II),
3.52 (dd, 1H, J2,3 = 9.18 Hz, H-2II), 3.44 (br t, 1H, J = 8.89 Hz, H-
3II), 3.42 (m, 1H, H-5II), 3.20 (s, 1H, H-2I). Anal. Calcd for
To a solution of 2 (809.3 mg, 1.53 mmol) in DMF (30 mL) were
added Ag2O (1.43 g, 6.12 mmol), KI (515.5 mg, 3.06 mmol) and
BnBr (0.70 mL, 6.12 mmol) with stirring at 0 °C in a brown flask.
After 6 h the reaction mixture was diluted with EtOAc. Insoluble
materials were filtered on Celite. The organic phase was washed
first with aq Na2S2O3 and treated as described in Section 4.1. The
volatiles were removed under diminished pressure and the residue
was subjected to a column of silica gel (7:1–2:1 n-hexane–EtOAc)
C
40H43N3O9: C, 67.68; H, 6.12; N, 5.92. Found: C, 67.59; H, 6.26;
to give 3 (1.00 g) in 93% yield as a syrup. [
a
]
ꢁ16 (c 0.65, CHCl3);
N, 5.70. Compound 6: [
a]
D
+11.0 (c 0.91, CHCl3); 1H NMR (CDCl3):
D
1H NMR (CDCl3): d 7.50–7.21 (m, 20H, Ar H), 5.56 (s, 1H, PhCH),
5.51 (s, 1H, H-1I), 4.96, 4.79 (ABq, 2H, J = 10.54 Hz, PhCH2), 4.92,
4.81 (ABq, 2H, J = 11.46 Hz, PhCH2), 4.67, 4.62 (ABq, 2H,
J = 11.91 Hz, PhCH2), 4.58 (br d, 1H, J = 6.42 Hz, H-5I), 4.54 (d, 1H,
J1,2 = 7.33 Hz, H-1II), 4.20 (dd, 1H, J5,6a = 5.04 Hz, Jgem = 10.54 Hz,
H-6aII), 4.09 (d, 1H, Jgem = 7.33 Hz, H-6aI), 3.89 (br s, 1H, H-3I),
3.75 (br t, 1H, J = 5.96 Hz, H-6bI), 3.74 (br t, 1H, J = 11.00 Hz, H-
6bII), 3.72 (s, 1H, H-4I), 3.72 (br t, 1H, J = 8.71 Hz, H-3II), 3.68 (t,
1H, J3,4 =J4,5 = 9.16 Hz, H-4II), 3.55 (br t, 1H, J = 8.03 Hz, H-2II),
3.30 (dd, 1H, H-5II), 3.27 (s, 1H, H-2I). Anal. Calcd for C40H41N3O9:
C, 67.87; H, 5.85; N, 5.94. Found: C, 67.95; H, 5.82; N, 5.64.
d 7.37–7.21 (m, 20H, Ar H), 5.84 (m, 1H, All), 5.51 (s, 1H, H-1I), 5.30
(d, 1H, J = 17.18 Hz, All), 5.19 (d, 1H, J = 10.31 Hz, All), 5.03, 4.73
(ABq, 2H, J = 10.54 Hz, PhCH2), 4.94, 4.81 (ABq, 2H, J = 11.00 Hz,
PhCH2), 4.81, 4.63 (ABq, 2H, J = 11.00 Hz, PhCH2), 4.64–4.56 (m,
6H, H-5I, 1II, All, PhCH2), 4.08 (d, 1H, Jgem = 7.33 Hz, H-6aI), 3.91–
3.87 (m, 2H, H-4I,II), 3.90 (s, 1H, H-3I), 3.76 (m, 1H, H-5II), 3.75
(m, 1H, H-6bI), 3.66 (t, 1H, J2,3 = J3,4 = 8.94 Hz, H-3II), 3.61 (t, 1H,
J1,2 = 8.94 Hz, H-2II), 3.26 (s, 1H, H-2I). Anal. Calcd for
C43H45N3O10ꢀ0.4H2O: C, 67.00; H, 6.17. Found: C, 67.34; H, 5.93.
4.1.4. (Allyl 2,3,4-tri-O-benzyl-b-D-glucopyranosyluronate)-(1?4)-
1,6-di-O-acetyl-2-azido-3-O-benzyl-2-deoxy-D-glucopyranose (7)
4.1.3. (2,3,4-Tri-O-benzyl-b-
dro-2-azido-3-O-benzyl-2-deoxy-b-
tri-O-benzyl-b- -glucopyranosyl)-(1?4)-1,6-anhydro-2-azido-
3-O-benzyl-2-deoxy-b- -glucopyranose (5) and (allyl 2,3,4-tri-O-
benzyl-b- -glucopyranosyluronate)-(1?4)-1,6-anhydro-2-
azido-3-O-benzyl-2-deoxy-b- -glucopyranose (6)
To a suspension of 3 (2.28 g, 3.23 mmol) and molecular sieves
4 Å (2.85 g) in CH2Cl2 (43 mL) and Et2O (8.6 mL) was added
BH3ꢀNMe3 (4.72 g, 64.6 mmol) with stirring at 0 °C. AlCl3 (1.75 g,
12.9 mmol) in Et2O (13 mL) was added to the suspension and stir-
ring was continued. After 30 min 1 M H2SO4 (108 mL) was added
D
-glucopyranosyl)-(1?4)-1,6-anhy
To a solution of 6 (502.7 mg, 0.66 mmol) in AcOH (0.4 mL) and
Ac2O (7.6 mL) was added TFA (1.1 mL) at 0 °C with stirring. The
temperature was raised to rt and stirring continued overnight. A
few portions of ice were added to the reaction mixture. The vola-
tiles were removed under diminished pressure and the residue
was subjected to a column of silica gel (10:1–8:1 n-hexane–EtOAc)
to give 7 (534.5 mg) in 94% yield as an anomeric mixture: 1H NMR
D-glucopyranose (4), (2,3,6-
D
D
D
D
(CDCl3) (selected): d 6.16 (d, 1H, J1,2 = 3.62 Hz, H-1
J1,2 = 8.25 Hz, H-1bI), 4.36–4.27 (m, 2H, H-6I), 2.18, 2.03 (2s, 6H,
2CH3CO- ), 2.15, 2.02 (2s, 6H, 2CH3CO-b). Anal. Calcd for
47H51N3O13ꢀ0.2H2O: C, 64.91; H, 5.97. Found: C, 64.69; H, 5.93.
aI), 5.39 (d, 1H,
a
C

18
N. Takeda et al. / Carbohydrate Research 353 (2012) 13–21
4.1.5. (Allyl 2,3,4-tri-O-benzyl-b-
D
-glucopyranosyluronate)-
4.1.9. (Methyl 2,3-di-O-benzyl-b-D-glucopyranosyluronate)-
(1?4)-6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-
D
-glucopyranose
(1?4)-1,6-anhydro-2-azido-3-O-benzyl-2-deoxy-b-D-glucopyr
(8)
anose (12)
To a solution of 7 (1.41 g, 1.63 mmol) in DMF (10.8 mL) was
added H2NNH2ꢀAcOH (226.0 mg, 2.45 mmol) with stirring for 1 h
at 50 °C. The reaction mixture was diluted with EtOAc. The organic
phase was treated as described in Section 4.1. The volatiles were
removed under diminished pressure and the residue was subjected
to a column of silica gel (10:1–2:1 toluene–EtOAc) to give 8
(1.24 g) in 93% yield as a syrup. The disappearance of one acetyl
signal was checked by 1H NMR and was used for the next reaction.
Anal. Calcd for C45H49N3O12: C, 65.59; H, 6.01. Found: C, 65.28; H,
5.94.
Diol (11, 122.0 mg, 0.172 mmol) was diluted with EtOAc (3 mL).
To this were added satd NaHCO3 (1.7 mL), KBr (2.6 mg, 22 mol),
n-Bu4NBr (4.0 mg, 12 mol), TEMPO (0.4 mg, 5 mol), 4% NaClO
l
l
l
soln (3 mL), satd NaHCO3 (1.5 mL) and satd NaCl (3.0 mL) with stir-
ring at 0 °C. One hour later, 1 M HCl, 1 M Na2S2O3 and satd NaCl
were added. The reaction mixture was extracted with CHCl3. The
organic phase was washed with brine and dried over MgSO4. The
volatiles were removed under diminished pressure. The residue
was dissolved in MeOH (0.75 mL) and toluene (0.25 mL). To the
solution was added 2 M solution of trimethylsilyldiazomethane
in Et2O portionwise. The volatiles were removed under diminished
pressure again, and the residue was subjected to a column of silica
gel (8:1–1:2 n-hexane–EtOAc) to give 12 (99.5 mg) in 78% yield (2
4.1.6. (Allyl 2,3,4-tri-O-benzyl-b-D-glucopyranosyluronate)-
(1?4)-6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-a-D-glucopyr
anosyltrichloroacetimidate (9)
steps) as a syrup: [a]
+11.7 (c 0.58, CHCl3); 1H NMR (CDCl3): d
D
To a solution of 8 (729.5 mg, 0.89 mmol) and CCl3CN (2.7 mL,
27 mmol) in CH2Cl2 (32 mL) was added DBU (68 L, 0.5 mmol)
7.39–7.26 (m, 15H, Ar H), 5.52 (s, 1H, H-1I), 5.01, 4.74 (ABq, 2H,
J = 10.54 Hz, PhCH2), 4.92, 4.83 (ABq, 2H, J = 11.23 Hz, PhCH2),
4.67, 4.64 (ABq, 2H, J = 11.69 Hz, PhCH2), 4.60 (br d, 1H,
J = 5.04 Hz, H-5I), 4.57 (d, 1H, J1,2 = 7.33 Hz, H-1II), 4.09 (d, 1H,
l
with stirring for 2 h at 0 °C. The reaction mixture was directly sub-
jected to a column of silica gel (30:1–1:4 n-hexane–EtOAc) to give
9 (742.8 mg) in 87% yield as a syrup: 1H NMR (CDCl3): d 8.73 (s, 1H,
NH), 7.30–7.26 (m, 20H, Ar H), 6.33 (d, 1H, J1,2 = 3.67 Hz, H-1I), 5.77
(m, 1H, All), 5.27 (m, 1H, All), 5.15 (m, 1H, All), 4.86, 4.83 (ABq, 2H,
J = 11.00 Hz, PhCH2), 4.80 (s, 2H, PhCH2), 4.78, 4.68 (ABq, 2H,
J = 10.44 Hz, PhCH2), 4.78, 4.60 (ABq, 2H, J = 10.77 Hz, PhCH2),
4.48 (m, 3H, H-1II, All), 4.37 (d, 1H, Jgem = 12.37 Hz, H-6aI), 4.31
(d, 1H, J5,6b = 2.29 Hz, H-6bI), 4.00 (m, 2H, H-4I, 5I), 3.95 (m, 1H,
H-3I), 3.88 (m, 2H, H-4II, 5II), 3.69 (br t, 1H, J = 8.60 Hz, H-3II),
3.62 (dd, 1H, J2,3 = 9.62 Hz, H-2I), 3.50 (br t, 1H, J = 8.48 Hz, H-2II),
2.02 (s, 3H, CH3CO). The product was used for the next reaction
without further purification.
J5,6a = 0.91 Hz,
J2,3 = J3,4 = 1.60 Hz,
Jgem = 7.33 Hz,
H-6aI),
(ddd,
3.96
1H,
(t,
1H,
H-3I),
3.90
J3,4 = 8.71 Hz,
J4,5 = 9.62 Hz, J4,OH = 2.53 Hz, H-4II), 3.79–3.74 (m, 6H, H-4I, 6bI,
5II, COOMe), 3.56 (dd, 1H, J2,3 = 9.73 Hz, H-2II), 3.51 (br t, 1H,
J = 8.94 Hz, H-3II), 3.21 (s, 1H, H-2I), 2.91 (d, 1H, OH-4II). Anal. Calcd
for C34H37N3O10: C, 63.04; H, 5.77; N, 6.49. Found: C, 62.89; H,
5.78; N, 6.14.
4.1.10. (Allyl 2,3,4-tri-O-benzyl-b-
(1?4)-(6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-
pyranosyl)-(1?3)-(methyl 2,3-di-O-benzyl-b- -glucopyranosy
luronate)-(1?4)-1,6-anhydro-2-azido-3-O-benzyl-2-deoxy-b-
glucopyranose (13 , 13b)
D
-glucopyranosyluronate)-
a
and b- -gluco
D
D
D
-
4.1.7. (Allyl 2,3,4-tri-O-benzyl-b-
D
-glucopyranosyluronate)-
a
(1?4)-6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-
anosyl chloride (10)
a
-
D
-glucopyr
Method A: Molecular sieves 4 Å (430 mg) was added to the solu-
tion of 9 (143.8 mg, 0.15 mmol) and 12 (88.5 mg, 0.14 mmol) in
toluene (7.0 mL) and stirred for 30 min at rt. To this was added
To a solution of 8 (218.4 mg, 0.27 mmol) in CH2Cl2 (2 mL) was
added 1-chloro-N,N,2-trimethylpropenylamine (40 L, 0.30 mmol)
with stirring. Additional reagents (71 and 54 L) were added after
l
TBDMSOTf (6.9
l
L, 30
l
mol) in CH2Cl2 (30
l
L) at ꢁ20 °C with stir-
l
ring gradually raising the temperature to rt for 7 h. The reaction
was quenched with DIPEA and the insoluble materials were fil-
tered on Celite. The organic phase was treated as described in Sec-
tion 4.1. The volatiles were removed under diminished pressure
and the residue was subjected to a column of gel permeation (S-
1 and 3 h, respectively. One hour later the volatiles were removed
under diminished pressure and the residue was directly subjected
to a column of silica gel (15:1–1:2 n-hexane–EtOAc) to give 10
(203.2 mg) in 89% yield as a syrup: 1H NMR (CDCl3): d 7.43–7.20
(m, 20H, Ar H), 6.00 (d, 1H, J1,2 = 3.67 Hz, H-1I), 5.76 (m, 1H, All),
5.26 (m, 1H, All), 5.16 (m, 1H, All), 5.12, 4.63 (ABq, 2H,
J = 10.54 Hz, PhCH2), 4.89, 4.82 (ABq, 2H, J = 11.00 Hz, PhCH2),
4.84, 4.77 (ABq, 2H, J = 11.46 Hz, PhCH2), 4.78, 4.60 (ABq, 2H,
J = 10.54 Hz, PhCH2), 4.47 (m, 2H, All), 4.45 (d, 1H, J1,2 = 8.02 Hz,
H-1II), 4.38 (dd, 1H, J5,6a = 1.60 Hz, Jgem = 12.60 Hz, H-6aI), 4.30
(dd, 1H, J5,6b = 3.41 Hz, H-6bI), 3.96 (m, 1H, H-5I), 3.92 (t, 1H,
J3,4 = J4,5 = 8.02 Hz, H-4I), 3.90 (br t, 1H, J = 9.16 Hz, H-4II), 3.86 (d,
1H, H-5II), 3.84 (t, 1H, H-3I), 3.68 (br t, 1H, J = 8.72 Hz, H-3II), 3.64
(dd, 1H, H-2I), 3.50 (br t, 1H, J = 8.48 Hz, H-2II), 2.04 (s, 3H, CH3CO).
The product was used for the next reaction without further
purification.
X1, toluene) and PTLC (7:1 toluene–EtOAc) to give 13
a (86.0 mg,
43%) and 13b (50.2 mg, 25%) as syrups. Method B: Molecular sieves
4 Å (799 mg), AgOTf (1.10 g, 4.26 mmol) and collidine (0.4 mL,
2.8 mmol) were added to the solution of 12 (233.0 mg,
0.360 mmol) in CH2Cl2 (10.5 mL) and stirred for 1 h at rt. The mix-
ture was cooled to ꢁ40 °C and 10 (593.9 mg, 0.705 mmol) in
CH2Cl2 (5.0 mL) and stirred overnight gradually raising the temper-
ature to rt. The reaction was quenched with aq NaHCO3 and brine.
The insoluble materials were filtered on Celite. The organic phase
was treated as described in Section 4.1. The volatiles were removed
under diminished pressure and the residue was subjected to col-
umns of gel permeation (S-X1, toluene) and silica gel (15:1–3:1
n-hexane–EtOAc) to give 13 (340.3 mg, 65%) and 13b (57.0 mg,
11%) as syrups. Compound 13a: [a]
+34 (c 0.60, CHCl3); 1H NMR
D
a
4.1.8. (2,3-Di-O-benzyl-b-
2-azido-3-O-benzyl-2-deoxy-b-
D
-glucopyranosyl)-(1?4)-1,6-anhydro-
D
-glucopyranose (11)
(CDCl3): d 7.42–7.12 (m, 35H, Ar H), 5.88 (m, 1H, All), 5.51 (m,
2H, H-1I, 1III), 5.25 (m, 1H, All), 5.15 (m, 1H, All), 5.10, 4.62 (ABq,
2H, J = 10.77 Hz, PhCH2), 5.03, 4.80 (ABq, 2H, J = 13.75 Hz, PhCH2),
5.03, 4.69 (ABq, 2H, J = 10.77 Hz, PhCH2), 4.91, 4.79 (ABq, 2H,
J = 11.46 Hz, PhCH2), 4.83, 4.78 (ABq, 2H, J = 11.00 Hz, PhCH2),
4.77, 4.58 (ABq, 2H, J = 7.33 Hz, PhCH2), 4.61, 4.58 (ABq, 2H,
J = 11.69 Hz, PhCH2), 4.58 (d, 1H, J1,2 = 7.56 Hz, H-1II), 4.58 (br d,
To a solution of 3 (2.89 g, 4.10 mmol) in CH2Cl2 (40 mL) and
MeOH (40 mL) was added camphorsulfonic acid (0.3 g, 1.3 mmol)
with stirring for 2 days. The reaction was quenched with Et3N.
The volatiles were removed under diminished pressure and the
residue was subjected to a column of silica gel (3:1–1:4 n-hex-
ane–EtOAc) to give 11 (2.25 g) in 92% yield as a syrup. Physical data
were in good agreement with those already reported.9
1H, J = 12.14 Hz, H-5I), 4.47 (m, 1H, All), 4.47 (d, 1H, J1,2
=

N. Takeda et al. / Carbohydrate Research 353 (2012) 13–21
19
8.02 Hz, H-1IV), 4.46 (m, 1H, All), 4.41 (dd, 1H, J5,6a = 2.06 Hz,
4.1.12. (Allyl 2,3,4-tri-O-benzyl-b-
(1?4)-(6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-
anosyl)-(1?3)-(methyl 2,3-di-O-benzyl-b- -glucopyranosylur
onate)-(1?4)-6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-D-gluco
pyranose (15)
D
-glucopyranosyluronate)-
Jgem = 12.60 Hz, H-6aIII), 4.35 (dd, 1H, J5,6b = 2.98 Hz, H-6bIII), 4.11
(dd, 1H, J3,4 = 8.71 Hz, J4,5 = 9.62 Hz, H-4II), 4.07 (d, 1H,
J5,6a = 6.64 Hz, H-6aI), 3.91 (d, 1H, H-5II), 3.88 (m, 1H, H-4IV), 3.87
(m, 1H, H-4III), 3.86 (br s, 1H, H-4I), 3.85 (d, 1H, J4,5 = 5.04 Hz, H-
5IV), 3.78 (br t, 1H, J2,3 = 10.31 Hz, J3,4 = 8.71 Hz, H-3III), 3.75 (m,
1H, H-6bI), 3.74 (m, 1H, H-3I), 3.73 (m, 1H, H-3II), 3.67 (br t, 1H,
J = 8.94 Hz, H-3IV), 3.66 (d, 1H, J4,5 = 8.71 Hz, H-5III), 3.61 (dd, 1H,
J2,3 = 8.94 Hz, H-2II), 3.47 (dd, 1H, J2,3 = 8.94 Hz, H-2IV), 3.43 (s,
3H, COOMe), 3.22 (br s, 1H, H-2I), 3.20 (dd, 1H, J1,2 = 3.90 Hz, H-
2III), 2.06 (s, 3H, CH3CO). Anal. Calcd for C79H84N6O21: C, 65.28;
H, 5.82; N, 5.78. Found: C, 65.25; H, 5.60; N, 5.40. Compound
a-D
-glucopyr
D
To a solution of 14 (141.6 mg, 91.0
lmol) in DMF (2 mL) was
added H2NNH2ꢀAcOH (17.4 mg, 189 mol) with stirring for 3 h at
l
50 °C. The reaction mixture was treated as described for the syn-
thesis of 8. The crude mixture was subjected to a column of silica
gel (50:1–3:1 toluene–EtOAc) to give 15 (126.1 mg) in 92% yield
as a syrup. The disappearance of one acetyl signal was checked
by 1H NMR and was used for the next reaction without further
purification.
13b: [
a]
ꢁ0.32 (c 0.62, CHCl3); 1H NMR (CDCl3): d 7.41–7.19 (m,
D
35H, Ar H), 5.80–5.72 (m, 1H, All), 5.49 (s, 1H, H-1I), 5.25 (d, 1H,
J = 17.41 Hz, All), 5.15 (d, 1H, J = 10.31 Hz, All), 4.99, 4.62 (ABq,
2H, J = 10.77 Hz, PhCH2), 4.95, 4.70 (ABq, 2H, J = 11.55 Hz, PhCH2),
4.89, 4.64 (ABq, 2H, J = 10.77 Hz, PhCH2), 4.85, 4.78 (ABq, 2H,
J = 11.00 Hz, PhCH2), 4.77, 4.57 (ABq, 2H, J = 10.77 Hz, PhCH2),
4.75 (s, 2H, PhCH2), 4.61 (m, 2H, PhCH2), 4.56 (m, 1H, H-5I), 4.55
(d, 1H, J1,2 = 9.79 Hz, H-1II), 4.47 (br t, 2H, J = 4.81 Hz, All), 4.38
(d, 1H, J1,2 = 7.79 Hz, H-1IV), 4.37 (d, 1H, J1,2 = 7.56 Hz, H-1III), 4.24
(d, 1H, Jgem = 10.77 Hz, H-6aIII), 4.15 (dd, 1H, J5,6b = 3.66 Hz, H-6bIII),
4.14 (t, 1H, J3,4 = J4,5 = 8.94 Hz, H-4II), 4.05 (d, 1H, Jgem = 7.33 Hz, H-
6aI), 3.91 (d, 1H, H-5II), 3.87 (br s, 1H, H-4I), 3.86 (t, 1H,
J3,4 = J4,5 = 8.94 Hz, H-4IV), 3.81 (d, 1H, H-5IV), 3.80 (t, 1H,
J3,4 = J4,5 = 9.39 Hz, H-4III), 3.78 (s, 3H, COOMe), 3.74 (d, 1H, H-
6bI), 3.73 (s, 1H, H-3I), 3.64 (t, 1H, H-3IV), 3.58 (t, 1H, H-3II), 3.52
(t, 1H, H-2II), 3.43 (br t, 1H, J = 8.14 Hz, H-2IV), 3.28 (br t, 1H,
J = 9.51 Hz, H-3III), 3.25 (br t, 1H, J = 8.70 Hz, H-2III), 3.23 (m, 1H,
H-5III), 3.19 (s, 1H, H-2I), 1.83 (s, 3H, CH3CO). Anal. Calcd for
4.1.13. (Allyl 2,3,4-tri-O-benzyl-b-
(1?4)-(6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-
anosyl)-(1?3)-(methyl 2,3-di-O-benzyl-b- -glucopyranosyluro
nate)-(1?4)-6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-a-D-gluco
pyranosyltrichloroacetimidate (16)
D
-glucopyranosyluronate)-
a-D
-glucopyr
D
To a solution of 15 (173.3 mg, 114
3.6 mmol) in CH2Cl2 (7.2 mL) was added DBU (9
l
mol) and CCl3CN (0.36 mL,
L, 0.06 mmol)
l
with stirring for 2 h at 0 °C and for more 2 h at rt. The reaction mix-
ture was directly subjected to a column of silica gel (100:1–6:1 tol-
uene–EtOAc) to give 16 (188.4 mg) in 98% yield as a syrup: 1H NMR
(CDCl3): d 8.74 (s, 1H, NH), 7.42–7.19 (m, 35H, Ar H), 6.33 (d, 1H,
J1,2 = 3.67 Hz, H-1I), 5.81–5.72 (m, 1H, All), 5.47 (d, 1H,
J1,2 = 3.90 Hz, H-1III), 5.27–5.23 (m, 1H, All), 5.16–5.14 (m, 1H,
All), 5.14, 4.66 (ABq, 2H, J = 10.54 Hz, PhCH2), 5.10, 4.62 (ABq, 2H,
J = 10.76 Hz, PhCH2), 4.99, 4.86 (ABq, 2H, J = 10.77 Hz, PhCH2),
4.91, 4.78 (ABq, 2H, J = 11.46 Hz, PhCH2), 4.83, 4.78 (ABq, 2H,
J = 11.23 Hz, PhCH2), 4.78, 4.59 (ABq, 2H, J = 11.00 Hz, PhCH2),
4.77 (m, 2H, PhCH2), 4.48 (d, 1H, H-1II), 4.46 (d, 1H, H-1IV), 4.46
(m, 2H, All), 4.41 (d, 1H, Jgem = 12.37 Hz, H-6aIII), 4.36 (d, 1H,
Jgem = 11.91 Hz, H-6aI), 4.34 (dd, 1H, J5,6b = 2.75 Hz, H-6bIII), 4.26
(dd, 1H, J5,6b = 3.44 Hz, H-6bI), 4.10 (br t, 1H, J = 9.28 Hz, H-4II),
3.97 (m, 1H, H-3I), 3.94 (m, 1H, H-4I), 3.87 (m, 4H, H-5II,IV, 4III,IV),
3.77 (br t, 1H, J = 9.28 Hz, H-3III), 3.75 (br t, 1H,
J2,3 = J3,4 = 8.71 Hz, H-3II), 3.67 (br t, 1H, J = 8.78 Hz, H-3IV), 3.63
(m, 1H, H-2I, 5I, III), 3.50 (br t, 1H, J = 8.85 Hz, H-2II), 3.47 (br t,
1H, J = 8.37 Hz, H-2IV), 3.24 (s, 3H, COOMe), 3.23 (m, 1H, H-
2III),2.06, 2.02 (2s, 3H ꢂ 2, 2CH3CO). The product was used for the
next reaction without further purification.
C79H84N6O21: C, 65.28; H, 5.82; N, 5.78. Found: C, 65.22; H, 5.85;
N, 5.39.
4.1.11. (Allyl 2,3,4-tri-O-benzyl-b-
(1?4)- (6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-
anosyl)-(1?3)-(methyl 2,3-di-O-benzyl-b-
D
-glucopyranosyluronate)-
-glucopyr
-glucopyranosylur
a-D
D
onate)-(1?4)-1,6-di-O-acetyl-2-azido-3-O-benzyl-2-deoxy-D-
glucopyranose (14)
To a solution of 13
a
(106.1 mg, 68.2
lmol) in AcOH (74 lL) and
Ac2O (1.4 mL) was added TFA (215
l
L) at 0 °C with stirring. The
temperature was raised to rt and stirring continued overnight. A
few portions of ice were added to the reaction mixture. The vola-
tiles were removed under diminished pressure and the residue
was subjected to a column of silica gel (50:1–9:1 toluene–EtOAc)
to give 14 (101.9 mg) in 90% yield as a syrup: 1H NMR (CDCl3): d
4.1.14. Octyl (allyl 2,3,4-tri-O-benzyl-b-
(1?4)-(6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-
syl)-(1?3)-(methyl 2,3-di-O-benzyl-b- -glucopyranosyluronate)-
(1?4)-6-O-acetyl-2-azido-3-O-benzyl-2-deoxy- and b- -gluco
pyranoside (17 , 17b)
Molecular sieves AW300 (556 mg) was added to the solution of
16 (188.4 mg,114 mol) and n-octanol (94 L, 597 mol) in CH2Cl2
(26 mL) and stirred for 30 min at rt. To this was added TMSOTf
(15 L, 83 mol) with stirring for 40 min. The reaction was
quenched with DIPEA (29 L) and diluted with CHCl3. Insoluble
D-glucopyranosyluronate)-
a-D-glucopyrano
D
7.43–7.19 (m, 35H, Ar H), 6.16 (d, 0.6H, J1,2 = 3.72 Hz, H-1aI),
a
D
5.78–5.71 (m, 1H, All), 5.46 (d, 1H, J1,2 = 3.90 Hz, H-1III), 5.39 (d,
0.4H, J1,2 = 8.47 Hz, H-1bI), 5.26–5.23 (m, 1H, All), 5.15–5.13 (m,
1H, All), 5.10, 4.63 (ABq, 2H, J = 10.50 Hz, PhCH2), 5.10, 4.61 (ABq,
2H, J = 10.38 Hz, PhCH2), 4.95, 4.84 (ABq, 2H, J = 10.63 Hz, PhCH2),
4.89, 4.77 (ABq, 2H, J = 11.48 Hz, PhCH2), 4.83, 4.78 (ABq, 2H,
J = 10.80 Hz, PhCH2), 4.81, 4.78 (ABq, 2H, J = 11.40 Hz, PhCH2),
a
l
l
l
l
l
l
4.77, 4.59 (ABq, 2H, J = 10.74 Hz, PhCH2), 4.46 (m, 4H, H-1II,IV
,
materials were filtered on Celite. The organic phase was treated
as described in Section 4.1. The volatiles were removed under
diminished pressure and the residue was subjected to a column
All), 4.41 (dd, 1H, J5,6a = 1.60 Hz, Jgem = 12.48 Hz, H-6aIII), 4.34 (dd,
1H, J5,6b = 1.92 Hz, H-6bIII), 4.33 (d, 1H, J5,6a = 2.88 Hz,
Jgem = 12.30 Hz, H-6aI), 4.25 (dd, 1H, J5,6b = 3.90 Hz, H-6bI), 4.09 (t,
1H, J3,4 = J4,5 = 9.24 Hz, H-4II), 3.90–3.83 (m, 5H, H-4I,III,IV, 5II,IV),
3.78–3.72 (m, 2H, H-3I,III), 3.74 (br t, 1H, J = 8.94 Hz, H-3II), 3.70
(m, 1H, H-5III), 3.67 (t, 1H, J2,3 = J3,4 = 8.91 Hz, H-3IV), 3.62 (m, 1H,
of silica gel (50:1–10:1 toluene–EtOAc) to give 17
67%) and 17b (35.1 mg, 20%) as syrups. Compound 17
a
(119.1 mg,
: [ +49
a
a]
D
(c 0.77, CHCl3); 1H NMR (CDCl3): d 7.47–7.19 (m, 35H, Ar H), 5.74
(m, 1H, All), 5.47 (d, 1H, J1,2 = 3.90 Hz, H-1III), 5.24 (m, 1H, All),
5.14 (m, 1H, All), 5.10, 4.62 (ABq, 2H, J = 10.54 Hz, PhCH2), 5.09,
4.61 (ABq, 2H, J = 10.77 Hz, PhCH2), 4.98, 4.84 (ABq, 2H,
J = 10.77 Hz, PhCH2), 4.94, 4.78 (ABq, 2H, J = 11.46 Hz, PhCH2),
4.80 (m, 4H, 2PhCH2), 4.77, 4.59 (ABq, 2H, J = 11.00 Hz, PhCH2),
H-5I), 3.53 (dd, 0.6H, J2,3 = 10.14 Hz, H-2
aI), 3.50–3.45 (m, 2.4H,
H-2bI, 2II,IV), 3.24 (dd, 1H, J2,3 = 10.44 Hz, H-2III), 3.23 (s, 3H,
COOMe), 2.18, 2.06, 2.04 (3s, 3H ꢂ 3, 3CH3CO). Anal. Calcd for
C83H90N6O24: C, 64.07; H, 5.84; N, 5.40. Found: C, 64.02; H, 5.78;
N, 5.04.

20
N. Takeda et al. / Carbohydrate Research 353 (2012) 13–21
4.82 (d, 1H, H-1I), 4.46 (m, 4H, H-1II,IV, All), 4.40 (dd, 1H,
J5,6a <1.0 Hz, Jgem = 12.37 Hz, H-6aIII), 4.36 (d, 1H, J5,6a = 2.06 Hz,
Jgem = 12.37 Hz, H-6aI), 4.33 (dd, 1H, J5,6b = 2.75 Hz, H-6bIII), 4.25
(dd, 1H, J5,6b = 3.90 Hz, H-6bI), 4.09 (br t, 1H, J = 9.28 Hz, H-4II),
3.88 (m, 1H, H-4IV), 3.87 (m, 1H, H-4III), 3.85 (m, 3H, H-3I, 5II,IV),
3.80 (br t, 1H, J = 9.46 Hz, H-4I), 3.76 (m, 1H, H-3III), 3.74 (m, 1H,
H-3II), 3.73 (m, 1H, H-5I), 3.66 (br t, 1H, J = 8.82 Hz, H-3IV), 3.62
(m, 2H, H-5III, 1/2OCH2), 3.50 (br t, 1H, J = 8.25 Hz, H-2II), 3.46 (br
t, 1H, J = 8.71 Hz, H-2IV), 3.44 (m, 1H, 1/2OCH2), 3.26 (dd, 1H,
J1,2 = 3.67 Hz, J2,3 = 9.85 Hz, H-2I), 3.23 (dd, 1H, J2,3 = 8.48 Hz, H-
2III), 3.22 (s, 3H, COOMe), 2.05, 2.03 (2s, 3H ꢂ 2, 2CH3CO), 1.63
(m, 2H, CH2), 1.38 (m, 2H, CH2), 1.28 (m, 8H, CH2), 0.86 (m, 3H,
CH3). Anal. Calcd for C89H104N6O23: C, 65.74; H, 6.46; N, 5.17.
3.25 (dd, 1H, J2,3 = 10.54 Hz, H-2III), 3.14 (dd, 1H, J2,3 = 10.31 Hz,
H-2I), 1.52 (m, 2H, CH2), 1.30 (m, 2H, CH2), 1.17 (m, 8H, CH2),
0.79 (m, 3H, CH3).
4.1.16. Octyl (sodium b-
amino-2-deoxy-6-O-sulfo-
b- -glucopyranosyluronate)-(1?4)-2-amino-2-deoxy-6-O-
sulfo- -glucopyranoside, disodium salt (20)
To a solution of 19 (22.4 mg, 12.9 mol) in EtOH (3 mL) was
D-glucopyranosyluronate)-(1?4)-(2-
a
-D
-glucopyranosyl)-(1?3)-(sodium
D
a-D
l
added a catalytic amount of Pd-C under H2-atmosphere with stir-
ring overnight at rt. H2O (2 mL) was added to the reaction mixture
after 1 d and the reaction was continued for 1 more d. Insoluble
materials were removed on Celite and the volatiles were removed
under diminished pressure. The crude mixture was subjected to a
column of C8 (H2O to H2O–MeOH 1:4) to give the hydrogenolyzed
product 20 (10.3 mg) in 76% yield. The product was characterized
by the following acetylation.
Found: C, 65.77; H, 6.45; N, 5.09. Compound 17b: [
a
]
D
+16 (c
0.77, CHCl3); 1H NMR (CDCl3): d 7.42–7.19 (m, 35H, Ar H), 5.75
(m, 1H, All), 5.47 (d, 1H, J1,2 = 3.90 Hz, H-1III), 5.25 (m, 1H, All),
5.15 (m, 1H, All), 5.10, 4.61 (ABq, 2H, J = 10.54 Hz, PhCH2), 4.97,
4.64 (ABq, 2H, J = 10.77 Hz, PhCH2), 4.96, 4.83 (ABq, 2H,
J = 10.77 Hz, PhCH2), 4.90, 4.78 (ABq, 2H, J = 11.46 Hz, PhCH2),
4.83, 4.78 (ABq, 2H, J = 11.00 Hz, PhCH2), 4.78, 4.73 (ABq, 2H,
J = 11.46 Hz, PhCH2), 4.77, 4.59 (ABq, 2H, J = 11.00 Hz, PhCH2),
4.1.17. Octyl (sodium b-
amido-2-deoxy-6-O-sulfo-
b- -glucopyranosyluronate)-(1?4)-2-acetamido -2-deoxy-6-O-
sulfo- -glucopyranoside, disodium salt (21)
To a solution of 20 (10.5 mg,10.0 mol) in H2O (1.0 mL) were
added 1 drop of Ac2O and Et3N (10 L) with stirring overnight for
D-glucopyranosyluronate)-(1?4)-(2-acet
a-D-glucopyranosyl)-(1?3)-(sodium
D
4.46 (m, 4H, H-1II,IV
,
All), 4.42 (dd, 1H, J5,6a = 2.06 Hz,
a-D
Jgem = 12.37 Hz, H-6aIII), 4.40 (br d, 1H, J = 9.62 Hz, H-6aI), 4.33
(br dd, 1H, J = 2.98 Hz, H-6bI), 4.20 (dd, 1H, J5,6b = 4.81 Hz, H-6bIII),
4.18 (d, 1H, J1,2 = 8.02 Hz, H-1I), 4.08 (br t, 1H, J = 9.28 Hz, H-4II),
3.89 (m, 1H, H-4IV), 3.87 (m, 1H, H-4III), 3.85 (m, 1H, H-5II), 3.83
(m, 2H, H-5IV, 1/2OCH2), 3.77 (m, 2H, H-4I, 3III), 3.73 (t, 1H,
J2,3 = J3,4 = 8.94 Hz, H-3II), 3.67 (t, 1H, J2,3 = J3,4 = 8.94 Hz, H-3IV),
3.63 (m, 1H, H-5III), 3.47 (t, 1H, J1,2 = 8.94 Hz, H-2IV), 3.46 (t, 1H,
J1,2 = 8.94 Hz, H-2II), 3.47 (m, 1H, 1/2OCH2), 3.34 (dd, 1H,
J2,3 = 9.85 Hz, H-2I), 3.29 (br t, 1H, J = 9.74 Hz, H-3I), 3.27 (m, 1H,
H-5I), 3.25 (s, 3H, COOMe), 3.23 (dd, 1H, J2,3 = 10.31 Hz, H-2III),
2.06, 2.02 (2s, 3H ꢂ 2, 2CH3CO), 1.61 (m, 2H, CH2), 1.34 (m, 2H,
CH2), 1.27 (m, 8H, CH2), 0.87 (m, 3H, CH3). Anal. Calcd for
l
l
6 h. Volatiles were removed under diminished pressure. The crude
mixture was subjected to a column of ion-exchange resin (Dowex
50Wx8, Na+ form, H2O) to give 21 (8.9 mg) in 78% yield: [
a]D +48.5
(c 0.88, H2O); 1H NMR (D2O): d 5.27 (d, 1H, J1,2 = 3.72 Hz, H-1III),
4.73 (d, 1H, J1,2 = 3.12 Hz, H-1I), 4.42 (d, 1H, J1,2 = 7.80 Hz, H-1II),
4.41 (d, 1H, J1,2 = 7.86 Hz, H-1IV), 4.30 (br d, 1H, J = 11.16 Hz, H-
6aIII), 4.20 (m, 2H, H-6I), 4.02 (br d, 1H, J = 11.16 Hz, H-6bIII), 3.89
(m, 2H, H-5I, III), 3.76 (m, 3H, H-2I,III, 3I), 3.69 (m, 2H, H-5II, 3III),
3.56 (m, 6H, H-4I,II,III, 3II, 5IV, 1/2OCH2), 3.36 (m, 3H, H-3IV, 4IV, 1/
2OCH2), 3.20 (m, 2H, H-2II,IV), 1.89 (s, 3H, NAc), 1.87 (s, 3H, NAc),
1.42 (m, 2H, CH2), 1.21–1.05 (m, 10H, CH2), 0.70 (t, 3H,
J = 13.92 Hz, CH3). MALDI-TOFMS: m/z calcd for C36H57N2O29S2Na4,
1137.20; found, 1137.33 [M+H+], C36H56N2O29S2Na5, 1159.19;
found, 1159.42 [M+Na+].
C89H104N6O23: C, 65.74; H, 6.46; N, 5.17. Found: C, 65.73; H,
6.41; N, 4.93.
4.1.15. Octyl (sodium 2,3,4-tri-O-benzyl-b-
nate)-(1?4)-(2-azido-3-O-benzyl-2-deoxy-6-O-sulfo-
pyranosyl)-(1?3)-(sodium 2,3-di-O-benzyl-b-
D
-glucopyranosyluro
-gluco
-glucopyranosy
a-D
D
4.1.18. Octyl (sodium b-
-2-sulfamido-6-O-sulfo-
glucopyranosyluronate)-(1?4)-2-deoxy-2-sulfamido-6-O-sulfo-
-glucopyranoside, tetrasodium salt (23) and octyl (sodium b- -glu
copyranosyluronate)-(1?4)-(2-deoxy-2-sulfamido-3,6-di-O-sulfo-
-glucopyranosyl)-(1?3)-(sodium b- -glucopyranosyluronate)-
-glucopyranoside, penta
D-glucopyranosyluronate)-(1?4)-(2-deoxy
luronate)-(1?4)- 2-azido-3-O-benzyl-2-deoxy-6-O-sulfo-
a
-D
-
a-D
-glucopyranosyl)-(1?3)-(sodium b-
D-
glucopyranoside, disodium salt (19)
a-
To a solution of 17
a
(24.4 mg, 15.0
lmol) in THF (1.5 mL)-H2O
D
D
(0.3 mL) was added 1.25 M LiOH (0.5 mL) with stirring at 0 °C
and the stirring was continued overnight at rt. The volatiles were
removed under diminished pressure. The residue was diluted with
MeOH (1.0 mL)–CH2Cl2 (0.3 mL). To this was added 0.5 M NaOH
(0.6 mL) with stirring for 4 h. The reaction was quenched with
50% AcOH, and the volatiles were removed under diminished pres-
sure. The residue was directly subjected to a column of gel-perme-
ation (LH-20, 1% AcOH) to give 18 (23.1 mg) all of which was
diluted with DMF (1.5 mL). To this solution was added SO3ꢀMe3N
(48.9 mg) with stirring at 60 °C for 1 d. All the reaction mixture
was subjected to columns of gel-permeation (LH-20, CHCl3–MeOH
1:1) and ion exchange resin (Dowex 50Wx8, Na+ form, MeOH–H2O
8:1) to give 19 (22.4 mg) in 85% yield for 3 steps. The product was
used for the next reaction without further purification. 1H NMR
(CD3OD) (selected): d 5.31 (d, 1H, J1,2 = 3.67 Hz, H-1III), 4.91 (m,
1H, H-1IV), 4.90 (m, 1H, H-1II), 4.74 (d, 1H, J1,2 = 3.21 Hz, H-1I),
4.46 (m, 1H, H-6aIII), 4.44 (m, 1H, H-6aI), 4.14 (br d, 1H,
J = 10.99 Hz, H-6bI), 4.04 (d, 1H, J4,5 = 9.63 Hz, H-5IV), 3.96 (m, 1H,
H-5II), 3.95 (m, 2H, H-4III, 6bIII), 3.94 (m, 2H, H-4I, 4IV), 3.90 (m,
1H, H-5III), 3.83 (br t, 1H, J = 9.05 Hz, H-3IV), 3.75 (m, 1H, H-3I),
3.72 (m, 1H, H-3II), 3.67 (br t, 1H, J = 9.16 Hz, H-3III), 4.66 (m, 1H,
H-4II), 3.60 (m, 1H, H-5I), 3.57 (m, 1H, 1/2OCH2), 3.39 (br t, 1H,
J = 8.48 Hz, H-2IV), 3.35 (m, 1H, 1/2OCH2), 3.30 (m, 1H, H-2II),
a-D
D
(1?4)-2-deoxy-2-sulfamido-6-O-sulfo-a-D
sodium salt (24)
Method A (via an N-sulfation in aq NaOH): The hydrogenolyzed
product 20 (10.3 mg) was diluted with H2O (2.4 mL). To this was
added SO3–pyridine complex (56.2 mg) and the pH was adjusted
at 9 with 0.5 M NaOH. After 2 h the reaction mixture was subjected
to a column of gel-permeation (LH-20, 1% AcOH). The residue was
subjected to columns of ion exchange resin (Dowex 50Wx8, Na+
form) and C8 (H2O to H2O–MeOH 1:4) to give a 3:1 mixture
(15.5 mg) of 23 and 24quantitatively. Method B (via an N-sulfation
in aq Et3N): the hydrogenolyzed product 20 (5.9 mg) was diluted
with H2O (1.4 mL). To this was added SO3-pyridine complex por-
tionwise (74.7 mg) adjusting pH at 10 with Et3N (112 lL total)
for 3d. The reaction mixture was subjected to a column of gel-per-
meation (LH-20, 1% AcOH). The residue was subjected to columns
of ion exchange resin (Dowex 50Wx8, Na+ form) to give 23
(5.5 mg) in 78% yield. Compound 23: [a]
+38.0 (c 0.41, H2O); 1H
D
NMR (D2O): d 5.56 (d, 1H, J1,2 = 3.67 Hz, H-1III), 5.12 (d, 1H,
J1,2 = 3.67 Hz, H-1I), 4.64 (d, 1H, J1,2 = 8.02 Hz, H-1II), 4.62 (d, 1H,
J1,2 = 5.96 Hz, H-1IV), 4.42 (br d, 1H, J = 10.99 Hz, H-6aIII), 4.34 (m,
2H, H-6I), 4.20 (br d, 1H, J = 12.15 Hz, H-6bIII), 4.04 (dd, 1H,

N. Takeda et al. / Carbohydrate Research 353 (2012) 13–21
21
J3,4 = 5.50 Hz, J4,5 = 8.71 Hz, H-4II), 3.98 (m, 1H, H-5I), 3.92 (m, 1H,
H-5III), 3.85 (d, 1H, H-5II), 3.85 (br t, 1H, J = 4.46 Hz, H-3II), 3.78
(m, 1H, H-4III), 3.73 (m, 3H, H-4I, 5IV, 1/2OCH2), 3.72 (m, 1H, H-
3I), 3.71 (m, 1H, H-3III), 3.55 (m, 3H, H-3IV, 4IV, 1/2OCH2), 3.40
(m, 1H, H-2II), 3.37 (m, 1H, H-2IV), 3.29 (dd, 1H, J2,3 = 10.77 Hz,
H-2III), 3.26 (dd, 1H, J2,3 = 9.39 Hz, H-2I), 1.65 (m, 2H, CH2), 1.39–
1.26 (m, 10H, CH2), 0.84 (m, 3H, CH3). MALDI-TOFMS: m/z calcd
Miyuki Tanmatsu for performing the elemental analysis measure-
ments (Research Center for Bioscience and Technology, Division
of Instrumental Analysis, Tottori University). This study was sup-
ported by a Grant-in-aid for Scientific Research (C) (20510200), a
Grant-in-aid for Scientific Research on Innovative Areas 3301,
MEXT, Japan and Daiwa Securities Science Foundation.
for
C
32H51N2O33S4Na6, 1257.06; found, 1257.09 [M+H+],
32H50N2O33S4Na7, 1279.05; found, 1279.11 [M+Na+]. Compound
References
C
24: 1H NMR (D2O): d 5.65 (d, 1H, J1,2 = 3.66 Hz, H-1III), 5.12 (d,
1H, J1,2 = 3.67 Hz, H-1I), 4.64 (d, 1H, J1,2 = 8.02 Hz, H-1II), 4.62 (d,
1H, J1,2 = 5.96 Hz, H-1IV), 4.60 (m, 1H, H-3III), 4.42 (br d, 1H,
J = 10.99 Hz, H-6aIII), 4.34 (m, 2H, H-6I), 4.18 (br d, 1H,
J = 11.68 Hz, H-6bIII), 4.04 (dd, 1H, J3,4 = 5.50 Hz, J4,5 = 8.71 Hz, H-
4II), 3.98 (m, 2H, H-5I,III), 3.92 3.85 (d, 1H, H-5II), 3.85 (br t, 1H,
J = 4.46 Hz, H-3II), 3.73 (m, 3H, H-4I, 5IV, 1/2OCH2), 3.72 (m, 1H,
H-3I), 3.55 (m, 3H, H-3IV, 4IV, 1/2OCH2), 3.40 (m, 1H, H-2II), 3.37
(m, 3H, H-2III,IV, 4III), 3.26 (dd, 1H, J2,3 = 9.39 Hz, H-2I), 1.65 (m,
2H, CH2), 1.39–1.26 (m, 10H, CH2), 0.84 (m, 3H, CH3). MALDI-TOF-
MS: m/z calcd for C32H50N2O36S5Na7, 1359.00; found, 1359.13
[M+H+].
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Acknowledgements
The authors are grateful to Professor Hiromune Ando (Gifu
University) for helpful suggestions. The authors also thank Ms.