Synthesis of the starfish ganglioside AG2 pentasaccharide

Article abstract of DOI:10.1016/j.tetlet.2009.08.071

This Letter reports the first synthesis of the AG2 pentasaccharide, using silylene-oxazolidinone double-locked sialic acid building blocks. The di-DTBS-protected sialic acid building block was easily prepared and readily activated with NIS and TfOH to pro

Full text of DOI:10.1016/j.tetlet.2009.08.071

Tetrahedron Letters 50 (2009) 6150–6153
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Synthesis of the starfish ganglioside AG2 pentasaccharide
a
b
c
Shinya Hanashima a, , Yoshiki Yamaguchi , Yukishige Ito , Ken-ichi Sato
*
a Structural Glycobiology Team, RIKEN Advanced Science Institute, Hirosawa 2-1, Wako-shi, Saitama 351-0198, Japan
b Synthetic Cellular Chemistry, RIKEN Advanced Science Institute, Hirosawa 2-1, Wako-shi, Saitama 351-0198, Japan
c Material and Life Chemistry, Faculty of Engineering, Kanagawa University, Rokkakubashi 3-27-1, Yokohama 221-8686, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 2 July 2009
Revised 20 August 2009
Accepted 21 August 2009
Available online 26 August 2009
This Letter reports the first synthesis of the AG2 pentasaccharide, using silylene-oxazolidinone double-
locked sialic acid building blocks. The di-DTBS-protected sialic acid building block was easily prepared
and readily activated with NIS and TfOH to provide the sialylated lactose unit in good yield with moder-
ate selectivity. After obtaining the trisaccharide unit, the oxazolidinone-protected C4–OH on the sialic
acid residue was readily deprotected by treatment with NaOMe. Coupling with the galactofurano-
sylb(1-3)galactopyranosyl fluoride building block produced the desired AG2 pentasaccharide in a highly
stereoselective manner. Finally, the desired AG2 pentasaccharide was obtained in good yield following
global deprotection.
Keywords:
Gangliosides
Sialic acid
Glycosylation
Acanthaster planci
Ó 2009 Elsevier Ltd. All rights reserved.
The structural diversity of sialyl-glycans on glycoproteins and
glycolipids is important because of their several functions in bio-
logical systems.1,2 Mammalian gangliosides mainly contain N-acet-
Recently, several echinoderm gangliosides involved in HLG-2
(isolated from sea cucumber Holothuria leucospilota; -Neu5Ac-
(2-4)- -Neu5Ac-b-(2-6)-Glc-R) and LLG-3 (isolated from starfish
Linckia laevigata; 8-O-Me- -Neu5Ac-(2-11)- -Neu5Gc-(2-3)-b-
a
a
ylneuraminic acid or N-glycolylneuraminic acid with
a(2-3/6)
a
a
binding modes on a penultimate galactose, glucosamine, or galac-
tosamine residue. Such gangliosides are involved in cell adhesion
and recognition, and in signal transduction through lipid rafts
and caveolae.3,4 The biosynthetic pathways of mammalian ganglio-
sides are currently being elucidated;5 the oligosaccharide chains of
these gangliosides are terminated with sialic acid residue(s).
Concurrent with these developments, unique echinoderm gan-
gliosides with potent mammalian nerve cell stimulating activity
have been identified from starfish and sea cucumbers.6,7 One of
these gangliosides, AG2 (isolated from the starfish Acanthaster
planci), has an unusual glycan sequence involving an inner sialic
acid residue with a galactofuranose capping moiety (Scheme 1).6a
In addition to this unique structure, AG2 exhibits potent nerve cell
stimulating activity toward the mouse PC-12 cell line that is com-
parable in magnitude to that of the mammalian GM1 ganglioside.7b
The GM1 receptor for nerve cell stimulation/suppression signals is
the myelin-associated glycoprotein, MAG/Siglec-4.8
Gal(1-4)-b-Glc-R) have been synthesized.11 These specific ganglio-
sides are of particular interest due to their unique sialyl-oligosac-
charide structures and potent nerve cell stimulating activity.11,12
In order to elucidate the nerve cell stimulation mechanism and
to investigate other biological functions of the unique AG2 struc-
ture, we synthesized the pentasaccharide part of AG2 1. The syn-
thesis of the AG2 oligosaccharide presented two problems,
namely, the introduction of a sialic acid residue onto the lactose
unit, and the further glycosylation of the sialic acid C4–OH with
the galactofuranosylb(1-3)galactopyranoside unit. Based on the
synthesis of HLG-2 and HPG-7 (isolated from sea cucumber Holoth-
uria pervicax;
a-Fuc-(1-4)-a-Neu5Ac-(2-11)-a-Neu5Gc(2-4)-a-
Neu5Ac-(2-6)-b-Glc-R) gangliosides, the observed low efficiency
of glycosylation of the sialic acid C4–OH resulted from low reactiv-
ity and the flexible side chain.11a,12 To overcome these issues, 1,5-
lactamized sialic acid acceptors have been developed for exposing
the C4–OH.12
The galactofuranose residue on AG2’s non-reducing terminus is
widespread throughout bacteria, protozoa, and marine organisms.9
However, galactofuranosylated glycans are not found in humans.
Recently, it has been reported that such galactofuranosylated gly-
cans on infectious organisms are recognized by human intelectin,
which is involved in immune defense and inflammation.10
Previously, we developed a novel sialic acid building block 5 for
efficient sialylation reactions. Compound 5 carries an oxazolidi-
none and di-tert-butylsilylene (DTBS) double-lock on the central
pyranose ring.13,14 We employed this building block for the synthe-
sis of AG2 because the 5,7-N,O-DTBS ring would expose the C4–OH
of the sialic acid, enhancing the reactivity of the hydroxy group.
Scheme 1 shows the plan for the synthesis of AG2 pentasaccharide
1. The galactofuranosylb(1-3)galactopyranose unit 2 on the non-
* Corresponding author. Tel.: +81 48 467 9619; fax: +81 48 467 9620.
E-mail address: shanashima@riken.jp (S. Hanashima).
reducing terminus and sialyla(2-3)lactose trisaccharide 3 were
0040-4039/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2009.08.071

S. Hanashima et al. / Tetrahedron Letters 50 (2009) 6150–6153
HO
6151
OH
COOH
O
OH
HO
HO
AcHN
O
O
O
OR
HO
O
OH
O
OH
HO
OH
O
OH
O
O
OH
AG2: R = ceramide
1: R = SE
OH
OH
OH
HO
t
Bu
O
Si
t
Bu
t
Bu
O
H
Si
O
t
Bu
O
COOMe
OBn
O
OBn
O
O
O
O
O
O
OSE
+
AcO
BnO
O
t
Bu Si
BnO
O
F
OBn
N
OBn
OH
t
Bu
O
OH
2
OMe
OAc
OAc
t
Bu
3
AcO
O
Si
t
Bu
O
O
COOMe
SPh
H
OBn
BnO
BnO
O
O
HO
O
OSE
+
O
t
Bu Si
OBn
N
O
OBn
OH
t
Bu
O
5
4
Scheme 1. Structure and synthetic scheme for starfish ganglioside AG2 pentasaccharide 1. SE = 2-(trimethylsilyl)ethyl.
coupled at the final glycosylation stage. Glycosylation of the rela-
selectivity (
a
:b = 51:32). The generated 6
a
and 6b were readily
tively exposed hydroxy group on 3 proceeded in good yield in a
separated by silica gel column chromatography (6a:
highly
a-selective manner due to 4,6-O-DTBS protection of the gal-
3JC1,H3 = 5.1 Hz). A single-step transformation with NaOMe pro-
vided the desired trisaccharide acceptor 3. Specifically, treatment
actose moiety on 2.15 The bulky t-butyl groups on the DTBS moiety
of 2 inhibited glycosylation from the b-face. The sialic acid building
block 5 was employed to introduce the sialic acid unit to the disac-
charide 4.16 The sialylation reaction resulted in regio-selective
opening of the oxazolidinone-ring, which liberated the C4–OH on
the sialic acid unit to yield 3. The acceptor hydroxy group of 3
was then sufficiently exposed to undergo further coupling with
disaccharide 2, since the DTBS-protecting group still fixes the side
chain.
of 6a with NaOMe smoothly provided lactone intermediate 7, then
the oxazolidinone moiety as well as the lactone moiety on 7 were
opened in a regio-selective manner to give 3 in 87% yield.14
Then, we synthesized galactofuranosylb(1-3)galactopyranose
unit 2, which represents the other half of the AG2 pentasaccharide
(Scheme 3). First, galactose triol 818 was treated with tBu2Si(OTf)2
in pyridine to produce 4,6-O-DTBS-protected galactose acceptor 9
in 84% yield. The subsequent glycosylation reaction between com-
The sialyla(2-3)lactose trisaccharide unit 3, which represents
pound
9
(1.0 equiv) and phenylthiogalactofuranoside 1019
half the AG2 molecule, was efficiently synthesized as depicted in
Scheme 2. First, the sialyllactose unit 3 was formed using the
highly reactive silylene-oxazolidinone double-locked sialic acid
building block 5.13 The sialylation reaction of lactose acceptor 4
with 5, activated with N-iodosuccinimide(NIS) and TfOH at
(1.2 equiv) in the presence of NIS and TfOH at 0 °C produced disac-
charide 11 in 96% yield as a single isomer. Next, oxidative removal
of the 4-methoxyphenyl functionality on the galactose residue of
11 using cerium ammonium nitrate (CAN) produced hemiacetal
12 in 55% yield. For further glycosylation reactions, disaccharide
12 was transformed into highly reactive trichloroacetimidate 13
À40 °C,17 produced trisaccharide 6 in 83% yield with moderate
a-
t
Bu
t
Bu
Si
NIS, TfOH
MS 4A
O Si
t
Bu
O
O
t
Bu
OBn
NaOMe,
MeOH
O
CH2Cl2
COOMe
O
H
OBn
O
O
O
O
OBn
O
O
O
4
+
5
H
O
O
3
O
O
−40 o
C
OSE
O
BnO
O
87%
t
Bu Si
OBn
t
Bu
N
O
OBn
Si
OH
t
Bu
N
O
OBn
t
Bu
+ β-anomer
6: 83% (α:β = 51:32)
Scheme 2. Synthesis of the trisaccharide acceptor 3.
O
7

6152
S. Hanashima et al. / Tetrahedron Letters 50 (2009) 6150–6153
t
Bu
Si
O
OH
t
Bu
t
Bu
SPh
O
HO
t
Bu Si(OTf)
2 2
O
NIS, TfOH (0.2 eq.)
Si
O
O
OAc
O
+
t
Bu
OMP
OMP
O
O
HO
MS 4A, CH Cl
2
2
pyridine, 84%
OBn
OBn
O
OAc
o
O
OAc
0
C, 96%
OAc
OAc
OMP
8
AcO
HO
OBn
9: 1.0 eq.
11
OAc
10: 1.2 eq.
AcO
t
Bu
t
Bu
Si
Si
O
O
CAN
toluene/CH CN/H O, 55%
t
Bu
O
t
Bu
CCl CN, Cs CO , CH Cl
O
3
2
3
2
2
3
2
or DAST, CH Cl
O
2
2
O
OH
O
O
R
BnO
O
OBn
O
OAc
OAc
OAc
OAc
13: R = CCl C(=NH)O (α), 71%
3
2: R = F (α:β = 58/42), 85%
12
OAc
OAc
AcO
AcO
Scheme 3. Synthesis of galactofuranosyl galactose donors 2 and 13. MP = 4-methoxyphenyl.
with CCl3CN and Cs2CO3 (71% yield), and into fluoride 2 with N,N-
diethylaminosulfur trifluoride (DAST, 85% yield; :b = 58:42). We
expected that the bulky 4,6-O-DTBS protection on the galactose
reactive glycosylfluoride 2. Upon activation of fluoride 2 with
Cp2HfCl2 and AgOTf at 0 °C,21 the desired AG2 pentasaccharide
a
14 was readily obtained in 83% yield with predominant
a-
moiety would favor
a
-selectivity due to the ‘DTBS effect’ in subse-
selectivity.
quent assembly with the sialyllactose trisaccharide.15a
To obtain fully deprotected AG2 pentasaccharide 1, a global
deprotection process was conducted. First, three silylene-protect-
ing groups on pentasaccharide 14 were simultaneously removed
by treatment with TBAF and AcOH in THF at 60 °C to give lactone
15 in 91% yield. Next, the N-methoxycarbonyl group on 15 was
hydrolyzed by 1.0 M aqueous LiOH with 1,4-dioxane at room tem-
perature, then N-acetylation with Ac2O in MeOH produced com-
pound 16 in 97% yield (2 steps). During the treatment with base,
the lactone moiety of 15 was simultaneously hydrolyzed to
To obtain fully protected AG2 pentasaccharide 14, coupling
reactions between trisaccharide acceptor 3 and disaccharide build-
ing blocks 2 and 13 were performed as shown in Scheme 4.
Initially, we employed the highly reactive Schmidt-type trichlo-
roacetimidate 13.20 Although imidate 13 (2 equiv) was readily
activated with a catalytic amount of TMSOTf at À40 °C, the desired
pentasaccharide 14 was obtained in an unsatisfactory 33% yield.
On the other hand, the best yield was obtained with the less
t
Bu
O
Si
t
Bu
13, TMSOTf, MS 4A,
O
CH2Cl2, −40 oC, 33%
COOMe
O
H
OBn
O
or
O
OBn
O
O
2, Cp2HfCl2, AgOTf, MS 4A,
CH2Cl2, 0 oC to r.t., 83%
O
TBAF, AcOH
THF, 60 oC
OSE
BnO
t
Bu Si
N
AcO
OBn
OBn
OH
t
Bu
OAc
3
O
OAc
MeO
O
BnO
91%
O
OAc
14
O
O
O
O
t
Bu Si
Bu
OH
OBn
t
OH
O
O
O
O
H
O
OBn
HO
OH
HO
O
O
COOH
OSE
BnO
OR
O
OR
O
HN
OBn
OBn
HO
HO
AcHN
O
O
OH
O
O
AcO
O
OSE
OH
RO
15
OAc
O
MeO
O
O
OAc
OR
OR
OH
1) 1.0 M LiOH, 1,4-dioxane, then AcOH
2) 10% Ac2O in CH3CN, MeOH
RO
O
O
OH
BnO
O
OAc
O
HO
OH
HO
OH
16: R = Bn, 97% (2 steps)
1: R = H, 97%
H2, 10% Pd-C
MeOH, H2O
Scheme 4. 2 + 3 coupling and deprotection for the synthesis of the AG2 pentasaccharide 1.

S. Hanashima et al. / Tetrahedron Letters 50 (2009) 6150–6153
6153
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Acknowledgments
We thank Dr. S. Akai (Kanagawa University) for helpful discus-
sions. This work was supported by the Science Frontier Project of
Kanagawa University and by Grants-in-Aid for Scientific Research
for Young Scientists B (No. 20710171 to S.H.) from the Japan Soci-
ety for the Promotion of Science. Global COE program of Osaka Uni-
versity from the Ministry of Education, Culture, Sports, Science, and
Technology of Japan supported this work, in part.
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Supplementary data
Synthetic procedures, spectroscopic data, and 1H and 13C NMR
spectra of compounds 1–3, 9, 11–16. Supplementary data associ-
ated with this article can be found, in the online version, at
doi:10.1016/j.tetlet.2009.08.071.
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