The total synthesis of the neurogenic ganglioside LLG-3 isolated from the starfish Linckia laevigata

Article abstract of DOI:10.1002/anie.201006035

(Figure Presented) A gift from the star: The title compound, which was identified in starfish, contains two sialic acid units and was constructed utilizing a sialic acid derivative as a pivotal precursor. The potentially difficult conjugation of the phyto

Full text of DOI:10.1002/anie.201006035

Communications
DOI: 10.1002/anie.201006035
Oligosaccharides
The Total Synthesis of the Neurogenic Ganglioside LLG-3 Isolated
from the Starfish Linckia laevigata**
Hideki Tamai, Hiromune Ando,* Hide-Nori Tanaka, Ritsuko Hosoda-Yabe, Tomio Yabe,
Hideharu Ishida, and Makoto Kiso*
Recently, echinodermatous gangliosides have attracted much
attention because of their characteristic structure and their
potent neurogenic activity towards neuron-like rat adrenal
pheochromocytoma (PC-12) cells in the presence of the nerve
growth factor (NGF); this activity can greatly exceed that of
the mammalian ganglioside GM1.[1] Therefore, it is of great
importance to cultivate their potential as drug leads for
combating neurological disorders at the molecular level.
However, the extensive structural diversity in echinoderma-
tous gangliosides has impeded studies into their structure–
activity relationships and the mechanism by which they
promote neurogenesis. Their structure characteristically con-
tains a range of modified sialic acid units connected by a
variety of linker groups, and such units are never seen in
mammalian gangliosides.[2] Previously, we carried out the first
synthesis of two of these structures, namely, Neu5Gca-
(2,4)Neu5Ac and 8-O-SO3H-Neu5Aca(2,8)Neu5Ac, by
developing the N-Troc sialyl (Neu5Troc) donor as a key
intermediate to modified sialic acid residues.[3] Recently, we
were the first to synthesize the neurogenic ganglioside HLG-
2, which is found in the sea cucumber.[4] Herein we report the
first total synthesis of ganglioside LLG-3 (1; Scheme 1),[5]
which contains the 8-O-Me-Neu5Aca(2,11)Neu5Ac struc-
ture, by using Neu5Troc chemistry combined with the glucosyl
ceramide (Glc-Cer) cassette approach.
NGF) (20.6 Æ 2.2)% of PC-12 cells were affected. Ganglio-
side 1 is the second most potent enchinoderm ganglioside
amongst the fifteen that have been examined. The most
potent activity [(64.8 Æ 7.6)%] was exhibited by the gangli-
oside SJG-2,[1,7] which is a heptasaccharide and contains a
trisialyl tetrasaccharide unit. Therefore, even though 1 is a
tetrasaccharide, it is considered to be more effective than
SJG-2 and has attracted much attention in the field of
medicinal chemistry.
The characteristic structure of modified sialic acids (8-O-
Me-Neu5Aca(2,11)Neu5Gc) that is presumably responsible
for the neurogenic activity, was expected to be efficiently
synthesized from the common Neu5Troc donor
4
(Scheme 1).[8] Thus, it was envisioned that the synthesis of
the 5-NH2-Neu intermediate and the 8-O-methyl-Neu glyco-
late derivative could be achieved from 4 through the cleavage
of the Troc group with subsequent migration of the acetyl
group from O8 to N5. To avoid the loss of the glycan portion
as a result of inefficient coupling with the lipid portion we
anticipated using the glycosyl ceramide cassette approach,
which was recently established in our laboratory.[9] Therefore,
1 was disconnected into the trisaccharide segment 2 and the
Glc-Cer cassette 3. Then, 3 was fragmented into the glucosyl
donor 5 and the phytoceramide 6 as an aglycon.
As depicted in Scheme 2, the synthesis of the 8-O-methyl
sialyl unit from 4 commenced with the stereoselective
incorporation of benzylglycolate at the anomeric position to
give 8[5b] in a 91% yield (a/b = 7.5:1). Next, 8 was treated with
zinc under acidic conditions to produce the 8-hydroxy-N-
acetyl sialyl derivative 9, through the migration of the acetyl
group from O8 to N5, in a high yield. The 8-O-methylation of
9 was attempted using two different methylating reagents
(Me3OBF4 and MeOTf) but each attempt was unsuccessful,
and only generated a complex mixture of 5-methylimidate
derivatives and N-acetyl-N-methyl derivatives. Therefore, we
masked the C8 hydroxy group with a chloroacetyl group
[(ClCH2CO)2O, cat. DMAP, THF],[10] and then protected the
C5 acetamide with an acetyl group (isopropenyl acetate,
TsOH),[11] thereby obtaining 11 in a high yield (93% over two
steps). Compound 11 was then subjected to a two-step
sequence for 8-O-methylation. 1-Selenocarbamoylpiperidine
(12), which was recently developed by our research group,[12]
was found to be the best reagent for the selective dechloro-
acetylation at C8. The chloroacetyl group could also be
removed by using the widely used DABCO method[13]
(DABCO, EtOH), however this strategy promoted the
migration of the acetyl group from N5 to O8, giving the
fully acetylated derivative as the major product. Removal of
the chloroacetyl group using 12 and 2,6-lutidine as an acid
Ganglioside LLG-3 (1) was identified in the starfish
Linckia laevigata by Higuchi and co-workers (Scheme 1).[6]
They revealed that at a concentration of 10 mm LLG-3 caused
neurogenesis of (63.1 Æ 6.3)% of PC-12 cells in the presence
of NGF (5 ngmLÀ1), whereas in the control experiment (only
[*] H. Tamai, Dr. H. Ando, Dr. H.-N. Tanaka, R. Hosoda-Yabe, Dr. T. Yabe,
Dr. H. Ishida, Dr. M. Kiso
Department of Applied Bioorganic Chemistry, Gifu University
1-1 Yanagido, Gifu-shi, Gifu 501-1193 (Japan)
Fax: (+81)58-293-3452
hando@ gifu-u.ac.jp
H. Tamai, Dr. H. Ando, Dr. H.-N. Tanaka, R. Hosoda-Yabe,
Dr. M. Kiso
Department of Applied Bioorganic Chemistry
Institute for Integrated Cell-Material Sciences (WPI program)
Kyoto University
Yoshida Ushinomiya-cho, Sakyo-ku, Kyoto 606-8501 (Japan)
[**] Part 152 of the series: Synthetic studies on sialoglycoconjugates.
This work was financially supported by MEXT of Japan (WPI
program and grant-in-aid for Scientific Research to M.K., No.
1701007 and No. 22380067). We thank Kiyoko Ito for technical
assistance.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201006035.
2330
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2011, 50, 2330 –2333

scavenger with subsequent
methylation using Meerweinꢀs
reagent,[14] successfully intro-
duced a methyl group at the
C8 hydroxy group, thus generat-
ing 13 in an 81% yield (two
steps). Upon hydrogenolysis, 13
was converted into the terminal
sialyl glycolic unit 14 in a quan-
titative yield.
To synthesize the 5-NH2-
sialyl unit 17, 4 was reacted
with the galactosyl acceptor 15
according to the procedure
reported in our previous
paper[8a,c] to give disaccharide
16 (Scheme 3). MeCN sup-
pressed the migration of the
acetyl group from O8 to N5
during the Troc removal, which
was achieved using zinc under
acidic conditions. This reaction
produced the 5-amino sialyl unit
17 in a satisfactory yield. The
carboxylic unit 14 and the amine
Scheme 1. Retrosynthetic scheme for target molecule 1. Ac=acetyl, LG=leaving group, P=protecting
group, PMB=p-methoxybenzyl, Troc=2,2,2-trichloroethoxycarbonyl.
unit 17 were successfully coupled to deliver the key terminal
trisaccharide 18 in an 88% yield; 18 was then manipulated in
a straightforward manner to furnish the imidate 19.[15]
The synthesis of the Glc-Cer portion (30) was conducted
in accordance with our earlier report.[4,9] The (R)-2-hydroxy-
tricosanoic acid derivative 23 was successfully assembled
Scheme 2. Synthesis of 8-O-Me-sialyl glycolic acid 14. a) 4, NIS, TfOH,
EtCN, molecular sieves (4 ꢀ), À808C, 91% (a/b=7.5:1); b) Zn, DMF/
AcOH (4:1), 84%; c) (ClCH2CO)2O, DMAP, THF, 93%; d) TsOH·H2O,
isopropenyl acetate, 858C, 98%; e) 12, 2,6-lutidine, DMF, 658C;
f) Me3OBF4, DTBMP, CH2Cl2, reflux, 81% (2 steps); g) H2, 20%
Pd(OH)2/C, EtOAc, quant. Bn=benzyl, DMAP=4-(dimethylamino)pyr-
idine, DMF=N,N’-dimethylformamide, DTBMP=2,6-di-tert-butyl-4-
methylpyridine, NIS=N-iodosuccinimide, Tf=trifluoromethanesul-
fonyl, THF=tetrahydrofuran, Ts=p-toluenesulfonyl.
Scheme 3. Synthesis of trisaccharide unit 19. a) See Ref 8a and 8c for
reaction conditions; b) Zn, MeCN/AcOH (4:1), 89%; c) 14, EDC·HCl,
HOBt, NaHCO3, MeCN, 88%; d) H2, 20% Pd(OH)2/C, EtOAc;
e) Bz2O, DMAP, Py, 408C, 96% (2 steps); f) CAN, toluene/MeCN/H2O
(5:6:3), 08C, 80%; g) CCl3CN, DBU, CH2Cl2, 08C, 84%. Bz=benzoyl,
CAN=cerium(IV) ammonium nitrate, DBU=1,8-diazabicyclo-
[5.4.0]undec-7-ene, EDC=1-ethyl-3-(3-dimethylaminopropyl)carbodi-
imide, HOBt=1-hydroxybenzotriazole, Imd=trichloroacetimidoyl,
PMP=p-methoxyphenyl, Py=pyridine.
Angew. Chem. Int. Ed. 2011, 50, 2330 –2333
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
2331

Communications
Scheme 4. Synthesis of the ceramide portion 25. a) 21, nBuLi, HMPA;
b) BzCl, DMAP, Py, 41% (2 steps); c) H2, 10% Pd(OH)2/C, EtOAc, RT;
d) DMP, NaHCO3, CH2Cl2, RT; e) NaClO2, NaH2PO4, 2-methyl-2-
butene, THF/tBuOH/H2O (4:5:1), RT, 84% (3 steps); f) 24, EDC·HCl,
HOBt, CH2Cl2, 61%; g) TrCl, Py, 508C; h) BzCl, DMAP, Py, 508C, 78%
(2 steps); i) H2, 10% Pd(OH)2/C, 88%. DMP=Dess–Martin perio-
dinane, HMPA=hexamethylphosphoric acid triamide, Tr=triphenyl-
methyl.
Scheme 5. Synthesis of the Glc-Cer cassette 30. a) 25 (1 equiv), NIS,
TfOH, molecular sieves (4 ꢀ), CH2Cl2, 08C; b) TBAF, THF, 308C, 93%.
Piv=pivaloyl, TBAF=tetra-n-butylammonium fluoride, TBS=tert-butyl-
dimethylsilyl.
from oxirane 20 and icos-1-yne (21) using a five-step reaction
sequence (Scheme 4). Phytosphingosine (24), which was
synthesized from 1,2:4,6-diacetone-d-mannose using proce-
dures reported by Tzou and Lin et al.,[16,17] was coupled with
23 using a carbodiimide (EDC·HCl, HOBt) to yield the
phytoceramide framework. In preparation for the subsequent
coupling with the Glc section, the hydroxy groups were
protected with benzoyl groups and the resulting product was
additionally manipulated to provide the 1-hydroxy derivative
25.
The protecting group at C4 of the Glc donor was shown to
strongly influence the yield of the coupling reaction with the
ceramide acceptor 25 (Scheme 5). As previously reported,[9]
the 4-chloroacetyl Glc donor 26[17] provided Glc-Cer 28 in
poor yield (23%). In contrast, the use of the TBS derivative
27[17] increased the yield to 81%. Upon exposure to TBAF,
the TBS group of 29 was removed to afford the Glc-Cer
acceptor 30, which was now ready for the assembly of the
glycolipid framework of LLG-3.
than twice the cell-body diameter varied depending on the
exogenous factors; for example, NGF at 5 ngmLÀ1 (0.57/cell),
NGF at high concentration (0.85/cell), and LLG-3 at 1 mm
(0.73/cell). These results suggest that in the presence of NGF
LLG-3 enhances the elongation of the neurites rather than
their formation.
In summary, we have presented the first synthesis of
ganglioside LLG-3 (1). The efficient assembly of the unusual
structure comprising of an 8-O-Me-sialic acid and an N-
glycolyl-sialic acid reinforces the utility of the N-Troc sialyl
donor 4 for the synthesis of different sialyl glycosides. The
Glc-Cer cassette approach provides an efficient route to
gangliosides that contain a phytoceramide moiety. It was also
revealed that silyl protection of the C4 hydroxy group of the
glucosyl donor remarkably improved the yield of the coupling
reaction with the phytoceramide acceptor. Finally, we could
demonstrate the neurogenic activity of the synthesized LLG-3
(1) towards the PC-12 cells. We are also investigating the
Pleasingly, the final glycosidation of the trisac-
charide donor 19 (1.0 equiv) with the Glc-Cer
acceptor 30 (1.0 equiv), promoted by TMSOTf in
CH2Cl2 at 08C, successfully produced the protected
LLG-3 31 in good yield (73%; Scheme 6). Treat-
ment of 31 with TFA resulted in removal of the
PMB groups, and subsequent treatment with LiCl
liberated the carboxylic acid, and then removal of
the acyl groups[5e] successfully delivered ganglioside
LLG-3 (1, 25 mg).
Examination of the neurite growth of PC-12
cells revealed that the synthesized ganglioside
LLG-3 (1) potentiated the neurite outgrowth in
the presence of NGF (5 ngmLÀ1).[18] LLG-3 (1 mm),
NGF (5 ngmLÀ1, control), and NGF at high con-
Scheme 6. Final coupling and global deprotection to deliver LLG-3 1. a) TMSOTf,
CH2Cl2, acid washed molecular sieves (4 ꢀ), 08C, 73%; b) TFA, CH2Cl2, 08C;
c) hydrazine acetate, THF, 08C; d) LiCl, Py, reflux; e) 0.1 m NaOH (aq.), 08C to
458C, 82% (4 steps). TFA=trifluoroacetic acid, TMSOTf=trimethylsilyl trifluoro-
centration (50 ngmLÀ1, positive control) all gave a
similar increase in the number of neurites that had a
length between one and two cell-body diameters. In
contrast, the number of neurites that were more methanesulfonate.
2332
www.angewandte.org
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2011, 50, 2330 –2333

[7] M. Kaneko, F. Kisa, K. Yamada, T. Miyamoto, R. Higuchi, Eur. J.
Org. Chem. 2003, 1004 – 1008.
neurogenic activity towards PC-12 cells by using other
synthesized gangliosides such as GM1,[19] GM2,[20] GQ1b,[5]
X1,[21] and HLG-2.[4] The detailed results of their activities will
be reported in due course.
[8] a) H. Ando, Y. Koike, H. Ishida, M. Kiso, Tetrahedron Lett. 2003,
44, 6883 – 6886; b) H. Tanaka, M. Adachi, T. Takahashi, Chem.
Eur. J. 2005, 11, 849 – 862; c) T. Fuse, H. Ando, A. Imamura, N.
Sawada, H. Ishida, M. Kiso, T. Ando, S.-C. Li, Y.-T. Li,
Glycoconjugate J. 2006, 23, 329 – 343.
[9] a) A. Imamura, H. Ando, H. Ishida, M. Kiso, J. Org. Chem. 2009,
74, 3009 – 3023; b) K. Fujikawa, T. Nohara, A. Imamura, H.
Ando, H. Ishida, M. Kiso, Tetrahedron Lett. 2010, 51, 1126 –
1130.
[10] A. Sakakura, K. Kawajiri, T. Ohkubo, Y. Kosugi, K. Ishihara, J.
Am. Chem. Soc. 2007, 129, 14775 – 14779.
[11] A. V. Demchenko, G.-J. Boons, Tetrahedron Lett. 1998, 39, 3065 –
3068.
[12] S. Sogabe, H. Ando, M. Koketsu, H. Ishihara, Tetrahedron Lett.
2006, 47, 6603 – 6606.
[13] D. J. Lefeber, J. P. Kamerling, J. F. G. Vliegenthart, Org. Lett.
2000, 2, 701 – 703.
[14] M. J. Diem, D. F. Burow, J. L. Fry, J. Org. Chem. 1977, 42, 1801 –
1802.
[15] a) R. R. Schmidt, Angew. Chem. 1986, 98, 213 – 236; Angew.
Chem. Int. Ed. Engl. 1986, 25, 212 – 235; b) R. R. Schmidt, J.
Michel, Angew. Chem. 1980, 92, 763 – 764; Angew. Chem. Int.
Ed. Engl. 1980, 19, 731 – 732.
Received: September 27, 2010
Revised: December 10, 2010
Published online: February 8, 2011
Keywords: carbohydrates · gangliosides · neurogenesis ·
.
sialic acids · total synthesis
[1] M. Kaneko, K. Yamada, T. Miyamoto, M. Inagaki, R. Higuchi,
Chem. Pharm. Bull. 2007, 55, 462 – 463.
[2] For reports on the synthesis of the glycan part of echinoderm-
atous gangliosides, see: a) H. Ando, H. Shimizu, Y. Katano, Y.
Koike, S. Koizumi, H. Ishida, M. Kiso, Carbohydr. Res. 2006, 341,
1522 – 1532; b) S. Hanashima, Y. Yamaguchi, Y. Ito, K.-I. Sato,
Tetrahedron Lett. 2009, 50, 6150 – 6153.
[3] H. Ando, Y. Koike, S. Koizumi, H. Ishida, M. Kiso, Angew.
Chem. 2005, 117, 6917 – 6921; Angew. Chem. Int. Ed. 2005, 44,
6759 – 6763.
[4] Y. Iwayama, H. Ando, H. Ishida, M. Kiso, Chem. Eur. J. 2009, 15,
4637 – 4648.
[16] H.-Y. Chiu, D.-L. M. Tzou, L. N. Patkar, C.-C. Lin, J. Org. Chem.
2003, 68, 5788 – 5791.
[5] For reports on the synthesis of the glycan part, see: a) S.
Hanashima, D. Ishikawa, S. Akai, K.-I. Sato, Carbohydr. Res.
2009, 344, 747 – 752; for preceding reports on the synthesis of
Neu5Aca(2,11)Neu linkage, see: b) C.-T. Ren, C.-S. Chen, S.-H.
Wu, J. Org. Chem. 2002, 67, 1376 – 1379; c) J. C. McAuliffe, D.
Rabuka, O. Hindsgaul, Org. Lett. 2002, 4, 3067 – 3069; d) G.-T.
Fan, C.-C. Lee, C.-C. Lin, J.-M. Fang, J. Org. Chem. 2002, 67,
7565 – 7568; e) C.-T. Ren, C.-S. Chen, Y.-P. Yu, Y.-F. Tsai, P.-Y.
Lin, Y.-J. Chen, W. Zou, S.-H. Wu, Chem. Eur. J. 2003, 9, 1085 –
1095.
[17] For the experimental procedures for the synthesis of compounds
24, 26, and 27 see the Supporting Information.
[18] For details, please see the Supporting Information.
[19] a) A. Hasegawa, T. Nagahama, M. Kiso, Carbohydr. Res. 1992,
235, C13-C17; b) A. Hasegawa, H. Ishida, T. Nagahama, M.
Kiso, J. Carbohydr. Chem. 1993, 12, 703 – 718.
[20] A. Hasegawa, T. Nagahama, H. Ohki, M. Kiso, J. Carbohydr.
Chem. 1992, 11, 699 – 714.
[21] S. Nakashima, H. Ando, A. Imamura, N. Yuki, H. Ishida, M.
Kiso, Chem. Eur. J. 2011, 17, 588 – 597.
[6] M. Inagaki, R. Isobe, R. Higuchi, Eur. J. Org. Chem. 1999, 771 –
774.
Angew. Chem. Int. Ed. 2011, 50, 2330 –2333
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
2333