Synthesis of β-d-GalNAc(4,6-diS)(1-4)[α-l-Fuc(2,4-diS)(1-3)]- β-d-GlcA, a novel trisaccharide unit of chondroitin sulfate with a fucose branch

Article abstract of DOI:10.1016/j.tetlet.2013.05.064

Novel glycosaminoglycans isolated from the body wall of sea cucumbers consist of chondroitin sulfates like polysaccharides. We have synthesized a representative repeating trisaccharide composed of a chondroitin backbone having a fucose (Fuc) branch at O-3 of glucuronic acid (GlcA), β-d-GalNAc(4,6- diS)(1-4)[α-l-Fuc(2,4-diS)(1-3)]-β-d-GlcA, in a stereocontrolled manner. Regiospecific sulfation at O-4,6 and O-2,4 of the N-acetylgalactosamine (GalNAc) and Fuc residues, respectively, was successfully achieved.

Full text of DOI:10.1016/j.tetlet.2013.05.064

Tetrahedron Letters 54 (2013) 3940–3943
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Synthesis of b-D-GalNAc(4,6-diS)(1–4)[
a-L-Fuc(2,4-diS)(1–3)]-b-D-GlcA,
a novel trisaccharide unit of chondroitin sulfate with a fucose branch
b
b
a
Jun-ichi Tamura a,b, , Haruna Tanaka , Ayumi Nakamura , Naoko Takeda
⇑
a Department of Chemistry and Biotechnology, Graduate School of Engineering, Tottori University, Koyamacho-Minami 4-101, Tottori 680-8552, Japan
b Department of Regional Environment, Faculty of Regional Sciences, Tottori University, Koyamacho-Minami 4-101, 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:
Novel glycosaminoglycans isolated from the body wall of sea cucumbers consist of chondroitin sulfates
like polysaccharides. We have synthesized a representative repeating trisaccharide composed of a
Received 1 April 2013
Revised 8 May 2013
Accepted 13 May 2013
Available online 24 May 2013
chondroitin backbone having a fucose (Fuc) branch at O-3 of glucuronic acid (GlcA), b-
D-GalNAc(4,6-
diS)(1–4)[ -Fuc(2,4-diS)(1–3)]-b- -GlcA, in a stereocontrolled manner. Regiospecific sulfation at O-
a
-L
D
4,6 and O-2,4 of the N-acetylgalactosamine (GalNAc) and Fuc residues, respectively, was successfully
achieved.
Keywords:
Ó 2013 Elsevier Ltd. All rights reserved.
Chondroitin sulfate
Glycosaminoglycan
Sea cucumber
Glycosylation
Chondroitin sulfates (CSs), important glycosaminoglycans, have
numerous biological activities represented by neuronal growth
control.1,2 Many animals produce CSs as linear polysaccharides
Based on the retrosynthetic analysis depicted in Scheme 1, we
planned the fucosylation of a suitably protected chondroitin disac-
charide precursor. Subsequent sulfation at specific positions was
expected to yield the target compound 1.
composed of a repeating disaccharide unit, -3)-b-
D-GalNAc(1-4)-
b- -GlcA(1-, some hydroxyl groups of which are often sulfated. A
D
The glycosyl acceptor for obtaining the disaccharide was syn-
thesized as shown in Scheme 2. First, the known diol (2)7 was
mono-acetylated with regulated amounts of reagents. The yield
was sufficient (73%), but no regioselectivity was shown and the
regioisomeric monoacetates (3a and 3b) could not be separated.
We tried to benzylate the mixture at the residual positions. To
our surprise, a sole compound (4) benzylated only at O-2 was
obtained in 97% yield. We rationalized that the result was due to
the basicity of Ag2O which moved the acetyl group to O-3 followed
by regioselective benzylation at O-2. Similar migration was not
observed in the case of the methylbenzoyl (MBz) analogue. The
acetate (4) was successfully converted to methylbenzoate (6) via
5 in 85 and 97% yield, respectively. The benzylidene acetal of 6
was removed in 81% yield and the primary alcohol was selectively
protected with TBDPS to yield the glycosyl acceptor (8) in 87%
yield. Galactosyl imidate (9)8 was stereoselectively coupled with
8 in the presence of BF3ÁOEt2 in toluene at À50 °C to afford the de-
sired disaccharide (10) in 40% yield (Scheme 2). The levulinoyl
group of 10 was removed with H2NNH2ÁAcOH and the liberated
O-3 was benzylated with BnBr in the presence of Ag2O to give 12
in 98 and 79% yield, respectively. Alternatively, we also adopted
galactosyl imidate (16) masked with a benzyl group at O-3 which
was obtained from the corresponding hemiacetal (15)9 in 83% yield
unique fucosyl chondroitin sulfate (FCS) has been isolated from
sea cucumber having a fucose branch at O-3 of the GlcA moiety
with a variety of sulfation patterns at GalNAc and Fuc residues.3
Di- or trimeric fucose branches have also been described.4 FCS
inhibits thrombin by activating heparin cofactor II, and also acti-
vates factor XII.5 FCS shows anticoagulant activities even when
delivered orally.6 This remarkable activity is completely different
from heparin which is injected intravenously. Medicines having
anticoagulant activities are available but can have significant side
effects. The sulfation patterns and the role of the fucosyl residues
of FCS might be closely related to its biological activities. This
prompted us to synthesize the repeating unit of FCS in order to elu-
cidate the relationship between its biological activities and facile
structure so as to develop an alternative type of medicine. Here
we describe the first synthesis of the FCS trisaccharide, b-D-Gal-
NAc(4,6-diS)(1–4)[a-L-Fuc(2,4-diS)(1–3)]-b-D-GlcA (1).
⇑
Corresponding author. Tel./fax: +81 857 31 5108.
E-mail address: jtamura@rstu.jp (J. Tamura).
0040-4039/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetlet.2013.05.064

J. Tamura et al. / Tetrahedron Letters 54 (2013) 3940–3943
3941
Scheme 1. Retrosynthetic analysis for the target trisaccharide 1. Abbreviations: MP, –C6H4OMe(p); Bn, –CH2Ph; All, –CH2CH@CH2; TBDPS, –Si(tert-Bu)Ph2; MBz, –
C(@O)C6H4Me(p); Lev, –C(@O)C2H4C(@O)Me.
with complete
a
-selectivity. The same acceptor (8) was coupled
Fucosyl fluoride (21) was synthesized from 1811 which was
converted to the allyl ether (19) with AllBr and NaH in 89%
yield and the product was subjected to acetolysis. The obtained
acetate was easily hydrolyzed at work-up to give 20 in 63%
yield. The hemiacetal was converted to the corresponding fluo-
ride (21) in 85% yield as an anomeric mixture (Scheme 3).
The OH-3 at the GlcA moiety was coupled with 21 in the pres-
ence of LiClO4 and CsF in CH2Cl2.12 The reaction afforded the de-
with 16 under similar reaction conditions as above. The b-linked
disaccharide (12) was stereoselectively obtained in 46% yield.
The first author has already reported an optimized coupling of 9
and 2-O-MBz analogue of 8 in higher yield (69%).10 The lower
coupling yields in this study might be due to the differences of
the protecting groups, but should be rationalized. TBDPS and
methylbenzoyl groups of 12 were removed stepwise via 13 in
97% and 86% yields, respectively, to give a diol (14) (Scheme 2).
Regioselective oxidation with TEMPO and NaClO and subsequent
TMSCHN2 treatment afforded a methyl ester (17) in 86% yield
(two steps).
sired trisaccharide (22) in 63% yield with complete
a-selectivity.
Corresponding fucosyl imidate and thioglycoside could not give
the desired glycoside at all. Allyl ethers of 22 were converted
to the corresponding vinyl ethers in the presence of iridium

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J. Tamura et al. / Tetrahedron Letters 54 (2013) 3940–3943
Scheme 2. Synthesis of the glycosyl acceptor 17 and the glycosylation toward the disaccharide moiety. Reagents and conditions: (a) AcCl, pyridine, 73%; (b) BnBr, Ag2O, KI,
DMF, 0 °C–rt, 97%; (c) Et3N, MeOH, H2O, 85%; (d) MBzCl, pyridine, 97%; (e) camphorsulfonic acid, CH2Cl2–MeOH, 81%; (f) TBDPSCl, imidazole, DMF, 87%; (g) BF3ÁOEt2, toluene,
À50 °C, 40%; (h) H2NNH2ÁAcOH, toluene–EtOH, 98%; (i) BnBr, Ag2O, KI, DMF, 0 °C–rt, 79%; (j) n-Bu4NF, THF, 97%; (k) NaOMe, MeOH, 86%; (l) CCl3CN, DBU, CH2Cl2, 0 °C–rt, 83%;
(m) BF3ÁOEt2, toluene, À78 °C, 46%; (n) TEMPO, NaClO, n-Bu4NBr, NaBr, NaHCO3, EtOAc–H2O, then TMSCHN2, toluene–MeOH, 86%.
complex and subsequent acidic hydrolysis gave 23 in 71% yield.
Further acidic hydrolysis afforded 24 in 54% yield. The obtained
tetraol was fully sulfated with SO3ÁNMe3 to give 25 in an almost
quantitative yield. Three benzyl ethers were removed by
hydrogenolysis in the presence of Pd/C in aq EtOH. The azide
was simultaneously reduced to amine which was subsequently
acetylated with Ac2O. Pd(OH)2-catalyzed hydrogenolysis
In summary, we synthesized a fucose-branched chondroitin
sulfate trisaccharide, b- -GalNAc(4,6-diS)(1–4)[ -Fuc(2,4-
diS)(1–3)]-b- -GlcA, for the first time. The stepwise coupling of
each monosaccharide moiety was achieved with complete
stereocontrol.
D
a-L
D
Acknowledgments
completely reduced the aglycon (4-methoxyphenyl) to
a
4-methoxycyclohexyl group. catalytic amount of AcOH in
A
We thank Mrs. Mayumi Ikenari (Research Center for Bioscience
and Technology, Division of Instrumental Analysis, Tottori Univer-
sity) for performing the ESI-MS measurement. This study was sup-
the presence of Pd/C cleaved the fucosyl residue from the CS
backbone. Final saponification of the product and gel-perme-
ation afforded the target compound 1 in 29% yield (from 25).
The final compound was fully characterized by 1H NMR and
ESI-MS.13
ported in part by
a Grant-in-aid for Scientific Research in
Innovative Areas 23110003, MEXT, Japan.

J. Tamura et al. / Tetrahedron Letters 54 (2013) 3940–3943
3943
Scheme 3. Coupling of the fucosyl residue and the conversion to the target compound. Reagents and conditions: (a) AllBr, NaH, DMSO, 89%; (b) Ac2O, AcOH, TFA, 0 °C–rt, 63%;
(c) DAST, CH2Cl2, À40 °C rt, 85%.; (d) LiClO4, CsF, CH2Cl2, À40 °C–rt, 63%; (e) [Ir(COD)(PPh2Me)2]PF6, THF, then CSA, CH2Cl2–MeOH, 71%; (f) CSA, CH2Cl2–MeOH, 54%; (g)
SO3ÁNMe3, DMF, 60 °C, quant; (h) Pd/C, H2, aq EtOH; (i) Ac2O, Et3N, aq EtOH; (j) aq NaOH, 29% (three steps).
9. Myers, A. G.; Gin, D. Y.; Rogers, D. H. J. Am. Chem. Soc. 1994, 116, 4697.
10. Tamura, J.; Neumann, K. W.; Ogawa, T. Liebigs Ann. 1996, 1239.
References and notes
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Carbohydr. Res. 1991, 211, 179.
T.; Santiago, L.; Mills, E. M.; Wang, Y.; Symes, A.; Geller, H. M. J. Cancer Sci. 2008,
12. Böhm, G.; Waldmann, H. Tetrahedron Lett. 1995, 36, 3843.
121, 3083.
13. 1H NMR assignments were confirmed by two-dimensional HH COSY
2. Mikami, T.; Yasunaga, D.; Kitagawa, H. J. Biol. Chem. 2009, 284, 4494.
experiments using BRUKER ADVANCE II 600 MHz spectrometers with tert-
3. (a) Yoshida, K.; Minami, Y.; Nemoto, H.; Numata, K.; Yamanaka, E. Tetrahedron
BuOH as an internal standard (1.23 ppm) in D2O. As an example of signal
Lett. 1992, 33, 4959; (b) Mourão, P. A. S.; Pereira, M. S.; Pavão, M. S. G.; Mulloy,
assignments, 1III stands for a proton at C-1 of sugar residue III. Positions of the
B.; Tollefsen, D. M.; Mowinckel, M.-C.; Abildgaard, U. J. Biol. Chem. 1996, 271,
protons in the aglycon are depicted in Scheme 1. Compound 1: [
a
]
À36 (c
D
23973; (c) Fonseca, R. J. C.; Oliveira, S.-N. M. C. G.; Pomin, V. H.; Mecawi, A. S.;
Araujo, I. G.; Mourão, P. A. S. Thromb. Haemost. 2010, 103, 994.
4. (a) Vieira, R. P.; Mourão, P. A. S. J. Biol. Chem. 1988, 263, 18176; (b) Kariya, Y.;
Watabe, S.; Hashimoto, K.; Yoshida, K. J. Biol. Chem. 1990, 265, 5081; (c) Kariya,
Y.; Watabe, S.; Kyogashima, M.; Ishihara, M.; Ishii, T. Carbohydr. Res. 1997, 297,
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829.
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0.29, H2O); 1H NMR: d 7.09–7.07 (m, 2H, Ph), 6.97–6.95 (m, 2H, Ph), 5.69 (d, 1H,
J1,2 = 3.54 Hz, H-1III), 5.07 (s, 1H, H-4III), 5.05 (m, 1H, H-5III), 5.02 (d, 1H,
J1,2 = 7.26 Hz, H-1I), 4.72 (d, 1H, J3,4 = 2.52 Hz, H-4II), 4.55 (d, 1H, J1,2 = 8.34 Hz,
H-1II), 4.50 (dd, 1H, J2,3 = 10.08 Hz, H-2III), 4.29 (dd, 1H, J5,6a = 4.50 Hz,
Jgem = 10.68 Hz, H-6IIa), 4.20 (dd, 1H, J5,6b = 7.44 Hz, H-6IIb), 4.13 (bd, 1H,
J = 9.90 Hz, H-3III), 4.08 (bt, 1H, J = 8.28 Hz, H-4I), 4.04 (d, 1H, J4,5 = 9.12 Hz, H-
5I), 4.00 (bt, 1H, J = 6.12 Hz, H-5II), 3.94 (dd, 1H, J2,3 = 10.86 Hz, H-2II), 3.90 (m,
2H, H-2I, 3I), 3.83 (dd, 1H, H-3II), 3.80 (s, 3H, OMe), 2.02 (s, 3H, NAc), 1.37 (d,
1H, J5,6 = 6.48 Hz, H-6III). ESI-MS: m/z calcd for C27H34NO29S4Na6, 1101.95;
found, 1101.94 [M+Na+].