Synthesis of the glycan moiety of ganglioside HPG-7 with an unusual trimer of sialic acid as the inner sugar residue

Article abstract of DOI:10.1039/c1cc13200h

The glycan moiety of ganglioside HPG-7, isolated from the sea cucumber (Holothuria pervicax), was synthesized for the first time. The characteristic substructure, a trisialic acid sequence embedded in the glycan, was deliberately constructed by utilizing

Full text of DOI:10.1039/c1cc13200h

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COMMUNICATION
Synthesis of the glycan moiety of ganglioside HPG-7 with an unusual
trimer of sialic acid as the inner sugar residuewzy
Yuki Iwayama,ab Hiromune Ando,*ab Hide-Nori Tanaka,ab Hideharu Ishidaa and
Makoto Kiso*ab
Received 31st May 2011, Accepted 11th July 2011
DOI: 10.1039/c1cc13200h
The glycan moiety of ganglioside HPG-7, isolated from the sea
cucumber (Holothuria pervicax), was synthesized for the first
time. The characteristic substructure, a trisialic acid sequence
embedded in the glycan, was deliberately constructed by utilizing
suitably differentiated sialyl units for various synthetic purposes.
Finally, a pentasaccharide was successfully delivered as the
hexyl glycoside.
comparable potency to that of other echinodermatous
gangliosides.1,2 Therefore, HPG-7, as well as other echino-
dermatous gangliosides, is considered a potential lead compound
for the development of carbohydrate-based therapeutics for
the treatment of neural disorders. However, structure–activity
relationships and the molecular basis underlying the neurite
outgrowth potentiated by gangliosides have not been elucidated
due to the lack of availability of homogenous gangliosides. To
address this issue, Hanashima et al. have achieved the synthesis
of the pentasaccharide of echinodermatous ganglioside AG-2,
which has an inner sialic acid residue within its glycan moiety,
and detailed the binding of the synthesized AG-2 glycan to
Siglec-2 by NMR spectroscopic analysis.3 On the other hand,
we have achieved the total synthesis of the echinodermatous
disialyl gangliosides HLG-24 and LLG-35 by developing new
methods for combining sialic acids through various linkages,
and the neurogenic activity of the synthetic LLG-3 was
demonstrated for the first time.5 Herein, as a new synthetic
challenge in our research group with ultimate aim of elucidating
the neurogenic activity of gangliosides, we report the synthesis
of the glycan moiety of HPG-7; the synthesis, based on
deliberate utilization of sialic acid chemistry, provides access
to the complex echinodermatous ganglioside.
Ganglioside HPG-7 1 was isolated from the sea cucumber,
Holothuria pervicax, together with a number of other new
ganglioside species.1 Detailed analysis of its structure revealed
an unusual trisialic acid residue embedded within the glycan
moiety that exhibited different inter-residual linkages between
the sialic acids (Scheme 1). To date, such an inner trisialic acid
residue has not been seen in either mammalian gangliosides or
other echinodermatous ganglioside species. In addition to this
rare structure, it remains of great interest that HPG-7 showed
neurogenic activity toward rat adrenal pheochromocytoma
(PC-12) cells in the presence of nerve growth factor with
Retrosynthetically, the pentasaccharide of HPG-7 was
fragmented into a terminal fucosyl-a(1,4)sialyl glycolic acid 2
and an inner amino-disialyl glucoside, and the inner segment
was further disconnected at the a(2,4)-glycosidic linkages
between sialic acid residues, according to the previous synthesis
of HLG-2 by our research group,4 and thus provided
a C5-modified sialyl donor 3 and a 1,5-lactamized sialyl
acceptor 4, which was suitable for the sialylation of the
inherently less reactive C4 hydroxyl group of sialic acid.
Scheme 1 Outline of retrosynthetic analysis of HPG glycan.
Scheme
2 shows the synthesis of the inner amino-
trisaccharide 9. By following the reported procedure,4b the
1,5-lactamized sialyl glucoside 5 was synthesized via a coupling
reaction between the N-TFAc sialyl donor6 and a suitably
protected glucose acceptor4b and lactamization under basic
conditions. The C9 hydroxyl group of sialic acid was then
selectively protected in the form of a bulky pivaloate ester to
afford 1,5-lactamized sialyl glucosyl acceptor 6 in quantitative
yield. To achieve amide formation between disaccharyl
carboxylic acid and amino-disialyl glucose with high efficiency
in the endgame of the synthesis, we designed a highly hydroxylated
a Department of Applied Bioorganic Chemistry, Gifu University,
1-1 Yanagido, Gifu-shi, Gifu 501-1193, Japan.
E-mail: hando@gifu-u.ac.jp; Fax: +81-58-293-3452;
Tel: +81-58-293-3452
b Institute for Integrated Cell-Material Sciences (WPI program),
Kyoto University, Yoshida Ushinomiya-cho, Sakyo-ku,
Kyoto 606-8501, Japan
w This article is part of the ‘‘Glycochemistry and glycobiology’’
web-theme issue for ChemComm.
z Part 154 of the series: Synthetic studies on sialoglycoconjugates.
y Electronic supplementary information (ESI) available. See DOI:
10.1039/c1cc13200h
c
9726 Chem. Commun., 2011, 47, 9726–9728
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Scheme 3 Synthesis of non-reducing end disaccharide unit 16.
(a) MsOH, MeOH, reflux, 36 h; (b) TrocOSu, NaHCO3/1,4-dioxane,
H2O, RT, 1 h; (c) DMP, CSA/MeCN, 15 min, 65% (3 steps);
(d) CAc2O, DMAP, K2CO3/CH2Cl2, ꢀ50 1C, 3 h, 96%; (e) Ac2O,
DMAP/THF, RT, 3.5 h, 99%; (f) 12, NIS, TfOH, MS3A/EtCN, ꢀ60 1C,
2 h, 93% (a : b = 58 : 35); (g) (NH2)2ꢁAcOH/THF–DMF (1 : 1),
RT, 3.5 h, 92%; (h) 14, NIS, TfOH, MS4A/tBuOMe-CH2Cl2 (1 : 1),
ꢀ40 1C, 2 h, 76%; (i) DDQ/ CH2Cl2–H2O (10 : 1), RT, 4 h, 95%;
(j) 80% aq. AcOH, 50 1C, 16 h; (k) Ac2O, DMAP/Py, RT, 1.5 h, 97%
(2 steps); (l) Zn, AcOH, RT, 4.5 h; (m) Ac2O, DMAP/Py, 2.5 h, 81%
(2 steps); (n) H2, Pd(OH)2–C/EtOAc, RT, 5 h, 99%.
derivative, which was subsequently converted into the
8,9-acetonide form. In the following step, to differentiate the
C4 and C7 hydroxyl groups, the group at C4 was selectively
protected with the chloroacetyl (CAc) group, while the other
group was acetylated, thereby yielding sialyl donor 11. Selective
deprotection of the CAc group that followed the glycosidation
with benzyl glycolate 12 furnished sialyl acceptor 13 for the
next fucosylation. As expected, fucosylation of 13 with fucosyl
donor 1412 successfully produced a Fuca(1,4)Neu sequence in
a complete stereoselective manner, aided by the synergic
solvent effect of tBuOMe-CH2Cl213 (76%). Next, the disaccharide
was converted into the fully acetylated derivative 15, the benzyl
group of which was cleaved, providing carboxylic unit 16.
As depicted in Scheme 4, we advanced the obtained 9 and 16
to the assembly of the HPG-7 glycan. Reduction of the
azide group of 9 was subsequently followed by dehydrative
condensation with 16, mediated by HBTU and HOBt, and
acetylation successfully yielded pentasaccharide 17 in 85%
(3 steps). Manipulation of the protecting groups of penta-
saccharide 17 (replacement of the MBn group with a benzoyl
group, conversion of carbamate to amide and introduction of
Scheme 2 Synthesis of trisaccharide unit 9. (a) PivCl/Py-CH2Cl2
(1 : 4), ꢀ40 1C, 30 min, quant.; (b) 7, NIS, TfOH, MS3A/EtCN,
ꢀ40 1C, 1 h, 50%; (c) Ac2O, DMAP/Py, RT, 2 h; (d) (NH2)2ꢁAcOH/
THF, RT, 12 h, 99% (2 steps); (e) Boc2O, DMAP/MeCN, RT, 20 min,
99%; (f) NaOMe, MeOH, RT, 2.5 h, 83%.
trisaccharide 9 as the precursor of the amino coupling partner
in order to prevent O to N acetyl migration and minimize
steric hampering during the coupling reaction. Next, C5-azido
sialyl thioglycoside derivative 7, which was developed by
Wong’s research group,7 was chosen as a terminal sialyl unit
to take advantage of the stability of the azide group under
reaction conditions for the conversion of trisaccharide into the
corresponding polyol derivative. Compound 6 was glycosylated
with the sialyl donor 7, promoted by NIS-Tf OH in EtCN8 at
ꢀ40 1C, to afford trisaccharide 8 in 50% yield together with
a complex mixture of byproducts, which included mainly
b-isomer with a slight amount of trisaccharide regioisomers
(20% total yield). The anomeric configuration of the newly
formed sialyl linkage was determined by the coupling constant
between C1 and H-3ax9 which was calculated as 6.2 Hz.
Thereafter, a Boc group was introduced to the lactam moiety,
and the amide bond was cleaved by the methoxide anion,
thereby furnishing compound 9 in high yield (81% from 8 over
4 steps).
a
trichloroacetimidoyl group at the anomeric hydroxyl
group)14 consequently yielded the pentasaccharide donor 18,
which is expected to function as a glycosyl donor for coupling
with ceramide in the total synthesis of HPG-7 in the near
future. In this study, the glycosyl donor 18 was reacted with
n-hexanol and catalyzed by TMSOTf to afford glycoside in
64% yield, which was finally deprotected to deliver the glycan
moiety of HPG-7 (19) for the first time.
The synthesis of carboxylic acid unit 16 commenced with
sialyl glycolate acceptor 13 (Scheme 3). Because the C4
hydroxyl group of N-acetyl sialic acid is less reactive as well
as the C8 hydroxyl group, the acetyl group at N5 was
In conclusion, the pentasaccharide of ganglioside HPG-7
was synthesized in a convergent and stereoselective manner.
The characteristic substructure, the trisialic acid sequence
embedded in the glycan, was achieved by exploiting the
suitably protected sialyl units 6, 7 and 9 for various synthetic
substituted with
a 2,2,2-trichloroethoxycarbamoyl (Troc)
group to ameliorate the reactivity of the adjacent C4 hydroxyl
group, according to the report by Takahashi et al.10 Subsequent
treatment of the known N-Ac sialyl thioglycoside 10
with MsOH in MeOH11 and TrocOSu produced an N-Troc
c
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Chem. Commun., 2011, 47, 9726–9728 9727

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Notes and references
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Scheme 4 Synthesis of the glycan part of HPG-7 19. (a) PPh3/
THF–H2O (98 : 2), 40 1C, 6 h; (b) 16, HBTU, HOBt, DIEA, RT,
10 h; (c) Ac2O, DMAP/Py, RT, 4 h, 85% (3 steps); (d) DDQ/
CH2Cl2–H2O (20 : 1), RT, 3.5 h; (e) Bz2O, DMAP/Py, RT, 15 h,
67% (2 steps); (f) TFA/CH2Cl2, 0 1C, 3 h; (g) Ac2O, DMAP/Py, RT, 1 h;
(h) (NH2)2ꢁAcOH/DMF, RT, 1 h; (i) CCl3CN, DBU/CH2Cl2, 0 1C,
45 min, 46% (4 steps); (j) n-hexanol, TMSOTf, MS4A (AW-300)/
CH2Cl2, 0 1C, 1 h, 64%; (k) H2, Pd(OH)2–C/EtOH, RT, 3.5 h; (l) LiCl/
Py, reflux, 14 h; (m) 0.1 M aq. NaOH, RT, 4 h, 95% (3 steps).
10 H. Tanaka, Y. Nishiura, M. Adachi and T. Takahashi, Hetero-
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purposes. Our research group is currently investigating the
neurogenic activity of 19 together with synthesized echinoderm
gangliosides. Total synthesis of HPG-7 is also in progress, and
results will be reported in due course.
11 T. Sugata and R. Higuchi, Tetrahedron Lett., 1996, 37, 2613–2614.
12 The synthesis of compound 14 is detailed in the ESIy.
13 A. Ishiwata, Y. Munemura and Y. Ito, Tetrahedron, 2008, 64,
92–102.
This work was financially supported in part by MEXT of
Japan (WPI program and a Grant-in-Aid for Scientific
Research (S) No.1701007 and (B) No. 22380067 to M.K.,
and a Grant-in-Aid for Young Scientists (A) No. 23688014 to
H.A.). We thank Ms Kiyoko Ito for providing technical
assistance.
14 (a) R. R. Schmidt, Angew. Chem., Int. Ed., 1986, 25, 212–235;
(b) R. R. Schmidt and J. Michel, Angew. Chem., Int. Ed., 1980, 19,
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15 For part 153 of the series Synthetic studies on sialoglycoconjugates,
see:K. Fujikawa, S. Nakashima, M. Konishi, T. Fuse, N. Komura,
T. Ando, H. Ando, N. Yuki, H. Ishida and M. Kiso, Chem.–Eur. J.,
2011, 17, 5641–5651.
c
9728 Chem. Commun., 2011, 47, 9726–9728
This journal is The Royal Society of Chemistry 2011