First synthesis of a pentasaccharide moiety of ganglioside GAA-7 containing unusually modified sialic acids through the use of N -Troc-sialic acid derivative as a key unit

Article abstract of DOI:10.1021/ol303122w

The pentasaccharide part of the potent neuritogenic ganglioside GAA-7 has been synthesized for the first time. The unique branched terminus constituting partially modified sialic acids and N-acetylgalactosamine was successfully established by stereoselective double-sialylation using 8-O-methyl-N-Troc-sialic acid as a donor. The final 4 + 1 coupling reaction provided a high yield of pentasaccharide, which was deprotected to deliver the target molecule.

Full text of DOI:10.1021/ol303122w

ORGANIC
LETTERS
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Vol. XX, No. XX
000–000
First Synthesis of a Pentasaccharide
Moiety of Ganglioside GAA‑7 Containing
Unusually Modified Sialic Acids through
the Use of N‑Troc-sialic Acid Derivative
as a Key Unit†
Hideki Tamai,‡,§ Hiromune Ando,*,‡,§ Hideharu Ishida,‡ and Makoto Kiso*,‡,§
Department of Applied Bioorganic Chemistry, Gifu University, 1-1 Yanagido, Gifu-shi,
Gifu 501-1193, Japan, and Institute for Integrated Cell-Material Sciences (WPI-iCeMS),
Kyoto University, Yoshida Ushinomiya-cho, Sakyo-ku, Kyoto 606-8501, Japan
hando@gifu-u.ac.jp; kiso@gifu-u.ac.jp
Received November 12, 2012
ABSTRACT
The pentasaccharide part of the potent neuritogenic ganglioside GAA-7 has been synthesized for the first time. The unique branched terminus constituting
partially modified sialic acids and N-acetylgalactosamine was successfully established by stereoselective double-sialylation using 8-O-methyl-N-Troc-
sialic acid as a donor. The final 4 þ 1 coupling reaction provided a high yield of pentasaccharide, which was deprotected to deliver the target molecule.
It is well-documented that gangliosides produced in
echinoderms (e.g., starfish and sea cucumber) show neuri-
togenic activity.2 Since the activity of some echinoderma-
tous gangliosides exceeds that of mammalian ganglioside
GM1, which has been used in developing a drug for
treating Alzheimer’s disease,3 echinoderms are considered
an important source of bioactive gangliosides that are
promising drug leads for combating neuronal disorders.
According to results from the evaluation of neuritogenic
activity toward PC-12 cells, four kinds of gangliosides
(SJG-2,4 LLG-5,5a LLG-3,5b and GAA-76) showed compara-
ble and highly potent activity.2 However, the mechanism
by which these gangliosides exert neuritogenic activity has
not been elucidated, mainly due to the lack of available
homogeneous gangliosides. The structures of echinoder-
matous gangliosides are substantially different from those
of mammalian gangliosides in that they bear partially
modified sialic acid residue(s) and/or repeated sequences
of sialic acid not found in mammals. Therefore, such
characteristic diversity in sialic acid structure is considered
to be correlated with the potent neuritogenic activity of
echinodermatous gangliosides. Our research group has
† Synthetic Studies on Sialoglycoconjugates. 158. For part 157, see ref 1.
‡ Gifu University.
§ Kyoto University.
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r
10.1021/ol303122w
XXXX American Chemical Society

previously reported the first total syntheses of HLG-27 and
LLG-3,8 which contain special tandem units consisting of
partially modified sialic acid. With the ultimate aim of
elucidating the neuritogenic activity of gangliosides, we
herein report the synthesis of the glycan moiety of gan-
glioside GAA-7 (1), which was isolated from the starfish
Asterias amurensis versicolor Sladen6 (Scheme 1).
by widening the differences in their mobility on silica gel.
As shown in Scheme 2, the 8-O-methyl-N-Troc-sialyl donor
was designed as triacetate derivative 9 carrying a phe-
nylthio group at the anomeric position based on the struc-
ture of the previously reported N-Troc sialyl donor.9a
To access compound 9 in a short sequence, known
compound 39b was first converted into 9-O-chloroacetyl
(CAc) derivative 4 in very high yield (97%).10 Next, the
CAc group of compound 4 was selectively cleaved by the
action of 1-selenocarbamoylpiperidine 511 in DMF at
65 °C to afford 6, which further underwent O8 to O9
acetyl migration promoted by an acidic tertiary ammo-
nium salt with hydrogen chloride 7, predominantly pro-
ducing 8-OH derivative 8 in 90% yield. Finally, the
hydroxyl group at C8 could be methylated most efficiently
with Me3OBF4 in the presence of tri-tert-butylpyrimidine
(TTBP) to give the 8-O-methyl sialyl donor 9 in 76% yield,
whereas MeOTf gave a complex mixture of products.
Scheme 1. Structure of Target Molecule 2 and Outline of
Synthetic Strategy toward 2
Scheme 2. Synthesis of 8-Methyl-N-Troc Sialyl Donor 9
The glycan moiety of GAA-7 contains unusual sialic
acid residues, which are methylated at the C8 hydroxyl
group and glycolylated at the C5 amino group. Further-
more, the modified sialic acids are linked at the C3 and C6
hydroxyl groups of the galactosamine (GalN) residue
through R-glycosidic bonds, forming a unique branched
substructure at the terminus. This terminal branch is seen
in only GAA-7 among all sialic acid-containing glycans
identified. The terminal branch is a challenging class of
molecule to synthesize because the reactivity of the C3
hydroxyl group is decreased by the adjacent acetamide
group, making it difficult to establish an R-glycoside of
sialic acid at the C3 position of GalN residue. Even if
sialylation of the C3 and C6 hydroxyl groups were possi-
ble, the chromatographic separation of stereoisomers of
sialyl oligosaccharides would generally be an arduous
procedure due to the similar mobility of the isomers in
silica gels, which impedes the purification of the desired
product. Keeping this in mind, we envisaged the synthetic
strategy depicted in Scheme 1. Target molecule 2 was
fragmented into modified sialic acids, disaccharide
(GalNAcβ(1,3)Gal), and glucose.
To introduce sialic acids at the two hydroxyl groups of
GalNAc residue in an efficient and stereoselective manner,
we chose a 2,2,2-trichloroethoxycarbonyl (Troc) group for
orthogonal modification of the C5 amino group because it
can be selectively removed and it endows high reactivity
and stereoselectivity on the sialic acid donor.9 According
to the results reported in our previous paper,9a the use of a
Troc group was also expected to facilitate the chromato-
graphic separation of the anomers of sialylated products
To perform an efficient double-sialylation of GalN-Gal
acceptor, it is crucial to boost the reactivity of the C3
hydroxyl group of the GalNresiduesincea hydroxyl group
adjacent to an acetamide group is generally unreactive,
likely due to hydrogen bonding. Because sialyl donor 9 is
equipped with a Troc group at the C5 amino group, a TCA
group, which is transformable into an acetyl group under
conditions for reducing a Troc group (zinc in acetic acid),12
was first selected as an orthogonal protecting group for the
C2 amino group of the GalN residue. Furthermore, we aimed
to deduce a suitable protection mode for the three hydroxyl
groups of the GalN residue for double-sialylation by compar-
ing a triol acceptor (10) and a diol acceptor (11) (Figure 1).13
It was difficult to obtain the disialylated product in the
glycosylation of GalN-Gal acceptors 10 and 11 with sialyl
(10) Sakakura, A.; Kawajiri, K.; Ohkubo, T.; Kosugi, Y.; Ishihara,
K. J. Am. Chem. Soc. 2007, 129, 14775–14779.
(11) Sogabe, S.; Ando, H.; Koketsu, M.; Ishihara, H. Tetrahedron
(8) Tamai, H.; Ando, H.; Tanaka, H.-N.; Hosoda-Yabe, R.; Yabe,
T.; Ishida, H.; Kiso, M. Angew. Chem., Int. Ed. 2011, 50, 2330–2333.
(9) (a) Ando, H.; Koike, Y.; Ishida, H.; Kiso, M. Tetrahedron Lett.
2003, 44, 6883–6886. (b) Tanaka, H.; Adachi, M.; Takahashi, T.
Chem.;Eur. J. 2005, 11, 849–862. (c) Hanashima, S.; Seeberger, P. H.
Chem. Asian J. 2007, 2, 1447–1459.
Lett. 2006, 47, 6603–6606.
(12) Ueki, A.; Nakahara, Y.; Hojo, H.; Nakahara, Y. Tetrahedron
2007, 63, 2170–2181.
(13) For the experimental procedure for the synthesis of compounds
10, 11, 12, and 16, see the Supporting Information.
B
Org. Lett., Vol. XX, No. XX, XXXX

Table 1. Examination of Double Sialylation of GalN-Gal Ac-
ceptor
Figure 1. GalN-Gal acceptors used in this study.
donor 9, which was promoted by N-iodosuccinimide (NIS)
and trifluororomethanesulfonic acid (TfOH) in nitrile
solvent.14 The results of glycosylation are summarized in
Table 1. In the case of triol acceptor 10, doubly sialylated
products were generated as a complex mixture of tetra-
saccharides 13 including stereoisomers and their 100,
40-lactonated derivatives in about 25% yield, accompa-
nied by 60-sialyl GalN-Gal as an anomeric mixture (59%)
(entry 1). Glycosylation of 40-benzylated acceptor 11 with
sialyl donor 9 did not produce tetrasaccharide 14 at all
(entry 2), again giving an anomeric mixture of 60-sialyl
GalN-Gals as the main products in 90% yield (R/β=2.5/1).
In contrast, an alternative GalN-Gal acceptor 12 bearing
an azido group at the C2 position of the GalN residue
provided a good yield (59%) of disialylated product 15 as a
mixture of stereoisomers (entry 3). The mixture consisted
of the desired R(2,3)/R(2,6)-disialyl product (41%) and
other stereoisomers (18%), namely, β(2,3)/R(2,6)-disialyl
(7%), R(2,3)/β(2,6)-disialyl (9%), and β(2,3)/β(2,6)-disia-
lyl isomers (2%) (R(2,3)/β(2,3) = 5.5:1; R(2,6)/β(2,6) =
4.3:1). As expected, all isomers were clearly separated on
TLC with different Rf values (ΔRf = 0.1ꢀ0.4), which
facilitated chromatographic separation of each isomer.15
This result indicated an important technical advantage in
the use of the N-Troc-sialyl donor for accomplishing the
synthesis of highly sialylated glycans. The anomeric con-
figuration of the newly formed glycosides was determined
on the basis of previous reports16 by measuring the long-
range 3JC‑1, H‑3ax coupling constants.
a The ratio of R:β was calculated on the basis of the isolated yield
unless otherwise specified. b Yield of the mixture of stereoisomers and
their lactonated derivatives, which were inseparable. c Yield of the
inseparable mixture of stereoisomers. The ratio of R:β could not be
estimated from the 1H NMR spectrum because of its complexity.
whereas at least one coupling constant of the other pro-
ducts was less than 1.0 Hz. Although we examined double-
sialylation using the N-acetylglycolyl sialyl donor 16,13,17
the disialylated product was obtained in poor yield (10%)
as a mixture of anomers, which were hardly separated by
column chromatography, owing to the same mobility of
each anomer on the silica gel15 (entry 4).
Scheme 3 shows the final part of the synthesis of target
molecule 2. To combine the tetrasaccharide part with a
glucose residue, tetrasaccharide 15 (R-isomer) was con-
verted into a suitable glycosyl donor. First, we screened
various reaction conditions for transforming the azido
group into an acetamide group in the presence of the Troc
group ((i) TMSCl, AcCl;18 (ii) Lindler catalyst; (iii) mod-
ified Staudinger reactions19). For compound 15, Stauding-
er reaction using Me2PPh, HOOBt, and acetic anhydride
For the main product (R(2,6)/R(2,3)-disialyl GalN-Gal)
in entry 3, the coupling constants were 6.3 and 6.0 Hz,
(14) (a) Veeneman, G. H.; van Leeuwen, S. H.; van Boom, J. H.
Tetrahedron Lett. 1990, 31, 1331–1334. (b) Konradsson, P.; Udodong,
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(c) Hasegawa, A.; Nagahama, T.; Ohki, H.; Hotta, K.; Ishida, H.; Kiso,
M. J. Carbohydr. Chem. 1991, 10, 493–498.
(15) For a detailed TLC profile of compounds 15 and 17, see Figure 1
in the Supporting Information.
(16) (a) Haverkamp, J.; Spoormaker, T.; Dorland, L.; Vliegenthart,
J. F. G.; Schauer, R. J. Am. Chem. Soc. 1979, 101, 4851–4853. (b) Hori,
H.; Nakajima, T.; Nishida, Y.; Ohrui, H.; Meguro, H. Tetrahedron Lett.
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Thiem, J. Carbohydr. Res. 1991, 215, 345–349.
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(a) Hasegawa, A.; Uchimura, A.; Ishida, H.; Kiso, M. Biosci. Biotech.
Biochem. 1995, 59, 1091–1094. (b) Sugata, T.; Higuchi, R. Tetrahedron
Lett. 1996, 37, 2613–2614. (c) Sherman, A. A.; Yudina, O. N.; Shashkov,
A. S.; Menshov, V. M.; Nifantiev, N. E. Carbohydr. Res. 2002, 337, 451–
457. (d) Ikeda, K.; Miyamoto, K.; Sato, M. Tetrahedron Lett. 2007, 48,
7431–7435. (e) Schroven, A.; Meinke, S.; Ziegelmuller, P.; Thiem, J.
Chem.;Eur. J. 2007, 13, 9012–9021. (f) Crich, D.; Wu, B. Org. Lett.
2008, 10, 4033–4035. (g) Hanashima, S.; Tomiya, T.; Ishikawa, D.; Akai,
S.; Sato, K.-I. Carbohydr. Res. 2009, 344, 959–965.
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(19) (a) Saxon, E.; Armstrong, J. I.; Bertozzi, C. R. Org. Lett. 2000, 2,
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Org. Lett., Vol. XX, No. XX, XXXX
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derivative in 69% yield together with a 100,20-lactam deri-
vative (29%). Next, the two Troc groups were cleaved by
the action of zinc in acetic acid21 and the resulting amine
group was subsequently acylated with acetoxyacetyl chlo-
ride (77% over two steps). Prior to the acidic cleavage of the
2-(trimethylsilyl)ethyl (SE) group attheanomericposition,
the acid-labile benzylidene acetal was hydrolyzed with
80% aqueous acetic acid, followed by the acetylation of
hydroxyl groups. Then, the benzyl group at the C4 hydrox-
yl group of the GalN residue was replaced with an acetyl
group by a conventional reaction sequence. To provide a
trichloroacetimidate group at the anomeric position as a
leaving group,22 the SE group was cleaved by exposure to
TFA in CH2Cl2, and the resulting hydroxyl group was
reacted with CCl3CN and DBU23 in CH2Cl2 at 0 °C. This
reaction sequence produced the tetrasaccharyl donor 18 in
93% yield over two steps. To our delight, the final coupling
of the tetrasaccharyl donor 18 (1.0 equiv) and glucosyl
acceptor 1924 (2.0 equiv) in CH2Cl2, which was catalyzed
with TMSOTf, provided pentasaccharide 20 in 73% yield.
Finally, the synthesis of target molecule 2 was completed
by full deprotection of compound 20 by hydrogenolysis on
Pd(OH)2 and subsequent removal of the ester groups.
In conclusion, the glycan moiety of ganglioside GAA-7
(2) has been synthesized for the first time. Taking advan-
tage of the site-specific migration of an acetyl group in the
glycerol moiety of sialic acid, key 8-O-Me-N-Troc sialic
acid donor 9 was efficiently synthesized. The results of the
double-sialylation of GalN-Gal acceptors demonstrated
the efficacy of N-Troc type sialic acid donors in terms of
glycosylation efficiency and facilitation of the chromato-
graphic separation of anomeric isomers of sialylated pro-
ducts. The total synthesis of ganglioside GAA-7 using
tetrasaccharide donor 18 and a glucosyl ceramide is under-
way and the results will be reported in due course.
Scheme 3. Assembly of the Target Molecule 2
Acknowledgment. The iCeMS is supported by World
Premier International Research Center Initiative (WPI),
MEXT, Japan. This work was financially supported in
part by MEXT of Japan (a Grant-in-Aid for Scientific
Research (B) No. 22380067 to M.K. and a Grant-in-Aid
for Young Scientists (A) No. 23688014 and Grant-in-Aid
on Innovative Areas No. 24110505, Deciphering sugar
chain-based signals regulating integrative neuronal func-
tions, to H.A.). We thank Ms. Kiyoko Ito (Gifu University)
for providing technical assistance.
in 1,4-dioxaneꢀH2O at 90 °C, which was reported by
Ito et al.,20 was the most effective, producing acetamide
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Y.; Ueki, A.; Hojo, H.; Nakahara, Y.; Ito, Y. J. Org. Chem. 2011, 76,
5229–5239.
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1379.
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235. (b) Schmidt, R. R.; Michel, J. Angew. Chem., Int. Ed. Engl. 1980, 19,
731–732.
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Kobata, A. Carbohydr. Res. 1986, 157, 101–123.
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Dahmen, J.; Noori, G.; Stenvall, K. J. Org. Chem. 1988, 53, 5629–5647.
Supporting Information Available. All experimental
procedures and 1H and 13C spectra of all new com-
pounds.This material is available free of charge via the
Internet at http://pubs.acs.org.
The authors declare no competing financial interest.
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